An Improved MLVF Method and Its Comparison with Traditional MLVF, spa Typing, MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

PubMed Central

Du, Xue-Fei; Xiao, Meng; Liang, Hong-Yan; Sun, Zhe; Jiang, Yue-Hong; Chen, Guo-Yu; Meng, Xiao-Yu; Zou, Gui-Ling; Zhang, Li; Liu, Ya-Li; Zhang, Hui; Sun, Hong-Li; Jiang, Xiao-Feng; Xu, Ying-Chun

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates. PMID:24406728

Molecular analysis of Acinetobacter baumannii strains isolated in Lebanon using four different typing methods.

PubMed

Rafei, Rayane; Dabboussi, Fouad; Hamze, Monzer; Eveillard, Matthieu; Lemarié, Carole; Gaultier, Marie-Pierre; Mallat, Hassan; Moghnieh, Rima; Husni-Samaha, Rola; Joly-Guillou, Marie-Laure; Kempf, Marie

2014-01-01

This study analyzed 42 Acinetobacter baumannii strains collected between 2009-2012 from different hospitals in Beyrouth and North Lebanon to better understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and blaOXA-51 sequence-based typing (SBT). Among 31 carbapenem resistant strains, we have detected three carbapenem resistance genes: 28 carried the blaOXA-23 gene, 1 the blaOXA-24 gene and 2 strains the blaOXA-58 gene. This is the first detection of blaOXA-23 and blaOXA-24 in Lebanon. PFGE identified 11 types and was the most discriminating technique followed by rep-PCR (9 types), blaOXA-51 SBT (8 types) and MLST (7 types). The PFGE type A'/ST2 was the dominant genotype in our collection present in Beyrouth and North Lebanon. The clustering agreement between all techniques was measured by adjust Wallace coefficient. An overall agreement has been demonstrated. High values of adjust Wallace coefficient were found with followed combinations: PFGE to predict MLST types  = 100%, PFGE to predict blaOXA-51 SBT = 100%, blaOXA-51 SBT to predict MLST = 100%, MLST to predict blaOXA-51 SBT = 84.7%, rep-PCR to predict MLST = 81.5%, PFGE to predict rep-PCR = 69% and rep-PCR to predict blaOXA-51 SBT = 67.2%. PFGE and MLST are gold standard methods for outbreaks investigation and population structure studies respectively. Otherwise, these two techniques are technically, time and cost demanding. We recommend the use of blaOXA-51 SBT as first typing method to screen isolates and assign them to their corresponding clonal lineages. Repetitive sequence-based PCR is a rapid tool to access outbreaks but careful interpretation of results must be always performed.

Application of a multilocus variable number of tandem repeats analysis to regional outbreak surveillance of Enterohemorrhagic Escherichia coli O157:H7 infections.

PubMed

Konno, Takayuki; Yatsuyanagi, Jun; Saito, Shioko

2011-01-01

A total of 18 strains of EHEC O157:H7 were isolated from distinct cases in Akita Prefecture, Japan from July to September 2007. The genetic relatedness of these isolates was investigated by performing a multilocus variable number of tandem repeats analysis (MLVA) and a pulsed-field gel electrophoresis (PFGE) analysis using XbaI. The PFGE analyses allowed us to group these 18 isolates into three major clusters. The MLVA results correlated closely with those obtained by PFGE, although some variants were found within the clusters obtained by PFGE, thus highlighting the utility of this technique for determining a precise classification when it is difficult to differentiate between isolates with indistinguishable or very similar PFGE patterns. In addition, MLVA is a much easier and more rapid method than PFGE for analysis of the genetic relatedness of strains. Thus, as a second molecular epidemiological subtyping method, MLVA is useful for the regional outbreak surveillance of EHEC O157:H7 infections.

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation

PubMed Central

Jackson, Brendan R.; Tarr, Cheryl; Strain, Errol; Jackson, Kelly A.; Conrad, Amanda; Carleton, Heather; Katz, Lee S.; Stroika, Steven; Gould, L. Hannah; Mody, Rajal K.; Silk, Benjamin J.; Beal, Jennifer; Chen, Yi; Timme, Ruth; Doyle, Matthew; Fields, Angela; Wise, Matthew; Tillman, Glenn; Defibaugh-Chavez, Stephanie; Kucerova, Zuzana; Sabol, Ashley; Roache, Katie; Trees, Eija; Simmons, Mustafa; Wasilenko, Jamie; Kubota, Kristy; Pouseele, Hannes; Klimke, William; Besser, John; Brown, Eric; Allard, Marc; Gerner-Smidt, Peter

2016-01-01

Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens. PMID:27090985

Inter-hospital outbreak of Klebsiella pneumoniae producing KPC-2 carbapenemase in Ireland.

PubMed

Morris, Dearbháile; Boyle, Fiona; Morris, Carol; Condon, Iris; Delannoy-Vieillard, Anne-Sophie; Power, Lorraine; Khan, Aliya; Morris-Downes, Margaret; Finnegan, Cathriona; Powell, James; Monahan, Regina; Burns, Karen; O'Connell, Nuala; Boyle, Liz; O'Gorman, Alan; Humphreys, Hilary; Brisse, Sylvain; Turton, Jane; Woodford, Neil; Cormican, Martin

2012-10-01

To describe an outbreak of KPC-2-producing Klebsiella pneumoniae with inter-hospital spread and measures taken to control transmission. Between January and March 2011, 13 K. pneumoniae isolates were collected from nine patients at hospital A and two patients at hospital B. Meropenem, imipenem and ertapenem MICs were determined by Etest, carbapenemase production was confirmed by the modified Hodge method and by a disc synergy test, and confirmed carbapenemase producers were tested for the presence of carbapenemase-encoding genes by PCR. PFGE, plasmid analysis, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST) analysis were performed on all or a subset of isolates. Meropenem, imipenem and ertapenem MICs were 4 to >32, 8-32 and >16 mg/L, respectively. PCR and sequencing confirmed the presence of bla(KPC-2). PFGE identified four distinguishable (≥88%) pulsed-field profiles (PFPs). Isolates distinguishable by PFGE had identical MLVA profiles, and MLST analysis indicated all isolates belonged to the ST258 clone. Stringent infection prevention and control measures were implemented. Over a period of almost 8 months no further carbapenemase-producing Enterobacteriaceae (CPE) were isolated. However, KPC-2-producing K. pneumoniae was detected in two further patients in hospital A in August (PFP indistinguishable from previous isolates) and October 2011 (PFP similar to but distinguishable from previous isolates). Stringent infection prevention and control measures help contain CPE in the healthcare setting; however, in the case of hospital A, where CPE appears to be established in the population served, it may be virtually impossible to achieve eradication or avoid reintroduction into the hospital.

A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in 2010

PubMed Central

Haendiges, Julie; Jones, Jessica; Myers, Robert A.; Mitchell, Clifford S.; Butler, Erin

2016-01-01

ABSTRACT In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations. IMPORTANCE Vibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations. PMID:26994080

A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in 2010.

PubMed

Haendiges, Julie; Jones, Jessica; Myers, Robert A; Mitchell, Clifford S; Butler, Erin; Toro, Magaly; Gonzalez-Escalona, Narjol

2016-06-01

In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations. Vibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sequence Typing Confirms that a Predominant Listeria monocytogenes Clone Caused Human Listeriosis Cases and Outbreaks in Canada from 1988 to 2010

PubMed Central

Reimer, Aleisha; Verghese, Bindhu; Lok, Mei; Ziegler, Jennifer; Farber, Jeffrey; Pagotto, Franco; Graham, Morag; Nadon, Celine A.

2012-01-01

Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades. PMID:22337989

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

PubMed

Guerrero, Abraham; Gómez Gil Rodríguez, Bruno; Wong-Chang, Irma; Lizárraga-Partida, Marcial Leonardo

2015-01-01

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing.

PubMed

Magnússon, S H; Guðmundsdóttir, S; Reynisson, E; Rúnarsson, A R; Harðardóttir, H; Gunnarson, E; Georgsson, F; Reiersen, J; Marteinsson, V Th

2011-10-01

Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans. A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database. The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

PubMed Central

Raphael, Brian H.; Joseph, Lavin A.; Meno, Sarah R.; Fernández, Rafael A.; Maslanka, Susan E.

2012-01-01

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE. PMID:23042179

Listeria monocytogenes Circulating in Rabbit Meat Products and Slaughterhouses in Italy: Prevalence Data and Comparison Among Typing Results.

PubMed

De Cesare, Alessandra; Parisi, Antonio; Mioni, Renzo; Comin, Damiano; Lucchi, Alex; Manfreda, Gerardo

2017-03-01

Rabbit meat has outstanding dietetic and nutritional properties. However, few data on microbiological hazards associated with rabbit productions are available. In this study, the presence of Listeria monocytogenes was determined in 430 rabbit carcasses, 256 rabbit meat cuts and products, and 599 environmental sponges collected from four Italian rabbit slaughterhouses over a period of 1 year. Prevalence of L. monocytogenes among the 1285 rabbit meat and environmental samples was 11%, with statistically significant differences between slaughterhouses. The highest prevalence (33.6%) was observed in rabbit meat cuts and products; the majority of positive environmental samples were collected from conveyor belts. Overall, 27.9% and 14.3% of rabbit cuts and carcasses, respectively, had L. monocytogenes counts higher than 1 colony-forming unit (CFU)/10 g. A selection of 123 isolates from positive samples was genotyped and serotyped to determine genetic profiles and diversity among L. monocytogenes isolates contaminating different slaughterhouses and classes of products investigated. Discriminatory power and concordance among the results obtained using multilocus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), automated EcoRI ribotyping, and serotyping were assessed. The isolates selected for typing were classified into serotypes 1/2a (52.8%), 1/2c (32.5%), and 1/2b (14.6%). The majority of the isolates were classified as ST14 (34.1%), ST9 (35.5%), ST121 (17.9%), and ST224 (14.6%). The greatest discriminatory power was observed with the MLVA typing, followed by MLST, PFGE, and ribotyping. The best bidirectional concordance was achieved between PFGE and MLST. There was 100% correlation between both MLST and MLVA with serotype. Moreover, a high unidirectional correspondence was observed between MLVA and both MLST and PFGE, as well as between PFGE and both MLST and serotyping. The results of this study show for the first time in Italy prevalence and genetic profiles of L. monocytogenes isolated in rabbit products and slaughterhouses.

Multilocus variable-number tandem repeat analysis distinguishes outbreak and sporadic Escherichia coli O157:H7 isolates.

PubMed

Noller, Anna C; McEllistrem, M Catherine; Pacheco, Antonio G F; Boxrud, David J; Harrison, Lee H

2003-12-01

Escherichia coli O157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by using XbaI and the confirmatory enzyme SpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method for E. coli O157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE.

Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains▿ †

PubMed Central

Faria, Nuno A.; Carrico, João A.; Oliveira, Duarte C.; Ramirez, Mário; de Lencastre, Hermínia

2008-01-01

Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies. PMID:17989188

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

PubMed

Jørgensen, H J; Mørk, T; Caugant, D A; Kearns, A; Rørvik, L M

2005-12-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

PubMed Central

Jørgensen, H. J.; Mørk, T.; Caugant, D. A.; Kearns, A.; Rørvik, L. M.

2005-01-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex. PMID:16332822

Emergence of Pseudomonas aeruginosa with class 1 integron carrying blaVIM-2 and blaVIM-4 in the University Clinical Hospital of Bialystok (northeastern Poland).

PubMed

Michalska-Falkowska, Anna; Sacha, Paweł Tomasz; Grześ, Henryk; Hauschild, Tomasz; Wieczorek, Piotr; Ojdana, Dominika; Tryniszewska, Elżbieta Anna

2017-07-11

The effectiveness of carbapenems, considered as last-resort antimicrobials in severe infections, becomes compromised by bacterial resistance. The production of metallo-β-lactamases (MBLs) is the most significant threat to carbapenems activity among Pseudomonas aeruginosa. The aim of this study was to assess the presence and type of MBLs genes in carbapenem-resistant P. aeruginosa clinical strains, to identify the location of MBLs genes and to determine genetic relatedness between MBL-producers using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The first identified MBL-positive (with blaVIM genes) P. aeruginosa strains were isolated from patients hospitalized in the University Clinical Hospital of Bialystok in the period from September 2012 to December 2013. Variants of MBLs genes and variable integron regions were characterized by PCR and sequencing. PFGE was performed after digesting of bacterial genomes by XbaI enzyme. By MLST seven housekeeping genes were analyzed for the determination of sequence type (ST). Three strains carried the blaVIM-2 gene and one harbored the blaVIM-4 gene. The blaVIM genes resided within class 1 integrons. PCR mapping of integrons revealed the presence of four different cassette arrays. Genetic relatedness analysis by PFGE classified VIM-positive strains into four unrelated pulsotypes (A-D). MLST demonstrated the presence of four (ST 111, ST27, and ST17) different sequence type including one previously undescribed new type of ST 2342. Antimicrobial susceptibility testing showed that VIM-positive strains were resistant to carbapenems, cephalosporins, aminoglycosides, and quinolones, intermediate to aztreonam, and susceptible only to colistin. Integrons mapping, PFGE, and MLST results may point to different origin of these strains and independent introduction into hospitalized patients.

Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

PubMed Central

Henri, Clémentine; Félix, Benjamin; Guillier, Laurent; Leekitcharoenphon, Pimlapas; Michelon, Damien; Mariet, Jean-François; Aarestrup, Frank M.; Mistou, Michel-Yves; Hendriksen, René S.

2016-01-01

ABSTRACT Listeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety. IMPORTANCE This study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices. PMID:27235443

Fluorescent Amplified-Fragment Length Polymorphism Genotyping of Neisseria meningitidis Identifies Clones Associated with Invasive Disease

PubMed Central

Goulding, Jonathan N.; Hookey, John V.; Stanley, John; Olver, Will; Neal, Keith R.; Ala'Aldeen, Dlawer A. A.; Arnold, Catherine

2000-01-01

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures. PMID:11101599

Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance.

PubMed

Bojarska, A; Molska, E; Janas, K; Skoczyńska, A; Stefaniuk, E; Hryniewicz, W; Sadowy, E

2016-06-01

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000-2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

PubMed Central

Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation.

PubMed

Jackson, Brendan R; Tarr, Cheryl; Strain, Errol; Jackson, Kelly A; Conrad, Amanda; Carleton, Heather; Katz, Lee S; Stroika, Steven; Gould, L Hannah; Mody, Rajal K; Silk, Benjamin J; Beal, Jennifer; Chen, Yi; Timme, Ruth; Doyle, Matthew; Fields, Angela; Wise, Matthew; Tillman, Glenn; Defibaugh-Chavez, Stephanie; Kucerova, Zuzana; Sabol, Ashley; Roache, Katie; Trees, Eija; Simmons, Mustafa; Wasilenko, Jamie; Kubota, Kristy; Pouseele, Hannes; Klimke, William; Besser, John; Brown, Eric; Allard, Marc; Gerner-Smidt, Peter

2016-08-01

Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Genetic Characterization of Atypical Enteropathogenic Escherichia coli Isolates from Ewes' Milk, Sheep Farm Environments, and Humans by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

PubMed Central

Otero, Verónica; Rodríguez-Calleja, José-María; Otero, Andrés; García-López, María-Luisa

2013-01-01

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water. PMID:23872571

Molecular and phenotypic characterization of Listeria monocytogenes from U.S. Department of Agriculture Food Safety and Inspection Service surveillance of ready-to-eat foods and processing facilities.

PubMed

Ward, Todd J; Evans, Peter; Wiedmann, Martin; Usgaard, Thomas; Roof, Sherry E; Stroika, Steven G; Hise, Kelley

2010-05-01

A panel of 501 Listeria monocytogenes isolates obtained from the U.S. Department of Agriculture Food Safety and Inspection Service monitoring programs for ready-to-eat (RTE) foods were subtyped by multilocus genotyping (MLGT) and by sequencing the virulence gene inlA, which codes for internalin. MLGT analyses confirmed that clonal lineages associated with previous epidemic outbreaks were rare (7.6%) contaminants of RTE meat and poultry products and their production environments. Conversely, sequence analyses revealed mutations leading to 11 different premature stop codons (PMSCs) in inlA, including three novel PMSC mutations, and revealed that the frequency of these virulence-attenuating mutations among RTE isolates (48.5%) was substantially higher than previously appreciated. Significant differences (P < 0.001) in the frequency of inlA PMSCs were observed between lineages and between major serogroups, which could partially explain differences in association of these subtypes with human listeriosis. Interrogation of single-nucleotide polymorphisms responsible for PMSCs in inlA improved strain resolution among isolates with the 10 most common pulsed-field gel electrophoresis (PFGE) patterns, 8 of which included isolates with a PMSC in inlA. The presence or absence of PMSCs in inlA accounted for significant differences (P < 0.05) in Caco-2 invasion efficiencies among isolates with identical PFGE patterns, and the proportion of PulseNet entries from clinical sources was significantly higher (P < 0.001) for PFGE patterns exclusively from isolates with full-length inlA. These results indicated that integration of PFGE and DNA sequence-based subtyping provides an improved framework for prediction of relative risk associated with L. monocytogenes strains from RTE foods.

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States.

PubMed

Chen, Yi; Luo, Yan; Curry, Phillip; Timme, Ruth; Melka, David; Doyle, Matthew; Parish, Mickey; Hammack, Thomas S; Allard, Marc W; Brown, Eric W; Strain, Errol A

2017-01-01

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents.

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States

PubMed Central

Chen, Yi; Luo, Yan; Curry, Phillip; Timme, Ruth; Melka, David; Doyle, Matthew; Parish, Mickey; Hammack, Thomas S.; Allard, Marc W.; Brown, Eric W.; Strain, Errol A.

2017-01-01

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from different outbreaks/incidents. PMID:28166293

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS.

PubMed

Stepan, Ryan M; Sherwood, Julie S; Petermann, Shana R; Logue, Catherine M

2011-06-27

Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar.

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

NASA Astrophysics Data System (ADS)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec.

PubMed

Vincent, Caroline; Usongo, Valentine; Berry, Chrystal; Tremblay, Denise M; Moineau, Sylvain; Yousfi, Khadidja; Doualla-Bell, Florence; Fournier, Eric; Nadon, Céline; Goodridge, Lawrence; Bekal, Sadjia

2018-08-01

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative study of all Salmonella enterica serovar Enteritidis strains isolated from food and food animals in Greece from 2008 to 2010 with clinical isolates.

PubMed

Papadopoulos, T; Petridou, E; Zdragas, A; Mandilara, G; Nair, S; Peters, T; Chattaway, M; de Pinna, E; Passiotou, M; Vatopoulos, A

2016-05-01

The aim of the present work was to study the epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) in Greece, comparing all the food and food animal isolates during a 3-year period with clinical isolates. Submission of the generated data to the PulseNet Europe database was carried out in order to study the population structure of this particular serovar and indicate possible connections with European strains. One hundred and sixty-eight (168) S. Enteritidis strains of human, animal, and food origin, isolated during the period 2008-2010 in Greece, were studied. Strains were characterized by phenotypic (antibiotic resistance) and molecular [pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)] methods. PFGE revealed 39 XbaI, 48 BlnI, and 80 XbaI-BlnI distinct pulsotypes, suggesting several clones circulating through the food chain and multiple sources of transmission. Submission to the PulseNet Europe database indicated that PFGE profile SENTXB.0001, the most common PFGE profile in Europe, was also predominant in Greece (33.3 %). MLST showed that all the strains studied shared the same sequence type (ST11), representing the most common ST in Europe. High rates of resistance to nalidixic acid were observed among human and poultry isolates (~25 %), indicating the potential fluoroquinolone treatment failure. Our data suggest that strains originating from multiple reservoirs circulated in Greece through the food chain during the study period. Predominant profiles in Greece were common to PulseNet Europe profiles, indicating similarities between the S. Enteritidis populations in Greece and Europe.

Genotyping of clinical and environmental multidrug resistant Enterococcus faecium strains.

PubMed

Shokoohizadeh, Leili; Mobarez, Ashraf Mohabati; Alebouyeh, Masoud; Zali, Mohammad Reza; Ranjbar, Reza

2017-01-01

Multidrug resistant (MDR) Enterococcus faecium is a nosocomial pathogen and clonal complex 17 (CC17) is the main genetic subpopulation of E. faecium in hospitals worldwide. There has thus far been no report of major E. faecium clones in Iranian hospitals. The present study analyzed strains of MDR E. faecium obtained from patients and the Intensive Care Unit environments using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine the antibiotic resistance patterns and genetic features of the dominant. clones of E. faecium. PFGE and MLST analysis revealed the presence of 17and 15 different subtypes, respectively. Of these, 18 (86%) isolates belonged toCC17. Most strains in this clonal complex harbored the esp gene and exhibited resistance to vancomycin, teicoplanin, ampicillin, ciprofloxacin, gentamicin, and erythromycin. The MLST results revealed 12 new sequence types (ST) for the first time. Approximately 50% of the STs were associated with ST203. Detection of E. faecium strains belonging to CC17 on medical equipment and in clinical specimens verified the circulation of high-risk MDR clones among the patients and in hospital environments in Iran.

Characterization of Streptococcus pyogenes from Animal Clinical Specimens, Spain

PubMed Central

Vela, Ana Isabel; Villalón, Pilar; Sáez-Nieto, Juan Antonio; Chacón, Gema; Domínguez, Lucas

2017-01-01

Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on isolation from animals. We provide a detailed characterization (emm typing, pulsed-field gel electrophoresis [PFGE], and multilocus sequence typing [MLST]) of 15 S. pyogenes isolates from animals associated with different clinical backgrounds. We also investigated erythromycin resistance mechanisms and phenotypes and virulence genes. We observed 2 emm types: emm12 (11 isolates) and emm77 (4 isolates). Similarly, we observed 2 genetic linages, sequence type (ST) 26 and ST63. Most isolates exhibited the M macrolide resistance phenotype and the mefA/ermB genotype. Isolates were grouped into 2 clones on the basis of emm-MLST-PFGE-virulence gene profile combinations: clone 1, characterized by the combined genotype emm12-ST36-pulsotype A-speG; and clone 2, characterized by the genotype emm77-ST63-pulsotype B-speC. Our results do not show conclusively that animals may represent a new reservoir of S. pyogenes but indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals. PMID:29148379

“Epidemic Clones” of Listeria monocytogenes Are Widespread and Ancient Clonal Groups

PubMed Central

Cantinelli, Thomas; Chenal-Francisque, Viviane; Diancourt, Laure; Frezal, Lise; Leclercq, Alexandre; Wirth, Thierry

2013-01-01

The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how “epidemic clones” should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the “epidemic clone” denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space. PMID:24006010

Whole genome sequencing as a tool to investigate a cluster of seven cases of listeriosis in Austria and Germany, 2011-2013.

PubMed

Schmid, D; Allerberger, F; Huhulescu, S; Pietzka, A; Amar, C; Kleta, S; Prager, R; Preußel, K; Aichinger, E; Mellmann, A

2014-05-01

A cluster of seven human cases of listeriosis occurred in Austria and in Germany between April 2011 and July 2013. The Listeria monocytogenes serovar (SV) 1/2b isolates shared pulsed-field gel electrophoresis (PFGE) and fluorescent amplified fragment length polymorphism (fAFLP) patterns indistinguishable from those from five food producers. The seven human isolates, a control strain with a different PFGE/fAFLP profile and ten food isolates were subjected to whole genome sequencing (WGS) in a blinded fashion. A gene-by-gene comparison (multilocus sequence typing (MLST)+) was performed, and the resulting whole genome allelic profiles were compared using SeqSphere(+) software version 1.0. On analysis of 2298 genes, the four human outbreak isolates from 2012 to 2013 had different alleles at ≤6 genes, i.e. differed by ≤6 genes from each other; the dendrogram placed these isolates in between five Austrian unaged soft cheese isolates from producer A (≤19-gene difference from the human cluster) and two Austrian ready-to-eat meat isolates from producer B (≤8-gene difference from the human cluster). Both food products appeared on grocery bills prospectively collected by these outbreak cases after hospital discharge. Epidemiological results on food consumption and MLST+ clearly separated the three cases in 2011 from the four 2012-2013 outbreak cases (≥48 different genes). We showed that WGS is capable of discriminating L. monocytogenes SV1/2b clones not distinguishable by PFGE and fAFLP. The listeriosis outbreak described clearly underlines the potential of sequence-based typing methods to offer enhanced resolution and comparability of typing systems for public health applications. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Prevalence of Diverse Clones of Vancomycin-Resistant Enterococcus faecium ST78 in a Chinese Hospital.

PubMed

Yang, Jiyong; Jiang, Yufeng; Guo, Ling; Ye, LIyan; Ma, Yanning; Luo, Yanping

2016-06-01

Vancomycin-resistant Enterococcus (VRE) has been identified in China. However, little is known about the spread of VRE isolates. The genetic relatedness of vancomycin-resistant Enterococcus faecium (VREfm) isolates was analyzed by pulsed-field gel electrophoresis (PFGE), their antimicrobial susceptibilities were analyzed by E-test and the VITEK 2 AST-GP67 test Kit, and their sequence types (STs) were investigated by multilocus sequence typing (MLST). S1-PFGE was used for plasmid profiling, and PCR and subsequent sequencing were performed to identify the virulence genes. A total of 96 nonduplicated VREfm isolates were obtained and categorized into 38 PFGE types (type 1-38). The predominant MLST type was ST78, while ST17, ST341, and ST342 were also sporadically identified. All types of clinical VREfm strains harbored the vanA gene; however, they carried plasmids of different sizes. While 92.1%, 71.1%, and 60.5% of VREfm strains carried hyl, scm, and ecbA genes, respectively, all of them were positive for esp, acm, sgrA, pilA, and pilB genes. Clonal VREfm spread was observed, and nonplasmid-mediated horizontal transfer of vancomycin-resistant gene might have conveyed resistance to some vancomycin-susceptible E. faecium strains. E. faecium ST78 carrying vanA gene was the most prevalent clone in this study. The high prevalence of virulence genes, including esp, hyl, acm, scm, ecbA, sgrA, pilA, and pilB, confirmed their important roles in the emergence of VREfm ST78 in nosocomial infections.

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda

PubMed Central

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R.

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island. PMID:28267763

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

PubMed

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri

PubMed Central

2009-01-01

Background Shigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri. Results Thirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b. Conclusions The 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable. PMID:20042119

Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran.

PubMed

Saffari, Fereshteh; Monsen, Tor; Karmostaji, Afsaneh; Azimabad, Fahimeh Bahadori; Widerström, Micael

2017-11-01

Infections associated with Acinetobacter baumannii represent an increasing threat in healthcare settings. Therefore, we investigated the epidemiological relationship between clinical isolates of A. baumannii obtained from patients in a university hospital in Bandar Abbas in southern Iran. Sixty-four consecutive non-duplicate clinical isolates collected during 2014-2015 were subjected to susceptibility testing, clonal relationship analysis using PFGE, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST), and examined for the presence of carbapenemases and integrons. Almost all A. baumannii isolates were extensively drug-resistant (XDR; 98 %) and carried an OXA carbapenemase gene (blaOXA-23-like; 98 %) and class 1 integrons (48 %). PFGE and MLST analysis identified three major genotypes, all belonging to clonal complex 92 (CC92): sequence type 848 (ST848) (n=23), ST451 (n=16) and ST195 (n=8). CC92 has previously been documented in the hospital setting in northern Iran, and ST195 has been reported in Arab States of the Persian Gulf. These data suggest national and global transmission of A. baumannii CC92. This report demonstrates the occurrence and potential spread of closely related XDR genotypes of A. baumannii CC92 within a university hospital in southern Iran. These genotypes were found in the majority of the investigated isolates, showed high prevalence of blaOXA-23 and integron class 1, and were associated with stay in the intensive care unit. Very few treatment options remain for healthcare-adapted XDR A. baumannii, and hence effective measures are desperately needed to reduce the spread of these strains and resultant infections in the healthcare setting.

Multilocus Sequence Analysis of Streptococcus canis Confirms the Zoonotic Origin of Human Infections and Reveals Genetic Exchange with Streptococcus dysgalactiae subsp. equisimilis

PubMed Central

Pinho, M. D.; Matos, S. C.; Pomba, C.; Lübke-Becker, A.; Wieler, L. H.; Preziuso, S.; Melo-Cristino, J.

2013-01-01

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection. PMID:23345291

Multilocus sequence analysis of Streptococcus canis confirms the zoonotic origin of human infections and reveals genetic exchange with Streptococcus dysgalactiae subsp. equisimilis.

PubMed

Pinho, M D; Matos, S C; Pomba, C; Lübke-Becker, A; Wieler, L H; Preziuso, S; Melo-Cristino, J; Ramirez, M

2013-04-01

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

PubMed Central

Drougka, E.; Foka, A.; Posantzis, D.; Giormezis, N.; Anastassiou, E.D.; Petinaki, E.; Spiliopoulou, I.

2015-01-01

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred. PMID:26623381

Dissemination of the ST-103 clonal complex serogroup C meningococci in Salvador, Brazil.

PubMed

Cordeiro, Soraia Machado; Cardoso, Cristiane Wanderley; de Araújo, Lorena Galvão; Ribeiro, Luis Eduardo; Azevedo, Jailton; Silva, Rita de Cassia Vilasboas; Dos Reis, Mitermayer Galvão; Ko, Albert Icksang; Reis, Joice Neves

2018-01-01

Invasive meningococcal disease (IMD) is a major public health problem worldwide. An epidemic of serogroup C (NmC) IMD occurred in 2010 in the city of Salvador. In this study, we describe the antigenic and genetic characterization of meningococcal isolates collected from meningitis cases in Salvador from 2001 to 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed for the analysis of IMD isolates. A total of 733 cases were identified, and the serogroup was determined for 391 (53.0%) of these. Most cases were caused by NmC (53%) or B (47%). The most prevalent strains were B:4,7:P1.19,15 (32.9%; 129/391) and C:23:P1.14-6 (28.6%; 112/391). Based on PFGE/MLST analysis, 71.3% (77/108 PFGE-tested isolates) clustered as two clones of sequence type ST-3779 and ST-3780, both belonging to the ST-103 clonal complex. ST-3779 has been detected in Salvador since 1996 and together with ST-3780 became predominant after 2005. There was a predominance of C:23:P1.14-6, ST-3779/3780 in Salvador during the period of 2007-2012, establishing a major clonal lineage, which remained in the community for a long time; this has serious implications for public health, particularly in terms of prevention and control strategies of IMD. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Molecular evidence of Burkholderia pseudomallei genotypes based on geographical distribution.

PubMed

Zulkefli, Noorfatin Jihan; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Chong, Chun Wie; Thong, Kwai Lin; Ponnampalavanar, Sasheela; Vadivelu, Jamuna; Teh, Cindy Shuan Ju

2016-01-01

Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters. Hierarchical clustering using UniFrac distance suggested that the isolates from Australia were genetically distinct from the Asian isolates. Nevertheless, statistical significant differences were detected between isolates from Malaysia, Thailand and Australia. Discussion. Overall, PFGE showed higher discriminative power in clustering the nine Malaysian B. pseudomallei isolates and indicated its suitability for localized epidemiological study. Compared to MLST, CIM genes showed higher resolution in distinguishing those non-related strains and better clustering of strains from different geographical regions. A closer genetic relatedness of Malaysian isolates with all Asian strains in comparison to Australian strains was observed. This finding was supported by UniFrac analysis which resulted in geographical segregation between Australia and the Asian countries.

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS

PubMed Central

2011-01-01

Background Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. Results The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. Conclusion The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar. PMID:21708021

First report of blaNDM-1-producing Acinetobacter baumannii isolated in Lebanon from civilians wounded during the Syrian war.

PubMed

Rafei, Rayane; Dabboussi, Fouad; Hamze, Monzer; Eveillard, Matthieu; Lemarié, Carole; Mallat, Hassan; Rolain, Jean-Marc; Joly-Guillou, Marie-Laure; Kempf, Marie

2014-04-01

The emergence of carbapenem-resistant Acinetobacter baumannii has been observed worldwide. We describe the first detection of A. baumannii carrying the blaNDM-1 gene in Lebanon, isolated from Syrian patients wounded during the civil war. Four carbapenem-resistant A. baumannii strains isolated in 2012 in the Tripoli Government Hospital, Lebanon, from civilians wounded during the Syrian war, were analysed. Susceptibility was determined by disk diffusion testing, and resistance to carbapenems was confirmed by Etest. The presence of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, and blaNDM was investigated by PCR. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and blaOXA-51 sequence-based typing. All isolates harboured the blaNDM-1 gene and were negative for other tested carbapenemases. They all belonged to the sequence type 85 and formed a single cluster by PFGE. Finally, blaOXA-51-like gene sequencing revealed the presence of the blaOXA-94 variant in all four isolates. These findings show that Syria constitutes a reservoir for NDM-1-producing bacteria. These results also highlight the need for effective measures to stop the threatening spread of such strains. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

The utility of multiple molecular methods including whole genome sequencing as tools to differentiate Escherichia coli O157:H7 outbreaks.

PubMed

Berenger, Byron M; Berry, Chrystal; Peterson, Trevor; Fach, Patrick; Delannoy, Sabine; Li, Vincent; Tschetter, Lorelee; Nadon, Celine; Honish, Lance; Louie, Marie; Chui, Linda

2015-01-01

A standardised method for determining Escherichia coli O157:H7 strain relatedness using whole genome sequencing or virulence gene profiling is not yet established. We sought to assess the capacity of either high-throughput polymerase chain reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or k-mer clustering to discriminate between outbreak-associated and sporadic E. coli O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the province of Alberta, Canada were included in the study. Two of the outbreaks occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were employed as comparator typing methods. The virulence gene profiles of isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic isolates were mostly identical; therefore the set of virulence genes chosen in this study were not discriminatory enough to distinguish between outbreak clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct events. k-mer phylogenies demonstrated increased discriminatory power compared with core SNV phylogenies. Prior to the widespread implementation of whole genome sequencing for routine public health use, issues surrounding cost, technical expertise, software standardisation, and data sharing/comparisons must be addressed.

Highly virulent M1 Streptococcus pyogenes isolates resistant to clindamycin.

PubMed

Plainvert, C; Martin, C; Loubinoux, J; Touak, G; Dmytruk, N; Collobert, G; Fouet, A; Ploy, M-C; Poyart, C

2015-01-01

Emm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. We aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. GAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. A total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. This is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Comparison and Evaluation of the Molecular Typing Methods for Toxigenic Vibrio cholerae in Southwest China.

PubMed

Liao, Feng; Mo, Zhishuo; Chen, Meiling; Pang, Bo; Fu, Xiaoqing; Xu, Wen; Jing, Huaiqi; Kan, Biao; Gu, Wenpeng

2018-01-01

Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains) isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and MLST of virulence gene (V-MLST) methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae , one was used seven housekeeping genes ( adk , gyrB , metE , pntA , mdh , purM , and pyrC ), and all the isolates belonged to ST69; another was used nine housekeeping genes ( cat , chi , dnaE , gyrB , lap , pgm , recA , rstA , and gmd ). A total of seven sequence types (STs) were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method ( ctxAB , tcpA , and toxR ) showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods could distinguish some epidemiological special strains in local area.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from retail meat and humans in Georgia.

PubMed

Jackson, Charlene R; Davis, Johnnie A; Barrett, John B

2013-04-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl(+). Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Retail Meat and Humans in Georgia

PubMed Central

Davis, Johnnie A.; Barrett, John B.

2013-01-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl+. Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat. PMID:23363837

Identification of Infantile Diarrhea Caused by Breast Milk-Transmitted Staphylococcus aureus Infection.

PubMed

Chen, Zhong; Pan, Wei-Guang; Xian, Wei-Yi; Cheng, Hang; Zheng, Jin-Xin; Hu, Qing-Hua; Yu, Zhi-Jian; Deng, Qi-Wen

2016-10-01

Staphylococcus aureus is a well-known organism which is responsible for a variety of human infectious diseases including skin infections, pneumonia, bacteremia, and endocarditis. Few of the microorganisms can be transmitted from mother to the newborn or infant by milk breastfeeding. This study aims to identify transmission of S. aureus from healthy, lactating mothers to their infants by breastfeeding. Stool specimens of diarrheal infants and breast milk of their mother (totally three pairs) were collected and six Staphylococcus aureus isolates were cultured positively. Homology and molecular characters of isolated strains were tested using pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Furthermore, toxin genes detection was also performed. Each pair of isolates has the same PFGE type and spa type. Four Sequence types (STs) were found among all the isolates; they are ST15, ST188, and ST59, respectively. Among the strains, seb, sec, and tst genes were found, and all were negative for pvl gene. The homology of the S. aureus strains isolated from the infants' stool and the mothers' milk was genetically demonstrated, which indicated that breastfeeding may be important in the transmission of S. aureus infection, and the character of S. aureus needed to be further evaluated.

High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses.

PubMed

Chen, Chih-Ming; Ke, Se-Chin; Li, Chia-Ru; Wu, Ying-Chen; Chen, Ter-Hsin; Lai, Chih-Ho; Wu, Xin-Xia; Wu, Lii-Tzu

2017-10-01

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to β-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum β-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Enterotoxin gene profile of methicillin-resistant Staphylococcus pseudintermedius isolates from dogs, humans and the environment.

PubMed

Phumthanakorn, Nathita; Fungwithaya, Punpichaya; Chanchaithong, Pattrarat; Prapasarakul, Nuvee

2018-06-01

This study aimed to detect and identify staphylococcal enterotoxin (SE) genes in methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains from different sources, and to investigate the relationship between their sequence types (STs) and SE gene patterns. The profiles of 17 SE genes in 93 MRSP strains isolated from dogs (n=43), humans (n=18) and the environment (n=32) were detected by PCR. Multilocus sequence typing (MLST), SCCmec typing and pulsed-field gel electrophoresis (PFGE) were used to analyse the clonal relatedness between the molecular type and SE gene profiles.Results/Key findings. The human MRSP strains harboured the greatest number of SE genes (12/17; sea, sec, seg, sei, sek, sel, sem, sen, seo, sep, seq and tst-1) compared to those from dogs (5/17; sec, sel, sem, seq and tst-1) and environmental sources (3/17; sec, seq and tst-1). Using MLST and PFGE, different SE gene profiles were found within the same clonal type. We show that isolates of MRSP vary in their virulence gene profiles, depending on the source from which they have been isolated. This insight should encourage the development of appropriate monitoring and mitigation strategies to reduce the transmission of MRSP in veterinary hospitals and households.

Genetic characteristics and molecular epidemiology of vancomycin-resistant Enterococci isolates from Caribbean countries

PubMed Central

Jayaratne, Padman; Wilson, Clyde; Golding, George R.; Nicholson, Alison M.; Lewis, Delores B.; Hermelijn, Sandra M.

2017-01-01

Emergence of vancomycin-resistant Enterococci (VRE) that first appeared on the stage about three decades ago is now a major concern worldwide as it has globally reached every continent. Our aim was to simply undertake a multinational study to delineate the resistance and virulence genes of clinical isolates of VRE isolates from the Caribbean. We employed both conventional (standard microbiological methods including use of E-test strips, chromogenic agar) and molecular methods (polymerase chain reactions–PCR, pulsed-field gel electrophoresis–PFGE and multilocus sequence typing–MLST) to analyze and characterize 245 Enterococci species and 77 VRE isolates from twelve hospitals from eight countries in the Caribbean. The PCR confirmed and demonstrated the resistance and virulence genes (vanA and esp) among all confirmed VRE isolates. The PFGE delineated clonally related isolates from patients from the same country and other countries in the region. The main sequence types of the VRE isolates from the region included STs 412, 750, 203, 736 and 18, all from the common ancestor for clonal complex 17 (CC17). Despite this common ancestor and association of outbreaks of this lineage clones, there has been no reports of outbreaks of infection by VRE in several hospitals in the Caribbean. PMID:29020115

First Description of KPC-2-Producing Escherichia coli and ST15 OXA-48-Positive Klebsiella pneumoniae in Tunisia.

PubMed

Ben Tanfous, Farah; Alonso, Carla Andrea; Achour, Wafa; Ruiz-Ripa, Laura; Torres, Carmen; Ben Hassen, Assia

2017-04-01

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011-2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The bla OXA-48 and bla KPC carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had bla OXA-48 gene, and 14/19 strains also harbored the bla CTX-M-15 gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. Two out of three R/IR-ERT E. coli isolates carried bla OXA-48 and one coproduced the bla CTX-M-15 gene. One E. coli strain, ascribed to the new sequence type ST5700, harbored the bla KPC-2 gene. E. coli isolates were not clonally related and belonged to different sequence types (ST5700, ST227, and ST58). To our knowledge, this is the first report in Tunisia of either KPC-2 carbapenemase in E. coli or OXA-48 carbapenemase in K. pneumoniae of lineage ST15.

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

PubMed Central

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J.; Chai, Yunlei; Li, Ran; Niu, Jieting

2015-01-01

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. PMID:26048942

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China.

PubMed

Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J; Chai, Yunlei; Li, Ran; Niu, Jieting; Jiang, Yujun

2015-08-15

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Extended-spectrum-β-lactamase-producing Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care unit outbreak due to a CTX-M-14-producing strain.

PubMed

Oteo, Jesús; Cercenado, Emilia; Fernández-Romero, Sara; Saéz, David; Padilla, Belén; Zamora, Elena; Cuevas, Oscar; Bautista, Verónica; Campos, José

2012-01-01

Little information is available about pediatric infections caused by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli. We characterized an outbreak caused by a CTX-M-14-producing E. coli isolate in a neonatal intensive care unit (NICU) and studied other infections caused by ESBL-producing E. coli in non-NICU pediatric units. All children ≤4 years old who were infected or colonized by ESBL-producing E. coli isolates between January 2009 and September 2010 were included. Molecular epidemiology was studied by phylogroup analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and by incompatibility group analysis using a PCR-based replicon-typing scheme. Of the ESBL-producing E. coli isolates colonizing or infecting the 30 newborns, identical PFGE results were observed for 21 (70%) isolates, which were classified as CTX-M-14-producing E. coli of ST23 phylogroup A. bla(CTX-M-14a) was linked to ISEcp1 and was carried on an ∼80-bp IncK plasmid. A smaller ongoing outbreak due to SHV-12-producing ST131 E. coli was also identified in the same NICU. Fifteen additional infections with ESBL-producing E. coli were identified in non-NICU pediatric units, but none was caused by the CTX-M-14-producing E. coli epidemic clone. Overall, CTX-M-14 (71.1%), CTX-M-15 (13.3%), and SHV-12 (13.3%) were the most important ESBLs causing pediatric infections in this study. Infections of newborns with CTX-M-14-producing E. coli were caused by both clonal and nonclonal isolates.

Molecular evidence for zoonotic transmission of an emergent, highly pathogenic Campylobacter jejuni clone in the United States.

PubMed

Sahin, Orhan; Fitzgerald, Collette; Stroika, Steven; Zhao, Shaohua; Sippy, Rachel J; Kwan, Patrick; Plummer, Paul J; Han, Jing; Yaeger, Michael J; Zhang, Qijing

2012-03-01

Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.

Molecular Evidence for Zoonotic Transmission of an Emergent, Highly Pathogenic Campylobacter jejuni Clone in the United States

PubMed Central

Sahin, Orhan; Fitzgerald, Collette; Stroika, Steven; Zhao, Shaohua; Sippy, Rachel J.; Kwan, Patrick; Plummer, Paul J.; Han, Jing; Yaeger, Michael J.

2012-01-01

Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health. PMID:22189122

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

PubMed

Pfeifer, Yvonne; Trifonova, Angelina; Pietsch, Michael; Brunner, Magdalena; Todorova, Iva; Gergova, Ivanka; Wilharm, Gottfried; Werner, Guido; Savov, Encho

2017-04-01

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla OXA-23 or bla OXA-72 ; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Wide spread of OXA-23-producing carbapenem-resistant Acinetobacter baumannii belonging to clonal complex II in different hospitals in Lebanon.

PubMed

Al Atrouni, Ahmad; Hamze, Monzer; Jisr, Tamima; Lemarié, Carole; Eveillard, Matthieu; Joly-Guillou, Marie-Laure; Kempf, Marie

2016-11-01

To investigate the molecular epidemiology of Acinetobacter baumannii strains isolated from different hospitals in Lebanon. A total of 119 non-duplicate Acinetobacter strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and partial rpoB gene sequencing. Antibiotic susceptibility testing was performed by disc diffusion method and all identified carbapenem-resistant isolates were investigated by PCR assays for the presence of the carbapenemase-encoding genes. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for molecular typing. Of the 119 A. baumannii isolates, 76.5% were resistant to carbapenems. The most common carbapenemase was the OXA-23-type, found in 82 isolates. The study of population structure using MLST revealed the presence of 30 sequence types (STs) including 18 new ones, with ST2 being the most commonly detected, accounting for 61% of the isolates typed. PFGE performed on all strains of ST2 identified a major cluster of 53 isolates, in addition to three other minor clusters and ten unique profiles. This study highlights the wide dissemination of highly related OXA-23-producing carbapenem-resistant A. baumannii belonging to the international clone II in Lebanon. Thus, appropriate infection control measures are recommended in order to control the geographical spread of this clone in this country. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genetic Analysis of Vibrio parahaemolyticus O3:K6 Strains That Have Been Isolated in Mexico Since 1998

PubMed Central

Licea-Navarro, Alexei Fedorovish; Revilla-Castellanos, Valeria Jeanette; Wong-Chang, Irma; González-Sánchez, Ricardo

2017-01-01

Vibrio parahaemolyticus is an important human pathogen that has been isolated worldwide from clinical cases, most of which have been associated with seafood consumption. Environmental and clinical toxigenic strains of V. parahaemolyticus that were isolated in Mexico from 1998 to 2012, including those from the only outbreak that has been reported in this country, were characterized genetically to assess the presence of the O3:K6 pandemic clone, and their genetic relationship to strains that are related to the pandemic clonal complex (CC3). Pathogenic tdh+ and tdh+/trh+ strains were analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Also, the entire genome of a Mexican O3:K6 strain was sequenced. Most of the strains were tdh/ORF8-positive and corresponded to the O3:K6 serotype. By PFGE and MLST, there was very close genetic relationship between ORF8/O3:K6 strains, and very high genetic diversities from non-pandemic strains. The genetic relationship is very close among O3:K6 strains that were isolated in Mexico and sequences that were available for strains in the CC3, based on the PubMLST database. The whole-genome sequence of CICESE-170 strain had high similarity with that of the reference RIMD 2210633 strain, and harbored 7 pathogenicity islands, including the 4 that denote O3:K6 pandemic strains. These results indicate that pandemic strains that have been isolated in Mexico show very close genetic relationship among them and with those isolated worldwide. PMID:28099500

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity.

PubMed

Conceição, Natália; da Silva, Lucas Emanuel Pinheiro; Darini, Ana Lúcia da Costa; Pitondo-Silva, André; de Oliveira, Adriana Gonçalves

2014-12-01

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The β-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing β-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573→Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573→Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated. Copyright © 2014 Elsevier B.V. All rights reserved.

Microbiological Features of KPC-Producing Enterobacter Isolates Identified in a U.S. Hospital System

PubMed Central

Ahn, Chulsoo; Syed, Alveena; Hu, Fupin; O’Hara, Jessica A.; Rivera, Jesabel I.; Doi, Yohei

2014-01-01

Microbiological data regarding KPC-producing Enterobacter spp. are scarce. In this study, 11 unique KPC-producing Enterobacter isolates were identified among 44 ertapenem-non-susceptible Enterobacter isolates collected between 2009 and 2013 at a hospital system in Western Pennsylvania. All cases were healthcare-associated and occurred in medically complex patients. While pulsed-field gel electrophoresis (PFGE) showed diverse restriction patterns overall, multilocus sequence typing (MLST) identified Enterobacter cloacae isolates with sequence types (STs) 93 and 171 from two hospitals each. The levels of carbapenem minimum inhibitory concentrations were highly variable. All isolates remained susceptible to colistin, tigecycline, and the majority to amikacin and doxycycline. A blaKPC-carrying IncN plasmid conferring trimethoprim-sulfamethoxazole resistance was identified in three of the isolates. Spread of blaKPC in Enterobacter spp. appears to be due to a combination of plasmid-mediated and clonal processes. PMID:25053203

International Life Science Institute North America Cronobacter (Formerly Enterobacter sakazakii) isolate set.

PubMed

Ivy, Reid A; Farber, Jeffrey M; Pagotto, Franco; Wiedmann, Martin

2013-01-01

Foodborne pathogen isolate collections are important for the development of detection methods, for validation of intervention strategies, and to develop an understanding of pathogenesis and virulence. We have assembled a publicly available Cronobacter (formerly Enterobacter sakazakii) isolate set that consists of (i) 25 Cronobacter sakazakii isolates, (ii) two Cronobacter malonaticus isolates, (iii) one Cronobacter muytjensii isolate, which displays some atypical phenotypic characteristics, biochemical profiles, and colony color on selected differential media, and (iv) two nonclinical Enterobacter asburiae isolates, which show some phenotypic characteristics similar to those of Cronobacter spp. The set consists of human (n = 10), food (n = 11), and environmental (n = 9) isolates. Analysis of partial 16S rDNA sequence and seven-gene multilocus sequence typing data allowed for reliable identification of these isolates to species and identification of 14 isolates as sequence type 4, which had previously been shown to be the most common C. sakazakii sequence type associated with neonatal meningitis. Phenotypic characterization was carried out with API 20E and API 32E test strips and streaking on two selective chromogenic agars; isolates were also assessed for sorbitol fermentation and growth at 45°C. Although these strategies typically produced the same classification as sequence-based strategies, based on a panel of four biochemical tests, one C. sakazakii isolate yielded inconclusive data and one was classified as C. malonaticus. EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with XbaI separated the set into 23 unique ribotypes and 30 unique PFGE types, respectively, indicating subtype diversity within the set. Subtype and source data for the collection are publicly available in the PathogenTracker database (www. pathogentracker. net), which allows for continuous updating of information on the set, including links to publications that include information on isolates from this collection.

The evaluation and application of multilocus variable number tandem repeat analysis (MLVA) for the molecular epidemiological study of Salmonella enterica subsp. enterica serovar Enteritidis infection.

PubMed

Liu, Yao; Shi, Xiaolu; Li, Yinghui; Chen, Qiongcheng; Jiang, Min; Li, Wanli; Qiu, Yaqun; Lin, Yiman; Jiang, Yixiang; Kan, Biao; Sun, Qun; Hu, Qinghua

2016-01-29

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent Salmonella serotypes that cause gastroenteritis worldwide and the most prevalent serotype causing Salmonella infections in China. A rapid molecular typing method with high throughput and good epidemiological discrimination is urgently needed for detecting the outbreaks and finding the source for effective control of S. Enteritidis infections. In this study, 194 strains which included 47 from six outbreaks that were well-characterized epidemiologically were analyzed with pulse field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA). Seven VNTR loci published by the US Center for Disease Control and Prevention (CDC) were used to evaluate and develop MLVA scheme for S. Enteritidis molecular subtyping by comparing with PFGE, and then MLVA was applied to the suspected outbreaks detection. All S. Enteritidis isolates were analyzed with MLVA to establish a MLVA database in Shenzhen, Guangdong province, China to facilitate the detection of S. Enteritidis infection clusters. There were 33 MLVA types and 29 PFGE patterns among 147 sporadic isolates. These two measures had Simpson indices of 0.7701 and 0.8043, respectively, which did not differ significantly. Epidemiological concordance was evaluated by typing 47 isolates from six epidemiologically well-characterized outbreaks and it did not differ for PFGE and MLVA. We applied the well established MLVA method to detect two S. Enteritidis foodborne outbreaks and find their sources successfully in 2014. A MLVA database of 491 S. Enteritidis strains isolated from 2004 to 2014 was established for the surveillance of clusters in the future. MLVA typing of S. Enteritidis would be an effective tool for early warning and epidemiological surveillance of S. Enteritidis infections.

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis.

PubMed

Prendergast, Deirdre M; Lendrum, Lynsey; Pearce, Rachel; Ball, Caroline; McLernon, Joanne; O'Grady, Don; Scott, Lourda; Fanning, Seamus; Egan, John; Gutierrez, Montserrat

2011-01-05

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n=407) and pooled carcass swabs (n=407) from 5 animals belonging to the same herd or flock and minced meat (n=91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n=77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection. Copyright © 2010 Elsevier B.V. All rights reserved.

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains.

PubMed

Rodriguez, Marcela; Hogan, Patrick G; Satola, Sarah W; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M; Burnham, Carey-Ann D; Fritz, Stephanie A

2015-09-01

Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections.

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains

PubMed Central

Rodriguez, Marcela; Hogan, Patrick G.; Satola, Sarah W.; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M.; Burnham, Carey-Ann D.; Fritz, Stephanie A.

2015-01-01

Abstract Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections. PMID:26376402

Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and veterinary staff in China.

PubMed

Zhang, Wanjiang; Hao, Zhihui; Wang, Yang; Cao, Xingyuan; Logue, Catherine M; Wang, Bing; Yang, Jing; Shen, Jianzhong; Wu, Congming

2011-11-01

The aim of this study was to determine the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolates from pet animals and veterinary staff and the characteristics of these isolates. A total of 22 MRSA isolates were isolated from nasal swabs from dogs, cats and veterinary staff in six pet hospitals in six cities, and examined for antimicrobial susceptibility, the presence of resistance genes, Panton-Valentine leukocidin gene lukF-lukS, staphylococcal chromosomal cassette (SCC) mec typing, spa tying, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Of 22 MRSA isolates, 21 were recovered from pet animals, and one was isolated from a member of sstaff. All 22 MRSA strains were resistant to penicillin, oxacillin, azithromycin, clindamycin and ceftriaxone, and harboured mecA, ermB and linA genes. The lukF-lukS gene was not detected in any of the MRSA isolates. Eighteen MRSA strains from Qingdao belonged to ST59-MRSA-IV-spa t437. Of four MRSA isolates from Beijing, one belonged to ST398-MRSA-V-spa t034, and three belonged to ST239-MRSA-III-spa t030 profiles. Two PFGE types (A and B) were identified. Two isolates originating from dogs and one isolate originating from a staff member in Beijing shared similar PFGE patterns. Our cumulative data suggested that cross-transmission of MRSA may have occurred between pet animals and veterinary staff. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk.

PubMed

Shome, Bibek Ranjan; Bhuvana, Mani; Mitra, Susweta Das; Krithiga, Natesan; Shome, Rajeswari; Velu, Dhanikachalam; Banerjee, Apala; Barbuddhe, Sukhadeo B; Prabhudas, Krishnamshetty; Rahman, Habibar

2012-12-01

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.

Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements

PubMed Central

Wardal, Ewa; Markowska, Katarzyna; Żabicka, Dorota; Wróblewska, Marta; Giemza, Małgorzata; Mik, Ewa; Połowniak-Pracka, Hanna; Woźniak, Agnieszka; Hryniewicz, Waleria; Sadowy, Ewa

2014-01-01

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated. PMID:25003118

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

PubMed

Quero, Sara; García-Núñez, Marian; Párraga-Niño, Noemí; Barrabeig, Irene; Pedro-Botet, Maria L; de Simon, Mercè; Sopena, Nieves; Sabrià, Miquel

2016-06-01

To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

Isolation and characterization of Listeria monocytogenes from the black-headed gull feces in Kunming, China.

PubMed

Cao, Xiaolong; Wang, Yi; Wang, Yan; Ye, Changyun

Listeria monocytogenes is among the most important foodborne pathogens, which can be isolated from bird feces. The aim of this study was to examine the prevalence, genotypes, virulence factors and drug-resistance characteristics of L. monocytogenes isolated from the black-headed gull in Kunming, China. Serotypes and virulence genes were identified by polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were used to describe the genetic characteristics of all positive isolates. Furthermore, all L. monocytogenes isolates were tested for antimicrobial susceptibility by disk diffusion method. All isolates were positive for inlA, inlB, plcB, prfA, iap, actA and hly genes. Serotyping showed the isolates belonged to serotype 1/2b (2/9, 22%), 1/2c (3/9, 33%), and 4a (4/9, 44%). PFGE showed 4 different pulsotypes (PTs), and MLST differentiated the 9 isolates into 4 sequence types (STs), ST3 (1), ST5 (1), ST35 (3) and ST201 (4). The result of antimicrobial susceptibility revealed that all isolates were naturally sensitive to most antibiotics. The L. monocytogenes isolates had pathogenic potential. There is a potential infection risk for people who come to frequent contact with black-headed gulls in Kunming. Hence, it is necessary to conduct surveillance of L. monocytogenes in the migratory black-headed gulls, which would be valuable to prevent listeriosis in local residents and tourists. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clonal diversity of extended-spectrum beta-lactamase producing Escherichia coli isolates in fecal samples of wild animals.

PubMed

Cristóvão, Filipe; Alonso, Carla Andrea; Igrejas, Gilberto; Sousa, Margarida; Silva, Vanessa; Pereira, José Eduardo; Lozano, Carmen; Cortés-Cortés, Gerardo; Torres, Carmen; Poeta, Patrícia

2017-03-01

The clonal diversity of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phenotypic characteristics and genotypic correlation between Salmonella isolates from a slaughterhouse and retail markets in Yangzhou, China.

PubMed

Cai, Yinqiang; Tao, Jing; Jiao, Yang; Fei, Xiao; Zhou, Le; Wang, Yan; Zheng, Huijuan; Pan, Zhiming; Jiao, Xinan

2016-04-02

An epidemiological investigation of Salmonella spp. in pig and pork samples from one slaughterhouse and its downstream retail markets in Yangzhou, Jiangsu Province, China, was conducted from October 2013 to March 2014. A total of 71.8% (155/216) and 70.9% (78/110), respectively, of the slaughterhouse and retail market samples were recovered positive for Salmonella. All Salmonella isolates were characterized using serotyping, antimicrobial resistance detection, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Seven serotypes were shared by isolates from the two sources, with the most common serotypes being Salmonella Derby, Typhimurium, and Uganda. Antimicrobial sensitivity testing revealed that the highest antimicrobial resistance rate was against tetracycline (49.7% and 37.2% in isolates from the slaughterhouse and retail market, respectively) with many multidrug-resistant (MDR) isolates in both sources. MLST analysis showed that eight sequence type (ST) patterns were shared, and ST40 occupied an absolute superiority among isolates from both sources. PFGE permitted the resolution of XbaI macrorestriction fragments of the selected 31 Salmonella Derby and 19 Salmonella Typhimurium into 30 and 10 distinct pulsotypes, displaying the high similarity between the isolates from the two sources. Our findings indicated that Salmonella isolates from a slaughterhouse and its downstream retail markets were phenotypically and genetically homologous. Additionally, Salmonella may propagate along the slaughter line and pork production chain from the slaughterhouse to retail markets. Copyright © 2016 Elsevier B.V. All rights reserved.

Dissemination of multidrug-resistant blaCTX-M-15/IncFIIk plasmids in Klebsiella pneumoniae isolates from hospital- and community-acquired human infections in Tunisia.

PubMed

Mansour, Wejdene; Grami, Raoudha; Ben Haj Khalifa, Anis; Dahmen, Safia; Châtre, Pierre; Haenni, Marisa; Aouni, Mahjoub; Madec, Jean-Yves

2015-11-01

This study investigated the molecular features of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae from hospital- and community-acquired (HA/CA) infections in the region of Mahdia, Tunisia. Among 336 K. pneumoniae isolates recovered from both clinical contexts between July 2009 and December 2011, 49 and 15 were ESBL producers and originated from clinical and community sources, respectively. All isolates produced the CTX-M-15 enzyme. As shown by Southern blot on S1 nuclease treatment followed by pulsed-field gel electrophoresis (PFGE) gels, the blaCTX-M-15 gene was carried on IncFII (n=4), IncFIIk (n=25), IncL/M (n=4), IncK (n=1), or untypeable (n=15) plasmids in HA isolates. In CA isolates, the blaCTX-M-15 gene was carried on IncFIIk (n=6), IncFII (n=1), IncHI1 (n=1), or untypeable (n=7) plasmids. In all, 23 and 11 PFGE types were found among the HA and CA isolates. Multilocus sequence typing on representative isolates shows diverse sequence types (STs), such as ST307, ST101, ST39, ST4, ST140, ST15, and ST307 in HA isolates and ST101, ST664, and ST323 in CA isolates. This study is the first comprehensive report of ESBL plasmids in K. pneumoniae from HA and CA infections in Tunisia. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

PubMed

Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

2012-09-01

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolated from a patient who never received vancomycin treatment.

PubMed

Zhu, Xuhui; Liu, Cailin; Gao, Sui; Lu, Yanfang; Chen, Zhongju; Sun, Ziyong

2015-04-01

With the abuse of antibiotics, the methicillin-resistant Staphylococcus aureus (MRSA) strain became prevalent. Furthermore, Staphylococcus aureus with a character of vancomycin intermediate-resistance (VISA) has been found globally since the first report in Japan. The main objectives of this study were to report a case of VISA isolated from a Chinese patient who had never undergone Vancomycin treatment, and to determine its molecular character. A total of 9 strains were recovered from a patient during the therapeutic process. Antimicrobial susceptibility testing was performed to determine their antibiotic susceptibility patterns. To detect the VISA strain's molecular epidemiological features, growth and morphological characters, we used multilocus sequence typing, autolysis assay and transmission electric microscope tests. Pulsed-field gel electrophoresis (PFGE) was performed to characterize the heterogeneities of all isolates. One isolate was found to exhibit vancomycin intermediated-resistant with MIC of 8 μg/ml. It was ST239-T030-agr-1, had thickened cell wall, and displayed a slower growth rate and reduced susceptibility to Triton X-100-induced autolysis than other strains. All 9 strains exhibited the same PFGE pattern. This is the first report of VISA found in central China from a patient who had never received vancomycin treatment. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Environmental Staphylococcus aureus contamination in a Tunisian hospital.

PubMed

Gharsa, Haythem; Dziri, Raoudha; Klibi, Naouel; Chairat, Sarra; Lozano, Carmen; Torres, Carmen; Bellaaj, Ridha; Slama, Karim Ben

2016-12-01

One hundred hospital environment samples were obtained in 2012 in a Tunisian hospital and tested for Staphylococcus aureus recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST), spa-typing, agr-typing and SmaI-pulsed-field gel electrophoresis (PFGE) were performed. Two methicillin-resistant S. aureus (MRSA) isolates typed as: ST247-t052-SCCmecI-agrI were recovered from the intensive care unit (ICU). Ten samples contained methicillin-susceptible S. aureus (MSSA) and these samples were collected in different services, highlighting the presence of the tst gene encoding the toxic shock syndrome toxin as well as the lukED, hla, hlb, hld and hlg v virulence genes in some of the isolates. In conclusion, we have shown that the hospital environment could be a reservoir contributing to dissemination of virulent S. aureus and MRSA.

High prevalence of EMRSA-15 in Portuguese public buses: a worrisome finding.

PubMed

Simões, Roméo Rocha; Aires-de-Sousa, Marta; Conceição, Teresa; Antunes, Filipa; da Costa, Paulo Martins; de Lencastre, Hermínia

2011-03-02

The nosocomial prevalence of methicillin resistant Staphylococcus aureus (MRSA) in Portugal remains one of the highest in Europe and is currently around 50%. Transmission of S. aureus, including MRSA, occurs principally by direct human-to-human skin contact. However, S. aureus can survive for long periods on inanimate objects, which may represent an important reservoir for dissemination as well. Between May 2009 and February 2010, handrails of 85 public urban buses circulating in Oporto, Portugal, were screened for the occurrence of MRSA. Twenty-two (26%) buses showed MRSA contamination. The molecular characterization of a total of 55 MRSA, by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome (SCC) mec typing, spa typing, and multilocus sequence typing (MLST), clustered the isolates into three clonal types. However, the overwhelming majority (n = 50; 91%) of the isolates belonged to a single clone (PFGE A, spa types t747, t032, t025 or t020, ST22, SCCmec type IVh) that exhibits the characteristics of the pandemic EMRSA-15, currently the major lineage circulating in Portuguese hospitals, namely in the Oporto region. Two additional clones were found but in much lower numbers: (i) PFGE B, ST5, spa type t002, SCCmec IVa (n = 3), and (ii) PFGE C, spa type t008, ST8, SCCmec IVa (n = 2). None of the 55 isolates was PVL positive. Public buses in Oporto seem to be an important reservoir of MRSA of nosocomial origin, providing evidence that the major hospital-associated MRSA clone in Portugal is escaping from the primary ecological niche of hospitals to the community environment. Infection control measures are urgently warranted to limit the spread of EMRSA-15 to the general population and future studies are required to assess the eventual increase of MRSA in the Portuguese community, which so far remains low.

Characterization of blaCTX-M IncFII plasmids and clones of Escherichia coli from pets in France.

PubMed

Dahmen, Safia; Haenni, Marisa; Châtre, Pierre; Madec, Jean-Yves

2013-12-01

To characterize bla(CTX-M) IncFII plasmids and clones of Escherichia coli from cats and dogs and to compare them with bla(CTX-M) IncFII plasmids reported in humans. From December 2006 to April 2010, 518 E. coli isolates from clinical infections in cats and dogs were screened for extended-spectrum β-lactamase (ESBL) production. Antimicrobial susceptibility was performed by disc diffusion and resistance genes were identified by PCR and sequencing. Plasmids were characterized using PCR-based replicon typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1-PFGE and Southern hybridization. Isolates were characterized by PFGE, phylogenetic grouping, O25b typing and multilocus sequence typing. Nineteen E. coli isolates (3.7%) produced ESBLs, of which 14 (74%) carried bla(CTX-M) IncFII plasmids. The bla(CTX-M) gene was predominant and located on F31:A4:B1, F36:A4:B1 or F36:A1:B20 plasmids, abundantly reported in humans. The bla(CTX-M) F22:A1:B20 or F2:A2:B20 plasmids were also found. Different sequence types (STs) were identified, such as ST10, ST410, ST359, ST617 and ST224. Only one E. coli isolate belonged to the ST131 E. coli clone and carried a bla(CTX-M) F2:A2:B20 plasmid. This is the first known extensive study on ESBL-producing E. coli isolates from pets in France. The ST131 clone was rare. However, the predominance of human-like bla(CTX-M) IncFII plasmids suggests exchanges of these plasmids with the human reservoir.

Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone.

PubMed

Shahsavan, Shadi; Jabalameli, Leila; Maleknejad, Parviz; Aligholi, Marzieh; Imaneini, Hossein; Jabalameli, Fereshteh; Halimi, Shahnaz; Taherikalani, Morovat; Khoramian, Babak; Eslampour, Mohammad Amin; Feizabadi, Mohammad Mehdi; Emaneini, Mohammad

2011-03-01

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.

Molecular characterization of fluoroquinolone and/or cephalosporin resistance in Shigella sonnei isolates from yaks.

PubMed

Zhu, Zhen; Shi, Yuxiang; Zhou, Xuzheng; Li, Bing; Zhang, Jiyu

2018-06-07

Members of the genus Shigella are intestinal pathogens and a major cause of seasonal outbreaks of bacterial diarrhea worldwide. Although humans are the conventional hosts of Shigella species, expansion of the Shigella host range to certain animals was recently reported. To investigate the prevalence of Shigella sonnei (S. sonnei) in yaks and perform molecular characterization, we analyzed 1132 fresh yak diarrheal stool samples and collected a total of 44 S. sonnei isolates. We performed multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with XbaI-digested DNA to study genetic relatedness among the 44 isolates, which were differentiated into 4 sequence types (STs) and 32 PFGE types (PTs). All isolates harbored virulence genes, and 87.36% tested positive for invasion plasmid antigen H (ipaH), invasion associated locus (ial) and the Shigella enterotoxin gene sen. According to the results of antimicrobial susceptibility tests, 45.45% (20/44) were resistant to fluoroquinolones and/or cephalosporin. By sequencing the quinolone resistance determining region (QRDR) genes, we identified double mutations in gyrA (Ser83-Leu and Asp87-Asn) and a single mutation in parC (Ser80-Ile). All 12 fluoroquinolone-resistant S. sonnei isolates tested positive for the aac(6')-Ib-cr gene but negative for qepA. Three isolates harbored qnr genes, including two with qnrS and one with qnrB. In addition, three types of β-lactamase genes, bla TEM-1 , bla OXA-1 and bla CTX-M-14/79 , were detected in cephalosporin-resistant isolates. The findings of this study have enriched our knowledge of fluoroquinolone- and/or cephalosporin-resistant S. sonnei isolates from yaks, which has important public health significance.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

PubMed

Usein, Codruţa-Romaniţa; Ciontea, Adriana Simona; Militaru, Cornelia Mãdãlina; Condei, Maria; Dinu, Sorin; Oprea, Mihaela; Cristea, Daniela; Michelacci, Valeria; Scavia, Gaia; Zota, Lavinia Cipriana; Zaharia, Alina; Morabito, Stefano

2017-11-01

IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae , ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae , with or without stx1a , while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016

PubMed Central

Usein, Codruţa-Romaniţa; Ciontea, Adriana Simona; Militaru, Cornelia Mãdãlina; Condei, Maria; Dinu, Sorin; Oprea, Mihaela; Cristea, Daniela; Michelacci, Valeria; Scavia, Gaia; Zota, Lavinia Cipriana; Zaharia, Alina; Morabito, Stefano

2017-01-01

Introduction At the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae, ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae, with or without stx1a, while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered. PMID:29183554

Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

PubMed

Oliveira, C J B; Tiao, N; de Sousa, F G C; de Moura, J F P; Santos Filho, L; Gebreyes, W A

2016-03-01

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted. © 2015 Blackwell Verlag GmbH.

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

PubMed Central

Cho, Seongbeom; Boxrud, David J; Bartkus, Joanne M; Whittam, Thomas S; Saeed, Mahdi

2007-01-01

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections. PMID:17692097

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

PubMed Central

2009-01-01

Background Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. Results We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Conclusion Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population. PMID:19573249

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

PubMed

Li, Zhen; Pérez-Osorio, Ailyn; Wang, Yu; Eckmann, Kaye; Glover, William A; Allard, Marc W; Brown, Eric W; Chen, Yi

2017-06-15

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

Dominance of serotype Ia among group B Streptococci causing invasive infections in nonpregnant adults in Portugal.

PubMed

Martins, E R; Melo-Cristino, J; Ramirez, M

2012-04-01

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

Isolation and characterization of Listeria species from rodents in natural environments in China.

PubMed

Wang, Yan; Lu, Liang; Lan, Ruiting; Salazar, Joelle K; Liu, Jingli; Xu, Jianguo; Ye, Changyun

2017-06-07

Listeria is ubiquitous in a variety of environments and can be isolated from a wide range of animal hosts. Rodents are capable of carrying pathogenic bacteria in their intestines, such as Listeria, and can disseminate those pathogens into the natural environment and to where human activity occurs. In this study, we investigated the occurrence and antimicrobial susceptibility of Listeria spp. isolated from wild rodents found in natural environments in China. We collected 341 intestinal fecal samples of rodents from five different regions of China, all representing different rodent habitats. The antimicrobial susceptibility of the Listeria spp. isolates obtained were firstly assessed using the Kirby-Bauer disk diffusion method. Thirty-one samples were positive for Listeria spp., of which 11 were positive for Listeria monocytogenes and seven were positive for Listeria ivanovii. Other species identified include Listeria innocua, Listeria fleischmannii and Listeria floridensis. All Listeria spp. isolates were sensitive to the majority of the antimicrobials tested, but largely resistant to oxacillin (94.1%) and cefuroxime (70.6%). All L. monocytogenes isolates were further characterized by serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). L. monocytogenes strains were grouped into three serotypes, five sequence types and five pulsotypes (PTs) by serotyping, MLST and PFGE, respectively. Almost half of the isolates (five of 11) belonged to serotype 1/2b, ST87 and PT1. This study determined that Listeria is carried in the intestinal tracts of wild rodents from multiple regions at a low rate, filling an epidemiological data gap on Listeria in natural environments in China.

Derivatives of a Vancomycin-Resistant Staphylococcus aureus Strain Isolated at Hershey Medical Center

PubMed Central

Bozdogan, Bülent; Ednie, Lois; Credito, Kim; Kosowska, Klaudia; Appelbaum, Peter C.

2004-01-01

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 μg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 μg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 μg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance. PMID:15561854

Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center.

PubMed

Bozdogan, Bülent; Ednie, Lois; Credito, Kim; Kosowska, Klaudia; Appelbaum, Peter C

2004-12-01

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

Isolation and characterization of Listeria species from rodents in natural environments in China

PubMed Central

Wang, Yan; Lu, Liang; Lan, Ruiting; Salazar, Joelle K; Liu, Jingli; Xu, Jianguo; Ye, Changyun

2017-01-01

Listeria is ubiquitous in a variety of environments and can be isolated from a wide range of animal hosts. Rodents are capable of carrying pathogenic bacteria in their intestines, such as Listeria, and can disseminate those pathogens into the natural environment and to where human activity occurs. In this study, we investigated the occurrence and antimicrobial susceptibility of Listeria spp. isolated from wild rodents found in natural environments in China. We collected 341 intestinal fecal samples of rodents from five different regions of China, all representing different rodent habitats. The antimicrobial susceptibility of the Listeria spp. isolates obtained were firstly assessed using the Kirby–Bauer disk diffusion method. Thirty-one samples were positive for Listeria spp., of which 11 were positive for Listeria monocytogenes and seven were positive for Listeria ivanovii. Other species identified include Listeria innocua, Listeria fleischmannii and Listeria floridensis. All Listeria spp. isolates were sensitive to the majority of the antimicrobials tested, but largely resistant to oxacillin (94.1%) and cefuroxime (70.6%). All L. monocytogenes isolates were further characterized by serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). L. monocytogenes strains were grouped into three serotypes, five sequence types and five pulsotypes (PTs) by serotyping, MLST and PFGE, respectively. Almost half of the isolates (five of 11) belonged to serotype 1/2b, ST87 and PT1. This study determined that Listeria is carried in the intestinal tracts of wild rodents from multiple regions at a low rate, filling an epidemiological data gap on Listeria in natural environments in China. PMID:28588285

Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

PubMed

Yanat, Betitera; Machuca, Jesús; Díaz-De-Alba, Paula; Mezhoud, Halima; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

2017-01-01

The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

Phylogenetic relationships of Shiga toxin-producing Escherichia coli isolated from Peruvian children

PubMed Central

Contreras, C. A.; Ruiz, J.; Lacher, D. W.; Rivera, F. P.; Saenz, Y.; Chea-Woo, E.; Zavaleta, N.; Gil, A. I.; Lanata, C. F.; Huicho, L.; Maves, R. C.; Torres, C.; DebRoy, C.; Cleary, T. G.

2011-01-01

The aim of this study was to determine the prevalence, virulence factors (stx, eae, ehxA and astA) and phylogenetic relationships [PFGE and multilocus sequence typing (MLST)] of Shiga toxin-producing Escherichia coli (STEC) strains isolated from four previous cohort studies in 2212 Peruvian children aged <36 months. STEC prevalence was 0.4 % (14/3219) in diarrhoeal and 0.6 % (15/2695) in control samples. None of the infected children developed haemolytic uraemic syndrome (HUS) or other complications of STEC. stx1 was present in 83 % of strains, stx2 in 17 %, eae in 72 %, ehxA in 59 % and astA in 14 %. The most common serotype was O26 : H11 (14 %) and the most common seropathotype was B (45 %). The strains belonged mainly to phylogenetic group B1 (52 %). The distinct combinations of alleles across the seven MLST loci were used to define 13 sequence types among 19 STEC strains. PFGE typing of 20 STEC strains resulted in 19 pulsed-field patterns. Comparison of the patterns revealed 11 clusters (I–XI), each usually including strains belonging to different serotypes; one exception was cluster VI, which gathered exclusively seven strains of seropathotype B, clonal group enterohaemorrhagic E. coli (EHEC) 2 and phylogenetic group B1. In summary, STEC prevalence was low in Peruvian children with diarrhoea in the community setting. The strains were phylogenetically diverse and associated with mild infections. However, additional studies are needed in children with bloody diarrhoea and HUS. PMID:21292859

Antibiotic resistance determinants and genetic analysis of Salmonella enterica isolated from food in Morocco.

PubMed

Murgia, Manuela; Bouchrif, Brahim; Timinouni, Mohammed; Al-Qahtani, Ahmed; Al-Ahdal, Mohammed N; Cappuccinelli, Pietro; Rubino, Salvatore; Paglietti, Bianca

2015-12-23

Antimicrobial-resistant non-typhoidal Salmonella (NTS) are an important cause of infection in Africa, but there is a lack of information on their molecular mechanisms of resistance and epidemiology. This study contributes to fill this gap through the characterization by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), plasmid profiling and analysis of antibiotic-resistance determinants of 94 Salmonella enterica strains isolated from food in Morocco. PFGE revealed considerable heterogeneity among the strains, showing 32 pulsotypes. MLST of strains representative of the different serovars evidenced 13 sequence types (STs), three of which were newly identified (ST1694, ST1768 and ST1818) and nine not previously reported in Morocco. Thirty-four strains harbored from one to four plasmids, of IncI1 group in S. Mbandaka, IncFIIA in S. Typhimurium, IncL/M in S. Hadar and S. Blockley. For the first time in Morocco an intact Salmonella Genomic Island 1 (SGI1) carrying the resistance genes aadA2, floR, tetG, blaPSE-1 and sul1 was detected in S. Typhimurium DT104. In serovar Hadar resistance to ampicillin, tetracycline and streptomycin was associated to blaTEM-1, tetA and strA genes respectively, whereas one mutation in gyrA (Asp87Asn) and one in parC (Thr54Ser) genes conferred resistance to nalidixic acid. These findings improve the information on foodborne Salmonella in Morocco, evidencing the presence of MDR strains potentially dangerous to humans, and provide useful data for future studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Group B streptococci causing neonatal infections in barcelona are a stable clonal population: 18-year surveillance.

PubMed

Martins, E R; Andreu, A; Correia, P; Juncosa, T; Bosch, J; Ramirez, M; Melo-Cristino, J

2011-08-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

Group B Streptococci Causing Neonatal Infections in Barcelona Are a Stable Clonal Population: 18-Year Surveillance▿

PubMed Central

Martins, E. R.; Andreu, A.; Correia, P.; Juncosa, T.; Bosch, J.; Ramirez, M.; Melo-Cristino, J.

2011-01-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years. PMID:21697333

Methicillin-Resistant Staphylococcus aureus Ocular Infection in Taiwan

PubMed Central

Kang, Yu-Chuan; Hsiao, Ching-Hsi; Yeh, Lung-Kun; Ma, David H.K.; Chen, Phil Y.F.; Lin, Hsin-Chiung; Tan, Hsin-Yuan; Chen, Hung-Chi; Chen, Shin-Yi; Huang, Yhu-Chering

2015-01-01

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important public health issue. This observational study aimed to characterize clinical features, antibiotic susceptibility, and genotypes of ocular infections caused by MRSA based on the clinical and molecular definitions of community-associated (CA) and healthcare-associated (HA) strains. Fifty-nine patients with culture-proven S aureus ocular infection were enrolled from January 1, 2010 to December 31, 2011 at Chang Gung Memorial Hospital, Taiwan. Antibiotic susceptibility was verified using disk diffusion/E test. For characterization, staphylococcal cassette chromosome mec (SCCmec), pulsed-field gel electrophoresis (PFGE), multilocus sequence type (MLST), and Panton–Valentine leukocidin (PVL) gene, were performed. MRSA isolates from the patients with HA factors were classified as clinically defined HA-MRSA, and those carrying SCCmec type I to III as molecularly defined HA-MRSA. Thirty-four patients with MRSA ocular infection were identified. The most common clone of CA-MRSA and HA-MRSA isolates was ST59/PFGE type D/SCCmec IV,VT/PVL (+) (n = 12) and CC 239/PFGE type A/SCCmec III, IIIA/PVL(−) (n = 10), respectively. All the 11 patients with molecularly defined HA-MRSA infections and 50% of the 22 patients with molecularly defined CA-MRSA infections were found to have HA factors (P = .005). CA-MRSA tended to cause lid infections, whereas HA-MRSA tended to cause corneal infections. Contrary to HA-MRSA isolates, nearly all the CA-MRSA isolates were susceptible to trimethoprim/sulfamethoxazole and fluoroquinolones under either clinical or molecular classifications. In Taiwan, CA-MRSA isolates exhibited considerably higher susceptibility to fluoroquinolones when compared with HA-MRSA isolates. A strong correlation was observed between the HA factors and molecularly defined HA-MRSA isolates. PMID:26496268

Subtyping of a Large Collection of Historical Listeria monocytogenes Strains from Ontario, Canada, by an Improved Multilocus Variable-Number Tandem-Repeat Analysis (MLVA)

PubMed Central

Saleh-Lakha, S.; Allen, V. G.; Li, J.; Pagotto, F.; Odumeru, J.; Taboada, E.; Lombos, M.; Tabing, K. C.; Blais, B.; Ogunremi, D.; Downing, G.; Lee, S.; Gao, A.; Nadon, C.

2013-01-01

Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario's food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson's diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario's food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks. PMID:23956391

Genetic background of novel sequence types of CTX-M-8- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil.

PubMed

Dropa, Milena; Lincopan, Nilton; Balsalobre, Livia C; Oliveira, Danielle E; Moura, Rodrigo A; Fernandes, Miriam Rodriguez; da Silva, Quézia Moura; Matté, Glavur R; Sato, Maria I Z; Matté, Maria H

2016-03-01

The release of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

[Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

PubMed

Samajpati, Sriparna; Das, Surojit; Ray, Ujjwayini; Dutta, Shanta

2018-05-24

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

First Report of cfr-Carrying Plasmids in the Pandemic Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Staphylococcal Cassette Chromosome mec Type IV Clone

PubMed Central

Shore, Anna C.; Lazaris, Alexandros; Kinnevey, Peter M.; Brennan, Orla M.; Brennan, Gráinne I.; O'Connell, Brian; Feßler, Andrea T.; Schwarz, Stefan

2016-01-01

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins; cfr, encoding phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance; its homologue cfr(B); or optrA, conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr is even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPSA susceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel electrophoresis (PFGE), plasmid curing, and conjugative transfer. Whole-genome sequencing was used for single-nucleotide variant (SNV) analysis, multilocus sequence typing, L protein mutation identification, cfr plasmid sequence analysis, and optrA and cfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles, compared to 1/5 in M13/0401. Both isolates were sequence type 22 MRSA staphylococcal cassette chromosome mec type IV (ST22-MRSA-IV)/spa type t032 isolates, harbored cfr, exhibited the PhLOPSA phenotype, and lacked optrA and cfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid. This is the first report of cfr in the pandemic ST22-MRSA-IV clone. Different cfr plasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlight that prudent management of linezolid use is essential. PMID:26953212

Dissemination of IMP-6 metallo-β-lactamase-producing Pseudomonas aeruginosa sequence type 235 in Korea.

PubMed

Seok, Yoonmi; Bae, Il Kwon; Jeong, Seok Hoon; Kim, Soo Hyun; Lee, Hyukmin; Lee, Kyungwon

2011-12-01

To investigate the epidemiological traits of Pseudomonas aeruginosa clinical isolates producing metallo-β-lactamases (MBLs) in Korea. A total of 386 non-duplicate P. aeruginosa clinical isolates were collected from Korea in 2009. Detection of MBL genes was performed by PCR. The genetic organization of class 1 integrons carrying the MBL gene cassette was investigated by PCR mapping and sequencing. The epidemiological relationships of the isolates were investigated by multilocus sequence typing and PFGE. Of 386 P. aeruginosa isolates, 30 (7.8%) isolates carried the bla(IMP-6) gene and 1 (0.3%) isolate carried the bla(VIM-2) gene. A probe specific for the bla(IMP-6) gene was hybridized to an ∼950 kbp I-CeuI-macrorestriction fragment from all 30 isolates and a probe specific for the bla(VIM-2) gene also hybridized to an ∼500 kbp I-CeuI-macrorestriction fragment from 1 isolate (BDC10). All 31 MBL-producing isolates shared an identical sequence type (ST), ST235, and they carried the same bla(OXA-50) allelic type, bla(OXA-50g). All MBL-producing isolates showed similar XbaI-macrorestriction patterns (similarity >85%), irrespective of MBL genotype. P. aeruginosa ST235 carrying the chromosomally located bla(IMP-6) gene is widely disseminated in Korea.

Emergence of carbapenem resistant Escherichia coli isolates producing blaNDM and blaOXA-48-like carried on IncA/C and IncL/M plasmids at two Iranian university hospitals.

PubMed

Solgi, Hamid; Giske, Christian G; Badmasti, Farzad; Aghamohammad, Shadi; Havaei, Seyed Asghar; Sabeti, Shahram; Mostafavizadeh, Kamyar; Shahcheraghi, Fereshteh

2017-11-01

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was bla OXA-48 (82.8%), followed by bla NDM-1 (31%), bla NDM-7 (6.9%) and bla OXA-181 (3.4%). Seven of the bla NDM positive isolates co-harbored bla OXA-48 carbapenemases. The bla NDM and bla OXA-48 were found in IncA/C and IncL/M conjugative plasmids, respectively. The bla CTX-M-15 , qnrA and intI1 genes were also present in most isolates. The PFGE revealed genetic diversity among the 28 E. coli isolates, which belonged to six minor PFGE clusters and 14 isolates were singletons. The 26 isolates were distributed into 18 STs, of which two were dominant (ST648 and ST167). We identified one bla NDM-1 -positive ST131 E. coli isolates that harbor the bla CTX-M-15 and bla TEM genes. Horizontal transfer of IncA/C and IncL/M plasmids has likely facilitated the spread of the bla OXA-48 and bla NDM genes among E. coli. Their clonal diversity and the presence of faecal carriers in isolates suggest an endemic spread of OXA-48 and NDM. Therefore, it emphasizes the critical importance of monitoring and controlling the spread of carbapenem resistant E. coli. Copyright © 2017. Published by Elsevier B.V.

Molecular characterization and antibiotic resistance of clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China.

PubMed

Lu, Binghuai; Fang, Yujie; Huang, Lei; Diao, Baowei; Du, Xiaoli; Kan, Biao; Cui, Yanchao; Zhu, Fengxia; Li, Dong; Wang, Duochun

2016-06-01

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is presently considered as a human pathogen associated with clinical infection. We characterized 56 SDSE isolates collected from two tertiary hospitals in Beijing, China. Sixteen distinct emm types/subtypes were detected, dominated by stG245.0 (32.1%), stG652.0 (10.7%), stG6.1 (10.7%) and stG485.0 (10.7%), and a novel stG840.0 variant type was identified. All isolates possessed virulence genes of sagA and scpA, and most carried slo (98.2%), ska (98.2%) and speG(dys) (35.7%). By multilocus sequence typing (MLST) analysis, 17 individual sequence types (STs) were distinguished, including 7 newly-identified STs (26.8% of isolates), of which ST127 (30.4%), ST7 (12.5%) and ST44 (10.7%) dominated. Meanwhile, pulsed-field gel electrophoresis (PFGE) analysis revealed 33 pattern types (PTs), which were further combined into 16 pattern clusters (PCs), and 59.3% of isolates were distributed into 2 dominant PCs. Notably, emm types had both close relationship and consistency with STs and PFGE PCs. Furthermore, of 56 SDSE isolates, the predominant antibiotic resistances were erythromycin (71.4%), clindamycin (71.4%) and tetracycline (60.7%). Correspondingly, the prevalent resistance genes of macrolide and tetracycline were erm(B) (78.6%) and tet(M) (73.2%). In addition, multiple point mutations of parC, one of fluoroquinolone resistance genes, were observed (accounting for 75%), and were divided into 12 types, with parC 07 as the predominant type. Our data suggested the wide molecular diversity and distinctive regional features of SDSE from clinical infection in Beijing, China. Copyright © 2016 Elsevier B.V. All rights reserved.

Foods from black market at EU border as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

PubMed

Oniciuc, Elena-Alexandra; Ariza-Miguel, Jaime; Bolocan, Andrei-Sorin; Diez-Valcarce, Marta; Rovira, Jordi; Hernández, Marta; Fernández-Natal, Isabel; Nicolau, Anca Ioana; Rodríguez-Lázaro, David

2015-09-16

The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all β-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community. Copyright © 2014 Elsevier B.V. All rights reserved.

Extensively Drug-Resistant Klebsiella pneumoniae Causing Nosocomial Bloodstream Infections in China: Molecular Investigation of Antibiotic Resistance Determinants, Informing Therapy, and Clinical Outcomes

PubMed Central

Bi, Wenzi; Liu, Haiyang; Dunstan, Rhys A.; Li, Bin; Torres, Von Vergel L.; Cao, Jianming; Chen, Lijiang; Wilksch, Jonathan J.; Strugnell, Richard A.; Lithgow, Trevor; Zhou, Tieli

2017-01-01

The rise in diversity of antimicrobial resistance phenotypes seen in Klebsiella pneumoniae is becoming a serious antibiotic management problem. We sought to investigate the molecular characteristics and clinical implications of extensively drug-resistant (XDR) K. pneumoniae isolated from different nosocomial bloodstream infections (BSIs) patients from July 2013 to November 2015. Even in combination treatment, meropenem did not protect against mortality of BSIs patients (P = 0.015). In contrast, tigecycline in combination with other antimicrobial agents significantly protected against mortality (P = 0.016). Antimicrobial susceptibility tests, molecular detection of antibiotic resistance determinants, conjugation experiments, multilocus sequence typing (MLST), S1-PFGE, Southern blot, SDS-PAGE, immunoblot analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize these isolates. These XDR K. pneumoniae strains were resistant to conventional antimicrobials except tigecycline and polymyxin B and co-harbored diverse resistance determinants. rmtB, blaKPC−2 as well as blaCTX−M−9 were located on a transferable plasmid of ~54.2 kb and the most predominant replicon type was IncF. 23 of the 35 isolates belonging the predominant clone were found to incorporate the globally-disseminated sequence type ST11, but others including a unique, previously undiscovered lineage ST2281 (allelic profile: 4-1-1-22-7-4-35) were also found and characterized. The porins OmpK35 and OmpK36 were deficient in two carbapenemase-negative carbapenem-resistant strains, suggesting decreased drug uptake as a mechanism for carbapenem resistance. This study highlights the importance of tracking hospital acquired infections, monitoring modes of antibiotic resistance to improve health outcomes of BSIs patients and to highlight the problems of XDR K. pneumoniae dissemination in healthcare settings. PMID:28713357

Dynamic of nasal colonization by methicillin-resistant Staphylococcus aureus ST398 and ST1 after mupirocin treatment in a family in close contact with pigs.

PubMed

Lozano, Carmen; Aspiroz, Carmen; Lasarte, Juan J; Gómez-Sanz, Elena; Zarazaga, Myriam; Torres, Carmen

2011-01-01

Nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) was evaluated after a mupirocin treatment in a family previously colonized by MRSA sequence type ST398 and ST1, who lived close to a pig farm. Eight nasal samples were swabbed from each of the four family members on different moments after mupirocin treatment. The efficacy of treatment was low in those family members who worked in the farm, and higher in the remaining two family members with sporadic contact with pigs. In addition, nasal and skin swabs from randomly selected pigs of the farm were taken. MRSA were detected in 33% of pigs tested. All MRSA isolates obtained were characterized by Staphylococcal-Cassette-Chromosome mec (SCCmec) determination, Multilocus-Sequence-Typing (MLST), spa- and agr-typing, Pulsed-field-gel-electrophoresis (PFGE), antimicrobial susceptibility, detection of antimicrobial resistance genes, and toxin gene profiling. Spa-types t011, t1255 and t1197 were detected in humans and animals, with indistinguishable PFGE patterns, suggesting animal to human MRSA transmission. Each spa-type was ascribed to a specific pulsotype. Spa-types t127 and t108 were only detected in MRSA isolates obtained from humans, and t012 only in those from animals. MRSA ST1-t127 isolates and some ST398-t011 and ST398-t1197 isolates presented a multiantimicrobial-resistance phenotype. None of them harbored lukF/lukS, tst, eta and etb virulence genes. This study showed that the efficacy of nasal MRSA decolonization in healthy people with very close contact with pigs is especially low. Copyright © 2010 Elsevier Ltd. All rights reserved.

Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens.

PubMed

Song, Minghui; Shi, Chunlei; Xu, Xuebing; Shi, Xianming

2016-11-01

The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.

Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

PubMed Central

Unemo, Magnus; Dillon, Jo-Anne R.

2011-01-01

Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

Molecular characterization and genetic diversity of ESBL-producing Escherichia coli colonizing the migratory Franklin's gulls (Leucophaeus pipixcan) in Antofagasta, North of Chile.

PubMed

Báez, John; Hernández-García, Marta; Guamparito, Constanza; Díaz, Sofía; Olave, Abdon; Guerrero, Katherine; Cantón, Rafael; Baquero, Fernando; Gahona, Joselyne; Valenzuela, Nicomedes; Del Campo, Rosa; Silva, Juan

2015-02-01

The role of wild animals, particularly migratory birds, in the dissemination of antibiotic-resistant bacteria between geographically distant ecosystems is usually underestimated. The aim of this work was to characterize the Escherichia coli population from Franklin's gull feces, focusing on the extended-spectrum β-lactamase (ESBL)-producing strains. In the summer of 2011, 124 fecal swabs from seagulls (1 of each) migrating from the United States and Canada to the coast of Antofagasta, north of Chile, were collected. Samples were seeded on MacConkey agar supplemented with 2 μg/ml of cefotaxime and a single colony from each plate was tested for ESBL production by the double-disk ESBL synergy test. Antibiotic susceptibility was determined by the disk diffusion method and blaESBL genes were amplified and sequenced. The genetic diversity of isolates was explored by pulsed-field gel electrophoresis (PFGE)-XbaI and multilocus sequence typing. A total of 91 E. coli isolates with high rates of antibiotic resistance were identified. Carbapenemase production was not detected, whereas 67 of the 91 (54%) isolates exhibited an ESBL phenotype due to the presence of CTX-M-15 (61.3%), CTX-M-2 (19.3%), CTX-M-22 (16.1%), and CTX-M-3 (1.6%) coding genes. High genetic diversity was observed, with 30 PFGE patterns and 23 sequence types (STs), including ST131 (18%), ST44 (15%), ST617 (9%), and ST10 (9%). Results presented here are complementary to those previously reported by Hernández et al. in the same gull species, but located in the Central Region of Chile. Differences observed between gulls from both areas lead us to hypothesize that gulls from the northern location retain, as gut carriers, those resistant bacteria acquired in the United States and/or Canada.

Spanish multicenter study of the epidemiology and mechanisms of amoxicillin-clavulanate resistance in Escherichia coli.

PubMed

Ortega, Adriana; Oteo, Jesús; Aranzamendi-Zaldumbide, Maitane; Bartolomé, Rosa M; Bou, Germán; Cercenado, Emilia; Conejo, M Carmen; González-López, Juan José; Marín, Mercedes; Martínez-Martínez, Luis; Merino, María; Navarro, Ferran; Oliver, Antonio; Pascual, Alvaro; Rivera, Alba; Rodríguez-Baño, Jesús; Weber, Irene; Aracil, Belén; Campos, José

2012-07-01

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

Demonstration of persistent contamination of a cooked egg product production facility with Salmonella enterica serovar Tennessee and characterization of the persistent strain.

PubMed

Jakočiūnė, D; Bisgaard, M; Pedersen, K; Olsen, J E

2014-08-01

The aim of this study was to investigate whether continuous contamination of light pasteurized egg products with Salmonella enterica serovar Tennessee (S. Tennessee) at a large European producer of industrial egg products was caused by persistent contamination of the production facility and to characterize the persistent strains. Seventy-three S. Tennessee isolates collected from products over a 3-year period with intermittent contamination, and 15 control strains were compared by pulsed field gel electrophoresis (PFGE) using two enzymes. Forty-five case isolates distributed throughout the full period were shown to belong to one profile type. Isolates representing different PFGE profiles were all assigned to ST 319 by multilocus sequence typing (MLST). The case isolates did not show a higher ability to form biofilm on a plastic surface than noncase isolates. Characteristically, members of the persistent clone were weak producers of H2 S in laboratory medium. S. Tennessee isolated from the case was able to grow better in pasteurized egg product compared with other serovars investigated. It was concluded that the contamination was caused by a persistent strain in the production facility and that this strain apparently had adapted to grow in the relevant egg product. S. Tennessee has previously been associated with persistence in hatching facilities. This is the first report of persistent contamination of an egg production facility with this serovar. © 2014 The Society for Applied Microbiology.

Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from zoo and wild animals.

PubMed

Feßler, Andrea T; Thomas, Patricia; Mühldorfer, Kristin; Grobbel, Mirjam; Brombach, Julian; Eichhorn, Inga; Monecke, Stefan; Ehricht, Ralf; Schwarz, Stefan

2018-05-01

Antimicrobial resistance of Staphylococcus aureus is a major problem in human and veterinary medicine. The aim of this study was to characterise S. aureus isolates from wild and zoo animals mainly associated with bacterial infections. In total, 23 S. aureus isolates, including nine from wild animals and 14 from zoo animals, were obtained during routine diagnostics. All isolates were subjected to multilocus sequence typing (MLST), spa typing, macrorestriction analysis with subsequent SmaI pulsed-field gelelectrophoresis (PFGE), antimicrobial susceptibility testing and S. aureus-specific DNA-microarray analysis. Resistant isolates were also tested for their respective resistance genes by PCR. Isolates from zoo animals and wildlife showed a high diversity of MLST types, spa types and PFGE patterns. Nineteen different spa types were identified, including three novel types and 16 main macrorestriction patterns. Only few isolates were resistant to members of four classes of antimicrobial agents and harboured the respective resistance genes (β-lactams [blaZ, mecA, mecC], tetracyclines [tet(K), tet(L)] and chloramphenicol [cat pC221 ]) or mutations (fluoroquinolones). The DNA microarray analysis identified one isolate from a zoo animal harbouring the toxic shock syndrome toxin gene tst1. Moreover, several enterotoxin genes were detected in five S. aureus isolates. All isolates were negative for Panton-Valentine leukocidin (PVL) genes, but the animal-associated leukocidin genes lukM/lukF-P83 were found in three isolates from two animals. Copyright © 2018 Elsevier B.V. All rights reserved.

Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates

PubMed Central

Anderson, Michele J.; Schaaf, Emily; Wallis, Heidi W.; Johnson, James R.; Tkaczyk, Christine; Sellman, Bret R.; Sun, Jisun; Peterson, Marnie L.

2018-01-01

Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections. PMID:29659477

Vibrio cholerae O1 El Tor from southern Vietnam in 2010 was molecularly distinct from that present from 1999 to 2004.

PubMed

Nguyen, V H; Pham, H T; Diep, T T; Phan, C D H; Nguyen, T Q; Nguyen, N T N; Ngo, T C; Nguyen, T V; Do, Q K; Phan, H C; Nguyen, B M; Ehara, M; Ohnishi, M; Yamashiro, T; Nguyen, L T P; Izumiya, H

2016-04-01

The Vibrio cholerae O1 (VCO1) El Tor biotype appeared during the seventh cholera pandemic starting in 1961, and new variants of this biotype have been identified since the early 1990s. This pandemic has affected Vietnam, and a large outbreak was reported in southern Vietnam in 2010. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analyses (MLVA) were used to screen 34 VCO1 isolates from the southern Vietnam 2010 outbreak (23 patients, five contact persons, and six environmental isolates) to determine if it was genetically distinct from 18 isolates from outbreaks in southern Vietnam from 1999 to 2004, and two isolates from northern Vietnam (2008). Twenty-seven MLVA types and seven PFGE patterns were identified. Both analyses showed that the 2008 and 2010 isolates were distinctly clustered and separated from the 1999-2004 isolates.

[Molecular types of group A Streptococcus isolated from scarlet fever patients and asymptomatic carriers in Shandong province, 2013].

PubMed

Liu, Zhenyan; Fang, Ming; Hu, Bin; Bi, Zhenwang; Kou, Zengqiang; Ren, Yanyan; Chen, Baoli; Bi, Zhenqiang

2014-12-01

To describe the molecular characteristics of group A Streptococcus (GAS) isolated from patients and asymptomatic carriers of scarlet fever in Shandong province, 2013, and to explore the relationships between emm types and other molecular types. 72 strains of GAS were isolated from throat swabs of children with scarlet fever or asymptomatic carriers of GAS. All the strains were typed by emm typing, multilocus sequence typing (MLST), super-antigen (SAg) genes detections and pulsed-field gel electrophoreses (PFGE). Among the 72 strains, emm1 (41.67%) and emm12 (56.94%) were the most common emm types. Two ST types were found, including ST28 (43.06%) and ST36 (56.94%). Additionally, emm1 was also found correlated to ST28, while emm12 was associated with ST36. Eight super-antigen genes were detected, including smeZ (100.00%), ssa (100.00%), speG (97.22%), speC (95.83%), speL (54.17%), speJ (41.67%), speA (38.89%) and speH (38.89%), while speK, speM, speL were not found (0%). Both speA and speJ genes were detected primarily in emm1 strains (all P < 0.05), while speH and speI genes were not detected in emm 1 strains (all P < 0.05). And emm12 strains were inclined to harbor speH and speL (all P < 0.05) but not speA or speJ (all P < 0.05). Twenty different genotypes were identified by PFGE. All the emm types of GAS isolated from scarlet fever patients and asymptomatic carriers in Shandong province 2013 were mainly emm1 and emm12 and carrying speC, speG and smeZ, ssa. ST types mainly exsited in ST28 and ST36. In addition, there were correlations between emm types and super-antigen genes, ST types, PFGE types.

Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans.

PubMed

Mora, Azucena; Viso, Susana; López, Cecilia; Alonso, María Pilar; García-Garrote, Fernando; Dabhi, Ghizlane; Mamani, Rosalía; Herrera, Alexandra; Marzoa, Juan; Blanco, Miguel; Blanco, Jesús E; Moulin-Schouleur, Maryvonne; Schouler, Catherine; Blanco, Jorge

2013-12-27

Escherichia coli strains O45:K1:H7 are implicated in severe human infections such as meningitis. Since an increasing prevalence of serogroup O45 among avian pathogenic (APEC) and human extraintestinal pathogenic (ExPEC) E. coli strains isolated in Spain have been noticed, the aims of the present study were to investigate similarities between poultry and human O45 isolates, and to investigate the evolutionary relationship of ST95 types. The genetic relatedness and virulence gene profiles of 55 O45 APEC obtained from an avian colibacillosis collection (1991-2011) and 19 human O45 ExPEC from a human septicemic/uropathogenic (UPEC) E. coli collection (1989-2010) were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based genotyping. Two main clonal groups were established. The most prevalent and highly pathogenic O45:K1:H7-B2-ST95 shows a successful persistence since the 90s to the present, with parallel evolution both in human and poultry, on the basis of their PFGE and virulence gene profile similarities (9 human strains and 15 avian strains showed ≥85% PFGE identity). Comparison of this group with other ST95 closely related members (O1:K1:H7 and O18:K1:H7 isolates from our collections) shows pathogenic specialization through conserved virulence genotypes. The other prevalent O45 clonal group characterized in this study, the O45:HNM/H19-D-ST371/ST2676 was only detected in APEC strains suggesting host specificity. In conclusion, poultry could be acting as a reservoir of O45:K1:H7-B2-ST95 and other pathogenic ST95 serotypes in humans. Further studies would be necessary to clarify if pathogenic mechanisms used by ST95 strains are the same in avian and human hosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid characterisation of Klebsiella oxytoca isolates from contaminated liquid hand soap using mass spectrometry, FTIR and Raman spectroscopy.

PubMed

Dieckmann, Ralf; Hammerl, Jens Andre; Hahmann, Hartmut; Wicke, Amal; Kleta, Sylvia; Dabrowski, Piotr Wojciech; Nitsche, Andreas; Stämmler, Maren; Al Dahouk, Sascha; Lasch, Peter

2016-06-23

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge

PubMed Central

Johansson, Anders; Aspan, Anna; Bagge, Elisabeth; Båverud, Viveca; Engström, Björn E; Johansson, Karl-Erik

2006-01-01

Background Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated. Results We used PFGE to examine the genetic diversity of 95 C. perfringens type A isolates from eight different sources. The isolates were also examined for the presence of the beta2 toxin gene (cpb2) and the enterotoxin gene (cpe). The cpb2 gene from the 28 cpb2-positive isolates was also partially sequenced (519 bp, corresponding to positions 188 to 706 in the consensus cpb2 sequence). The results of PFGE revealed a wide genetic diversity among the C. perfringens type A isolates. The genetic relatedness of the isolates ranged from 58 to 100% and 56 distinct PFGE types were identified. Almost all clusters with similar patterns comprised isolates with a known epidemiological correlation. Most of the isolates from pig, horse and sheep carried the cpb2 gene. All isolates originating from food poisoning outbreaks carried the cpe gene and three of these also carried cpb2. Two evolutionary different populations were identified by sequence analysis of the partially sequenced cpb2 genes from our study and cpb2 sequences previously deposited in GenBank. Conclusion As revealed by PFGE, there was a wide genetic diversity among C. perfringens isolates from different sources. Epidemiologically related isolates showed a high genetic similarity, as expected, while isolates with no obvious epidemiological relationship expressed a lesser degree of genetic similarity. The wide diversity revealed by PFGE was not reflected in the 16S rRNA sequences, which had a considerable degree of sequence similarity. Sequence comparison of the partially sequenced cpb2 gene revealed two genetically different populations. This is to our knowledge the first study in which the genetic diversity of C. perfringens isolates both from different animals species, from food poisoning outbreaks and from sludge has been investigated. PMID:16737528

Dissemination of blaNDM-5 gene via an IncX3-type plasmid among non-clonal Escherichia coli in China.

PubMed

Li, Xi; Fu, Ying; Shen, Mengyuan; Huang, Danyan; Du, Xiaoxing; Hu, Qingfeng; Zhou, Yonglie; Wang, Dairong; Yu, Yunsong

2018-01-01

The emergence and spread of New Delhi metallo-β-lactamase-producing Enterobacteriaceae has been a serious challenge to manage in the clinic due to its rapid dissemination of multi-drug resistance worldwide. As one main type of carbapenemases, New Delhi metallo-β-lactamase (NDM)is able to confer resistance to almost all β-lactams, including carbapenems, in Enterobacteriaceae . Recently, New Delhi metallo-β-lactamase-5 attracted extensive attention because of increased resistance to carbapenems and widespread dissemination. However, the dissemination mechanism of bla NDM-5 gene remains unclear. A total of 224 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected from different hospitals in Zhejiang province. NDM-5-positive isolates were identified and subjected to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of bla NDM-5 with southern blot hybridisation, and analysed plasmids containing bla NDM-5 with filter mating and DNA sequencing. Eleven New Delhi metallo-β-lactamase-5 (NDM-5)-producing strains were identified, including 9 Escherichia coli strains, 1 Klebsiella pneumoniae strain, and 1 Citrobacter freundii strain. No epidemiological links for E. coli isolates were identified by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). S1-PFGE and southern blot suggested that the bla NDM-5 gene was located on a 46-kb IncX3-type plasmid in all isolates. Nine of the 11 isolates (81.8%) tested could successfully transfer their carbapenem-resistant phenotype to E. coli strain C600. Moreover, sequence analysis further showed that this plasmid possessed high sequence similarity to most of previously reported bla NDM-5 -habouring plasmids in China. The present data in this study showed the IncX3 type plasmid played an important role in the dissemination of bla NDM-5 in Enterobacteriaceae . In addition, to the best of our knowledge, this report is the first to isolate both E. coli and C. freundii strains carrying bla NDM-5 from one single patient, which further indicated the possibility of bla NDM-5 transmission among diverse species. Close surveillance is urgently needed to monitor the further dissemination of NDM-5-producing isolates.

Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus

PubMed Central

Jolley, Keith A.; Reed, Elizabeth; Martinez-Urtaza, Jaime

2017-01-01

ABSTRACT Vibrio parahaemolyticus is an important human foodborne pathogen whose transmission is associated with the consumption of contaminated seafood, with a growing number of infections reported over recent years worldwide. A multilocus sequence typing (MLST) database for V. parahaemolyticus was created in 2008, and a large number of clones have been identified, causing severe outbreaks worldwide (sequence type 3 [ST3]), recurrent outbreaks in certain regions (e.g., ST36), or spreading to other regions where they are nonendemic (e.g., ST88 or ST189). The current MLST scheme uses sequences of 7 genes to generate an ST, which results in a powerful tool for inferring the population structure of this pathogen, although with limited resolution, especially compared to pulsed-field gel electrophoresis (PFGE). The application of whole-genome sequencing (WGS) has become routine for trace back investigations, with core genome MLST (cgMLST) analysis as one of the most straightforward ways to explore complex genomic data in an epidemiological context. Therefore, there is a need to generate a new, portable, standardized, and more advanced system that provides higher resolution and discriminatory power among V. parahaemolyticus strains using WGS data. We sequenced 92 V. parahaemolyticus genomes and used the genome of strain RIMD 2210633 as a reference (with a total of 4,832 genes) to determine which genes were suitable for establishing a V. parahaemolyticus cgMLST scheme. This analysis resulted in the identification of 2,254 suitable core genes for use in the cgMLST scheme. To evaluate the performance of this scheme, we performed a cgMLST analysis of 92 newly sequenced genomes, plus an additional 142 strains with genomes available at NCBI. cgMLST analysis was able to distinguish related and unrelated strains, including those with the same ST, clearly showing its enhanced resolution over conventional MLST analysis. It also distinguished outbreak-related from non-outbreak-related strains within the same ST. The sequences obtained from this work were deposited and are available in the public database (http://pubmlst.org/vparahaemolyticus). The application of this cgMLST scheme to the characterization of V. parahaemolyticus strains provided by different laboratories from around the world will reveal the global picture of the epidemiology, spread, and evolution of this pathogen and will become a powerful tool for outbreak investigations, allowing for the unambiguous comparison of strains with global coverage. PMID:28330888

Ready-to-eat street-vended food as a potential vehicle of bacterial pathogens and antimicrobial resistance: An exploratory study in Porto region, Portugal.

PubMed

Campos, Joana; Gil, Joana; Mourão, Joana; Peixe, Luísa; Antunes, Patrícia

2015-08-03

The ready-to-eat street vending commerce, as street mobile food vendors, has grown exponentially worldwide, representing in some countries a significant proportion of food consumed by the urban population. However, the microbiological food safety hazards of mobile vending units in industrialized countries are scarcely evaluated. To assess the microbiological quality and safety of this type of food and try to achieve the connection of its contamination with hygienic conditions of food-handlers, we analyzed hotdogs (n = 10), hamburgers (n = 10) and hands (n = 9) from ten street-vending trailers in the Porto region. Food and food-handler samples were tested for Enterobacteriaceae and coliform counts, Escherichia coli and coagulase-positive staphylococci counts/detection and presence of Salmonella. Aerobic mesophilic counts and detection of Listeria monocytogenes (Pulsed Field Gel Electrophoresis-PFGE and serotyping) were also tested in food samples. E. coli isolates were confirmed by MALDI-TOF and characterized for clonality (phylogenetic groups-PhG, PFGE and Multilocus Sequence Typing), antibiotic resistance (disk diffusion, PCR/sequencing) and intestinal pathogenic virulence factors (PCR/sequencing). All food samples presented poor microbiological quality (100% Enterobacteriaceae and coliforms; 20% E. coli (4 hamburgers, 4 trailers) and 20% (2 hamburgers/2 hotdogs, 3 trailers) were positive for L. monocytogenes (2 PFGE-types belonging to serotype 1/2a and 4b). Salmonella and coagulase-positive staphylococci were not detected. Food-handlers carried Enterobacteriaceae and coliforms (100%), E. coli (11%) and/or coagulase-positive staphylococci (44%). E. coli was detected in 12 samples (n = 30-food/food-handlers; phylogenetic groups A0/A1/B1) with 33% resistant to one or more antibiotics. Two multidrug resistant atypical E. coli pathotype strains (astA-ST165(CC165)/food-handler, eaeA-ST327/food) were detected. Three out of eight E. coli clonal lineages [ST409/ST976(CC10)/ST297] and the two L. monocytogenes clones were spread in different samples/trailers, suggesting cross-contamination or a common source of contamination. This exploratory study, in Porto region, showed ready-to-eat street foods from vending trailers as potential vehicles of clinically relevant L. monocytogenes serotypes and/or E. coli carrying clinically relevant virulence/antibiotic resistance features, and food-handlers as a critical risk factor in this expanding food sector. Copyright © 2015 Elsevier B.V. All rights reserved.

Successful infection control for a vancomycin-intermediate Staphylococcus aureus outbreak in an advanced emergency medical service centre.

PubMed

Sakai, Y; Qin, L; Miura, M; Masunaga, K; Tanamachi, C; Iwahashi, J; Kida, Y; Takasu, O; Sakamoto, T; Watanabe, H

2016-04-01

A vancomycin-intermediate Staphylococcus aureus (VISA) (vancomycin minimum inhibitory concentration: 4mg/L) outbreak occurred in an advanced emergency medical service centre [hereafter referred to as the intensive care unit (ICU)] between 2013 and 2014. Our objective was to evaluate the infection control measures that were successful. Seventeen VISA strains were isolated from the sputum of 15 inpatients and the skin of two inpatients. Fourteen VISA strains were recognized as colonization. However, three VISA strains were isolated from the sputum of three inpatients with pneumonia. Environmental cultures were performed and VISA strains were detected in five of 65 sites. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) was performed on 21 VISA strains. Molecular typing including PFGE and MLST showed that the patterns of 19 VISA strains were identical and those of the other two VISA strains were possibly related. This meant that a horizontal transmission of VISA strains had occurred in the ICU. In August 2013, the infection control team began interventions. However, new inpatients with VISA strains continued to appear. Therefore, in October 2013, the ICU was partially closed in order to try to prevent further horizontal transmission, and existing inpatients with the VISA strain were isolated. Although new cases quickly dissipated after the partial closure, it took approximately five months to eradicate the VISA outbreak. Our data suggest that despite the employment of various other infection control measures, partial closure of the ICU was essential in terminating this VISA outbreak. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Comparison of Genotypes of Salmonella enterica Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds▿

PubMed Central

Parker, Craig T.; Huynh, Steven; Quiñones, Beatriz; Harris, Linda J.; Mandrell, Robert E.

2010-01-01

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized l-aspartic acid, l-glutamic acid, l-proline, l-alanine, and d-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations. PMID:20363782

Comparative evaluation of an automated repetitive-sequence-based PCR instrument versus pulsed-field gel electrophoresis in the setting of a Serratia marcescens nosocomial infection outbreak.

PubMed

Ligozzi, Marco; Fontana, Roberta; Aldegheri, Marco; Scalet, Giovanna; Lo Cascio, Giuliana

2010-05-01

A semiautomated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with pulsed-field gel electrophoresis (PFGE) to investigate an outbreak of Serratia marcescens infections in a neonatal intensive care unit (NICU). A selection of 36 epidemiologically related and 8 epidemiologically unrelated isolates was analyzed. Among the epidemiologically related isolates, PFGE identified five genetically unrelated patterns. Thirty-two isolates from patients and wet nurses showed the same PFGE profile (pattern A). Genetically unrelated PFGE patterns were found in one patient (pattern B), in two wet nurses (patterns C and D), and in an environmental isolate from the NICU (pattern G). Rep-PCR identified seven different patterns, three of which included the 32 isolates of PFGE type A. One or two band differences in isolates of these three types allowed isolates to be categorized as similar and included in a unique cluster. Isolates of different PFGE types were also of unrelated rep-PCR types. All of the epidemiologically unrelated isolates were of different PFGE and rep-PCR types. The level of discrimination exhibited by rep-PCR with the DiversiLab system allowed us to conclude that this method was able to identify genetic similarity in a spatio-temporal cluster of S. marcescens isolates.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1

PubMed Central

2014-01-01

Background Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. Methods V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). Results All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498–511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. Conclusions In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. PMID:25022982

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

PubMed

Leekitcharoenphon, Pimlapas; Friis, Carsten; Zankari, Ea; Svendsen, Christina Aaby; Price, Lance B; Rahmani, Maral; Herrero-Fresno, Ana; Fashae, Kayode; Vandenberg, Olivier; Aarestrup, Frank M; Hendriksen, Rene S

2013-10-15

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Reassessment of Cronobacter spp. originally isolated as Enterobacter sakazakii from infant food.

PubMed

Akineden, Ömer; Heinrich, Vanessa; Gross, Madeleine; Usleber, Ewald

2017-08-01

Cronobacter spp. cause infant disease, several cases have been associated with powdered infant formulae (PIF). In the early 2000s, contamination of German PIF with these opportunistic pathogens was quite common. Before 2008, all isolates Cronobacter spp. had been classified as Enterobacter sakazakii, therefore little is known about species diversity within such isolates. Genetic, serologic, and biochemical traits of 80 Cronobacter isolates, originally obtained 2003-2006 within infant food surveys in Germany, were reassessed in this study. By sequencing of the fusA gene, all isolates were unambiguously assigned to two species, C. sakazakii (n = 73) and C. malonaticus (n = 7). PCR serotyping identified five C. sakazakii serotypes and two C. malonaticus serotypes, biochemical profiling yielded five biogroups. PFGE analysis also showed high heterogeneity in both species. Multilocus sequence typing of 26 selected isolates yielded 16 different sequence types (ST), including C. sakazakii ST 1 (n = 6) and the highly virulent ST 4 (n = 2). The results suggest that just two, but highly heterogeneous species were responsible for the Cronobacter contamination problem which challenged the German PIF industry in the beginning of this century. This fact may have influenced the success of efforts to identify and eliminate sources of contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain.

PubMed

Solà-Ginés, Marc; Cameron-Veas, Karla; Badiola, Ignacio; Dolz, Roser; Majó, Natalia; Dahbi, Ghizlane; Viso, Susana; Mora, Azucena; Blanco, Jorge; Piedra-Carrasco, Nuria; González-López, Juan José; Migura-Garcia, Lourdes

2015-01-01

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6')-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.

Clinical and epidemiological analysis of Campylobacter fetus subsp. fetus infections in humans and comparative genetic analysis with strains isolated from cattle.

PubMed

Escher, Robert; Brunner, Colette; von Steiger, Niklaus; Brodard, Isabelle; Droz, Sara; Abril, Carlos; Kuhnert, Peter

2016-05-14

Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated. The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians. PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance. Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts.

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecium: a Prospective, Multicenter Study in South American Hospitals▿

PubMed Central

Panesso, Diana; Reyes, Jinnethe; Rincón, Sandra; Díaz, Lorena; Galloway-Peña, Jessica; Zurita, Jeannete; Carrillo, Carlos; Merentes, Altagracia; Guzmán, Manuel; Adachi, Javier A.; Murray, Barbara E.; Arias, Cesar A.

2010-01-01

Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years. PMID:20220167

Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level.

PubMed

Mahmmod, Y S; Klaas, I C; Katholm, J; Lutton, M; Zadoks, R N

2015-10-01

Host-adaptation of Streptococcus agalactiae subpopulations has been described whereby strains that are commonly associated with asymptomatic carriage or disease in people differ phenotypically and genotypically from those causing mastitis in dairy cattle. Based on multilocus sequence typing (MLST), the most common strains in dairy herds in Denmark belong to sequence types (ST) that are also frequently found in people. The aim of this study was to describe epidemiological and diagnostic characteristics of such strains in relation to bovine mastitis. Among 1,199 cattle from 6 herds, cow-level prevalence of S. agalactiae was estimated to be 27.4% based on PCR and 7.8% based on bacteriological culture. Quarter-level prevalence was estimated at 2.8% based on bacteriological culture. Per herd, between 2 and 26 isolates were characterized by pulsed-field gel electrophoresis (PFGE) and MLST. Within each herd, a single PFGE type and ST predominated, consistent with a contagious mode of transmission or point source infection within herds. Evidence of within-herd evolution of S. agalactiae was detected with both typing methods, although ST belonged to a single clonal complex (CC) per herd. Detection of CC23 (3 herds) was associated with significantly lower approximate count (colony-forming units) at the quarter level and significantly lower cycle threshold value at the cow level than detection of CC1 (2 herds) or CC19 (1 herd), indicating a lower bacterial load in CC23 infections. Median values for the number of infected quarters and somatic cell count (SCC) were numerically but not significantly lower for cows infected with CC23 than for cows with CC1 or CC19. For all CC, an SCC threshold of 200,000 cells/mL was an unreliable indicator of infection status, and prescreening of animals based on SCC as part of S. agalactiae detection and eradication campaigns should be discouraged. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

PubMed Central

Solà-Ginés, Marc; Cameron-Veas, Karla; Badiola, Ignacio; Dolz, Roser; Majó, Natalia; Dahbi, Ghizlane; Viso, Susana; Mora, Azucena; Blanco, Jorge; Piedra-Carrasco, Nuria; González-López, Juan José; Migura-Garcia, Lourdes

2015-01-01

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained bla CTX-M-14, two bla SHV-12, two bla CMY-2 and one bla SHV-2. Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured bla CMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health. PMID:26600205

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

PubMed

Cunha, Marcos Paulo Vieira; Saidenberg, Andre Becker; Moreno, Andrea Micke; Ferreira, Antonio José Piantino; Vieira, Mônica Aparecida Midolli; Gomes, Tânia Aparecida Tardelli; Knöbl, Terezinha

2017-01-01

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.

Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results

PubMed Central

Shariat, Nikki; Kirchner, Margaret K.; Sandt, Carol H.; Trees, Eija; Barrangou, Rodolphe

2013-01-01

Salmonella enterica subsp. enterica serovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains of S. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR–multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set of S. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38 S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recent S. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20 S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping for S. Newport. PMID:23678062

Antimicrobial Resistance and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus Isolates from Child Patients of High-Risk Wards in Shenzhen, China.

PubMed

Qin, Yang; Wen, Feiqiu; Zheng, Yuejie; Zhao, Ruizhen; Hu, Qinghua; Zhang, Renli

2017-09-25

Methicillin-resistant Staphylococcus aureus (MRSA) strains are responsible for high rates of mortality and thus pose a substantial burden to public health worldwide. Here, we investigated the antimicrobial susceptibility and molecular characteristics of MRSA isolated from child patients at Shenzhen Children's Hospital. We characterized 140 MRSA strains through antimicrobial susceptibility testing. We further performed spa typing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) analysis, pvl gene analysis, and pulsed-field gel electrophoresis (PFGE). The analyzed MRSA strains were found to be sensitive to most non-β-lactam antimicrobial agents. Sequence type (ST) 59 was found to be the most common MLST lineage (54.3%). Most MRSA isolates belonged to the SCCmec IV (64.3%) and V (22.8%) types. The MRSA-ST59-SCCmec IV-t437 clone was the most predominant strain that infected 28.6% of all patients studied. Moreover, 50.7% of MRSA isolates were found to be pvl-positive. We report preliminary data on the prevalence and distribution of MRSA genotypes in Shenzhen Children's Hospital. We characterized MRSA colonization dynamics in child patients in China, and our findings can serve as the basis for the development of strategies to prevent MRSA infection and transmission.

Defining a Core Genome Multilocus Sequence Typing Scheme for the Global Epidemiology of Vibrio parahaemolyticus.

PubMed

Gonzalez-Escalona, Narjol; Jolley, Keith A; Reed, Elizabeth; Martinez-Urtaza, Jaime

2017-06-01

Vibrio parahaemolyticus is an important human foodborne pathogen whose transmission is associated with the consumption of contaminated seafood, with a growing number of infections reported over recent years worldwide. A multilocus sequence typing (MLST) database for V. parahaemolyticus was created in 2008, and a large number of clones have been identified, causing severe outbreaks worldwide (sequence type 3 [ST3]), recurrent outbreaks in certain regions (e.g., ST36), or spreading to other regions where they are nonendemic (e.g., ST88 or ST189). The current MLST scheme uses sequences of 7 genes to generate an ST, which results in a powerful tool for inferring the population structure of this pathogen, although with limited resolution, especially compared to pulsed-field gel electrophoresis (PFGE). The application of whole-genome sequencing (WGS) has become routine for trace back investigations, with core genome MLST (cgMLST) analysis as one of the most straightforward ways to explore complex genomic data in an epidemiological context. Therefore, there is a need to generate a new, portable, standardized, and more advanced system that provides higher resolution and discriminatory power among V. parahaemolyticus strains using WGS data. We sequenced 92 V. parahaemolyticus genomes and used the genome of strain RIMD 2210633 as a reference (with a total of 4,832 genes) to determine which genes were suitable for establishing a V. parahaemolyticus cgMLST scheme. This analysis resulted in the identification of 2,254 suitable core genes for use in the cgMLST scheme. To evaluate the performance of this scheme, we performed a cgMLST analysis of 92 newly sequenced genomes, plus an additional 142 strains with genomes available at NCBI. cgMLST analysis was able to distinguish related and unrelated strains, including those with the same ST, clearly showing its enhanced resolution over conventional MLST analysis. It also distinguished outbreak-related from non-outbreak-related strains within the same ST. The sequences obtained from this work were deposited and are available in the public database (http://pubmlst.org/vparahaemolyticus). The application of this cgMLST scheme to the characterization of V. parahaemolyticus strains provided by different laboratories from around the world will reveal the global picture of the epidemiology, spread, and evolution of this pathogen and will become a powerful tool for outbreak investigations, allowing for the unambiguous comparison of strains with global coverage. Copyright © 2017 Gonzalez-Escalona et al.

Characterization of Staphylococcus aureus faecal isolates associated with food-borne disease in Korea.

PubMed

Shin, E; Hong, H; Park, J; Oh, Y; Jung, J; Lee, Y

2016-07-01

To characterize Staphylococcus aureus faecal isolates from people suspected to be infected with food poisoning by using antimicrobial susceptibility testing and molecular techniques. A total of 340 Staph. aureus isolates from 6226 people suspected to be infected with food poisoning were identified and characterized by biochemical methods, antimicrobial susceptibility testing and PCR. Samples were obtained from January 2006 to December 2008 from the National Notifiable Diseases Surveillance System at the Research Institute of Public Health and Environment in Seoul Metropolitan, Korea. All strains carried at least one of the eight staphylococcal enterotoxin (se) genes tested and a total of 27 se profiles were produced; the most frequent se profile was seg-sei and the next was sea. Among the total isolates, 36 methicillin-resistant Staphylococcus aureus (MRSAs) isolates were further analysed by multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE) and PCR detection for pvl. ST72-SCCmec type IV was the most predominant clone (27 isolates, 75%) followed by ST1-SCCmec type IV (five isolates, 13·8%), ST20-SCCmec type IV (one isolate, 2·8%), ST493-SCCmec type IV (one isolate, 2·8%), ST903-SCCmec type IV (one isolate, 2·8%) and ST5-SCCmec type II (one isolate, 2·8%). By PFGE typing, MRSAs isolated during the same period were grouped together although they were isolated from different regions. None of MRSAs had PVL gene and nine MRSAs were multidrug resistant. Analysis of MRSAs by MLST, SCCmec typing, PFGE and pvl detection showed that the majority of strain associated with food-borne diseases belonged to a Korean community-acquired (CA) MRSA clone with ST72-SCCmec type IV-PVL negative-SEG/SEI and its variations while one strain was hospital-acquired (HA) MRSA. CA-MRSA clone which possessed ST72-SCCmec type IV-PVL negative-SEG/SEI was spread most commonly among MRSAs that were associated with food-borne diseases. This is the first report of ST903 strain in Korea. © 2016 The Society for Applied Microbiology.

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr)

USDA-ARS?s Scientific Manuscript database

A Multilocus Sequence Typing (MLST) method based on allelic variation of 7 chromosomal loci was developed for characterizing genotypes within the genus Bradyrhizobium. With the method 29 distinct multilocus genotypes (GTs) were identified among 191 culture collection soybean strains. The occupancy ...

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013

PubMed Central

Schjørring, Susanne; Niskanen, Taina; Torpdahl, Mia; Björkman, Jonas T; Nielsen, Eva Møller

2016-01-01

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector. PMID:28006653

Comparative genomics of Vibrio cholerae from Haiti, Asia, and Africa.

PubMed

Reimer, Aleisha R; Van Domselaar, Gary; Stroika, Steven; Walker, Matthew; Kent, Heather; Tarr, Cheryl; Talkington, Deborah; Rowe, Lori; Olsen-Rasmussen, Melissa; Frace, Michael; Sammons, Scott; Dahourou, Georges Anicet; Boncy, Jacques; Smith, Anthony M; Mabon, Philip; Petkau, Aaron; Graham, Morag; Gilmour, Matthew W; Gerner-Smidt, Peter

2011-11-01

Cholera was absent from the island of Hispaniola at least a century before an outbreak that began in Haiti in the fall of 2010. Pulsed-field gel electrophoresis (PFGE) analysis of clinical isolates from the Haiti outbreak and recent global travelers returning to the United States showed indistinguishable PFGE fingerprints. To better explore the genetic ancestry of the Haiti outbreak strain, we acquired 23 whole-genome Vibrio cholerae sequences: 9 isolates obtained in Haiti or the Dominican Republic, 12 PFGE pattern-matched isolates linked to Asia or Africa, and 2 nonmatched outliers from the Western Hemisphere. Phylogenies for whole-genome sequences and core genome single-nucleotide polymorphisms showed that the Haiti outbreak strain is genetically related to strains originating in India and Cameroon. However, because no identical genetic match was found among sequenced contemporary isolates, a definitive genetic origin for the outbreak in Haiti remains speculative.

Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms.

PubMed

Solà-Ginés, Marc; González-López, Juan José; Cameron-Veas, Karla; Piedra-Carrasco, Nuria; Cerdà-Cuéllar, Marta; Migura-Garcia, Lourdes

2015-06-01

Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained bla(CTX-M-1), 18 contained bla(CTX-M-14), and 1 contained bla(CTX-M-9). ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Wide geographical dissemination of the multiresistant Staphylococcus capitis NRCS-A clone in neonatal intensive-care units.

PubMed

Butin, M; Rasigade, J-P; Martins-Simões, P; Meugnier, H; Lemriss, H; Goering, R V; Kearns, A; Deighton, M A; Denis, O; Ibrahimi, A; Claris, O; Vandenesch, F; Picaud, J-C; Laurent, F

2016-01-01

Nosocomial late-onset sepsis represents a frequent cause of morbidity and mortality in preterm neonates. The Staphylococcus capitis clone NRCS-A has been previously described as an emerging cause of nosocomial bacteraemia in French neonatal intensive-care units (NICUs). In this study, we aimed to explore the possible unrecognized dissemination of this clone on a larger geographical scale. One hundred methicillin-resistant S. capitis strains isolated from neonates (n = 86) and adult patients (n = 14) between 2000 and 2013 in four different countries (France, Belgium, the UK, and Australia) were analysed with SmaI pulsed-field gel electrophoresis (PFGE) and dru typing. The vast majority of NICU strains showed the NRCS-A pulsotype and the dt11c type (96%). We then randomly selected 14 isolates (from neonates, n = 12, three per country; from adult patients, n = 2), considered to be a subset of representative isolates, and performed further molecular typing (SacII PFGE, SCCmec typing, and multilocus sequence typing-like analysis), confirming the clonality of the S. capitis strains isolated from neonates, despite their distant geographical origin. Whole genome single-nucleotide polymorphism-based phylogenetic analysis of five NICU isolates (from the different countries) attested to high genetic relatedness within the NRCS-A clone. Finally, all of the NRCS-A strains showed multidrug resistance (e.g. methicillin and aminoglycoside resistance, and decreased vancomycin susceptibility), with potential therapeutic implications for infected neonates. In conclusion, this study represents the first report of clonal dissemination of methicillin-resistant coagulase-negative Staphylococcus clone on a large geographical scale. Questions remain regarding the origin and means of international spread, and the reasons for this clone's apparent predilection for neonates. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Emergence of Salmonella enterica serovar Indiana and California isolates with concurrent resistance to cefotaxime, amikacin and ciprofloxacin from chickens in China.

PubMed

Wang, Yongxiang; Zhang, Anyun; Yang, Yongqiang; Lei, Changwei; Jiang, Wei; Liu, Bihui; Shi, Hongping; Kong, Linghan; Cheng, Guangyang; Zhang, Xiuzhong; Yang, Xin; Wang, Hongning

2017-12-04

The aim of this study was to investigate the prevalence and characterization of Salmonella concerning the poultry industry in China. A total of 170 non-duplicate Salmonella isolates were recovered from the 1540 chicken samples. Among the Salmonella isolates from chickens, the predominant serovars were S. enterica serovar Enteritidis (S. Enteritidis) (49/170, 28.8%), S. enterica serovar Indiana (S. Indiana) (37/170, 21.8%) and S. enterica serovar California (S. California) (34/170, 20.0%). High antimicrobial resistance was observed for ciprofloxacin (68.2%), amikacin (48.2%) and cefotaxime (44.7%). Of particular concerns were the 18 S. Indiana and 17 S. California isolates, which were concurrently resistant to cefotaxime, amikacin and ciprofloxacin. The bla CTX-M genes, 16S rRNA methylase genes (armA, rmtD or rmtC) and five plasmid-mediated quinolone resistance (PMQR) determinants (aac(6')-Ib-cr, oqxAB, qnrB, qepA and qnrD) were identified in 18 S. Indiana and 17 S. California isolates. To clarify their genetic correlation, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were further conducted. PFGE profiles showed that the majority of S. Indiana and S. California isolates were clonally unrelated with a standard cut-off of 85%. The results of MLST demonstrated that ST17 and ST40 were the most common ST types in S. Indiana and S. California isolates, respectively. Our findings indicated that the multiple antibiotic resistant S. Indiana and S. California isolates were widespread in chicken in China and might pose a potential threat to public health. Copyright © 2017 Elsevier B.V. All rights reserved.

Antimicrobial resistance and population structure of Staphylococcus epidermidis recovered from animals and humans.

PubMed

Argudín, M Angeles; Vanderhaeghen, Wannes; Vandendriessche, Stien; Vandecandelaere, Ilse; André, François-Xavier; Denis, Olivier; Coenye, Tom; Butaye, Patrick

2015-07-09

While Staphylococcus epidermidis, as part of the commensal flora, is a well-known human opportunistic pathogen, only little is known about the genetic relatedness of S. epidermidis carriage isolates from animal and human origin. This study aimed to compare S. epidermidis recovered from livestock, livestock-farmers and humans associated with the hospital environment. A total of 193 S. epidermidis isolates from three populations [animals (n=33), farmers (n=86) and hospital-associated (n=74)] were characterized by broth microdilution antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The overall S. epidermidis nasal colonization rate was low in animals (1-9%) but high among farmers (75%). High levels of multi-resistance were found in all populations. Tetracycline resistance was high in animal and farmer isolates; resistance to erythromycin, clindamycin and trimethoprim was high in animal and hospital-associated isolates. Methicillin-resistant S. epidermidis - MRSE isolates were found in all collections, with 22 (67%) MRSE in animals, 44 (51%) MRSE in farmers and 42 (57%) MRSE associated with the hospital-setting. Known SCCmec types and variants were detected in 79% of MRSE; the rest were non-typeable cassettes. In total 79 PFGE-types were found, of which 22 were shared between livestock, farmers and the hospital settings. Clonal complex 2 was predominant in all three populations and most STs corresponded to types previously observed in community and nosocomial S. epidermidis populations. S. epidermidis isolates from livestock, farmers and hospital-setting showed a high level of diversity, but some clones can be found in humans as well as in animals. Copyright © 2015 Elsevier B.V. All rights reserved.

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014

PubMed Central

Sekirov, Inna; Croxen, Matthew A.; Ng, Corrinne; Azana, Robert; Chang, Yin; Mataseje, Laura; Boyd, David; Mangat, Chand; Mack, Benjamin; Tadros, Manal; Brodkin, Elizabeth; Kibsey, Pamela; Stefanovic, Aleksandra; Champagne, Sylvie; Mulvey, Michael R.

2015-01-01

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated. PMID:26607987

Streptococcus agalactiae isolates of serotypes Ia, III and V from human and cow are able to infect tilapia.

PubMed

Chen, Ming; Wang, Rui; Luo, Fu-Guang; Huang, Yan; Liang, Wan-Wen; Huang, Ting; Lei, Ai-Ying; Gan, Xi; Li, Li-Ping

2015-10-22

Recent studies have shown that group B streptococcus (GBS) may be infectious across hosts. The purpose of this study is to investigate the pathogenicity of clinical GBS isolates with serotypes Ia, III and V from human and cow to tilapia and the evolutionary relationship among these GBS strains of different sources. A total of 27 clinical GBS isolates from human (n=10), cow (n=2) and tilapia (n=15) were analyzed using serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Among them, 15 isolates were tested for their pathogenicity to tilapia. The results showed that five human GBS strains (2 serotype III, 2 serotype Ia and 1 serotype V) infected tilapia with mortality rate ranging from 56.67% to 100%, while the other five human GBS strains tested were unable to infect tilapia. In addition, two cow GBS strains C001 and C003 of serotype III infected tilapia. However, they had significantly lower pathogenicity than the five human strains. Furthermore, human GBS strains H005 and H008, which had very strong ability to infect tilapia, had the same PFGE pattern. MLST analysis showed that the five human and the two cow GBS strains that were able to infect tilapia belonged to clonal complexes CC19, CC23 and CC103. The study for the first time confirmed that human or cow GBS clonal complexes CC19, CC23 and CC103 containing strains with serotypes Ia, III and V could infect tilapia and induce clinical signs under experimental conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

PubMed Central

Ghosh, Anuradha; Dowd, Scot E.; Zurek, Ludek

2011-01-01

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks. PMID:21811613

Strains of the Group I Lineage of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops, are Predominant in Brazil.

PubMed

Silva, Gustavo M; Souza, Ricardo M; Yan, Lichun; Júnior, Rui S; Medeiros, Flavio H V; Walcott, Ron R

2016-12-01

Bacterial fruit blotch (BFB), caused by the seedborne bacterium Acidovorax citrulli, is an economically important threat to cucurbitaceous crops worldwide. Since the first report of BFB in Brazil in 1990, outbreaks have occurred sporadically on watermelon and, more frequently, on melon, resulting in significant yield losses. At present, the genetic diversity and the population structure of A. citrulli strains in Brazil remain unclear. A collection of 74 A. citrulli strains isolated from naturally infected tissues of different cucurbit hosts in Brazil between 2000 and 2014 and 18 A. citrulli reference strains from other countries were compared by pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes, and pathogenicity tests on seedlings of different cucurbit species. The Brazilian population comprised predominantly group I strains (98%), regardless of the year of isolation, geographical region, or host. Whole-genome restriction digestion and PFGE analysis revealed that three unique and previously unreported A. citrulli haplotypes (assigned as haplotypes B22, B23, and B24) occurred in Brazil. The greatest diversity of A. citrulli (four haplotypes) was found among strains collected from the northeastern region of Brazil, which accounts for more than 90% of the country's melon production. MLSA clearly distinguished A. citrulli strains into two well-supported clades, in agreement with observations based on PFGE analysis. Five Brazilian A. citrulli strains, representing different group I haplotypes, were moderately aggressive on watermelon seedlings compared with four group II strains that were highly aggressive. In contrast, no significant differences in BFB severity were observed between group I and II A. citrulli strains on melon and squash seedlings. Finally, we observed a differential effect of temperature on in vitro growth of representative group I and II A. citrulli haplotypes. Specifically, of 18 group II strains tested, all grew at 40 and 41°C, whereas only 3 of 15 group I strains (haplotypes B8[P], B3[K], and B15) grew at 40°C. Three strains representing haplotype B8(P) were the only group I strains that grew at 41°C. These results contribute to a better understanding of the genetic diversity of A. citrulli associated with BFB outbreaks in Brazil, and reinforce the efficiency of MLSA and PFGE analysis for assessing population structure. This study also provides the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations.

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

PubMed

Mosiej, Ewa; Krysztopa-Grzybowska, Katarzyna; Polak, Maciej; Prygiel, Marta; Lutyńska, Anna

2017-06-01

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

First Report of Group CTX-M-9 Extended Spectrum Beta-Lactamases in Escherichia coli Isolates from Pediatric Patients in Mexico

PubMed Central

Merida-Vieyra, Jocelin; De Colsa, Agustin; Calderon Castañeda, Yair; Arzate Barbosa, Patricia; Aquino Andrade, Alejandra

2016-01-01

The aim of this study was to identify the presence of group CTX-M-9 extended spectrum beta-lactamases (ESBL) in clinical Escherichia coli isolates from pediatric patients. A total of 404 non-repeated positive ESBL E. coli isolates were collected from documented clinical infections in pediatric patients over a 2-year period. The identification and susceptibility profiles were determined using an automated system. Isolates that suggested ESBL production based on their resistance profiles to third and fourth generation cephalosporin and monobactam were selected. ESBL production was phenotypically confirmed using a diffusion method with cefotaxime and ceftazidime discs alone and in combination with clavulanic acid. blaESBL gene identification was performed through PCR amplification and sequencing. Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) were performed to establish the clonal relationships of the E. coli isolates. CTX-M-9-type ESBLs were detected in 2.5% of the isolates. The subtypes corresponded to blaCTX-M-14 (n = 4) and blaCTX-M-27 (n = 6). Additionally, coexistence with other beta-lactamases was observed. A clonal relationship was established in three isolates; the rest were classified as non-related. We found seven different sequence type (ST) in CTX-M-9- producing E. coli isolates. ST38 was the most frequent. This study is the first report in Mexico to document the presence of group CTX-M-9 ESBLs in E. coli isolates from pediatric patients. PMID:27992527

Analysis of Haemophilus influenzae serotype f isolated from three Japanese children with invasive H. influenzae infection.

PubMed

Hoshino, Tadashi; Hachisu, Yushi; Kikuchi, Takashi; Tokutake, Shoko; Okui, Hideyuki; Kutsuna, Satoru; Fukasawa, Chie; Murayama, Kei; Oohara, Asami; Shimizu, Hiroyuki; Ito, Midori; Takahashi, Yoshiko; Ishiwada, Naruhiko

2015-04-01

In Japan, publicly subsidized Haemophilus influenzae serotype b vaccines became available in 2011; consequently, the incidence of invasive H. influenzae infection in paediatric patients of less than 5 years of age decreased dramatically. In 2013, the first case of H. influenzae serotype f (Hif) meningitis in a Japanese infant was reported, and another case of Hif meningitis in a Japanese infant was observed in 2013. We experienced a fatal paediatric case of Hif bacteraemia in 2004; therefore, we conducted an analysis of the three Hif strains isolated from these three Japanese children with invasive Hif infections. All three strains were β-lactamase-non-producing, ampicillin-sensitive strains, with MICs of 1 µg ml(-1) or less. However, one of the three strains showed slightly elevated MICs for ampicillin (1 µg ml(-1)), cefotaxime (0.25 µg ml(-1)) and meropenem (0.13 µg ml(-1)). A molecular analysis by multilocus sequence typing identified all three strains as sequence type (ST) 124, which is a predominant invasive Hif strain in many countries. SmaI-digested PFGE showed variable DNA fragmentation patterns among the strains, suggesting that some highly virulent strains have originated from a single ST124 clone and caused invasive Hif infections in Japan. Additional studies are needed to determine the factors that have led to the clonal expansion of virulent ST124 strains. © 2015 The Authors.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

PubMed

Cavanagh, Jorunn Pauline; Klingenberg, Claus; Hanssen, Anne-Merethe; Fredheim, Elizabeth Aarag; Francois, Patrice; Schrenzel, Jacques; Flægstad, Trond; Sollid, Johanna Ericson

2012-06-01

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Public health significance of major genotypes of Salmonella enterica serovar Enteritidis present in both human and chicken isolates in Korea.

PubMed

Kang, Min-Su; Oh, Jae-Young; Kwon, Yong-Kuk; Lee, Deog-Yong; Jeong, Ok-Mi; Choi, Byung-Kook; Youn, So-Youn; Jeon, Byung-Woo; Lee, Hye-Jin; Lee, Hee-Soo

2017-06-01

Salmonella enterica serovar Enteritidis is one of the most common serotypes implicated in Salmonella infections in both humans and poultry worldwide. It has been reported that human salmonellosis is mainly associated with the consumption of poultry products contaminated with serovar Enteritidis. The present study was to extensively analyze the public health risk of serovar Enteritidis isolates from chickens in Korea. A total of 127 chicken isolates were collected from clinical cases, on-farm feces, and chicken meat between 1998 and 2012 and 20 human clinical isolates were obtained from patients with diarrhea between 2000 and 2006 in Korea. To characterize the isolates from chickens and humans, we compared the pulsed-field gel electrophoresis (PFGE) patterns and multilocus variable-number tandem-repeat analysis (MLVA) profiles of the isolates. We further characterized representative isolates of different genotypes using a DNA microarray. PFGE revealed 28 patterns and MLVA identified 16 allelic profiles. The DNA microarray showed high genetic variability in plasmid regions and other fimbrial subunits of the isolates although the virulence gene contents of isolates from the same source and/or of the same genotype were unrelated. PFGE and MLVA showed that major genotypes were present in both human and chicken isolates. This result suggests that chickens in Korea pose a significant risk to public health as a source of serovar Enteritidis as has been noted in other countries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces

USDA-ARS?s Scientific Manuscript database

The identification and classification of species within the genus Streptomyces is difficult because there are presently 576 validly described species and this number increases every year. The value of the application of multilocus sequence analysis scheme to the systematics of Streptomyces species h...

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples

PubMed Central

Esposito, Eliana P.; Gaiarsa, Stefano; Del Franco, Mariateresa; Crivaro, Valeria; Bernardo, Mariano; Cuccurullo, Susanna; Pennino, Francesca; Triassi, Maria; Marone, Piero; Sassera, Davide; Zarrilli, Raffaele

2017-01-01

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim–sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2′-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples. PMID:29163422

Genetic diversity of Flavobacterium psychrophilum isolates from three Oncorhynchus spp. in the United States, as revealed by multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Flavobacterium psychrophilum is an important pathogen of salmonids worldwide. Multilocus sequence typing (MLST) has identified a recombinogenic population structure from which emerged a few epidemic clonal complexes particularly threatening for salmonid aquaculture. To date, MLST genotypes for this ...

Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

PubMed

Gogoi, Purnima; Borah, Probodh; Hussain, Iftikar; Das, Leena; Hazarika, Girin; Tamuly, Shantanu; Barkalita, Luit Moni

2018-05-01

A total of 12 Salmonella isolates belonging to different serovars, viz , Salmonella enterica serovar Enteritidis ( n = 4), Salmonella enterica serovar Weltevreden ( n = 4), Salmonella enterica serovar Newport ( n = 1), Salmonella enterica serovar Litchifield ( n = 1), and untypeable strains ( n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz , repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR. Copyright © 2018 American Society for Microbiology.

A molecular surveillance reveals the prevalence of Vibrio cholerae O139 isolates in China from 1993 to 2012.

PubMed

Zhang, Ping; Zhou, Haijian; Diao, Baowei; Li, Fengjuan; Du, Pengcheng; Li, Jie; Kan, Biao; Morris, J Glenn; Wang, Duochun

2014-04-01

Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates. Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing (MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates, in contrast, generally lacked VSP-I and -II, and fell within 13 additional sequence types in two clonal complexes distinct from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity, have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited greater diversity, with multiple clonal lineages, than did their toxigenic counterparts.

Genetic diversity of Streptococcus suis isolated from three pig farms of China obtained by acquiring antibiotic resistance genes.

PubMed

Huang, Jinhu; Shang, Kexin; Kashif, Jam; Wang, Liping

2015-05-01

Acquiring antibiotic resistance genes may change an organism's genetic characteristics and the effect of antibiotics, resulting in a rapid transmission of microbial pathogens. The objectives of this experiment were to identify the features of Streptococcus suis (S. suis) isolated from three pig farms in China which are geographically isolated. Among the isolates, 56.52% were sequence type 7 (ST7), followed by ST1 (26.09%), indicating that ST7 prevails in China, as revealed by multi-locus sequence typing (MLST). Statistical analysis indicated an association between geography, sequence types and antibiotic resistance genotypes. 66.67% of the isolates in Sichuan province presented a (ermB(-) + mefA(-) + tetO(-) + tetM(-)) + ST7 type. The tetM(+) +ST7 type was the most prevalent in Jiangsu province, whereas the strains from Hebei province had a phenotype ermB(+) +tetO(+) +ST1 (63.64%). Pulsed-field gel electrophoresis (PGFE) pattern A2 with 100% similarity reflected the clonal dissemination between Sichuan and Jiangsu provinces. Strains carrying or not carrying antibiotic resistance genes presented different PFGE patterns in Hebei province. ST7 is widespread in many regions of China and a clonal dissemination occurred between Sichuan and Jiangsu provinces in diseased pigs. However, ST1 strains with macrolide and tetracycline resistance (ermB(+) +tetO(+) +ST1) isolated from a farm in Hebei province demonstrated that the genetic diversity was contributed by horizontal acquiring of ermB and tetO carrying elements. © 2014 Society of Chemical Industry.

Characterization of Vibrio parahaemolyticus clinical strains from Maryland (2012-2013) and comparisons to a locally and globally diverse V. parahaemolyticus strains by whole-genome sequence analysis.

PubMed

Haendiges, Julie; Timme, Ruth; Allard, Marc W; Myers, Robert A; Brown, Eric W; Gonzalez-Escalona, Narjol

2015-01-01

Vibrio parahaemolyticus is the leading cause of foodborne illnesses in the US associated with the consumption of raw shellfish. Previous population studies of V. parahaemolyticus have used Multi-Locus Sequence Typing (MLST) or Pulsed Field Gel Electrophoresis (PFGE). Whole genome sequencing (WGS) provides a much higher level of resolution, but has been used to characterize only a few United States (US) clinical isolates. Here we report the WGS characterization of 34 genomes of V. parahaemolyticus strains that were isolated from clinical cases in the state of Maryland (MD) during 2 years (2012-2013). These 2 years saw an increase of V. parahaemolyticus cases compared to previous years. Among these MD isolates, 28% were negative for tdh and trh, 8% were tdh positive only, 11% were trh positive only, and 53% contained both genes. We compared this set of V. parahaemolyticus genomes to those of a collection of 17 archival strains from the US (10 previously sequenced strains and 7 from NCBI, collected between 1988 and 2004) and 15 international strains, isolated from geographically-diverse environmental and clinical sources (collected between 1980 and 2010). A WGS phylogenetic analysis of these strains revealed the regional outbreak strains from MD are highly diverse and yet genetically distinct from the international strains. Some MD strains caused outbreaks 2 years in a row, indicating a local source of contamination (e.g., ST631). Advances in WGS will enable this type of analysis to become routine, providing an excellent tool for improved surveillance. Databases built with phylogenetic data will help pinpoint sources of contamination in future outbreaks and contribute to faster outbreak control.

Characterization of Vibrio parahaemolyticus clinical strains from Maryland (2012–2013) and comparisons to a locally and globally diverse V. parahaemolyticus strains by whole-genome sequence analysis

PubMed Central

Haendiges, Julie; Timme, Ruth; Allard, Marc W.; Myers, Robert A.; Brown, Eric W.; Gonzalez-Escalona, Narjol

2015-01-01

Vibrio parahaemolyticus is the leading cause of foodborne illnesses in the US associated with the consumption of raw shellfish. Previous population studies of V. parahaemolyticus have used Multi-Locus Sequence Typing (MLST) or Pulsed Field Gel Electrophoresis (PFGE). Whole genome sequencing (WGS) provides a much higher level of resolution, but has been used to characterize only a few United States (US) clinical isolates. Here we report the WGS characterization of 34 genomes of V. parahaemolyticus strains that were isolated from clinical cases in the state of Maryland (MD) during 2 years (2012–2013). These 2 years saw an increase of V. parahaemolyticus cases compared to previous years. Among these MD isolates, 28% were negative for tdh and trh, 8% were tdh positive only, 11% were trh positive only, and 53% contained both genes. We compared this set of V. parahaemolyticus genomes to those of a collection of 17 archival strains from the US (10 previously sequenced strains and 7 from NCBI, collected between 1988 and 2004) and 15 international strains, isolated from geographically-diverse environmental and clinical sources (collected between 1980 and 2010). A WGS phylogenetic analysis of these strains revealed the regional outbreak strains from MD are highly diverse and yet genetically distinct from the international strains. Some MD strains caused outbreaks 2 years in a row, indicating a local source of contamination (e.g., ST631). Advances in WGS will enable this type of analysis to become routine, providing an excellent tool for improved surveillance. Databases built with phylogenetic data will help pinpoint sources of contamination in future outbreaks and contribute to faster outbreak control. PMID:25745421

Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance

PubMed Central

Martinez, Teresa; Martinez, Idali; Vazquez, Guillermo J.; Aquino, Edna E.

2016-01-01

Carbapenems are considered the last-resort antibiotics to treat infections caused by multidrug-resistant Gram-negative bacilli. The Klebsiella pneumoniae carbapenemase (KPC) enzyme hydrolyses β-lactam antibiotics including the carbapenems. KPC has been detected worldwide in Enterobacteriaceae and Pseudomonas aeruginosa isolates associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen. KPC-producing A. baumannii has been reported to date only in Puerto Rico. The objective of this study was to determine the whole genomic sequence of a KPC-producing A. baumannii in order to (i) define its allelic diversity, (ii) identify the location and genetic environment of the blaKPC and (iii) detect additional mechanisms of antimicrobial resistance. Next-generation sequencing, Southern blot, PFGE, multilocus sequence typing and bioinformatics analysis were performed. The organism was assigned to the international ST2 clone. The blaKPC-2 was identified on a novel truncated version of Tn4401e (tentatively named Tn4401h), located in the chromosome within an IncA/C plasmid fragment derived from an Enterobacteriaceae, probably owing to insertion sequence IS26. A chromosomally located truncated Tn1 transposon harbouring a blaTEM-1 was found in a novel genetic environment within an antimicrobial resistance cluster. Additional resistance mechanisms included efflux pumps, non-β-lactam antibiotic inactivating enzymes within and outside a resistance island, two class 1 integrons, In439 and the novel In1252, as well as mutations in the topoisomerase and DNA gyrase genes which confer resistance to quinolones. The presence of the blaKPC in an already globally disseminated A. baumannii ST2 presents a serious threat of further dissemination. PMID:27259867

Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil

PubMed Central

Baio, Paulo Victor Pereira; Mota, Higor Franceschi; Freitas, Andréa D'avila; Gomes, Débora Leandro Rama; Ramos, Juliana Nunes; Sant'Anna, Lincoln Oliveira; Souza, Mônica Cristina; Camello, Thereza Cristina Ferreira; Hirata, Raphael; Vieira, Verônica Viana; Mattos-Guaraldi, Ana Luíza

2013-01-01

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases. PMID:23440110

Rapid pulsed-field gel electrophoresis protocol for subtyping of Streptococcus suis serotype 2.

PubMed

Luey, Cindy K Y; Chu, Yiu Wai; Cheung, Terence K M; Law, Catherine C P; Chu, Man Yu; Cheung, Danny T L; Kam, Kai Man

2007-03-01

A rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Streptococcus suis serotype 2 was developed and evaluated using 27 clinical isolates from 22 epidemiologically unrelated patients. Results were matched against antibiogram, virulence genotyping and multi locus sequence typing (MLST). PFGE appeared to be the most discriminatory with numerical index of discrimination (D) equal to 0.87.

Antimicrobial Resistance and Molecular Investigation of H2S-Negative Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China.

PubMed

Xie, Jing; Yi, Shengjie; Zhu, Jiangong; Li, Peng; Liang, Beibei; Li, Hao; Yang, Xiaoxia; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Wu, Zhihao; Qiu, Shaofu; Song, Hongbin

2015-01-01

Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H2S-negative S. Choleraesuis isolates and two H2S-positive isolates. This is the first report of H2S-negative S. Choleraesuis isolated from humans. The majority of H2S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H2S-negative and H2S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H2S-negative S. Choleraesuis isolates belonging to Group I and H2S-positive isolates belonging to Group II. By MLST analysis, the H2S-negative isolates were all found to belong to ST68 and H2S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H2S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H2S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H2S-positive isolates, which may be responsible for the H2S-negative phenotype. Moreover, the 19 H2S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these results indicated that these H2S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H2S-negative S. Choleraesuis in China.

Antimicrobial Resistance and Molecular Investigation of H2S-Negative Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China

PubMed Central

Li, Peng; Liang, Beibei; Li, Hao; Yang, Xiaoxia; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Wu, Zhihao; Qiu, Shaofu; Song, Hongbin

2015-01-01

Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H2S-negative S. Choleraesuis isolates and two H2S-positive isolates. This is the first report of H2S-negative S. Choleraesuis isolated from humans. The majority of H2S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H2S-negative and H2S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H2S-negative S. Choleraesuis isolates belonging to Group I and H2S-positive isolates belonging to Group II. By MLST analysis, the H2S-negative isolates were all found to belong to ST68 and H2S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H2S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H2S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H2S-positive isolates, which may be responsible for the H2S-negative phenotype. Moreover, the 19 H2S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these results indicated that these H2S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H2S-negative S. Choleraesuis in China. PMID:26431037

Taxonomic evaluation of putative Streptomyces scabiei strains held in the ARS (NRRL) Culture Collection using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

Multi-locus sequence analysis has been demonstrated to be a useful tool for identification of Streptomyces species and was previously applied to phylogenetically differentiate the type strains of species pathogenic on potatoes (Solanum tuberosum L.). The ARS Culture Collection (NRRL) contains 43 str...

Fluoroquinolone Resistance Mechanisms and population structure of Enterobacter cloacae non-susceptible to Ertapenem in North-Eastern France

PubMed Central

Guillard, Thomas; Cholley, Pascal; Limelette, Anne; Hocquet, Didier; Matton, Lucie; Guyeux, Christophe; Lebreil, Anne-Laure; Bajolet, Odile; Brasme, Lucien; Madoux, Janick; Vernet-Garnier, Véronique; Barbe, Coralie; Bertrand, Xavier; de Champs on behalf of CarbaFrEst Group, Christophe

2015-01-01

Fluoroquinolone (FQ) agents are a potential resort to treat infection due to Enterobacteriaceae producing extended spectrum β-lactamase and susceptible to FQ. In a context of increase of non-susceptibility to carbapenems among Enterobacteriaceae, we characterized FQ resistance mechanisms in 75 Enterobacter cloacae isolates non-susceptible to ertapenem in North-Eastern France in 2012 and describe the population structure by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Among them, 14.7% (12/75) carried a carbapenemase-encoding gene. Except one isolate producing VIM-1, the carbapenemase-producing isolates carried the well-known IncL/M pOXA48a plasmid. Most of the isolates (59/75) harbored at least a FQ-R determinant. qnr genes were predominant (40%, 30/75). The MLST study revealed that E. cloacae isolates’ clonality was wide [24 different sequence types (STs)]. The more widespread STs were ST74, ST101, ST110, ST114, and ST133. Carbapenem MICs were higher for E. cloacae ST74 than for other E. cloacae isolates. Plasmid-mediated quinolone resistance determinants were more often observed in E. cloacae ST74 isolates. These findings showed that (i) pOXA-48a is spreading in North-Eastern France, (ii) qnr is preponderant in E. cloacae, (iii) E. cloacae comprised a large amount of lineages spreading in North-Eastern France, and (iv) FQ as an alternative to β-lactams to treat ertapenem non-susceptible Enterobacteriaceae are compromised. PMID:26557115

Rickettsia asembonensis Characterization by Multilocus Sequence Typing of Complete Genes, Peru.

PubMed

Loyola, Steev; Flores-Mendoza, Carmen; Torre, Armando; Kocher, Claudine; Melendrez, Melanie; Luce-Fedrow, Alison; Maina, Alice N; Richards, Allen L; Leguia, Mariana

2018-05-01

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

PubMed Central

Feltrin, Fabiola; Alba, Patricia; Kraushaar, Britta; Ianzano, Angela; Argudín, María Angeles; Di Matteo, Paola; Porrero, María Concepción; Aarestrup, Frank M.; Butaye, Patrick; Franco, Alessia

2015-01-01

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas. PMID:26590279

Global Spread of the hylEfm Colonization-Virulence Gene in Megaplasmids of the Enterococcus faecium CC17 Polyclonal Subcluster▿

PubMed Central

Freitas, Ana R.; Tedim, Ana P.; Novais, Carla; Ruiz-Garbajosa, Patricia; Werner, Guido; Laverde-Gomez, Jenny A.; Cantón, Rafael; Peixe, Luísa; Baquero, Fernando; Coque, Teresa M.

2010-01-01

Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumptively associated with the expansion of a human-associated Enterococcus faecium polyclonal subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin and vancomycin) and virulence (espEfm, hylEfm, and fms) traits. We analyzed the presence and the location of a putative glycoside hydrolase hylEfm gene among E. faecium strains obtained from hospitalized patients (255 patients; outbreak, bacteremic, and/or disseminated isolates from 23 countries and five continents; 1986 to 2009) and from nonclinical origins (isolates obtained from healthy humans [25 isolates], poultry [30], swine [90], and the environment [55]; 1999 to 2007). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid analysis included determination of content and size (S1-PFGE), transferability (filter mating), screening of Rep initiator proteins (PCR), and location of vanA, vanB, ermB, and hylEfm genes (S1/I-CeuI hybridization). Most E. faecium isolates contained large plasmids (>150 kb) and showed variable contents of van, hylEfm, or espEfm. The hylEfm gene was associated with megaplasmids (170 to 375 kb) of worldwide spread (ST16, ST17, and ST18) or locally predominant (ST192, ST203, ST280, and ST412) ampicillin-resistant CC17 clones collected in the five continents since the early 1990s. All but one hylEfm-positive isolate belonged to the CC17 polyclonal subcluster. The presence of hylEfm megaplasmids among CC17 from Europe, Australia, Asia, and Africa since at least the mid-1990s was documented. This study further demonstrates the pandemic expansion of particular CC17 clones before acquisition of vancomycin resistance and putative virulence traits and describes the presence of megaplasmids in most of the contemporary E. faecium isolates with different origins. PMID:20385861

Phenotypic and molecular characterization of antimicrobial resistance in Enterobacter spp. isolates from companion animals in Japan.

PubMed

Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Kajino, Akari; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi

2017-01-01

The emergence of antimicrobial resistance among Enterobacter spp., including resistance to extended-spectrum cephalosporins (ESC), is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance among 60 isolates of Enterobacter spp., including E. cloacae (n = 44), E. aerogenes (n = 10), and E. asburiae (n = 6), from clinical specimens of dogs and cats from 15 prefectures in Japan. Furthermore, we characterized the resistance mechanisms harbored by these isolates, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR); and assessed the genetic relatedness of ESC-resistant Enterobacter spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated the resistance rates to ampicillin (93.3%), amoxicillin-clavulanic acid (93.3%), cefmetazole (93.3%), chloramphenicol (46.7%), ciprofloxacin (43.3%), tetracycline (40.0%), ceftazidime (33.3%), cefotaxime (33.3%), trimethoprim/sulfamethoxazole (28.3%), gentamicin (23.3%), and meropenem (0%). Phenotypic testing detected ESBLs in 16 of 18 ESC-resistant E. cloacae isolates but not in the other species. The most frequent ESBL was CTX-M-15 (n = 8), followed by SHV-12 (n = 7), and CTX-M-3 (n = 1). As for AmpC β-lactamases, CMY-2 (n = 2) and DHA-1 (n = 2) were identified in ESC-resistant E. cloacae strains with or without ESBLs. All of the ESC-resistant E. cloacae strains also harbored one or two PMQRs, including qnrB (n = 15), aac(6')-Ib-cr (n = 8), and qnrS (n = 2). Based on MLST and PFGE analysis, E. cloacae clones of ST591-SHV-12, ST171-CTX-M-15, and ST121-CTX-M-15 were detected in one or several hospitals. These results suggested intra- and inter-hospital dissemination of E. cloacae clones co-harboring ESBLs and PMQRs among companion animals. This is the first report on the large-scale monitoring of antimicrobial-resistant isolates of Enterobacter spp. from companion animals in Japan.

Development of new multilocus variable number of tandem repeat analysis (MLVA) for Listeria innocua and its application in a food processing plant.

PubMed

Takahashi, Hajime; Ohshima, Chihiro; Nakagawa, Miku; Thanatsang, Krittaporn; Phraephaisarn, Chirapiphat; Chaturongkasumrit, Yuphakhun; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2014-01-01

Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility.

PubMed

Hower, Suzanne; Phillips, Matthew C; Brodsky, Micah; Dameron, Adrienne; Tamargo, Manuel A; Salazar, Norma C; Jackson, Charlene R; Barrett, John B; Davidson, Maureen; Davis, Johnnie; Mukherjee, Sampa; Ewing, Ruth Y; Gidley, Maribeth L; Sinigalliano, Christopher D; Johns, Lisa; Johnson, Frank E; Adebanjo, Olufunmilola; Plano, Lisa R W

2013-05-01

In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

Characteristics and management of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in a tertiary hospital.

PubMed

Pang, Feng; Jia, Xiu-Qin; Song, Zhen-Zhu; Li, Yan-Hua; Wang, Bin; Zhao, Qi-Gang; Wang, Chuan-Xin; Zhang, Yi; Wang, Le-Xin

2016-03-01

The emergence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases is rare. We report an occurrence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases in a Chinese tertiary care hospital from November 2010 to December 2012. The clinical characteristics of 30 patients were described. The genetic relationship of isolates was determined by pulsed-field gel electrophoresis (PFGE). Carbapenemases were detected by modified Hodge test (MHT) and polymerase chain reactions (PCRs). Amplicons were sequenced and blasted to determine the genotype. Most infected patients were from intensive care unit and had complex and serious underlying illnesses requiring mechanical ventilation. PFGE revealed that Klebsiella pneumoniae showed two major PFGE types. Two Klebsiella oxytoca had an indistinguishable PFGE pattern, while four Enterobacter cloacae were different strains. The sequencing studies showed Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in the 23 infected patients. The majority of patients had infections with the carbapenemase-producing Enterobacteriaceae (CPE) strain, most were successfully treated with a range of antibiotics and discharged. It is important to maintain a high index of suspicion to screen for carbapenemase-producing Enterobacteriaceae strains. Rapid identification of these strains and implementation of stringent procedures are the key to prevent major outbreaks in a hospital setting.

Low prevalence of methicillin-resistant Staphylococcus aureus nasal carriage in urban and rural community settings in Bolivia and Peru.

PubMed

Bartoloni, Alessandro; Pallecchi, Lucia; Fernandez, Connie; Mantella, Antonia; Riccobono, Eleonora; Magnelli, Donata; Mannini, Dario; Strohmeyer, Marianne; Bartalesi, Filippo; Segundo, Higinio; Monasterio, Joaquin; Rodriguez, Hugo; Cabezas, César; Gotuzzo, Eduardo; Rossolini, Gian Maria

2013-05-01

To investigate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage in rural and urban community settings of Bolivia and Peru. MRSA nasal carriage was investigated in 585 individuals living in rural and urban areas of Bolivia and Peru (one urban area, one small rural village, and two native communities, one of which was highly isolated). MRSA isolates were subjected to molecular analysis for the detection of virulence genes, characterization of the staphylococcal cassette chromosome mec (SCCmec), and genotyping (multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)). An overall very low prevalence of MRSA nasal carriage was observed (0.5%), with MRSA carriers being detected only in a small rural village of the Bolivian Chaco. The three MRSA isolates showed the characteristics of community-associated MRSA (being susceptible to all non-beta-lactam antibiotics and harboring the SCCmec type IV), were clonally related, and belonged to ST1649. This study provides an insight into the epidemiology of MRSA in community settings of Bolivia and Peru. Reliable, time-saving, and low-cost methods should be implemented to encourage continued surveillance of MRSA dissemination in resource-limited countries. Copyright © 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Molecular epidemiology, antimicrobial susceptibility, and characterization of macrolide-resistant Streptococcus pyogenes in Japan.

PubMed

Tanaka, Yuhei; Gotoh, Kenji; Teramachi, Mariko; Ishimoto, Kazuhisa; Tsumura, Naoki; Shindou, Shizuo; Yamashita, Yushiro

2016-11-01

Here we report the molecular epidemiology of macrolide-resistant Streptococcus pyogenes (group A streptococci, GAS) isolated from children with pharyngotonsillitis between 2011 and 2013 in Japan. In 299 isolates, 124 (41.5%) isolates were macrolide-resistant. We characterized the isolates by emm typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Of 299 isolates, 124 (41.5%) were macrolide-resistant isolates, 76 (61.3%) possessed mefA and 46 (37.1%) possessed ermB. All 76 isolates with mefA possessed msrD. There were no isolates possessed ermTR in this study. Eight emm/MLST types were observed. The predominant type was emm1/ST28 (57 isolates, 46.0%), which possessed the mefA/msrD complex, presenting as the M phenotype. The second most predominant type was emm12/ST467, which possessed ermB, presenting as the cMLS B phenotype. Of the cMLS B phenotype isolates, types emm28/ST52 and emm12/ST36 had multiple genetic backgrounds. We found high proportions of macrolide-resistant GAS in the southwestern areas of Japan. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Direct Repeat Unit (dru) Typing of Methicillin-Resistant Staphylococcus pseudintermedius from Dogs and Cats.

PubMed

Kadlec, Kristina; Schwarz, Stefan; Goering, Richard V; Weese, J Scott

2015-12-01

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged in a remarkable manner as an important problem in dogs and cats. However, limited molecular epidemiological information is available. The aims of this study were to apply direct repeat unit (dru) typing in a large collection of well-characterized MRSP isolates and to use dru typing to analyze a collection of previously uncharacterized MRSP isolates. Two collections of MRSP isolates from dogs and cats were included in this study. The first collection comprised 115 well-characterized MRSP isolates from North America and Europe. The data for these isolates included multilocus sequence typing (MLST) and staphylococcal protein A gene (spa) typing results as well as SmaI macrorestriction patterns after pulsed-field gel electrophoresis (PFGE). The second collection was a convenience sample of 360 isolates from North America. The dru region was amplified by PCR, sequenced, and analyzed. For the first collection, the discriminatory indices of the typing methods were calculated. All isolates were successfully dru typed. The discriminatory power for dru typing (D = 0.423) was comparable to that of spa typing (D = 0.445) and of MLST (D = 0.417) in the first collection. Occasionally, dru typing was able to further discriminate between isolates that shared the same spa type. Among all 475 isolates, 26 different dru types were identified, with 2 predominant types (dt9a and dt11a) among 349 (73.4%) isolates. The results of this study underline that dru typing is a useful tool for MRSP typing, being an objective, standardized, sequence-based method that is relatively cost-efficient and easy to perform. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Linezolid-Resistant Staphylococcus aureus Strain 1128105, the First Known Clinical Isolate Possessing the cfr Multidrug Resistance Gene

PubMed Central

Zuill, Douglas E.; Scharn, Caitlyn R.; Deane, Jennifer; Sahm, Daniel F.; Denys, Gerald A.; Goering, Richard V.; Shaw, Karen J.

2014-01-01

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZDr clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination. PMID:25155597

Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

PubMed Central

Johnsen, P. J.; Østerhus, J. I.; Sletvold, H.; Sørum, M.; Kruse, H.; Nielsen, K.; Simonsen, G. S.; Sundsfjord, A.

2005-01-01

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments. PMID:15640183

Evaluation of Fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for sub-typing Escherichia coli O157:H7 isolates.

PubMed

Davis, R; Paoli, G; Mauer, L J

2012-09-01

The importance of tracking outbreaks of foodborne illness and the emergence of new virulent subtypes of foodborne pathogens have created the need for rapid and reliable sub-typing methods for Escherichia coli O157:H7. Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate statistical analyses was used for sub-typing 30 strains of E. coli O157:H7 that had previously been typed by multilocus variable number tandem repeat analysis (MLVA) and pulsed field gel electrophoresis (PFGE). Hierarchical cluster analysis (HCA) and canonical variate analysis (CVA) of the FT-IR spectra resulted in the clustering of the same or similar MLVA types and separation of different MLVA types of E. coli O157:H7. The developed FT-IR method showed better discriminatory power than PFGE in sub-typing E. coli O157:H7. Results also indicated the spectral relatedness between different outbreak strains. However, the grouping of some strains was not in complete agreement with the clustering based on PFGE and MLVA. Additionally, HCA of the spectra differentiated the strains into 30 sub-clusters, indicating the high specificity and suitability of the method for strain level identification. Strains were also classified (97% correct) based on the type of Shiga toxin present using CVA of the spectra. This study demonstrated that FT-IR spectroscopy is suitable for rapid (≤16 h) and economical sub-typing of E. coli O157:H7 with comparable accuracy to MLVA typing. This is the first report of using an FT-IR-based method for sub-typing E. coli O157:H7. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diversity of Escherichia coli strains involved in vertebral osteomyelitis and arthritis in broilers in Brazil.

PubMed

Braga, Juliana Fortes Vilarinho; Chanteloup, Nathalie Katy; Trotereau, Angélina; Baucheron, Sylvie; Guabiraba, Rodrigo; Ecco, Roselene; Schouler, Catherine

2016-07-14

Locomotor disorders and infections by Escherichia coli represent major concerns to the poultry industry worldwide. Avian pathogenic E. coli (APEC) is associated with extraintestinal infections leading to respiratory or systemic disease known as colibacillosis. The most common lesions seen in cases of colibacillosis are perihepatitis, airsacculitis, pericarditis, peritonitis/salpingitis and arthritis. These diseases are responsible for significant economic losses in the poultry industry worldwide. E. coli has been recently isolated from vertebral osteomyelitis cases in Brazil and there are no data on molecular and phenotypic characteristics of E. coli strains isolated from lesions in the locomotor system of broilers. This raised the question whether specific E. coli strains could be responsible for bone lesions in broilers. The aim of this study was to assess these characteristics of E. coli strains isolated from broilers presenting vertebral osteomyelitis and arthritis in Brazil. Fifteen E. coli strains from bone lesions were submitted to APEC diagnosis and setting of ECOR phylogenic group, O serogroup, flagella type, virulence genes content, genetic patterns by Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). In addition, bacterial isolates were further characterized through a lethality test, serum resistance test and antibiotic resistance profile. E. coli strains harbored different genetic pattern as assessed by PFGE, regardless of flock origin and lesion site. The strains belonged to seven sequence types (STs) previously described (ST117, ST101, ST131, ST 371 and ST3107) or newly described in this study (ST5766 and ST5856). ECOR group D (66.7 %) was the most frequently detected. The strains belonged to diverse serogroups (O88, O25, O12, and O45), some of worldwide importance. The antibiotic resistance profile confirmed strains' diversity and revealed a high proportion of multidrug-resistant strains (73 %), mainly to quinolones and beta-lactams, including third generation cephalosporin. The percentage of resistance to tetracycline was moderate (33 %) but always associated with multidrug resistance. Our results demonstrated that vertebral osteomyelitis and arthritis in broilers can be associated with highly diverse E. coli based on molecular and phenotypic characteristics. There was no specific virulence patterns of the E. coli strains associated with vertebral osteomyelitis or arthritis. Also, E. coli strains were frequently multidrug resistant and belonged to STs commonly shared by APEC and human ExPEC strains.

Genotyping of Campylobacter coli strains isolated in Brazil suggests possible contamination amongst environmental, human, animal and food sources.

PubMed

Gomes, Carolina N; Souza, Roberto A; Passaglia, Jaqueline; Duque, Sheila S; Medeiros, Marta I C; Falcão, Juliana P

2016-01-01

Campylobacter coli and Campylobacter jejuni are two of the most common causative agents of food-borne gastroenteritis in numerous countries worldwide. In Brazil, campylobacteriosis is under diagnosed and under-reported, and few studies have molecularly characterized Campylobacter spp. in this country. The current study genotyped 63 C. coli strains isolated from humans (n512), animals (n521), food (n510) and the environment (n520) between 1995 and 2011 in Brazil. The strains were genotyped using pulsed-field gel electrophoresis (PFGE), sequencing the short variable region (SVR) of the flaA gene ( flaA-SVR) and high-resolution melting analysis (HRMA) of the clustered regularly interspaced short palindromic repeat (CRISPR) locus to better understand C. coli genotypic diversity and compare the suitability of these three methods for genotyping this species. Additionally, the discrimination index (DI) of each of these methods was assessed. Some C. coli strains isolated from clinical and non-clinical origins presented ≥80 % genotypic similarity by PFGE and flaA-SVR sequencing. HRMA of the CRISPR locus revealed only four different melting profiles. In total, 22 different flaA-SVR alleles were detected. Of these, seven alleles, comprising gt1647–gt1653, were classified as novel. The most frequent genotypes were gt30 and gt1647. This distribution reveals the diversity of selected Brazilian isolates in comparison with the alleles described in the PubMLST database. The DIs for PFGE, flaA–SVR sequencing and CRISPR-HRMA were 0.986, 0.916 and 0.550, respectively. PFGE and flaA-SVR sequencing were suitable for subtyping C. coli strains, in contrast to CRISPR-HRMA. The high genomic similarity amongst some C. coli strains confirms the hypothesis that environmental and food sources potentially lead to human and animal contamination in Brazil.

Microbiological Quality of Raw Dried Pasta from the German Market, with Special Emphasis on Cronobacter Species.

PubMed

Akineden, Ömer; Murata, Kristina Johanna; Gross, Madeleine; Usleber, Ewald

2015-12-01

The microbiological quality of 132 dried pasta products available on the German market, originating from 11 different countries, was studied. Sample materials included soft or durum wheat products, some of which produced with other ingredients such as eggs, spices, or vegetables. Parameters included hygiene indicators (aerobic plate count, mold count, the presence of Enterobacteriaceae) and pathogenic/toxinogenic bacterial species (Salmonella spp., Staphylococcus aureus, presumptive Bacillus cereus, and Cronobacter spp.). The overall results of hygiene parameters indicated a satisfactory quality. Salmonella was not found in any sample. Three samples were positive for S. aureus (10(2) to 10(4) colony forming unit (CFU)/g). Presumptive B. cereus at levels of 10(3) to 10(4) CFU/g were detected in 3 samples. Cronobacter spp. were isolated from 14 (10.6%) products. Of these, 9 isolates were identified as C. sakazakii, 2 each as C. turicensis and C. malonaticus, and 1 as C. muytjensii. The isolates were assigned to 9 multilocus sequence typing (MLST) sequence types and to 14 different PFGE profiles. Although pasta products are typically cooked before consumption, some consumers, and children in particular, may also eat raw pasta as nibbles. Raw pasta seems to be a relevant source of exposure to dietary Cronobacter spp., although health risks are probably restricted to vulnerable consumers. High numbers of presumptive B. cereus as found in some samples may be a risk after improper storage of cooked pasta products because toxinogenic strains are frequently found within this species. © 2015 Institute of Food Technologists®

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis.

PubMed

Lindahl, Susanne; Söderlund, Robert; Frosth, Sara; Pringle, John; Båverud, Viveca; Aspán, Anna

2011-11-21

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi. Copyright © 2011 Elsevier B.V. All rights reserved.

Sinorhizobium meliloti strains TII7 and A5 by Multilocus Sequence Typing (MLST) have chromsomes identical with Rm1021 and form an effective and ineffective symbiosis with Medicago truncatula line Jemalong A17, respectively

USDA-ARS?s Scientific Manuscript database

The strains TII7 and A5 formed an effective and ineffective symbiosis with Medicago truncatula Jemalong A17, respectively. Both were shown to have identical chromsomes with strains Rm1021 and RCR2011 using a Multilocus Sequence Typing method. The 2260 bp segments of DNA stretching from the 3’ end ...

Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes.

PubMed

García-Alvarez, Andrés; Fernández-Garayzábal, José Francisco; Chaves, Fernando; Pinto, Chris; Cid, Dolores

2018-06-01

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep. Copyright © 2018 Elsevier B.V. All rights reserved.

Taxonomic evaluation of putative Streptomyces scabiei strains held in the ARS Culture Collection (NRRL) using multi-locus sequence analysis.

PubMed

Labeda, David P

2016-03-01

Multi-locus sequence analysis has been demonstrated to be a useful tool for identification of Streptomyces species and was previously applied to phylogenetically differentiate the type strains of species pathogenic on potatoes (Solanum tuberosum L.). The ARS Culture Collection (NRRL) contains 43 strains identified as Streptomyces scabiei deposited at various times since the 1950s and these were subjected to multi-locus sequence analysis utilising partial sequences of the house-keeping genes atpD, gyrB, recA, rpoB and trpB. Phylogenetic analyses confirmed the identity of 17 of these strains as Streptomyces scabiei, 9 of the strains as the potato-pathogenic species Streptomyces europaeiscabiei and 6 strains as potentially new phytopathogenic species. Of the 16 other strains, 12 were identified as members of previously described non-pathogenic Streptomyces species while the remaining 4 strains may represent heretofore unrecognised non-pathogenic species. This study demonstrated the value of this technique for the relatively rapid, simple and sensitive molecular identification of Streptomyces strains held in culture collections.

spads 1.0: a toolbox to perform spatial analyses on DNA sequence data sets.

PubMed

Dellicour, Simon; Mardulyn, Patrick

2014-05-01

SPADS 1.0 (for 'Spatial and Population Analysis of DNA Sequences') is a population genetic toolbox for characterizing genetic variability within and among populations from DNA sequences. In view of the drastic increase in genetic information available through sequencing methods, spads was specifically designed to deal with multilocus data sets of DNA sequences. It computes several summary statistics from populations or groups of populations, performs input file conversions for other population genetic programs and implements locus-by-locus and multilocus versions of two clustering algorithms to study the genetic structure of populations. The toolbox also includes two MATLAB and r functions, GDISPAL and GDIVPAL, to display differentiation and diversity patterns across landscapes. These functions aim to generate interpolating surfaces based on multilocus distance and diversity indices. In the case of multiple loci, such surfaces can represent a useful alternative to multiple pie charts maps traditionally used in phylogeography to represent the spatial distribution of genetic diversity. These coloured surfaces can also be used to compare different data sets or different diversity and/or distance measures estimated on the same data set. © 2013 John Wiley & Sons Ltd.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

PubMed

Sharma, Anshul; Kaur, Jasmine; Lee, Sulhee; Park, Young-Seo

2018-06-01

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

Outbreak of Salmonella Oslo Infections Linked to Persian Cucumbers - United States, 2016.

PubMed

Bottichio, Lyndsay; Medus, Carlota; Sorenson, Alida; Donovan, Danielle; Sharma, Reeti; Dowell, Natasha; Williams, Ian; Wellman, Allison; Jackson, Alikeh; Tolar, Beth; Griswold, Taylor; Basler, Colin

2016-12-30

In April 2016, PulseNet, the national molecular subtyping network for foodborne disease surveillance, detected a multistate cluster of Salmonella enterica serotype Oslo infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern (XbaI PFGE pattern OSLX01.0090).* This PFGE pattern was new in the database; no previous infections or outbreaks have been identified. CDC, state and local health and agriculture departments and laboratories, and the Food and Drug Administration (FDA) conducted epidemiologic, traceback, and laboratory investigations to identify the source of this outbreak. A total of 14 patients in eight states were identified, with illness onsets occurring during March 21-April 9, 2016. Whole genome sequencing, a highly discriminating subtyping method, was used to further characterize PFGE pattern OSLX01.0090 isolates. Epidemiologic evidence indicates Persian cucumbers as the source of Salmonella Oslo infections in this outbreak. This is the fourth identified multistate outbreak of salmonellosis associated with cucumbers since 2013. Further research is needed to understand the mechanism and factors that contribute to contamination of cucumbers during growth, harvesting, and processing to prevent future outbreaks.

Molecular Phylogenetics and Systematics of the Bivalve Family Ostreidae Based on rRNA Sequence-Structure Models and Multilocus Species Tree

PubMed Central

Salvi, Daniele; Macali, Armando; Mariottini, Paolo

2014-01-01

The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML) and Bayesian (BA) methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA) and coalescent based (BA) approaches and consistently supported three main clades: (i) Crassostrea, (ii) Saccostrea, and (iii) an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassotreinae (including Crassostrea), Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea) and Ostreinae (including Ostreinae and Lophinae taxa) are recognized. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics. PMID:25250663

Molecular phylogenetics and systematics of the bivalve family Ostreidae based on rRNA sequence-structure models and multilocus species tree.

PubMed

Salvi, Daniele; Macali, Armando; Mariottini, Paolo

2014-01-01

The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML) and Bayesian (BA) methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA) and coalescent based (BA) approaches and consistently supported three main clades: (i) Crassostrea, (ii) Saccostrea, and (iii) an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassostreinae (including Crassostrea), Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea) and Ostreinae (including Ostreinae and Lophinae taxa) are recognized [corrected]. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

PubMed

Watahiki, Masanori; Isobe, Junko; Kimata, Keiko; Shima, Tomoko; Kanatani, Jun-ichi; Shimizu, Miwako; Nagata, Akihiro; Kawakami, Keiko; Yamada, Mikiko; Izumiya, Hidemasa; Iyoda, Sunao; Morita-Ishihara, Tomoko; Mitobe, Jiro; Terajima, Jun; Ohnishi, Makoto; Sata, Tetsutaro

2014-08-01

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Phylogenetic relationships of Malassezia species based on multilocus sequence analysis.

PubMed

Castellá, Gemma; Coutinho, Selene Dall' Acqua; Cabañes, F Javier

2014-01-01

Members of the genus Malassezia are lipophilic basidiomycetous yeasts, which are part of the normal cutaneous microbiota of humans and other warm-blooded animals. Currently, this genus consists of 14 species that have been characterized by phenetic and molecular methods. Although several molecular methods have been used to identify and/or differentiate Malassezia species, the sequencing of the rRNA genes and the chitin synthase-2 gene (CHS2) are the most widely employed. There is little information about the β-tubulin gene in the genus Malassezia, a gene has been used for the analysis of complex species groups. The aim of the present study was to sequence a fragment of the β-tubulin gene of Malassezia species and analyze their phylogenetic relationship using a multilocus sequence approach based on two rRNA genes (ITS including 5.8S rRNA and D1/D2 region of 26S rRNA) together with two protein encoding genes (CHS2 and β-tubulin). The phylogenetic study of the partial β-tubulin gene sequences indicated that this molecular marker can be used to assess diversity and identify new species. The multilocus sequence analysis of the four loci provides robust support to delineate species at the terminal nodes and could help to estimate divergence times for the origin and diversification of Malassezia species.

DNA gel electrophoresis: the reptation model(s).

PubMed

Slater, Gary W

2009-06-01

DNA gel electrophoresis has been the most important experimental tool to separate DNA fragments for several decades. The introduction of PFGE in the 1980s and capillary gel electrophoresis in the 1990s made it possible to study, map and sequence entire genomes. Explaining how very large DNA molecules move in a gel and why PFGE is needed to separate them has been an active field of research ever since the launch of the journal Electrophoresis. This article presents a personal and historical overview of the development of the theory of gel electrophoresis, focusing on the reptation model, the band broadening mechanisms, and finally the factors that limit the read length and the resolution of electrophoresis-based sequencing systems. I conclude with a short discussion of some of the questions that remain unanswered.

Phenotypic and molecular characterization of antimicrobial resistance in Enterobacter spp. isolates from companion animals in Japan

PubMed Central

Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Kajino, Akari; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi

2017-01-01

The emergence of antimicrobial resistance among Enterobacter spp., including resistance to extended-spectrum cephalosporins (ESC), is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance among 60 isolates of Enterobacter spp., including E. cloacae (n = 44), E. aerogenes (n = 10), and E. asburiae (n = 6), from clinical specimens of dogs and cats from 15 prefectures in Japan. Furthermore, we characterized the resistance mechanisms harbored by these isolates, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR); and assessed the genetic relatedness of ESC-resistant Enterobacter spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated the resistance rates to ampicillin (93.3%), amoxicillin-clavulanic acid (93.3%), cefmetazole (93.3%), chloramphenicol (46.7%), ciprofloxacin (43.3%), tetracycline (40.0%), ceftazidime (33.3%), cefotaxime (33.3%), trimethoprim/sulfamethoxazole (28.3%), gentamicin (23.3%), and meropenem (0%). Phenotypic testing detected ESBLs in 16 of 18 ESC-resistant E. cloacae isolates but not in the other species. The most frequent ESBL was CTX-M-15 (n = 8), followed by SHV-12 (n = 7), and CTX-M-3 (n = 1). As for AmpC β-lactamases, CMY-2 (n = 2) and DHA-1 (n = 2) were identified in ESC-resistant E. cloacae strains with or without ESBLs. All of the ESC-resistant E. cloacae strains also harbored one or two PMQRs, including qnrB (n = 15), aac(6’)-Ib-cr (n = 8), and qnrS (n = 2). Based on MLST and PFGE analysis, E. cloacae clones of ST591-SHV-12, ST171-CTX-M-15, and ST121-CTX-M-15 were detected in one or several hospitals. These results suggested intra- and inter-hospital dissemination of E. cloacae clones co-harboring ESBLs and PMQRs among companion animals. This is the first report on the large-scale monitoring of antimicrobial-resistant isolates of Enterobacter spp. from companion animals in Japan. PMID:28328967

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

PubMed Central

Flores-Páez, Luis A.; Zenteno, Juan C.; Alcántar-Curiel, María D.; Vargas-Mendoza, Carlos F.; Rodríguez-Martínez, Sandra; Cancino-Diaz, Mario E.; Jan-Roblero, Janet; Cancino-Diaz, Juan C.

2015-01-01

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates. PMID:26275056

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins.

PubMed

Hammerum, Anette M; Larsen, Jesper; Andersen, Vibe D; Lester, Camilla H; Skovgaard Skytte, Timmy S; Hansen, Frank; Olsen, Stefan S; Mordhorst, Hanne; Skov, Robert L; Aarestrup, Frank M; Agersø, Yvonne

2014-10-01

To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Phylogeny and strain typing of Escherichia coli, inferred from variation at mononucleotide repeat loci.

PubMed

Diamant, Eran; Palti, Yniv; Gur-Arie, Riva; Cohen, Helit; Hallerman, Eric M; Kashi, Yechezkel

2004-04-01

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Phylogeny and Strain Typing of Escherichia coli, Inferred from Variation at Mononucleotide Repeat Loci

PubMed Central

Diamant, Eran; Palti, Yniv; Gur-Arie, Riva; Cohen, Helit; Hallerman, Eric M.; Kashi, Yechezkel

2004-01-01

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria. PMID:15066845

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries.

PubMed

Ko, Kwan Soo; Oh, Won Sup; Peck, Kyong Ran; Lee, Jang Ho; Lee, Nam Yong; Song, Jae-Hoon

2005-07-01

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.

Genetic characterization of Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic Escherichia coli (EPEC) isolates from goat's milk and goat farm environment.

PubMed

Álvarez-Suárez, María-Elena; Otero, Andrés; García-López, María-Luisa; Dahbi, Ghizlane; Blanco, Miguel; Mora, Azucena; Blanco, Jorge; Santos, Jesús A

2016-11-07

The aim of this study was to characterize a collection of 44 Shiga toxin-producing (STEC) and enteropathogenic Escherichia coli (EPEC) isolated from goat milk and goat farm environment. Of the 19 STEC isolates, five (26.3%) carried the stx1 gene, four (21.1%) the stx2 gene and 10 (52.6%) presented both stx genes. Six (31.6%) STEC strains were eae-positive and belonged to serotypes related to severe human disease (O157:H7 and O5:HNM). Another seven STEC strains were of serotype O146:H21 and three of serotype O166:H28, also linked to human disease. The STEC strains isolated from goat milk were of serotypes potentially pathogenic for humans. All the 25 EPEC isolates were considered atypical (aEPEC) and one aEPEC strain was of serotype O26:H11, a serotype frequently isolated in children with diarrhea. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 23 sequence types (ST) were detected, 14 of them newly described. Twelve STs grouped STEC isolates and 11 STs grouped EPEC isolates. Genetic typing by pulsed field gel electrophoresis (PFGE) resulted in 38 patterns which grouped in 10 clusters. Well-defined groups were also observed for strains of pathogenic serotypes. In conclusion, strains of STEC and aEPEC belonging to serotypes related to severe human disease have been detected in goat milk and the goat farm environment. Ruminants are an important reservoir of STEC strains and the role of these animals as carriers of other pathogenic types of E. coli seems to be an emerging concern. Copyright © 2016 Elsevier B.V. All rights reserved.

Rise and fall of outbreak-specific clone inside endemic pulsotype of Salmonella 4,[5],12:i:-; insights from high-resolution molecular surveillance in Emilia-Romagna, Italy, 2012 to 2015.

PubMed

Morganti, Marina; Bolzoni, Luca; Scaltriti, Erika; Casadei, Gabriele; Carra, Elena; Rossi, Laura; Gherardi, Paola; Faccini, Fabio; Arrigoni, Norma; Sacchi, Anna Rita; Delledonne, Marco; Pongolini, Stefano

2018-03-01

Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions : The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.

Novel sequence types (STs) of Staphylococcus aureus isolates causing clinical and subclinical mastitis in flocks of sheep in the northeast of Brazil.

PubMed

de Almeida, Lara M; de Almeida, Mayra Zilta P R B; de Mendonça, Carla L; Mamizuka, Elsa M

2011-08-01

Staphylococcus aureus is one of the most important infectious mastitis causative agents in small ruminants. In order to know the distribution of Staph. aureus strains associated with infectious mastitis in flocks of sheep in the northeast of Brazil and establish whether these clones are related to the strains distributed internationally, this study analysed the genetic diversity of Staph. aureus isolates from cases of clinical and subclinical mastitis in ewes by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this research, 135 ewes with mastitis from 31 sheep flocks distributed in 15 districts were examined. Staph. aureus was isolated from sheep milk in 9 (29%) out of 31 herds located in 47% of the districts surveyed. MLST analysis allowed the identification of four STs (ST750, ST1728, ST1729 and ST1730). The last three with their respective novel alleles (glp-220; pta-182 and yqil-180) were recently reported in the Staph. aureus MLST database (http://www.mlst.net). Each novel allele showed only a nucleotide different from those already described. The occurrence of CC133 (ST750 and ST1729) in this study is in agreement with other reports that only a few clones of Staph. aureus seem to be responsible for most cases of mastitis in dairy farms and that some of these clones may have broad geographic distribution. However, the prevalence of CC5 (ST1728 and ST1730)--an important group related to cases of colonization or infection in humans--differs from previous studies by its widespread occurrence and may suggest human contamination followed by selective pressures of the allelic diversifications presented for these STs.

Detection of NDM-2-producing Acinetobacter baumannii and VIM-producing Pseudomonas aeruginosa in Palestine.

PubMed

Sjölander, Isabella; Hansen, Frank; Elmanama, Abdelraouf; Khayyat, Rasha; Abu-Zant, Alaeddin; Hussein, Ayman; Taha, Adham Abu; Hammerum, Anette M; Ciofu, Oana

2014-06-01

The aim of this study was to screen for carbapenem-resistant Gram-negative bacteria in Palestine and subsequently to identify and investigate the mechanisms of resistance. For a period of 6 weeks, all Gram-negative isolates were collected from six Palestinian hospital laboratories and were tested for susceptibility using 10μg meropenem disks. Isolates showing resistance to meropenem were further investigated. The presence of carbapenemases was assessed by PCR. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing carbapenemases were further investigated by multilocus sequence typing (MLST). In total, 248 Gram-negative isolates were collected from the six laboratories. Among the 248 tested isolates, 15 Acinetobacter baumannii and 6 Pseudomonas aeruginosa were resistant to meropenem. One A. baumannii from Gaza produced NDM-2 and belonged to ST103. Thirteen of the carbapenem-resistant A. baumannii isolates possessed the intrinsic upregulated bla OXA-66 gene and one isolate carried bla OXA-51 . All but one of the OXA-66-producing A. baumannii belonged to ST2; the remaining isolate belonged to ST183. One of the carbapenem-resistant P. aeruginosa was classified as VIM-4-producing and three were VIM-2-producing isolates. The three VIM-2-producing isolates belonged to three new sequences types (ST1562, ST1563 and ST1564). All of the carbapenemase-producing isolates were multiresistant non-fermenters. To the best of our knowledge, this is the first report on NDM-producing A. baumannii and VIM-producing P. aeruginosa from Palestine. Copyright © 2013 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Characterization of Shigella sonnei isolates from travel-associated cases in Japan.

PubMed

Izumiya, Hidemasa; Tada, Yuki; Ito, Kenichiro; Morita-Ishihara, Tomoko; Ohnishi, Makoto; Terajima, Jun; Watanabe, Haruo

2009-11-01

Shigella sonnei infection in industrialized countries is often associated with foreign travel. A total of 195 S. sonnei isolates in Japan, isolated from cases associated with foreign travel, were analysed by biotyping and molecular typing using PFGE and multilocus variable-number tandem-repeat analysis (MLVA); their antimicrobial susceptibilities were also evaluated. The isolates were from 26 countries, most of which were Asian. Molecular typing revealed a correlation among the genotypes, biotypes and their geographical areas of origin. The isolates were classified into two biotypes, a and g. Biotype g isolates (n=178) were further divided into distinct clusters mainly on the basis of their geographical areas of origin by both PFGE and MLVA. Isolates from South Asian countries constituted one of the distinct clusters. Biotype g isolates from countries other than South Asia constituted other distinct clusters. Most of the isolates from other countries and continents, excluding the South Asian countries, were included in one major cluster by PFGE analysis. However, by MLVA, they were further divided into minor subclusters mainly on the basis of their countries of origin. MLVA was also demonstrated to be useful in molecular epidemiological analysis, even when only seven loci were applied, resulting in a high resolution with Simpson's index of diversity (D) of 0.993. A core drug-resistance pattern of streptomycin, sulfisoxazole, tetracycline and trimethoprim-sulfamethoxazole was observed in 108 isolates, irrespective of their geographical areas of origin, but the frequency of resistance to nalidixic acid was high among the South Asian and East Asian isolates. Two isolates from China and India were resistant to cefotaxime and harboured the bla(CTX-M-14) and bla(CTX-M-15) genes, respectively; these isolates were also resistant to nalidixic acid, which is a matter of concern in terms of shigellosis treatment. Use of a combination of methods was found to be effective for epidemiological investigation in the case of S. sonnei infection.

[The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei].

PubMed

Antonov, V A; Altukhova, V V; Savchenko, S S; Zamaraev, V S; Iliukhin, V I; Alekseev, V V

2007-01-01

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

PubMed Central

de Gier, Camilla; Kirkham, Lea-Ann S.

2015-01-01

Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment. PMID:26378279

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

PubMed

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

OXA-Carbapenemases Present in Clinical Acinetobacter baumannii-calcoaceticus Complex Isolates from Patients in Kurdistan Region, Iraq.

PubMed

Ganjo, Aryann R; Maghdid, Delshad M; Mansoor, Isam Y; Kok, Dik J; Severin, Juliette A; Verbrugh, Henri A; Kreft, Deborah; Fatah, M H; Alnakshabandi, A A; Dlnya, Asad; Hammerum, Anette M; Ng, Kim; Goessens, Wil

2016-12-01

In addition to intrinsic resistance in Acinetobacter baumannii, many different types of acquired resistance mechanisms have been reported, including the presence of VIM and IMP metallo β-lactamases and also of bla OXA-23-like and bla OXA-58-like enzymes. In the Kurdistan region of Iraq, the multiresistant A. baumannii-calcoaceticus complex is prevalent. We characterized the different mechanisms of resistance present in clinical isolates collected from different wards and different hospitals from the Kurdistan region. One hundred twenty clinical nonduplicate A. baumannii-calcoaceticus complex isolates were collected from four hospitals between January 2012 and October 2013. The identification of the isolates was confirmed by MALDI-TOF. The susceptibility to different antibiotics was determined by disk diffusion and analyzed in accordance to EUCAST guidelines. By PCR, the presence of bla OXA-51-like , bla OXA-23-like , bla OXA-24-like , and bla OXA-58-like genes was determined as well as the presence of the insertion element ISAba1. Clonal diversity was analyzed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme ApaI and, in addition, multilocus sequence typing (MLST) was performed on a selected subset of 15 isolates. All 120 A. baumannii isolates harbored bla OXA-51-like genes. One hundred one out of 110 (92%) imipenem (IMP)-resistant A. baumannii-calcoaceticus complex isolates additionally carried the bla OXA-23-like gene and four isolates (3%) were positive for bla OXA-24-like. All 101 bla OXA-23-like -positive isolates had the ISAba1 insertion sequence, 1,600 bp upstream of the bla OXA-23-like gene. The bla OXA-58-like gene was not detected in any of the 110 IMP-resistant strains. Eight different PFGE clusters were identified and distributed over the different hospitals. MLST analysis performed on a subset of 15 representative isolates revealed the presence of the international clone ST2 (Pasteur). Besides ST2 (Pasteur), also many other STs (Pasteur) were encountered such as ST136, ST94, ST623, ST792, and ST793, all carrying the bla OXA-23 gene. In clinical A. baumannii-calcoaceticus complex isolates from Kurdistan-Iraq, the bla OXA-23 gene in combination with the upstream ISAba1 insertion element is largely responsible for carbapenem resistance. Several small clusters of identical genotypes were found from patients admitted to the same ward and during overlapping time periods, suggesting transmission within the hospital. Identification of source(s) and limiting the transmission of these strains to patients needs to be prioritized.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing.

PubMed

Lindstedt, Bjørn-Arne; Tham, Wilhelm; Danielsson-Tham, Marie-Louise; Vardund, Traute; Helmersson, Seved; Kapperud, Georg

2008-02-01

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing.

PubMed

Ziebell, Kim; Chui, Linda; King, Robin; Johnson, Suzanne; Boerlin, Patrick; Johnson, Roger P

2017-08-01

Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE. Three subtyping methods, PFGE, MLVA and phage typing were compared for their discriminatory power when applied to three panels of Canadian SE isolates: Panel 1: 70 isolates representing the diversity of phage types (PTs) and PFGE subtypes within these PTs; Panel 2: 214 apparently unrelated SE isolates of the most common PTs; and Panel 3: 27 isolates from 10 groups of epidemiologically related strains. For Panel 2 isolates, four MLVA subtypes were shared among 74% of unrelated isolates and in Panel 3 isolates, one MLVA subtype accounted for 62% of the isolates. For all panels, combining results from PFGE, MLVA and PT gave the best discrimination, except in Panel 1, where the combination of PT and PFGE was equally as high, due to the selection criteria for this panel. However, none of these methods is sufficiently discriminatory alone for reliable outbreak detection or source attribution, and must be applied together to achieve sufficient discrimination for practical purposes. Even then, some large clusters were not differentiated adequately. More discriminatory methods are required for reliable subtyping of this genetically highly homogeneous serovar. This need will likely be met by whole genome sequence analysis given the recent promising reports and as more laboratories implement this tool for outbreak response and surveillance. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Mycoplasma synoviae (MS) core genome multilocus sequence typing (cgMLST) scheme.

PubMed

Ghanem, Mostafa; El-Gazzar, Mohamed

2018-05-01

Mycoplasma synoviae (MS) is a poultry pathogen with reported increased prevalence and virulence in recent years. MS strain identification is essential for prevention, control efforts and epidemiological outbreak investigations. Multiple multilocus based sequence typing schemes have been developed for MS, yet the resolution of these schemes could be limited for outbreak investigation. The cost of whole genome sequencing became close to that of sequencing the seven MLST targets; however, there is no standardized method for typing MS strains based on whole genome sequences. In this paper, we propose a core genome multilocus sequence typing (cgMLST) scheme as a standardized and reproducible method for typing MS based whole genome sequences. A diverse set of 25 MS whole genome sequences were used to identify 302 core genome genes as cgMLST targets (35.5% of MS genome) and 44 whole genome sequences of MS isolates from six countries in four continents were used for typing applying this scheme. cgMLST based phylogenetic trees displayed a high degree of agreement with core genome SNP based analysis and available epidemiological information. cgMLST allowed evaluation of two conventional MLST schemes of MS. The high discriminatory power of cgMLST allowed differentiation between samples of the same conventional MLST type. cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation between MS isolates. Like conventional MLST, it provides stable and expandable nomenclature, allowing for comparing and sharing the typing results between different laboratories worldwide. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

[Etiological and molecular characteristics of diarrhea caused Proteus mirabilis].

PubMed

Shi, Xiaolu; Hu, Qinghua; Lin, Yiman; Qiu, Yaqun; Li, Yinghui; Jiang, Min; Chen, Qiongcheng

2014-06-01

To analyze the etiological characteristics, virulence genes and plasmids that carrying diarrhea-causing Proteus mirabilis and to assess their relationship with drug resistance and pathogenicity. Proteus mirabilis coming from six different sources (food poisoning, external environment and healthy people) were analyzed biochemically, on related susceptibility and pulsed-field gel electrophoresis (PFGE). Virulence genes were detected by PCR. Plasmids were extracted and sequenced after gel electrophoresis purification. The biochemical characteristics of Proteus mirabilis from different sources seemed basically the same, and each of them showed having common virulence genes, as ureC, rsmA, hpmA and zapA. However, the PFGE patterns and susceptibility of these strains were different, so as the plasmids that they carried. Plasmid that presented in the sequenced strain showed that the 2 683 bp length plasmid encodes qnrD gene was associated with the quinolone resistance. Etiological characteristics and molecular characteristics of Proteus mirabilis gathered from different sources, were analyzed. Results indicated that traditional biochemical analysis and common virulence gene identification might be able to distinguish the strains with different sources. However, PFGE and plasmids analysis could distinguish the sources of strains and to identify those plasmids that commonly carried by the drug-resistant strains. These findings also provided theoretical basis for further study on the nature of resistance and pathogenicity in Proteus mirabilis.

Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis.

PubMed

Perrig, Melina S; Ambroggio, María B; Buzzola, Fernanda R; Marcipar, Iván S; Calvinho, Luis F; Veaute, Carolina M; Barbagelata, María Sol

2015-01-01

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6% concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8% (134/137) and 94.9% (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95% and 100% with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

An empirical evaluation of two-stage species tree inference strategies using a multilocus dataset from North American pines

Treesearch

Michael DeGiorgio; John Syring; Andrew J. Eckert; Aaron Liston; Richard Cronn; David B. Neale; Noah A. Rosenberg

2014-01-01

Background: As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models,...

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination.

PubMed

Perez, Federico; Endimiani, Andrea; Ray, Amy J; Decker, Brooke K; Wallace, Christopher J; Hujer, Kristine M; Ecker, David J; Adams, Mark D; Toltzis, Philip; Dul, Michael J; Windau, Anne; Bajaksouzian, Saralee; Jacobs, Michael R; Salata, Robert A; Bonomo, Robert A

2010-08-01

Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained bla(OXA-23), and four isolates possessed bla(OXA-24/40). Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing bla(KPC-2) or bla(KPC-3) were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination

PubMed Central

Perez, Federico; Endimiani, Andrea; Ray, Amy J.; Decker, Brooke K.; Wallace, Christopher J.; Hujer, Kristine M.; Ecker, David J.; Adams, Mark D.; Toltzis, Philip; Dul, Michael J.; Windau, Anne; Bajaksouzian, Saralee; Jacobs, Michael R.; Salata, Robert A.; Bonomo, Robert A.

2010-01-01

Background Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Methods Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Results Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). Conclusion In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes. PMID:20513702

Outbreak of Carbapenem-Resistant Pseudomonas aeruginosa Producing VIM-8, a Novel Metallo-β-Lactamase, in a Tertiary Care Center in Cali, Colombia

PubMed Central

Crespo, M. P.; Woodford, N.; Sinclair, A.; Kaufmann, M. E.; Turton, J; Glover, J.; Velez, J. D.; Castañeda, C. R.; Recalde, M.; Livermore, D. M.

2004-01-01

The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and blaIMP and blaVIM genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 μg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with blaVIM-specific primers, and sequencing of DNA from a representative isolate revealed blaVIM-8, a novel allele with three polymorphisms compared with the sequence of blaVIM-2. Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried blaVIM alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, ≤32 μg/ml) which was not significantly reduced by EDTA. No blaIMP alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with blaVIM were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced. PMID:15528701

Distribution of Campylobacter jejuni isolates from turkey farms and different stages at slaughter using pulsed-field gel electrophoresis and flaA-short variable region sequencing.

PubMed

Perko-Mäkelä, P; Alter, T; Isohanni, P; Zimmermann, S; Lyhs, U

2011-09-01

The aim of this study was to assess the diversity of thermotolerant Campylobacter spp. isolated from turkey flocks at six rearing farms 1-2 weeks prior to slaughter (360 faecal swab samples) and from 11 different stages at the slaughterhouse (636 caecal, environmental, neck skin and meat samples). A total of 121 Campylobacter isolates were identified to species level using a multiplex PCR assay and were typed by pulsed-field gel electrophoresis (PFGE) and flaA-short variable region (SVR) sequencing. All Campylobacter isolates were identified as Campylobacter jejuni. PFGE analysis with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA SVR typing yielded in nine flaA-SVR alleles. The Campylobacter-positive turkey flocks A, C and E were colonized by a limited number of Campylobacter clones at the farm and slaughter. The present study confirms the traceability of flock-specific strains (PFGE types I, V and IX; flaA types 21, 36 and 161) from the farm along the entire processing line to meat cuts. It seems that stress factors such as high temperature of the defeathering water (54-56 °C), drying of the carcass skin during air chilling (24 h at 2 °C), and oxygen in the air could not eliminate Campylobacter completely. Campylobacter-negative flocks became contaminated during processing by the same subtypes of Campylobacter introduced into the slaughter house by preceeding positive flocks even if they were slaughtered on subsequent days. Proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination, especially in countries with a low prevalence of Campylobacter spp. The majority of flaA SVR alleles displayed a distinct association with a specific PFGE type. However, a linear relationship for all strains among both typing methods could not be established. To specify genetic relatedness of strains, a combination of different genotyping methods, is needed. © 2011 Blackwell Verlag GmbH.

Whole-Genome Sequencing Reveals Laboratory Salmonella Typhimurium Strain 14028s as a Likely Source of Outbreak Isolates from 2009-2010

USDA-ARS?s Scientific Manuscript database

Background: Next-generation sequencing (NGS) of bacterial isolates has emerged as valuable tool for tracking of an outbreak source. Between 2009 and 2011, clinical isolates of Salmonella Typhimurium sharing the JPXX01.0014 (XbaI) PFGE type were isolated across the U.S. The initial isolates were asso...

Whole-Genome Sequences of Two Listeria monocytogenes Serovar 1/2a Strains Responsible for a Severe Listeriosis Outbreak in Central Italy.

PubMed

Orsini, Massimiliano; Cornacchia, Alessandra; Patavino, Claudio; Torresi, Marina; Centorame, Patrizia; Acciari, Vicdalia Aniela; Ruolo, Anna; Marcacci, Maurilia; Ancora, Massimo; Di Domenico, Marco; Mangone, Iolanda; Blasi, Giuliana; Duranti, Anna; Cammà, Cesare; Pomilio, Francesco; Migliorati, Giacomo

2018-06-14

We report the whole-genome sequences of two Listeria monocytogenes strains responsible for a severe invasive listeriosis outbreak in central Italy that occurred in 2015 and 2016. These two strains differ by a single band in their pulsed-field gel electrophoresis (PFGE) profiles. Copyright © 2018 Orsini et al.

Molecular characterization of Giardia psittaci by multilocus sequence analysis.

PubMed

Abe, Niichiro; Makino, Ikuko; Kojima, Atsushi

2012-12-01

Multilocus sequence analyses targeting small subunit ribosomal DNA (SSU rDNA), elongation factor 1 alpha (ef1α), glutamate dehydrogenase (gdh), and beta giardin (β-giardin) were performed on Giardia psittaci isolates from three Budgerigars (Melopsittacus undulates) and four Barred parakeets (Bolborhynchus lineola) kept in individual households or imported from overseas. Nucleotide differences and phylogenetic analyses at four loci indicate the distinction of G. psittaci from the other known Giardia species: Giardia muris, Giardia microti, Giardia ardeae, and Giardia duodenalis assemblages. Furthermore, G. psittaci was related more closely to G. duodenalis than to the other known Giardia species, except for G. microti. Conflicting signals regarded as "double peaks" were found at the same nucleotide positions of the ef1α in all isolates. However, the sequences of the other three loci, including gdh and β-giardin, which are known to be highly variable, from all isolates were also mutually identical at every locus. They showed no double peaks. These results suggest that double peaks found in the ef1α sequences are caused not by mixed infection with genetically different G. psittaci isolates but by allelic sequence heterogeneity (ASH), which is observed in diplomonad lineages including G. duodenalis. No sequence difference was found in any G. psittaci isolates at the gdh and β-giardin, suggesting that G. psittaci is indeed not more diverse genetically than other Giardia species. This report is the first to provide evidence related to the genetic characteristics of G. psittaci obtained using multilocus sequence analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

PubMed

Ture, M; Altinok, I; Capkin, E

2015-01-01

Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. © 2014 John Wiley & Sons Ltd.

Characterization of carbapenem resistance mechanisms and integrons in Pseudomonas aeruginosa strains from blood samples in a French hospital.

PubMed

Rojo-Bezares, Beatriz; Cavalié, Laurent; Dubois, Damien; Oswald, Eric; Torres, Carmen; Sáenz, Yolanda

2016-04-01

Metallo-β-lactamases (MBLs), porin OprD, integrons, virulence factors and the clonal relationships were characterized in imipenem-resistant Pseudomonas aeruginosa (IRPA) isolates. Fifty-six IRPA strains were recovered from blood samples of different patients at a Toulouse teaching hospital from 2011 to 2013. Susceptibility testing of 14 antibiotics was performed by the disc diffusion method. Detection and characterization of MBLs, the oprD gene and integrons were studied by PCR and sequencing. Thirteen genes involved in the virulence of P. aeruginosa were analysed. Molecular typing of IRPA strains was performed by PFGE and multilocus sequence typing. In this study, 61 % of the IRPA isolates showed a multi-resistance phenotype. The MBL phenotype, detected in three isolates (5.4 %), was linked to the blaVIM-2 gene. The oprD gene was amplified in 55 (98.2 %) IRPA strains, and variations were observed in 54 of them. Insertion sequences (IS) truncating oprD were detected in eight IRPA strains, with the novel ISPa56 identified in two strains. Class 1 integrons were detected in 24 (42.9 %) IRPA strains. The blaVIM-2 gene was found inside the class 1 integron arrangements. The new integrons In1054 (intI1-aacA56-qacEΔ1-sul1) and In1160 (intI1-aacA4-aacC1d-ISKpn4-gcuE-qacEΔ1-sul1) have been described for the first time, to the best of our knowledge, in this study. A high clonal diversity was found in our strains. Among the variety of sequence types (STs) found, ST175, ST233, ST235, ST244 and ST654 were noteworthy as epidemic clones. In conclusion, 5.4 % of IRPA strains showed an MBL phenotype linked to the blaVIM-2 gene. The identified oprD high polymorphism could be implicated in the variable resistance to carbapenems in IRPA strains. The dissemination of high-risk clones is a cause of concern.

A web-based genomic sequence database for the Streptomycetaceae: a tool for systematics and genome mining

USDA-ARS?s Scientific Manuscript database

The ARS Microbial Genome Sequence Database (http://199.133.98.43), a web-based database server, was established utilizing the BIGSdb (Bacterial Isolate Genomics Sequence Database) software package, developed at Oxford University, as a tool to manage multi-locus sequence data for the family Streptomy...

New Insights on Taxonomy, Phylogeny and Population Genetics of Leishmania (Viannia) Parasites Based on Multilocus Sequence Analysis

PubMed Central

Boité, Mariana C.; Mauricio, Isabel L.; Miles, Michael A.; Cupolillo, Elisa

2012-01-01

The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)—extensively used for bacteria—has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes—glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)—from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences. PMID:23133690

Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data.

PubMed

Tanabe, Akifumi S

2011-09-01

Proportional and separate models able to apply different combination of substitution rate matrix (SRM) and among-site rate variation model (ASRVM) to each locus are frequently used in phylogenetic studies of multilocus data. A proportional model assumes that branch lengths are proportional among partitions and a separate model assumes that each partition has an independent set of branch lengths. However, the selection from among nonpartitioned (i.e., a common combination of models is applied to all-loci concatenated sequences), proportional and separate models is usually based on the researcher's preference rather than on any information criteria. This study describes two programs, 'Kakusan4' (for DNA sequences) and 'Aminosan' (for amino-acid sequences), which allow the selection of evolutionary models based on several types of information criteria. The programs can handle both multilocus and single-locus data, in addition to providing an easy-to-use wizard interface and a noninteractive command line interface. In the case of multilocus data, SRMs and ASRVMs are compared at each locus and at all-loci concatenated sequences, after which nonpartitioned, proportional and separate models are compared based on information criteria. The programs also provide model configuration files for mrbayes, paup*, phyml, raxml and Treefinder to support further phylogenetic analysis using a selected model. When likelihoods are optimized by Treefinder, the best-fit models were found to differ depending on the data set. Furthermore, differences in the information criteria among nonpartitioned, proportional and separate models were much larger than those among the nonpartitioned models. These findings suggest that selecting from nonpartitioned, proportional and separate models results in a better phylogenetic tree. Kakusan4 and Aminosan are available at http://www.fifthdimension.jp/. They are licensed under gnugpl Ver.2, and are able to run on Windows, MacOS X and Linux. © 2011 Blackwell Publishing Ltd.

Population structure of Lactobacillus helveticus isolates from naturally fermented dairy products based on multilocus sequence typing.

PubMed

Sun, Zhihong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Yu, Jie; Bilige, Menghe; Zhang, Heping; Chen, Yongfu

2015-05-01

Lactobacillus helveticus is an economically important lactic acid bacterium used in industrial dairy fermentation. In the present study, the population structure of 245 isolates of L. helveticus from different naturally fermented dairy products in China and Mongolia were investigated using an multilocus sequence typing scheme with 11 housekeeping genes. A total of 108 sequence types were detected, which formed 8 clonal complexes and 27 singletons. Results from Structure, SplitsTree, and ClonalFrame software analyses demonstrated the presence of 3 subpopulations in the L. helveticus isolates used in our study, namely koumiss, kurut-tarag, and panmictic lineages. Most L. helveticus isolates from particular ecological origins had specific population structures. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157 isolates

USDA-ARS?s Scientific Manuscript database

The PCR-based Escherichia coli O157 (O157) strain typing system, Polymorphic Amplified Typing Sequences (PATS), targets insertions-deletions (Indels) and single nucleotide polymorphisms (SNPs) at the XbaI and AvrII(BlnI) restriction enzyme sites, respectively, besides amplifying four known virulenc...

Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

PubMed

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genotype diversity of Escherichia coli isolates in natural waters determined by PFGE and ERIC-PCR.

PubMed

Casarez, Elizabeth A; Pillai, Suresh D; Di Giovanni, George D

2007-08-01

Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1-12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product-moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2-5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.

Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157).

PubMed

Hyytiä-Trees, Eija; Smole, Sandra C; Fields, Patricia A; Swaminathan, Bala; Ribot, Efrain M

2006-01-01

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci.

PubMed

Brassac, Jonathan; Blattner, Frank R

2015-09-01

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

Population structure of Salmonella enterica subspecies enterica (subspecies 1)

USDA-ARS?s Scientific Manuscript database

We sequenced and assembled 354 new Salmonella enterica ssp. enterica genomes. These genomes were chosen to maximize genetic diversity, representing at least 100 different serovars and distinct PFGE patterns within these serovars. 119 of the strains were of known antibiotic resistance,...

Characterization of Colistin-Resistant Escherichia coli Isolated from Diseased Pigs in France

PubMed Central

Delannoy, Sabine; Le Devendec, Laetitia; Jouy, Eric; Fach, Patrick; Drider, Djamel; Kempf, Isabelle

2017-01-01

We studied a collection of 79 colistin-resistant Escherichia coli isolates isolated from diseased pigs in France between 2009 and 2013. We determined a number of phenotypic and genetic characters using broth microdilution to characterize their antimicrobial susceptibility. We performed pulse field gel electrophoresis (PFGE) to assess their genetic diversity and assign them to phylogroups. High-throughput real-time PCR micro-array was used to screen for a selection of genetic markers of virulence, and PCR and sequencing of the main recognized resistance genes allowed us to investigate the mechanisms of colistin resistance. Results showed that isolates belonged to several phylogroups and most had a unique PFGE profile. More than 50% of the isolates were also resistant to sulfonamides, trimethoprim, tetracycline, ampicillin or chloramphenicol. The mcr-1 gene was detected in 70 out of 79 isolates and was transferred by conjugation in 33 of them, sometimes together with resistance to sulfonamides, trimethoprim, tetracycline, ampicillin, chloramphenicol, cefotaxime, or gentamicin. Mutations in the amino-acid sequences of proteins MgrB, PhoP, PhoQ, PmrB, but not PmrA, were detected in isolates with or without the mcr-1 gene. More than one-third of the isolates harbored the F18, F4, astA, hlyA, estI, estII, elt, stx2e, iha, orfA, orfB, paa, terE, ecs1763, or ureD virulence markers. In conclusion, although most isolates had a unique PFGE profile, a few particular combinations of phylogenetic groups, virulence genes and mutations in the sequenced genes involved in colistin resistance were identified on a number of occasions, suggesting the persistence of certain isolates over several years. PMID:29209292

Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray

PubMed Central

2012-01-01

Background Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF) patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF) patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT) multimarker microarray (Alere Technologies GmbH, Jena, Germany), a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were employed as reference genotyping techniques to estimate the ArrayTube resolution power. Results 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU) whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient), respectively. AT typing of this Italian collection could be easily integrated with the global P. aeruginosa AT-typed population, uncovering that most AT-genotypes identified (> 80%) belonged to two large clonal clusters, and included 12 among the most abundant clones of the global population. Conclusions The ArrayTube (AT) multimarker array represented a robust and portable alternative to reference techniques for performing P. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an Italian clinical collection from chronic and acute infections. PMID:22840192

Pneumocystis jirovecii multilocus gene sequencing: findings and implications.

PubMed

Matos, Olga; Esteves, Francisco

2010-08-01

Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunocompromised patients, especially patients infected with HIV, but it has also been isolated from immunocompetent persons. This article discusses the application of multilocus genotyping analysis to the study of the genetic diversity of P. jirovecii and its epidemiological and clinical parameters, and the important concepts achieved to date with these approaches. The multilocus typing studies performed until now have shown that there is an important genetic diversity of stable and ubiquitous P. jirovecii genotypes; infection with P. jirovecii is not necessarily clonal, recombination between some P. jirovecii multilocus genotypes has been suggested. P. jirovecii-specific multilocus genotypes can be associated with severity of PcP. Patients infected with P. jirovecii, regardless of the form of infection they present with, are part of a common human reservoir for future infections. The CYB, DHFR, DHPS, mtLSU rRNA, SOD and the ITS loci are suitable genetic targets to be used in further epidemiological studies focused on the identification and characterization of P. jirovecii haplotypes correlated with drug resistance and PcP outcome.

Molecular Properties of Salmonella enterica Serotype Paratyphi B Distinguish between Its Systemic and Its Enteric Pathovars

PubMed Central

Prager, Rita; Rabsch, Wolfgang; Streckel, Wiebke; Voigt, Wolfgang; Tietze, Erhardt; Tschäpe, Helmut

2003-01-01

Salmonella enterica serotype O1,4,5,12:Hb:1,2, designated according to the current Kauffmann-White scheme as S. enterica serotype Paratyphi B, is a very diverse serotype with respect to its clinical and microbiological properties. PCR and blot techniques, which identify the presence, polymorphism, and expression of various effector protein genes, help to distinguish between strains with systemic and enteric outcomes of disease. All serotype Paratyphi B strains from systemic infections have been found to be somewhat genetically related with respect to the pattern of their virulence genes sopB, sopD, sopE1, avrA, and sptP as well as other molecular properties (multilocus enzyme electrophoresis type, pulsed-field gel electrophoresis [PFGE] type, ribotype, and IS200 type). They have been classified as members of the systemic pathovar (SPV). All these SPV strains possess a new sopE1-carrying bacteriophage (designated ΦSopE309) with high SopE1 protein expression but lack the commonly occurring avrA determinant. They exhibit normal SopB protein expression but lack SopD protein production. In contrast, strains from enteric infections classified as belonging to the enteric pathovar possess various combinations of the respective virulence genes, PFGE pattern, and ribotypes. We propose that the PCR technique for testing for the presence of the virulence genes sopE1 and avrA be used as a diagnostic tool for identifying both pathovars of S. enterica serotype Paratyphi B. This will be of great public health importance, since strains of serotype Paratyphi B have recently reemerged worldwide. PMID:12958256

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

PubMed

Very, K J; Kirchner, M K; Shariat, N; Cottrell, W; Sandt, C H; Dudley, E G; Kariyawasam, S; Jayarao, B M

2016-05-01

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella. © 2015 Blackwell Verlag GmbH.

Clinical Manifestations and Molecular Characterization of Pertactin-Deficient and Pertactin-Producing Bordetella pertussis in Children, Philadelphia 2007-2014.

PubMed

Vodzak, Jennifer; Queenan, Anne Marie; Souder, Emily; Evangelista, Alan T; Long, Sarah S

2017-01-01

 Bordetella pertussis strains lacking expression of pertactin, a bacterial adhesin and vaccine target, are emerging. There are limited data on disease manifestations of mutant strains in children. We sought to compare clinical manifestations of pertactin-deficient and pertactin-producing B. pertussis infection in infants and describe corresponding molecular characteristics.  Molecular characterization of archived B. pertussis isolates (collected January 2007 to March 2014) included Western blot analysis, pulsed-field gel electrophoresis (PFGE), polymerase chain reaction, and pertactin gene sequencing. Medical record review compared epidemiologic and clinical courses of pertactin-producing and pertactin-deficient B. pertussis infections.  Sixty of 72 B. pertussis isolates were viable for analysis. Within the cohort of infants, the median age was 95 days, 90% received ≤1 dose of vaccine, and 72% were hospitalized. Pertactin deficiency was first noted in 2008, and its prevalence increased over time (68% overall prevalence). There were no statistically significant differences in presenting symptoms or signs, hospitalization, intensive care, respiratory support, or laboratory results related to pertactin expression. Illness length was shorter in pertactin-deficient group (mean difference, 3.2 days; P = .04); no difference was noted in the subgroup of infants <4 months old. Molecular analyses identified 11 PFGE profiles (Centers for Disease Control and Prevention profile No. 002 predominant, 47%). In 41 pertactin-deficient strains, sequencing identified 2 stop codon and 3 IS481 locations disrupting the prn gene. Mutations and nucleotide positions were not unique to PFGE type, nor were they clustered in time.  In this cohort of predominantly unimmunized infants, clinical disease did not differ between infection with pertactin-deficient and those with pertactin-producing B. pertussis. Molecular analyses demonstrated remarkable PFGE strain diversity, with multiple mechanisms and molecular sites of pertactin inactivation. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Emergence of a multiresistant KPC-3 and VIM-1 carbapenemase-producing Escherichia coli strain in Spain.

PubMed

Porres-Osante, Nerea; Azcona-Gutiérrez, Jose Manuel; Rojo-Bezares, Beatriz; Undabeitia, Esther; Torres, Carmen; Sáenz, Yolanda

2014-07-01

To characterize the mechanisms involved in carbapenem resistance, as well as the genetic elements supporting their mobilization, in a multidrug-resistant Escherichia coli isolate. The E. coli isolate was obtained from a patient with fatal urinary sepsis. Antimicrobial susceptibility testing was performed by the disc diffusion and agar dilution methods. The E. coli molecular type and phylogroup were determined using multilocus sequence typing and the triple PCR technique, respectively. PCR and sequencing were used for virulence and resistance genotype characterization. Plasmid content and gene location were analysed by S1-PFGE, I-Ceu1-PFGE and hybridization experiments. Transformation assays were performed. The E. coli strain, typed as ST448 and phylogroup B1, was resistant to all tested antibiotics except fosfomycin, tigecycline and tetracycline. The following resistance and virulence genetic structures were obtained: ISKpn7 + bla(KPC-3) + ISKpn6 linked to Tn4401; tnpR + aac(6')-Ib'-9 + aadA1 + bla(OXA-9) + tnpR + bla(TEM-1a) + tnpB + strB + strA + sul2; intI1 + bla(VIM-1) + aac(6')-Ib' + aphA15 + aadA1 + catB2 + qacEΔ1-sul1 + orf5; ISEcp1 + bla(CMY-2); IS26 + bla(SHV-12); aph(3')-I; aac(3)-IV; floR; catA; and fimA. Mutations in the ampC promoter (-18, -1 and +58) and substitutions in the GyrA (Ser-83→Leu and Asp-87→Asn) and ParC (Ser-80→Ile) proteins were observed. IncFII (ST2), IncA/C and ColE(TP) plasmids of 145.5, 87 and <2 kb, respectively, were found. The bla(VIM-1) gene was located in a non-typeable plasmid of >300 kb, and the bla(KPC-3) gene in the 145.5 kb IncFII plasmid. Transformant strains carried the IncFII and ColE(TP) plasmids, and the bla(KPC-3), bla(TEM-1a), bla(OXA-9), aadA1, aac(6')-Ib'-9, aac(3)-IV and floR genes. This is the first report of the co-production of KPC-3, VIM-1, SHV-12, OXA-9 and CMY-2 in a unique clinical multiresistant E. coli isolate. The dissemination of these genes on mobile genetic elements is alarming and complicates antimicrobial therapies. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution.

PubMed

Mora, Azucena; López, Cecilia; Dabhi, Ghizlane; Blanco, Miguel; Blanco, Jesús E; Alonso, María Pilar; Herrera, Alexandra; Mamani, Rosalía; Bonacorsi, Stéphane; Moulin-Schouleur, Maryvonne; Blanco, Jorge

2009-07-07

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence.APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.

Taxonomic evaluation of unidentified Streptomyces isolates in the ARS Culture Collection (NRRL) using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

The ARS Culture Collection (NRRL) currently contains 7569 strains within the family Streptomycetaceae but 4368 of them have not been characterized to the species level. A gene sequence database using the Bacterial Isolate Genomic Sequence Database package (BIGSdb) (Jolley & Maiden, 2010) is availabl...

Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis

USDA-ARS?s Scientific Manuscript database

In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811T formed a cluster with 5 other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these ot...

Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

PubMed Central

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-01-01

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations. PMID:24556669

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

PubMed

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-02-19

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

USA300-related methicillin-resistant Staphylococcus aureus clone is the predominant cause of community and hospital MRSA infections in Colombian children.

PubMed

Márquez-Ortiz, Ricaurte Alejandro; Álvarez-Olmos, Martha I; Escobar Pérez, Javier Antonio; Leal, Aura Lucia; Castro, Betsy Esperanza; Mariño, Ana Cristina; Barrero, Esther Rocio; Mujica, Sandra Celina; Gaines, Sebastián; Vanegas, Natasha

2014-08-01

Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) isolates are known to be more virulent and clinically aggressive in children. The goal of the present study was characterize the molecular epidemiology of MRSA isolates causing infections in Colombian children. An observational and prospective study was conducted between April 2009 and June 2011 at 15 hospitals in Bogotá, Colombia. A detailed epidemiological profile was made of 162 children infected with MRSA. The isolates were subjected to antimicrobial susceptibility testing, molecular characterization including 21 virulence genes, SCCmec, spa and agr typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Among all isolates included in the study, 85.8% were obtained from patients whose infectious process was initiated in the community; of these, 69,8% occurred in patients without healthcare-associated risk factors. The molecular characterization of the isolates showed a high proportion (95.1%) containing a community-genotype profile with a high prevalence of SCCmec type IV, PVL-positives, and also related to CC8. Most CG-MRSA isolates (143, 92.9%) were genetically related to the pandemic clone USA300, differing by the presence of SCCmec IVc and the absence of the arginine catabolic mobile element (ACME). An increase in the frequency of CG-MRSA infections has been reported worldwide. In this study we found that almost all MRSA infections in our pediatric population were caused by community-genotype isolates, supporting the success of the CG-MRSA clones. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular epidemiology of an outbreak of clinical mastitis in sheep caused by Mannheimia haemolytica.

PubMed

Omaleki, Lida; Browning, Glenn F; Allen, Joanne L; Markham, Philip F; Barber, Stuart R

2016-08-15

The aetiology and epidemiology of outbreaks of clinical mastitis in sheep under extensive pastoral conditions are incompletely understood. The objective of this study was to conduct a detailed investigation of a clinical mastitis outbreak that affected more than 10% of 230 at-risk ewes on a sheep and grain producing property in south east Australia during drought conditions in 2009. Milk samples were collected aseptically from all affected ewes and plated on sheep blood agar for bacterial identification. M. haemolytica was isolated from 80% of the samples that yielded cultivable microorganisms and thus was the main microorganism responsible for the outbreak. Analysis of the restriction endonuclease cleavage patterns of the isolates using pulsed field gel electrophoresis revealed some evidence of clonality, suggesting the possibility of horizontal transmission, but there was also considerable diversity between the clusters of closely related isolates. Multilocus sequence typing of the M. haemolytica isolates revealed most of the isolates belonged to ST1 with no association between the PFGE and MLST fingerprints of the isolates. Resistance to neomycin, streptomycin and sulphafurazole was detected in some of the isolates, but they were all susceptible to penicillin, ampicillin, ceftiofur, amoxycillin/clavulanic acid, ciprofloxacin, tetracycline, erythromycin and trimethoprim. This is the first published record of a comparison of the strains of M. haemolytica involved in a clinical mastitis outbreak in sheep and demonstrates the importance of this pathogen in sheep production systems, particularly during adverse climatic conditions and increased stocking rate. Copyright © 2016 Elsevier B.V. All rights reserved.

Selection and Validation of a Multilocus Variable-Number Tandem-Repeat Analysis Panel for Typing Shigella spp.▿ †

PubMed Central

Gorgé, Olivier; Lopez, Stéphanie; Hilaire, Valérie; Lisanti, Olivier; Ramisse, Vincent; Vergnaud, Gilles

2008-01-01

The Shigella genus has historically been separated into four species, based on biochemical assays. The classification within each species relies on serotyping. Recently, genome sequencing and DNA assays, in particular the multilocus sequence typing (MLST) approach, greatly improved the current knowledge of the origin and phylogenetic evolution of Shigella spp. The Shigella and Escherichia genera are now considered to belong to a unique genomospecies. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses of highly homogeneous bacterial pathogens. Here, we assess the capability of MLVA for Shigella typing. Thirty-two potentially polymorphic VNTRs were selected by analyzing in silico five Shigella genomic sequences and subsequently evaluated. Eventually, a panel of 15 VNTRs was selected (i.e., MLVA15 analysis). MLVA15 analysis of 78 strains or genome sequences of Shigella spp. and 11 strains or genome sequences of Escherichia coli distinguished 83 genotypes. Shigella population cluster analysis gave consistent results compared to MLST. MLVA15 analysis showed capabilities for E. coli typing, providing classification among pathogenic and nonpathogenic E. coli strains included in the study. The resulting data can be queried on our genotyping webpage (http://mlva.u-psud.fr). The MLVA15 assay is rapid, highly discriminatory, and reproducible for Shigella and Escherichia strains, suggesting that it could significantly contribute to epidemiological trace-back analysis of Shigella infections and pathogenic Escherichia outbreaks. Typing was performed on strains obtained mostly from collections. Further studies should include strains of much more diverse origins, including all pathogenic E. coli types. PMID:18216214

[Standard algorithm of molecular typing of Yersinia pestis strains].

PubMed

Eroshenko, G A; Odinokov, G N; Kukleva, L M; Pavlova, A I; Krasnov, Ia M; Shavina, N Iu; Guseva, N P; Vinogradova, N A; Kutyrev, V V

2012-01-01

Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

Genome Sequences of 228 Shiga Toxin-Producing Escherichia coli Isolates and 12 Isolates Representing Other Diarrheagenic E. coli Pathotypes

PubMed Central

Strockbine, Nancy; Changayil, Shankar; Ranganathan, Satishkumar; Zhao, Kun; Weil, Ryan; MacCannell, Duncan; Sabol, Ashley; Schmidtke, Amber; Martin, Haley; Stripling, Devon; Ribot, Efrain M.; Gerner-Smidt, Peter

2014-01-01

Shiga toxin-producing Escherichia coli (STEC) are a common cause for food-borne diarrheal illness outbreaks and sporadic cases. Here, we report the availability of the draft genome sequences of 228 STEC strains representing 32 serotypes with known pulsed-field gel electrophoresis (PFGE) types and epidemiological relationships, as well as 12 strains representing other diarrheagenic E. coli pathotypes. PMID:25103754

Exploration of the Genomic Diversity and Core Genome of the Bifidobacterium adolescentis Phylogenetic Group by Means of a Polyphasic Approach

PubMed Central

Duranti, Sabrina; Turroni, Francesca; Milani, Christian; Foroni, Elena; Bottacini, Francesca; Dal Bello, Fabio; Ferrarini, Alberto; Delledonne, Massimo; van Sinderen, Douwe

2013-01-01

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays. PMID:23064340

Taxonomic evaluation of Streptomyces hirsutus and related species using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

Phylogenetic analyses of species of Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100% bootstrap value) containing 8 species having very similar gross morphology. These species, including Streptomyces bambergiensis, Streptomyces chlorus, Streptomyces...

[Multilocus Sequence Typing analysis of human Campylobacter coli in Granada (Spain)].

PubMed

Carrillo-Ávila, J A; Sorlózano-Puerto, A; Pérez-Ruiz, M; Gutiérrez-Fernández, J

2016-12-01

Different subtypes of Campylobacter spp. have been associated with diarrhoea and a Multilocus Sequence Typing (MLST) method has been performed for subtyping. In the present work, MLST was used to analyse the genetic diversity of eight strains of Campylobacter coli. Nineteen genetic markers were amplified for MLST analysis: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. After comparing the obtained sequences with the Campylobacter MLST database, the allele numbers, sequence types (STs) and clonal complexes (CCs) were assigned. The 8 C. coli isolates yielded 4 different STs belonging to 2 CCs. Seven isolates belong to ST-828 clonal complex and only one isolate belong to ST-21. Two samples came from the same patient, but were isolated in two different periods of time. MLST can be useful for taxonomic characterization of C. coli isolates.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

PubMed

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie; Maubon, Danièle

2017-08-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1

PubMed Central

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie

2017-01-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus. PMID:28726611

The rise of ampicillin-resistant Enterococcus faecium high-risk clones as a frequent intestinal colonizer in oncohaematological neutropenic patients on levofloxacin prophylaxis: a risk for bacteraemia?

PubMed

Sánchez-Díaz, A M; Cuartero, C; Rodríguez, J D; Lozano, S; Alonso, J M; Rodríguez-Domínguez, M; Tedim, A P; Del Campo, R; López, J; Cantón, R; Ruiz-Garbajosa, P

2016-01-01

Levofloxacin extended prophylaxis (LEP), recommended in oncohaematological neutropenic patients to reduce infections, might select resistant bacteria in the intestine acting as a source of endogenous infection. In a prospective observational study we evaluated intestinal emergence and persistence of ampicillin-resistant Enterococcus faecium (AREfm), a marker of hospital adapted high-risk clones. AREfm was recovered from the faeces of 52 patients with prolonged neutropenia after chemotherapy, at admission (Basal), during LEP, and twice weekly until discharge (Pos-LEP). Antibiotic susceptibility, virulence traits and population structure (pulsed-field gel electrophoresis and multilocus sequence typing) were determined and compared with bacteraemic isolates. Gut enterococcal population was monitored using a quantitative PCR quantification approach. AREfm colonized 61.4% of patients (194/482 faecal samples). Sequential AREfm acquisition (25% Basal, 36.5% LEP, 50% Pos-LEP) and high persistent colonization rates (76.9-89.5%) associated with a decrease in clonal diversity were demonstrated. Isolates were clustered into 24 PFGE-patterns within 13 sequence types, 95.8% of them belonging to hospital-associated Bayesian analysis of population structure subgroups 2.1a and 3.3a. Levofloxacin resistance and high-level streptomycin resistance were a common trait of these high-risk clones. AREfm-ST117, the most persistent clone, was dominant (60.0% isolates, 32.6% patients). It presented esp gene and caused 18.2% of all bacteraemia episodes in 21% of patients previously colonized by this clone. In AREfm-colonized patients, intestinal enrichment in the E. faecium population with a decline in total bacterial load was observed. AREfm intestinal colonization increases during hospital stay and coincides with enterococci population enrichment in the gut. Dominance and intestinal persistence of the ST117 clone might increase the risk of bacteraemia. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

L. monocytogenes in a cheese processing facility: Learning from contamination scenarios over three years of sampling.

PubMed

Rückerl, I; Muhterem-Uyar, M; Muri-Klinger, S; Wagner, K-H; Wagner, M; Stessl, B

2014-10-17

The aim of this study was to analyze the changing patterns of Listeria monocytogenes contamination in a cheese processing facility manufacturing a wide range of ready-to-eat products. Characterization of L. monocytogenes isolates included genotyping by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Disinfectant-susceptibility tests and the assessment of L. monocytogenes survival in fresh cheese were also conducted. During the sampling period between 2010 and 2013, a total of 1284 environmental samples were investigated. Overall occurrence rates of Listeria spp. and L. monocytogenes were 21.9% and 19.5%, respectively. Identical L. monocytogenes genotypes were found in the food processing environment (FPE), raw materials and in products. Interventions after the sampling events changed contamination scenarios substantially. The high diversity of globally, widely distributed L. monocytogenes genotypes was reduced by identifying the major sources of contamination. Although susceptible to a broad range of disinfectants and cleaners, one dominant L. monocytogenes sequence type (ST) 5 could not be eradicated from drains and floors. Significantly, intense humidity and steam could be observed in all rooms and water residues were visible on floors due to increased cleaning strategies. This could explain the high L. monocytogenes contamination of the FPE (drains, shoes and floors) throughout the study (15.8%). The outcome of a challenge experiment in fresh cheese showed that L. monocytogenes could survive after 14days of storage at insufficient cooling temperatures (8 and 16°C). All efforts to reduce L. monocytogenes environmental contamination eventually led to a transition from dynamic to stable contamination scenarios. Consequently, implementation of systematic environmental monitoring via in-house systems should either aim for total avoidance of FPE colonization, or emphasize a first reduction of L. monocytogenes to sites where contamination of the processed product is unlikely. Drying of surfaces after cleaning is highly recommended to facilitate the L. monocytogenes eradication. Copyright © 2014 Elsevier B.V. All rights reserved.

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

PubMed

Bouchez, Valérie; Guglielmini, Julien; Dazas, Mélody; Landier, Annie; Toubiana, Julie; Guillot, Sophie; Criscuolo, Alexis; Brisse, Sylvain

2018-06-01

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.

Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

The family Streptomycetaceae, notably species in the genus Streptomyces, have long been the subject of investigation due to their well-known ability to produce secondary metabolites. The emergence of drug resistant pathogens and the relative ease of producing genome sequences has renewed the importa...

The multilocus sequence typing network: mlst.net.

PubMed

Aanensen, David M; Spratt, Brian G

2005-07-01

The unambiguous characterization of strains of a pathogen is crucial for addressing questions relating to its epidemiology, population and evolutionary biology. Multilocus sequence typing (MLST), which defines strains from the sequences at seven house-keeping loci, has become the method of choice for molecular typing of many bacterial and fungal pathogens (and non-pathogens), and MLST schemes and strain databases are available for a growing number of prokaryotic and eukaryotic organisms. Sequence data are ideal for strain characterization as they are unambiguous, meaning strains can readily be compared between laboratories via the Internet. Laboratories undertaking MLST can quickly progress from sequencing the seven gene fragments to characterizing their strains and relating them to those submitted by others and to the population as a whole. We provide the gateway to a number of MLST schemes, each of which contain a set of tools for the initial characterization of strains, and methods for relating query strains to other strains of the species, including clustering based on differences in allelic profiles, phylogenetic trees based on concatenated sequences, and a recently developed method (eBURST) for identifying clonal complexes within a species and displaying the overall structure of the population. This network of MLST websites is available at http://www.mlst.net.

Molecular Phylogenetic Diversity of Dermatologic and Other Human Pathogenic Fusaria from Hospitals in Northern Italy

USDA-ARS?s Scientific Manuscript database

Fifty-eight fusaria isolated from 52 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusari...

Pathogenic Leptospira Species in Insectivorous Bats, China, 2015.

PubMed

Han, Hui-Ju; Wen, Hong-Ling; Liu, Jian-Wei; Qin, Xiang-Rong; Zhao, Min; Wang, Li-Jun; Luo, Li-Mei; Zhou, Chuan-Min; Zhu, Ye-Lei; Qi, Rui; Li, Wen-Qian; Yu, Hao; Yu, Xue-Jie

2018-06-01

PCR amplification of the rrs2 gene indicated that 50% (62/124) of insectivorous bats from eastern China were infected with Leptospira borgpetersenii, L. kirschneri, and several potentially new Leptospira species. Multilocus sequence typing defined 3 novel sequence types in L. kirschneri, suggesting that bats are major carriers of Leptospira.

Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

Aspergillus section Versicolores, nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

Characterization of Encapsulated and Noncapsulated Haemophilus influenzae and Determination of Phylogenetic Relationships by Multilocus Sequence Typing

PubMed Central

Meats, Emma; Feil, Edward J.; Stringer, Suzanna; Cody, Alison J.; Goldstein, Richard; Kroll, J. Simon; Popovic, Tanja; Spratt, Brian G.

2003-01-01

A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net, it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet. PMID:12682154

Pan-genome multilocus sequence typing and outbreak-specific reference-based single nucleotide polymorphism analysis to resolve two concurrent Staphylococcus aureus outbreaks in neonatal services.

PubMed

Roisin, S; Gaudin, C; De Mendonça, R; Bellon, J; Van Vaerenbergh, K; De Bruyne, K; Byl, B; Pouseele, H; Denis, O; Supply, P

2016-06-01

We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7 months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

New system for multilocus variable-number tandem-repeat analysis of the enterohemorrhagic Escherichia coli strains belonging to three major serogroups: O157, O26, and O111.

PubMed

Izumiya, Hidemasa; Pei, Yingxin; Terajima, Jun; Ohnishi, Makoto; Hayashi, Tetsuya; Iyoda, Sunao; Watanabe, Haruo

2010-10-01

Enterohemorrhagic Escherichia coli (EHEC), a food- and waterborne pathogen, causes diarrhea, hemorrhagic colitis, and life-threatening HUS. MLVA is a newly developed and widely accepted genotyping tool. An MLVA system for EHEC O157 involving nine genomic loci has already been established. However, the present study revealed that the above-mentioned MLVA system cannot analyze EHEC O26 and O111 isolates-the second and third most dominant EHEC serogroups in Japan, respectively. Therefore, with several modifications to the O157 system and the use of nine additional loci, we developed an expanded MLVA system applicable to EHEC O26, O111, and O157. Our MLVA system had a relatively high resolution power for each of the three serogroups: Simpson's index of diversity was 0.991 (95% CI = 0.989-0.993), 0.988 (95% CI, 0.986-0.990), and 0.986 (95% CI, 0.979-0.993) for O26, O111, and O157, respectively. This system also detected outbreak-related isolates; the isolates collected during each of the 12 O26 and O111 outbreaks formed unique clusters, and most of the repeat copy numbers among the isolates collected during the same outbreak exhibited no or single-locus variations. These results were comparable to those of cluster analyses based on PFGE profiles. Therefore, our system can complement PFGE analysis-the current golden method. Because EHEC strains of three major serogroups can be rapidly analyzed on a single platform with our expanded MLVA system, this system could be widely used in molecular epidemiological studies of EHEC infections. © 2010 The Societies and Blackwell Publishing Asia Pty Ltd.

Whole genome sequencing may not be adequate to determine genome relatedness for surveillance and outbreak investigation of foodborne pathogens

USDA-ARS?s Scientific Manuscript database

Introduction: Advances in genomic technologies have improve the speed and precision of foodborne disease outbreak detection and response. For the past two decades, pulsed field gel electrophoresis (PFGE) has been the method of choice for surveillance and outbreak investigation with foodborne pathoge...

Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb.

PubMed

Lopez-Valladares, Gloria; Danielsson-Tham, Marie-Louise; Goering, Richard V; Tham, Wilhelm

2017-01-01

Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958-2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and Ö-6 to Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advantageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.

Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis

PubMed Central

Sévellec, Yann; Vignaud, Marie-Léone; Granier, Sophie A.; Lailler, Renaud; Feurer, Carole; Le Hello, Simon; Mistou, Michel-Yves; Cadel-Six, Sabrina

2018-01-01

In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen. PMID:29867804

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

PubMed

Tellapragada, Chaitanya; Kamthan, Aayushi; Shaw, Tushar; Ke, Vandana; Kumar, Subodh; Bhat, Vinod; Mukhopadhyay, Chiranjay

2016-01-01

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016.

PubMed

Saavedra, Sandra Yamile; Diaz, Lorena; Wiesner, Magdalena; Correa, Adriana; Arévalo, Stefany Alejandra; Reyes, Jinnethe; Hidalgo, Andrea Melissa; de la Cadena, Elsa; Perenguez, Marcela; Montaño, Lucy Angeline; Ardila, Javier; Ríos, Rafael; Ovalle, María Victoria; Díaz, Paula; Porras, Paola; Villegas, Maria V; Arias, Cesar A; Beltrán, Mauricio; Duarte, Carolina

2017-12-01

Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1 -positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1 , including 8 Escherichia coli , 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate . E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10 -5 to 2.07 × 10 -1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids. Copyright © 2017 American Society for Microbiology.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016

PubMed Central

Diaz, Lorena; Wiesner, Magdalena; Correa, Adriana; Arévalo, Stefany Alejandra; Reyes, Jinnethe; Hidalgo, Andrea Melissa; de la Cadena, Elsa; Perenguez, Marcela; Montaño, Lucy Angeline; Ardila, Javier; Ríos, Rafael; Ovalle, María Victoria; Díaz, Paula; Porras, Paola; Villegas, Maria V.; Arias, Cesar A.; Beltrán, Mauricio

2017-01-01

ABSTRACT Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae. Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1-positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1, including 8 Escherichia coli, 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate. E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S. Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10−5 to 2.07 × 10−1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids. PMID:28893788

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics.

PubMed

Couto, Natacha; Chlebowicz, Monika A; Raangs, Erwin C; Friedrich, Alex W; Rossen, John W

2018-04-05

The emergence of nosocomial infections by multidrug-resistant Staphylococcus haemolyticus isolates has been reported in several European countries. Here, we report the first two complete genome sequences of S. haemolyticus sequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics. Copyright © 2018 Couto et al.

Molecular characterisation and control of Acinetobacter baumannii isolates resistant to multi-drugs emerging in inter-intensive care units.

PubMed

Ertürk, Ayşe; Çiçek, Ayşegül Çopur; Gümüş, Aziz; Cüre, Erkan; Şen, Ahmet; Kurt, Aysel; Karagöz, Alper; Aydoğan, Nebahat; Sandallı, Cemal; Durmaz, Rıza

2014-07-22

A nosocomial outbreak of Acinetobacter baumannii (AB) infections occurred among intensive care units (ICU) (surgery, medical, cardiovascular surgery, coronary unit) of Recep Tayyip Erdogan University Medical School (Rize, Turkey) between January 2011 and May 2012. The identification of isolates and clonal relation among them were investigated by molecular techniques. A total of 109 AB isolates were obtained from 64 clinical materials from 54 ICU patients and 3 from the hands of healthcare workers (HCWs) of 42 environmental samples. The isolates were identified by 16S rDNA sequencing and OXA- specific PCR. The clonal relation between isolates was investigated by PFGE methods using ApaI restriction enzyme. All isolates were determined as AB by 16S rDNA sequencing and OXA-spesific PCR. While the blaOXA-51-like gene was amplified in all isolates, the blaOXA-23-like gene was amplified from 103 isolates. The PFGE pattern generated 9 pulsotypes and showed that the isolates from patients, HCWs, and the environment were genetically related. In 7 of these pulsotypes, there were 107 strains (98%) showing similar PFGE profiles that cannot be distinguished from each other, ranging from 2 to 53. The remaining 2 pulsotypes were comprised of strains closely associated with the main cluster. Two major groups were discovered with similarity coefficient of 85% and above. The first group consisted of 97 strains that are similar to each other at 92.7% rate, and the second group consisted of 12 strains that are 100% identical. The common utilization of the blood gas device among ICU was the reason for the contamination. AB strains can remain stable for a long period of time, although due to the disinfection procedures applied in hospitals, there is a small chance that the same clone might reappear and cause another epidemic. For that reason, the resistance profiles of the strains must be continuously followed with amplification-based methods, and these methods should be used to support the PFGE method in the short term.

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

PubMed

Mulet, M; Gomila, M; Ramírez, A; Cardew, S; Moore, E R B; Lalucat, J; García-Valdés, E

2017-02-01

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

Detection and characterization of Histoplasma capsulatum in a German badger (Meles meles) by ITS sequencing and multilocus sequencing analysis

USDA-ARS?s Scientific Manuscript database

A wild badger (Meles meles) with a severe nodular dermatitis was presented for post mortem examination. Numerous cutaneous granulomas with superficial ulceration were present especially on head, dorsum, and forearms were found at necropsy. Histopathological examination of the skin revealed a severe ...

Diversity of the Cronobacter Genus as Revealed by Multilocus Sequence Typing

PubMed Central

Joseph, S.; Sonbol, H.; Hariri, S.; Desai, P.; McClelland, M.

2012-01-01

Cronobacter (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti. In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis, including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis. PMID:22785185

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

PubMed

Barony, Gustavo M; Tavares, Guilherme C; Pereira, Felipe L; Carvalho, Alex F; Dorella, Fernanda A; Leal, Carlos A G; Figueiredo, Henrique C P

2017-10-19

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

PubMed Central

Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

2014-01-01

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

Comparison of a newly developed binary typing with ribotyping and multilocus sequence typing methods for Clostridium difficile.

PubMed

Li, Zhirong; Liu, Xiaolei; Zhao, Jianhong; Xu, Kaiyue; Tian, Tiantian; Yang, Jing; Qiang, Cuixin; Shi, Dongyan; Wei, Honglian; Sun, Suju; Cui, Qingqing; Li, Ruxin; Niu, Yanan; Huang, Bixing

2018-04-01

Clostridium difficile is the causative pathogen for antibiotic-related nosocomial diarrhea. For epidemiological study and identification of virulent clones, a new binary typing method was developed for C. difficile in this study. The usefulness of this newly developed optimized 10-loci binary typing method was compared with two widely used methods ribotyping and multilocus sequence typing (MLST) in 189 C. difficile samples. The binary typing, ribotyping and MLST typed the samples into 53 binary types (BTs), 26 ribotypes (RTs), and 33 MLST sequence types (STs), respectively. The typing ability of the binary method was better than that of either ribotyping or MLST expressed in Simpson Index (SI) at 0.937, 0.892 and 0.859, respectively. The ease of testing, portability and cost-effectiveness of the new binary typing would make it a useful typing alternative for outbreak investigations within healthcare facilities and epidemiological research. Copyright © 2018 Elsevier B.V. All rights reserved.

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China.

PubMed

Yang, Yong; Yu, Xiaofeng; Zhan, Li; Chen, Jiancai; Zhang, Yunyi; Zhang, Junyan; Chen, Honghu; Zhang, Zheng; Zhang, Yanjun; Lu, Yiyu; Mei, Lingling

2017-04-01

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multilocus Sequence Types of Campylobacter jejuni Isolates from Different Sources in Eastern China.

PubMed

Zhang, Gong; Zhang, Xiaoyan; Hu, Yuanqing; Jiao, Xin-An; Huang, Jinlin

2015-09-01

Campylobacter jejuni is a major food-borne pathogen that causes human gastroenteritis in many developed countries. In our study, we applied multilocus sequence typing (MLST) technology to 167 C. jejuni isolates from diverse sources in Eastern China to examine their genetic diversity. MLST defined 94 sequence types (STs) belonging to 18 clonal complexes (CCs). Forty-five STs from 60 isolates (36%) and 22 alleles have not been previously documented in an international database. One hundred and two isolates, accounting for 61.1% of all isolates, belonged to eight clonal complexes. The eight major CCs were also the most common complexes from different sources. The most common ST type of isolates from human and food was ST-353. The dominant ST type in chicken and foods was ST-354. Among 21 STs that contained two or more different sources isolates, 15 STs contained human isolates and isolates from other sources, suggesting that potentially pathogenic strains are not restricted to specific lineages.

Streptococcus iniae outbreaks in Brazilian Nile tilapia (Oreochromis niloticus L:) farms

PubMed Central

Figueiredo, H. C. P.; Netto, L. Nobrega; Leal, C. A. G.; Pereira, Ulisses P.; Mian, Glaúcia F.

2012-01-01

This is the first report of outbreaks of Streptococcus iniae in Nile tilapia farms in South America. Seven isolates were identified by biochemical, serological and molecular tests. Their 16S rRNA gene sequences showed 100% similarity with S. iniae ATCC 29178 and two distinct PFGE patterns were observed for Brazilian isolates. PMID:24031866

Prevalence of methicillin-resistant Staphylococcus aureus in meat.

PubMed

de Boer, E; Zwartkruis-Nahuis, J T M; Wit, B; Huijsdens, X W; de Neeling, A J; Bosch, T; van Oosterom, R A A; Vila, A; Heuvelink, A E

2009-08-31

Recently the isolation of methicillin-resistant Staphylococcus aureus (MRSA) strains from several food-producing animals has been reported. During slaughtering of MRSA-positive animals, contamination of carcasses with MRSA may occur and consequently the meat of these animals may get contaminated. The aim of this study was to estimate the prevalence of MRSA in raw meat samples from the retail trade. Samples of raw beef, pork, veal, lamb/mutton, chicken, turkey, fowl and game were collected from the retail trade. A detection method including a two-step enrichment in Mueller-Hinton broth+6.5% NaCl and phenol red mannitol broth containing ceftizoxime and aztreonam, followed by isolation on MRSA ID agar (bioMérieux) was evaluated and subsequently applied for the detection of MRSA in samples of raw meats. MRSA strains were isolated from 264 (11.9%) of 2217 samples analyzed. Isolation percentages for the meat species were: beef (10.6%), veal (15.2%), lamb and mutton (6.2%), pork (10.7%), chicken (16.0%), turkey (35.3%), fowl (3.4%) and game (2.2%). The majority (85%) of the isolated strains belonged to spa-types of pulsed-field gel electrophoresis (PFGE) non-typeable (NT)-MRSA, corresponding to the multilocus sequence type ST398, a type also recently isolated in the Netherlands from pigs. However, a smaller part of these strains were found to be of other ST's, possibly of human origin. Further studies are needed to elucidate transmission routes of MRSA in relation to meat and other foods and to provide the tools for preventing the spread of MRSA. At present the high prevalence of MRSA in meat has not been shown to contribute significantly to the dissemination of MRSA to humans and the possible health hazard for consumers of the presence of MRSA in foods should be further elucidated.

Genetically similar strains of Escherichia coli O157:H7 isolated from sheep, cattle and human patients.

PubMed

Söderlund, Robert; Hedenström, Ingela; Nilsson, Anna; Eriksson, Erik; Aspán, Anna

2012-10-24

Comparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients. Verotoxin-producing O157:H7 was found in 11/597 (1.8%) of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8%) and 2/105 ear samples (1.9%). All positive sheep were < 6 months old. Pulsed field gel electrophoresis typing revealed exact matches between isolates from the sheep prevalence study and human patients as well as between isolates from sheep and cattle. In one case, matching isolates were found in sheep, cattle, and a human patient in the same municipality. Identical PFGE profiles generally corresponded to similar but non-identical multi-locus VNTR profiles. In one sheep sample, SNP-typing found the highly virulent clade 8 variant of O157:H7. The virulence gene profiles of sheep isolates from the prevalence study and three sheep farms linked to cases of human illness were investigated by PCR detection (eaeA, hlyA, cdtV-B, vtx1), and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously. The same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two-year period in a tertiary teaching hospital in Malaysia.

PubMed

Sit, Pik San; Teh, Cindy Shuan Ju; Idris, Nuryana; Sam, I-Ching; Syed Omar, Sharifah Faridah; Sulaiman, Helmi; Thong, Kwai Lin; Kamarulzaman, Adeeba; Ponnampalavanar, Sasheela

2017-04-13

Methicillin-resistant Staphylococcus aureus (MRSA) is an established pathogen that causes hospital- and community-acquired infections worldwide. The prevalence rate of MRSA infections were reported to be the highest in Asia. As there is limited epidemiological study being done in Malaysia, this study aimed to determine the prevalence of MRSA infection and the molecular characteristics of MRSA bacteraemia. Two hundred and nine MRSA strains from year 2011 to 2012 were collected from a tertiary teaching hospital in Malaysia. The strains were characterized by antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) typing, detection of Panton-Valentine leukocidin (PVL) gene, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Patient's demographic and clinical data were collected and correlated with molecular data by statistical analysis. Male gender and patient >50 years of age (p < 0.0001) were significantly associated with the increased risk of MRSA acquisition. Fifty-nine percent of MRSA strains were HA-MRSA that carried SCCmec type II, III, IV and V while 31% were CA-MRSA strains with SCCmec III, IV and V. The prevalence of PVL gene among 2011 MRSA strains was 5.3% and no PVL gene was detected in 2012 MRSA strains. All of the strains were sensitive to vancomycin. However, vancomycin MIC creep phenomenon was demonstrated by the increased number of MRSA strains with MIC ≥1.5 μg/mL (p = 0.008) between 2011 and 2012. Skin disease (p = 0.034) and SCCmec type III (p = 0.0001) were found to be significantly associated with high vancomycin MIC. Forty-four percent of MRSA strains from blood, were further subtyped by MLST and PFGE. Most of the bacteraemia cases were primary bacteraemia and the common comorbidities were diabetes, hypertension and chronic kidney disease. The predominant pulsotype was pulsotype C exhibited by SCCmec III-ST239. This is a first study in Malaysia that reported the occurrence of MRSA clones such as SCCmec V-ST5, untypeable-ST508, SCCmec IV-ST1 and SCCmec IV-ST1137. SCCmec type III remained predominant among the MRSA strains in this hospital. The occurrence of SCCmec IV and V among hospital strains and the presence of SCCmec III in CA-MRSA strains are increasing. MRSA strains causing bacteraemia over the two-year study period were found to be genetically diverse.

Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov

USDA-ARS?s Scientific Manuscript database

In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811T forms a cluster with 5 other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these oth...

Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

DTIC Science & Technology

2010-05-12

Sequence Types. The evolutionary history of all the B. cereus MLST concatenated Sequence Types (545 taxa, 2,394 nucleotide positions) was inferred using...the Neighbor-Joining method [28]. The bootstrap consensus tree inferred from 100 replicates was taken to represent the evolutionary history of the... Chlamydia (manuscript in preparation) and performed pilot studies on Staphylococcus aureus and Streptoccus pneumoniae (Data S4 and Text S2). Another potential

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household.

PubMed

Staples, M; Graham, R M A; Doyle, C J; Smith, H V; Jennison, A V

2012-05-01

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters. © 2012 QUEENSLAND HEALTH. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Genetic Characterization of Clostridium botulinum Associated with Type B Infant Botulism in Japan▿

PubMed Central

Umeda, Kaoru; Seto, Yoshiyuki; Kohda, Tomoko; Mukamoto, Masafumi; Kozaki, Shunji

2009-01-01

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause. PMID:19571018

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle.

PubMed

Pringle, Märit; Bergsten, Christer; Fernström, Lise-Lotte; Höök, Helena; Johansson, Karl-Erik

2008-10-20

Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

Long-Term Care Facilities Are Reservoirs for Antimicrobial-Resistant Sequence Type 131 Escherichia coli

PubMed Central

Burgess, Mary J.; Johnson, James R.; Porter, Stephen B.; Johnston, Brian; Clabots, Connie; Lahr, Brian D.; Uhl, James R.; Banerjee, Ritu

2015-01-01

Background. Emerging data implicate long-term care facilities (LTCFs) as reservoirs of fluoroquinolone-resistant (FQ-R) Escherichia coli of sequence type 131 (ST131). We screened for ST131 among LTCF residents, characterized isolates molecularly, and identified risk factors for colonization. Methods. We conducted a cross-sectional study using a single perianal swab or stool sample per resident in 2 LTCFs in Olmsted County, Minnesota, from April to July 2013. Confirmed FQ-R E. coli isolates underwent polymerase chain reaction-based phylotyping, detection of ST131 and its H30 and H30-Rx subclones, extended virulence genotyping, and pulsed-field gel electrophoresis (PFGE) analysis. Epidemiological data were collected from medical records. Results. Of 133 fecal samples, 33 (25%) yielded FQ-R E. coli, 32 (97%) of which were ST131. The overall proportion with ST131 intestinal colonization was 32 of 133 (24%), which differed by facility: 17 of 41 (42%) in facility 1 vs 15 of 92 (16%) in facility 2 (P = .002). All ST131 isolates represented the H30 subclone, with virulence gene and PFGE profiles resembling those of previously described ST131 clinical isolates. By PFGE, certain isolates clustered both within and across LTCFs. Multivariable predictors of ST131 colonization included inability to sign consent (odds ratio [OR], 4.16 [P = .005]), decubitus ulcer (OR, 4.87 [ P = .04]), and fecal incontinence (OR, 2.59 [P = .06]). Conclusions. Approximately one fourth of LTCF residents carried FQ-R ST131 E. coli resembling ST131 clinical isolates. Pulsed-field gel electrophoresis suggested intra- and interfacility transmission. The identified risk factors suggest that LTCF residents who require increased nursing care are at greatest risk for ST131 colonization, possibly due to healthcare-associated transmission. PMID:26034762

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PubMed

Bedenić, Branka; Slade, Mia; Starčević, Lidija Žele; Sardelić, Sanda; Vranić-Ladavac, Mirna; Benčić, Ana; Zujić Atalić, Vlasta; Bogdan, Maja; Bubonja-Šonje, Marina; Tomić-Paradžik, Maja; Tot, Tatjana; Lukić-Grlić, Amarela; Drenjančević, Domagoj; Varda-Brkić, Dijana; Bandić-Pavlović, Daniela; Mihaljević, Slobodan; Zarfel, Gernot; Gužvinec, Marija; Conzemius, Rick; Barišić, Ivan; Tambić-Andraševic, Arjana

2018-06-21

A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Molecular sequence typing reveals genotypic diversity among Escherichia coli isolates recovered from a cantaloupe packinghouse in Northwestern Mexico

USDA-ARS?s Scientific Manuscript database

The increase in the consumption of fresh produce in the United States has correlated with a rise in the number of reported foodborne illnesses. To identify potential risk factors associated with post-harvest practices, the present study employed multilocus sequence typing (MLST) for the genotypic c...

Taxonomic evaluation of species in the Streptomyces hirsutus clade using multi-locus sequence analysis and proposals to reclassify several species in this clade

USDA-ARS?s Scientific Manuscript database

Previous phylogenetic analyses of species of Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100% bootstrap value) containing 8 species that exhibited very similar gross morphology in producing open looped (Retinaculum-Apertum) to spiral (Spira) chains...

Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areas.

PubMed

Sanchez, Inma; Garcia-Nuñez, Marian; Ragull, Sonia; Sopena, Nieves; Pedro-Botet, Maria Luisa; Estere, Maria; Rey-Joly, Celestino; Sabria, Miquel; Esteve, Maria

2008-02-01

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed >or= 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed >or= 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.

Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain.

PubMed

Hughes, L A; Wigley, P; Bennett, M; Chantrey, J; Williams, N

2010-10-01

Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing.

PubMed

Parcell, B J; Oravcova, K; Pinheiro, M; Holden, M T G; Phillips, G; Turton, J F; Gillespie, S H

2018-03-01

Pseudomonas aeruginosa healthcare outbreaks can be time consuming and difficult to investigate. Guidance does not specify which typing technique is most practical for decision-making. To explore the usefulness of whole-genome sequencing (WGS) in the investigation of a P. aeruginosa outbreak, describing how it compares with pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) analysis. Six patient isolates and six environmental samples from an intensive care unit (ICU) positive for P. aeruginosa over two years underwent VNTR, PFGE and WGS. VNTR and PFGE were required to fully determine the potential source of infection and rule out others. WGS results unambiguously distinguished linked isolates, giving greater assurance of the transmission route between wash-hand basin water and two patients, supporting the control measures employed. WGS provided detailed information without the need for further typing. When allied to epidemiological information, WGS can be used to understand outbreak situations rapidly and with certainty. Implementation of WGS in real-time would be a major advance in day-to-day practice. It could become a standard of care as it becomes more widespread due to its reproducibility and lower costs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

PubMed Central

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

1999-01-01

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

PubMed

Caruso, Paola; Biosca, Elena G; Bertolini, Edson; Marco-Noales, Ester; Gorris, María Teresa; Licciardello, Concetta; López, María M

2017-12-01

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Human isolates of Listeria monocytogenes in Sweden during half a century (1958-2010).

PubMed

Lopez-Valladares, G; Tham, W; Parihar, V Singh; Helmersson, S; Andersson, B; Ivarsson, S; Johansson, C; Ringberg, H; Tjernberg, I; Henriques-Normark, B; Danielsson-Tham, M-L

2014-11-01

Isolates of Listeria monocytogenes (n = 932) isolated in Sweden during 1958-2010 from human patients with invasive listeriosis were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) (AscI). Of the 932 isolates, 183 different PFGE types were identified, of which 83 were each represented by only one isolate. In all, 483 serovar 1/2a isolates were distributed over 114 PFGE types; 90 serovar 1/2b isolates gave 32 PFGE types; 21 serovar 1/2c isolates gave nine PFGE types; three serovar 3b isolates gave one PFGE type; and, 335 serovar 4b isolates gave 31 PFGE types. During the 1980s in Sweden, several serovar 4b cases were associated with the consumption of European raw soft cheese. However, as cheese-production hygiene has improved, the number of 4b cases has decreased. Since 1996, serovar 1/2a has been the dominant L. monocytogenes serovar in human listeriosis in Sweden. Therefore, based on current serovars and PFGE types, an association between human cases of listeriosis and the consumption of vacuum-packed gravad and cold-smoked salmon is suggested.

Phene Plate (PhP) biochemical fingerprinting. A screening method for epidemiological typing of enterococcal isolates.

PubMed

Saeedi, B; Tärnberg, M; Gill, H; Hällgren, A; Jonasson, J; Nilsson, L E; Isaksson, B; Kühn, I; Hanberger, H

2005-09-01

Pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard for genotyping of enterococci. However, PFGE is both expensive and time-consuming. The purpose of this study was to investigate whether the PhP system can be used as a reliable clinical screening method for detection of genetically related isolates of enterococci. If so, it should be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money. Ninety-nine clinical enterococcal isolates were analysed by PhP (similarity levels 0.90-0.975) and PFGE (similarity levels < or =3 and < or =6 bands) and all possible pairs of isolates were cross-classified as matched or mismatched. We found that the probability that a pair of isolates (A and B) belonging to the same type according to PhP also belong to the same cluster according to PFGE, i.e. p(A(PFGE)=B(PFGE) * A(PhP)=B(PhP)), and the probability that a pair of isolates of different types according to PhP also belong to different clusters according to PFGE, i.e. p(A(PFGE) not equalB(PFGE) * A(PhP) not equalB(PhP)), was relatively high for E. faecalis (0.86 and 0.96, respectively), but was lower for E. faecium (0.51 and 0.77, respectively). The concordance which shows the probability that PhP and PFGE agree on match or mismatch was 86%-93% for E. faecalis and 54%-66% for E. faecium, which indicates that the PhP method may be useful for epidemiological typing of E. faecalis in the current settings but not for E. faecium.

Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

PubMed

Park, Miseon; Deck, Joanna; Foley, Steven L; Nayak, Rajesh; Songer, J Glenn; Seibel, Janice R; Khan, Saeed A; Rooney, Alejandro P; Hecht, David W; Rafii, Fatemeh

2016-04-01

Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes. Published by Elsevier Ltd.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

PubMed

Talukder, Kaisar A; Khajanchi, Bijay K; Islam, M Aminul; Dutta, Dilip K; Islam, Zhahirul; Safa, Ashrafus; Khan, G Y; Alam, Khorshed; Hossain, M A; Malla, Sarala; Niyogi, S K; Rahman, Mustafizur; Watanabe, Haruo; Nair, G Balakrish; Sack, David A

2004-10-01

The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Multistate Outbreak of Salmonella Anatum Infections Linked to Imported Hot Peppers - United States, May-July 2016.

PubMed

Hassan, Rashida; Rounds, Joshua; Sorenson, Alida; Leos, Greg; Concepción-Acevedo, Jeniffer; Griswold, Taylor; Tesfai, Adiam; Blessington, Tyann; Hardy, Cerise; Basler, Colin

2017-06-30

Foodborne salmonellosis causes an estimated 1 million illnesses and 400 deaths annually in the United States (1). Salmonella Anatum is one of the top 20 Salmonella serotypes in the United States. During 2013-2015 there were approximately 300-350 annual illnesses reported to PulseNet, the national molecular subtyping network for foodborne disease surveillance. In June 2016, PulseNet identified a cluster of 16 Salmonella Anatum infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern from four states.* In April 2016, the same PFGE pattern had been uploaded to PulseNet from an isolate obtained from an Anaheim pepper, a mild to medium hot pepper. Hot peppers include many pepper varieties, such as Anaheim, jalapeño, poblano, and serrano, which can vary in heat level from mild to very hot depending on the variety and preparation. This rare PFGE pattern had been seen only 24 times previously in the PulseNet database, compared with common PFGE patterns for this serotype which have been seen in the database hundreds of times. Local and state health departments, CDC, and the Food and Drug Administration (FDA) investigated to determine the cause of the outbreak. Thirty-two patients in nine states were identified with illness onsets from May 6-July 9, 2016. Whole-genome sequencing (WGS) was performed to characterize clinical isolates and the Anaheim pepper isolate further. The combined evidence indicated that fresh hot peppers were the likely source of infection; however, a single pepper type or source farm was not identified. This outbreak highlights challenges in reconciling epidemiologic and WGS data, and the difficulties of identifying ingredient-level exposures through epidemiologic investigations alone.

Two Atypical Cases of Kingella kingae Invasive Infection with Concomitant Human Rhinovirus Infection

PubMed Central

Basmaci, Romain; Ilharreborde, Brice; Doit, Catherine; Presedo, Ana; Lorrot, Mathie; Alison, Marianne; Mazda, Keyvan; Bidet, Philippe

2013-01-01

We describe two atypical cases of Kingella kingae infection in children diagnosed by PCR, one case involving a soft tissue abscess and one case a femoral Brodie abscess. Both patients had concomitant human rhinovirus infection. K. kingae strains, isolated from an oropharyngeal swab, were characterized by multilocus sequence typing and rtxA sequencing. PMID:23784119

Multilocus Sequence Typing of Bartonella henselae in the United Kingdom Indicates that Only a Few, Uncommon Sequence Types Are Associated with Zoonotic Disease▿†

PubMed Central

Chaloner, Gemma L.; Harrison, Timothy G.; Coyne, Karen P.; Aanensen, David M.; Birtles, Richard J.

2011-01-01

Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections. PMID:21471345

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa.

PubMed

Nunney, L; Elfekih, S; Stouthamer, R

2012-05-01

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.

Beta-hemolytic Streptococcus dysgalactiae strains isolated from horses are a genetically distinct population within the Streptococcus dysgalactiae taxon.

PubMed

Pinho, Marcos D; Erol, Erdal; Ribeiro-Gonçalves, Bruno; Mendes, Catarina I; Carriço, João A; Matos, Sandra C; Preziuso, Silvia; Luebke-Becker, Antina; Wieler, Lothar H; Melo-Cristino, Jose; Ramirez, Mario

2016-08-17

The pathogenic role of beta-hemolytic Streptococcus dysgalactiae in the equine host is increasingly recognized. A collection of 108 Lancefield group C (n = 96) or L (n = 12) horse isolates recovered in the United States and in three European countries presented multilocus sequence typing (MLST) alleles, sequence types and emm types (only 56% of the isolates could be emm typed) that were, with few exceptions, distinct from those previously found in human Streptococcus dysgalactiae subsp. equisimilis. Characterization of a subset of horse isolates by multilocus sequence analysis (MLSA) and 16S rRNA gene sequence showed that most equine isolates could also be differentiated from S. dysgalactiae strains from other animal species, supporting the existence of a horse specific genomovar. Draft genome information confirms the distinctiveness of the horse genomovar and indicates the presence of potentially horse-specific virulence factors. While this genomovar represents most of the isolates recovered from horses, a smaller MLST and MLSA defined sub-population seems to be able to cause infections in horses, other animals and humans, indicating that transmission between hosts of strains belonging to this group may occur.

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

PubMed

Li, Xiang; Tambong, James; Yuan, Kat Xiaoli; Chen, Wen; Xu, Huimin; Lévesque, C André; De Boer, Solke H

2018-01-01

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses

PubMed Central

Li, Xiang; Tambong, James; Yuan, Kat (Xiaoli); Chen, Wen; Xu, Huimin; Lévesque, C. André; De Boer, Solke H.

2018-01-01

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA–DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov. PMID:29160202

The importance of molecular analyses for understanding the genetic diversity of Histoplasma capsulatum: an overview.

PubMed

Vite-Garín, Tania; Estrada-Bárcenas, Daniel Alfonso; Cifuentes, Joaquín; Taylor, Maria Lucia

2014-01-01

Advances in the classification of the human pathogen Histoplasma capsulatum (H. capsulatum) (ascomycete) are sustained by the results of several genetic analyses that support the high diversity of this dimorphic fungus. The present mini-review highlights the great genetic plasticity of H. capsulatum. Important records with different molecular tools, mainly single- or multi-locus sequence analyses developed with this fungus, are discussed. Recent phylogenetic data with a multi-locus sequence analysis using 5 polymorphic loci support a new clade and/or phylogenetic species of H. capsulatum for the Americas, which was associated with fungal isolates obtained from the migratory bat Tadarida brasiliensis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Streptococcus mutans clonal variation revealed by multilocus sequence typing.

PubMed

Nakano, Kazuhiko; Lapirattanakul, Jinthana; Nomura, Ryota; Nemoto, Hirotoshi; Alaluusua, Satu; Grönroos, Lisa; Vaara, Martti; Hamada, Shigeyuki; Ooshima, Takashi; Nakagawa, Ichiro

2007-08-01

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.

16S rRNA Gene Sequencing, Multilocus Sequence Analysis, and Mass Spectrometry Identification of the Proposed New Species “Clostridium neonatale”

PubMed Central

Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose

2014-01-01

In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, “Clostridium neonatale.” To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies. PMID:25232167

[Genotypic variability and persistence of Legionella pulsed-field gel electrophoresis patterns in 16 cooling towers in Shanghai, China].

PubMed

Chen, Ming-liang; Wang, Gang-yi; Chen, Min; Zhou, Hai-jian; Shao, Zhu-jun; Zhang, Xi; Wu, Fan

2010-07-01

To investigate the genotypic characteristics and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns in 16 air-conditioner cooling towers in six different public sites of Shanghai. From May to October, continuous sampling was operated once per month in 2007. Legionella strains isolated from the 16 cooling towers were confirmed by serological and latex agglutination. PFGE was applied for the fingerprinting of the isolates, while the cluster results of PFGE were analyzed by BioNumerics software. 131 strains of Legionella were isolated, including L. pneumophila, L. bozemanae, L. micdadei and L. anisa. 52 distinguishable PFGE patterns were differentiated among the 16 cooling towers, with 37 patterns were owned by just one cooling tower, which was not shared with other cooling towers, while 15 patterns were shared by more than 2 cooling towers. All the cooling towers had ≥ 2 PFGE patterns, while in 13 cooling towers the same PFGE patterns were recovered during the six months. From June to October of 2007, 18 strains of Legionella belonging to the PFGE pattern of LPAs.SH0078 were isolated continuously from 6 cooling towers. This study demonstrated great genotypic diversity and complexity of Legionella in cooling towers. Persistence of the PFGE patterns was observed in 81.25% of the cooling towers. The PFGE pattern of LPAs. SH0078 was distributed widely, suggesting it might be the dominate strain in Shanghai.

Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio

PubMed Central

Gabriel, Michael W.; Matsui, George Y.; Friedman, Robert

2014-01-01

Multilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. The recA, rpoA, gapA, 16S rRNA gene, gyrB, and ftsZ sequences from 56 species of the genus Vibrio were used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employing recA and rpoA in both possible gene orders were different. The addition of the gapA gene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification of Vibrio species. PMID:24951781

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil.

PubMed

Ferreira, Joseane Cristina; Penha Filho, Rafael Antonio Casarin; Kuaye, Ana Paula Yorika; Andrade, Leonardo Neves; Berchieri Junior, Angelo; Darini, Ana Lúcia da Costa

2018-06-01

The expression of plasmid-mediated quinolone resistance (PMQR) genes confers low-level quinolone and fluoroquinolones resistance alone. However, the association to chromosomal resistance mechanisms determines an expressively higher resistance in Enterobacteriaceae. These mechanisms are horizontally disseminated within plasmids and have contributed to the emergence of bacteria with reduced susceptibility or resistant to therapies worldwide. The epidemiological characterization of PMQR dissemination is highly relevant in the scientific and medical context, to investigate the dissemination within enterobacteria, from different populations, including humans and food-producing animals. In the present study, 200 Enterobacteriaceae isolates were harvested from poultry with cloacal swabs and identified as Escherichia coli (90.5%), Escherichia fergusonii (5.5%), Klebsiella oxytoca (2.5%) and Klebsiella pneumoniae (1.5%). Among isolates evaluated, 46 (23%) harboured PMQR genes including qnrB (43/200), qnrS (2/200) and aac(6')-Ib-cr (1/200). All isolates carrying PMQR genes showed multidrug-resistance phenotype. The 36 E. coli isolates showed 18 different PFGE types. All E. fergusonii isolates showed the same PFGE type. The two Klebsiella oxytoca belonged to two different PFGE types. The phylogenetic groups A, B1, and D were found among the E. coli harboring PMQR genes. Based on the phylogenetic analysis and PFGE, the population structure of E. coli isolates was diverse, even within the same farm. All isolates carrying qnrB and qnrS genes also harboured ColE-like plasmids. The Southern blot hybridization using the S1-PFGE revealed that the qnrB genes were located on low molecular weight plasmids, smaller than 10Kb. Resistance plasmids were sequenced and showed 100% identity with plasmid pPAB19-3. The association of PMQR genes with mobile genetic elements, such as transferable plasmids, favours the selection and dissemination of (fluoro) quinolones resistant bacteria among food-producing animals, and may play an important role in the current increased prevalence of resistant bacteria in different environments reported worldwide. Copyright © 2018. Published by Elsevier B.V.

Multilocus sequence typing of Scedosporium apiospermum and Pseudallescheria boydii isolates from cystic fibrosis patients.

PubMed

Bernhardt, A; Sedlacek, L; Wagner, S; Schwarz, C; Würstl, B; Tintelnot, K

2013-12-01

Scedosporium and Pseudallescheria species are the second most common lung-colonising fungi in cystic fibrosis (CF) patients. For epidemiological reasons it is important to trace sources of infection, routes of transmission and to determine whether these fungi are transient or permanent colonisers of the respiratory tract. Molecular typing methods like multilocus sequence typing (MLST) help provide this data. Clinical isolates of the P. boydii complex (including S. apiospermum and P. boydii) from CF patients in different regions of Germany were studied using MLST. Five gene loci, ACT, CAL, RPB2, BT2 and SOD2, were analysed. The S. apiospermum isolates from 34 patients were assigned to 32 sequence types (STs), and the P. boydii isolates from 14 patients to 8 STs. The results revealed that patients can be colonised by individual strains for years. The MLST scheme developed for S. apiospermum and P. boydii is a highly effective tool for epidemiologic studies worldwide. The MLST data are accessible at http://mlst.mycologylab.org/. Copyright © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains.

PubMed

Klein, Günter

2011-07-01

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible. Copyright ©, International Association for Food Protection

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

PubMed

Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

2006-08-01

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Veterinary Fusarioses within the United States

USDA-ARS?s Scientific Manuscript database

Multilocus DNA sequence data was used to retrospectively assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically dist...

Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community

PubMed Central

2014-01-01

Background Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Enterobacteriaeceae. CTX-M type extended-spectrum β- lactamases, of which there are now over 90 variants, are distributed globally, yet appear to vary in regional distribution. AmpC β-lactamases hydrolyze third generation cephalosporins, but are resistant to inhibition by clavulanate or other β-lactamase inhibitors in vitro. Fecal carriage and rates of colonization by bacteria harboring these resistance mechanisms have been reported in patients with community-acquired infections and in healthy members of their households. Expression of these ESBLs compromises the efficacy of current antibacterial therapies, potentially increasing the seriousness of hospital- and community-acquired Escherichia coli (E. coli) infections. To investigate the occurrence of ESBL-producing E. coli in human fecal flora isolated from two pediatric populations residing in the Libyan cities Zleiten and Abou El Khoms. Isolates were further studied to characterize genes encoding β-lactam resistance, and establish genetic relationships. Methods Antibiotic resistance profiles of phenotypically characterized E. coli isolates recovered from the stools of 243 Libyan children during two surveillance periods in 2001 and 2007 were determined by the disk diffusion method. ESBL-screening was performed using the cephalosporin/clavulanate double synergy disc method, and the AmpC-phenotype was confirmed by the aminophenyl-boronic acid test. ESBL genes were molecularly characterized. Phylogenetic group and multilocus sequence typing (MLST) were determined for ESBL-producing isolates and PFGE was performed to compare banding profiles of some dominant strains. Results ESBLs were identified in 13.4% (18/134) of E. coli isolates, and nine isolates (6.7%) demonstrated AmpC activity; all 18 isolates contained a CTX-M gene. Three CTX-M gene families (CTX-M-1, n = 9; CTX-M-15, n = 8 and CTX-M-3, n = 1) were distributed in diverse E. coli backgrounds (phylogenetic group D, 39%; B2, 28%; B1, 22% and A, 11%). MLST analysis revealed 14 sequence type (ST) with six new sequence types. The gene encoding the CMY-2 enzyme was detected in five AmpC-positive E. coli. Conclusions These results identified heterogeneous clones of CTX-M-producing E. coli in the fecal isolates, indicating that the intestinal tract acts as a reservoir for ESBL-producing organisms, and a trafficker of antibiotic resistance genes. PMID:24934873

Strain-specific transmission in an outbreak of ESBL-producing Enterobacteriaceae in the hemato-oncology care unit: a cohort study.

PubMed

Uemura, Makiko; Imataki, Osamu; Uchida, Shumpei; Nakayama-Imaohji, Haruyuki; Ohue, Yukiko; Matsuka, Harumi; Mori, Hatsune; Dobashi, Hiroaki; Kuwahara, Tomomi; Kadowaki, Norimitsu

2017-01-05

Extended-spectrum β-lactamase (ESBL)-producing bacteria are resistant to several types of antibiotics excluding carbapenems. A transmissibility of ESBL-producing Enterobacteriaceae would be depending on each bacterial property, however, that has not been elucidated in clinical setting. In this study, we attempted to identify the source of an outbreak of ESBL-producing bacteria in a medical oncology and immunology care unit. An ESBL-producing Enterobacteriaceae (ESBL-E) outbreak observed between July 2012 and August 2012 in Kagawa University Hospital was surveyed using various molecular microbiology techniques. We used Pulsed-field gel electrophoresis (PFGE), PCR-based ESBL gene typing, and direct sequence of ESBL gene as molecular microbiology typing method to distinguish each strain. The typical prevalence of ESBL-E isolation in the unit was 7.0 per month (1.7 per week). The prevalence of ESBL-E isolation during the target research period was 20.0 per month (5.0 per week). In total, 19 isolates (11 K. pneumoniae and 8 E. coli) were obtained from clinical samples, including four control strains (two each of both bacteria), that were physically different from those obtained from other inpatient units in our hospital. Pulsed-field gel electrophoresis (PFGE) for K. pneumoniae (digested by XbaI) produced similar patterns excluding one control strain. PCR classification of the ESBL gene for K. pneumoniae revealed that all strains other than the control strain carried SHV and CTX-M-9. This result was reconfirmed by direct DNA sequencing. Although the outbreak of K. pneumoniae was considered to be "clonal," PFGE and PCR classification of the ESBL genes for E. coli uncovered at least six different "non-clonal" strains possessing individual ESBL gene patterns. According to the result of an antibiogram, the pattern of antimicrobial susceptibility was more variable for K. pneumoniae than for E. coli. Typing by PFGE and ESBL gene PCR analysis is practical for discriminating various organisms. In our cohort, two outbreaks were concomitantly spread with different transmission strategies, namely clonal and non-clonal, in the same unit. This might represent clinical evidence that transmissibility differs according to the type of strain. We speculated that patient-to-patient transmission of ESBL-E occurred according to the properties of each individual strain.

PFGE MAPPER and PFGE READER: two tools to aid in the analysis and data input of pulse field gel electrophoresis maps.

PubMed Central

Shifman, M. A.; Nadkarni, P.; Miller, P. L.

1992-01-01

Pulse field gel electrophoresis mapping is an important technique for characterizing large segments of DNA. We have developed two tools to aid in the construction of pulse field electrophoresis gel maps: PFGE READER which stores experimental conditions and calculates fragment sizes and PFGE MAPPER which constructs pulse field gel electrophoresis maps. PMID:1482898

Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

PubMed Central

Mora, Azucena; Blanco, Miguel; Blanco, Jesús E.; Alonso, M. Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A.; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

2004-01-01

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused. PMID:15364983

Vancomycin-resistant enterococci with vanA gene in treated municipal wastewater and their association with human hospital strains.

PubMed

Oravcova, Veronika; Mihalcin, Matus; Zakova, Jana; Pospisilova, Lucie; Masarikova, Martina; Literak, Ivan

2017-12-31

Vancomycin-resistant enterococci (VRE) are pathogens of increasing medical importance. In Brno, Czech Republic, we collected 37 samples from the effluent of a wastewater treatment plant (WWTP), 21 surface swabs from hospital settings, and 59 fecal samples from hospitalized patients and staff. Moreover, we collected 284 gull cloacal swabs from the colony situated 35km downstream the WWTP. Samples were cultured selectively. Enterococci were identified using MALDI-TOF MS, phenotypically tested for susceptibility to antibiotics, and by PCR for occurrence of resistance and virulence genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to examine genotypic diversity. VRE carrying the vanA gene were found in 32 (86%, n=37) wastewater samples, from which we obtained 49 isolates: Enterococcus faecium (44) and Enterococcus gallinarum (2), Enterococcus casseliflavus (2), and Enterococcus raffinosus (1). From 33 (69%) of 48 inpatient stool samples, we obtained 39 vanA-carrying VRE, which belonged to E. faecium (33 isolates), Enterococcus faecalis (4), and Enterococcus raffinosus (2). Nearly one-third of the samples from hospital surfaces contained VRE with the vanA gene. VRE were not detected among gulls. Sixty-seven (84%, n=80) E. faecium isolates carried virulence genes hyl and/or esp. Virulence of E. faecalis was encoded by gelE, asa1, and cylA genes. A majority of the E. faecium isolates belonged to the clinically important sequence types ST17 (WWTP: 10 isolates; hospital: 4 isolates), ST18 (9;8), and ST78 (5;0). The remaining isolates belonged to ST555 (2;0), ST262 (1;6), ST273 (3;0), ST275 (1;0), ST549 (2;0), ST19 (0;1), ST323 (3;0), and ST884 (7;17). Clinically important enterococci carrying the vanA gene were almost continually detectable in the effluent of the WWTP, indicating insufficient removal of VRE during wastewater treatment and permanent shedding of these antibiotic resistant pathogens into the environment from this source. This represents a risk of their transmission to the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

High Prevalence of Escherichia coli-Producing CTX-M-15 Extended-Spectrum Beta-Lactamases in Poultry and Human Clinical Isolates in Romania.

PubMed

Maciuca, Iuliana E; Williams, Nicola J; Tuchilus, Cristina; Dorneanu, Olivia; Guguianu, Eleonora; Carp-Carare, Catalin; Rimbu, Cristina; Timofte, Dorina

2015-12-01

Use of antibiotics in food animals may contribute to development and spread of resistant organisms, particularly so in some countries. The aim of this study was two-fold; first, to establish the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chicken production in a region within Romania. Second, to study the relatedness of ESBL-producing E. coli isolates recovered from broilers, abattoir workers where the chickens were slaughtered and from the human clinical specimens from two regional hospitals. The results indicated a very high (69%) rate of carriage of ESBL and AmpC-producing E. coli in chickens with 36% CTX-M producers. Sequencing showed that chickens in Romania have the highest worldwide prevalence (53%) of blaCTX-M-15 reported in poultry E. coli isolates. The majority (53%) of the extended-spectrum cephalosporin-resistant E. coli carried plasmid-mediated blaampC genes, mostly blaCMY-2 type, one of the highest prevalences reported in Europe. The predominant CTX-M type found in the human clinical E. coli isolates was blaCTX-M-15 and most isolates coharbored blaOXA-1, blaTEM, and aac(6')-ib-cr. The majority (60%) of the human clinical isolates belonged to the pandemic virulent clone B2-ST131. The clonal relationship between broiler and the human CTX-M-producing E. coli isolates was assessed by macrorestriction pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which indicated strain diversity with no common STs found between human and poultry isolates. Moreover, IncI1 was the most prevalent replicon found in broiler ESBL-producing E. coli isolates and also in transconjugants, indicating that plasmids and not clonal spread may play a role in the transfer of blaCTX-M genes. This study identifies a high prevalence of ESBL-producing E. coli from broiler chickens in Romania with a high occurrence incidence of blaCTX-M-15, which reflects the main ESBL type found in human E. coli infections in this country.

Emergence of a Staphylococcus aureus Clone Resistant to Mupirocin and Fusidic Acid Carrying Exotoxin Genes and Causing Mainly Skin Infections

PubMed Central

Spiliopoulou, Iris; Spyridis, Nikolaos; Giormezis, Nikolaos; Kopsidas, John; Militsopoulou, Maria; Lebessi, Evangelia; Tsolia, Maria

2017-01-01

ABSTRACT Skin and soft tissue infections (SSTIs) caused by mupirocin-resistant Staphylococcus aureus strains have recently increased in number in our settings. We sought to evaluate the characteristics of these cases over a 43-month period. Data for all community-acquired staphylococcal infections caused by mupirocin-resistant strains were retrospectively reviewed. Genes encoding products producing high-level resistance (HLR) to mupirocin (mupA), fusidic acid resistance (fusB), resistance to macrolides and lincosamides (ermC and ermA), Panton-Valentine leukocidin (PVL) (lukS/lukF-PV), exfoliative toxins (eta and etb), and fibronectin binding protein A (fnbA) were investigated by PCRs in 102 selected preserved strains. Genotyping was performed by SCCmec and agr typing, whereas clonality was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 437 cases among 2,137 staphylococcal infections were recorded in 2013 to 2016; they were all SSTIs with the exception of 1 case of primary bacteremia. Impetigo was the predominant clinical entity (371 cases [84.9%]), followed by staphylococcal scalded skin syndrome (21 cases [4.8%]), and there were no abscesses. The number of infections detected annually increased during the study years. All except 3 isolates were methicillin susceptible. The rates of HLR to mupirocin and constitutive resistance to clindamycin were 99% and 20.1%, respectively. Among the 102 tested strains, 100 (98%) were mupA positive and 97 (95%) were fusB positive, 26/27 clindamycin-resistant strains (96.3%) were ermA positive, 83 strains (81.4%) were lukS/lukF positive, 95 (93%) carried both eta and etb genes, and 99 (97%) were fnbA positive. Genotyping of methicillin-sensitive S. aureus (MSSA) strains revealed that 96/99 (96.7%) belonged to one main pulsotype, pulsotype 1, classified as sequence type 121 (ST121). The emergence of a single MSSA clone (ST121) causing impetigo was documented. Resistance to topical antimicrobials and a rich toxinogenic profile confer to this clone adaptability for spread in the community. PMID:28592549

Acinetobacter baumannii: Epidemiological and Beta-Lactamase Data From Two Tertiary Academic Hospitals in Tshwane, South Africa

PubMed Central

Lowe, Michelle; Ehlers, Marthie M.; Ismail, Farzana; Peirano, Gisele; Becker, Piet J.; Pitout, Johann D. D.; Kock, Marleen M.

2018-01-01

Acinetobacter baumannii is an opportunistic pathogen that is increasingly responsible for hospital-acquired infections. The increasing prevalence of carbapenem resistant A. baumannii has left clinicians with limited treatment options. Last line antimicrobials (i.e., polymyxins and glycylcyclines) are often used as treatment options. The aim of this study was to determine the prevalence of selected β-lactamase genes from A. baumannii isolates obtained from patients with hospital-acquired infections and to determine the genetic relationship and epidemiological profiles among clinical A. baumannii isolates collected from two tertiary academic hospitals in the Tshwane region, South Africa (SA). Multiplex-PCR (M-PCR) assays were performed to detect selected resistance genes. The collected isolates’ genetic relatedness was determined by using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The acquired oxacillinase (OXA) genes, notably blaOXA-23-like were prevalent in the A. baumannii isolates. The M-PCR assays showed that the isolates collected from hospital A contained the OXA-23-like (96%; n = 69/72) genes and the isolates collected from hospital B contained the OXA-23-like (91%; n = 63/69) and OXA-58-like (4%; n = 3/69) genes. Colistin resistance was found in 1% of the isolates (n = 2/141) and tigecycline intermediate resistance was found in 6% of the isolates (n = 8/141). The A. baumannii isolates were genetically diverse. Molecular epidemiological data showed that specific sequence types (STs) (ST106, ST229, ST258 and ST208) were established in both hospitals, while ST848 was established in hospital A and ST502, ST339 and the novel ST1552 were established in hospital B. ST848 (established in hospital A) was predominately detected in ICU wards whereas ST208, ST339 and the novel ST1552 (established in hospital B) were detected in ICUs and the general wards. The origin of the A. baumannii isolates in the hospitals may be due to the dissemination and adaptation of a diverse group of successful clones. Poor infection control and prevention strategies and possibly the overuse of antimicrobials contributed to the establishment of these A. baumannii clones in the studied hospitals. PMID:29946315

Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing.

PubMed Central

Barrett, T J; Lior, H; Green, J H; Khakhria, R; Wells, J G; Bell, B P; Greene, K D; Lewis, J; Griffin, P M

1994-01-01

Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates. Images PMID:7883892

Cost-Effectiveness and Efficacy of spa, SCCmec, and PVL Genotyping of Methicillin-Resistant Staphylococcus aureus as Compared to Pulsed-Field Gel Electrophoresis

PubMed Central

Li, Vincent; Chui, Linda; Louie, Lisa; Simor, Andrew; Golding, George R.; Louie, Marie

2013-01-01

Pulsed-field gel electrophoresis (PFGE) is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA) surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab) for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa), Staphylococcal cassette chromosome (SCCmec), and Panton-Valentine leukocidin (PVL) typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type. PMID:24244440

[Molecular typing characterization of food-borne methicillin-resistant Staphylococcus aureus in China].

PubMed

Bai, Y; Wang, W; Yan, L; Yang, S R; Yan, S F; Dong, Y P; Zhao, B C; Zhao, Y Y; Xu, J; Hu, Y J; Li, F Q

2018-04-06

Objective: To analyses the antimicrobial resistance and molecular characterization of 21 MRSA isolates cultured from retail foods from different provinces in China, and evaluate the molecular typing methods. Methods: Twenty-one MRSA isolates were obtained from national foodborne pathogen surveillance network in 2012 (Chinese salad, n= 3; milk, n= 1; cake, n= 2; rice, n= 1; cold noodle, n= 1; spiced beef, n= 1; dumpling, n= 1; packed meal, n= 1; salad, n= 1; raw pork, n= 9). The antimicrobial resistance of 21 strains to 12 antimicrobial agents was tested by broth dilution method. Polymerase chain reaction (PCR) and DNA sequencing were performed to obtain the genetic types of MLST (ST) and spa typing. The clonal complex (CC) was assigned by eBURST soft and the MLVA type (MT) and MLVA complex (MC) were identified via the database of the MLVA website (http://www.mlva.net). Sma I pulsed-field gel electrophoresis ( Sma Ⅰ-PFGE) was also carried out to obtain the PFGE patterns of 21 strains. The genetic diversity and discriminatory power of typing were calculated by the Simpson's index of diversity (diversity index, DI) to find out the best genotyping method for MRSA. Results: All MRSA isolates showed multi-drug resistance(MDR), and were resistant to oxacillin, benzylpenicillin, clindamycin and erythromycin, and 71.4% (15/21), 47.6% (10/21), 42.9% (9/21) and 28.6% (6/21) of the MRSA isolates were resistant to tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and gentamicin, respectively. Moreover, one strain was found to be resistant to all three antimicrobials of levofloxacin, moxifloxacin and rifampicin. Great diversity was found in these food-associated MRSA (6 STs, 7 spa types, and 9 MTs). PFGE patterns were more diverse than those of other three molecular typing methods (19 pulse types). The index of diversity (DI) of PFGE, MLVA, spa typing and MLST was 0.99, 0.80, 0.73, and 0.61, respectively. Among the MRSA isolates, CC9-ST9-t899-MT929-MC2236 (PFGE Cluster Ⅴ) was the most prevalent clone, which were all cultured from raw pork (9 isolates). Besides, two MRSA were identified as CC59-ST338-t437-MT621-MC621 (PFGE Cluster Ⅳ). Different clone had their own resistance spectrum profiles. Conclusion: The food-borne MRSA isolates were all MDR in this study. Different clones had their own resistance spectrum profiles. MLVA represented a promising tool for molecular epidemiology tracing of MRSA in foodborne disease events.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Characterization of carbapenem nonsusceptible Pseudomonas aeruginosa in Denmark: a nationwide, prospective study.

PubMed

Hansen, Frank; Johansen, Helle Krogh; Østergaard, Claus; Arpi, Magnus; Hansen, Dennis Schrøder; Littauer, Pia; Holm, Anette; Heltberg, Ole; Schumacher, Helga; Fuursted, Kurt; Lykke, Mari-Ann Domar; Tønning, Birgitte; Hammerum, Anette M; Justesen, Ulrik Stenz

2014-02-01

From January 1st 2011 through June 30th 2011, 116 nonreplicate, noncystic fibrosis-related Pseudomonas aeruginosa isolates with reduced carbapenem susceptibility were collected from 12 out of 13 Danish departments of clinical microbiology. The presence of acquired β-lactamases was assessed with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi locus sequence typing. Eight isolates produced the metallo-β-lactamase (MBL) VIM-2, and one isolate produced OXA-10 and VEB-1-like extended-spectrum beta-lactamase (ESBL). Phenotypic indications of derepressed AmpC and efflux pump were seen in 56 and 43 isolates, respectively. Overall, the results indicate that mutational factors related to permeability--often combined with derepressed, chromosomal AmpC--is the main factor behind carbapenem nonsusceptibility in Danish P. aeruginosa isolates. The ESBL producer and all the VIM producers belonged to international clones. PFGE revealed that most of the isolates were unrelated, but clonal spread was seen; the 116 isolates distributed in 97 PFGE types, with the largest cluster consisting of 4 isolates (including three isolates from the same hospital with 100% similarity). Thirty-two isolates were pair-wise related, while the remaining isolates were clonally unrelated, as were all nine ESBL/MBL producers.

Entomopathogen ID: A multi-locus sequence alignment resource for entomopathogenic fungi

USDA-ARS?s Scientific Manuscript database

The ability to correctly identify entomopathogenic fungi is an important step in developing biopesticides and effectively communicating research results. Over the years, identifying entomopathogenic fungi has evolved from a system based on diagnostic morphological and physiological characters to mol...

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii.

PubMed

Tanigawa, Kana; Watanabe, Koichi

2011-03-01

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

Multilocus Sequence Typing Analysis of Staphylococcus lugdunensis Implies a Clonal Population Structure

PubMed Central

Chassain, Benoît; Lemée, Ludovic; Didi, Jennifer; Thiberge, Jean-Michel; Brisse, Sylvain; Pons, Jean-Louis

2012-01-01

Staphylococcus lugdunensis is recognized as one of the major pathogenic species within the genus Staphylococcus, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87 S. lugdunensis isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL). The number of alleles ranged from four (gmk and ldh) to nine (yqiL). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within S. lugdunensis. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for S. lugdunensis. This first MLST scheme for S. lugdunensis provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative Staphylococcus. PMID:22785196

Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

PubMed

Macheras, Edouard; Konjek, Julie; Roux, Anne-Laure; Thiberge, Jean-Michel; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby E; Bodmer, Thomas; Jarlier, Vincent; Cambau, Emmanuelle; Brisse, Sylvain; Caro, Valérie; Rastogi, Nalin; Gaillard, Jean-Louis; Heym, Beate

2014-01-01

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

PubMed

Dan, Tong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Menghe, Bilige; Zhang, Heping; Sun, Zhihong

2015-05-20

Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.

First isolation of Actinobacillus genomospecies 2 in Japan.

PubMed

Murakami, Miyuki; Shimonishi, Yoshimasa; Hobo, Seiji; Niwa, Hidekazu; Ito, Hiroya

2016-05-03

We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization.

Clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

PubMed Central

Momeni, Stephanie S.; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A.; Childers, Noel K.

2015-01-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African American children was examined using MLST. Serotype and presence of collagen-binding proteins (CBP) cnm/cbm were also assessed. One hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using START2 and MEGA. Thirty-four sequence types (ST) were identified of which 27 were unique to this population. Seventy-five percent of the isolates clustered into 16 clonal groups. Serotypes observed were c (n=84), e (n=3), and k (n=11). The prevalence of S. mutans isolates serotype k was notably high at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized populations studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study is higher than reported in most populations and is the first report of S. mutans serotype k in a US population. PMID:26443288

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b].

PubMed

Platonov, A E; Mironov, K O; Iatsyshina, S B; Koroleva, I S; Platonova, O V; Gushchin, A E; Shipulin, G A

2003-01-01

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

PubMed

El-Gazzar, Mohamed; Ghanem, Mostafa; McDonald, Kristina; Ferguson-Noel, Naola; Raviv, Ziv; Slemons, Richard D

2017-03-01

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

STBase: one million species trees for comparative biology.

PubMed

McMahon, Michelle M; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J

2015-01-01

Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user's query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees.

Imbalanced presence of Borrelia burgdorferi s.l. multilocus sequence types in clinical manifestations of Lyme borreliosis.

PubMed

Coipan, E Claudia; Jahfari, Setareh; Fonville, Manoj; Oei, G Anneke; Spanjaard, Lodewijk; Takumi, Katsuhisa; Hovius, Joppe W R; Sprong, Hein

2016-08-01

In this study we used typing based on the eight multilocus sequence typing scheme housekeeping genes (MLST) and 5S-23S rDNA intergenic spacer (IGS) to explore the population structure of Borrelia burgdorferi sensu lato isolates from patients with Lyme borreliosis (LB) and to test the association between the B. burgdorferi s.l. sequence types (ST) and the clinical manifestations they cause in humans. Isolates of B. burgdorferi from 183 LB cases across Europe, with distinct clinical manifestations, and 257 Ixodes ricinus lysates from The Netherlands, were analyzed for this study alone. For completeness, we incorporated in our analysis also 335 European B. burgdorferi s.l. MLST profiles retrieved from literature. Borrelia afzelii and Borrelia bavariensis were associated with human cases of LB while Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana were associated with questing I. ricinus ticks. B. afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. The samples in our study belonged to 251 different STs, of which 94 are newly described, adding to the overall picture of the genetic diversity of Borrelia genospecies. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and Borrelia spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). We found six multilocus sequence types that were significantly associated to clinical manifestations in humans and five IGS haplotypes that were associated with the human LB cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

PubMed

Blanchard, Adam M; Jolley, Keith A; Maiden, Martin C J; Coffey, Tracey J; Maboni, Grazieli; Staley, Ceri E; Bollard, Nicola J; Warry, Andrew; Emes, Richard D; Davies, Peers L; Tötemeyer, Sabine

2018-01-01

Dichelobacter nodosus ( D. nodosus ) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

PubMed Central

Pringle, Märit; Bergsten, Christer; Fernström, Lise-Lotte; Höök, Helena; Johansson, Karl-Erik

2008-01-01

Background Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Methods Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Results Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. Conclusion This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates. PMID:18937826

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

PubMed

Denagamage, Thomas N; Patterson, Paul; Wallner-Pendleton, Eva; Trampel, Darrell; Shariat, Nikki; Dudley, Edward G; Jayarao, Bhushan M; Kariyawasam, Subhashinie

2016-11-01

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

Spatio-temporal scan statistics for the detection of outbreaks involving common molecular subtypes: using human cases of Escherichia coli O157:H7 provincial PFGE pattern 8 (National Designation ECXAI.0001) in Alberta as an example.

PubMed

So, H C; Pearl, D L; von Königslöw, T; Louie, M; Chui, L; Svenson, L W

2013-08-01

Molecular typing methods have become a common part of the surveillance of foodborne pathogens. In particular, pulsed-field gel electrophoresis (PFGE) has been used successfully to identify outbreaks of Escherichia coli O157:H7 in humans from a variety of food and environmental sources. However, some PFGE patterns appear commonly in surveillance systems, making it more difficult to distinguish between outbreak and sporadic cases based on molecular data alone. In addition, it is unknown whether these common patterns might have unique epidemiological characteristics reflected in their spatial and temporal distributions. Using E. coli O157:H7 surveillance data from Alberta, collected from 2000 to 2002, we investigated whether E. coli O157:H7 with provincial PFGE pattern 8 (national designation ECXAI.0001) clustered in space, time and space-time relative to other PFGE patterns using the spatial scan statistic. Based on our purely spatial and temporal scans using a Bernoulli model, there did not appear to be strong evidence that isolates of E. coli O157:H7 with provincial PFGE pattern 8 are distributed differently from other PFGE patterns. However, we did identify space-time clusters of isolates with PFGE pattern 8, using a Bernoulli model and a space-time permutation model, which included known outbreaks and potentially unrecognized outbreaks or additional outbreak cases. There were differences between the two models in the space-time clusters identified, which suggests that the use of both models could increase the sensitivity of a quantitative surveillance system for identifying outbreaks involving isolates sharing a common PFGE pattern. © 2012 Blackwell Verlag GmbH.

Phenotypic and Molecular Characterization of Antimicrobial Resistance in Klebsiella spp. Isolates from Companion Animals in Japan: Clonal Dissemination of Multidrug-Resistant Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae

PubMed Central

Harada, Kazuki; Shimizu, Takae; Mukai, Yujiro; Kuwajima, Ken; Sato, Tomomi; Usui, Masaru; Tamura, Yutaka; Kimura, Yui; Miyamoto, Tadashi; Tsuyuki, Yuzo; Ohki, Asami; Kataoka, Yasushi

2016-01-01

The emergence of antimicrobial resistance in Klebsiella spp., including resistance to extended-spectrum cephalosporins (ESC) and fluoroquinolones, is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance in a total of 103 Klebsiella spp. isolates, consisting of Klebsiella pneumoniae complex (KP, n = 89) and K. oxytoca (KO, n = 14) from clinical specimens of dogs and cats in Japan. Furthermore, we characterized the resistance mechanisms, including extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and assessed genetic relatedness of ESC-resistant Klebsiella spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated that resistance rates to ampicillin, cephalothin, enrofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, cefotaxime, gentamicin, tetracycline, chloramphenicol, amoxicillin-clavulanic acid, and cefmetazole were 98.1, 37.9, 37.9, 35.9, 35.0, 34.0, 31.1, 30.1, 28.2, 14.6, and 6.8%, respectively. Phenotypic testing detected ESBLs and/or AmpC β-lactamases in 31 of 89 (34.8%) KP isolates, but not in KO isolates. Resistances to 5 of the 12 antimicrobials tested, as well as the three PMQRs [qnrB, qnrS, and aac(6′)-Ib-cr], were detected significantly more frequently in ESBL-producing KP, than in non-ESBL-producing KP and KO. The most frequent ESBL was CTX-M-15 (n = 13), followed by CTX-M-14 (n = 7), CTX-M-55 (n = 6), SHV-2 (n = 5), CTX-M-2 (n = 2), and CTX-M-3 (n = 2). Based on the rpoB phylogeny, all ESBL-producing strains were identified as K. pneumoniae, except for one CTX-M-14-producing strain, which was identified as K. quasipneumoniae. All of AmpC β-lactamase positive isolates (n = 6) harbored DHA-1, one of the PABLs. Based on MLST and PFGE analysis, ST15 KP clones producing CTX-M-2, CTX-M-15, CTX-M-55, and/or SHV-2, as well as KP clones of ST1844-CTX-M-55, ST655-CTX-M-14, and ST307-CTX-M-15, were detected in one or several hospitals. Surprisingly, specific clones were detected in different patients at an interval of many months. These results suggest that multidrug-resistant ESBL-producing KP were clonally disseminated among companion animals via not only direct but also indirect transmission. This is the first report on large-scale monitoring of antimicrobial-resistant Klebsiella spp. isolates from companion animals in Japan. PMID:27446056

Development and application of SINE multilocus and quantitative genetic markers to study oilseed rape (Brassica napus L.) crops.

PubMed

Allnutt, T R; Roper, K; Henry, C

2008-01-23

A genetic marker system based on the S1 Short Interspersed Elements (SINEs) in the important commercial crop, oilseed rape ( Brassica napus L.) has been developed. SINEs provided a successful multilocus, dominant marker system that was capable of clearly delineating winter- and spring-type crop varieties. Sixteen of 20 varieties tested showed unique profiles from the 17 polymorphic SINE markers generated. The 3' or 5' flank region of nine SINE markers were cloned, and DNA was sequenced. In addition, one putative pre-transposition SINE allele was cloned and sequenced. Two SINE flanking sequences were used to design real-time PCR assays. These quantitative SINE assays were applied to study the genetic structure of eight fields of oilseed rape crops. Studied fields were more genetically diverse than expected for the chosen loci (mean H T = 0.23). The spatial distribution of SINE marker frequencies was highly structured in some fields, suggesting locations of volunteer impurities within the crop. In one case, the assay identified a mislabeling of the crop variety. SINE markers were a useful tool for crop genetics, phylogenetics, variety identification, and purity analysis. The use and further application of quantitative, real-time PCR markers are discussed.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

PubMed

Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

2016-10-01

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

Genotypic Characterization of Enterococci Collected from a Coastal Area Using PFGE

NASA Astrophysics Data System (ADS)

Furukawa, Takashi; Yoshida, Terutoyo; Suzuki, Yoshihiro

Development of microbial source tracking (MST) is crucial to ensure public health and bacteriological safety in coastal areas. In this study, pulsed-field gel electrophoresis (PFGE) , utilized for pollution source tracking of hospital infections, was applied to the aquatic environment as a MST method. Enterococcus faecium and Entecococcus faecalis, fecal indicator bacterias, were isolated and identified from a port area, and the genotype was analyzed by PFGE. A total of 230 enterococcal strains were isolated by PFGE, and forty strains were identified as E. faecium using both PCR and the Api 20 Strep test. Dendrogram analysis of the PFGE types revealed that all the 40 E. faecium strains can be classified separately into 7 different groups at a 0.7 similarity level. The genotypic characterization of E. faecium from the coastal port area revealed diverse types. It is suggested that PFGE can be used to differentiate and characterize fecal pollution indicating bacteria by applying it to the aquatic environment, as well as provide detailed information to specify the pollution source.

Genotyping of Indian antigenic, vaccine, and field Brucella spp. using multilocus sequence typing.

PubMed

Shome, Rajeswari; Krithiga, Natesan; Shankaranarayana, Padmashree B; Jegadesan, Sankarasubramanian; Udayakumar S, Vishnu; Shome, Bibek Ranjan; Saikia, Girin Kumar; Sharma, Narendra Kumar; Chauhan, Harshad; Chandel, Bharat Singh; Jeyaprakash, Rajendhran; Rahman, Habibur

2016-03-31

Brucellosis is one of the most important zoonotic diseases that affects multiple livestock species and causes great economic losses. The highly conserved genomes of Brucella, with > 90% homology among species, makes it important to study the genetic diversity circulating in the country. A total of 26 Brucella spp. (4 reference strains and 22 field isolates) and 1 B. melitensis draft genome sequence from India (B. melitensis Bm IND1) were included for sequence typing. The field isolates were identified by biochemical tests and confirmed by both conventional and quantitative polymerase chain reaction (qPCR) targeting bcsp 31Brucella genus-specific marker. Brucella speciation and biotyping was done by Bruce ladder, probe qPCR, and AMOS PCRs, respectively, and genotyping was done by multilocus sequence typing (MLST). The MLST typing of 27 Brucella spp. revealed five distinct sequence types (STs); the B. abortus S99 reference strain and 21 B. abortus field isolates belonged to ST1. On the other hand, the vaccine strain B. abortus S19 was genotyped as ST5. Similarly, B. melitensis 16M reference strain and one B. melitensis field isolate were grouped into ST7. Another B. melitensis field isolate belonged to ST8 (draft genome sequence from India), and only B. suis 1330 reference strain was found to be ST14. The sequences revealed genetic similarity of the Indian strains to the global reference and field strains. The study highlights the usefulness of MLST for typing of field isolates and validation of reference strains used for diagnosis and vaccination against brucellosis.

Diversity of plasmids and transmission of high-level mupirocin mupA resistance gene in Staphylococcus haemolyticus.

PubMed

do Carmo Ferreira, Natália; Schuenck, Ricardo P; dos Santos, Kátia Regina Netto; de Freire Bastos, Maria do Carmo; Giambiagi-deMarval, Marcia

2011-03-01

The coagulase-negative staphylococci are known for their ability to acquire resistance genes, which limits the choice of therapeutic options for the treatment of infections caused by these microorganisms. In this study, the diversity of high-level mupirocin resistance plasmids (Mup(R) ) was investigated in four strains of Staphylococcus haemolyticus belonging to different pulsed-field gel electrophoresis (PFGE) types or subtypes, isolated in a Brazilian hospital. These strains harbor the mupA gene in large plasmids. In addition, the presence of IS257 sequences flanking the mupA gene was also shown. Two isolates belonging to two different PFGE types exhibited a similar polymorphism for a fragment of the mupA gene and the closest proximal flanking copies of the IS257, suggesting horizontal transmission of S. haemolyticus mupirocin resistance plasmids in the environment and a role of this species as a reservoir of the mupA gene. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates in Japan.

PubMed

Harada, Sohei; Ishii, Yoshikazu; Saga, Tomoo; Aoki, Kotaro; Tateda, Kazuhiro

2018-03-20

Although severe infections caused by hypervirulent Klebsiella pneumoniae isolates, such as K1 isolates belonging to sequence type (ST) 23, have been a significant problem in Asian countries, epidemiology of these isolates in Japan remains unclear. We performed a nationwide molecular epidemiological study of K. pneumoniae K1 and K2 isolates in Japan. Of the 259K. pneumoniae isolates collected, 14 and 16 isolates were identified as capsular genotypes K1 and K2, respectively. All K1 isolates were ST23 or its closely related clones and showed high genetic similarity by pulsed-field gel electrophoresis (PFGE) and the DiversiLab system (DL). K2 isolates, belonging to ST14, ST25, ST65, ST86, and ST110, were more genetically diverse than K1 isolates. Isolates belonging to a specific ST showed identical virulence gene profiles with a few exceptions. PFGE and DL results using K1 and K2 isolates were generally in agreement. Copyright © 2018. Published by Elsevier Inc.

Rapid Detection & Identification of Bacillus Species using MALDI-TOF/TOF and Biomarker Database

DTIC Science & Technology

2006-06-01

rRNA sequence analysis. Multilocus enzyme electrophoresis ( MEE ) and comparative DNA sequence analysis suggest that they may represent a single species...adaptation of the MEE method [63] but with greater discrimination [64]. All of these new PCR-based subtyping methods are certainly superior and more...Demirev, P.A., Lin, J.S., Pineda , F.J., and Fenselau, C. (2001). Bioinformatics and mass spectrometry for microorganism identification: proteome-wide

First isolation of Actinobacillus genomospecies 2 in Japan

PubMed Central

MURAKAMI, Miyuki; SHIMONISHI, Yoshimasa; HOBO, Seiji; NIWA, Hidekazu; ITO, Hiroya

2015-01-01

We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization. PMID:26668165

Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan.

PubMed

Chiou, Chien-Shun; Wang, You-Wun; Chen, Pei-Ling; Wang, Wan-Ling; Wu, Ping-Fuai; Wei, Hsiao-Lun

2009-06-01

The number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE) method. National weekly report data were collected for investigating epidemiological trends. A total of 23 emm types were identified in 1,218 S. pyogenes isolates. The five most prevalent emm types were emm12 (50.4%), emm4 (23.2%), emm1 (16.4%), emm6 (3.8%) and emm22 (3.0%). PFGE analysis with SmaI suggested that, with a few exceptions, strains with a common emm type belonged to the same clone. There were two large emm12 clones, one with DNA resistant to cleavage by SmaI. Each prevalent emm clone had major PFGE strain(s) and many minor strains. Most of the minor strains emerged in the population and disappeared soon after. Even some major strains remained prevalent for only 2-3 years before declining. The large fluctuation of scarlet fever cases between 2000 and 2006 was associated with the shuffling of six prevalent emm clones. In 2003, the dramatic drop in scarlet fever cases in central Taiwan and throughout the whole country was associated with the occurrence of a severe acute respiratory syndrome (SARS) outbreak that occurred between late-February and mid-June in Taiwan. The occurrences of scarlet fever in central Taiwan in 2000-2006 were primarily caused by five emm types, which accounted for 96.8% of the isolates collected. Most of the S. pyogenes strains (as defined by PFGE genotypes) emerged and lasted for only a few years. The fluctuation in the number of scarlet fever cases during the seven years can be primarily attributed to the shuffling of six prevalent emm clones and to the SARS outbreak in 2003.

Mel-36 – preliminary description of a new morel species

USDA-ARS?s Scientific Manuscript database

A pilot survey of true morels (Morchella) of Newfoundland and Labrador (NL), employing phylogenetic analyses of multilocus DNA sequence data, resulted in the discovery of a novel species that is currently only known from NL and New Brunswick. This unnamed species was informally designated Morchella ...

Resolving the Mortierellaceae phylogeny through Multi-Locus Sequence Typing (MLST) and phylogenomics

USDA-ARS?s Scientific Manuscript database

The Mortierellaceae (Mortierellomycotina) are a diverse family of fungi that are of evolutionary and ecological relevance. They are the closest lineage to the arbuscular mycorrhizae (Glomeromycotina) and include some of the first species to evolve fruiting body production. The Mortierellaceae are es...

Trichoderma asperellum reconsidered: two cryptic species

USDA-ARS?s Scientific Manuscript database

Analysis of a world-wide collection of strains of Trichoderma asperellum using multilocus genealogies of four genomic regions (tef1, rbp2, act, ITS1, 2, 5.8s), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF MS) of ...

Multi-locus analysis of Giardia duodenalis from nonhuman primates kept in zoos in China: geographical segregation and host-adaptation of assemblage B isolates.

PubMed

Karim, Md Robiul; Wang, Rongjun; Yu, Fuchang; Li, Tongyi; Dong, Haiju; Li, Dezhong; Zhang, Longxian; Li, Junqiang; Jian, Fuchun; Zhang, Sumei; Rume, Farzana Islam; Ning, Changshen; Xiao, Lihua

2015-03-01

Only a few studies based on single locus characterization have been conducted on the molecular epidemiology of Giardia duodenalis in nonhuman primates (NHPs). The present study was conducted to examine the occurrence and genotype identity of G. duodenalis in NHPs based on multi-locus analysis of the small-subunit ribosomal RNA (SSU rRNA), triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh), and beta-giardin (bg) genes. Fecal specimens were collected from 496 animals of 36 NHP species kept in seven zoos in China and screened for G. duodenalis by tpi-based PCR. G. duodenalis was detected in 92 (18.6%) specimens from 18 NHP species, belonging to assemblage A (n=4) and B (n=88). In positive NHP species, the infection rates ranged from 4.8% to 100%. In tpi sequence analysis, the assemblage A included subtypes A1, A2 and one novel subtype. Multi-locus analysis of the tpi, gdh, and bg genes detected 11 (8 known and 3 new), 6 (3 known and 3 new) and 9 (2 known and 7 new) subtypes in 88, 47 and 35 isolates in assemblage B, respectively. Thirty-two assemblage B isolates with data at all three loci yielded 15 multi-locus genotypes (MLGs), including 2 known and 13 new MLGs. Phylogenetic analysis of concatenated sequences of assemblage B showed that MLGs found here were genetically different from those of humans, NHPs, rabbit and guinea pig in Italy and Sweden. It further indicated that assemblage B isolates in ring-tailed lemurs and squirrel monkeys might be genetically different from those in other NHPs. These data suggest that NHPs are mainly infected with G. duodenalis assemblage B and there might be geographical segregation and host-adaptation in assemblage B in NHPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Prevalence of Extended-Spectrum β-Lactamases CTX-M-8 and CTX-M-2-Producing Salmonella Serotypes from Clinical and Nonhuman Isolates in Brazil.

PubMed

Fernandes, Sueli Aparecida; Camargo, Carlos Henrique; Francisco, Gabriela Rodrigues; Bueno, Maria Fernanda Campagnari; Garcia, Doroti Oliveira; Doi, Yohei; Casas, Monique Ribeiro Tiba

2017-07-01

We characterized extended-spectrum β-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. β-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. bla CTX-M-8 and bla CTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. bla SHV-5 and bla SHV-2 were also detected but in lower frequencies (4%, 2%). bla TEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective bla ESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing bla CTX-M-2 , bla CTX-M-8 , bla TEM-1 , and bla SHV-2 genes. Salmonella Muenchen (harboring bla CTX-M-2 ) and Salmonella Corvallis (bla CTX-M-8 and bla SHV-5 ) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing.

PubMed

Keys, C; Kemper, S; Keim, P

2005-01-01

Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.

Major outbreak of toxic shock-like syndrome caused by Streptococcus mitis.

PubMed

Lu, Hong-Zhou; Weng, Xin-Hua; Zhu, Bai; Li, Haijing; Yin, You-Kuan; Zhang, Yong-Xin; Haas, David W; Tang, Yi-Wei

2003-07-01

Severe illness caused by viridans streptococci rarely occurs in immunocompetent hosts. Between December 1990 and May 1991, thousands of patients in the YangZi River Delta area of Jiangsu Province, China, suffered from scarlet fever-like pharyngitis. Fewer cases occurred in subsequent years with the same seasonality. Approximately half of the cases developed complications characteristic of streptococcal toxic shock-like syndrome (TSLS). Throat cultures yielded predominant growth of alpha-hemolytic streptococci. All cases admitted to Haian People's Hospital were investigated. Clinical specimens were collected, medical records were reviewed, and bacterial isolates were identified phenotypically and analyzed by 16S rRNA gene sequencing and pulsed-field gel electrophoresis (PFGE). Proteins were purified from culture supernatants by extraction, ammonium sulfate precipitation, and fast-protein liquid chromatography. Biological activities of protein components were determined by subcutaneous inoculation into rabbits. A total of 178 cases of non-beta-hemolytic streptococcal scarlet fever-like pharyngitis were studied. In 88 (79.3%) of 111 patients, oropharyngeal swab cultures grew morphologically identical alpha-hemolytic streptococci. A protein in culture supernatants was pyrogenic in rabbits, was mitogenic for splenocytes, and enhanced rabbit susceptibility to endotoxin challenge. The N-terminal amino acid sequence of this 34-kDa protein showed no homology with known Streptococcus pyrogenic exotoxins. The organism was identified as Streptococcus mitis based on biochemical and 16S rRNA sequence analyses. Representative outbreak isolates from 1990 to 1995 displayed identical PFGE patterns. This TSLS outbreak in southeastern China was caused by a toxigenic clone of S. mitis. An apparently novel toxin may explain the unusual virulence of this organism.

Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France.

PubMed

Calvez, Ségolène; Fournel, Catherine; Douet, Diane-Gaëlle; Daniel, Patrick

2015-06-23

Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.

Genotypic and phenotypic characterization of Cryptococcus flavescens, beneficial biocontrol agents for controlling Fusarium head blight of wheat

USDA-ARS?s Scientific Manuscript database

Cryptococcus flavescens strain OH182.9_3C (3C) previously displayed significant biological control activity against Fusarium head blight, a globally important disease of wheat; however, the diversity within C. flavescens has not been previously characterized. Multilocus sequence typing was performed...

Molecular characterization of Xanthomonas strains responsible for bacterial leaf spot of tomato in Ethiopia

USDA-ARS?s Scientific Manuscript database

Bacterial spot of tomato (BST) is a major constraint to tomato production in Ethiopia and many other countries leading to significant crop losses. In the present study, using pathogenicity tests, sensitivity to copper and streptomycin, and multilocus sequence analysis, a diverse group of Xanthomonas...

Prevalence and molecular typing of Vibrio parahaemolyticus isolated from seafood in Shanghai using multilocus sequence typing (MLST)

USDA-ARS?s Scientific Manuscript database

Vibrio parahaemolyticus is a gram-negative bacterium that inhabits coastal and marine environments. Thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and the type III secretion system are considered the potential virulent factors of pathogenic V. parahaemolyticus. The frequency of str...

Phylogenetic distribution of phenotypic traits in bacillus thuringiensis analyzed by multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Strains from a collection of 3,639 diverse Bacillus thuringiensis isolates were classified based on phenotypic profiles resulting from six biochemical tests, including production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). St...

Variation in Campylobacter multilocus sequence subtypes from chickens as detected on three plating media

USDA-ARS?s Scientific Manuscript database

The objective of this study was to compare subtypes of Campylobacter jejuni and coli detected on three discreet selective Campylobacter plating media to determine if different media select for different subtypes. Fifty ceca and fifty carcasses (n=100, representing 50 flocks) were collected from the...

Multilocus sequence typing of Mycoplasma bovis reveals host-specific genotypes in cattle versus bison

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant health problem in bison, causing necro...

A multilocus sequence typing method and curated database for Mycoplasma bovis

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant problem in bison, causing necrotic pha...

Campylobacter multi-locus sequence typing subtypes detected on chicken livers available at retail.

USDA-ARS?s Scientific Manuscript database

Foodborne campylobacteriosis has been traced to undercooked chicken liver. It is not known what prevalence of Campylobacter to expect on fresh chicken livers available at retail. The objectives of this study were to measure prevalence of Campylobacter associated with chicken livers at retail and d...

Population sub-structuring among Trypanosoma evansi stocks.

PubMed

Njiru, Z K; Constantine, C C

2007-10-01

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (IA=0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed Fst=0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei's genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.

Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

PubMed

Santos-Sanches, Ilda; Chambel, Lélia; Tenreiro, Rogério

2015-01-01

Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

Genetic patterns of Streptococcus uberis isolated from bovine mastitis.

PubMed

Reinoso, Elina B; Lasagno, Mirta C; Odierno, Liliana M

2015-01-01

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

PubMed

Deshpande, Ashutosh; Inkster, Teresa; Hamilton, Kate; Litt, David; Fry, Norman; Kennedy, Iain T R; Shookhye-Dickson, Jacqueline; Hill, Robert L R

2015-01-01

On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

Epidemiological characterization of a nosocomial outbreak of extended spectrum β-lactamase Escherichia coli ST-131 confirms the clinical value of core genome multilocus sequence typing.

PubMed

Woksepp, Hanna; Ryberg, Anna; Berglind, Linda; Schön, Thomas; Söderman, Jan

2017-12-01

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST-131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST-131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

PubMed

Momeni, Stephanie S; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A; Childers, Noel K

2015-12-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population. © 2015 Eur J Oral Sci.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

PubMed Central

Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

High frequency of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in children under 1 year old with skin and soft tissue infections.

PubMed

Salazar-Ospina, Lorena; Jiménez, Judy Natalia

2017-09-21

Staphylococcus aureus is responsible for a large number of infections in pediatric population; however, information about the behavior of such infections in this population is limited. The aim of the study was to describe the clinical, epidemiological, and molecular characteristics of infections caused by methicillin-susceptible and resistant S. aureus (MSSA-MRSA) in a pediatric population. A cross-sectional descriptive study in patients from birth to 14 years of age from three high-complexity institutions was conducted (2008-2010). All patients infected with methicillin-resistant S. aureus and a representative sample of patients infected with methicillin-susceptible S. aureus were included. Clinical and epidemiological information was obtained from medical records and molecular characterization included spa typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). In addition, staphylococcal cassette chromosome mec (SCCmec) and virulence factor genes were detected. A total of 182 patients, 65 with methicillin-susceptible S. aureus infections and 117 with methicillin-resistant S. aureus infections, were included in the study; 41.4% of the patients being under 1 year. The most frequent infections were of the skin and soft tissues. Backgrounds such as having stayed in day care centers and previous use of antibiotics were more common in patients with methicillin-resistant S. aureus infections (p≤0.05). Sixteen clonal complexes were identified and methicillin-susceptible S. aureus strains were more diverse. The most common cassette was staphylococcal cassette chromosomemec IVc (70.8%), which was linked to Panton-Valentine leukocidin (pvl). In contrast with other locations, a prevalence of infections in children under 1 year of age in the city could be observed; this emphasizes the importance of epidemiological knowledge at the local level. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Effect of lactation therapy on Staphylococcus aureus transmission dynamics in two commercial dairy herds

PubMed Central

2013-01-01

Background Treatment of subclinical mastitis during lactation can have both direct (individual animal level) and indirect (population level) effects. With a few exceptions, prior research has focused on evaluating the direct effects of mastitis treatment, and to date no controlled field trials have been conducted to test whether beneficial indirect effects of lactation treatment strategies targeting subclinical mastitis can be demonstrated on commercial dairy farms. Furthermore, there is limited knowledge on the impact of such interventions on the population dynamics of specific bacterial strains. The purpose of this study was to test the hypothesis that lactation therapy targeting S. aureus subclinical intramammary infection reduces transmission of S. aureus strains within dairy herds. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to determine strain specific infection dynamics in treated and control groups in a split herd trial conducted on 2 commercial dairy farms. Results The direct effect of 8 days intramammary lactation therapy with pirlimycin hydrochloride was demonstrated by an increased proportion of cure and a reduction in duration of infection in quarters receiving treatment compared to untreated controls. The indirect effect of lactation therapy was demonstrated by reduction of new S. aureus intramammary infections (IMI) caused by the dominant strain type in both herds. Strain typing of representative isolates taken over the duration of all IMI, including pre- and post-treatment isolates, provided more precise estimates of new infection, cure, and re-infection rates. New S. aureus infections in recovered susceptible quarters and the emergence of a new strain type in one herd influenced incidence measures. Conclusion In addition to demonstrating positive direct effects of lactation therapy, this study provides evidence that treatment of subclinical S. aureus mastitis during lactation can have indirect effects including preventing new IMI and reducing incidence of clinical mastitis within dairy herds. Strain specific transmission parameter estimates for S. aureus MLST clonal complexes 5, 97 and 705 in 2 commercial dairy herds are also reported. PMID:23398676

Effect of lactation therapy on Staphylococcus aureus transmission dynamics in two commercial dairy herds.

PubMed

Barlow, John W; Zadoks, Ruth N; Schukken, Ynte H

2013-02-11

Treatment of subclinical mastitis during lactation can have both direct (individual animal level) and indirect (population level) effects. With a few exceptions, prior research has focused on evaluating the direct effects of mastitis treatment, and to date no controlled field trials have been conducted to test whether beneficial indirect effects of lactation treatment strategies targeting subclinical mastitis can be demonstrated on commercial dairy farms. Furthermore, there is limited knowledge on the impact of such interventions on the population dynamics of specific bacterial strains. The purpose of this study was to test the hypothesis that lactation therapy targeting S. aureus subclinical intramammary infection reduces transmission of S. aureus strains within dairy herds. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to determine strain specific infection dynamics in treated and control groups in a split herd trial conducted on 2 commercial dairy farms. The direct effect of 8 days intramammary lactation therapy with pirlimycin hydrochloride was demonstrated by an increased proportion of cure and a reduction in duration of infection in quarters receiving treatment compared to untreated controls. The indirect effect of lactation therapy was demonstrated by reduction of new S. aureus intramammary infections (IMI) caused by the dominant strain type in both herds. Strain typing of representative isolates taken over the duration of all IMI, including pre- and post-treatment isolates, provided more precise estimates of new infection, cure, and re-infection rates. New S. aureus infections in recovered susceptible quarters and the emergence of a new strain type in one herd influenced incidence measures. In addition to demonstrating positive direct effects of lactation therapy, this study provides evidence that treatment of subclinical S. aureus mastitis during lactation can have indirect effects including preventing new IMI and reducing incidence of clinical mastitis within dairy herds. Strain specific transmission parameter estimates for S. aureus MLST clonal complexes 5, 97 and 705 in 2 commercial dairy herds are also reported.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates.

PubMed

Galán-Sánchez, Fátima; Pérez-Eslava, Maria; Machuca, Jesús; Trujillo-Soto, Teresa; Arca-Suarez, Jorge; Rodríguez-Iglesias, Manuel

2018-06-20

The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Clinical and Microbiological Characteristics of Heteroresistant and Vancomycin-Intermediate Staphylococcus aureus from Bloodstream Infections in a Brazilian Teaching Hospital

PubMed Central

da Costa, Thaina Miranda; Morgado, Priscylla Guimarães Migueres; Cavalcante, Fernanda Sampaio; Damasco, Andreia Paredes; Nouér, Simone Aranha; dos Santos, Kátia Regina Netto

2016-01-01

This study analyzed clinical and microbiological characteristics of heteroresistant (hVISA) and vancomycin-intermediate Staphylococcus aureus (VISA) from bloodstream infections (BSI) in a Brazilian teaching hospital, between 2011 and 2013. Minimum inhibitory concentrations (MIC) of antimicrobials were determined by broth microdilution method and SCCmec was detected by PCR. Isolates with a vancomycin MIC ≥ 2mg/L were cultured on BHI agar with 3, 4 or 6 mg/L (BHIa3, BHIa4 or BHIa6) of vancomycin and BHIa4 with casein (BHIa4ca). Macromethod Etest® and Etest® Glicopeptides Resistance Detection were also used. VISA and hVISA isolates were confirmed by the population analysis profile then typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Medical data from the patients were obtained from their medical records. Among 110 consecutive isolates, 31 (28%) were MRSA and carried the SCCmec type II (15 isolates) or IV (16 isolates). Vancomycin MIC50 and MIC90 were 1 and 2 mg/L, respectively. MRSA isolates had increased non-susceptibility to daptomycin (p = 0.0003). Six (5%) isolates were VISA, four of which were MRSA, three SCCmec type II/USA100/ST5 and one type IV/USA800/ST3192. One MRSA SCCmec II isolate grew on agar BHIa3, BHIa4 and BHIa4ca, and it was confirmed as hVISA. Among the six VISA isolates, five (83%) grew on BHIa3 and three (50%) on BHI4ca. Four of the six VISA isolates and the one hVISA isolate were from patients who had undergone dialysis. Thus, a possible dissemination of the SCCmec II/USA100/ST5 lineage may have occurred in the hospital comprising the VISA, hVISA and daptomycin non-susceptible S. aureus Brazilian isolates from health care associated bloodstream infections. PMID:27575698

Clinical and Microbiological Characteristics of Heteroresistant and Vancomycin-Intermediate Staphylococcus aureus from Bloodstream Infections in a Brazilian Teaching Hospital.

PubMed

da Costa, Thaina Miranda; Morgado, Priscylla Guimarães Migueres; Cavalcante, Fernanda Sampaio; Damasco, Andreia Paredes; Nouér, Simone Aranha; Dos Santos, Kátia Regina Netto

2016-01-01

This study analyzed clinical and microbiological characteristics of heteroresistant (hVISA) and vancomycin-intermediate Staphylococcus aureus (VISA) from bloodstream infections (BSI) in a Brazilian teaching hospital, between 2011 and 2013. Minimum inhibitory concentrations (MIC) of antimicrobials were determined by broth microdilution method and SCCmec was detected by PCR. Isolates with a vancomycin MIC ≥ 2mg/L were cultured on BHI agar with 3, 4 or 6 mg/L (BHIa3, BHIa4 or BHIa6) of vancomycin and BHIa4 with casein (BHIa4ca). Macromethod Etest® and Etest® Glicopeptides Resistance Detection were also used. VISA and hVISA isolates were confirmed by the population analysis profile then typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Medical data from the patients were obtained from their medical records. Among 110 consecutive isolates, 31 (28%) were MRSA and carried the SCCmec type II (15 isolates) or IV (16 isolates). Vancomycin MIC50 and MIC90 were 1 and 2 mg/L, respectively. MRSA isolates had increased non-susceptibility to daptomycin (p = 0.0003). Six (5%) isolates were VISA, four of which were MRSA, three SCCmec type II/USA100/ST5 and one type IV/USA800/ST3192. One MRSA SCCmec II isolate grew on agar BHIa3, BHIa4 and BHIa4ca, and it was confirmed as hVISA. Among the six VISA isolates, five (83%) grew on BHIa3 and three (50%) on BHI4ca. Four of the six VISA isolates and the one hVISA isolate were from patients who had undergone dialysis. Thus, a possible dissemination of the SCCmec II/USA100/ST5 lineage may have occurred in the hospital comprising the VISA, hVISA and daptomycin non-susceptible S. aureus Brazilian isolates from health care associated bloodstream infections.

Epidemiologic and Clinical Impact of Acinetobacter baumannii Colonization and Infection

PubMed Central

Villar, Macarena; Cano, María E.; Gato, Eva; Garnacho-Montero, José; Miguel Cisneros, José; Ruíz de Alegría, Carlos; Fernández-Cuenca, Felipe; Martínez-Martínez, Luis; Vila, Jordi; Pascual, Alvaro; Tomás, María; Bou, Germán; Rodríguez-Baño, Jesús

2014-01-01

Abstract Acinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p < 0.001). The number of non-nosocomial health care-associated cases increased from 1.2% to 14.2%, respectively (p < 0.001). Previous exposure to carbapenems increased in 2010 (16.9% in 2000 vs 27.3% in 2010, p = 0.03). The drugs most frequently used for definitive treatment of patients with infections were carbapenems in 2000 (45%) and colistin in 2010 (50.3%). There was molecular-typing evidence of an increase in the frequency of A. baumannii acquisition in non-intensive care unit wards in 2010 (7.6% in 2000 vs 19.2% in 2010, p = 0.01). By MSLT, the ST2 clonal group predominated and increased in 2010. This epidemic clonal group was more frequently resistant to imipenem and was associated with an increased risk of sepsis, although not with severe sepsis or mortality. Some significant changes were noted in the epidemiology of A. baumannii, which is increasingly affecting patients admitted to conventional wards and is also the cause of non-nosocomial health care-associated infections. Epidemic clones seem to combine antimicrobial resistance and the ability to spread, while maintaining their clinical virulence. PMID:25181313

Dissemination of a Multidrug-Resistant VIM-1- and CMY-99-Producing Proteus mirabilis Clone in Bulgaria.

PubMed

Markovska, Rumyana; Schneider, Ines; Keuleyan, Emma; Ivanova, Dobrinka; Lesseva, Magdalena; Stoeva, Temenuga; Sredkova, Mariya; Bauernfeind, Adolf; Mitov, Ivan

2017-04-01

The aim of this study was to analyze the beta-lactamases and the molecular epidemiology of 19 clinically significant isolates of Proteus mirabilis with decreased susceptibility to imipenem, which have been collected from seven hospitals, located in different Bulgarian towns (Sofia, Varna, and Pleven). The isolates were obtained from blood, urine, tracheal and wound specimens. One additional isolate from hospital environment was included. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and PFGE. Integron mapping was performed by PCR and sequencing. All isolates showed a multidrug-resistance profile, but remained susceptible to piperacillin/tazobactam, cefepime, meropenem, and fosfomycin. They produced identical beta-lactamases, namely: TEM-1, VIM-1, and CMY-99. PCR mapping revealed that the bla VIM-1 gene was part of a class 1 integron that additionally included the aac(6')-I, dhfrA1, and ant(3″)-Ia genes. In addition, 17 of the isolates carried the armA gene. Conjugation experiments and plasmid replicon typing were unsuccessful. The isolates were clonally related according to RAPD and PFGE typing. This study reveals the nationwide distribution of a multidrug-resistant P. mirabilis clone producing VIM-1 and CMY-99 along with the presence of different aminoglycoside resistance mechanisms.

Intestinal Escherichia coli colonization in a mallard duck population over four consecutive winter seasons.

PubMed

Rödiger, Stefan; Kramer, Toni; Frömmel, Ulrike; Weinreich, Jörg; Roggenbuck, Dirk; Guenther, Sebastian; Schaufler, Katharina; Schröder, Christian; Schierack, Peter

2015-09-01

We report the population structure and dynamics of one Escherichia coli population of wild mallard ducks in their natural environment over four winter seasons, following the characterization of 100 isolates each consecutive season. Macro-restriction analysis was used to define isolates variously as multi- or 1-year pulsed-field gel electrophoresis (PFGE) types. Isolates were characterized genotypically based on virulence-associated genes (VAGs), phylogenetic markers, and phenotypically based on haemolytic activity, antimicrobial resistance, adhesion to epithelial cells, microcin production, motility and carbohydrate metabolism. Only 12 out of 220 PFGE types were detectable over more than one winter, and classified as multi-year PFGE types. There was a dramatic change of PFGE types within two winter seasons. Nevertheless, the genetic pool (VAGs) and antimicrobial resistance pattern remained remarkably stable. The high diversity and dynamics of this E. coli population were also demonstrated by the occurrence of PFGE subtypes and differences between isolates of one PFGE type (based on VAGs, antimicrobial resistance and adhesion rates). Multi- and 1-year PFGE types differed in antimicrobial resistance, VAGs and adhesion. Other parameters were not prominent colonization factors. In conclusion, the high diversity, dynamics and stable genetic pool of an E. coli population seem to enable their successful colonization of host animal population over time. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Variable number of tandem repeats and pulsed-field gel electrophoresis cluster analysis of enterohemorrhagic Escherichia coli serovar O157 strains.

PubMed

Yokoyama, Eiji; Uchimura, Masako

2007-11-01

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157.

Enterobacter muelleri sp. nov., isolated from the rhizosphere of Zea mays.

PubMed

Kämpfer, Peter; McInroy, John A; Glaeser, Stefanie P

2015-11-01

A beige-pigmented, oxidase-negative bacterial strain (JM-458T), isolated from a rhizosphere sample, was studied using a polyphasic taxonomic approach. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain JM-458T with sequences of the type strains of closely related species of the genus Enterobacter showed that it shared highest sequence similarity with Enterobacter mori (98.7 %), Enterobacter hormaechei (98.3 %), Enterobacter cloacae subsp. dissolvens, Enterobacter ludwigii and Enterobacter asburiae (all 98.2 %). 16S rRNA gene sequence similarities to all other Enterobacter species were below 98 %. Multilocus sequence analysis based on concatenated partial rpoB, gyrB, infB and atpD gene sequences showed a clear distinction of strain JM-458T from its closest related type strains. The fatty acid profile of the strain consisted of C16 : 0, C17 : 0 cyclo, iso-C15 : 0 2-OH/C16 : 1ω7c and C18 : 1ω7c as major components. DNA-DNA hybridizations between strain JM-458T and the type strains of E. mori, E. hormaechei and E. ludwigii resulted in relatedness values of 29 % (reciprocal 25 %), 24 % (reciprocal 43 %) and 16 % (reciprocal 17 %), respectively. DNA-DNA hybridization results together with multilocus sequence analysis results and differential biochemical and chemotaxonomic properties showed that strain JM-458T represents a novel species of the genus Enterobacter, for which the name Enterobacter muelleri sp. nov. is proposed. The type strain is JM-458T ( = DSM 29346T = CIP 110826T = LMG 28480T = CCM 8546T).

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

PubMed

Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

2011-07-01

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

SNP-Based Typing: A Useful Tool to Study Bordetella pertussis Populations

PubMed Central

van der Heide, Han G. J.; Heuvelman, Kees J.; Kallonen, Teemu; He, Qiushui; Mertsola, Jussi; Advani, Abdolreza; Hallander, Hans O.; Janssens, Koen; Hermans, Peter W.; Mooi, Frits R.

2011-01-01

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in the Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis. PMID:21647370

PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland.

PubMed

Fox, Edward M; deLappe, Niall; Garvey, Patricia; McKeown, Paul; Cormican, Martin; Leonard, Nola; Jordan, Kieran

2012-04-01

Listeria monocytogenes is an important foodborne human pathogen. Human infection is associated with high mortality rates. Epidemiological investigation and molecular subtyping can be useful in linking human illness with specific sources of infection. This retrospective study describes the use of PFGE to examine relationships of 222 isolates from human and non-human sources in Ireland. Human clinical isolates from other countries were also examined. Eight small clusters of human and non-human isolates (mostly serotype 4b) that were indistinguishable from one another were detected, suggesting potential sources for human infection. For non-human isolates, some PFGE types appeared to be exclusively associated with a single source, whereas other PFGE-types appeared to be more widely disseminated. Indistinguishable, or highly related clusters of isolates of Irish and non-Irish origin suggest that some PFGE patterns may be globally distributed.

High-resolution typing of Chlamydia trachomatis: epidemiological and clinical uses.

PubMed

de Vries, Henry J C; Schim van der Loeff, Maarten F; Bruisten, Sylvia M

2015-02-01

A state-of-the-art overview of molecular Chlamydia trachomatis typing methods that are used for routine diagnostics and scientific studies. Molecular epidemiology uses high-resolution typing techniques such as multilocus sequence typing, multilocus variable number of tandem repeats analysis, and whole-genome sequencing to identify strains based on their DNA sequence. These data can be used for cluster, network and phylogenetic analyses, and are used to unveil transmission networks, risk groups, and evolutionary pathways. High-resolution typing of C. trachomatis strains is applied to monitor treatment efficacy and re-infections, and to study the recent emergence of lymphogranuloma venereum (LGV) amongst men who have sex with men in high-income countries. Chlamydia strain typing has clinical relevance in disease management, as LGV needs longer treatment than non-LGV C. trachomatis. It has also led to the discovery of a new variant Chlamydia strain in Sweden, which was not detected by some commercial C. trachomatis diagnostic platforms. After a brief history and comparison of the various Chlamydia typing methods, the applications of the current techniques are described and future endeavors to extend scientific understanding are formulated. High-resolution typing will likely help to further unravel the pathophysiological mechanisms behind the wide clinical spectrum of chlamydial disease.

Whole genome sequencing of Salmonella Typhimurium illuminates distinct outbreaks caused by an endemic multi-locus variable number tandem repeat analysis type in Australia, 2014.

PubMed

Phillips, Anastasia; Sotomayor, Cristina; Wang, Qinning; Holmes, Nadine; Furlong, Catriona; Ward, Kate; Howard, Peter; Octavia, Sophie; Lan, Ruiting; Sintchenko, Vitali

2016-09-15

Salmonella Typhimurium (STM) is an important cause of foodborne outbreaks worldwide. Subtyping of STM remains critical to outbreak investigation, yet current techniques (e.g. multilocus variable number tandem repeat analysis, MLVA) may provide insufficient discrimination. Whole genome sequencing (WGS) offers potentially greater discriminatory power to support infectious disease surveillance. We performed WGS on 62 STM isolates of a single, endemic MLVA type associated with two epidemiologically independent, food-borne outbreaks along with sporadic cases in New South Wales, Australia, during 2014. Genomes of case and environmental isolates were sequenced using HiSeq (Illumina) and the genetic distance between them was assessed by single nucleotide polymorphism (SNP) analysis. SNP analysis was compared to the epidemiological context. The WGS analysis supported epidemiological evidence and genomes of within-outbreak isolates were nearly identical. Sporadic cases differed from outbreak cases by a small number of SNPs, although their close relationship to outbreak cases may represent an unidentified common food source that may warrant further public health follow up. Previously unrecognised mini-clusters were detected. WGS of STM can discriminate foodborne community outbreaks within a single endemic MLVA clone. Our findings support the translation of WGS into public health laboratory surveillance of salmonellosis.

Population genetic and evolution analysis of controversial genus Edwardsiella by multilocus sequence typing.

PubMed

Buján, Noemí; Balboa, Sabela; L Romalde, Jesús; E Toranzo, Alicia; Magariños, Beatriz

2018-05-08

At present, the genus Edwardsiella compiles five species: E. tarda, E. hoshinae, E. ictaluri, E. piscicida and E. anguillarum. Some species of this genus such us E. ictaluri and E. piscicida are important pathogens of numerous fish species. With the description of the two latter species, the phylogeny of Edwardsiella became more complicated. With the aim to clarify the relationships among all species in the genus, a multilocus sequence typing (MLST) approach was developed and applied to characterize 56 isolates and 6 reference strains belonging to the five Edwardsiella species. Moreover, several analyses based on the MLST scheme were performed to investigate the evolution within the genus, as well as the influence of recombination and mutation in the speciation. Edwardsiella isolates presented a high genetic variability reflected in the fourteen sequence types (ST) represented by a single isolates out of eighteen total ST. Mutation events were considerably more frequent than recombination, although both approximately equal influenced the genetic diversification. However, the speciation among species occurred mostly by recombination. Edwardsiella genus displays a non-clonal population structure with some degree of geographical isolation followed by a population expansion of E. piscicida. A database from this study was created and hosted on pubmlst.org (http://pubmlst.org/edwardsiella/). Copyright © 2018 Elsevier Inc. All rights reserved.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

PubMed

Danet, Jean Luc; Balakishiyeva, Gulnara; Cimerman, Agnès; Sauvion, Nicolas; Marie-Jeanne, Véronique; Labonne, Gérard; Lavina, Amparo; Batlle, Assumpcio; Krizanac, Ivana; Skoric, Dijana; Ermacora, Paolo; Serçe, Cigdem Ulubas; Caglayan, Kadriye; Jarausch, Wolfgang; Foissac, Xavier

2011-02-01

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Population structure of the Yersinia pseudotuberculosis complex according to multilocus sequence typing

PubMed Central

Laukkanen-Ninios, Riikka; Didelot, Xavier; Jolley, Keith A.; Morelli, Giovanna; Sangal, Vartul; Kristo, Paula; Imori, Priscilla F. M.; Fukushima, Hiroshi; Siitonen, Anja; Tseneva, Galina; Voskressenskaya, Ekaterina; Falcao, Juliana P.; Korkeala, Hannu; Maiden, Martin C. J.; Mazzoni, Camila; Carniel, Elisabeth; Skurnik, Mikael; Achtman, Mark

2014-01-01

Summary Multilocus sequence analysis of 417 strains of Yersinia pseudotuberculosis revealed that it is a complex of four populations, three of which have been previously assigned species status [Y. pseudotuberculosis sensu stricto (s.s.), Yersinia pestis and Yersinia similis] and a fourth population, which we refer to as the Korean group, which may be in the process of speciation. We detected clear signs of recombination within Y. pseudotuberculosis s.s. as well as imports from Y. similis and the Korean group. The sources of genetic diversification within Y. pseudotuberculosis s.s. were approximately equally divided between recombination and mutation, whereas recombination has not yet been demonstrated in Y. pestis, which is also much more genetically monomorphic than is Y. pseudotuberculosis s.s. Most Y. pseudotuberculosis s.s. belong to a diffuse group of sequence types lacking clear population structure, although this species contains a melibiose-negative clade that is present globally in domesticated animals. Yersinia similis corresponds to the previously identified Y. pseudotuberculosis genetic type G4, which is probably not pathogenic because it lacks the virulence factors that are typical for Y. pseudotuberculosis s.s. In contrast, Y. pseudotuberculosis s.s., the Korean group and Y. pestis can all cause disease in humans. PMID:21951486

Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings▿

PubMed Central

Freitas, Ana R.; Novais, Carla; Ruiz-Garbajosa, Patricia; Coque, Teresa M.; Peixe, Luísa

2009-01-01

The population structure of 56 Enterococcus faecium isolates selected from a collection of enterococci from humans, animals, and the environment in Portugal (1997 to 2007) was analyzed by multilocus sequence typing. We identified 41 sequence types clustering into CC17, CC5, CC9, CC22 and CC94, all clonal lineages comprising isolates from different hosts. Our findings highlight the role of community-associated hosts as reservoirs of enterococci able to cause human infections. PMID:19447948

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification.

USDA-ARS?s Scientific Manuscript database

Since 2002, severe leaf spotting on parsley (Petroselinum crispum L.) has occurred in Monterey County, California. One of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from seven distinct outbreaks and twice from the same outbreak (2002 and 2009). Frag...

Typing of Lymphogranuloma Venereum Chlamydia trachomatis Strains

PubMed Central

Christerson, Linus; de Vries, Henry J.C.; de Barbeyrac, Bertille; Gaydos, Charlotte A.; Henrich, Birgit; Hoffmann, Steen; Schachter, Julius; Thorvaldsen, Johannes; Vall-Mayans, Martí; Klint, Markus; Morré, Servaas A.

2010-01-01

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe. PMID:21029543

Streptococcus agalactiae serotype Ib as an agent of meningitis in two adult nonpregnant women.

PubMed

Martins, E R; Florindo, C; Martins, F; Aldir, I; Borrego, M J; Brum, L; Ramirez, M; Melo-Cristino, J

2007-11-01

Two temporally and geographically clustered cases of meningitis caused by Streptococcus agalactiae expressing the infrequent Ib serotype are reported. Characterization by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the isolates were identical and represented the widely distributed ST10/ST8 lineage associated with serotype Ib.

Clonality, recombination, and hybridization in the plumbing-inhabiting human pathogen Fusarium keratoplasticum inferred from multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Recent work has shown that Fusarium species and genotypes most commonly associated with human infections, particularly of the cornea (mycotic keratitis), are the same as those most commonly isolated from plumbing systems. The species most dominant in plumbing biofilms is Fusarium keratoplasticum, a ...

Multi-locus sequence subtypes of Campylobacter detected on the surface and from internal tissue of retail chicken livers

USDA-ARS?s Scientific Manuscript database

Foodborne campylobacteriosis has been traced to undercooked chicken liver. The objectives of this study were to measure prevalence of Campylobacter associated with chicken livers at retail and determine which subtypes are detected on the surface and inner tissue of livers. Fifteen packages of fres...

Multilocus sequence analysis reveals extensive genetic variety within Tenacibaculum spp. associated with ulcers in sea-farmed fish in Norway.

PubMed

Olsen, Anne Berit; Gulla, Snorre; Steinum, Terje; Colquhoun, Duncan J; Nilsen, Hanne K; Duchaud, Eric

2017-06-01

Skin ulcer development in sea-reared salmonids, commonly associated with Tenacibaculum spp., is a significant fish welfare- and economical problem in Norwegian aquaculture. A collection of 89 Tenacibaculum isolates was subjected to multilocus sequence analysis (MLSA). The isolates were retrieved from outbreaks of clinical disease in farms spread along the Norwegian coast line from seven different fish species over a period of 19 years. MLSA analysis reveals considerable genetic diversity, but allows identification of four main clades. One clade encompasses isolates belonging to the species T. dicentrarchi, whereas three clades encompass bacteria that likely represent novel, as yet undescribed species. The study identified T. maritimum in lumpsucker, T. ovolyticum in halibut, and has extended the host and geographic range for T. soleae, isolated from wrasse. The overall lack of clonality and host specificity, with some indication of geographical range restriction argue for local epidemics involving multiple strains. The diversity of Tenacibaculum isolates from fish displaying ulcerative disease may complicate vaccine development. Copyright © 2017 Elsevier B.V. All rights reserved.

The Evolution of Advanced Molecular Diagnostics for the Detection and Characterization of Mycoplasma pneumoniae.

PubMed

Diaz, Maureen H; Winchell, Jonas M

2016-01-01

Over the past decade there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis (MLVA), Multi-locus sequence typing (MLST), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen.

Molecular Epidemiologic Analysis of Enterococcus faecalis Isolates in Cuba by Multilocus Sequence Typing

PubMed Central

Kobayashi, Nobumichi; Nagashima, Shigeo

2009-01-01

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6′)-aph(2″), aph(3′), ant(6), ant(3″)(9), aph(2″)-Id, aph(2″)-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6′)-Ie-aph(2″)-Ia, aph(3′), ant(6), ant(3″)(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits. PMID:19857135

Ongoing outbreak of invasive listeriosis, Germany, 2012 to 2015.

PubMed

Ruppitsch, Werner; Prager, Rita; Halbedel, Sven; Hyden, Patrick; Pietzka, Ariane; Huhulescu, Steliana; Lohr, Dorothee; Schönberger, Katharina; Aichinger, Elisabeth; Hauri, Anja; Stark, Klaus; Vygen, Sabine; Tietze, Erhard; Allerberger, Franz; Wilking, Hendrik

2015-01-01

Listeriosis patient isolates in Germany have shown a new identical pulsed-field gel electrophoresis (PFGE) pattern since 2012 (n = 66). Almost all isolates (Listeria monocytogenes serotype 1/2a) belonged to cases living in southern Germany, indicating an outbreak with a so far unknown source. Case numbers in 2015 are high (n = 28). No outbreak cases outside Germany have been reported. Next generation sequencing revealed the unique cluster type CT1248 and confirmed the outbreak. Investigations into the source are ongoing.

STBase: One Million Species Trees for Comparative Biology

PubMed Central

McMahon, Michelle M.; Deepak, Akshay; Fernández-Baca, David; Boss, Darren; Sanderson, Michael J.

2015-01-01

Comprehensively sampled phylogenetic trees provide the most compelling foundations for strong inferences in comparative evolutionary biology. Mismatches are common, however, between the taxa for which comparative data are available and the taxa sampled by published phylogenetic analyses. Moreover, many published phylogenies are gene trees, which cannot always be adapted immediately for species level comparisons because of discordance, gene duplication, and other confounding biological processes. A new database, STBase, lets comparative biologists quickly retrieve species level phylogenetic hypotheses in response to a query list of species names. The database consists of 1 million single- and multi-locus data sets, each with a confidence set of 1000 putative species trees, computed from GenBank sequence data for 413,000 eukaryotic taxa. Two bodies of theoretical work are leveraged to aid in the assembly of multi-locus concatenated data sets for species tree construction. First, multiply labeled gene trees are pruned to conflict-free singly-labeled species-level trees that can be combined between loci. Second, impacts of missing data in multi-locus data sets are ameliorated by assembling only decisive data sets. Data sets overlapping with the user’s query are ranked using a scheme that depends on user-provided weights for tree quality and for taxonomic overlap of the tree with the query. Retrieval times are independent of the size of the database, typically a few seconds. Tree quality is assessed by a real-time evaluation of bootstrap support on just the overlapping subtree. Associated sequence alignments, tree files and metadata can be downloaded for subsequent analysis. STBase provides a tool for comparative biologists interested in exploiting the most relevant sequence data available for the taxa of interest. It may also serve as a prototype for future species tree oriented databases and as a resource for assembly of larger species phylogenies from precomputed trees. PMID:25679219

Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics.

PubMed

Delgado, Susana; Arroyo, Rebeca; Jiménez, Esther; Marín, Maria L; del Campo, Rosa; Fernández, Leonides; Rodríguez, Juan M

2009-05-07

Although Staphylococcus aureus is considered the main etiological agent of infectious mastitis, recent studies have suggested that coagulase-negative staphylococci (CNS) may also play an important role in such infections. The aims of this work were to isolate staphylococci from milk of women with lactational mastitis, to select and characterize the CNS isolates, and to compare such properties with those displayed by CNS strains isolated from milk of healthy women. The milk of 30 women was collected and bacterial growth was noted in 27 of them, of which Staphylococcus epidermidis was isolated from 26 patients and S. aureus from 8. Among the 270 staphylococcal isolates recovered from milk of women with mastitis, 200 were identified as Staphylococcus epidermidis by phenotypic assays, species-specific PCR and PCR sequencing. They were typified by pulsed field gel electrophoresis (PFGE) genotyping. The PFGE profiles of the S. epidermidis strains were compared with those of 105 isolates from milk of healthy women. A representative of the 76 different PFGE profiles was selected to study the incidence of virulence factors and antibiotic resistance. The number of strains that contained the biofilm-related icaD gene and that showed resistance to oxacillin, erythromycin, clindamycin and mupirocin was significantly higher among the strains isolated from mastitic milk. S. epidermidis may be a frequent but largely underrated cause of infectious mastitis in lactating women. The resistance to diverse antibiotics and a higher ability to form biofilms found among the strains isolated from milk of women suffering mastitis may explain the chronic and/or recurrent nature of this infectious condition.

Multilocus inference of species trees and DNA barcoding.

PubMed

Mallo, Diego; Posada, David

2016-09-05

The unprecedented amount of data resulting from next-generation sequencing has opened a new era in phylogenetic estimation. Although large datasets should, in theory, increase phylogenetic resolution, massive, multilocus datasets have uncovered a great deal of phylogenetic incongruence among different genomic regions, due both to stochastic error and to the action of different evolutionary process such as incomplete lineage sorting, gene duplication and loss and horizontal gene transfer. This incongruence violates one of the fundamental assumptions of the DNA barcoding approach, which assumes that gene history and species history are identical. In this review, we explain some of the most important challenges we will have to face to reconstruct the history of species, and the advantages and disadvantages of different strategies for the phylogenetic analysis of multilocus data. In particular, we describe the evolutionary events that can generate species tree-gene tree discordance, compare the most popular methods for species tree reconstruction, highlight the challenges we need to face when using them and discuss their potential utility in barcoding. Current barcoding methods sacrifice a great amount of statistical power by only considering one locus, and a transition to multilocus barcodes would not only improve current barcoding methods, but also facilitate an eventual transition to species-tree-based barcoding strategies, which could better accommodate scenarios where the barcode gap is too small or inexistent.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

Molecular Epidemiology of Human Oral Chagas Disease Outbreaks in Colombia

PubMed Central

Ramírez, Juan David; Montilla, Marleny; Cucunubá, Zulma M.; Floréz, Astrid Carolina; Zambrano, Pilar; Guhl, Felipe

2013-01-01

Background Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes. Methodology and Principal Findings High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission. Conclusions These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed. PMID:23437405

Characterization of the Salmonella enterica Serotype Isangi Isolated from Patients for the First Time in China.

PubMed

Li, Xin-Peng; Gao, Ri-Hong; Hou, Pei-Bin; Ren, Yan-Yan; Zhang, Hua-Ning; Jiang, Kui-Ying; Chen, Yu-Zhen; Qi, Zi-Gang; Xu, Min; Bi, Zhen-Wang

2017-08-01

No studies have reported the isolation of serotype Salmonella Isangi from cases of salmonellosis in mainland China. We investigated an outbreak of foodborne disease with salmonella and collected the samples from the patients and surplus foods. Salmonella strains were isolated and the serotype was identified according to the Kauffmann-White scheme. The relatedness of the isolates was determined using pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). Antimicrobial susceptibility was conducted by the broth microdilution method. There were 74 diners in the case, 33 of which got ill, with an attack rate of 44.6% (33/74). A total of 24 samples were collected from the outbreak cases, six Salmonella Isangi strains were isolated and susceptible to all tested drugs. PFGE and WGS analysis suggested that the pathogen dissemination through a single or limited vector(s), the steamed fish and mixed food (fry spicy chicken, braised pork ribs, and goose leg), may be the source of infection or be cross-contaminated. We first report the characteristics of an outbreak and molecular strain relatedness of Salmonella Isangi in mainland China.

Regional Spread of CTX-M-2-Producing Proteus mirabilis with the Identical Genetic Structure in Japan.

PubMed

Kato, Karin; Matsumura, Yasufumi; Yamamoto, Masaki; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

2017-07-01

In this study, we analyzed the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Proteus mirabilis isolates collected from the central region of Japan. Between 2005 and 2012, 820 clinical P. mirabilis isolates were obtained from ten acute care hospitals in Japan. We characterized ESBL confirmatory test-positive isolates by sequencing the ESBL genes and their flanking regions, detecting plasmid replicons, and performing pulsed-field gel electrophoresis (PFGE). Ninety-six isolates (12%) were positive according to the ESBL confirmatory test; all these isolates possessed bla CTX-M-2 with the same flanking structure of upstream ΔISEcp1 and a downstream region identical to downstream bla KLUA-1 . IncT was the prevalent, and only, replicon found in 63 isolates. PFGE analysis detected eight clusters with more than one isolate, among which three included 56 isolates and six included isolates from multiple hospitals. CTX-M-2-producing P. mirabilis with an identical genetic structure flanking bla CTX-M-2 is dominant in this Japanese region, and there is evidence for the clonal spread of isolates.

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae.

PubMed

Dijkman, R; Feberwee, A; Landman, W J M

2016-08-01

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

PubMed

Desoubeaux, Guillaume; Debourgogne, Anne; Wiederhold, Nathan P; Zaffino, Marie; Sutton, Deanna; Burns, Rachel E; Frasca, Salvatore; Hyatt, Michael W; Cray, Carolyn

2018-07-01

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

Emergence of multidrug-resistant Proteus mirabilis in a long-term care facility in Croatia.

PubMed

Bedenić, Branka; Firis, Nataša; Elveđi-Gašparović, Vesna; Krilanović, Marija; Matanović, Krešimir; Štimac, Iva; Luxner, Josefa; Vraneš, Jasmina; Meštrović, Tomislav; Zarfel, Gernot; Grisold, Andrea

2016-06-01

An increased frequency of Proteus mirabilis isolates resistant to expanded-spectrum cephalosporins was observed recently in a long-term care facility in Zagreb (Godan). The aim of this study was the molecular characterization of resistance mechanisms to new cephalosporins in P. mirabilis isolates from this nursing home. Thirty-eight isolates collected from 2013-2015 showing reduced susceptibility to ceftazidime were investigated. Antibiotic susceptibilities were determined by broth microdilution method. Inhibitor-based tests were performed to detect extended-spectrum (ESBLs) and AmpC β-lactamases. AmpC β-lactamases were characterized by polymerase chain reaction (PCR) followed by sequencing of bla ampC genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of the isolates was performed by repetitive element sequence (rep)-PCR and pulsed-field gel electrophoresis (PFGE). Presence of an AmpC β-lactamase was confirmed in all isolates by combined-disk test with phenylboronic acid. All isolates were resistant to amoxicillin alone and combined with clavulanate, cefotaxime, ceftriaxone, cefoxitin, and ciprofloxacin; but susceptible to cefepime, imipenem, and meropenem. PCR followed by sequencing using primers targeting bla ampc genes revealed CMY-16 β-lactamase in all but one strain. Bla cmy-16 was carried by a non-conjugative plasmid which did not belong to any known plasmid-based replicon typing (PBRT) group. Rep-PCR identified one large clone consisting of 15 isolates, three pairs or related isolates, one triplet, and four singletons. PFGE confirmed the clonality of the isolates. This is the first report of multidrug resistant P. mirabilis in a nursing home in Croatia. Cephalosporin resistance was due to plasmid-mediated AmpC β-lactamase CMY-16.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

PubMed Central

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

PubMed Central

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis

2014-01-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. PMID:25078912

Molecular characterization and epidemiology of cefoxitin resistance among Enterobacteriaceae lacking inducible chromosomal ampC genes from hospitalized and non-hospitalized patients in Algeria: description of new sequence type in Klebsiella pneumoniae isolates.

PubMed

Gharout-Sait, Alima; Touati, Abdelaziz; Guillard, Thomas; Brasme, Lucien; de Champs, Christophe

2015-01-01

In this study, 922 consecutive non-duplicate clinical isolates of Enterobacteriaceae obtained from hospitalized and non-hospitalized patients at Bejaia, Algeria were analyzed for AmpC-type β-lactamases production. The ampC genes and their genetic environment were characterized using polymerase chain reaction (PCR) and sequencing. Plasmid incompatibility groups were determined by using PCR-based replicon typing. Phylogenetic grouping and multilocus sequence typing were determined for molecular typing of the plasmid-mediated AmpC (pAmpC) isolates. Of the isolates, 15 (1.6%) were identified as AmpC producers including 14 CMY-4-producing isolates and one DHA-1-producing Klebsiella pneumoniae. All AmpC-producing isolates co-expressed the broad-spectrum TEM-1 β-lactamase and three of them co-produced CTX-M and/or SHV-12 ESBL. Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to groups D and B1. Multilocus sequence typing analysis of K. pneumoniae isolates identified four different sequence types (STs) with two new sequences: ST1617 and ST1618. Plasmid replicon typing indicates that blaCMY-4 gene was located on broad host range A/C plasmid, while LVPK replicon was associated with blaDHA-1. All isolates carrying blaCMY-4 displayed the transposon-like structures ISEcp1/ΔISEcp1-blaCMY-blc-sugE. Our study showed that CMY-4 was the main pAmpC in the Enterobacteriaceae isolates in Algeria. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia.

PubMed

McMillan, Kate; Moore, Sean C; McAuley, Catherine M; Fegan, Narelle; Fox, Edward M

2016-07-29

Highly pathogenic strains of Staphylococcus aureus can cause disease in both humans and animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. The aim of this study was to use genotypic and immunological methods to characterize S. aureus isolates from milk-related samples collected from 7 dairy farms across Victoria. A total of 30 S. aureus isolates were collected from milk and milk filter samples from 3 bovine, 3 caprine and 1 ovine dairy farms across Victoria, Australia. Pulsed Field Gel Electrophoresis (PFGE) identified 11 distinct pulsotypes among isolates; all caprine and ovine isolates shared greater than 80 % similarity regardless of source. Conversely, bovine isolates showed higher diversity. Multi-Locus Sequence Typing (MLST) identified 5 different sequence types (STs) among bovine isolates, associated with human or ruminant lineages. All caprine and ovine isolates were ST133, or a single allele variant of ST133. Two new novel STs were identified among isolates in this study (ST3183 and ST3184). With the exception of these 2 new STs, eBURST analysis predicted all other STs to be founding members of their associated clonal complexes (CCs). Analysis of genetic markers revealed a diverse range of classical staphylococcal enterotoxins (SE) among isolates, with 11 different SEs identified among bovine isolates, compared with just 2 among caprine and ovine isolates. None of the isolates contained mecA, or were resistant to oxacillin. The only antibiotic resistance identified was that of a single isolate resistant to penicillin; this isolate also contained the penicillin resistance gene blaZ. Production of SE was observed at 16 °C and/or 37 °C in milk, however no SE production was detected at 12 °C. Although this study characterized a limited number of isolates, bovine-associated isolates showed higher genetic diversity than their caprine or ovine counterparts. This was also reflected in a more diverse SE repertoire among bovine isolates. Very little antibiotic resistance was identified among isolates in this study. These results suggest maintaining the milk cold chain will minimise any risk from SE production and highlights the need to prevent temperature abuse.

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012

PubMed Central

Vignaud, Marie-Léone; Cherchame, Emeline; Marault, Muriel; Chaing, Emilie; Le Hello, Simon; Michel, Valerie; Jourdan-Da Silva, Nathalie; Lailler, Renaud; Brisabois, Anne; Cadel-Six, Sabrina

2017-01-01

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations. PMID:28289408

Characterization of Escherichia coli O157:H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis.

PubMed

Dyet, K H; Robertson, I; Turbitt, E; Carter, P E

2011-03-01

Recently, multiple-locus variable-number tandem-repeat analysis (MLVA) has been proposed as an alternative to pulsed-field gel electrophoresis (PFGE) for characterization of Escherichia coli O157:H7. In this study we characterized 118 E. coli O157:H7 isolates from cases of gastrointestinal disease in New Zealand using XbaI PFGE profiles and a MLVA scheme that assessed variability in eight polymorphic loci. The 118 isolates characterized included all 80 E. coli O157:H7 referred to New Zealand's Enteric Reference Laboratory in 2006 and 29 phage-type 2 isolates from 2005. When applied to these isolates the discriminatory power of PFGE and MLVA was not significantly different. However, MLVA data may be more epidemiologically relevant as isolates from family clusters of disease had identical MLVA profiles, even when the XbaI PFGE profiles differed slightly. Furthermore, most isolates with indistinguishable XbaI PFGE profiles that did not appear to be epidemiologically related had distinct MLVA profiles.

Class 1 integrons characterization and multilocus sequence typing of Salmonella spp. from swine production chains in Chiang Mai and Lamphun provinces, Thailand.

PubMed

Boonkhot, Phacharaporn; Tadee, Pakpoom; Yamsakul, Panuwat; Pocharoen, Chairoj; Chokesajjawatee, Nipa; Patchanee, Prapas

2015-05-01

Pigs and pork products are well known as an important source of Salmonella, one of the major zoonotic foodborne pathogens. The emergence and spread of antimicrobial resistance is becoming a major public health concern worldwide. Integrons are genetic elements known to have a role in the acquisition and expression of genes conferring antibiotic resistance. This study focuses on the prevalence of class 1 integrons-carrying Salmonella, the genetic diversity of strains of those organisms obtained from swine production chains in Chiang Mai and Lamphun provinces, Thailand, using multilocus sequence typing (MLST) and comparison of genetic diversity of sequence types of Salmonella from this study with pulsotypes identified in previous study. In 175 Salmonella strains, the overall prevalence of class 1 integrons-carrying-Salmonella was 14%. The gene cassettes array pattern "dfrA12-orfF-aadA2" was the most frequently observed. Most of the antimicrobial resistance identified was not associated with related gene cassettes harbored by Salmonella. Six sequence types were generated from 30 randomly selected strains detected by MLST. Salmonella at the human-animal-environment interface was confirmed. Linkages both in the farm to slaughterhouse contamination route and the horizontal transmission of resistance genes were demonstrated. To reduce this problem, the use of antimicrobials in livestock should be controlled by veterinarians. Education and training of food handlers as well as promotion of safe methods of food consumption are important avenues for helping prevent foodborne illness.

Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis

PubMed Central

Joseph, Susan; Forsythe, Stephen J.

2012-01-01

Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognized route of infection being the consumption of contaminated infant formula. As a recently recognized bacterial pathogen of considerable importance and regulatory control, appropriate detection, and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB, and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognized genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health. PMID:23189075

Multi-Locus Sequence Typing of Bartonella henselae Isolates from Three Continents Reveals Hypervirulent and Feline-Associated Clones

PubMed Central

Arvand, Mardjan; Feil, Edward J.; Giladi, Michael; Boulouis, Henri-Jean; Viezens, Juliane

2007-01-01

Bartonella henselae is a zoonotic pathogen and the causative agent of cat scratch disease and a variety of other disease manifestations in humans. Previous investigations have suggested that a limited subset of B. henselae isolates may be associated with human disease. In the present study, 182 human and feline B. henselae isolates from Europe, North America and Australia were analysed by multi-locus sequence typing (MLST) to detect any associations between sequence type (ST), host species and geographical distribution of the isolates. A total of 14 sequence types were detected, but over 66% (16/24) of the isolates recovered from human disease corresponded to a single genotype, ST1, and this type was detected in all three continents. In contrast, 27.2% (43/158) of the feline isolates corresponded to ST7, but this ST was not recovered from humans and was restricted to Europe. The difference in host association of STs 1 (human) and 7 (feline) was statistically significant (P≤0.001). eBURST analysis assigned the 14 STs to three clonal lineages, which contained two or more STs, and a singleton comprising ST7. These groups were broadly consistent with a neighbour-joining tree, although splits decomposition analysis was indicative of a history of recombination. These data indicate that B. henselae lineages differ in their virulence properties for humans and contribute to a better understanding of the population structure of B. henselae. PMID:18094753

A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a Chromosomal cpe Gene Belong to One Multilocus Sequence Typing Cluster

PubMed Central

Xiao, Yinghua; Wagendorp, Arjen; Moezelaar, Roy; Abee, Tjakko

2012-01-01

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)5 fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods. PMID:22865060

Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure

PubMed Central

Cooper, Vaughn S.; Hatcher, Philip J.; Verheyde, Bart; Carlier, Aurélien; Vandamme, Peter

2017-01-01

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence. PMID:28430818

Biogeography of Burkholderia pseudomallei in the Torres Strait Islands of Northern Australia

PubMed Central

Baker, Anthony; Mayo, Mark; Owens, Leigh; Burgess, Graham; Norton, Robert; McBride, William John Hannan; Currie, Bart J.

2013-01-01

It has been hypothesized that biogeographical boundaries are a feature of Burkholderia pseudomallei ecology, and they impact the epidemiology of melioidosis on a global scale. This study examined the relatedness of B. pseudomallei sourced from islands in the Torres Strait of Northern Australia to determine if the geography of isolated island communities is a determinant of the organisms' dispersal. Environmental sampling on Badu Island in the Near Western Island cluster recovered a single clone. An additional 32 clinical isolates from the region were sourced. Isolates were characterized using multilocus sequence typing and a multiplex PCR targeting the flagellum gene cluster. Gene cluster analysis determined that 69% of the isolates from the region encoded the ancestral Burkholderia thailandensis-like flagellum and chemotaxis gene cluster, a proportion significantly lower than that reported from mainland Australia and consistent with observations of isolates from southern Papua New Guinea. A goodness-of-fit test indicated that there was geographic localization of sequence types throughout the archipelago, with the exception of Thursday Island, the economic and cultural hub of the region. Sequence types common to mainland Australia and Papua New Guinea were identified. These findings demonstrate for the first time an environmental reservoir for B. pseudomallei in the Torres Strait, and multilocus sequence typing suggests that the organism is not randomly distributed throughout this region and that seawater may provide a barrier to dispersal of the organism. Moreover, these findings support an anthropogenic dispersal hypothesis for the spread of B. pseudomallei throughout this region. PMID:23698533

Prevalence of three campylobacter species, C. jejuni, C. coli, and C. lari, using multilocus sequence typing in wild birds of the Mid-Atlantic region, USA.

PubMed

Keller, Judith I; Shriver, W Gregory

2014-01-01

Campylobacter jejuni is responsible for the majority of bacterial foodborne gastroenteritis in the US, usually due to the consumption of undercooked poultry. Research on which avian species transmit the bacterium is limited, especially in the US. We sampled wild birds in three families-Anatidae, Scolopacidae, and Laridae-in eastern North America to determine the prevalence and specific strains of Campylobacter. The overall prevalence of Campylobacter spp. was 9.2% for all wild birds sampled (n = 781). Campylobacter jejuni was the most prevalent species (8.1%), while Campylobacter coli and Campylobacter lari prevalence estimates were low (1.4% and 0.3%, respectively). We used multilocus sequence typing PCR specific to C. jejuni to characterize clonal complexes and sequence types isolated from wild bird samples and detected 13 novel sequence types, along with a clonal complex previously only associated with human disease (ST-658). Wild birds share an increasing amount of habitat with humans as more landscapes become fragmented and developed for human needs. Wild birds are and will remain an important aspect of public health due to their ability to carry and disperse emerging zoonotic pathogens or their arthropod vectors. As basic information such as prevalence is limited or lacking from a majority of wild birds in the US, this study provides further insight into Campylobacter epidemiology, host preference, and strain characterization of C. jejuni.

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

PubMed Central

Sulakvelidze, Alexander; Kekelidze, Merab; Gomelauri, Tsaro; Deng, Yingkang; Khetsuriani, Nino; Kobaidze, Ketino; De Zoysa, Aruni; Efstratiou, Androulla; Morris, J. Glenn; Imnadze, Paata

1999-01-01

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates. PMID:10488190

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis.

PubMed

Pei, Yingxin; Terajima, Jun; Saito, Yasunori; Suzuki, Reiko; Takai, Nobuko; Izumiya, Hidemasa; Morita-Ishihara, Tomoko; Ohnishi, Makoto; Miura, Masashi; Iyoda, Sunao; Mitobe, Jiro; Wang, Binyou; Watanabe, Haruo

2008-01-01

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.

Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons.

PubMed

Rojo-Bezares, Beatriz; Estepa, Vanesa; Cebollada, Rocío; de Toro, María; Somalo, Sergio; Seral, Cristina; Castillo, Francisco Javier; Torres, Carmen; Sáenz, Yolanda

2014-05-01

Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46. Copyright © 2014 Elsevier GmbH. All rights reserved.

Cluster of Serogroup W135 Meningococci, Southeastern Florida, 2008–2009

PubMed Central

Mejia-Echeverry, Alvaro; Fiorella, Paul; Leguen, Fermin; Livengood, John; Kay, Robyn; Hopkins, Richard

2010-01-01

Recently, 14 persons in southeastern Florida were identified with Neisseria meningitidis serogroup W135 invasive infections. All isolates tested had matching or near-matching pulsed-field gel electrophoresis patterns and belonged to the multilocus sequence type 11 clonal complex. The epidemiologic investigation suggested recent endemic transmission of this clonal complex in southeastern Florida. PMID:20031054

Clonal origins of Vibrio cholerae O1 El Tor strains, Papua New Guinea, 2009-2011.

PubMed

Horwood, Paul F; Collins, Deirdre; Jonduo, Marinjho H; Rosewell, Alexander; Dutta, Samir R; Dagina, Rosheila; Ropa, Berry; Siba, Peter M; Greenhill, Andrew R

2011-11-01

We used multilocus sequence typing and variable number tandem repeat analysis to determine the clonal origins of Vibrio cholerae O1 El Tor strains from an outbreak of cholera that began in 2009 in Papua New Guinea. The epidemic is ongoing, and transmission risk is elevated within the Pacific region.

Chlamydia pecorum Infection in Free-ranging Koalas ( Phascolarctos cinereus ) on French Island, Victoria, Australia.

PubMed

Legione, Alistair R; Amery-Gale, Jemima; Lynch, Michael; Haynes, Leesa; Gilkerson, James R; Sansom, Fiona M; Devlin, Joanne M

2016-04-28

We detected Chlamydia pecorum in two koalas ( Phascolarctos cinereus ) from a closed island population in Victoria, Australia, previously free of Chlamydia infection. The ompA and multilocus sequence type were most closely related to published isolates of livestock rather than koala origin, suggesting potential cross-species transmission of C. pecorum .

Campylobacter multi-locus sequence types and antimicrobial susceptibility of broiler cecal isolates: a two year study of 143 commercial flocks

USDA-ARS?s Scientific Manuscript database

The objective of this study was to assess genetic diversity and antimicrobial susceptibility of Campylobacter jejuni and coli recovered from broiler ceca at slaughter. Ceca from one broiler were collected from the evisceration line in a commercial processing plant, once or twice weekly for two year...

Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan

PubMed Central

2009-01-01

Background The number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE) method. National weekly report data were collected for investigating epidemiological trends. Results A total of 23 emm types were identified in 1,218 S. pyogenes isolates. The five most prevalent emm types were emm12 (50.4%), emm4 (23.2%), emm1 (16.4%), emm6 (3.8%) and emm22 (3.0%). PFGE analysis with SmaI suggested that, with a few exceptions, strains with a common emm type belonged to the same clone. There were two large emm12 clones, one with DNA resistant to cleavage by SmaI. Each prevalent emm clone had major PFGE strain(s) and many minor strains. Most of the minor strains emerged in the population and disappeared soon after. Even some major strains remained prevalent for only 2–3 years before declining. The large fluctuation of scarlet fever cases between 2000 and 2006 was associated with the shuffling of six prevalent emm clones. In 2003, the dramatic drop in scarlet fever cases in central Taiwan and throughout the whole country was associated with the occurrence of a severe acute respiratory syndrome (SARS) outbreak that occurred between late-February and mid-June in Taiwan. Conclusion The occurrences of scarlet fever in central Taiwan in 2000–2006 were primarily caused by five emm types, which accounted for 96.8% of the isolates collected. Most of the S. pyogenes strains (as defined by PFGE genotypes) emerged and lasted for only a few years. The fluctuation in the number of scarlet fever cases during the seven years can be primarily attributed to the shuffling of six prevalent emm clones and to the SARS outbreak in 2003. PMID:19486515

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans

PubMed Central

Carter, Stuart D.; Birtles, Richard J.; Brown, Jennifer M.; Hart, C. Anthony; Evans, Nicholas J.

2016-01-01

ABSTRACT Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used “-like” suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. PMID:27208135

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

PubMed

Clegg, Simon R; Carter, Stuart D; Birtles, Richard J; Brown, Jennifer M; Hart, C Anthony; Evans, Nicholas J

2016-08-01

Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. Copyright © 2016 Clegg et al.

Genetic characteristics of Streptococcus dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.).

PubMed

Tsai, M-A; Wang, P-C; Yoshida, T; Chen, S-C

2015-12-01

Disease outbreaks occurred during 2007-2013 in Taiwan with 2.5-10% mortality among the cage cultured cobia, Rachycentron canadum (L.), characterized by the presence of polyserositis, pericarditis and peritonitis. The micro-organisms isolated from internal organs were Gram-positive cocci. The isolates were confirmed as Streptococcus dysgalactiae by a polymerase chain reaction assay that yielded the expected specific 259 bp amplicon. Additionally, partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish was also compared and produced 100% sequence identity with S. dysgalactiae (GenBank accession number AB252398). The genetic characterization was then determined by pulsed-field gel electrophoresis (PFGE) analysis. Based on PFGE, the Apa I or Sma I digestion patterns of chromosomal DNA of these isolates were grouped into three main clusters. Taiwanese strains were divided into two clusters, and the tet(M) gene was detected in cluster 1 (pulsotypes: A1-A2 and S1-S3), but not in cluster 2 strains (pulsotypes: A3-A4 and S4-S5). Three Japanese strains from amberjack, Seriola dumerili (Risso), were grouped into cluster 3 (pulsotypes: A5-A7 and S6-S8) and displayed no mortality to cobia in the challenge experiment. Conversely, Taiwanese strains from cobia and snubnose pompano, Trachinotus blochii (L.), displayed a mortality rate of 50-87.5% in cobia. © 2014 John Wiley & Sons Ltd.

Surface Microflora of Four Smear-Ripened Cheeses

PubMed Central

Mounier, Jérôme; Gelsomino, Roberto; Goerges, Stefanie; Vancanneyt, Marc; Vandemeulebroecke, Katrien; Hoste, Bart; Scherer, Siegfried; Swings, Jean; Fitzgerald, Gerald F.; Cogan, Timothy M.

2005-01-01

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses. PMID:16269673

Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli causing urinary tract infection in the Auckland community likely to be resistant to commonly prescribed antimicrobials.

PubMed

Drinkovic, Dragana; Morris, Arthur J; Dyet, Kristin; Bakker, Sarah; Heffernan, Helen

2015-03-13

To estimate the prevalence and characterise plasmid-mediated AmpC beta-lactamase (PMACBL)- producing Escherichia coli in the Auckland community. All cefoxitin non-susceptible (NS) E. coli identified at the two Auckland community laboratories between 1 January and 31 August 2011 were referred to ESR for boronic acid double-disc synergy testing, to detect the production of AmpC beta-lactamase, and polymerase chain reaction (PCR) to identify the presence of PMACBL genes. PMACBL-producing isolates were typed using pulsed-field gel electrophoresis (PFGE), and PCR was used to determine their phylogenetic group and to identify multilocus sequence type (ST)131. Antimicrobial susceptibility testing and detection of extended-spectrum beta-lactamases (ESBLs) were performed according to the Clinical and Laboratory Standards Institute recommendations. 101 (51%) and 74 (37%) of 200 non-duplicate cefoxitin-NS E. coli were PMACBL producers or assumed hyper-producers of chromosomal AmpC beta-lactamase, respectively. The prevalence of PMACBL-producing E. coli was 0.4%. PMACBL-producing E. coli were significantly less susceptible to norfloxacin, trimethoprim and nitrofurantoin than E. coli that produced neither a PMACBL nor an ESBL. Very few (4%) PMACBL-producing E. coli co-produced an ESBL. Most (88%) of the PMACBL-producing isolates had a CMY-2-like PMACBL. The PMACBL-producing E. coli isolates were diverse based on their PFGE profiles, 44% belonged to phylogenetic group D, and only four were ST131. 100 of the 101 PMACBL-producing E. coli were cultured from urine, and were causing urinary tract infection (UTI) in the majority of patients. The median patient age was 56 years and most (94%) of the patients were women. A greater proportion of patients with community-acquired UTI caused by PMACBL-producing E. coli received a beta-lactam antimicrobial than patients with community-acquired UTI caused by other non-AmpC, non-ESBL-producing E. coli. Thirty-six (43%) patients with community-acquired UTI due to PMACBL-producing E. coli were neither hospitalised nor had any antimicrobial treatment in the previous 6 months. The prevalence of PMACBL-producing E. coli was relatively low in the Auckland community, but has increased in recent years. Typing revealed that the majority of the PMACBL-producing E. coli in the Auckland region were genetically unrelated meaning that a point source or direct person to person transmission are not drivers of local community spread currently. The isolates were more resistant to non-beta-lactam antimicrobials than other non-AmpC, non-ESBL-producing E. coli, leaving few treatment options. The majority of the PMACBL-producing E. coli isolates seemed to be acquired in the community and were most frequently isolated from women with UTI. A large proportion of patients with community-acquired UTI had not been hospitalised nor had any antimicrobial treatment in the previous 6 months.

Diversity, Frequency, and Persistence of Escherichia coli O157 Strains from Range Cattle Environments†

PubMed Central

Renter, David G.; Sargeant, Jan M.; Oberst, Richard D.; Samadpour, Mansour

2003-01-01

Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E. coli O157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to be E. coli O157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E. coli O157. By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample. Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E. coli O157 isolates (124 of 235 isolates). Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.). The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E. coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments. PMID:12514039

A pulsed field gel electrophoresis (PFGE) study that suggests a major world-wide clone of Salmonella enterica serovar Enteritidis.

PubMed

Pang, Jen-Chieh; Chiu, Tsai-Hsin; Helmuth, Reiner; Schroeter, Andreas; Guerra, Beatriz; Tsen, Hau-Yang

2007-05-30

Since human infections by Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) have been increasing world-wide over the past years and epidemiological studies have implicated the consumption of meat, poultry, eggs and egg products, elucidation of the predominant subtypes for this Salmonella spp. is important. In this study, 107 poultry and food isolates of Salmonella Enteritidis obtained from Germany were analyzed by pulsed field gel electrophoresis (PFGE), and the subtypes were compared with those of the 124 human isolates obtained in Taiwan. Results showed that for these 107 poultry and food isolates, when XbaI, SpeI and NotI were used for chromosomal DNA digestion followed by PFGE analysis, a total of 19, 20 and 19 PFGE patterns, respectively, were identified. Of them, 51 (47.7%), 52 (48.6%) and 42 (39.3%) strains belong to a single pattern of X3, S3 and N3, respectively, and 34 strains belong to a pattern combination of X3S3N3, which was the major subtype. When PFGE patterns of these 107 German isolates were compared with those of the 124 human isolates obtained in Taiwan, pattern combination of X3S3N3 was found as the most common pattern shared by isolates from both areas. PT4 is a major phage type for German and Taiwan isolates. Although most of the X3S3N3 strains are of this phage type, some strains of other PFGE patterns are also of this phage type. Since strains used in this study were unrelated, i.e., they were isolated from different origins in areas geographically far apart from each other, the PFGE study suggests a major world-wide clone of S. enterica serovar Enteritidis.

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States

PubMed Central

O'Hara, F. Patrick; Suaya, Jose A.; Ray, G. Thomas; Baxter, Roger; Brown, Megan L.; Mera, Robertino M.; Close, Nicole M.; Thomas, Elizabeth

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants. PMID:26669861

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

PubMed

O'Hara, F Patrick; Suaya, Jose A; Ray, G Thomas; Baxter, Roger; Brown, Megan L; Mera, Robertino M; Close, Nicole M; Thomas, Elizabeth; Amrine-Madsen, Heather

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

PubMed

Nakano, V; Ignacio, A; Llanco, L; Bueris, V; Sircili, M P; Avila-Campos, M J

2017-04-01

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates.

PubMed

Rodriguez, C; Taminiau, B; Brévers, B; Avesani, V; Van Broeck, J; Leroux, A A; Amory, H; Delmée, M; Daube, G

2014-08-06

Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual. Copyright © 2014 Elsevier B.V. All rights reserved.

Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students.

PubMed

Roberts, Marilyn C; Soge, Olusegun O; Horst, Jeremy A; Ly, Kiet A; Milgrom, Peter

2011-10-01

Methicillin-resistant Staphylococcus aureus (MRSA) isolated from frequently touched dental school clinic surfaces were compared with MRSA isolated nasal cultures of dental students. Sixty-one dental students and 95 environmental surfaces from 7 clinics were sampled using SANICULT (Starplex Scientific Inc, Etobicoke, Ontario, Canada) swabs. Antimicrobial susceptibility testing was performed, and pulsed-field gel electrophoresis analysis, the mecA gene, multilocus sequence type, and SCCmec type were determined by polymerase chain reaction and sequencing. Thirteen (21%) dental students and 8 (8.4%) surfaces were MRSA positive. Three MRSA strains were SCCmec type IV, whereas 3 were nontypeable isolates and Panton-Valentine leukocidin positive (PVL+), and none were USA300. One surface and 1 student isolate shared the same multilocus sequence type ST 8 and were 75% related. Two groups of students carried the same MRSA strains. The MRSA-positive samples were from 4 of 7 dental clinics. In addition, 21% of the dental students carried MRSA, which is > 10 times higher than the general public and twice as frequent as in other university students. This is the first study to characterize MRSA from dental clinic surfaces and dental students and suggests that both may be reservoirs for MRSA. Further studies are needed to verify this premise. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Serotypes, antibiotic susceptibilities, and multi-locus sequence type profiles of Streptococcus agalactiae isolates circulating in Beijing, China.

PubMed

Wang, Ping; Tong, Jing-jing; Ma, Xiu-hua; Song, Feng-li; Fan, Ling; Guo, Cui-mei; Shi, Wei; Yu, Sang-jie; Yao, Kai-hu; Yang, Yong-hong

2015-01-01

To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections. All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles. In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB. S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.

[Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates with toxic shock syndrome toxin and staphylococcal enterotoxin C genes].

PubMed

Kim, Jae Seok; Kim, Han Sung; Song, Wonkeun; Cho, Hyoun Chan; Lee, Kyu Man; Kim, Eui Chong

2007-04-01

Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.

Coagulase-Negative Staphylococci in Human Milk From Mothers of Preterm Compared With Term Neonates.

PubMed

Soeorg, Hiie; Metsvaht, Tuuli; Eelmäe, Imbi; Metsvaht, Hanna Kadri; Treumuth, Sirli; Merila, Mirjam; Ilmoja, Mari-Liis; Lutsar, Irja

2017-05-01

Human milk is the preferred nutrition for neonates and a source of bacteria. Research aim: The authors aimed to characterize the molecular epidemiology and genetic content of staphylococci in the human milk of mothers of preterm and term neonates. Staphylococci were isolated once per week in the 1st month postpartum from the human milk of mothers of 20 healthy term and 49 preterm neonates hospitalized in the neonatal intensive care unit. Multilocus variable-number tandem-repeats analysis and multilocus sequence typing were used. The presence of the mecA gene, icaA gene of the ica-operon, IS 256, and ACME genetic elements was determined by PCR. The human milk of mothers of preterm compared with term neonates had higher counts of staphylococci but lower species diversity. The human milk of mothers of preterm compared with term neonates more often contained Staphylococcus epidermidis mecA (32.7% vs. 2.6%), icaA (18.8% vs. 6%), IS 256 (7.9% vs. 0.9%), and ACME (15.4% vs. 5.1%), as well as Staphylococcus haemolyticus mecA (90.5% vs. 10%) and IS 256 (61.9% vs. 10%). The overall distribution of multilocus variable-number tandem-repeats analysis (MLVA) types and sequence types was similar between the human milk of mothers of preterm and term neonates, but a few mecA-IS 256-positive MLVA types colonized only mothers of preterm neonates. Maternal hospitalization within 1 month postpartum and the use of an arterial catheter or antibacterial treatment in the neonate increased the odds of harboring mecA-positive staphylococci in human milk. Limiting exposure of mothers of preterm neonates to the hospital could prevent human milk colonization with more pathogenic staphylococci.

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

PubMed Central

Fu, Ying; Du, Xiaoxing; Shen, Yuqin; Yu, Yunsong

2015-01-01

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla KPC, bla IMP, bla VIM, bla OXA-48 and bla NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla KPC-2 genes, and five bla NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla NDM-1 (bla NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla NDM-1 plasmids contained a common gene environment around bla NDM-1 (IS5-bla NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla NDM-1 gene among the CRE. PMID:26047502

Clonal Origins of Vibrio cholerae O1 El Tor Strains, Papua New Guinea, 2009–2011

PubMed Central

Collins, Deirdre; Jonduo, Marinjho H.; Rosewell, Alexander; Dutta, Samir R.; Dagina, Rosheila; Ropa, Berry; Siba, Peter M.; Greenhill, Andrew R.

2011-01-01

We used multilocus sequence typing and variable number tandem repeat analysis to determine the clonal origins of Vibrio cholerae O1 El Tor strains from an outbreak of cholera that began in 2009 in Papua New Guinea. The epidemic is ongoing, and transmission risk is elevated within the Pacific region. PMID:22099099

Influence of Seasonality on the Genetic Diversity of Vibrio parahaemolyticus in New Hampshire Shellfish Waters as Determined by Multilocus Sequence Analysis

PubMed Central

Ellis, Crystal N.; Schuster, Brian M.; Striplin, Megan J.; Jones, Stephen H.; Whistler, Cheryl A.

2012-01-01

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed. PMID:22407686

Influence of seasonality on the genetic diversity of Vibrio parahaemolyticus in New Hampshire shellfish waters as determined by multilocus sequence analysis.

PubMed

Ellis, Crystal N; Schuster, Brian M; Striplin, Megan J; Jones, Stephen H; Whistler, Cheryl A; Cooper, Vaughn S

2012-05-01

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed.

Multilocus sequence typing of Metarhizium anisopliae var acridum isolates as microbial agents for locust and grasshopper control. Genbank Accession numbers FJ787311 to FJ787325

USDA-ARS?s Scientific Manuscript database

A growing interest in the biological control of locusts and grasshoppers (Acrididae) has led to the development of biopesticides based on naturally occurring pathogens which offers an environmentally safe alternative to chemical pesticides. However, the fungal strains which are being sought for biop...

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

PubMed

Bleidorn, Christoph; Gerth, Michael

2018-01-01

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multilocus sequence identification of Penicillium species in cork bark during plank preparation for the manufacture of stoppers.

PubMed

Serra, Rita; Peterson, Stephen; Venâncio, Armando

2008-04-01

Despite several studies reporting Penicillium as one of the most frequent fungal genera in cork planks, the isolates were rarely identified to species level. We conducted a detailed study to identify Penicillium species from the field to the factory environment prior to and after boiling the cork planks. A total of 84 samples were analyzed. Of the 486 Penicillium isolates phenotypically identified, 32 representative or unusual strains were selected for identification by multilocus DNA sequence type. Cork proved to be a rich source of Penicillium biodiversity. A total of 30 taxa were recognized from cork including rarely seen species and 6 phylogenetically unique groups. Spores of some species lodged deep in cork can survive the boiling process. P. glabrum, P. glandicola and P. toxicarium, species with high CFU numbers in the field, are still frequently present in cork after boiling. Other species are killed by the boiling treatment and replaced by Penicillium species originating from the factory environment. Species known to contribute to cork taint were isolated at all stages. Good manufacturing practices are necessary at all stages in the preparation of cork planks to minimize the load of Penicillium species that produce cork taint.