Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

PubMed Central

Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter

2017-01-01

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other’s behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved. PMID:28263995

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

PubMed Central

Mansson, Maria; Nielsen, Anita; Kjærulff, Louise; Gotfredsen, Charlotte H.; Wietz, Matthias; Ingmer, Hanne; Gram, Lone; Larsen, Thomas O.

2011-01-01

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium. PMID:22363239

Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes.

PubMed

Kamiya, Regianne Umeko; Höfling, José Francisco; Gonçalves, Reginaldo Bruno

2008-05-01

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I-IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=-0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

Involvement of methicillin-susceptible Staphylococcus aureus related to sequence type 25 and harboring pvl genes in a case of carotid cavernous fistula after community-associated sepsis.

PubMed

Damasco, Paulo V; Chamon, Raiane C; Barbosa, Angélica T L; da Cunha, Sérgio; Aquino, José H W; Cavalcante, Fernanda S; Dos Santos, Kátia R N

2012-01-01

Staphylococcus aureus encoding Panton-Valentine leukocidin (PVL) genes has become the cause of life-threatening infections. We describe a case of carotid cavernous fistula after bacteremia in a 12-year-old male, caused by a methicillin-susceptible S. aureus isolate carrying the pvl, fnbA, and ebpS genes and related to sequence type 25 (ST25). The patient's condition was complicated by pleural empyema and osteomyelitis in the right femur. The patient was discharged in good clinical condition after 160 days of hospitalization.

Identification of the novel lincosamide resistance gene lnu(E) truncated by ISEnfa5-cfr-ISEnfa5 insertion in Streptococcus suis: de novo synthesis and confirmation of functional activity in Staphylococcus aureus.

PubMed

Zhao, Qin; Wendlandt, Sarah; Li, Hui; Li, Jun; Wu, Congming; Shen, Jianzhong; Schwarz, Stefan; Wang, Yang

2014-01-01

The novel lincosamide resistance gene lnu(E), truncated by insertion of an ISEnfa5-cfr-ISEnfa5 segment, was identified in Streptococcus suis. The gene lnu(E) encodes a 173-amino-acid protein with ≤69.4% identity to other lincosamide nucleotidyltransferases. The lnu(E) gene and its promoter region were de novo synthesized, and Staphylococcus aureus RN4220 carrying a shuttle vector with the cloned lnu(E) gene showed a 16-fold increase in the lincomycin MIC. Mass spectrometry experiments demonstrated that Lnu(E) catalyzed the nucleotidylation of lincomycin.

Identification of the Novel Lincosamide Resistance Gene lnu(E) Truncated by ISEnfa5-cfr-ISEnfa5 Insertion in Streptococcus suis: De Novo Synthesis and Confirmation of Functional Activity in Staphylococcus aureus

PubMed Central

Zhao, Qin; Wendlandt, Sarah; Li, Hui; Li, Jun; Wu, Congming; Shen, Jianzhong

2014-01-01

The novel lincosamide resistance gene lnu(E), truncated by insertion of an ISEnfa5-cfr-ISEnfa5 segment, was identified in Streptococcus suis. The gene lnu(E) encodes a 173-amino-acid protein with ≤69.4% identity to other lincosamide nucleotidyltransferases. The lnu(E) gene and its promoter region were de novo synthesized, and Staphylococcus aureus RN4220 carrying a shuttle vector with the cloned lnu(E) gene showed a 16-fold increase in the lincomycin MIC. Mass spectrometry experiments demonstrated that Lnu(E) catalyzed the nucleotidylation of lincomycin. PMID:24366733

Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species

PubMed Central

Ussery, David; Nielsen, Lene N.; Ingmer, Hanne

2015-01-01

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera. PMID:25883793

Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species.

PubMed

Wassenaar, Trudy M; Ussery, David; Nielsen, Lene N; Ingmer, Hanne

2015-03-01

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera.

Occurrence of virulence-associated genes among Staphylococcus saprophyticus isolated from different sources.

PubMed

de Paiva-Santos, Weslley; de Sousa, Viviane Santos; Giambiagi-deMarval, Marcia

2018-03-28

Staphylococcus saprophyticus is an important pathogen responsible for community urinary tract infections (UTI). Besides composing the human microbiota, this species is widely distributed in the environment and the origins of this organism for human infection is not fully characterized. Although some virulence determinants are known, such as d-serine deaminase (DsdA), urease and cell-wall associated proteins, few studies investigated the distribution of virulence-associated genes and analyzed the pathogenic potential of S. saprophyticus strains from different sources. The aim of the present study was to detect the presence of S. saprophyticus genes encoding surface proteins UafA, Aas, Ssp, SdrI, SssF as well as the DsdA and urease enzymes. A total of 142 S. saprophyticus strains were obtained from four sources: UTI, colonization, water and food. It was found, in every tested strain, the presence of genes encoding the surface proteins UafA, Aas, Ssp and SssF and the DsdA and urease enzymes. In contrast, the gene encoding SdrI surface protein was not detected in any of the strains of S. saprophyticus. These results provide a better understanding of the characteristics of S. saprophyticus strains and suggest that isolates from non-human sources have a potential to colonize the urinary tract. Copyright © 2018 Elsevier Ltd. All rights reserved.

Occurrence of Salmonella, Listeria monocytogenes, and enterotoxigenic Staphylococcus in goat milk from small and medium-sized farms located in Minas Gerais State, Brazil.

PubMed

Cavicchioli, V Q; Scatamburlo, T M; Yamazi, A K; Pieri, F A; Nero, L A

2015-12-01

Consumption of goat milk has been increasing due to its nutritional characteristics and health benefits. Therefore, assessment of the presence of foodborne pathogens in this product is critical to ensure its safety to consumers. The present study aimed to identify common foodborne pathogens in raw goat milk. Fifty-three samples of raw goat milk from 11 farms were collected and cultured for the presence of Salmonella spp. and Listeria monocytogenes, as well as for enumeration and isolation of coagulase-positive and coagulase-negative Staphylococcus (CPS and CNS, respectively). All samples tested negative for Salmonella spp. and L. monocytogenes. The CPS counts in raw goat milk samples were predominantly less than 2 log cfu/mL (n=39), and CNS counts were predominantly higher than 3 log cfu/mL (n=42). Based on Staphylococcus counts, 51 isolates were selected (CPS=26; CNS=25) and tested by PCR for the presence of classic enterotoxin-encoding genes (sea, seb, sec, sed, and see). Only 3 isolates (CPS=2, CNS=1) were negative for all enterotoxin-encoding genes tested, and the genotype sec and see was the most frequent (n=16), followed by sea, sec, and see (n=13) and sec (n=13); sed was not detected in any isolate. The frequencies of enterotoxin-encoding genes for CPS and CNS were similar, demonstrating the equivalence of both groups in harboring these virulent markers. These results suggest that Salmonella and L. monocytogenes are not frequent contaminants of raw goat milk, but that Staphylococcus spp. that are capable of producing enterotoxins are prevalent; therefore, consumers of raw goat milk and products made from raw milk are at risk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A study of Staphylococcus aureusnasal carriage, antibacterial resistance and virulence factor encoding genes in a tertiary care hospital, Kayseri, Turkey.

PubMed

Oguzkaya-Artan, M; Artan, C; Baykan, Z; Sakalar, C; Turan, A; Aksu, H

2015-01-01

This study was to determine the virulence encoding genes, and the antibiotic resistance patterns of the Staphylococcus aureus isolates, which were isolated from the nasal samples of chest clinic patients. The nasal samples of the in-patients (431) and out-patients (1857) in Kayseri Training and Research Hospital's Chest Clinic, Kayseri, Turkey, were cultured on CHROMagar (Biolife, Italiana) S. aureus, and subcultured on sheep blood agar for the isolation of S. aureus. Disc diffusion method was used for antimicrobial susceptibility testing. The occurrence of the staphylococcal virulence encoding genes (enterotoksins [sea, seb, sec, see, seg, seh, sei, sej], fibronectin-binding proteins A, B [fnbA, fnbB], toxic shock syndrome toxin-1 [tst]) were detected by polymerase chain reaction. Forty-five of the 55 (81.8%) S. aureus isolates from inpatients, and 319 (90.6%) isolates from tested 352 out-patient's isolates were suspected to all the antibiotics tested. methicillin-resistant S. aureus (MRSA) was detected in 1.2% of S. aureus isolates. Rifampin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, gentamicin resistance rates were 1.2%, 1.7%, 2.0%, 8.8%, and 1.2%, respectively. The isolates were susceptible to teicoplanin and vancomycin. The genes most frequently found were tst (92.7%), seg (85.8%), sea (83.6%), fnbA (70.9%). There was no statistical significance detected between MRSA and mecA-negative S. aureus isolates in encoding genes distribution (P > 0.05). Our results show that virulence factor encoding genes were prevalent in patients with S. aureus carriage, whereas antibiotic resistance was low. These virulence determinants may increase the risk for subsequent invasive infections in carriers.

Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters.

PubMed

Marty, Esther; Bodenmann, Chantal; Buchs, Jasmin; Hadorn, Ruedi; Eugster-Meier, Elisabeth; Lacroix, Christophe; Meile, Leo

2012-10-01

To provide new meat starter strains lacking antibiotic (AB) resistances, we explored the AB susceptibility in 116 coagulase-negative Staphylococcus (CNS) isolates from traditionally fermented sausages (n=40) manufactured with meat from conventional animal breeding, and from meat products (n=76) made from meat of animals raised in natural habitats under low- or no-antibiotic pressure. Less than 50% of these CNS isolates showed phenotypic resistances to at least one antibiotic (AB) by using microdilution assay. Resistances to penicillins and tetracycline were most often observed and could be traced back to blaZ and tet(K) genes. Prevalence of AB resistances was species-dependent and mainly found in isolates of Staphylococcus warneri (78%), Staphylococcus capitis (75%) and Staphylococcus epidermidis (67%), but only sporadically detected in Staphylococcus carnosus (27%) and Staphylococcus equorum (18%). AB resistances were more often observed in S. xylosus isolates originating from natural habitats compared to traditionally fermented sausages made from conventional meat. A selection of 101 isolates belonging to S. xylosus (n=63), S. carnosus (n=21) and S. equorum (n=17) were subsequently grouped by pulsed-field gel electrophoresis (PFGE) into strain clusters. No S. carnosus and only five S. xylosus strains were lacking AB resistances and exhibited a PFGE genotype different from commercial starters. These strains, together with 17 S. equorum strains, were further studied for safety and technological characteristics. The ability to produce biogenic amines was not detected in any strain. PCR amplifications for enterotoxin encoding genes seg-sej were detected in one, and for δ-hemolysin encoding gene hld in four S. equorum strains, but phenotypic hemolytic activity was visible for three S. xylosus and 15 S. equorum strains. Catalase and nitrate reductase activity was observed in all isolates tested; particularly S. equorum showed high nitrate reduction. In conclusion, we were able to select four new meat starter strains (two S. xylosus and two S. equorum strains) out of 116 investigated CNS, fulfilling all safety criteria including the absence of AB resistances, production of biogenic amines and genes encoding virulence factors but exhibiting high nitrate reductase and catalase activity as suitable technological characteristics. Thus, S. equorum isolates, often the dominant species in spontaneously fermented meat products, provided a prospective meat starter species exhibiting high nitrate reduction and low prevalence of AB resistances. Copyright © 2012 Elsevier B.V. All rights reserved.

Involvement of Methicillin-Susceptible Staphylococcus aureus Related to Sequence Type 25 and Harboring pvl Genes in a Case of Carotid Cavernous Fistula after Community-Associated Sepsis

PubMed Central

Damasco, Paulo V.; Chamon, Raiane C.; Barbosa, Angélica T. L.; da Cunha, Sérgio; Aquino, José H. W.; Cavalcante, Fernanda S.

2012-01-01

Staphylococcus aureus encoding Panton-Valentine leukocidin (PVL) genes has become the cause of life-threatening infections. We describe a case of carotid cavernous fistula after bacteremia in a 12-year-old male, caused by a methicillin-susceptible S. aureus isolate carrying the pvl, fnbA, and ebpS genes and related to sequence type 25 (ST25). The patient's condition was complicated by pleural empyema and osteomyelitis in the right femur. The patient was discharged in good clinical condition after 160 days of hospitalization. PMID:22090398

Adaptation of Staphylococcus xylosus to Nutrients and Osmotic Stress in a Salted Meat Model

PubMed Central

Vermassen, Aurore; Dordet-Frisoni, Emilie; de La Foye, Anne; Micheau, Pierre; Laroute, Valérie; Leroy, Sabine; Talon, Régine

2016-01-01

Staphylococcus xylosus is commonly used as starter culture for meat fermentation. Its technological properties are mainly characterized in vitro, but the molecular mechanisms for its adaptation to meat remain unknown. A global transcriptomic approach was used to determine these mechanisms. S. xylosus modulated the expression of about 40–50% of the total genes during its growth and survival in the meat model. The expression of many genes involved in DNA machinery and cell division, but also in cell lysis, was up-regulated. Considering that the S. xylosus population remained almost stable between 24 and 72 h of incubation, our results suggest a balance between cell division and cell lysis in the meat model. The expression of many genes encoding enzymes involved in glucose and lactate catabolism was up-regulated and revealed that glucose and lactate were used simultaneously. S. xylosus seemed to adapt to anaerobic conditions as revealed by the overexpression of two regulatory systems and several genes encoding cofactors required for respiration. In parallel, genes encoding transport of peptides and peptidases that could furnish amino acids were up-regulated and thus concomitantly a lot of genes involved in amino acid synthesis were down-regulated. Several genes involved in glutamate homeostasis were up-regulated. Finally, S. xylosus responded to the osmotic stress generated by salt added to the meat model by overexpressing genes involved in transport and synthesis of osmoprotectants, and Na+ and H+ extrusion. PMID:26903967

Staphylococcus aureus nasal carriage in Ukraine: antibacterial resistance and virulence factor encoding genes.

PubMed

Netsvyetayeva, Irina; Fraczek, Mariusz; Piskorska, Katarzyna; Golas, Marlena; Sikora, Magdalena; Mlynarczyk, Andrzej; Swoboda-Kopec, Ewa; Marusza, Wojciech; Palmieri, Beniamino; Iannitti, Tommaso

2014-03-05

The number of studies regarding the incidence of multidrug resistant strains and distribution of genes encoding virulence factors, which have colonized the post-Soviet states, is considerably limited. The aim of the study was (1) to assess the Staphylococcus (S.) aureus nasal carriage rate, including Methicillin Resistant S. aureus (MRSA) strains in adult Ukrainian population, (2) to determine antibiotic resistant pattern and (3) the occurrence of Panton Valentine Leukocidine (PVL)-, Fibronectin-Binding Protein A (FnBPA)- and Exfoliative Toxin (ET)-encoding genes. Nasal samples for S. aureus culture were obtained from 245 adults. The susceptibility pattern for several classes of antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The virulence factor encoding genes, mecA, lukS-lukF, eta, etb, etd, fnbA, were detected by Polymerase Chain Reaction (PCR). The S. aureus nasal carriage rate was 40%. The prevalence of nasal MRSA carriage in adults was 3.7%. LukS-lukF genes were detected in over 58% of the strains. ET-encoding genes were detected in over 39% of the strains and the most prevalent was etd. The fnbA gene was detected in over 59% of the strains. All MRSA isolates tested were positive for the mecA gene. LukS-lukF genes and the etd gene were commonly co-present in MRSA, while lukS-lukF genes and the fnbA gene were commonly co-present in Methicillin Sensitive S. aureus (MSSA) isolates. No significant difference was detected between the occurrence of lukS-lukF genes (P > 0.05) and the etd gene (P > 0.05) when comparing MRSA and MSSA. The occurrence of the fnbA gene was significantly more frequent in MSSA strains (P < 0.05). In Ukraine, S. aureus is a common cause of infection. The prevalence of S. aureus nasal carriage in our cohort of patients from Ukraine was 40.4%. We found that 9.1% of the strains were classified as MRSA and all MRSA isolates tested positive for the mecA gene. We also observed a high prevalence of PVL- and ET- encoding genes among S. aureus nasal carriage strains. A systematic surveillance system can help prevent transmission and spread of drug resistant toxin producing S. aureus strains.

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

PubMed

Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

2016-08-01

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

PubMed

Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

PubMed

Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

PubMed Central

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models. PMID:28859130

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.

Methicillin-Susceptible Teicoplanin-Resistant Staphylococcus haemolyticus Isolate from a Bloodstream Infection with Novel Mutations in the tcaRAB Teicoplanin Resistance Operon.

PubMed

Bakthavatchalam, Yamuna Devi; Sudarsanam, Thambu David; Babu, Priyanka; Munuswamy, Elakkiya; Muthuirulandi Sethuvel, Dhiviya Prabaa; Devanga Ragupathi, Naveen Kumar; Veeraraghavan, Balaji

2017-07-24

Staphylococcus haemolyticus is a coagulase-negative staphylococcus that is frequently isolated from blood cultures. Here, we report a case of methicillin-susceptible S. haemolyticus that is resistant to teicoplanin (TEC) and heteroresistant to vancomycin (VAN). The isolate was susceptible to cefoxitin and resistant to TEC by Etest. Population analysis profile-area under the curve analysis confirmed the presence of a VAN heteroresistant subpopulation. Next-generation sequencing analysis of the genome revealed the presence of blaZ and msr(A), which encode cross-resistance to macrolide, lincosamide, and streptogramin B, and the quinolone resistance-conferring gene norA. In addition, several amino acid substitutions were observed in the TEC resistance operon tcaRAB, including I3N, I390N, and L450I in tcaA and L44V, G52V, and S87P in tcaR, as well as in the transpeptidase encoding gene walK (D336Y, R375L, and V404A) and L315 and P316 in graS. We hypothesized that this combination of mutations could confer TEC resistance and reduced VAN susceptibility.

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus.

PubMed

Chatterjee, Som S; Chen, Liang; Joo, Hwang-Soo; Cheung, Gordon Y C; Kreiswirth, Barry N; Otto, Michael

2011-01-01

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.

Distribution of the ACME-arcA gene among methicillin-resistant Staphylococcus aureus from England and Wales.

PubMed

Ellington, Matthew J; Yearwood, Lianne; Ganner, Mark; East, Claire; Kearns, Angela M

2008-01-01

The ST8-SCCmecIVa (USA300) methicillin-resistant Staphylococcus aureus (MRSA) clone can harbour the arginine catabolic mobile element (ACME). The arc gene cluster within the ACME may function as a virulence or strain survival factor. We determined the distribution of the ACME-associated arcA gene among genetically diverse MRSA from around England and Wales. MRSA isolates (n = 203) of diverse genetic types, referred to the England and Wales Staphylococcus reference laboratory, were tested for the presence of the ACME-arcA gene. ACME-arcA-positive isolates were characterized by toxin gene profiling, PFGE and spa sequence typing. MICs of a range of antimicrobials were also determined. The ACME-arcA gene was detected in 17 isolates. Twelve were related to known ST8-MRSA-SCCmecIVa isolates of the USA300 lineage by pulsotype and were resistant to oxacillin, with variable ciprofloxacin and erythromycin resistance. Outside the USA300 lineage, four of the remaining five ACME-arcA isolates were closely related ST97-MRSA-SCCmecV, Panton-Valentine leucocidin (PVL)-negative, resistant to oxacillin and variously resistant to erythromycin, ciprofloxacin, clindamycin, gentamicin, tetracycline and fusidic acid. The remaining isolate was ST1, PVL-positive and resistant to fusidic acid as well as oxacillin. Thirteen out of the 17 isolates were associated with skin and soft tissue infections. The detection of ACME-arcA in diverse MRSA types highlights the mobility of the elements encoding ACME-arcA genes. The diversity of strain types and resistance profiles among ACME-arcA-encoding MRSA is a cause for public-health concern and demands continued surveillance and close monitoring.

Effect of antibiotic prophylaxis on Coagulase-negative Staphylococcus virulence factor profiles in patients undergoing cataract surgery.

PubMed

López, Yolanda; Samudio, Margarita; Fariña, Norma; Castillo, Verónica; Abente, Sonia; Nentwich, Martin M; González-Britez, Nilsa; Laspina, Florentina; Carron, Agustín; Cibils, Diógenes; de Kaspar, Herminia Miño

2017-08-01

In this prospective study, multiplex polymerase chain reaction (PCR) was used to identify genes encoding virulence factors (ica, atlE and mecA) in Coagulase-negative Staphylococcus (CNS) isolates from the ocular microbiota of patients undergoing cataract surgery and to investigate possible changes in the CNS profile due to antibiotic prophylaxis. Between 09/2011 and 08/2013, patients undergoing cataract surgery were recruited at the Department of Ophthalmology, National University of Asuncion, Paraguay. In the eye to be operated on, patients received moxifloxacin 0.5 % eye drops four times at the day before surgery and a last drop 1 hour before surgery (T1). The other eye remained as control (T0). Conjunctival swabs were taken from both eyes 1 hour after the last drop. The presence of genes encoding biofilm formation (ica and atlE) and methicillin resistance (mecA) was detected by a multiplex PCR. Of the 162 patients (162 study eyes, 162 fellow eye as control group), 87 (53.7 %) eyes were positive for CNS at T0 yielding 96 CNS isolates; 70 eyes (43.2 %) were positive at T1 yielding 77 CNS isolates. For this study, 43 CNS isolates (44.8 %) from T0 and 45 (64.3 %) from T1 were used. Of the total isolates, 81.8 % (72/88) had at least one virulence factor gene (37/43 from T0 and 35/45 from T1) (p = 0.314). Simultaneous detection of ica and atlE genes was higher in T0 (58.0 %) than T1 (46.7 %), but the difference was not significant (p = 0.28). A high frequency of genes encoding virulence factors was observed in the coagulase-negative Staphylococcus isolates. The use of moxifloxacin did not significantly modify the CNS virulence factor profiles.

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia.

PubMed

Jiménez, Judy Natalia; Ocampo, Ana María; Vanegas, Johanna Marcela; Rodríguez, Erika Andrea; Garcés, Carlos Guillermo; Patiño, Luz Adriana; Ospina, Sigifredo; Correa, Margarita María

2011-12-01

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.

The effect of antimicrobial photodynamic therapy on the expression of novel methicillin resistance markers determined using cDNA-AFLP approach in Staphylococcus aureus.

PubMed

Hoorijani, Mohammad Neshvan; Rostami, Hosein; Pourhajibagher, Maryam; Chiniforush, Nasim; Heidari, Mansour; Pourakbari, Babak; Kazemian, Hossein; Davari, Kambiz; Amini, Vahid; Raoofian, Reza; Bahador, Abbas

2017-09-01

Widespread methicillin resistant Staphylococcus aureus (MRSA) and absence of effective antimicrobial agents has led to limited therapeutic options for treating MRSA infection. We aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the expression of novel identified methicillin resistance markers (NIMRMs) in S. aureus using complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) approaches to address the therapeutic alternatives for MRSA infections. We used cDNA-AFLP to compare MRSA and methicillin susceptible S. aureus (MSSA) for identification of target genes implicated in methicillin resistance. To determine the sub-lethal aPDT (sPDT), MRSA and MSSA clinical isolates photosensitized with toluidine blue O (TBO), and then were irradiated with diode laser. After sPDT, the colony forming units/mL was quantified. Antimicrobial susceptibility against methicillin was assessed for cell-surviving aPDT. Effects of sPDT on the expression of NIMRMs were evaluated by real-time quantitative reverse transcription PCR. According to our results, serine hydrolase family protein (Shfp) encoding gene and a gene encoding a conserved hypothetical protein (Chp) were implicated in methicillin resistance in MRSA. sPDT reduced the minimum inhibitory concentrations of methicillin by 3-fold in MRSA. sPDT could lead to about 10- and 6.2- fold suppression of expression of the Chp and Shfp encoding genes, respectively. sPDT would lead to reduction in resistance to methicillin of MRSA in surviving cells by suppressing the expression of the Shfp and Chp encoding genes associated with methicillin resistance. This may have potential implications of aPDT for the treatment of MRSA infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Staphylococcus aureus toxin gene hitchhikes on a transferable antibiotic resistance element.

PubMed

Otto, Michael

2010-01-01

Virulence and antibiotic resistance of the dangerous human pathogen Staphylococcus aureus are to large extent determined by the acquisition of mobile genetic elements (MGEs). Up to now, these elements were known to comprise either resistance or virulence determinants, but not a mixture of the two. Queck et al. now found a cytolysin gene of the phenol-soluble modulin (PSM) family within SCCmec elements, which contain methicillin resistance genes and are largely responsible for the spread of methicillin-resistant S. aureus (MRSA). The novel gene, called psm-mec, had a significant impact on virulence in MRSA strains that do not produce high levels of genome-encoded PSMs. This first example of a combination of toxin and resistance genes on one staphylococcal MGE shows that such bundling is possible and may lead to an even faster acquisition of toxin and resistance genes by S. aureus and other staphylococcal pathogens.

Biofilm density and detection of biofilm-producing genes in methicillin-resistant Staphylococcus aureus strains.

PubMed

Szczuka, Ewa; Urbańska, Katarzyna; Pietryka, Marta; Kaznowski, Adam

2013-01-01

Many serious diseases caused by Staphylococcus aureus appear to be associated with biofilms. Therefore, we investigated the biofilm-forming ability of the methicillin-resistant S. aureus (MRSA) isolates collected from hospitalized patients. As many as 96 % strains had the ability to form biofilm in vitro. The majority of S. aureus strains formed biofilm in ica-dependent mechanism. However, 23 % of MRSA isolates formed biofilm in ica-independent mechanism. Half of these strains carried fnbB genes encoding surface proteins fibronectin-binding protein B involved in intercellular accumulation and biofilm development in S. aureus strains. The biofilm structures were examined via confocal laser scanning microscopy (CLSM) and three-dimensional structures were reconstructed. The images obtained in CLSM revealed that the biofilm created by ica-positive strains was different from biofilm formed by ica-negative strains. The MRSA population showed a large genetic diversity and we did not find a single clone that occurred preferentially in hospital environment. Our results demonstrated the variation in genes encoding adhesins for the host matrix proteins (elastin, laminin, collagen, fibronectin, and fibrinogen) and in the gene involved in biofilm formation (icaA) within the majority of S. aureus clones.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

PubMed

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE PAGES

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.; ...

2017-08-31

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Cell Density Control of Staphylococcal Virulence Mediated by an Octapeptide Pheromone

NASA Astrophysics Data System (ADS)

Ji, Guangyong; Beavis, Ronald C.; Novick, Richard P.

1995-12-01

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

PubMed

Zuniga, Eveline; Melville, Priscilla A; Saidenberg, André B S; Laes, Marco A; Gonsales, Fernanda F; Salaberry, Sandra R S; Gregori, Fabio; Brandão, Paulo E; dos Santos, Franklin G B; Lincopan, Nilton E; Benites, Nilson R

2015-12-01

This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

PubMed

Santosaningsih, Dewi; Santoso, Sanarto; Setijowati, Nanik; Rasyid, Harun A; Budayanti, Nyoman S; Suata, Ketut; Widhyatmoko, Dicky B; Purwono, Priyo B; Kuntaman, Kuntaman; Damayanti, Damayanti; Prakoeswa, Cita R S; Laurens, Mitchell; van Nierop, Josephine W I; Nanninga, Geraldine L; Oudenes, Neline; de Regt, Michelle; Snijders, Susan V; Verbrugh, Henri A; Severin, Juliëtte A

2018-01-01

To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community. © 2017 John Wiley & Sons Ltd.

Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.

PubMed

Queck, Shu Y; Khan, Burhan A; Wang, Rong; Bach, Thanh-Huy L; Kretschmer, Dorothee; Chen, Liang; Kreiswirth, Barry N; Peschel, Andreas; Deleo, Frank R; Otto, Michael

2009-07-01

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

PubMed

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes

PubMed Central

Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E.

2018-01-01

We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes. PMID:25720844

Virulence properties of methicillin-susceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene.

PubMed

Sudagidan, Mert; Aydin, Ali

2010-04-15

In this study, three Panton-Valentine Leukocidin gene carrying methicillin-susceptible Staphylococcus aureus (MSSA) strains (M1-AAG42B, PY30C-b and YF1B-b) were isolated from different food samples in Kesan-Edirne, Turkey. These strains were characterized on the basis of MLST type, spa type, virulence factor gene contents, antibiotic susceptibilities against 21 antibiotics and biofilm formation. The genetic relatedness of the strains was determined by PFGE. In addition, the complete gene sequences of lukS-PV and lukF-PV were also investigated. All strains were found to be susceptible to tested antibiotics and they were mecA negative. Three strains showed the same PFGE band pattern, ST152 clonal type and t355 spa type. In the detection of virulence factor genes, sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, seu, eta, etb, set1, geh and tst genes were not detected. All strains showed the positive results for alpha- and beta-haemolysin genes (hla and hlb), protease encoding genes (sspA, sspB and aur), lukE and lukD leukocidin genes (lukED). The strains were found to be non-biofilm formers. By this study, the virulence properties of the strains were described and this is one of the first reports regarding PVL-positive MSSA strains from food. (c) 2010 Elsevier B.V. All rights reserved.

Draft genome sequences of 50 MRSA ST5 isolates obtained from a U.S. hospital

USDA-ARS?s Scientific Manuscript database

Methicillin resistant Staphylococcus aureus (MRSA) can be a commensal or pathogen in humans. Pathogenicity and disease are related to the acquisition of mobile genetic elements encoding virulence and antimicrobial resistance genes. Here, we report draft genome sequences for 50 clinical MRSA isolates...

Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri.

PubMed

Iwase, Tadayuki; Seki, Keiko; Shinji, Hitomi; Mizunoe, Yoshimitsu; Masuda, Shogo

2007-10-01

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3' end of the primer (3' mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (10(1)-10(7) cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-04-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

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.

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

PubMed Central

Goerke, Christiane; Köller, Johanna; Wolz, Christiane

2006-01-01

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization.

PubMed

Mahmmod, Yasser S; Klaas, Ilka Christine; Svennesen, Line; Pedersen, Karl; Ingmer, Hanne

2018-05-16

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative β-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding protein A of S. aureus, respectively. Our NAS isolates showed 3 main patterns: (1) downregulation effect such as S. chromogenes (milk) and Staphylococcus xylosus (milk and teat), (2) no effect such as Staphylococcus sciuri (teat) and S. vitulinus (teat), and the third pattern (c) variable effect such as S. epidermidis (milk and teat) and S. equorum (milk and teat). The pattern of cross-talk between NAS species and S. aureus virulence genes varied according to the involved NAS species, habitat type, and herd factors. The knowledge of how NAS influences S. aureus virulence factor expression could explain the varying protective effect of NAS on S. aureus intramammary infections. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Phenotypic and Molecular Antibiotic Resistance Determination of Airborne Coagulase Negative Staphylococcus spp. Strains from Healthcare Facilities in Southern Poland.

PubMed

Lenart-Boroń, Anna; Wolny-Koładka, Katarzyna; Stec, Joanna; Kasprowic, Andrzej

2016-10-01

This study assessed the antimicrobial resistance of airborne Staphylococcus spp. strains isolated from healthcare facilities in southern Poland. A total of 55 isolates, belonging to 10 coagulase-negative staphylococci (CoNS) species, isolated from 10 healthcare facilities (including hospitals and outpatient units) were included in the analysis. The most frequently identified species were Staphylococcus saprophyticus and Staphylococcus warneri, which belong to normal human skin flora, but can also be the cause of common and even severe nosocomial infections. Disk diffusion tests showed that the bacterial strains were most frequently resistant to erythromycin and tetracycline and only 18% of strains were susceptible to all tested antimicrobials. Polymerase chain reaction amplification of specific gene regions was used to determine the presence of the Macrolide-Lincosamide-Streptogramin resistance mechanisms in CoNS. The molecular analysis, conducted using specific primer pairs, identified the msrA1 gene, encoding active efflux pumps in bacterial cells, as the most frequent resistance gene. As many as seven antibiotic resistance genes were found in one isolate, whereas the most common number of resistance genes per isolate was five (n = 17). It may be concluded that drug resistance was widely spread among the tested strains, but the resulting antimicrobial resistance profile indicates that in the case of infection, the use of antibiotics from the basic antibiogram group will be effective in therapy. However, before administering treatment, determination of the specific antimicrobial resistance should be conducted, particularly in the case of hospitalized patients.

eap Gene as novel target for specific identification of Staphylococcus aureus.

PubMed

Hussain, Muzaffar; von Eiff, Christof; Sinha, Bhanu; Joost, Insa; Herrmann, Mathias; Peters, Georg; Becker, Karsten

2008-02-01

The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n = 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.

First description of PVL-positive methicillin-resistant Staphylococcus aureus (MRSA) in wild boar meat.

PubMed

Kraushaar, Britta; Fetsch, Alexandra

2014-09-01

Staphylococcus aureus is an important food-borne pathogen due to the ability of enterotoxigenic strains to produce staphylococcal enterotoxins (SEs) in food. Methicillin-resistant S. aureus (MRSA) is also an important pathogen for humans, causing severe and hard to treat diseases in hospitals and in the community due to its multiresistance against antimicrobials. In particular, strains harbouring genes encoding for the Panton-Valentine leukocidin (PVL) toxin are of concern from a public health perspective as they are usually capable of causing severe skin and soft tissue infections (sSSTIs) and occasionally necrotizing pneumonia which is associated with high mortality. This is the first report on the detection of MRSA with genes encoding for PVL in wild boar meat. Among the 28 MRSA isolated from wild boar meat in the course of a national monitoring programme in Germany, seven harboured PVL-encoding genes. Six of the isolates were identical according to the results of spa-, MLST-, microarray- and PFGE-typing. They could be assigned to the epidemic MRSA clone USA300. Epidemiological investigations revealed that people handling the food were the most likely common source of contamination with these MRSA. These findings call again for suitable hygienic measures at all processing steps of the food production chain. The results of the study underline that monitoring along the food chain is essential to closely characterise the total burden of MRSA for public health. Copyright © 2014. Published by Elsevier B.V.

Genetic linkage between resistance to quaternary ammonium compounds and beta-lactam antibiotics in food-related Staphylococcus spp.

PubMed

Sidhu, M S; Heir, E; Sørum, H; Holck, A

2001-01-01

Little is known about the occurrence of antimicrobial resistance determinants in staphylococci isolated from food and food processing industries. Quaternary ammonium compound (QAC)-resistant coagulase-negative staphylococci (CNS) isolated from food and food-processing industries were investigated for the presence of genetic determinants (qacA/B and qacC/smr) encoding resistance to the QAC benzalkonium chloride (BC), several antibiotic resistance genes, and staphylococcal insertion sequences IS257 and IS256. Six qacA/B-harboring strains were resistant to penicillin and hybridized to a blaZ probe. The qacA/B and blaZ probes hybridized to plasmids of similar size in three isolates. Molecular and genetic characterization of the 23-kb plasmid (pST6) of Staphylococcus epidermidis St.6 revealed the presence of qacB adjacent to an incomplete beta-lactamase transposon Tn552 encoding the gene cluster blaZ, blaR, and blaI. Sequence analysis of flanking regions and the intergenic region between blaZ and qacB revealed the presence of IS257 downstream of blaZ as well as sin and binR between blaZ and qacB. In the three other BC and penicillin-resistant strains, the qacA/B and blaZ genes were located on separate plasmids. A qacC harboring S. epidermidis strain (St.17) also hybridized to tetK (tetracycline resistance) and ermB (erythromycin resistance) genes. The individual genes were located on separate plasmids, suggesting no linkage between QAC and antibiotic resistance determinants. Plasmid-free Staphylococcus aureus RN4220 allowed uptake of the pST6 plasmid DNA, indicating that the resistance genes could potentially be transferred to pathogens under selective stress. In conclusion, presence of both resistance determinants could lead to co-selection during antimicrobial therapy or disinfection in hospitals or in food industries.

Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species.

PubMed Central

Choi, Su Mi; Kim, Seung-Han; Kim, Hee-Jung; Lee, Dong-Gun; Choi, Jung-Hyun; Yoo, Jin-Hong; Kang, Jin-Han; Shin, Wan-Shik; Kang, Moon-Won

2003-01-01

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method. PMID:14555812

Mrp--a new auxiliary gene essential for optimal expression of methicillin resistance in Staphylococcus aureus.

PubMed

Wu, S W; De Lencastre, H

1999-01-01

Screening of a library of Tn551 insertional mutants selected for reduction in the methicillin resistance level of the parental Staphylococcus aureus strain COL resulted in the isolation of mutant RUSA266 in which the minimal inhibitory concentration (MIC) of the parent was reduced from 1,600 to 1.5 micrograms/mL. Cloning and sequencing of the vicinity of the insertion site omega 726 identified an open reading frame (orf1365) encoding a very large polypeptide of more than 1,365 amino acids. A unique feature of the deduced amino acid sequence was the presence of multiple tandem repeats of 75 amino acids in the polypeptide, reminiscent of the structure of high-molecular-weight cell-surface proteins EF* and Emb identified in some streptococcal strains. Mutant RUSA266 with the inactivated gene, which we shall provisionally refer to as mrp (for multiple repeat polypeptide), produced a peptidoglycan with altered muropeptide composition, and both the reduced antibiotic resistance and the altered cell wall composition were co-transduced in back-crosses into the parental strain COL. Additional sequencing upstream of mrp has revealed that this gene was part of a five-gene cluster occupying a 9.2-kb region of the staphylococcal chromosome and was composed of glmM (directly upstream of mrp), two open reading frames orf310 and orf269 coding for two hypothetical proteins, and the gene encoding the staphylococcal arginase (arg). Transcriptional analysis demonstrated that the five genes in the cluster were transcribed together.

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

PubMed

Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

2014-04-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin--phenotypic and genotypic antibiotic resistance.

PubMed

Chajęcka-Wierzchowska, Wioleta; Zadernowska, Anna; Nalepa, Beata; Sierpińska, Magda; Łaniewska-Trokenheim, Łucja

2015-04-01

The aim of this work was to study the pheno- and genotypical antimicrobial resistance profile of coagulase negative staphylococci (CoNS) isolated from 146 ready-to-eat food of animal origin (cheeses, cured meats, sausages, smoked fishes). 58 strains were isolated, they were classified as Staphylococcus xylosus (n = 29), Staphylococcus epidermidis (n = 16); Staphylococcus lentus (n = 7); Staphylococcus saprophyticus (n = 4); Staphylococcus hyicus (n = 1) and Staphylococcus simulans (n = 1) by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin, clindamycin, gentamicin, cefoxitin, norfloxacin, ciprofloxacin, tetracycline, tigecycline, rifampicin, nitrofurantoin, linezolid, trimetoprim, sulphamethoxazole/trimethoprim, chloramphenicol, quinupristin/dalfopristin by the disk diffusion method. PCR was used for the detection of antibiotic resistance genes encoding: methicillin resistance--mecA; macrolide resistance--erm(A), erm(B), erm(C), mrs(A/B); efflux proteins tet(K) and tet(L) and ribosomal protection proteins tet(M). For all the tet(M)-positive isolates the presence of conjugative transposons of the Tn916-Tn1545 family was determined. Most of the isolates were resistant to cefoxitin (41.3%) followed by clindamycin (36.2%), tigecycline (24.1%), rifampicin (17.2%) and erythromycin (13.8%). 32.2% staphylococcal isolates were multidrug resistant (MDR). All methicillin resistant staphylococci harboured mecA gene. Isolates, phenotypic resistant to tetracycline, harboured at least one tetracycline resistance determinant on which tet(M) was most frequent. All of the isolates positive for tet(M) genes were positive for the Tn916-Tn1545 -like integrase family gene. In the erythromycin-resistant isolates, the macrolide resistance genes erm(C) or msr(A/B) were present. Although coagulase-negative staphylococci are not classical food poisoning bacteria, its presence in food could be of public health significance due to the possible spread of antibiotic resistance. Copyright © 2014 Elsevier Ltd. All rights reserved.

Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

PubMed

Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

2016-01-01

The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12.5% exhibiting the presence of egc-like variants. In two animals, we also noted the gene encoding the TSST-1 toxin. The results of the study showed that free-living animals may be a significant reservoir of bacteria that are potentially pathogenic for humans. The results of the statistical analysis revealed that, among the animals species studied, the red fox constitutes the most important source of infections.

Induction of Virulence Gene Expression in Staphylococcus aureus by Pulmonary Surfactant

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi

2014-01-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal. PMID:24452679

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program

PubMed Central

2010-01-01

Background The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. Findings We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Conclusions Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features. PMID:21047438

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program.

PubMed

Sheraba, Norhan S; Yassin, Aymen S; Amin, Magdy A

2010-11-04

The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features.

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone.

PubMed

Ji, Guangyong; Pei, Wuhong; Zhang, Linsheng; Qiu, Rongde; Lin, Jianqun; Benito, Yvonne; Lina, Gerard; Novick, Richard P

2005-05-01

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.

Mobile genetic elements of Staphylococcus aureus.

PubMed

Malachowa, Natalia; DeLeo, Frank R

2010-09-01

Bacteria such as Staphylococcus aureus are successful as commensal organisms or pathogens in part because they adapt rapidly to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics, including the gene that confers resistance to beta-lactam antibiotics in methicillin-resistant S. aureus (MRSA). Inasmuch as MRSA infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to improve diagnostic assays and to develop new antimicrobial agents for treatment of disease. Our understanding of S. aureus MGEs and the molecules they encode has played an important role toward these ends and has provided detailed insight into the evolution of antimicrobial resistance mechanisms and virulence.

Staphylococcus aureus genomics and the impact of horizontal gene transfer.

PubMed

Lindsay, Jodi A

2014-03-01

Whole genome sequencing and microarrays have revealed the population structure of Staphylococcus aureus, and identified epidemiological shifts, transmission routes, and adaptation of major clones. S. aureus genomes are highly diverse. This is partly due to a population structure of conserved lineages, each with unique combinations of genes encoding surface proteins, regulators, immune evasion and virulence pathways. Even more variable are the mobile genetic elements (MGE), which encode key proteins for antibiotic resistance, virulence and host-adaptation. MGEs can transfer at high frequency between isolates of the same lineage by horizontal gene transfer (HGT). There is increasing evidence that HGT is key to bacterial adaptation and success. Recent studies have shed light on new mechanisms of DNA transfer such as transformation, the identification of receptors for transduction, on integration of DNA pathways, mechanisms blocking transfer including CRISPR and new restriction systems, strategies for evasion of restriction barriers, as well as factors influencing MGE selection and stability. These studies have also lead to new tools enabling construction of genetically modified clinical S. aureus isolates. This review will focus on HGT mechanisms and their importance in shaping the evolution of new clones adapted to antibiotic resistance, healthcare, communities and livestock. Copyright © 2013 Elsevier GmbH. All rights reserved.

Staphylococcus aureus Isolates Carrying Panton-Valentine Leucocidin Genes: Their Frequency, Antimicrobial Patterns, and Association With Infectious Disease in Shahrekord City, Southwest Iran.

PubMed

Shariati, Laleh; Validi, Majid; Hasheminia, Ali Mohammad; Ghasemikhah, Reza; Kianpour, Fariborz; Karimi, Ali; Nafisi, Mohammad Reza; Tabatabaiefar, Mohammad Amin

2016-01-01

A diversity of virulence factors work together to create the pathogenicity of Staphylococcus aureus. These factors include cell surface components that promote adherence to surfaces as well as exoproteins such as Panton-Valentine leukocidin (PVL), encoded by the luk-PV genes, that invade or bypass the immune system and are toxic to the host, thereby enhancing the severity of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to determine the frequency of PVL-positive MRSA strains by real-time PCR and their antibiotic susceptibility patterns by phenotypic test. In total, 284 Staphylococcus isolates, identified by phenotypic methods from clinical samples of Shahrekord University Hospitals, Shahrekord, Iran, were tested for nuc, mecA, and PVL genes by TaqMan real-time PCR. The antibiotic susceptibility patterns of PVL-containing MRSA strains were determined via the disk diffusion method. In total, 196 isolates (69%) were nuc positive (i.e., S. aureus); of those isolates, 96 (49%) were mecA positive (MRSA). Eighteen (18.8%) of the 96 MRSA positive and 3 (3%) of the 100 methicillin-susceptible Staphylococcus aureus (MSSA) strains were PVL positive. PVL-positive MRSA strains were mostly recovered from tracheal specimens. Eight PVL-positive MRSA strains were resistant to all the tested antibiotics except vancomycin. A significant correlation (P = 0.001) was found between the mecA positivity and the presence of luk-PV genes. Community acquired (CA)-MRSA is becoming a public health concern in many parts of the world, including Asian countries. The variable prevalence of luk-PV-positive MRSA isolates in different regions and their rather high frequency in pneumonia necessitate the application of rapid diagnostic methods such as real-time PCR to improve treatment effectiveness.

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

PubMed Central

Ito, Teruyo; Ma, Xiao Xue; Takeuchi, Fumihiko; Okuma, Keiko; Yuzawa, Harumi; Hiramatsu, Keiichi

2004-01-01

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome. PMID:15215121

Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity

PubMed Central

Dittmann, Karen K.; Chaul, Luíza T.; Lee, Sarah H. I.; Corassin, Carlos H.; Fernandes de Oliveira, Carlos A.; Pereira De Martinis, Elaine C.; Alves, Virgínia F.; Gram, Lone; Oxaran, Virginie

2017-01-01

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0 to 63.3% between dairies. Sixty-six isolates from the 31 samples were typed by Multi-Locus Sequence Typing to determine if these isolates were persistent or continuously reintroduced. Seven known sequence types (STs), ST1, ST5, ST30, ST97, ST126, ST188 and ST398, and four new ST were identified, ST3531, ST3540, ST3562 and ST3534. Clonal complex (CC) 1 (including the four new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance toward 11 antibiotic compounds was assessed. Twelve profiles were generated with 75.8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains (4.5%) harbored seg and two of these also harbored sea. Despite the isolates being Methicillin-sensitive S. aureus (MSSA), the presence of CC1 clones in the processing environment, including some harboring enterotoxin encoding genes, is of concern and hygiene must have high priority to reduce contamination. PMID:29123505

Genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and dairy products in South Italy.

PubMed

Basanisi, M G; La Bella, G; Nobili, G; Franconieri, I; La Salandra, G

2017-04-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen emerging in hospitals as well as community and livestock. MRSA is a significant and costly public health concern because it may enter the human food chain and contaminate milk and dairy products causing foodborne illness. This study aimed to determine the occurrence and the characteristics of MRSA isolated from 3760 samples of milk and dairy products in a previous survey conducted in southern Italy during 2008-2014. Overall out of 484 S. aureus strains isolated, 40 (8.3%) were MRSA and were characterized by spa-typing, Multi-Locus Sequence Typing, SCCmec typing, Staphylococcal enterotoxins (SEs) genes, Panton-Valentine Leukocidin (PVL) genes and ability to form biofilm. The most frequently recovered STs were ST152 (t355-67.5%), followed by ST398 (t899, t108-25%), ST1 (t127-5%) and ST5 (t688-2.5%). All isolates harboured the SCCmec type V (92.5%) or IVa (25%). In one isolate (2.5%), ST398/t899, the SCCmec resulted not detected. Three isolates (7.5%) carried one or more enterotoxin encoding genes (one strain had seg, sei, sem, sen and seo genes; two strains had seh gene). The 50% of isolated strains harboured PVL-encoding genes. Molecular analysis for icaA and icaD genes showed: 72.5% icaA and icaD positive, 25% only icaD gene and one icaA and icaD negative. The detection of MRSA in food of animal origin is a potential health hazard, thus it is necessary monitoring of food-producing animals and improving hygiene standards in food practices in order to reduce the microbiological risk to minimum. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

PubMed Central

Dean, Melissa A.; Olsen, Randall J.; Long, S. Wesley; Rosato, Adriana E.

2014-01-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice. PMID:24452687

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

PubMed Central

Rodriguez, Michelle D.; Paul, Zubin; Wood, Charles E.; Rice, Kelly C.; Triplett, Eric W.

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources. PMID:29312199

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

PubMed

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

PubMed

Ciupescu, Laurentiu-Mihai; Auvray, Frederic; Nicorescu, Isabela Madalina; Meheut, Thomas; Ciupescu, Veronica; Lardeux, Anne-Laure; Tanasuica, Rodica; Hennekinne, Jacques-Antoine

2018-06-05

To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis (PFGE) techniques. Nineteen CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene (se). The Calarași outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Pitești outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

[Investigation of biofilm formation properties of staphylococcus isolates].

PubMed

Öcal, Duygu Nilüfer; Dolapçı, İştar; Karahan, Zeynep Ceren; Tekeli, Alper

2017-01-01

Biofilm production is an important virulence factor which allows staphylococci to adhere to medical devices. The principal component of biofilm is a "polysaccharide intercellular adhesin (PIA)" which is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme (N-acetylglucosamine transferase) encoded by the ica operon found on the bacterial chromosome. This operon is composed of four genes (A, B, C, and D), and a transposable element IS256. In this study, we aimed to determine the biofilm production characteristics of invasive/non-invasive staphylococcus isolates and different staphylococcus species. Biofilm production of 166 staphylococci was phenotypically investigated on Congo Red Agar (CRA); the presence of icaA, icaD and IS256 genes were investigated by polymerase chain reaction (PCR). 74 of the isolates (44.6%) were identified as methicillin resistant Staphylococcus aureus (MRSA), 25 (15.1%) as methicillin sensitive S.aureus (MSSA), 25 (37.3%) as Staphylococcus hominis, 20 (12%) as S.epidermidis, ten (15%) as Staphylococcus haemolyticus, nine (13.4%) as Staphylococcus capitis, two (3%) Staphylococcus saprophyticus and one (1.5%) as Staphylococcus warnerii. Of the MRSA strains, 52 were isolated from blood and 22 from nose; all MSSA strains were isolated from nose cultures. Coagulase-negative staphylococci (CoNS) strains were composed of invasive and non-invasive strains isolated from nose, catheter tip and blood cultures from patients with catheter. Production with CRA method was found to be statistically significant in invasive isolates (p< 0.001). It is concluded that; as the biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation in staphylococci. 40.3% of the CoNS isolates, and 85.8% of S.aureus isolates produced biofilm on CRA (p< 0.001) and with PCR method the ratio of carrying three genes was found to be statistically important in S.aureus when compared with CoNS. Carriage of three genes and biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation in staphylococci.

Low Sensitivity of Listeria monocytogenes to Quaternary Ammonium Compounds

PubMed Central

Mereghetti, L.; Quentin, R.; Marquet-Van Der Mee, N.; Audurier, A.

2000-01-01

Ninety-seven epidemiologically unrelated strains of Listeria monocytogenes were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the mdrL gene, which encodes a multidrug efflux pump, and the orfA gene, a putative transcriptional repressor of mdrL. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the qacA and smr genes, encoding multidrug efflux systems previously described for the genus Staphylococcus. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall. PMID:11055967

Old Drugs To Treat Resistant Bugs: Methicillin-Resistant Staphylococcus aureus Isolates with mecC Are Susceptible to a Combination of Penicillin and Clavulanic Acid

PubMed Central

Ba, Xiaoliang; Lovering, Andrew L.; Gleadall, Nicholas; Zadoks, Ruth; Peacock, Sharon J.; Holden, Matthew T. G.; Paterson, Gavin K.; Holmes, Mark A.

2015-01-01

β-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum β-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct β-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the β-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections. PMID:26392513

Comprehensive identification of mutations responsible for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)-to-VISA conversion in laboratory-generated VISA strains derived from hVISA clinical strain Mu3.

PubMed

Matsuo, Miki; Cui, Longzhu; Kim, Jeeyoung; Hiramatsu, Keiichi

2013-12-01

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

Distribution of genes encoding resistance to aminoglycoside modifying enzymes in methicillin-resistant Staphylococcus aureus (MRSA) strains.

PubMed

Khosravi, Azar Dokht; Jenabi, Atefeh; Montazeri, Effat Abbasi

2017-12-01

Today Methicillin-Resistant Staphylococcus aureus (MRSA) have acquired multiple resistance to a wide range of antibiotics including aminoglycosides. So, this study was aimed to investigate the rate of aminoglycoside resistance and the frequency of aminoglycoside resistance mediated genes of aac(Ia)-2, aph(3)-IIIa and ant(4')-Ia among MRSA strains. A total of 467 staphylococci isolates were collected from various clinical samples. S. aureus strains were identified by standard culture and identification criteria and investigating of presence of 16S rRNA and nuc genes. Cefoxitin disk diffusion, and oxacillin-salt agar screening methods were used to detect the MRSA strains with subsequent molecular identification for the presence of mecA gene. Antibiotic susceptibility of MRSA strains against aminoglycoside antibiotics was evaluated by using agar disk diffusion method. Multiplex PCR for the presence of aac(Ia)-2, aph(3)-IIIa and ant(4')-Ia encoding genes for aminoglycosides were performed for MRSA strains. From total staphylococci tested isolates, 262 (56.1%) were identified as S. aureus, of which 161 (61.45%) were detected as MRSA and all comprised mecA gene. The resistance pattern of MRSA strains to aminoglycoside antibiotics were: gentamicin 136 (84.5%); amikacin 125 (77.6%); kanamycin 139 (86.3%); tobramycin 132 (82%); and neomycin 155 (96.3%). The frequency of aac(Ia)-2, aph(3)-IIIa, and ant(4')-Ia genes among MRSA strains, were 64%, 42% and 11.8% respectively. In conclusion, as MRSA strains are of great concern in human infections, the results of present study could provide a useful resource for health sectors for choosing appropriate antibiotics for the effective treatment of infections due to MRSA strains. Copyright © 2017. Published by Elsevier Taiwan.

Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

PubMed

Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

2016-05-01

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

PubMed

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-06-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products.

PubMed

Podkowik, Magdalena; Seo, Keun Seok; Schubert, Justyna; Tolo, Isaiah; Robinson, D Ashley; Bania, Jacek; Bystroń, Jarosław

2016-07-16

We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes. In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36μg/mL, to milk ranging from 6 to 9ng/mL, to beef meat juice from 2 to 3μg/mL and to pork meat juice from 1 to 2μg/mL after 24 and 48h of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to Staphylococcus aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

PubMed Central

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

Comparative analysis of agr groups and virulence genes among subclinical and clinical mastitis Staphylococcus aureus isolates from sheep flocks of the Northeast of Brazil.

PubMed

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

2013-01-01

Staphylococcus aureus is one of the most frequent mastitis causative agents in small ruminants. The expression of most virulence genes of S. aureus is controlled by an accessory gene regulator (agr) locus. This study aimed to ascertain the prevalence of the different agr groups and to evaluate the occurrence of encoding genes for cytotoxin, adhesins and toxins with superantigen activity in S. aureus isolates from milk of ewes with clinical and subclinical mastitis in sheep flocks raised for meat production The agr groups I and II were identified in both cases of clinical and subclinical mastitis. Neither the arg groups III and IV nor negative agr were found. The presence of cflA gene was identified in 100% of the isolates. The frequency of hla and lukE-D genes was high - 77.3 and 82.8%, respectively and all isolates from clinical mastitis presented these genes. The sec gene, either associated to tst gene or not, was identified only in isolates from subclinical mastitis. None of the following genes were identified: bbp, ebpS, cna, fnbB, icaA, icaD, bap, hlg, lukM-lukF-PV and se-a-b-d-e.

Phenotypic and molecular characterization of resistance to macrolides, lincosamides and type B streptogramin of clinical isolates of Staphylococcus spp. of a university hospital in Recife, Pernambuco, Brazil.

PubMed

Pereira, Jussyêgles Niedja da Paz; Rabelo, Marcelle Aquino; Lima, Jailton Lobo da Costa; Neto, Armando Monteiro Bezerra; Lopes, Ana Catarina de Souza; Maciel, Maria Amélia Vieira

2016-01-01

There is a mechanism of macrolide resistance in Staphylococcus spp. which also affects the lincosamides and type B streptogramins characterizing the so-called MLSB resistance, whose expression can be constitutive (cMLSB) or inducible (iMLSB) and is encoded mainly by ermA and ermC genes. The cMLSB resistance is easily detected by susceptibility testing used in the laboratory routine, but iMLSB resistance is not. Therapy with clindamycin in cases of infection with isolated iMLSB resistance may fail. To characterize the phenotypic (occurrence of cMLSB and iMLSB phenotypes) and molecular (occurrence of ermA and ermC genes) profiles of MLSB resistance of clinical isolates of susceptible and methicillin-resistant Staphylococcus aureus and CNS (coagulase-negative Staphylococcus) from patients of a university hospital, in Pernambuco. The antimicrobial susceptibility of 103 isolates was determined by the disk diffusion technique in Mueller-Hinton agar followed by oxacillin screening. The iMLSB phenotype was detected by D test. Isolates with cMLSB and iMLSB phenotypes were subjected to polymerase chain reaction (PCR) for the detection of ermA and ermC genes. The cMLSB and iMLSB phenotypes were respectively identified in 39 (37.9%) and five (4.9%) isolates. The iMLSB phenotype was found only in four (10.8%) methicillin-susceptible S. aureus and one (4.5%) methicillin-resistant S. aureus. In the 44 isolates subjected to PCR, four (9.1%) only ermA gene was detected, a lower frequency when compared to only ermC 17 (38.6%) gene and to one (2.3%) isolate presenting both genes. In the Staphylococcus spp. analyzed, the ermC gene was found more often than the ermA, although the iMLSB phenotype had been less frequent than the cMLSB. It was important to perform the D test for its detection to guide therapeutic approaches. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.

PubMed

Grossoehme, Nicholas; Kehl-Fie, Thomas E; Ma, Zhen; Adams, Keith W; Cowart, Darin M; Scott, Robert A; Skaar, Eric P; Giedroc, David P

2011-04-15

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.

Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus*

PubMed Central

Grossoehme, Nicholas; Kehl-Fie, Thomas E.; Ma, Zhen; Adams, Keith W.; Cowart, Darin M.; Scott, Robert A.; Skaar, Eric P.; Giedroc, David P.

2011-01-01

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes. PMID:21339296

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus

PubMed Central

Sekine, Miwa; Hishinuma, Tomomi; Aiba, Yoshifumi; Hiramatsu, Keiichi

2016-01-01

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology. PMID:27067329

Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products.

PubMed

Morandi, S; Brasca, M; Lodi, R; Cremonesi, P; Castiglioni, B

2007-09-20

Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

Genome-wide analysis of the regulatory function mediated by the small regulatory psm-mec RNA of methicillin-resistant Staphylococcus aureus.

PubMed

Cheung, Gordon Y C; Villaruz, Amer E; Joo, Hwang-Soo; Duong, Anthony C; Yeh, Anthony J; Nguyen, Thuan H; Sturdevant, Daniel E; Queck, S Y; Otto, M

2014-07-01

Several methicillin resistance (SCCmec) clusters characteristic of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains harbor the psm-mec locus. In addition to encoding the cytolysin, phenol-soluble modulin (PSM)-mec, this locus has been attributed gene regulatory functions. Here we employed genome-wide transcriptional profiling to define the regulatory function of the psm-mec locus. The immune evasion factor protein A emerged as the primary conserved and strongly regulated target of psm-mec, an effect we show is mediated by the psm-mec RNA. Furthermore, the psm-mec locus exerted regulatory effects that were more moderate in extent. For example, expression of PSM-mec limited expression of mecA, thereby decreasing methicillin resistance. Our study shows that the psm-mec locus has a rare dual regulatory RNA and encoded cytolysin function. Furthermore, our findings reveal a specific mechanism underscoring the recently emerging concept that S. aureus strains balance pronounced virulence and high expression of antibiotic resistance. Published by Elsevier GmbH.

Distinct Bacteriophages Encoding Panton-Valentine Leukocidin (PVL) among International Methicillin-Resistant Staphylococcus aureus Clones Harboring PVL▿

PubMed Central

Boakes, E.; Kearns, A. M.; Ganner, M.; Perry, C.; Hill, R. L.; Ellington, M. J.

2011-01-01

Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains. PMID:21106787

Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

PubMed

Krzymińska, Sylwia; Szczuka, Ewa; Dudzińska, Kinga; Kaznowski, Adam

2015-04-01

We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6'-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6')/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4'-adenylyltransferase (ant(4')-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6')/aph(2″)) and (aph(3')-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the development of S. haemolyticus-associated infections. The bacteria adhered and invaded to non-professional phagocytes. The invasion of epithelial cells by S. haemolyticus could be similar to phagocytosis that requires polymerization of the actin cytoskeleton. The process is inhibited by cytochalasin D. Moreover, they survived within the cells by residing in membrane bound compartments and induced apoptotic cell death.

Structural and Biological Characterization of a Capsular Polysaccharide Produced by Staphylococcus haemolyticus▿

PubMed Central

Flahaut, Sigrid; Vinogradov, Evgeny; Kelley, Kathryn A.; Brennan, Shannon; Hiramatsu, Keiichi; Lee, Jean C.

2008-01-01

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFGSh) showed ≥57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLMSh genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: −3-α-l-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-β-d-Glc)-4-α-d-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent. PMID:18165309

Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus.

PubMed

Flahaut, Sigrid; Vinogradov, Evgeny; Kelley, Kathryn A; Brennan, Shannon; Hiramatsu, Keiichi; Lee, Jean C

2008-03-01

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFG(Sh)) showed > or = 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLM(Sh) genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: -3-alpha-L-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-beta-D-Glc)-4-alpha-D-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

[Multiplex PCR strategy for the simultaneous identification of Staphylococcus aureus and detection of staphylococcal enterotoxins in isolates from food poisoning outbreaks].

PubMed

Brizzio, Aníbal A; Tedeschi, Fabián A; Zalazar, Fabián E

2013-01-01

Staphylococcal food poisoning is the most frequent type of food poisoning around the world. Staphylococcus aureus enterotoxins cause significant loss of water in the intestinal lumen, followed by vomiting and diarrhea. To report a fast, reliable and inexpensive strategy based on multiplex PCR for the simultaneous identification of S. aureus and detection of five classical S. aureus enterotoxin genes ( sea, seb, sec, sed, see ) in Staphylococcus spp. strains isolated from food poisoning outbreaks. We analyzed isolates from 12 food poisoning outbreaks occurred in Santa Fe province (Argentina). Isolation and phenotypic characterization were carried out by standard procedures. Genotypic analysis was performed by multiplex PCR, using primers for nuc , sea-see and 16S rRNA genes simultaneously. Of all the strains tested, 58% were found to carry toxigenic genes. Sea and seb toxins were found at the same percentage (29%) while sec, sed and see genes were found in a lower and identical proportion (14%). We did not find more than one different type of S. aureus enterotoxin in the isolates analyzed. The multiplex PCR strategy designed in this work has enabled us to identify strains of S. aureus and detect -at the same time- their enterotoxigenic ability. At present, our efforts are devoted to the detection of genes encoding enterotoxins other than the classical ones, in order to know their impact on staphylococcal food poisoning, as well as to investigate their relevance to our country's public health.

Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species.

PubMed

Ogaki, Mayara Baptistucci; Rocha, Katia Real; Terra, MÁrcia Regina; Furlaneto, MÁrcia Cristina; Maia, Luciana Furlaneto

2016-06-28

In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

Genetic diversity of methicillin resistant Staphylococcus aureus strains isolated from burn patients in Iran: ST239-SCCmec III/t037 emerges as the major clone.

PubMed

Goudarzi, Mehdi; Bahramian, Mahnaz; Satarzadeh Tabrizi, Mahboobeh; Udo, Edet E; Figueiredo, Agnes Marie Sá; Fazeli, Maryam; Goudarzi, Hossein

2017-04-01

Methicillin-resistant Staphylococcus aureus (MRSA) as a major cause of infection in health care, hospital and community settings is a global health concern. The purpose of this study was to determine the antibiotic susceptibility pattern and distribution of circulating molecular types of MRSA in a burn hospital in Tehran, the capital of Iran. During a 10-month study period, 106 Staphylococcus aureus isolates were assessed. Isolates were subjected to susceptibility testing using the disk diffusion method and Polymerase Chain Reaction (PCR) for detection of mecA, fem and nuc genes. The presence of PVL and tst encoding genes were determined by PCR method. All the MRSA isolates were genotyped by multilocus sequence typing (MLST), spa typing, SCCmec typing and agr typing. The presence of mecA gene was confirmed in all the Staphylococcus aureus isolates. Antimicrobial susceptibility testing revealed a high resistance rate (90.6%) to ampicillin, tetracycline, and erythromycin. The rates of resistance to remaining antibiotics tested varied between 18.9% and 84.9%. The high- level of resistance to mupirocin was confirmed in 19.8% of MRSA strains isolated from burn patients. Multi-drug resistance was observed in 90.6% of isolates. Sixteen of the 106 MRSA isolates (15.1%) harbored PVL-encoding genes. The majority of our MRSA strains carried SCCmec III (71.7%). ST239-SCCmec III/t037 (34%) was the most common genotype followed by ST239-SCCmec III/t030 (24.5%), ST15-SCCmec IV/t084 (15.1%), ST22-SCCmec IV/t790 (13.2%), and ST239-SCCmec III/t631 (13.2%). Mupirocin resistant MRSA isolates belonged to ST15-SCCmec IV/t084 (40%), ST22-SCCmec IV/t790 (23.3%), ST239-SCCmec III/t631 (20%), and ST239-SCCmec III/t030 (16.7%) clones. The results showed that genetically diverse strains of MRSA are circulating in our burn hospitals with relatively high prevalence of ST239-SCCmec III/t037 clone. The findings support the need for regular surveillance of MRSA to determine the distribution of existing MRSA clones and to detect the emergence of new MRSA clones. Copyright © 2017 Elsevier Ltd. All rights reserved.

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients.

PubMed

Amissah, Nana Ama; Chlebowicz, Monika A; Ablordey, Anthony; Tetteh, Caitlin S; Prah, Isaac; van der Werf, Tjip S; Friedrich, Alex W; van Dijl, Jan Maarten; Stienstra, Ymkje; Rossen, John W

2017-06-01

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like α-hemolysin, and the α- and β-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (SCC) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply that the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Old Drugs To Treat Resistant Bugs: Methicillin-Resistant Staphylococcus aureus Isolates with mecC Are Susceptible to a Combination of Penicillin and Clavulanic Acid.

PubMed

Ba, Xiaoliang; Harrison, Ewan M; Lovering, Andrew L; Gleadall, Nicholas; Zadoks, Ruth; Parkhill, Julian; Peacock, Sharon J; Holden, Matthew T G; Paterson, Gavin K; Holmes, Mark A

2015-12-01

β-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum β-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct β-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the β-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phenotypical and Genotypical Antimicrobial Resistance of Coagulase-negative staphylococci Isolated from Cow Mastitis.

PubMed

Klimiene, I; Virgailis, M; Pavilonis, A; Siugzdiniene, R; Mockeliunas, R; Ruzauskas, M

2016-09-01

The objectives of this study were to determine the prevalence and antimicrobial resistance of coagulase-negative staphylococci (CNS) isolated from dairy cows with subclinical mastitis. Antimicrobial resistance in staphylococci were evaluated by breakpoint values specific to the species (EU-CAST). The presence of resistance-encoding genes was detected by multiplex PCR. A total of 191 CNS isolates were obtained. The CNS isolates were typically resistant to penicillin (67.4%), tetracyc-line (18.9%), and erythromycin (13.7%). CNS isolates (78.0%) were resistant to at least one antimicrobial compound, and 22.0% were multiresistant. The multiresistant isolates were predominantly Staphylococcus chromogenes (28.6%), Staphylococcus warneri (19%) and Staphylococcus haemolyticus (14.3%). According to MIC pattern data, multiresistant isolates showed the highest resistance (p<0.05) rates to penicillin (85.7%), tetracycline (66.7%), and erythromycin (48.2%), but all of them were sensitive to daptomycin, oxacillin, qiunupristin/dalfopristin, and vancomycin. S. chromogenes (9.5%), S. haemolyticus (4.8%), and S. capitis ss capitis (2.4%) strains were resistant to methicillin; their resistance to oxacillin and penicillin was more than 8 mg/l. A high rate of resistance to penicillin was linked to a blaZ gene found in 66.6% of the isolated multiresistant CNS strains. Resistance to tetracycline via the tetK (38.1%) gene and penicillin via the mecA (23.8%) gene were detected less frequently. Gene msrAB was responsible for macrolides and lincosamides resistance and detected in 28.6% of the CNS isolates. Antimicrobial resistance genes were identified more frequently in S. epidermidis, S. chromogenes, and S. warneri.

Staphylococcus aureus Isolates Carrying Panton-Valentine Leucocidin Genes: Their Frequency, Antimicrobial Patterns, and Association With Infectious Disease in Shahrekord City, Southwest Iran

PubMed Central

Shariati, Laleh; Validi, Majid; Hasheminia, Ali Mohammad; Ghasemikhah, Reza; Kianpour, Fariborz; Karimi, Ali; Nafisi, Mohammad Reza; Tabatabaiefar, Mohammad Amin

2016-01-01

Background: A diversity of virulence factors work together to create the pathogenicity of Staphylococcus aureus. These factors include cell surface components that promote adherence to surfaces as well as exoproteins such as Panton-Valentine leukocidin (PVL), encoded by the luk-PV genes, that invade or bypass the immune system and are toxic to the host, thereby enhancing the severity of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Objectives: The aim of this study was to determine the frequency of PVL-positive MRSA strains by real-time PCR and their antibiotic susceptibility patterns by phenotypic test. Materials and Methods: In total, 284 Staphylococcus isolates, identified by phenotypic methods from clinical samples of Shahrekord University Hospitals, Shahrekord, Iran, were tested for nuc, mecA, and PVL genes by TaqMan real-time PCR. The antibiotic susceptibility patterns of PVL-containing MRSA strains were determined via the disk diffusion method. Results: In total, 196 isolates (69%) were nuc positive (i.e., S. aureus); of those isolates, 96 (49%) were mecA positive (MRSA). Eighteen (18.8%) of the 96 MRSA positive and 3 (3%) of the 100 methicillin-susceptible Staphylococcus aureus (MSSA) strains were PVL positive. PVL-positive MRSA strains were mostly recovered from tracheal specimens. Eight PVL-positive MRSA strains were resistant to all the tested antibiotics except vancomycin. A significant correlation (P = 0.001) was found between the mecA positivity and the presence of luk-PV genes. Conclusions: Community acquired (CA)-MRSA is becoming a public health concern in many parts of the world, including Asian countries. The variable prevalence of luk-PV-positive MRSA isolates in different regions and their rather high frequency in pneumonia necessitate the application of rapid diagnostic methods such as real-time PCR to improve treatment effectiveness. PMID:27099685

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

PubMed

Azara, E; Longheu, C; Sanna, G; Tola, S

2017-08-01

To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis. © 2017 The Society for Applied Microbiology.

Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

PubMed Central

O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

2015-01-01

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

PubMed Central

Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

2010-01-01

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

PubMed

Tan, Honglue; Peng, Zhaoxiang; Li, Qingtian; Xu, Xiaofen; Guo, Shengrong; Tang, Tingting

2012-01-01

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.

PubMed

Song, Jae-Hoon; Ko, Kwan Soo; Lee, Ji-Young; Baek, Jin Yang; Oh, Won Sup; Yoon, Ha Sik; Jeong, Jin-Yong; Chun, Jongsik

2005-06-30

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes.

PubMed

Ni, Lisheng; Jensen, Slade O; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M; Guan, Fiona H X; Brown, Melissa H; Skurray, Ronald A; Firth, Neville; Schumacher, Maria A

2009-11-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

PubMed Central

Ni, Lisheng; Jensen, Slade O.; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M.; Guan, Fiona H. X.; Brown, Melissa H.; Skurray, Ronald A.; Firth, Neville; Schumacher, Maria A.

2009-01-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes. PMID:19759211

Virulence Factor Genes in Staphylococcus aureus Isolated From Diabetic Foot Soft Tissue and Bone Infections.

PubMed

Víquez-Molina, Gerardo; Aragón-Sánchez, Javier; Pérez-Corrales, Cristian; Murillo-Vargas, Christian; López-Valverde, María Eugenia; Lipsky, Benjamin A

2018-03-01

The aim of this study is to describe the presence of genes encoding for 4 virulence factors (pvl, eta, etb, and tsst), as well as the mecA gene conferring resistance to beta-lactam antibiotics, in patients with diabetes and a staphylococcal foot infection. We have also analyzed whether isolates of Staphylococcus aureus from bone infections have a different profile for these genes compared with those from exclusively soft tissue infections. In this cross-sectional study of a prospectively recruited series of patients admitted to the Diabetic Foot Unit, San Juan de Dios Hospital, San José, Costa Rica with a moderate or severe diabetic foot infection (DFI), we collected samples from infected soft tissue and from bone during debridement. During the study period (June 1, 2014 to May 31, 2016), we treated 379 patients for a DFI. S aureus was isolated from 101 wound samples, of which 43 were polymicrobial infections; we only included the 58 infections that were monomicrobial S aureus for this study. Infections were exclusively soft tissue in 17 patients (29.3%) while 41 (70.7%) had bone involvement (osteomyelitis). The mecA gene was detected in 35 cases (60.3%), pvl gene in 4 cases (6.9%), and tsst gene in 3 (5.2%). We did not detect etA and etB in any of the cases. There were no differences in the profile of S aureus genes encoding for virulence factors (pvl, etA, etB, and tsst) recovered from DFIs between those with just soft tissue compared to those with osteomyelitis. However, we found a significantly higher prevalence of pvl+ strains of S aureus associated with soft tissue compared with bone infections. Furthermore, we observed a significantly longer time to healing among patients infected with mecA+ (methicillin-resistant) S aureus (MRSA).

Emerging Functions for the Staphylococcus aureus RNome

PubMed Central

Felden, Brice

2013-01-01

Staphylococcus aureus is a leading pathogen for animals and humans, not only being one of the most frequently isolated bacteria in hospital-associated infections but also causing diseases in the community. To coordinate the expression of its numerous virulence genes for growth and survival, S. aureus uses various signalling pathways that include two-component regulatory systems, transcription factors, and also around 250 regulatory RNAs. Biological roles have only been determined for a handful of these sRNAs, including cis, trans, and cis-trans acting RNAs, some internally encoding small, functional peptides and others possessing dual or multiple functions. Here we put forward an inventory of these fascinating sRNAs; the proteins involved in their activities; and those involved in stress response, metabolisms, and virulence. PMID:24348246

Role of the Stringent Stress Response in the Antibiotic Resistance Phenotype of Methicillin-Resistant Staphylococcus aureus

PubMed Central

Aedo, Sandra

2016-01-01

Resistance to beta-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) requires the presence of an acquired genetic determinant, mecA or mecC, which encode penicillin-binding protein PBP2A or PBP2A′, respectively. Although all MRSA strains share a mechanism of resistance, the phenotypic expression of beta-lactam resistance shows considerable strain-to-strain variation. The stringent stress response, a stress response that results from nutrient limitation, was shown to play a key role in determining the resistance level of an MRSA strain. In the present study, we validated the impact of the stringent stress response on transcription and translation of mecA in the MRSA clinical isolate strain N315, which also carries known regulatory genes (mecI/mecR1/mecR2 and blaI/blaR1) for mecA transcription. We showed that the impact of the stringent stress response on the resistance level may be restricted to beta-lactam resistance based on a “foreign” determinant such as mecA, as opposed to resistance based on mutations in the native S. aureus determinant pbpB (encoding PBP2). Our observations demonstrate that high-level resistance mediated by the stringent stress response follows the current model of beta-lactam resistance in which the native PBP2 protein is also essential for expression of the resistance phenotype. We also show that the Staphylococcus sciuri pbpD gene (also called mecAI), the putative evolutionary precursor of mecA, confers oxacillin resistance in an S. aureus strain, generating a heterogeneous phenotype that can be converted to high and homogenous resistance by induction of the stringent stress response in the bacteria. PMID:26833147

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T).

PubMed

Iwamoto, Kazuaki; Tsuruta, Hiroki; Nishitaini, Yosuke; Osawa, Ro

2008-09-01

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

PubMed Central

Matsuo, Miki; Cui, Longzhu; Kim, Jeeyoung

2013-01-01

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10−6 or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate. PMID:24018261

Prevalence, antibiotic resistance, virulence traits and genetic lineages of Staphylococcus aureus in healthy sheep in Tunisia.

PubMed

Gharsa, Haythem; Ben Slama, Karim; Lozano, Carmen; Gómez-Sanz, Elena; Klibi, Naouel; Ben Sallem, Rym; Gómez, Paula; Zarazaga, Myriam; Boudabous, Abdellatif; Torres, Carmen

2012-05-04

Nasal swabs of 163 healthy sheep were obtained from two farms and one abattoir in Tunisia during 2010. Samples were inoculated in Baird Parker agar and ORSAB medium for Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) recovery, respectively. MRSA was detected in 5 of these 163 samples (3%) in ORSAB medium, and one isolate per sample was further studied. MRSA isolates were mecA-positive, typed as ST153-CC80-t044-agrIII, and contained blaZ, ant(6)-Ia, aph(3')-IIIa, erm(C), tet(K), and fusB genes encoding penicillin, streptomycin, kanamycin, erythromycin, tetracycline and fusidic acid resistance, respectively. These MRSA isolates showed indistinguishable or closely related PFGE-patterns and harboured the lukF/lukS gene encoding the Panton-Valentine leukocidin and the luk-ED, hla, hld, and hlg(v) genes. Methicillin-susceptible S. aureus (MSSA) were recovered in 68 of the 163 samples (41.7%) and one isolate per sample was characterized. Most of MSSA (82.4%) showed susceptibility to the tested antibiotics with exceptions: penicillin (6%, carrying blaZ gene), tetracycline (19%, carrying tet(K) gene) and fusidic acid (9%). The following toxin-genes were identified among MSSA: tst (53 isolates), luk-M (52), luk-ED, hla, hlb, hld and hlg(v) (67), hlg (1), sec (49), sel (52), and the egc-cluster-like sen-sem-sei-seo-seg (1). Ten spa-types (two of them new ones) and nine sequence types (six new ones) were detected among the 73 S. aureus isolates, and they were ascribed to agr types I and III. All MRSA and MSSA isolates were able to coagulate bovine plasma and MRSA harboured the immune-evasion-gene-cluster type E. Conclusions. Nares of healthy sheep could be a reservoir of PVL-positive community-associated-MRSA and also of TSST-positive S. aureus isolates, with potential implications in public health. Copyright © 2011 Elsevier B.V. All rights reserved.

The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

PubMed Central

Puri, Sumant; O'Brian, Mark R.

2006-01-01

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a Kd value of 0.8 μM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria. PMID:16952937

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

Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893

PubMed Central

Yu, Li; Mok, Hoyin; Tkaczyk, Christine; Sellman, Bret R.; Wu, Yuling; Oganesyan, Vaheh; Slidel, Tim; Jafri, Hasan; McCarthy, Michael; Bradford, Patricia; Esser, Mark T.

2016-01-01

Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development. PMID:27324766

Metabolism of azo dyes by human skin microbiota

PubMed Central

Stingley, Robin L.; Zou, Wen; Heinze, Thomas M.; Chen, Huizhong; Cerniglia, Carl E.

2018-01-01

Reduction of Methyl Red (MR) and Orange II (Or II) by 26 human skin bacterial species was monitored by a rapid spectrophotometric assay. The analysis indicated that skin bacteria, representing the genera Staphylococcus, Corynebacterium, Micrococcus, Dermacoccus and Kocuria, were able to reduce MR by 74–100 % in 24 h, with only three species unable to reduce completely the dye in that time. Among the species tested, only Corynebacterium xerosis was unable to reduce Or II to any degree by 24 h, and only Staphylococcus delphini, Staphylococcus sciuri subsp. sciuri and Pseudomonas aeruginosa were able to reduce completely this dye within 24 h. MR reduction started with early-exponential growth in Staphylococcus aureus and Staphylococcus epidermidis, and around late-exponential/early-stationary growth in P. aeruginosa. Reduction of Or II, Ponceau S and Ponceau BS started during late-exponential/early-stationary growth for all three species. Using liquid chromatography/electrospray ionization mass spectrometry analyses, MR metabolites produced by Staph. aureus, Staph. epidermidis and P. aeruginosa were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. Searches of available genomic and proteomic data revealed that at least four of the staphylococci in this study, Staphylococcus haemolyticus, Staph. epidermidis, Staphylococcus cohnii and Staphylococcus saprophyticus, have hypothetical genes with 77, 76, 75 and 74 % sequence identity to azo1 encoding an azoreductase from Staph. aureus and hypothetical proteins with 82, 80, 72 and 74 % identity to Azo1, respectively. In addition, Staphylococcus capitis has a protein with 79 % identity to Azo1. Western analysis detected proteins similar to Azo1 in all the staphylococci tested, except Staph. delphini, Staph. sciuri subsp. sciuri and Staphylococcus auricularis. The data presented in this report will be useful in the risk assessment process for evaluation of public exposure to products containing these dyes. PMID:19729456

Metabolism of azo dyes by human skin microbiota.

PubMed

Stingley, Robin L; Zou, Wen; Heinze, Thomas M; Chen, Huizhong; Cerniglia, Carl E

2010-01-01

Reduction of Methyl Red (MR) and Orange II (Or II) by 26 human skin bacterial species was monitored by a rapid spectrophotometric assay. The analysis indicated that skin bacteria, representing the genera Staphylococcus, Corynebacterium, Micrococcus, Dermacoccus and Kocuria, were able to reduce MR by 74-100 % in 24 h, with only three species unable to reduce completely the dye in that time. Among the species tested, only Corynebacterium xerosis was unable to reduce Or II to any degree by 24 h, and only Staphylococcus delphini, Staphylococcus sciuri subsp. sciuri and Pseudomonas aeruginosa were able to reduce completely this dye within 24 h. MR reduction started with early-exponential growth in Staphylococcus aureus and Staphylococcus epidermidis, and around late-exponential/early-stationary growth in P. aeruginosa. Reduction of Or II, Ponceau S and Ponceau BS started during late-exponential/early-stationary growth for all three species. Using liquid chromatography/electrospray ionization mass spectrometry analyses, MR metabolites produced by Staph. aureus, Staph. epidermidis and P. aeruginosa were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. Searches of available genomic and proteomic data revealed that at least four of the staphylococci in this study, Staphylococcus haemolyticus, Staph. epidermidis, Staphylococcus cohnii and Staphylococcus saprophyticus, have hypothetical genes with 77, 76, 75 and 74 % sequence identity to azo1 encoding an azoreductase from Staph. aureus and hypothetical proteins with 82, 80, 72 and 74 % identity to Azo1, respectively. In addition, Staphylococcus capitis has a protein with 79 % identity to Azo1. Western analysis detected proteins similar to Azo1 in all the staphylococci tested, except Staph. delphini, Staph. sciuri subsp. sciuri and Staphylococcus auricularis. The data presented in this report will be useful in the risk assessment process for evaluation of public exposure to products containing these dyes.

A novel hybrid SCCmec-mecC region in Staphylococcus sciuri

PubMed Central

Harrison, Ewan M.; Paterson, Gavin K.; Holden, Matthew T. G.; Ba, Xiaoliang; Rolo, Joana; Morgan, Fiona J. E.; Pichon, Bruno; Kearns, Angela; Zadoks, Ruth N.; Peacock, Sharon J.; Parkhill, Julian; Holmes, Mark A.

2014-01-01

Objectives Methicillin resistance in Staphylococcus spp. results from the expression of an alternative penicillin-binding protein 2a (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA known as mecC (formerly mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified two Staphylococcus sciuri subsp. carnaticus isolates from bovine infections that harbour three different mecA homologues: mecA, mecA1 and mecC. Methods We subjected the two isolates to whole-genome sequencing to further understand the genetic context of the mec-containing region. We also used PCR and RT–PCR to investigate the excision and expression of the SCCmec element and mec genes, respectively. Results Whole-genome sequencing revealed a novel hybrid SCCmec region at the orfX locus consisting of a class E mec complex (mecI-mecR1-mecC1-blaZ) located immediately downstream of a staphylococcal cassette chromosome mec (SCCmec) type VII element. A second SCCmec attL site (attL2), which was imperfect, was present downstream of the mecC region. PCR analysis of stationary-phase cultures showed that both the SCCmec type VII element and a hybrid SCCmec-mecC element were capable of excision from the genome and forming a circular intermediate. Transcriptional analysis showed that mecC and mecA, but not mecA1, were both expressed in liquid culture supplemented with oxacillin. Conclusions Overall, this study further highlights that a range of staphylococcal species harbour the mecC gene and furthers the view that coagulase-negative staphylococci associated with animals may act as reservoirs of antibiotic resistance genes for more pathogenic staphylococcal species. PMID:24302651

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein.

PubMed

Bleiziffer, Isabelle; Eikmeier, Julian; Pohlentz, Gottfried; McAulay, Kathryn; Xia, Guoqing; Hussain, Muzaffar; Peschel, Andreas; Foster, Simon; Peters, Georg; Heilmann, Christine

2017-01-01

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encode...

Characterization of a complex context containing mecA but lacking genes encoding cassette chromosome recombinases in Staphylococcus haemolyticus

PubMed Central

2013-01-01

Background Methicillin resistance determinant mecA is generally transferred by SCCmec elements. However, the mecA gene might not be carried by a SCCmec in a Staphylococcus haemolyticus clinical isolate, WCH1, as no cassette chromosome recombinase genes were detected. Therefore, the genetic context of mecA in WCH1 was investigated. Results A 40-kb region containing mecA was obtained from WCH1, bounded by orfX at one end and several orfs of S. haemolyticus core chromosome at the other. This 40-kb region was very complex in structure with multiple genetic components that appeared to have different origins. For instance, the 3.7-kb structure adjacent to orfX was almost identical to that on the chromosome of Staphylococcus epidermidis RP62a but was absent from S. haemolyticus JCSC1435. Terminal inverted repeats of SCC were found but no ccr genes could be detected. mecA was bracketed by two copies of IS431, which was flanked by 8-bp direct target repeat sequence (DR). Conclusions The presence of 8-bp DR suggests that the two copies of IS431 might have formed a composite transposon for mobilizing mecA. This finding is of significance as multiple copies of IS431 are commonly present in the contexts of mecA, which might have the potential to form various composite transposons that could mediate the mobilization of mecA. This study also provides an explanation for the absence of ccr in some staphylococci isolates carrying mecA. PMID:23521926

Characterization of a complex context containing mecA but lacking genes encoding cassette chromosome recombinases in Staphylococcus haemolyticus.

PubMed

Zong, Zhiyong

2013-03-22

Methicillin resistance determinant mecA is generally transferred by SCCmec elements. However, the mecA gene might not be carried by a SCCmec in a Staphylococcus haemolyticus clinical isolate, WCH1, as no cassette chromosome recombinase genes were detected. Therefore, the genetic context of mecA in WCH1 was investigated. A 40-kb region containing mecA was obtained from WCH1, bounded by orfX at one end and several orfs of S. haemolyticus core chromosome at the other. This 40-kb region was very complex in structure with multiple genetic components that appeared to have different origins. For instance, the 3.7-kb structure adjacent to orfX was almost identical to that on the chromosome of Staphylococcus epidermidis RP62a but was absent from S. haemolyticus JCSC1435. Terminal inverted repeats of SCC were found but no ccr genes could be detected. mecA was bracketed by two copies of IS431, which was flanked by 8-bp direct target repeat sequence (DR). The presence of 8-bp DR suggests that the two copies of IS431 might have formed a composite transposon for mobilizing mecA. This finding is of significance as multiple copies of IS431 are commonly present in the contexts of mecA, which might have the potential to form various composite transposons that could mediate the mobilization of mecA. This study also provides an explanation for the absence of ccr in some staphylococci isolates carrying mecA.

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

PubMed

Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

2016-09-25

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

Tailor-made gene silencing of Staphylococcus aureus clinical isolates by CRISPR interference

PubMed Central

Sato’o, Yusuke; Hisatsune, Junzo; Yu, Liansheng; Sakuma, Tetsushi; Yamamoto, Takashi

2018-01-01

Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for Staphylococcus aureus. The transformation efficiency of S. aureus was ~104 CFU/μg DNA using a vector extracted from dcm negative, which encoded one of DNA modification genes, E. coli. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. dcas9 in the vector was expressed throughout the growth phases of S. aureus and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains’ phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from S. aureus and other Gram-positive bacteria. PMID:29377933

Effects of Subinhibitory Concentrations of Antibiotics on Alpha-Toxin (hla) Gene Expression of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Isolates

PubMed Central

Ohlsen, Knut; Ziebuhr, Wilma; Koller, Klaus-Peter; Hell, Wolfgang; Wichelhaus, Thomas A.; Hacker, Jörg

1998-01-01

Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of β-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 μg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections. PMID:9797209

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi

PubMed Central

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-01-01

The PKS-NRPS-derived tetramic acid equisetin and its N-desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus. The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum, a species distantly related to the notorious rice pathogen Fusarium fujikuroi. Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi. Bioinformatic analysis revealed that this cluster does not contain the equisetin N-methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi. Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22, led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23, encoding a second Zn(II)2Cys6 TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T. TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus. PMID:28379186

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi.

PubMed

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-04-05

The PKS-NRPS-derived tetramic acid equisetin and its N -desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus . The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum , a species distantly related to the notorious rice pathogen Fusarium fujikuroi . Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi . Bioinformatic analysis revealed that this cluster does not contain the equisetin N -methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi . Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22 , led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23 , encoding a second Zn(II)₂Cys₆ TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T . TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus.

Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus from Retail Raw Chicken Meat.

PubMed

Li, Suixia; Wang, Panpan; Zhao, Jialin; Zhou, Luhong; Zhang, Pengfei; Fu, Chengyu; Meng, Jianghong; Wang, Xin

2018-04-01

The aim of this study was to investigate the toxin gene profile and antimicrobial resistance of Staphylococcus aureus isolates from raw chicken in the People's Republic of China. In total, 289 S. aureus isolates were characterized by antimicrobial susceptibility testing, and genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin were revealed by PCR. Overall, 46.0% of the isolates were positive for one or more toxin genes. A high proportion of toxin genes were pvl (26.6%), followed by sej (12.5%), sea (9.0%), seh (8.3%), seb (6.9%), sec (6.9%), sed (4.8%), sei (3.1%), and see (2.4%). None of the isolates harbored seg, tsst-1, or exfoliative toxin genes. In total, 29 toxin gene profiles were obtained, and pvl (10.7%) was the most frequent genotype, followed by sea (5.9%), seb (4.8%), and sej (4.2%). Furthermore, 99.7% of the strains were resistant to at least one of the tested antimicrobial agents, and 87.2% of them displayed multidrug resistance. Resistance was most frequently observed to trimethoprim-sulfamethoxazole and erythromycin (86.2% for each), followed by tetracycline (69.9%), amoxicillin-clavulanic acid (45.0%), and ampicillin (42.6%). None of the strains were resistant to vancomycin. This study indicates that S. aureus isolates from raw chicken harbored multiple toxin genes and exhibited multiple antimicrobial resistance, which represents a potential health hazard for consumers.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka

2016-08-29

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-familymore » replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.« less

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.

The Catabolite Control Protein E (CcpE) Affects Virulence Determinant Production and Pathogenesis of Staphylococcus aureus*

PubMed Central

Hartmann, Torsten; Baronian, Grégory; Nippe, Nadine; Voss, Meike; Schulthess, Bettina; Wolz, Christiane; Eisenbeis, Janina; Schmidt-Hohagen, Kerstin; Gaupp, Rosmarie; Sunderkötter, Cord; Beisswenger, Christoph; Bals, Robert; Somerville, Greg A.; Herrmann, Mathias; Molle, Virginie; Bischoff, Markus

2014-01-01

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. PMID:25193664

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus

PubMed Central

Neamah, Maan M.; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F.

2017-01-01

Abstract DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. PMID:28475766

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

Genome comparisons of two Taiwanese community-associated methicillin-resistant Staphylococcus aureus ST59 clones support the multi-origin theory of CA-MRSA.

PubMed

Feng, Ye; Chen, Hsiu-Ling; Chen, Chih-Jung; Chen, Chyi-Liang; Chiu, Cheng-Hsun

2017-10-01

Sequence type (ST) 59 is an epidemic lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Two ST59 clones are prevalent in Taiwan: the Taiwan clone (TW) causes severe infections, whereas the Asian-Pacific clone (AP) is usually commensal. In this study, we sequenced the genome and transcriptome of the representative strains of these two clones and found their differences to focus on three mobile genetic elements: TW carries SCCmec Type V T , Panton-Valentine leucocidin (PVL)-encoding prophage ΦSa2, whereas AP carries SCCmec Type IV and staphylokinase (SAK)-encoding prophage ΦSa3. The anti-virulent role of SAK was confirmed using murine skin and bloodstream infection models. ΦSa3 usually integrates into the hlb gene, but in AP was found to be integrated at the genomic island νSaβ. The mutation of the attB site "TGTATCCAAACTGG" to "TGTATCCGAATTGG" led to a failure in the integration of ΦSa3 in hlb, prompting atypical integration at other sites. The sak gene possessed remarkably different patterns of distribution among the different STs of S. aureus. We conclude that the atypical integration of ΦSa3 may help S. aureus adapt to the human host habitat and that the subsequent loss of ΦSa3 contributes toward the development of a virulent CA-MRSA lineage for wider horizontal transmission. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequence of the toxic shock syndrome toxin gene (tstH) borne by strains of Staphylococcus aureus isolated from patients with Kawasaki syndrome.

PubMed Central

Deresiewicz, R L; Flaxenburg, J; Leng, K; Kasper, D L

1996-01-01

To explore whether a novel staphylococcal clone or structural variant of toxic shock syndrome toxin 1 is associated with Kawasaki syndrome, six toxigenic strains of Staphylococcus aureus from Kawasaki syndrome patients were studied. The strains were divisible into two groups based on phenotypic and genotypic characteristics and are therefore unequivocally not clonal. Portions of the tstH genes of each strain were sequenced. Three were sequenced in their entirety, while the remainder were sequenced from codon 66 to codon 137 of the mature protein only. Two of the former group differed slightly in the sequences of their signal peptides relative to the sequence published for the tstH signal peptide. Those differences did not affect toxin processing or secretion. The sequenced portions of the regions encoding mature toxic shock syndrome toxin 1 were identical in all six strains and corresponded exactly to the published sequence of tstH. No evidence was found for the existence of a structural variant of tstH uniquely associated with Kawasaki syndrome. PMID:8757881

Genetic Organization of the Chromosome Region Surrounding mecA in Clinical Staphylococcal Strains: Role of IS431-Mediated mecI Deletion in Expression of Resistance in mecA-Carrying, Low-Level Methicillin- Resistant Staphylococcus haemolyticus

PubMed Central

Katayama, Yuki; Ito, Teruyo; Hiramatsu, Keiichi

2001-01-01

We report on the structural diversity of mecA gene complexes carried by 38 methicillin-resistant Staphylococcus aureus and 91 methicillin-resistant coagulase-negative Staphylococcus strains of seven different species with a special reference to its correlation with phenotypic expression of methicillin resistance. The most prevalent and widely disseminated mec complex had the structure mecI-mecR1-mecA-IS431R (or IS431mec), designated the class A mecA gene complex. In contrast, in S. haemolyticus, mecA was bracketed by two copies of IS431, forming the structure IS431L-mecA-IS431R. Of the 38 S. haemolyticus strains, 5 had low-level methicillin resistance (MIC, 1 to 4 mg/liter) and characteristic heterogeneous methicillin resistance as judged by population analysis. In these five strains, IS431L was located to the left of an intact mecI gene, forming the structure IS431L-class A mecA-gene complex. In other S. haemolyticus strains, IS431L was associated with the deletion of mecI and mecR1, forming the structure IS431L-ΔmecR1-mecA-IS431mec, designated the class C mecA gene complex. Mutants with the class C mecA gene complex were obtained in vitro by selecting strain SH621, containing the IS431L-class A mecA gene complex with low concentrations of methicillin (1 and 3 mg/liter). The mutants had intermediate level of methicillin resistance (MIC, 16 to 64 mg/liter). The mecA gene transcription was shown to be derepressed in a representative mutant strain, SH621-37. Our study indicated that the mecI-encoded repressor function is responsible for the low-level methicillin resistance of some S. haemolyticus clinical strains and that the IS431-mediated mecI gene deletion causes the expression of methicillin resistance through the derepression of mecA gene transcription. PMID:11408208

Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers.

PubMed

Okolie, Charles E; Wooldridge, Karl G; Turner, David P; Cockayne, Alan; James, Richard

2015-06-01

Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 10(1) to 3.0 × 10(1) colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hypoxia-Inducible Factor 1-Regulated Lysyl Oxidase Is Involved in Staphylococcus aureus Abscess Formation

PubMed Central

Beerlage, Christiane; Greb, Jessica; Kretschmer, Dorothee; Assaggaf, Mohammad; Trackman, Philip C.; Hansmann, Martin-Leo; Bonin, Michael; Eble, Johannes A.; Peschel, Andreas; Brüne, Bernhard

2013-01-01

Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor involved in the adaptation of mammals to hypoxia and plays a crucial role in cancer angiogenesis. Recent evidence suggests a leading role for HIF-1 in various inflammatory and infectious diseases. Here we describe the role of HIF-1 in Staphylococcus aureus infections by investigating the HIF-1-dependent host cell response. For this purpose, transcriptional profiling of HIF-1α-deficient HepG2 and control cells, both infected with Staphylococcus aureus, was performed. Four hours after infection, the expression of 190 genes, 24 of which were regulated via HIF-1, was influenced. LOX (encoding lysyl oxidase) was one of the upregulated genes with a potential impact on the course of S. aureus infection. LOX is an amine oxidase required for biosynthetic cross-linking of extracellular matrix components. LOX was upregulated in vitro in different cell cultures infected with S. aureus and also in vivo, in kidney abscesses of mice intravenously infected with S. aureus and in clinical skin samples from patients with S. aureus infections. Inhibition of LOX by β-aminopropionitrile (BAPN) did not affect the bacterial load in kidneys or blood but significantly influenced abscess morphology and collagenization. Our data provide evidence for a crucial role of HIF-1-regulated LOX in abscess formation. PMID:23649089

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections.

PubMed

Campoccia, Davide; Speziale, Pietro; Ravaioli, Stefano; Cangini, Ilaria; Rindi, Simonetta; Pirini, Valter; Montanaro, Lucio; Arciola, Carla Renata

2009-12-01

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

Molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bacteremia based on MLST, SCCmec, spa, and agr locus types analysis.

PubMed

Goudarzi, Mehdi; Seyedjavadi, Sima Sadat; Nasiri, Mohammad Javad; Goudarzi, Hossein; Sajadi Nia, Raheleh; Dabiri, Hossein

2017-03-01

The widespread emergence of methicillin resistant Staphylococcus aureus, as a common cause of nosocomial infections, is becoming a serious concern in global public health. The objective of the present study was to investigate antimicrobial susceptibility pattern, frequency of virulence genes and molecular characteristics of methicillin-resistant Staphylococcus aureus strains isolated from patients with bacteremia. A total of 128 methicillin-resistant Staphylococcus aureus isolates were collected during February 2015 to January 2016. In vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion method. Conventional PCR was performed for the detection of adhesion (can, bbp, ebp, fnbB, fnbA, clfB, clfA) and toxin (etb, eta, pvl, tst) encoding genes, determining the agr type, SCCmec, MLST and spa typing of the isolates. All the methicillin-resistant Staphylococcus aureus isolates were found to be sensitive to linezolid, teicoplanin, and vancomycin. Resistance to the tested antibiotics varied from 97.7% for penicillin to 24.2% for mupirocin. The rate of multi drug resistance (MDR) in the present study was 97.7%. The most commonly detected toxin and adhesion genes were tst (58.6%), and clfB (100%), respectively. The majority of SCCmec III isolates were found in agr group I while SCCmec IV and II isolates were distributed among agr group III. Multilocus Sequence Typing (MLST) of the MRSA isolates showed five different sequence types: ST239 (43%), ST22 (39.8%), ST585 (10.9%), ST45 (3.9%) and ST240 (2.3%). All of the pvl positive strains belonged to ST22-SCCmec IV/t790 clone and were MDR. Among different 7 spa types, the most common were t790 (27.3%), t037 (21.9%), and t030 (14.1%). spa types t016, t924 and spa type t383 were reported for the first time from Asia and Iran, respectively. It was shown that spa types circulating in the studied hospitals varied which support the need to perform future surveillance studies in order to understand methicillin-resistant Staphylococcus aureus distribution and thus more effective infection control. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Staphylococcus aureus.

PubMed

Hong, Seung Kon; Kim, Kook Han; Kim, Eunice EunKyeong

2010-01-01

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 A, alpha = gamma = 90, beta = 106.330 degrees . The asymmetric unit contains one SaMCAT molecule.

Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products

PubMed Central

Podkowik, Magdalena; Seo, Keun Seok; Schubert, Justyna; Tolo, Isaiah; Robinson, D. Ashley; Bania, Jacek; Bystroń, Jarosław

2016-01-01

We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes.In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36 μg/mL, to milk ranging from 6-9 ng/mL, to beef meat juice from 2-3 μg/mL and to pork meat juice from 1-2 μg/mL after 24 and 48 hours of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to S. aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety. PMID:27105039

Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398.

PubMed

Gómez, Paula; Lozano, Carmen; Benito, Daniel; Estepa, Vanesa; Tenorio, Carmen; Zarazaga, Myriam; Torres, Carmen

2016-05-01

The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1-5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that Staphylococcus spp. are normal contaminants of urban wastewater, including different lineages of S. aureus and a high diversity of coagulase-negative species. The presence of multiple resistance and virulence genes, including mecA, in staphylococci of wastewater can be a concern for the public health. Copyright © 2016 Elsevier Ltd. All rights reserved.

[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.

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

PubMed Central

Rosey, E L; Oskouian, B; Stewart, G C

1991-01-01

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus. PMID:1655695

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

PubMed

Yeswanth, Sthanikam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-12-01

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin.

PubMed

Guzmán-Rodríguez, Jaquelina J; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Ochoa-Zarzosa, Alejandra; Suárez-Rodríguez, Luis María; Rodríguez-Zapata, Luis C; Salgado-Garciglia, Rafael; Jimenez-Moraila, Beatriz; López-Meza, Joel E; López-Gómez, Rodolfo

2013-09-01

Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis▿†

PubMed Central

Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K.; Park, Yong Ho; Deobald, Claudia F.; Wang, Dan; Liu, Song; Daugherty, Sean C.; Gill, Ann Lindley; Bohach, Gregory A.; Gill, Steven R.

2011-01-01

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

PubMed Central

Jagusztyn-Krynicka, E K; Hansen, J B; Crow, V L; Thomas, T D; Honeyman, A L; Curtiss, R

1992-01-01

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis. Images PMID:1328153

Staphylococci on ICE: Overlooked agents of horizontal gene transfer.

PubMed

Sansevere, Emily A; Robinson, D Ashley

2017-01-01

Horizontal gene transfer plays a significant role in spreading antimicrobial resistance and virulence genes throughout the genus Staphylococcus , which includes species of clinical relevance to humans and animals. While phages and plasmids are the most well-studied agents of horizontal gene transfer in staphylococci, the contribution of integrative conjugative elements (ICEs) has been mostly overlooked. Experimental work demonstrating the activity of ICEs in staphylococci remained frozen for years after initial work in the 1980s that showed Tn 916 was capable of transfer from Enterococcus to Staphylococcus . However, recent work has begun to thaw this field. To date, 2 families of ICEs have been identified among staphylococci - Tn 916 that includes the Tn 5801 subfamily, and ICE 6013 that includes at least 7 subfamilies. Both Tn 5801 and ICE 6013 commonly occur in clinical strains of S. aureus . Tn 5801 is the most studied of the Tn 916 family elements in staphylococci and encodes tetracycline resistance and a protein that, when expressed in Escherichia coli , inhibits restriction barriers to incoming DNA. ICE 6013 is among the shortest known ICEs, but it still includes many uncharacterized open reading frames. This element uses an IS 30 -like transposase as its recombinase, providing some versatility in integration sites. ICE 6013 also conjugatively transfers among receptive S. aureus strains at relatively higher frequency than Tn 5801 . Continued study of these mobile genetic elements may reveal the full extent to which ICEs impact horizontal gene transfer and the evolution of staphylococci.

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

PubMed

Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

2015-12-01

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

PubMed

Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

2015-06-12

The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97

PubMed Central

Chang, Yoonjee; Shin, Hakdong; Lee, Ju-Hoon; Park, Chul Jong; Paik, Soon-Young; Ryu, Sangryeol

2015-01-01

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus. PMID:26437428

Comparative analysis of USA300 virulence determinants in a rabbit model of skin and soft tissue infection.

PubMed

Kobayashi, Scott D; Malachowa, Natalia; Whitney, Adeline R; Braughton, Kevin R; Gardner, Donald J; Long, Dan; Bubeck Wardenburg, Juliane; Schneewind, Olaf; Otto, Michael; Deleo, Frank R

2011-09-15

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are frequently associated with strains harboring genes encoding Panton-Valentine leukocidin (PVL). The role of PVL in the success of the epidemic CA-MRSA strain USA300 remains unknown. Here we developed a skin and soft tissue infection model in rabbits to test the hypothesis that PVL contributes to USA300 pathogenesis and compare it with well-established virulence determinants: alpha-hemolysin (Hla), phenol-soluble modulin-alpha peptides (PSMα), and accessory gene regulator (Agr). The data indicate that Hla, PSMα, and Agr contribute to the pathogenesis of USA300 skin infections in rabbits, whereas a role for PVL could not be detected.

Community-associated MRSA: what makes them special?

PubMed Central

Otto, Michael

2013-01-01

Summary While infections with methicillin-resistant Staphylococcus aureus (MRSA) were traditionally restricted to the hospital setting, novel MRSA strains emerged over the last two decades that have the capacity to infect otherwise healthy people outside of the hospital setting. These communityassociated (CA-) MRSA strains combine methicillin resistance with enhanced virulence and fitness. Interestingly, CA-MRSA strains emerged globally and from different backgrounds, indicating that the “trade-off” between maintaining sufficient levels of methicillin resistance and obtaining enhanced virulence at a low fitness cost was achieved on several occasions in convergent evolution. However, frequently this process comprised similar changes. First and foremost, all CA-MRSA strains typically carry a novel type of methicillin resistance locus that appears to cause less of a fitness burden. Additionally, acquisition of specific toxin genes, most notably that encoding Panton-Valentine leukocidin (PVL), and adaptation of gene expression of genome-encoded toxins, such as alpha-toxin and phenol-soluble modulins (PSMs), further contributed to the evolution of CA-MRSA. Finally, the exceptional epidemiological success of the USA300 CA-MRSA clone in particular may have been due to yet another gene acquisition, namely that of the speG gene, which is located on the arginine catabolic mobile element (ACME) and involved in detoxifying harmful host-derived polyamines. PMID:23517691

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus.

PubMed

Neamah, Maan M; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F; Marina, Alberto; Ayora, Silvia; Penadés, José R

2017-06-20

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

PubMed Central

Farrand, Allison J.; Haley, Kathryn P.; Lareau, Nichole M.; Heilbronner, Simon; McLean, John A.; Foster, Timothy

2015-01-01

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection. PMID:26123800

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus

PubMed Central

Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C. S.; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

2016-01-01

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ. PMID:26999783

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

PubMed

Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

2016-01-01

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

2012-04-01

Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

Extensive Horizontal Gene Transfer during Staphylococcus aureus Co-colonization In Vivo

PubMed Central

McCarthy, Alex J.; Loeffler, Anette; Witney, Adam A.; Gould, Katherine A.; Lloyd, David H.; Lindsay, Jodi A.

2014-01-01

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro. PMID:25260585

The Major Cold Shock Gene, cspA, Is Involved in the Susceptibility of Staphylococcus aureus to an Antimicrobial Peptide of Human Cathepsin G

PubMed Central

Katzif, Samuel; Danavall, Damien; Bowers, Samera; Balthazar, Jacqueline T.; Shafer, William M.

2003-01-01

A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment. Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively. This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S. aureus to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system. PMID:12874306

The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G.

PubMed

Katzif, Samuel; Danavall, Damien; Bowers, Samera; Balthazar, Jacqueline T; Shafer, William M

2003-08-01

A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment. Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively. This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S. aureus to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.

A cis-antisense RNA acts in trans in Staphylococcus aureus to control translation of a human cytolytic peptide.

PubMed

Sayed, Nour; Jousselin, Ambre; Felden, Brice

2011-12-25

Antisense RNAs (asRNAs) pair to RNAs expressed from the complementary strand, and their functions are thought to depend on nucleotide overlap with genes on the opposite strand. There is little information on the roles and mechanisms of asRNAs. We show that a cis asRNA acts in trans, using a domain outside its target complementary sequence. SprA1 small regulatory RNA (sRNA) and SprA1(AS) asRNA are concomitantly expressed in S. aureus. SprA1(AS) forms a complex with SprA1, preventing translation of the SprA1-encoded open reading frame by occluding translation initiation signals through pairing interactions. The SprA1 peptide sequence is within two RNA pseudoknots. SprA1(AS) represses production of the SprA1-encoded cytolytic peptide in trans, as its overlapping region is dispensable for regulation. These findings demonstrate that sometimes asRNA functional domains are not their gene-target complementary sequences, suggesting there is a need for mechanistic re-evaluation of asRNAs expressed in prokaryotes and eukaryotes.

Effect of Mild Acid on Gene Expression in Staphylococcus aureus

PubMed Central

Weinrick, Brian; Dunman, Paul M.; McAleese, Fionnuala; Murphy, Ellen; Projan, Steven J.; Fang, Yuan; Novick, Richard P.

2004-01-01

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, ∼5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus. PMID:15576791

Identification and Characterization of the Anti-Methicillin-Resistant Staphylococcus aureus WAP-8294A2 Biosynthetic Gene Cluster from Lysobacter enzymogenes OH11 ▿ †

PubMed Central

Zhang, Wei; Li, Yaoyao; Qian, Guoliang; Wang, Yan; Chen, Haotong; Li, Yue-Zhong; Liu, Fengquan; Shen, Yuemao; Du, Liangcheng

2011-01-01

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter. PMID:21930890

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300.

PubMed

Rozo, Zayda Lorena Corredor; Márquez-Ortiz, Ricaurte Alejandro; Castro, Betsy Esperanza; Gómez, Natasha Vanegas; Escobar-Pérez, Javier

2017-07-01

Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Genetic and biochemical characterization of hyicin 3682, the first bacteriocin reported for Staphylococcus hyicus.

PubMed

Carlin Fagundes, Patrícia; Nascimento de Sousa Santos, Ilana; Silva Francisco, Márcia; Mattos Albano, Rodolpho; de Freire Bastos, Maria do Carmo

2017-05-01

Hyicin 3682, the first bacteriocin reported for Staphylococcus hyicus, is a Bsa COL variant produced by S. hyicus 3682, a strain isolated from bovine milk. Hyicin 3682 is found in the culture supernatant, is bactericidal and its producing strain exhibits a much broader spectrum of antimicrobial activity than the producing strain of Bsa COL against several Gram-positive bacteria, which include foodborne pathogens, food-spoilage microorganisms and bacterial species of medical and veterinary importance. Sequencing of the genome of S. hyicus 3682 provided the nucleotide sequence of the entire gene cluster involved in hyicin 3682 production, which seems to be located on pRJ109, the single plasmid carried by this strain. This gene cluster is expressed and consists of 8525bp and of eight genes (hyiA, hyiB, hyiC, hyiD, hyiP, hyiF, hyiE and hyiG) encoded on the same DNA strand. The mature lantibiotic exhibits 91% identity to Bsa COL and its molecular mass was found to be ∼26Da higher due to two amino acid substitutions. S. hyicus 3682 proved to be only partially immune to its cognate bacteriocin up to 1024 AU/ml. Therefore, hyicin 3682, the first Bsa variant reported in coagulase-negative staphylococci, does exhibit antimicrobial and siblicidal activities. Copyright © 2017 Elsevier GmbH. All rights reserved.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

Presence of Genes Encoding Panton-Valentine Leukocidin Is Not the Primary Determinant of Outcome in Patients with Hospital-Acquired Pneumonia Due to Staphylococcus aureus

PubMed Central

Sharma-Kuinkel, Batu K.; Ahn, Sun H.; Rude, Thomas H.; Zhang, Yurong; Tong, Steven Y. C.; Ruffin, Felicia; Genter, Fredric C.; Braughton, Kevin R.; DeLeo, Frank R.; Barriere, Steven L.

2012-01-01

The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus. PMID:22205797

Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic.

PubMed

Fitzgerald, J R; Sturdevant, D E; Mackie, S M; Gill, S R; Musser, J M

2001-07-17

An emerging theme in medical microbiology is that extensive variation exists in gene content among strains of many pathogenic bacterial species. However, this topic has not been investigated on a genome scale with strains recovered from patients with well-defined clinical conditions. Staphylococcus aureus is a major human pathogen and also causes economically important infections in cows and sheep. A DNA microarray representing >90% of the S. aureus genome was used to characterize genomic diversity, evolutionary relationships, and virulence gene distribution among 36 strains of divergent clonal lineages, including methicillin-resistant strains and organisms causing toxic shock syndrome. Genetic variation in S. aureus is very extensive, with approximately 22% of the genome comprised of dispensable genetic material. Eighteen large regions of difference were identified, and 10 of these regions have genes that encode putative virulence factors or proteins mediating antibiotic resistance. We find that lateral gene transfer has played a fundamental role in the evolution of S. aureus. The mec gene has been horizontally transferred into distinct S. aureus chromosomal backgrounds at least five times, demonstrating that methicillin-resistant strains have evolved multiple independent times, rather than from a single ancestral strain. This finding resolves a long-standing controversy in S. aureus research. The epidemic of toxic shock syndrome that occurred in the 1970s was caused by a change in the host environment, rather than rapid geographic dissemination of a new hypervirulent strain. DNA microarray analysis of large samples of clinically characterized strains provides broad insights into evolution, pathogenesis, and disease emergence.

Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus.

PubMed

Wang, Yi; Yan, Weiqiang; Fu, Shanshan; Hu, Shoukui; Wang, Yan; Xu, Jianguo; Ye, Changyun

2018-01-01

Staphylococcus aureus ( S. aureus ), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus . Multiplex MCDA (m-MCDA), which targets the nuc gene ( S. aureus -specific gene) and mecA gene (encoding penicillin-binding protein-2'), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non- S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.

Presence of Classical Enterotoxin Genes, agr Typing, Antimicrobial Resistance, and Genetic Diversity of Staphylococcus aureus from Milk of Cows with Mastitis in Southern Brazil.

PubMed

Kroning, Isabela S; Iglesias, Mariana A; Mendonça, Karla S; Lopes, Graciela V; Silva, Wladimir P

2018-05-01

Staphylococcus aureus is a common causative agent of bovine mastitis in dairy cows and commonly associated with foodborne disease outbreaks. The aim of this study was to evaluate the presence of enterotoxin genes, agr typing, antimicrobial resistance, and genetic diversity of S. aureus isolated from milk of cows with mastitis in dairy farms from southern Brazil. Results showed that 7 (22.6%) of 31 S. aureus isolates were positive for enterotoxin genes. Specifically, the genes encoding for enterotoxins A ( n = 4), C ( n = 2), and B ( n = 1) were detected. Isolates belonging to the agr group III (10 of 31, 32.2%) and agr group I (7 of 31, 22.5%) were the most common. To our knowledge, this is the first report of both agr I and III in the same S. aureus isolate from milk of cows with bovine mastitis. The antimicrobial resistance test showed that 54% of the isolates were multiresistant to antimicrobial agents. The macrorestriction analysis produced 16 different major SmaI pulsed-field gel electrophoresis patterns, with up to two subpatterns. Moreover, the presence of some S. aureus clones in a distinct area was observed. Although this study characterized a limited number of S. aureus isolates, the presence of classical enterotoxin genes and resistance to multiple antimicrobial agents reinforces the importance of this microorganism to animal and human health. In addition, similar genetic profiles have been identified in distinct geographic areas, suggesting clonal dissemination of S. aureus in dairy herds from southern Brazil.

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

PubMed

Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

2014-04-01

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p < 0.05), while the frequency of sea, sed, eta, and etb genes were higher among MRSA isolates (p > 0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

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

Changes in the Staphylococcus aureus Transcriptome during Early Adaptation to the Lung

PubMed Central

Chaffin, Donald O.; Taylor, Destry; Skerrett, Shawn J.; Rubens, Craig E.

2012-01-01

Staphylococcus aureus is a common inhabitant of the human nasopharynx. It is also a cause of life-threatening illness, producing a potent array of virulence factors that enable survival in normally sterile sites. The transformation of S. aureus from commensal to pathogen is poorly understood. We analyzed S. aureus gene expression during adaptation to the lung using a mouse model of S. aureus pneumonia. Bacteria were isolated by bronchoalveolar lavage after residence in vivo for up to 6 hours. S. aureus in vivo RNA transcription was compared by microarray to that of shake flask grown stationary phase and early exponential phase cells. Compared to in vitro conditions, the in vivo transcriptome was dramatically altered within 30 minutes. Expression of central metabolic pathways changed significantly in response to the lung environment. Gluconeogenesis (fbs, pckA) was down regulated, as was TCA cycle and fermentation pathway gene expression. Genes associated with amino acid synthesis, RNA translation and nitrate respiration were upregulated, indicative of a highly active metabolic state during the first 6 hours in the lung. Virulence factors regulated by agr were down regulated in vivo and in early exponential phase compared to stationary phase cells. Over time in vivo, expression of ahpCF, involved in H2O2 scavenging, and uspA, which encodes a universal stress regulator, increased. Transcription of leukotoxic α and β-type phenol-soluble modulins psmα1-4 and psmβ1-2 increased 13 and 8-fold respectively; hld mRNA, encoding δ-hemolysin, was increased 9-fold. These were the only toxins to be significantly upregulated in vivo. These data provide the first complete survey of the S. aureus transcriptome response to the mammalian airway. The results present intriguing contrasts with previous work in other in vitro and in vivo models and provide novel insights into the adaptive and temporal response of S. aureus early in the pathogenesis of pneumonia. PMID:22876285

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients.

PubMed

de Oliveira, Caio Ferreira; Morey, Alexandre Tadachi; Santos, Jussevania Pereira; Gomes, Ludmila Vilela Pereira; Cardoso, Juscélio Donizete; Pinge-Filho, Phileno; Perugini, Márcia Regina Eches; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2015-07-30

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

PubMed

Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P H; Savelkoul, Paul H M; Jansen, Kathrin U; Anderson, Annaliesa S; Kluytmans, Jan

2015-01-01

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.

PubMed

Kilic, Abdullah; Basustaoglu, A Celal

2011-12-01

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min. Copyright © 2011 Institut Pasteur. All rights reserved.

Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis

PubMed Central

Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Sotto, Albert; Lavigne, Jean-Philippe

2016-01-01

Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds. PMID:27399775

Ascorbic acid augments colony spreading by reducing biofilm formation of methicillin-resistant Staphylococcus aureus.

PubMed

Ali Mirani, Zulfiqar; Khan, Muhammad Naseem; Siddiqui, Anila; Khan, Fouzia; Aziz, Mubashir; Naz, Shagufta; Ahmed, Ayaz; Khan, Seema Ismat

2018-02-01

Staphylococcus aureus is a Gram-positive pathogen, well known for its resistance and versatile lifestyle. Under unfavourable conditions, it adapts biofilm mode of growth. For staphylococcal biofilm formation, production of extracellular polymeric substances (EPS) is a pre-requisite, which is regulated by ica operon-encoded enzymes. This study was designed to know the impact of ascorbic acid on biofilm formation and colony spreading processes of S. aureus and MRSA. The isolates of methicillin-resistant S. aureus (MRSA) used in present study, were recovered from different food samples. Various selective and differential media were used for identification and confirmation of S. aureus . Agar dilution method was used for determination of oxacillin and ascorbic acid resistance level. MRSA isolates were re-confirmed by E-test and by amplification of mecA gene. Tube methods and Congo-Red agar were used to study biofilm formation processes. Gene expression studies were carried on real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results revealed the presence of mecA gene belonging to SCC mecA type IV along with agr type II in the isolates. In vitro studies showed the sub-inhibitory concentration of oxacillin induced biofilm production. However, addition of sub-inhibitory dose of ascorbic acid was found to inhibit EPS production, biofilm formation and augment colony spreading on soft agar plates. The inhibition of biofilm formation and augmentation of colony spreading observed with ascorbic acid alone or in combination with oxacillin. Moreover, gene expression studies showed that ascorbic acid increases agr expression and decreases icaA gene expression. The present study concluded that ascorbic acid inhibits biofilm formation, promotes colony spreading and increases agr gene expression in MRSA.

Novel Genes Encoding Hexadecanoic Acid Δ6-Desaturase Activity in a Rhodococcus sp.

PubMed

Araki, Hiroyuki; Hagihara, Hiroshi; Takigawa, Hirofumi; Tsujino, Yukiharu; Ozaki, Katsuya

2016-11-01

cis-6-Hexadecenoic acid, a major component of human sebaceous lipids, is involved in the defense mechanism against Staphylococcus aureus infection in healthy skin and closely related to atopic dermatitis. Previously, Koike et al. (Biosci Biotechnol Biochem 64:1064-1066, 2000) reported that a mutant strain of Rhodococcus sp. produced cis-6-hexadecenoate derivatives from palmitate alkyl esters. From the mutant Rhodococcus strain, we identified and sequenced two open reading frames present in an amplified 5.7-kb region; these open reading frames encoded tandemly repeated Δ6-desaturase-like genes, Rdes1 and Rdes2. A phylogenetic tree indicated that Rdes1 and Rdes2 were different from previously known Δ6-desaturase genes, and that they formed a new cluster. Rdes1 and Rdes2 were each introduced into vectors and then expressed separately in Escherichia coli, and the fatty acid composition of the transformed cells was analyzed by gas chromatography and mass spectrometry. The amount of cis-6-hexadecenoic acid was significantly higher in Rdes1- or Rdes2-transformed E. coli cells (twofold and threefold, respectively) than in vector-only control cells. These results showed that cis-6-hexadecenoic acid was produced in E. coli cells by the rhodococcal Δ6-desaturase-like proteins.

Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

PubMed

Osman, Kamelia; Alvarez-Ordóñez, Avelino; Ruiz, Lorena; Badr, Jihan; ElHofy, Fatma; Al-Maary, Khalid S; Moussa, Ihab M I; Hessain, Ashgan M; Orabi, Ahmed; Saad, Alaa; Elhadidy, Mohamed

2017-05-10

The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

PubMed

Luebke, Justin L; Shen, Jiangchuan; Bruce, Kevin E; Kehl-Fie, Thomas E; Peng, Hui; Skaar, Eric P; Giedroc, David P

2014-12-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. © 2014 John Wiley & Sons Ltd.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus

PubMed Central

Luebke, Justin L.; Shen, Jiangchuan; Bruce, Kevin E.; Kehl-Fie, Thomas E.; Peng, Hui; Skaar, Eric P.; Giedroc, David P.

2014-01-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR (Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor) represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60’ interprotomer crosslinks, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. PMID:25318663

Antibiotic resistance assessment in S. aureus strains isolated from raw sheep's milk cheese.

PubMed

Spanu, V; Virdis, S; Scarano, C; Cossu, F; De Santis, E P L; Cosseddu, A M

2010-06-01

In vitro activities of 16 antibiotics were tested against 36 Staphylococcus aureus (SA) strains isolated from raw sheep's milk cheese from six dairies. The minimum inhibitory concentration (MIC) was determined using a broth microdilution method (CLSI). All 36 isolates were analyzed for the presence of the accessory gene regulator gene, agr (I-IV), and genes encoding resistance to methicillin (mecA), erythromycin (ermA), penicillin (blaZ), and vancomycin (vanA-B). The isolates were also analyzed for similarities in pulsed-field gel electrophoresis (PFGE) patterns. SA strains showed resistance to ampicillin (36.1%), penicillin (33.3%), tetracycline (11.1%), and cloxacillin (2.8%) but were susceptible (>or=94.4%) to 12 out of 16 tested antimicrobials. The overall susceptibility of the strains to oxacillin, vancomycin, and erythromycin was confirmed by the absence of the mecA, vanA-B, and ermA genes. The PFGE results showed that 32 strains belonged to 10 different clusters (P1-P10) while four strains were untypeable.

Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo.

PubMed

McCarthy, Alex J; Loeffler, Anette; Witney, Adam A; Gould, Katherine A; Lloyd, David H; Lindsay, Jodi A

2014-09-25

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.

PubMed

Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes ( sea , seb , sec , sed , see , seg , seh , sei , sej ), the exfoliative toxin genes ( eta and etb ), the toxic shock syndrome toxin-1 gene ( tst ), and the Panton-Valentine leucocidin-encoding gene ( pvl ). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes ( sea - see ), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl , eta , etb , and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China

PubMed Central

Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, sej), the exfoliative toxin genes (eta and etb), the toxic shock syndrome toxin-1 gene (tst), and the Panton-Valentine leucocidin-encoding gene (pvl). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes (sea–see), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies. PMID:29662467

Complete genome sequences of two Staphylococcus aureus ST5 isolates from California, USA

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a bacteria that can cause disease in humans and animals. S. aureus bacteria can transfer or exchange segments of genetic material with other bacteria. These segments are known as mobile genetic elements and in some instances they can encode for factors that increase the abil...

Draft genome sequences of 14 Staphylococcus aureus ST5 isolates from California, USA

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a bacteria that can cause disease in humans and animals. S. aureus bacteria can transfer or exchange segments of genetic material with other bacteria. These segments are known as mobile genetic elements and in some instances they can encode for factors that increase the abil...

Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

DOE Office of Scientific and Technical Information (OSTI.GOV)

Safo,M.; Ko, T.; Musayev, F.

The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of {beta}-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Angstroms resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtualmore » DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Staphylococcus agnetis, a potential pathogen in broiler breeders.

PubMed

Poulsen, Louise Ladefoged; Thøfner, Ida; Bisgaard, Magne; Olsen, Rikke Heidemann; Christensen, Jens Peter; Christensen, Henrik

2017-12-01

In this study, four broiler parent flocks have been followed from the onset of the production period (week 20) until slaughter (week 60). Every week, approximately ten dead broiler breeders, randomly selected among birds dead on their own, were collected and subjected to a full post mortem analysis including bacteriological examination. In total 997 breeders were investigated and for the first time Staphylococcus agnetis was isolated in pure culture from cases of endocarditis and septicemia from 16 broiler breeders. In addition, the cloacal flora from newly hatched chickens originating from the same four flocks were characterized and S. agnetis was found in pure culture of several newly hatched chickens (n=12) and only in one case in combination with another species. Clonality of the isolates was examined by pulsed-field-gel-electrophoresis which showed indistinguishable patterns in isolates from both broiler breeders and broilers. Three isolates were whole genome sequenced to obtain knowledge on virulence genes. The isolates harbored a number of genes encoding different fibrinogen binding proteins and toxins which might be important for virulence. The present findings demonstrate that S. agnetis may be associated with mortality in broiler breeders. No disease was associated with the broilers which were found positive for S. agnetis in the cloaca. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation of MgrA and its effect on expression of the NorA and NorB efflux pumps of Staphylococcus aureus.

PubMed

Truong-Bolduc, Que Chi; Hooper, David C

2010-05-01

MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrA(S110A-S113A) bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

PubMed

Sayari, Adel; Mosbah, Habib; Gargouri, Youssef

2007-05-01

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

First report of qacG, qacH and qacJ genes in Staphylococcus haemolyticus human clinical isolates.

PubMed

Correa, J E; De Paulis, A; Predari, S; Sordelli, D O; Jeric, P E

2008-11-01

To investigate phenotypically and genotypically the presence of MDR efflux pumps in 21 clinical isolates of Staphylococcus haemolyticus collected over a period of 10 years. MICs of different antibiotics and biocides were determined by the broth dilution method in the presence/absence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an efflux pump inhibitor. PCR followed by sequencing was performed to detect the qac genes that encode for antiseptic resistance. Clonal relationships were determined by PFGE SmaI patterns using a standard protocol. All the isolates were resistant to gentamicin, 15 to erythromycin, 18 to ciprofloxacin, 7 to chloramphenicol and 1 to tetracycline. They showed higher susceptibility to antibiotics when they were exposed to CCCP. The MICs of ethidium bromide, SDS and benzalkonium chloride were also decreased, whereas the MIC of triclosan was decreased in only four isolates in the presence CCCP. Of the 21 isolates, qacA/B was detected in 5 isolates, smr in all of the isolates, qacG in 11 isolates, qacH in 10 isolates and qacJ in 4 isolates. PFGE analysis of the 21 isolates clustered them into 14 clones at 90% similarity corresponding to differences of between 7 and 16 bands among the clones. The efflux mechanism seems to be an important mechanism to confer resistance to antibiotics and biocides through MDR pumps. It was observed that several qac genes coexist in some of the isolates and seem to act simultaneously in the removal of different compounds out of the bacterial cell. The qac genes are horizontally spread among different clones.

Molecular characterization of endocarditis-associated Staphylococcus aureus.

PubMed

Nethercott, Cara; Mabbett, Amanda N; Totsika, Makrina; Peters, Paul; Ortiz, Juan C; Nimmo, Graeme R; Coombs, Geoffrey W; Walker, Mark J; Schembri, Mark A

2013-07-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

PubMed Central

Nethercott, Cara; Mabbett, Amanda N.; Totsika, Makrina; Peters, Paul; Ortiz, Juan C.; Nimmo, Graeme R.; Coombs, Geoffrey W.

2013-01-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted. PMID:23616460

Association of Recurrent Furunculosis with Panton-Valentine Leukocidin and the Genetic Background of Staphylococcus aureus▿ †

PubMed Central

Masiuk, Helena; Kopron, Katarzyna; Grumann, Dorothee; Goerke, Christiane; Kolata, Julia; Jursa-Kulesza, Joanna; Giedrys-Kalemba, Stefania; Bröker, Barbara M.; Holtfreter, Silva

2010-01-01

Staphylococcus aureus is a major cause of skin and soft tissue infections, such as furuncles, carbuncles, and abscesses, but it also frequently colonizes the human skin and mucosa without causing clinical symptoms. Panton-Valentine leukocidin (PVL) is a pore-forming toxin that has been associated with soft tissue infections and necrotizing pneumonia. We have compared the genotypes, virulence gene repertoires, and phage patterns of 74 furunculosis isolates with those of 108 control strains from healthy nasal carriers. The large majority of furunculosis strains were methicillin sensitive. Clonal cluster (CC) 121 (CC121) and CC22 accounted for 70% of the furunculosis strains but for only 8% of the nasal isolates. The PVL-encoding genes luk-PV were detected in 85% of furunculosis strains, while their prevalence among colonizing S. aureus strains was below 1%. luk-PV genes were distributed over several lineages (CCs 5, 8, 22, 30, and 121 and sequence type 59). Even within the same lineages, luk-PV-positive phages characterized furunculosis strains, while their luk-PV-negative variants were frequent among nasal strains. The very tight epidemiological linkage between luk-PV and furunculosis, which could be separated from the genetic background of the S. aureus strain as well as from the gene makeup of the luk-PV-transducing phage, lends support to the notion of an important role for PVL in human furunculosis. These results make a case for the determination of luk-PV in recurrent soft tissue infections with methicillin-sensitive as well as methicillin-resistant S. aureus. PMID:20200289

Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

PubMed Central

Canovas, Jaime; Baldry, Mara; Bojer, Martin S.; Andersen, Paal S.; Gless, Bengt H.; Grzeskowiak, Piotr K.; Stegger, Marc; Damborg, Peter; Olsen, Christian A.; Ingmer, Hanne

2016-01-01

Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources of compounds that can be applied as anti-virulence therapy for combating S. aureus infections. PMID:27877157

Evidence for Horizontal Gene Transfer in Evolution of Elongation Factor Tu in Enterococci

PubMed Central

Ke, Danbing; Boissinot, Maurice; Huletsky, Ann; Picard, François J.; Frenette, Johanne; Ouellette, Marc; Roy, Paul H.; Bergeron, Michel G.

2000-01-01

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes. PMID:11092850

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

PubMed Central

Lioliou, Efthimia; Sharma, Cynthia M.; Caldelari, Isabelle; Helfer, Anne-Catherine; Fechter, Pierre; Vandenesch, François; Vogel, Jörg; Romby, Pascale

2012-01-01

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. PMID:22761586

Identification and Characterization of Staphylococcus aureus Strains with an Incomplete Hemolytic Phenotype.

PubMed

Zhang, Haifang; Zheng, Yi; Gao, Huasheng; Xu, Ping; Wang, Min; Li, Aiqing; Miao, Minhui; Xie, Xiaofang; Deng, Yimai; Zhou, Huiqin; Du, Hong

2016-01-01

Staphylococcus aureus is a common pathogen causing both hospital and community-acquired infections. Hemolysin is one of the important virulence factors for S. aureus and causes the typical β-hemolytic phenotype which is called complete hemolytic phenotype as well. Recently, S. aureus with an incomplete hemolytic phenotype (SIHP) was isolated from clinical samples. To study the microbiologic characteristics of SIHP, the special hemolytic phenotype of SIHP was verified on the sheep blood agar plates supplied by different manufacturers. Expression of hemolysin genes hla, hlb, hlgC , and hld of SIHP was detected by qRT-PCR and it was showed that expression of hlb in SIHP was obviously increased compared to the control S. aureus strains with complete hemolytic phenotype (SCHP), while the expression of hla, hlgC , and hld in SIHP was significantly decreased. In addition, the α-hemolysin encoded by gene hla was decreased obviously in SIHP compared to SCHP by western blot. All 60 SIHP strains were identified to be the methicillin resistant S. aureus (MRSA), and moreover these SIHP strains all contains mecA gene. The virulence gene tst were all present in SIHP, and the intracellular survival ability of SIHP was much greater than that of the gene tst negative S. aureus . We also found that IL-2, IL-6, and IL-17A secreted in the supernatant of SIHP infected macrophages increased significantly compared to tst negative control strains infected ones. MLST analysis showed that all of SIHP strains were classified into ST5 clone. To our knowledge, this study firstly showed that SIHP strains are a kind of methicillin resistant strains which express β-hemolysin highly and possess a potential high virulence, and it was suggested that SIHP should be paid more attention in hospital.

Transcriptional response of Musca domestica larvae to bacterial infection.

PubMed

Tang, Ting; Li, Xiang; Yang, Xue; Yu, Xue; Wang, Jianhui; Liu, Fengsong; Huang, Dawei

2014-01-01

The house fly Musca domestica, a cosmopolitan dipteran insect, is a significant vector for human and animal bacterial pathogens, but little is known about its immune response to these pathogens. To address this issue, we inoculated the larvae with a mixture of Escherichia coli and Staphylococcus aureus and profiled the transcriptome 6, 24, and 48 h thereafter. Many genes known to controlling innate immunity in insects were induced following infection, including genes encoding pattern recognition proteins (PGRPs), various components of the Toll and IMD signaling pathways and of the proPO-activating and redox systems, and multiple antimicrobial peptides. Interestingly, we also uncovered a large set of novel immune response genes including two broad-spectrum antimicrobial peptides (muscin and domesticin), which might have evolved to adapt to house-fly's unique ecological environments. Finally, genes mediating oxidative phosphorylation were repressed at 48 h post-infection, suggesting disruption of energy homeostasis and mitochondrial function at the late stages of infection. Collectively, our data reveal dynamic changes in gene expression following bacterial infection in the house fly, paving the way for future in-depth analysis of M. domestica's immune system.

Full-Genome Sequencing Identifies in the Genetic Background Several Determinants That Modulate the Resistance Phenotype in Methicillin-Resistant Staphylococcus aureus Strains Carrying the Novel mecC Gene

PubMed Central

de Lencastre, Hermínia; Tomasz, Alexander

2017-01-01

ABSTRACT Most methicillin-resistant Staphylococcus aureus (MRSA) strains are resistant to beta-lactam antibiotics due to the presence of the mecA gene, encoding an extra penicillin-binding protein (PBP2A) that has low affinity for virtually all beta-lactam antibiotics. Recently, a new resistance determinant—the mecC gene—was identified in S. aureus isolates recovered from humans and dairy cattle. Although having typically low MICs to beta-lactam antibiotics, MRSA strains with the mecC determinant are also capable of expressing high levels of oxacillin resistance when in an optimal genetic background. In order to test the impact of extensive beta-lactam selection on the emergence of mecC-carrying strains with high levels of antibiotic resistance, we exposed the prototype mecC-carrying MRSA strain, LGA251, to increasing concentrations of oxacillin. LGA251 was able to rapidly adapt to high concentrations of oxacillin in growth medium. In such laboratory mutants with increased levels of oxacillin resistance, we identified mutations in genes with no relationship to the mecC regulatory system, indicating that the genetic background plays an important role in the establishment of the levels of oxacillin resistance. Our data also indicate that the stringent stress response plays a critical role in the beta-lactam antibiotic resistance phenotype of MRSA strains carrying the mecC determinant. PMID:28069659

Reconstruction of mreB Expression in Staphylococcus aureus via a Collection of New Integrative Plasmids

PubMed Central

Yepes, Ana; Koch, Gudrun; Waldvogel, Andrea; Garcia-Betancur, Juan-Carlos

2014-01-01

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models Escherichia coli and Bacillus subtilis have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium Staphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the S. aureus chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of mreB in S. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in S. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use S. aureus as a model system in exploring diverse aspects of cellular microbiology. PMID:24747904

Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

PubMed

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu; He, Chuan

2012-04-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

PubMed Central

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu

2012-01-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl3 repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus. PMID:22267518

East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

PubMed

Jans, Christoph; Merz, Axel; Johler, Sophia; Younan, Mario; Tanner, Sabine A; Kaindi, Dasel Wambua Mulwa; Wangoh, John; Bonfoh, Bassirou; Meile, Leo; Tasara, Taurai

2017-08-01

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Tracing to the source of staphylococcus aureus isolates from ice cream].

PubMed

Zhang, Yan-Jun; Xu, Dan-Ge; Fang, Ye-Zhen; Gong, Pu; Zhu, Min; Bao, Fang-Zhen

2008-07-01

To investigate the contamination of Staphylococcus aureus isolates in ice cream by phenotypic typing and molecular typing. The Staphylococcus aureus isolates were separated from ice cream, filler, cutter, salves and material. The separated isolates were characterized by drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E, G-J) genes and pulsed-field gel electrophoresis (PFGE) types. Two Staphylococcus aureus isolates were separated, one from ice cream, another from cutter. Their characteristics of drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E,G-J) genes and PFGE type were the same. The two Staphylococcus aureus isolates were the same clone. The contaminated Staphylococcus aureus isolates could be traced to the contaminated cutters.

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis.

PubMed

Rall, V L M; Miranda, E S; Castilho, I G; Camargo, C H; Langoni, H; Guimarães, F F; Araújo Júnior, J P; Fernandes Júnior, A

2014-02-01

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes.

PubMed

Pinheiro, Luiza; Brito, Carla Ivo; de Oliveira, Adilson; Martins, Patrícia Yoshida Faccioli; Pereira, Valéria Cataneli; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-09-14

Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly producing the non-classical enterotoxins seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates.

Identification, Characterization, and Recombinant Expression of Epidermicin NI01, a Novel Unmodified Bacteriocin Produced by Staphylococcus epidermidis That Displays Potent Activity against Staphylococci

PubMed Central

Sandiford, Stephanie

2012-01-01

We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide. PMID:22155816

Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes

PubMed Central

Pinheiro, Luiza; Ivo Brito, Carla; de Oliveira, Adilson; Yoshida Faccioli Martins, Patrícia; Cataneli Pereira, Valéria; Ribeiro de Souza da Cunha, Maria de Lourdes

2015-01-01

Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly containing the non-classical enterotoxin genes seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates. PMID:26389954

Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus.

PubMed

Truong-Bolduc, Que Chi; Ding, Yanpeng; Hooper, David C

2008-11-01

MgrA is a global regulator in Staphylococcus aureus. Differences in the effects of MgrA on norA expression have been reported for different strains, which varied in rsbU, a gene that affects the expression of sigB, which encodes an alternative sigma factor involved in stress responses. We hypothesized that MgrA was modified by sigB-dependent factors that affected its ability to control the expression of the norA efflux pump. Heterologously expressed MgrA purified from Escherichia coli was incubated with crude extracts (CE) from strains RN6390 (rsbU) and SH1000 (rsbU(+)) and tested for binding to the norA promoter. Purified MgrA exhibited greater binding to norA promoter DNA after being incubated with SH1000 CE than MgrA incubated with the RN6390 CE. Phosphorylation of MgrA occurring in cell extracts caused it to lose the ability to bind norA promoter DNA. Overexpression of pknB, encoding a candidate serine/threonine kinase, produced increased phospho-MgrA and led to a fivefold increase in the transcript level of norA for both RN6390 and SH1000, as well as a fourfold increase in the MICs of norfloxacin and ciprofloxacin for these two strains. The levels of expression of pknB in RN6390 and SH1000, however, indicated that additional factors related to rsbU or sigB contribute to the differential regulatory effects of MgrA on norA expression.

Community-associated methicillin-resistant Staphylococcus aureus causing chronic pneumonia.

PubMed

Enayet, Iram; Nazeri, Ali; Johnson, Leonard B; Riederer, Kathleen; Pawlak, Joan; Saravolatz, Louis D

2006-04-01

A young woman presented with pneumonia of a 3-month duration with predominantly nodular pulmonary infiltrates. Methicillin-resistant Staphylococcus aureus was identified in multiple cultures of sputum specimens. According to findings of pulsed-field gel electrophoresis, the isolate was identical to USA 300 and carried a type IV Staphylococcus cassette chromosome mec type IV gene and the genes for Panton-Valentine leukocidin.

Species diversity and antibiotic resistance properties of Staphylococcus of farm animal origin in Nkonkobe Municipality, South Africa.

PubMed

Adegoke, Anthony A; Okoh, Anthony I

2014-03-01

The occurrence and antibiotic susceptibility profile of Staphylococcus isolates of healthy farm animal origin in Nkonkobe Municipality as well as the prevalence of putative antibiotic resistance genes were investigated using phenotypic and molecular methods. A total of 120 Staphylococcus isolates were isolated from 150 animal samples and consisted of Staphylococcus haemolyticus (30 %) and Staphylococcus aureus (23.3 %) from pig, Staphylococcus capitis (15 %) from goat, S. haemolyticus (5 %) and Staphylococcus xylosus (15 %) from cattle, and other staphylococci (11.7 %) from dead chicken and pigs. Besides this, the presence of these isolates was observed from the animal dung, showing that the organisms are shed to the environment. About 23.3 % of these isolates were coagulase-positive and 76.7 % were coagulase-negative Staphylococcus. Between 75 and 100 % of the isolates were resistant to penicillin G, tetracycline, sulfamethoxazole, and nalidixic acid; about 38 % were methicillin-resistant staphylococci, including 12.6 % methicillin-resistant S. aureus from pigs. In total, 12 % of all isolates were vancomycin resistant. Also, 12 % of the isolates were erythromycin resistant, while 40.2 % were resistant to ceftazidime. Only the genes mecA and mphC could be confirmed, whereas the genes vanA, vanB, ermA, ermB, and ermC could not be detected. The high phenotypic antibiotic resistance and the presence of some associated resistance genes is a potential threat to public health and suggest the animals to be important reservoirs of antibiotic resistance determinants in the environment.

Genomic and transcriptomic differences in community acquired methicillin resistant Staphylococcus aureus USA300 and USA400 strains.

PubMed

Jones, Marcus B; Montgomery, Christopher P; Boyle-Vavra, Susan; Shatzkes, Kenneth; Maybank, Rosslyn; Frank, Bryan C; Peterson, Scott N; Daum, Robert S

2014-12-19

Staphylococcus aureus is a human pathogen responsible for substantial morbidity and mortality through its ability to cause a number of human infections including bacteremia, pneumonia and soft tissue infections. Of great concern is the emergence and dissemination of methicillin-resistant Staphylococcus aureus strains (MRSA) that are resistant to nearly all β-lactams. The emergence of the USA300 MRSA genetic background among community associated S. aureus infections (CA-MRSA) in the USA was followed by the disappearance of USA400 CA-MRSA isolates. To gain a greater understanding of the potential fitness advantages and virulence capacity of S. aureus USA300 clones, we performed whole genome sequencing of 15 USA300 and 4 USA400 clinical isolates. A comparison of representative genomes of the USA300 and USA400 pulsotypes indicates a number of differences in mobile genome elements. We examined the in vitro gene expression profiles by microarray hybridization and the in vivo transcriptomes during lung infection in mice of a USA300 and a USA400 MRSA strain by performing complete genome qRT-PCR analysis. The unique presence and increased expression of 6 exotoxins in USA300 (12- to 600-fold) compared to USA400 may contribute to the increased virulence of USA300 clones. Importantly, we also observed the up-regulation of prophage genes in USA300 (compared with USA400) during mouse lung infection (including genes encoded by both prophages ΦSa2usa and ΦSa3usa), suggesting that these prophages may play an important role in vivo by contributing to the elevated virulence characteristic of the USA300 clone. We observed differences in the genetic content of USA300 and USA400 strains, as well as significant differences of in vitro and in vivo gene expression of mobile elements in a lung pneumonia model. This is the first study to document the global transcription differences between USA300 and USA400 strains during both in vitro and in vivo growth.

A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

PubMed

Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

2012-11-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

Characterization of a Prenyltransferase for Iso-A82775C Biosynthesis and Generation of New Congeners of Chloropestolides.

PubMed

Pan, Yuanyuan; Liu, Ling; Guan, Feifei; Li, Erwei; Jin, Jin; Li, Jinyang; Che, Yongsheng; Liu, Gang

2018-03-16

Chloropupukeananin and chloropestolides are novel metabolites of the plant endophyte Pestalotiopsis fici, showing antimicrobial, antitumor, and anti-HIV activities. Their highly complex and unique skeletons were generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on our previous studies. Here, we identified the biosynthetic gene cluster iac of 10 and characterized an iacE encoded prenyltransferase. Deletion of iacE abolished iso-A82775C production, accumulated the prenyl group-lacking siccayne (2), and generated four new chloropestolides (3-6). Compounds 5 and 6 showed antibacterial effects against Staphylococcus aureus and Bacillus subtilis, and 5 was also cytotoxic to human tumor cell lines HeLa, MCF-7, and SW480. These results provided the first genetic and biochemical insights into the biosynthesis of natural prenylepoxycyclohexanes and demonstrated the feasibility for generation of diversified congeners by manipulating the biosynthetic genes of 10.

Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis.

PubMed

Salaberry, Sandra Renata Sampaio; Saidenberg, André Becker Simões; Zuniga, Eveline; Melville, Priscilla Anne; Santos, Franklin Gerônimo Bispo; Guimarães, Ednaldo Carvalho; Gregori, Fábio; Benites, Nilson Roberti

2015-08-01

The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

PubMed Central

2013-01-01

Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

PubMed

Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed polysaccharide intercellular adhesin, which appears to have an important role in pathogenic Staph. aureus infections. The blaZ gene encodes the presence of the penicillin resistance in the strain. The EBS assay together with the logistic regression model revealed that the duration of therapy is the most important factor of therapy outcome in this in vitro model. Furthermore, the effect of genotypic differences seems to be more important for therapy outcome than the antimicrobial used in this model. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model.

PubMed

Capparelli, Rosanna; De Chiara, Francesco; Nocerino, Nunzia; Montella, Rosa Chiara; Iannaccone, Marco; Fulgione, Andrea; Romanelli, Alessandra; Avitabile, Concetta; Blaiotta, Giuseppe; Capuano, Federico

2012-11-17

Antimicrobial peptides (AMPs) are an ancient group of defense molecules. AMPs are widely distributed in nature (being present in mammals, birds, amphibians, insects, plants, and microorganisms). They display bactericidal as well as immunomodulatory properties. The aim of this study was to investigate the antimicrobial and anti-inflammatory activities of a combination of two AMPs (temporin B and the royal jellein I) against Staphylococcus epidermidis. The temporin B (TB-KK) and the royal jelleins I, II, III chemically modified at the C terminal (RJI-C, RJII-C, RJIII-C), were tested for their activity against 10 different Staphylococcus epidermidis strains, alone and in combination. Of the three royal jelleins, RJI-C showed the highest activity. Moreover, the combination of RJI-C and TB-KK (MIX) displayed synergistic activity. In vitro, the MIX displayed low hemolytic activity, no NO2- production and the ability to curb the synthesis of the pro-inflammatory cytokines TNF-α and IFN-γ to the same extent as acetylsalicylic acid. In vivo, the MIX sterilized mice infected with Staphylococcus epidermidis in eleven days and inhibited the expression of genes encoding the prostaglandin-endoperoxide synthase 2 (COX-2) and CD64, two important parameters of inflammation. The study shows that the MIX - a combination of two naturally occurring peptides - displays both antimicrobial and anti-inflammatory activities.

Rapid detection and differentiation of Staphylococcus colonies using an optical scattering technology.

PubMed

Alsulami, Tawfiq S; Zhu, Xingyue; Abdelhaseib, Maha Usama; Singh, Atul K; Bhunia, Arun K

2018-05-24

Staphylococcus species are a major pathogen responsible for nosocomial infections and foodborne illnesses. We applied a laser-based BARDOT (bacterial rapid detection using optical scattering technology) for rapid colony screening and detection of Staphylococcus on an agar plate and differentiate these from non-Staphylococcus spp. Among the six growth media tested, phenol red mannitol agar (PRMA) was found most suitable for building the Staphylococcus species scatter image libraries. Scatter image library for Staphylococcus species gave a high positive predictive value (PPV 87.5-100%) when tested against known laboratory strains of Staphylococcus spp., while the PPV against non-Staphylococcus spp. was 0-38%. A total of nine naturally contaminated bovine raw milk and ready-to-eat chicken salad samples were tested, and BARDOT detected Staphylococcus including Staphylococcus aureus with 80-100% PPV. Forty-five BARDOT-identified bacterial isolates from naturally contaminated foods were further confirmed by tuf and nuc gene-specific PCR and 16S rRNA gene sequence. This label-free, non-invasive on-plate colony screening technology can be adopted by the food industries, biotechnology companies, and public health laboratories for Staphylococcus species detection including S. aureus from various samples for food safety and public health management. Graphical abstract.

Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits.

PubMed

Roetzer, Andreas; Gruener, Corina S; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M

2016-10-28

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SE l N, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SE l N led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSE l N led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens.

Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits

PubMed Central

Roetzer, Andreas; Gruener, Corina S.; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M.

2016-01-01

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SElN, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SElN led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSElN led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens. PMID:27801832

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

PubMed Central

Denayer, Sarah; Nia, Yacine; Botteldoorn, Nadine

2017-01-01

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented. PMID:29261162

Phloretin derived from apple can reduce alpha-hemolysin expression in methicillin-resistant Staphylococcus aureus USA300.

PubMed

Zhou, Xuan; Liu, Shui; Li, Wenhua; Zhang, Bing; Liu, Bowen; Liu, Yan; Deng, Xuming; Peng, Liping

2015-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly important because it is the most common cause of hospital-acquired infections, which have become globally epidemic. Our study specifically focused on the MRSA strain USA300, which was shown in 2014 to be responsible for the most current pandemic of highly virulent MRSA in the United States. We aimed to evaluate the in vitro effect of phloretin on USA300. Susceptibility testing, western blotting assays, hemolysis assays and real-time RT-PCR were employed to examine the in vitro effects of phloretin on alpha-hemolysin (Hla) production when the bacterium was co-cultured with phloretin. The protective effect of phloretin against the USA300-mediated injury of human alveolar epithelial cells (A549) was tested using the live/dead analysis and cytotoxicity assays. We showed that sub-inhibitory concentrations of phloretin have no effect on bacterial viability; however, they can markedly inhibit the production of Hla in culture supernatants and the transcriptional levels of hla (the gene encoding Hla) and agrA (the accessory gene regulator). Phloretin, at a final concentration of 16 µg/ml, could protect A549 cells from injury caused by USA300 in the co-culture system. Our study suggests that phloretin might have a potential application in the development of treatment for MRSA infections.

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

DOE Office of Scientific and Technical Information (OSTI.GOV)

Safo, Martin K., E-mail: msafo@vcu.edu; Ko, Tzu-Ping; Musayev, Faik N.

The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor. The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA,more » and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Crystal structures of MW1337R and lin2004: Representatives of a novel protein family that adopt a four-helical bundle fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kozbial, Piotr; Xu, Qingping; Chiu, Hsiu-Ju

2009-08-28

To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs,more » which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similarity to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures of MW1337R and lin2004, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG), part of the National Institute of General Medical Sciences Protein Structure Initiative.« less

Reconstruction of mreB expression in Staphylococcus aureus via a collection of new integrative plasmids.

PubMed

Yepes, Ana; Koch, Gudrun; Waldvogel, Andrea; Garcia-Betancur, Juan-Carlos; Lopez, Daniel

2014-07-01

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models Escherichia coli and Bacillus subtilis have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium Staphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the S. aureus chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of mreB in S. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in S. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use S. aureus as a model system in exploring diverse aspects of cellular microbiology. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10.

PubMed

Bari, S M Nayeemul; Walker, Forrest C; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma

2017-12-15

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

Nosocomial outbreak of staphyloccocal scalded skin syndrome in neonates in England, December 2012 to March 2013.

PubMed

Paranthaman, K; Bentley, A; Milne, L M; Kearns, A; Loader, S; Thomas, A; Thompson, F; Logan, M; Newitt, S; Puleston, R

2014-08-21

Staphylococcal scalded skin syndrome (SSSS) is a blistering skin condition caused by exfoliative toxin-producing strains of Staphylococcus aureus. Outbreaks of SSSS in maternity settings are rarely reported. We describe an outbreak of SSSS that occurred among neonates born at a maternity unit in England during December 2012 to March 2013. Detailed epidemiological and microbiological investigations were undertaken. Eight neonates were found to be infected with the outbreak strain of S. aureus, of spa type t346, representing a single pulsotype. All eight isolates contained genes encoding exfoliative toxin A (eta) and six of them contained genes encoding toxin B (etb). Nasal swabs taken during targeted staff screening yielded a staphylococcal carriage rate of 21% (17/80), but none contained the outbreak strain. Mass screening involving multi-site swabbing and pooled, enrichment culture identified a healthcare worker (HCW) with the outbreak strain. This HCW was known to have a chronic skin condition and their initial nasal screen was negative. The outbreak ended when they were excluded from work. This outbreak highlights the need for implementing robust swabbing and culture methodswhen conventional techniques are unsuccessful in identifying staff carrier(s). This study adds to the growing body of evidence on the role of HCWs in nosocomial transmission of S. aureus.

Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae

PubMed Central

2012-01-01

Background Staphylococcus belongs to the Gram-positive low G + C content group of the Firmicutes division of bacteria. Staphylococcus aureus is an important human and veterinary pathogen that causes a broad spectrum of diseases, and has developed important multidrug resistant forms such as methicillin-resistant S. aureus (MRSA). Staphylococcus simiae was isolated from South American squirrel monkeys in 2000, and is a coagulase-negative bacterium, closely related, and possibly the sister group, to S. aureus. Comparative genomic analyses of closely related bacteria with different phenotypes can provide information relevant to understanding adaptation to host environment and mechanisms of pathogenicity. Results We determined a Roche/454 draft genome sequence for S. simiae and included it in comparative genomic analyses with 11 other Staphylococcus species including S. aureus. A genome based phylogeny of the genus confirms that S. simiae is the sister group to S. aureus and indicates that the most basal Staphylococcus lineage is Staphylococcus pseudintermedius, followed by Staphylococcus carnosus. Given the primary niche of these two latter taxa, compared to the other species in the genus, this phylogeny suggests that human adaptation evolved after the split of S. carnosus. The two coagulase-positive species (S. aureus and S. pseudintermedius) are not phylogenetically closest but share many virulence factors exclusively, suggesting that these genes were acquired by horizontal transfer. Enrichment in genes related to mobile elements such as prophage in S. aureus relative to S. simiae suggests that pathogenesis in the S. aureus group has developed by gene gain through horizontal transfer, after the split of S. aureus and S. simiae from their common ancestor. Conclusions Comparative genomic analyses across 12 Staphylococcus species provide hypotheses about lineages in which human adaptation has taken place and contributions of horizontal transfer in pathogenesis. PMID:22272658

Prevalence, antimicrobial susceptibility, and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China.

PubMed

Liu, Huimin; Li, Songli; Meng, Lu; Dong, Lei; Zhao, Shengguo; Lan, Xinyi; Wang, Jiaqi; Zheng, Nan

2017-11-01

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2", ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Evolutionary blueprint for host- and niche-adaptation in Staphylococcus aureus clonal complex CC30

PubMed Central

McGavin, Martin J.; Arsic, Benjamin; Nickerson, Nicholas N.

2012-01-01

Staphylococcus aureus clonal complex CC30 has caused infectious epidemics for more than 60 years, and, therefore, provides a model system to evaluate how evolution has influenced the disease potential of closely related strains. In previous multiple genome comparisons, phylogenetic analyses established three major branches that evolved from a common ancestor. Clade 1, comprised of historic pandemic phage type 80/81 methicillin susceptible S. aureus (MSSA), and Clade 2 comprised of contemporary community acquired methicillin resistant S. aureus (CA-MRSA) were hyper-virulent in murine infection models. Conversely, Clade 3 strains comprised of contemporary hospital associated MRSA (HA-MRSA) and clinical MSSA exhibited attenuated virulence, due to common single nucleotide polymorphisms (SNP's) that abrogate production of α-hemolysin Hla, and interfere with signaling of the accessory gene regulator agr. We have now completed additional in silico genome comparisons of 15 additional CC30 genomes in the public domain, to assess the hypothesis that Clade 3 has evolved to favor niche adaptation. In addition to SNP's that influence agr and hla, other common traits of Clade 3 include tryptophan auxotrophy due to a di-nucleotide deletion within trpD, a premature stop codon within isdH encoding an immunogenic cell surface protein involved in iron acquisition, loss of a genomic toxin–antitoxin (TA) addiction module, acquisition of S. aureus pathogenicity islands SaPI4, and SaPI2 encoding toxic shock syndrome toxin tst, and increased copy number of insertion sequence ISSau2, which appears to target transcription terminators. Compared to other Clade 3 MSSA, S. aureus MN8, which is associated with Staphylococcal toxic shock syndrome, exhibited a unique ISSau2 insertion, and enhanced production of toxic shock syndrome toxin encoded by SaPI2. Cumulatively, our data support the notion that Clade 3 strains are following an evolutionary blueprint toward niche-adaptation. PMID:22919639

Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

PubMed

Deshpande, Lalitagauri M; Ashcraft, Deborah S; Kahn, Heather P; Pankey, George; Jones, Ronald N; Farrell, David J; Mendes, Rodrigo E

2015-10-01

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 μg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of Staphylococcus epidermidis with transferrable mupirocin resistance from canine skin.

PubMed

Rossi, C C; Salgado, B A B; Barros, E M; de Campos Braga, P A; Eberlin, M N; Lilenbaum, W; Giambiagi-deMarval, M

2018-05-01

Resistance to mupirocin was analysed in Staphylococcus spp. isolated from healthy dogs (n=21) and dogs with pyoderma (n=47) or otitis externa (n=52). Isolates were identified to species level by MALDI-TOF and PCR-RFLP of the groEL gene. One isolate of Staphylococcus epidermidis from the skin of a healthy dog, which harboured a plasmid carrying the mupA gene, was resistant to mupirocin. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Differentiation of spa types and staphylococcal cassette chromosome mec (SCCmec) in clinical methicillin-resistant Staphylococcus aureus isolated in medical sites of Gdańsk region].

PubMed

Kasprzyk, Joanna; Piechowicz, Lidia; Wiśniewska, Katarzyna; Dziewit, Łukasz; Bronk, Marek; Świeć, Krystyna

2015-01-01

Methicillin-resistant Staphylococcus aureus bacteria are one of the key etiological factors of hospital-acquired and community-acquired infections. MRSA strains have an ability of causing a broad spectrum infections: from a relatively mild skin infections to severe life-threatening systemic infections. They are characterized by multi-drug resistance, virulence of a number of factors, may clonally spread within the hospitals and between hospitals. The study embraced a number of 75 isolates of MRSA isolated from patients of 7 medical sites of the Gdansk region within the period of six months (June to December 2013). Strains have derived from various clinical materials, both of hospitalized patients (n=59) and outpatient (n=16). The isolates were tested for the susceptibility to antimicrobial agents accordance with the guidelines EUCAST. To estimate of the variability of occurrence of S. aureus clones used were standard spa gene, consisting in the amplified polymorphic region of the X gene encoding the protein A gene (spa). After receiving the results, a spa types were identified using international database Ridom Spa Server (www.spaserver.ridom.de). To determine the polymorphism cassette carrying the inecA gene from MRSA strains, used typing five major chromosomal cassette SCCmec (I-V) by multiplex PCR. MRSA population genetic analysis carried out on the basis of typing SCCmec cassettes and spa gene has showed a predominance of strains with SCCmec type II casette (46.7%) and SCCmec IV casette (38.7%). Less frequently detected were strains containing SCCmec I cassette (12.0%) and SCCmec III cassette (2.6%). Spa typing revealed the presence of 13 gene types in MRSA. The most frequently observed spa types were: t151 (24.0%), t003 (16.0%) in strains of the SCCmec II cassette and t437 (16.0%) and t008 (14.8%) in the isolates with SCCmec cassette IV, whereas staphylococcus with the type of spa t011 (12.0%) had SCCmec cassette I. In our population most frequent strains cassette SCCmec II (46.7%), in most representing types of spa t151 (51.4%) and t003 (34.3%), generally resistant not only to β-lactam antibiotics, but as erythromycin, clindamycin and norfloxacin (82.8%), the more frequently they were isolated from patients than a hospital outpatient centers. The strains SCCmec IV that represent the majority of outpatient centers (68.8%), the most represented type t437 (41.4%) and often occurred in hospital centers.

[Genetic characterization and antimicrobial susceptibility analysis of methicillin-resistant Staphylococcus aureus isolated from ready-to-eat food and pig-related sources in China].

PubMed

Wang, Wei; Guo, Yunchang; Pei, Xiaoyan; Hu, Yujie; Bai, Li; Sun, Aiping; Liu, Jikai; Fu, Ping; Li, Fengqin

2013-11-01

To study the mecA gene distribution in 877 strains of Staphylococcus aureus isolated from the environment of pig farm and slaughter house, pig carcass and its iliac lymph nodes, and ready-to-eat foods in China as to screen the methicillin-resistant Staphylococcus aureus (MRSA), and to evaluate the antimicrobial susceptibility of MRSA. A total of 877 strains of S. aureus that had been phenotypically identified by Gram staining, catalase test, ability to coagulate rabbit plasma, API STAPH as well as analysis of nuc gene, encoding for a S. aureus specific thermonu-clease were screened for MRSA by characterizing the mecA gene. The antimicrobial susceptibility of MRSA was tested in accordance with the broth microdilution method recommended by the Clinical and Laboratory Standards Institute. Of 877 S. aureus strains tested, 71 (8.1%, 71/887) were mecA positive and identified as MRSA, among which, 48 isolates were pig-associated and 23 isolates were ready-to-eat food-associated. The frequency of pig-associated MRSA was significantly higher than that of food-associated one (chi2 = 53.040, P < 0.01). All MRSA were susceptible to linezolid, vancomycin, tigecycline, and nitrofurantoin but resistant to cefoxitin, oxacillin and benzylpenicillin. Meanwhile, 98.6% (70 strains), 95.8% (68 strains), 88.7% (63 strains), 80.3% (57 strains), 80.3% (57 strains) and 32.4% (23 strains) MRSA exhibited the resistance to clindamycin, erythromycin, tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole, and gentamicin, respectively. Besides, one strain was resistant to each of antibiotics including levofloxacin, moxifloxacin, rifampicin, and quinupristin/dalfopristin. It was worth noting that the frequency of resistance to ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole of pig-associated MRSA was significantly higher than that of food-associated MRSA (CIP: chi2 = 29.110, P < 0.01, TET: chi2 = 18.816, P < 0.01, TMP/ SMZ: chi2 = 36.394, P < 0.01). It should be pointed out that 70 (98.6%) strains of MRSA were multi-drug resistant and eight spectrums of antimicrobial susceptibility were observed. The multi-drug resistant MRSA isolated from pig- and food-associated matrixin China is very serious.

Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

PubMed

Teng, Zihao; Shi, Dongxue; Liu, Huanyu; Shen, Ziying; Zha, Yonghong; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

2017-09-01

α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

Biofilm formation by Staphylococcus hominis strains isolated from human clinical specimens.

PubMed

Szczuka, Ewa; Telega, Kinga; Kaznowski, Adam

2015-01-01

Staphylococcus hominis is the third species of coagulase-negative staphylococci (CoNS) most frequently isolated from specimens of patients with hospital-acquired infections. Many infections caused by CoNS appeared to be associated with biofilms. Nevertheless, the knowledge of the ability of S. hominis to form a biofilm is limited. The aim of this study was to analyze the formation of the biofilm by 56 S. hominis strains isolated from clinical cases. The biofilm three-dimensional structure was reconstructed by confocal laser scanning microscopy. We found that most of S. hominis strains carried icaADBC genes encoding polysaccharide intercellular adhesin (PIA), which plays a crucial role in the formation of biofilms in staphylococci strains. However, only a half of the ica-positive strains had an ability to form a biofilm in vitro. In this study, we also accessed the sensitivity of biofilms of S. hominis strains to sodium metaperiodate, proteinase K and DNase. We found that polysaccharides and proteins are the major components of the extracellular matrix of the biofilm formed by S. hominis. DNase did not have a significant effect on biofilms, which suggested that nucleic acid plays a minor role in the mature biofilm.

Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcus aureus Mediated Killing of Human Neutrophils

PubMed Central

Nielsen, Anita; Månsson, Maria; Bojer, Martin S.; Gram, Lone; Larsen, Thomas O.; Novick, Richard P.; Frees, Dorte; Frøkiær, Hanne; Ingmer, Hanne

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus. PMID:24416329

The Staphylococcus aureus leucine aminopeptidase LAP is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine

PubMed Central

Carroll, Ronan K.; Veillard, Florian; Gagne, Danielle T.; Lindenmuth, Jarrod M.; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N.

2013-01-01

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part, due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase - LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria, however, in systemic and localized infection models the pepZ mutant was significantly attenuated in virulence. Recently, a contradictory report has been published, suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition we conduct a biochemical investigation of purified recombinant LAP identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine and that leucine is not the primary target of LAP. PMID:23241672

Co-location of the oxazolidinone resistance genes optrA and cfr on a multiresistance plasmid from Staphylococcus sciuri.

PubMed

Li, Dexi; Wang, Yang; Schwarz, Stefan; Cai, Jiachang; Fan, Run; Li, Jun; Feßler, Andrea T; Zhang, Rong; Wu, Congming; Shen, Jianzhong

2016-06-01

To identify and characterize the oxazolidinone/phenicol resistance gene optrA in Staphylococcus isolates. Fifty porcine staphylococci with florfenicol MICs of ≥16 mg/L were screened by PCR for the presence of the optrA gene. Transferability of optrA was examined by transformation and conjugation. Functionality of this gene was confirmed by cloning and expression in a susceptible Staphylococcus host. The optrA-carrying plasmid was completely sequenced and analysed. A single Staphylococcus sciuri was optrA positive. This isolate carried the optrA gene on the 60 563 bp multiresistance plasmid pWo28-3, which also harboured the resistance genes, cfr, fexA, aadD, ble and aacA-aphD. Plasmid pWo28-3 is composed of three regions (A, B and C). Region A, which harboured all resistance genes except optrA, showed ≥99.8% nucleotide sequence identity to the corresponding region of plasmids pJP1 and pJP1-like from Jeotgalicoccus pinnipedialis and Staphylococcus lentus, respectively. The optrA gene located in region B differed from the optrA gene of the Enterococcus faecalis plasmid pE349 by four nucleotide substitutions, which also resulted in amino acid substitutions. This optrA variant also conferred resistance to oxazolidinones and phenicols in staphylococci. The 28 genes in region C represent the plasmid backbone and were apparently acquired from staphylococci, enterococci and nosocomiicocci. This is the first report of the optrA gene in staphylococci and of the coexistence of optrA and cfr on the same plasmid. Dissemination of this plasmid will substantially limit the efficacy of oxazolidinones. Surveillance of optrA in staphylococci of both human and animal origin is urgently warranted. © The Author 2016. 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.

Evaluation of the biofilm forming ability and its associated genes in Staphylococcus species isolates from bovine mastitis in Argentinean dairy farms.

PubMed

Felipe, Verónica; Morgante, Carolina A; Somale, Paola S; Varroni, Florencia; Zingaretti, María L; Bachetti, Romina A; Correa, Silvia G; Porporatto, Carina

2017-03-01

Staphylococcus aureus and coagulase-negative staphylococci (CNS) are important causes of intramammary infection in dairy cattle, and their ability to produce biofilm is considered an important virulence property in the pathogenesis of mastitis. However, the published date on mechanisms and factors involved in infection persistence in the mammary gland remains unclear. The aim of this study was to investigate whether the main Staphylococcus species involved in bovine intramammary infections possess specific characteristics that promote colonization of the udder. We evaluated the biofilm-forming ability and distribution of adhesion- and biofilm-associated genes of Staphylococcus spp. isolated from bovine mastitis infected animals in Argentinean dairy farms. For this purpose, the phenotypic biofilm formation ability of 209 Staphylococcus spp. from bovine mastitis was investigated. All isolates produced biofilm in vitro, being 35,0% and 45,0% of the 127 S. aureus or 51,0% and 29,0% of the 82 CNS strong and moderate biofilm producers respectively. All S. aureus samples were PCR-positive for icaA, icaD, clfA, clfB and fnbpA genes, 76.3% were positive for fnbpB gene and 11.0% were positive for bap gene. In CNS isolates, the positive rates for icaA and icaD were 73.2%, while for clfA, clfB, fnbpA fnbpB and bap genes the percentage were lower. The results demonstrate that in Staphylococcus spp. biofilm formation, the polysaccharide and the adhesion- and biofilm-associated genes are of overall importance on bovine mastitis in Argentina. Therefore, future works should focus on these pathogenic specific factors for the development of more effective therapies of control, being essential to consider the ability of isolates to produce biofilm. Copyright © 2017 Elsevier Ltd. All rights reserved.

From the ORFeome concept to highly comprehensive, full-genome screening libraries.

PubMed

Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

2013-02-01

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

The agr Locus Regulates Virulence and Colonization Genes in Clostridium difficile 027

PubMed Central

Martin, Melissa J.; Clare, Simon; Goulding, David; Faulds-Pain, Alexandra; Barquist, Lars; Browne, Hilary P.; Pettit, Laura; Dougan, Gordon; Lawley, Trevor D.

2013-01-01

The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage. PMID:23772065

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

PubMed Central

Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

2004-01-01

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

PubMed Central

Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

2018-01-01

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

First report of sasX-positive methicillin-resistant Staphylococcus aureus in Japan.

PubMed

Nakaminami, Hidemasa; Ito, Teruyo; Han, Xiao; Ito, Ayumu; Matsuo, Miki; Uehara, Yuki; Baba, Tadashi; Hiramatsu, Keiichi; Noguchi, Norihisa

2017-09-01

SasX is a known virulence factor of Staphylococcus aureus involved in colonisation and immune evasion of the bacterium. The sasX gene, which is located on the ϕSPβ prophage, is frequently found in the sequence type (ST) 239 S. aureus lineage, which is the predominant healthcare-associated clone in Asian countries. In Japan, ST239 clones have rarely been identified, and sasX-positive strains have not been reported to date. Here, we report the first identification of 18 sasX-positive methicillin-resistant S. aureus (MRSA) strains in Japanese hospitals between 2009 and 2011. All sasX-positive isolates belonged to an ST239-staphylococcal cassette chromosome mec type III (ST239-III) lineage. However, we were unable to identify additional sasX-positive MRSA strains from 2012 to 2016, indicating that the small epidemic of sasX-positive isolates observed in this study was temporary. The sequence surrounding sasX in the strain TOHH628 lacked 51 genes that encode phage packaging and structural proteins, and no bacteriophage was induced by mitomycin C. Additionally, in the TOHH628 strain, the region (64.6 kb) containing sasX showed high identity to the ϕSPβ-like element (71.3 kb) of the Taiwanese MRSA strain Z172. The data strongly suggest that the present sasX-positive isolates found in Japanese hospitals were transmitted incidentally from other countries. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Vancomycin Resistance in Staphylococcus aureus


PubMed Central

McGuinness, Will A.; Malachowa, Natalia; DeLeo, Frank R.

2017-01-01

The evolution of Staphylococcus aureus during the modern antibiotic era has been delineated by distinct strain emergence events, many of which include acquisition of antibiotic resistance. The relative high burden of methicillin-resistant S. aureus (MRSA) in healthcare and community settings is a major concern worldwide. Vancomycin, a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe MRSA infections. S. aureus strains exhibiting increased resistance to vancomycin, known as vancomycin intermediate-resistant S. aureus (VISA) (MIC = 4-8 µg/mL), were discovered in the 1990s. The molecular basis of resistance in VISA is polygenic and involves stepwise mutations in genes encoding molecules predominantly involved in cell envelope biosynthesis. S. aureus isolates with complete resistance to vancomycin (MIC ≥ 16 µg/mL) are termed vancomycin-resistant S. aureus (VRSA)—they were first reported in the U.S. in 2002. Resistance in VRSA is conferred by the vanA gene and operon, which is present on a plasmid. Although treatment of VRSA infections is challenging, the total number of human VRSA infections to date is limited (14 in the U.S.). By comparison, the burden of VISA is relatively high and the molecular mechanisms of resistance are less well-defined. VISA are associated with persistent infections, vancomycin treatment failure, and poor clinical outcomes. Here, we review in brief progress made toward understanding the acquisition of antibiotic resistance in S. aureus, with an emphasis on the molecular mechanisms underlying vancomycin resistance. PMID:28656013

Vancomycin Resistance in Staphylococcus aureus
.

PubMed

McGuinness, Will A; Malachowa, Natalia; DeLeo, Frank R

2017-06-01

The evolution of Staphylococcus aureus during the modern antibiotic era has been delineated by distinct strain emergence events, many of which include acquisition of antibiotic resistance. The relative high burden of methicillin-resistant S. aureus (MRSA) in healthcare and community settings is a major concern worldwide. Vancomycin, a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe MRSA infections. S. aureus strains exhibiting increased resistance to vancomycin, known as vancomycin intermediate-resistant S. aureus (VISA) (MIC = 4-8 µg/mL), were discovered in the 1990s. The molecular basis of resistance in VISA is polygenic and involves stepwise mutations in genes encoding molecules predominantly involved in cell envelope biosynthesis. S. aureus isolates with complete resistance to vancomycin (MIC ≥ 16 µg/mL) are termed vancomycin-resistant S. aureus (VRSA)-they were first reported in the U.S. in 2002. Resistance in VRSA is conferred by the vanA gene and operon, which is present on a plasmid. Although treatment of VRSA infections is challenging, the total number of human VRSA infections to date is limited (14 in the U.S.). By comparison, the burden of VISA is relatively high and the molecular mechanisms of resistance are less well-defined. VISA are associated with persistent infections, vancomycin treatment failure, and poor clinical outcomes. Here, we review in brief progress made toward understanding the acquisition of antibiotic resistance in S. aureus , with an emphasis on the molecular mechanisms underlying vancomycin resistance.

Staphylococci isolated from ready-to-eat meat - Identification, antibiotic resistance and toxin gene profile.

PubMed

Fijałkowski, Karol; Peitler, Dorota; Karakulska, Jolanta

2016-12-05

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevalence of β-lactamase genes in domestic washing machines and dishwashers and the impact of laundering processes on antibiotic-resistant bacteria.

PubMed

Rehberg, L; Frontzek, A; Melhus, Å; Bockmühl, D P

2017-12-01

To investigate the prevalence of β-lactamase genes in domestic washing machines and dishwashers, and the decontamination efficacy of laundering. For the first investigation, swab samples from washing machines (n = 29) and dishwashers (n = 24) were analysed by real-time quantitative PCR to detect genes encoding β-lactamases. To test the impact of laundering on resistant bacteria, cotton test swatches were artificially contaminated with susceptible and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus within a second investigation. They were washed in a domestic washing machine with or without activated oxygen bleach (AOB)-containing detergent at 20-50°C. β-Lactamase genes (most commonly of the AmpC- and OXA-type) were detected in 79% of the washing machines and in 96% of the dishwashers and Pseudomonadaceae dominated the microbiota. The level of bacterial reduction after laundering was ≥80% for all Ps. aeruginosa and Kl. pneumoniae strains, while it was only 37-61% for the methicillin-resistant Staph. aureus outbreak strain. In general, the reduction was tendentially higher for susceptible bacteria than for the resistant outbreak strains, especially for Staph. aureus. β-Lactamase genes seem to be frequently present in domestic appliances and may pose a potential risk for cross-contamination and horizontal transfer of genes encoding resistance against clinically important β-lactams. In general, higher temperatures and the use of AOB can improve the reduction of antibiotic-resistant bacteria, including Staph. aureus which appears to be less susceptible to the decontamination effect of laundering. Data on the presence of antibiotic-resistant bacteria in the domestic environment are limited. This study suggests that β-lactamase genes in washing machines and dishwashers are frequent, and that antibiotic-resistant strains are generally more resistant to the used washing conditions. © 2017 The Society for Applied Microbiology.

Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

PubMed

Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

2016-07-01

The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.

Wild rodents and shrews are natural hosts of Staphylococcus aureus.

PubMed

Mrochen, Daniel M; Schulz, Daniel; Fischer, Stefan; Jeske, Kathrin; El Gohary, Heba; Reil, Daniela; Imholt, Christian; Trübe, Patricia; Suchomel, Josef; Tricaud, Emilie; Jacob, Jens; Heroldová, Marta; Bröker, Barbara M; Strommenger, Birgit; Walther, Birgit; Ulrich, Rainer G; Holtfreter, Silva

2017-09-22

Laboratory mice are the most commonly used animal model for Staphylococcus aureus infection studies. We have previously shown that laboratory mice from global vendors are frequently colonized with S. aureus. Laboratory mice originate from wild house mice. Hence, we investigated whether wild rodents, including house mice, as well as shrews are naturally colonized with S. aureus and whether S. aureus adapts to the wild animal host. 295 animals of ten different species were caught in different locations over four years (2012-2015) in Germany, France and the Czech Republic. 45 animals were positive for S. aureus (15.3%). Three animals were co-colonized with two different isolates, resulting in 48 S. aureus isolates in total. Positive animals were found in Germany and the Czech Republic in each studied year. The S. aureus isolates belonged to ten different spa types, which grouped into six lineages (clonal complex (CC) 49, CC88, CC130, CC1956, sequence type (ST) 890, ST3033). CC49 isolates were most abundant (17/48, 35.4%), followed by CC1956 (14/48, 29.2%) and ST890 (9/48, 18.8%). The wild animal isolates lacked certain properties that are common among human isolates, e.g., a phage-encoded immune evasion cluster, superantigen genes on mobile genetic elements and antibiotic resistance genes, which suggests long-term adaptation to the wild animal host. One CC130 isolate contained the mecC gene, implying wild rodents might be both reservoir and vector for methicillin-resistant S. aureus. In conclusion, we demonstrated that wild rodents and shrews are naturally colonized with S. aureus, and that those S. aureus isolates show signs of host adaptation. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Outbreak of Staphylococcal food poisoning due to SEA-producing Staphylococcus aureus.

PubMed

Johler, Sophia; Tichaczek-Dischinger, Petra S; Rau, Jörg; Sihto, Henna-Maria; Lehner, Angelika; Adam, Maja; Stephan, Roger

2013-09-01

In 2008, 150 people gathered for a wedding celebration in Baden-Württemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy. Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transform-infrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determine the presence/absence of genes involved in virulence and antimicrobial resistance. We were able to match an enterotoxigenic strain from the stool sample of a patient to isolates of the same strain obtained from food and the nasal cavity of a food handler. The strain produced the enterotoxin SEA and the toxic shock syndrome toxin-1, and was also found to exhibit the genes encoding enterotoxins SEG and SEI, as well as the enterotoxin gene cluster egc. This is one of only a few studies that were able to link a staphylococcal food poisoning outbreak to its source.

Antibiotic resistance and clonal diversity of invasive Staphylococcus aureus in the rural Ashanti Region, Ghana.

PubMed

Dekker, Denise; Wolters, Manuel; Mertens, Eva; Boahen, Kennedy Gyau; Krumkamp, Ralf; Eibach, Daniel; Schwarz, Norbert G; Adu-Sarkodie, Yaw; Rohde, Holger; Christner, Martin; Marks, Florian; Sarpong, Nimako; May, Jürgen

2016-11-29

Staphylococcus aureus is among the most common pathogens isolated from blood cultures in Ghana; yet the epidemiology of blood infections in rural settings is poorly described. This study aims to investigate antimicrobial susceptibility and clonal diversity of S. aureus causing bloodstream infections in two hospitals in the Ashanti Region, Ghana. Blood cultures were performed for all febrile patients (≥37.5 °C) on hospital admission. Antibiotic susceptibility testing for S. aureus isolates was carried out by the VITEK 2 system. Multiplex polymerase chain reaction (PCR) was used to detect S. aureus-specific nuc gene, Panton-Valentine leukocidin (PVL), and methicillin-resistant S. aureus (MRSA)-specific mecA and mecC genes. The population structure of S. aureus was assessed by spa typing. In total, 9,834 blood samples were cultured, out of which 0.6% (n = 56) were positive for S. aureus. Multidrug resistance (MDR) was detected in 35.7% (n = 20) of the S. aureus strains, of which one was a MRSA. The highest rate of antibiotic resistance was seen for commonly available antibiotics, including penicillin (n = 55; 98.2%), tetracycline (n = 32; 57.1%) and trimethoprim/sulfamethoxazole (n = 26; 46.4%). Of all S. aureus strains, 75.0% (n = 42) carried the PVL-encoding genes. We found 25 different spa types with t355 (n = 11; 19.6%), t314 (n = 8; 14.3%), t084 (n = 8; 14.3%) and t311 (n = 5; 8.9%) being predominant. The study exhibited an alarmingly large level of antibiotic resistance to locally available antibiotics. The frequency of genetically diverse and PVL-positive methicillin-sensitive S. aureus (MSSA) was high and could represent a reservoir for the emergence of virulent PVL-positive MRSA clones.

Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin

PubMed Central

Palomo, Sara; González, Ignacio; de la Cruz, Mercedes; Martín, Jesús; Tormo, José Rubén; Anderson, Matthew; Hill, Russell T.; Vicente, Francisca; Reyes, Fernando; Genilloud, Olga

2013-01-01

Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs. PMID:23538871

Sponge-derived Kocuria and Micrococcus spp. as sources of the new thiazolyl peptide antibiotic kocurin.

PubMed

Palomo, Sara; González, Ignacio; de la Cruz, Mercedes; Martín, Jesús; Tormo, José Rubén; Anderson, Matthew; Hill, Russell T; Vicente, Francisca; Reyes, Fernando; Genilloud, Olga

2013-03-28

Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.

Analysis of Staphylococcal cassette chromosome mec in Staphylococcus haemolyticus and Staphylococcus sciuri: identification of a novel ccr gene complex with a newly identified ccrA allotype (ccrA7).

PubMed

Urushibara, Noriko; Paul, Shyamal Kumar; Hossain, Mohammad Akram; Kawaguchiya, Mitsuyo; Kobayashi, Nobumichi

2011-06-01

Methicillin resistance in staphylococci is conferred by the acquisition in its chromosome of the mecA gene, which is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). Genetic type of SCCmec is defined by combination of mec gene complex class and cassette chromosome recombinase gene (ccr) allotype. In this study, we analyzed genetic diversity of the SCCmec in 11 Staphylococcus haemolyticus strains and a Staphylococcus sciuri strain, which were recently isolated from clinical specimens in Bangladesh. Among these strains, only two S. haemolyticus strains were proved to have the known types of SCCmec, that is, SCCmec V (class C2 mec-ccrC) and VII (class C1 mec-ccrC). Five S. haemolyticus strains were assigned two unique mec-ccr gene complexes combination; that is, class C1 mec-ccrA4B4 (four isolates) and class A mec-ccrC (one isolate). In the remaining four S. haemolyticus strains with class C1 mec, no known ccr allotypes could be detected. A single S. sciuri strain with class A mec complex carried a ccrA gene belonging to a novel allotype designated ccrA7, together with ccrB3. The ccrA7 gene in the S. sciuri strain showed 61.7%-82.7% sequence identity to the ccrA gene sequences published so far, and 75.3% identity to ccrA3, which is a component of the type 3 ccr complex (ccrA3-ccrB3) in methicillin-resistant Staphylococcus aureus. The results of the present study indicated that mec gene complex and ccr genes in coagulase-negative staphylococci are highly divergent, and distinct from those of common methicillin-resistant S. aureus. Identification of the novel ccrA7 allotype combined with ccrB3 suggested an occurrence of recombination between different ccr complexes in nature.

Comparison of the gentamicin resistance transposon Tn5281 with regions encoding gentamicin resistance in Enterococcus faecalis isolates from diverse geographic locations.

PubMed Central

Hodel-Christian, S L; Murray, B E

1992-01-01

The genetic determinant encoding gentamicin resistance (Gmr) on the beta-lactamase encoding plasmid pBEM10 of Enterococcus faecalis HH22 is carried on a transposon, termed Tn5281, that is highly related to the staphylococcal Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in United States isolates of Staphylococcus epidermidis. We have now studied plasmid DNA from Gmr strains of E. faecalis isolated from diverse geographical locations (Houston, Pennsylvania, Thailand, and Chile) by using restriction endonuclease analysis and DNA-DNA hybridization to determine whether other Gmr E. faecalis carry Tn5281 or a similar type of element. We also compared these enterococci to several United States isolates of Staphylococcus aureus with nonmobile Gmr determinants. Three E. faecalis isolates (from Houston and Chile) carried Tn5281-like elements, whereas two isolates (from Houston and Pennsylvania) had restriction endonuclease and DNA-DNA hybridization patterns more similar to those of the Tn4001-IS257 hybrid found in the nonmobile Gmr determinants in United States isolates of S. aureus. A strain from Thailand had a third pattern unrelated to either Tn5281 or the nonmobile Gmr determinants present in United States isolates of S. aureus. Our results demonstrate that there is both similarity and diversity between the Gmr determinant of strains of E. faecalis isolated in diverse geographic locations. Images PMID:1332593

The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

PubMed

Xu, Jia; Tan, Xiao; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2015-11-01

Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese dairy herd are different from the previously reported, and that the herd- or farm-based diagnosis of staphylococcal bovine mastitis is required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prevalence and Genetic Characteristics of Staphylococcus aureus and Staphylococcus argenteus Isolates Harboring Panton-Valentine Leukocidin, Enterotoxins, and TSST-1 Genes from Food Handlers in Myanmar.

PubMed

Aung, Meiji Soe; San, Thida; Aye, Mya Mya; Mya, San; Maw, Win Win; Zan, Khin Nyein; Htut, Wut Hmone Win; Kawaguchiya, Mitsuyo; Urushibara, Noriko; Kobayashi, Nobumichi

2017-08-04

Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes ( pvl ) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa -VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl -positive clinical isolates in Myanmar. A pvl -positive, ST2250 nasal isolate was identified as S. argenteus , a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl -negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw . The present study revealed the relatively high rate of pvl , as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar.

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

PubMed

Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

2016-11-18

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

Characterisation of nasal Staphylococcus delphini and Staphylococcus pseudintermedius isolates from healthy donkeys in Tunisia.

PubMed

Gharsa, H; Slama, K Ben; Gómez-Sanz, E; Gómez, P; Klibi, N; Zarazaga, M; Boudabous, A; Torres, C

2015-07-01

Staphylococcus intermedius group (SIG) bacteria can colonise the nares of some animals but are also emerging pathogens in humans and animals. To analyse SIG nasal carriage in healthy donkeys destined for food consumption in Tunisia and to characterise recovered isolates. Nasal swabs from 100 healthy donkeys were tested for SIG recovery, and isolates were identified by biochemical and molecular methods. Antimicrobial susceptibility of isolates was tested and detection of antimicrobial resistance and virulence genes was performed. Isolates were typed at the clonal level by multilocus sequence typing and SmaI pulsed-field gel electrophoresis. Staphylococcus delphini and Staphylococcus pseudintermedius (included in SIG) were obtained in 19% and 2% of the tested samples, respectively, and one isolate per sample was characterised. All isolates were meticillin susceptible and mecA negative. Most S. delphini and S. pseudintermedius isolates showed susceptibility to all antimicrobials tested, with the exception of 2 isolates resistant to tetracycline (tet(M) gene) or fusidic acid. The following toxin genes were identified (percentage of isolates): lukS-I (100%), lukF-I (9.5%), siet (100%), se-int (90%), seccanine (19%) and expA (9.5%). Thirteen different pulsed-field gel electrophoresis profiles were identified among the 21 SIG isolates. Additionally, the following 9 different sequence types (STs) were detected by multilocus sequence typing, 6 of them new: ST219 (6 isolates), ST12 (5 isolates), ST220 (3 isolates), ST13, ST50, ST193, ST196, ST218 and ST221 (one isolate each). Staphylococcus delphini and S. pseudintermedius are common nasal colonisers of donkeys, generally susceptible to the antimicrobials tested; nevertheless, these SIG isolates contain virulence genes, including the recently described exfoliative gene (expA) and several enterotoxin genes, with potential implications for public health. This is the first description of S. delphini in Tunisia. The Summary is available in Chinese - see Supporting information. © 2014 EVJ Ltd.

Detection of mecC-Positive Staphylococcus aureus (CC130-MRSA-XI) in Diseased European Hedgehogs (Erinaceus europaeus) in Sweden

PubMed Central

Monecke, Stefan; Gavier-Widen, Dolores; Mattsson, Roland; Rangstrup-Christensen, Lena; Lazaris, Alexandros; Coleman, David C.; Shore, Anna C.; Ehricht, Ralf

2013-01-01

Recently, a novel mec gene conferring beta-lactam resistance in Staphylococcus aureus has been discovered. This gene, mecC, is situated on a SCCmec XI element that has to date been identified in clonal complexes 49, 130, 425, 599 and 1943. Some of the currently known isolates have been identified from animals. This, and observations of mecA alleles that do not confer beta-lactam resistance, indicate that mec genes might have a reservoir in Staphylococcus species from animals. Thus it is important also to screen wildlife isolates for mec genes. Here, we describe mecC-positive Staphylococcus aureus (ST130-MRSA-XI) and the lesions related to the infection in two diseased free-ranging European hedgehogs (Erinaceus europaeus). One was found dead in 2003 in central Sweden, and suffered from S. aureus septicaemia. The other one, found on the island of Gotland in the Baltic Sea in 2011, showed a severe dermatitis and was euthanised. ST130-MRSA-XI isolates were isolated from lesions from both hedgehogs and were essentially identical to previously described isolates from humans. Both isolates carried the complete SCCmec XI element. They lacked the lukF-PV/lukS-PV and lukM/lukF-P83 genes, but harboured a gene for an exfoliative toxin homologue previously described from Staphylococcus hyicus, Staphylococcus pseudintermedius and other S. aureus of the CC130 lineage. To the best of our knowledge, these are the first reported cases of CC130-MRSA-XI in hedgehogs. Given that one of the samples was taken as early as 2003, this was the earliest detection of this strain and of mecC in Sweden. This and several other recent observations suggest that CC130 might be a zoonotic lineage of S. aureus and that SCCmec XI/mecC may have originated from animal pathogens. PMID:23776626

Detection of mecC-positive Staphylococcus aureus (CC130-MRSA-XI) in diseased European hedgehogs (Erinaceus europaeus) in Sweden.

PubMed

Monecke, Stefan; Gavier-Widen, Dolores; Mattsson, Roland; Rangstrup-Christensen, Lena; Lazaris, Alexandros; Coleman, David C; Shore, Anna C; Ehricht, Ralf

2013-01-01

Recently, a novel mec gene conferring beta-lactam resistance in Staphylococcus aureus has been discovered. This gene, mecC, is situated on a SCCmec XI element that has to date been identified in clonal complexes 49, 130, 425, 599 and 1943. Some of the currently known isolates have been identified from animals. This, and observations of mecA alleles that do not confer beta-lactam resistance, indicate that mec genes might have a reservoir in Staphylococcus species from animals. Thus it is important also to screen wildlife isolates for mec genes. Here, we describe mecC-positive Staphylococcus aureus (ST130-MRSA-XI) and the lesions related to the infection in two diseased free-ranging European hedgehogs (Erinaceus europaeus). One was found dead in 2003 in central Sweden, and suffered from S. aureus septicaemia. The other one, found on the island of Gotland in the Baltic Sea in 2011, showed a severe dermatitis and was euthanised. ST130-MRSA-XI isolates were isolated from lesions from both hedgehogs and were essentially identical to previously described isolates from humans. Both isolates carried the complete SCCmec XI element. They lacked the lukF-PV/lukS-PV and lukM/lukF-P83 genes, but harboured a gene for an exfoliative toxin homologue previously described from Staphylococcus hyicus, Staphylococcus pseudintermedius and other S. aureus of the CC130 lineage. To the best of our knowledge, these are the first reported cases of CC130-MRSA-XI in hedgehogs. Given that one of the samples was taken as early as 2003, this was the earliest detection of this strain and of mecC in Sweden. This and several other recent observations suggest that CC130 might be a zoonotic lineage of S. aureus and that SCCmec XI/mecC may have originated from animal pathogens.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

PubMed

Sanchini, A; Del Grosso, M; Villa, L; Ammendolia, M G; Superti, F; Monaco, M; Pantosti, A

2014-11-01

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

PubMed

Zeden, Merve S; Schuster, Christopher F; Bowman, Lisa; Zhong, Qiyun; Williams, Huw D; Gründling, Angelika

2018-03-02

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping.

PubMed

Gallina, S; Bianchi, D M; Bellio, A; Nogarol, C; Macori, G; Zaccaria, T; Biorci, F; Carraro, E; Decastelli, L

2013-12-01

In June 2011, an outbreak of Staphylococcus aureus enterotoxin food poisoning gastroenteritis occurred in Turin, Italy, following a catered dinner party at a private home. Within a few hours, 26 of the 47 guests experienced gastrointestinal illness, and 9 were hospitalized. A retrospective cohort study using a standardized questionnaire was carried out, and the risk ratios for each food item were calculated. The analysis indicated consumption of seafood salad as the most probable cause of the outbreak (risk ratio = 11.72; 95 % confidence interval, 1.75 to 78.54). Biological samples were collected from four of the hospitalized guests (stool and vomit), nasal mucosa swabs from three food handlers employed with the caterer, and available food residuals. All stool and vomit samples tested positive for enterotoxigenic S. aureus. As residues of the seafood salad were no longer available for sampling, suspected contamination could not be verified. However, no other food was found contaminated by S. aureus or its enterotoxins. All isolates from the biological samples were characterized at the genomic level by means of two multiplex PCR protocols to determine the presence of genes encoding staphylococcal enterotoxins, pulsed-field gel electrophoresis and staphylococcal protein A gene (spa) typing to describe their genetic profiles. All the isolates presented genes encoding SEA and SEI; the pulsed-field gel electrophoresis genetic profiles revealed the same pulsotype in the microorganism isolated from the hospitalized guests as in one of the isolates from a food handler's nasal mucosa, and the spa typing analysis reported two closely related spa types (t701 and t267), implicating the food handler as the most likely outbreak source.

Successful Multiresistant Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from Taipei, Taiwan, That Carries Either the Novel Staphylococcal Chromosome Cassette mec (SCCmec) Type VT or SCCmec Type IV

PubMed Central

Boyle-Vavra, Susan; Ereshefsky, Ben; Wang, Chih-Chien; Daum, Robert S.

2005-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) isolates carry the methicillin resistance gene (mecA) on a horizontally transferred genetic element called the staphylococcal chromosome cassette mec (SCCmec). Community-acquired MRSA (CAMRSA) isolates usually carry SCCmec type IV. We previously reported that 76% of 17 CAMRSA isolates (multilocus sequence type 59) obtained from pediatric patients with skin and soft tissue infections (SSTI) from Taipei did not carry SCCmec types I to IV. We used DNA sequence analysis to determine that the element harbored by these nontypeable isolates is a novel subtype of SCCmec V called SCCmec VT. It contains a ccrC recombinase gene variant (ccrC2) and mec complex C2. One SSTI isolate contained molecular features of SCCmec IV but also contained ccrC2 (a feature of SCCmec VT), suggesting that it may harbor a composite SCCmec element. The genes lukS-PV and lukF-PV encoding the Panton-Valentine leukocidin (PVL) were present in all CAMRSA SSTI isolates whether they contained SCCmec type IV or VT. SCCmec VT was also present in 5 of 34 (14.7%) CAMRSA colonization isolates collected from healthy children from Taipei who lacked MRSA risk factors. Four (80%) of the these isolates contained lukS-PV and lukF-PV, as did 1 of 27 (3.7%) SCCmec IV-containing colonization isolates. A total of 63% (10 of 16) of the SSTI isolates and 61.7% (21 of 34) of the colonization isolates tested were resistant to at least four classes of non-β-lactam antimicrobials. SCCmec VT is a novel SCCmec variant that is found in multiply resistant CAMRSA strains with sequence type 59 in Taipei in association with the PVL leukotoxin genes. PMID:16145133

Whole-genome comparison of meticillin-resistant Staphylococcus aureus CC22 SCCmecIV from people and their in-contact pets.

PubMed

Loeffler, Anette; McCarthy, Alex; Lloyd, David H; Musilová, Eva; Pfeiffer, Dirk U; Lindsay, Jodi A

2013-10-01

Meticillin-resistant Staphylococcus aureus (MRSA) infections remain important medical and veterinary challenges. The MRSA isolated from dogs and cats typically belong to dominant hospital-associated clones, in the UK mostly EMRSA-15 (CC22 SCCmecIV), suggesting original human-to-animal transmission. Nevertheless, little is known about host-specific genetic variation within the same S. aureus lineage. To identify host-specific variation amongst MRSA CC22 SCCmecIV by comparing isolates from pets with those from in-contact humans using whole-genome microarray. Six pairs of MRSA CC22 SCCmecIV from human carriers (owners and veterinary staff) and their respective infected in-contact pets were compared using a 62-strain whole-genome S. aureus microarray (SAM-62). The presence of putative host-specific genes was subsequently determined in a larger number of human (n = 47) and pet isolates (n = 93) by PCR screening. Variation in mobile genetic elements (MGEs) occurred frequently and appeared largely independent of host and in-contact pair. A plasmid (SAP078A) encoding heavy-metal resistance genes (arsR, arsA, cadA, cadC, mco and copB) was found in three of six human and none of six animal isolates. However, only two of four resistance genes were associated with human hosts (P = 0.015 for arsA and cadA). The variation found amongst MGEs highlights that genetic adaptation in MRSA continues. However, host-specific MGEs were not detected, which supports the hypothesis that pets may not be natural hosts of MRSA CC22 and emphasizes that rigorous hygiene measures are critical to prevent contamination and infection of dogs and cats. The host specificity of individual heavy-metal resistance genes warrants further investigation into different selection pressures in humans and animals. © 2013 ESVD and ACVD.

Sanitary quality, occurrence and identification of Staphylococcus sp. in food services.

PubMed

de Mello, Jozi Fagundes; da Rocha, Laura Braga; Lopes, Ester Souza; Frazzon, Jeverson; da Costa, Marisa

2014-01-01

Sanitary conditions are essential for the production of meals and control of the presence of pathogensis important to guarantee the health of customers. The aim of this study was to evaluate the sanitary quality of food services by checking the presence of thermotolerant coliforms, Staphylococcus sp. and evaluate the toxigenic potential from the latter. The analysis was performed on water, surfaces, equipment, ready-to-eat foods, hands and nasal cavity of handlers in seven food services. The water used in food services proved to be suitable for the production of meals. Most food, equipment and surfaces showed poor sanitary conditions due to the presence of thermotolerant coliforms (60.6%). Twenty-six Staphylococcus species were identified from the 121 Staphylococcus isolates tested. Staphylococci coagulase-negative species were predominant in the foods, equipment and surfaces. In food handlers and foods, the predominant species was Staphylococcus epidermidis. Twelve different genotypes were found after PCR for the classical enterotoxin genes. The seb gene (19.8%) was the most prevalent among all Staphylococcus sp. Both coagulase-positive and coagulase-negative Staphylococci showed some of the genes of the enterotoxins tested. We conclude that there are hygienic and sanitary deficiencies in the food services analyzed. Although coagulase-positive Staphylococci have not been present in foods there is a wide dispersion of enterotoxigenic coagulase-negative Staphylococci in the environment and in the foods analyzed, indicating a risk to consumer health.

Sanitary quality, occurrence and identification of Staphylococcus sp. in food services

PubMed Central

de Mello, Jozi Fagundes; da Rocha, Laura Braga; Lopes, Ester Souza; Frazzon, Jeverson; da Costa, Marisa

2014-01-01

Sanitary conditions are essential for the production of meals and control of the presence of pathogensis important to guarantee the health of customers. The aim of this study was to evaluate the sanitary quality of food services by checking the presence of thermotolerant coliforms, Staphylococcus sp. and evaluate the toxigenic potential from the latter. The analysis was performed on water, surfaces, equipment, ready-to-eat foods, hands and nasal cavity of handlers in seven food services. The water used in food services proved to be suitable for the production of meals. Most food, equipment and surfaces showed poor sanitary conditions due to the presence of thermotolerant coliforms (60.6%). Twenty-six Staphylococcus species were identified from the 121 Staphylococcus isolates tested. Staphylococci coagulase-negative species were predominant in the foods, equipment and surfaces. In food handlers and foods, the predominant species was Staphylococcus epidermidis. Twelve different genotypes were found after PCR for the classical enterotoxin genes. The seb gene (19.8%) was the most prevalent among all Staphylococcus sp. Both coagulase-positive and coagulase-negative Staphylococci showed some of the genes of the enterotoxins tested. We conclude that there are hygienic and sanitary deficiencies in the food services analyzed. Although coagulase-positive Staphylococci have not been present in foods there is a wide dispersion of enterotoxigenic coagulase-negative Staphylococci in the environment and in the foods analyzed, indicating a risk to consumer health. PMID:25477940

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

PubMed Central

Knobloch, Johannes K.-M.; Nedelmann, Max; Kiel, Kathrin; Bartscht, Katrin; Horstkotte, Matthias A.; Dobinsky, Sabine; Rohde, Holger; Mack, Dietrich

2003-01-01

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis. PMID:14532029

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

PubMed

Pereira, Eliezer M; Schuenck, Ricardo P; Malvar, Karoline L; Iorio, Natalia L P; Matos, Pricilla D M; Olendzki, André N; Oelemann, Walter M R; dos Santos, Kátia R N

2010-03-31

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

PubMed

Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

2016-10-01

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

Methicillin-resistant Staphylococcus argenteus misidentified as methicillin-resistant Staphylococcus aureus emerging in western Sweden.

PubMed

Tång Hallbäck, Erika; Karami, Nahid; Adlerberth, Ingegerd; Cardew, Sofia; Ohlén, Maria; Engström Jakobsson, Hedvig; Svensson Stadler, Liselott

2018-05-17

Two strains included in a whole-genome sequencing project for methicillin-resistant Staphylococcus aureus (MRSA) were identified as non-Staphylococcus aureus when the sequences were analysed using the bioinformatics software ALEX (www.1928diagnostics.com, Gothenburg, Sweden). Sequencing of the sodA gene of these strains identified them as Staphylococcus argenteus. The collection of MRSA in western Sweden was checked for additional strains of this species. A total of 18 strains of S. argenteus isolated between 2011 and December 2017 were identified.

Shifted T Helper Cell Polarization in a Murine Staphylococcus aureus Mastitis Model.

PubMed

Zhao, Yanqing; Zhou, Ming; Gao, Yang; Liu, Heyuan; Yang, Wenyu; Yue, Jinhua; Chen, Dekun

2015-01-01

Mastitis, one of the most costly diseases in dairy ruminants, is an inflammation of the mammary gland caused by pathogenic infection. The mechanisms of adaptive immunity against pathogens in mastitis have not been fully elucidated. To investigate T helper cell-mediated adaptive immune responses, we established a mastitis model by challenge with an inoculum of 4 × 106 colony-forming units of Staphylococcus aureus in the mammary gland of lactating mice, followed by quantification of bacterial burden and histological analysis. The development of mastitis was accompanied by a significant increase in both Th17 and Th1 cells in the mammary gland. Moreover, the relative expression of genes encoding cytokines and transcription factors involved in the differentiation and function of these T helper cells, including Il17, Rorc, Tgfb, Il1b, Il23, Ifng, Tbx21, and Il12, was greatly elevated in the infected mammary gland. IL-17 is essential for neutrophil recruitment to infected mammary gland via CXC chemokines, whereas the excessive IL-17 production contributes to tissue damage in mastitis. In addition, a shift in T helper cell polarization toward Th2 and Treg cells was observed 5 days post-infection, and the mRNA expression of the anti-inflammatory cytokine Il10 was markedly increased at day 7 post-infection. These results indicate that immune clearance of Staphylococcus aureus in mastitis is facilitated by the enrichment of Th17, Th1 and Th2 cells in the mammary gland mediated by pro-inflammatory cytokine production, which is tightly regulated by Treg cells and the anti-inflammatory cytokine IL-10.

Shifted T Helper Cell Polarization in a Murine Staphylococcus aureus Mastitis Model

PubMed Central

Zhao, Yanqing; Zhou, Ming; Gao, Yang; Liu, Heyuan; Yang, Wenyu; Yue, Jinhua; Chen, Dekun

2015-01-01

Mastitis, one of the most costly diseases in dairy ruminants, is an inflammation of the mammary gland caused by pathogenic infection. The mechanisms of adaptive immunity against pathogens in mastitis have not been fully elucidated. To investigate T helper cell-mediated adaptive immune responses, we established a mastitis model by challenge with an inoculum of 4 × 106 colony-forming units of Staphylococcus aureus in the mammary gland of lactating mice, followed by quantification of bacterial burden and histological analysis. The development of mastitis was accompanied by a significant increase in both Th17 and Th1 cells in the mammary gland. Moreover, the relative expression of genes encoding cytokines and transcription factors involved in the differentiation and function of these T helper cells, including Il17, Rorc, Tgfb, Il1b, Il23, Ifng, Tbx21, and Il12, was greatly elevated in the infected mammary gland. IL-17 is essential for neutrophil recruitment to infected mammary gland via CXC chemokines, whereas the excessive IL-17 production contributes to tissue damage in mastitis. In addition, a shift in T helper cell polarization toward Th2 and Treg cells was observed 5 days post-infection, and the mRNA expression of the anti-inflammatory cytokine Il10 was markedly increased at day 7 post-infection. These results indicate that immune clearance of Staphylococcus aureus in mastitis is facilitated by the enrichment of Th17, Th1 and Th2 cells in the mammary gland mediated by pro-inflammatory cytokine production, which is tightly regulated by Treg cells and the anti-inflammatory cytokine IL-10. PMID:26230498

Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis

DTIC Science & Technology

2002-07-01

DAAT and 45% identical to the Staphylococcus haemolyticus DAAT. The ybgE and ywaA sequences were found in the Illa subfamily, and were 59% identical to...halodurans BH1060 gene product. The two sequences also had a respective 40% and 37% identity to the Staphylococcus aureuts SAV2560 gene product. The 6

Draft genome sequence of multidrug-resistant Staphylococcus haemolyticus IPK_TSA25 harbouring a Staphylococcus aureus plasmid, pS0385-1.

PubMed

Kim, Hyung Jun; Jang, Soojin

2017-12-01

Staphylococcus haemolyticus is the second most frequently isolated coagulase-negative staphylococci from blood cultures. Moreover, multidrug resistance associated with the genome flexibility of S. haemolyticus has been increasingly reported worldwide. Here we report the draft genome sequence of multidrug-resistant S. haemolyticus IPK_TSA25 isolated from a building surface in South Korea. Genomic DNA of S. haemolyticus IPK_TSA25 was sequenced using the PacBio RS II sequencing platform. Generated reads were assembled using PacBio SMRT Analysis 2.3.0. The draft genome was annotated and antibiotic resistance genes were identified. The genome of 2517398bp contains various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides and macrolides. Genome analysis also revealed chromosomal integration of the full-length Staphylococcus aureus plasmid pS0385-1 containing a tetracycline resistance gene. The genome sequence reported in this study will provide valuable information to understand the flexibility of the S. haemolyticus genome, which facilitates acquisition of antibiotic resistance genes and contributes to the dissemination of antibiotic resistance by this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation

PubMed Central

Das, Sudip; Lindemann, Claudia; Young, Bernadette C.; Muller, Julius; Österreich, Babett; Ternette, Nicola; Winkler, Ann-Cathrin; Paprotka, Kerstin; Reinhardt, Richard; Allen, Elizabeth; Flaxman, Amy; Yamaguchi, Yuko; Rollier, Christine S.; van Diemen, Pauline; Blättner, Sebastian; Remmele, Christian W.; Selle, Martina; Dittrich, Marcus; Müller, Tobias; Vogel, Jörg; Ohlsen, Knut; Crook, Derrick W.; Massey, Ruth; Wilson, Daniel J.; Rudel, Thomas; Wyllie, David H.; Fraunholz, Martin J.

2016-01-01

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection. PMID:27185949

The Staphylococcus aureus ArlRS Two-Component System Is a Novel Regulator of Agglutination and Pathogenesis

PubMed Central

Walker, Jennifer N.; Crosby, Heidi A.; Spaulding, Adam R.; Salgado-Pabón, Wilmara; Malone, Cheryl L.; Rosenthal, Carolyn B.; Schlievert, Patrick M.; Boyd, Jeffrey M.; Horswill, Alexander R.

2013-01-01

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis. PMID:24367264

A Novel ESAT-6 Secretion System-Secreted Protein EsxX of Community-Associated Staphylococcus aureus Lineage ST398 Contributes to Immune Evasion and Virulence.

PubMed

Dai, Yingxin; Wang, Yanan; Liu, Qian; Gao, Qianqian; Lu, Huiying; Meng, Hongwei; Qin, Juanxiu; Hu, Mo; Li, Min

2017-01-01

The ESAT-6 secretion system (ESS) has been reported to contribute to the virulence and pathogenicity of several Staphylococcus aureus strains such as USA300 and Newman. However, the role of the ESS in community-associated S. aureus (CA-SA) lineage ST398 in China is not well understood. By comparing the ess locus of ST398 with the published S. aureus sequence in the NCBI database, we found one gene in the ess locus encoding a novel WXG superfamily protein that is highly conserved only in ST398. LC-MS/MS and Western blot analysis revealed that this protein is a novel secreted protein controlled by the ST398 ESS, and we named the protein EsxX. Although EsxX was not under the control of the accessory gene regulator like many other virulence factors and had no influence on several phenotypes of ST398, such as growth, hemolysis, and biofilm formation, it showed important impacts on immune evasion and virulence in ST398. An esxX deletion mutant led to significantly reduced resistance to neutrophil killing and decreased virulence in murine skin and blood infection models, indicating its essential contribution to the evasion of innate host defense and virulence to support the pathogenesis of ST398 infections. The function of this novel secreted protein EsxX might help us better understand the role of the ESS in the virulence and epidemic success of the CA-SA lineage ST398.

[Community-acquired methicillin-resistant Staphylococcus aureus infections in children].

PubMed

Frick, Marie Antoinette; Moraga-Llop, Fernando A; Bartolomé, Rosa; Larrosa, Nieves; Campins, Magda; Roman, Yuani; Vindel, Ana; Figueras, Concepció

2010-12-01

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections were first reported in the 1990s. Young, healthy individuals are frequently affected. The incidence of CA-MRSA in Spain is increasing. All children seen between August 2006 and January 2009 with CA-MRSA infections were included. The S. aureus isolates were studied by conventional techniques, their antibiotic susceptibility by agar disk diffusion, the presence of mecA gene was detected by multiplex polymerase chain reaction (PCR) and the gene encoding the Panton-Valentine leukocidin (PVL) by conventional PCR. CA-MRSA colonization was studied both in patients and their family members. CA-MRSA was isolated in 15 samples from 12 patients, aged between 6 days and 14 years. Half of them were not native. Eight patients required hospital admission. The most common clinical presentation was skin and soft tissue infection (92%). Secondary CA-MRSA bacteraemia was present in two patients. All strains were PVL producers and two were resistant to macrolides associated to methicillin resistance and one of them was also resistant to lincosamides. An intra-familial transmission was identified. The clinical outcome was favourable in all patients. CA-MRSA infections are emerging in Spain. Empirical treatment of skin and soft tissue infections should not be changed, since their incidence is still low. The drainage of CA-MRSA suppurative infections plays an important role in their treatment. Clindamycin or trimethoprim-sulfamethoxazole should be used for mild or moderate skin and soft tissue infections. Controlling the spread of these strains presents a challenge in the community today. Copyright © 2009 Elsevier España, S.L. All rights reserved.

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

PubMed Central

McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M.

2017-01-01

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. PMID:28381601

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

PubMed

McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

Molecular characterization of antimicrobial resistance genes against Staphylococcus aureus isolates from Trinidad and Tobago.

PubMed

Akpaka, Patrick E; Roberts, Rashida; Monecke, Stefan

Staphylococcus aureus continues to pose major public health challenges in many areas because of antibiotic resistance problems. In the Caribbean, especially Trinidad and Tobago, the challenge is not different. This study was performed to evaluate the antimicrobial resistance gene prevalence among S. aureus isolates in Trinidad and Tobago. Standard and molecular microbiological methods, including the Microscan automated system, DNA microarray and multi locus sequence typing (MLST) analysis, were performed on 309 clinical S. aureus isolates recovered from patients who were treated at three of the country's main health institutions. S. aureus exhibited susceptibilities ≥80% to eleven of the 19 antimicrobials tested against it, and these belong to the most commonly used and available antibiotics in the country. While the antibiotic to which it was most susceptible of the commonly used antibiotics was trimethoprim/sulfamethoxazole, the antibiotics to which it was least susceptible or most resistant to were ampicillin and penicillin. S. aureus isolates from the pediatric ward produced the greatest rate of susceptibility among the isolates recovered from patients admitted into hospitals, while isolates from Accident and Emergency rooms displayed the greatest susceptibilities among patients from the community. S. aureus isolates from the country did not harbor acquired resistant genes targeting clindamycin/macrolides (ermB), linezolid (cfr) or vancomycin (vanA). The blaZ gene, which is the most common beta lactam (Penicillinase) resistance mechanism for S. aureus, was observed in 88.7% of the methicillin susceptible S. aureus, while methicillin resistance mediated by the mec gene was present in 13.6%. Most of the resistance markers found in MRSA isolates were significantly associated with the ST239-MRSA-III strain in this study, and all isolates that belonged to the USA300 strain, which additionally encoded both the PVL gene and ACME cluster, belonged to CC8. Several resistant genes, such as vanA, cfr and ermB, mediating resistance in S. aureus, are currently non-existent in Trinidad and Tobago. However, the majority of SCCmec genes were observed, suggesting that there is ongoing nosocomial transmission with minimal community transmission. This calls for stringent antibiotic stewardship and policies in the country. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.

PubMed

Frey, Yvonne; Rodriguez, Joan Peña; Thomann, Andreas; Schwendener, Sybille; Perreten, Vincent

2013-04-01

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster.

PubMed

Mattheus, Wesley; Gao, Ling-Jie; Herdewijn, Piet; Landuyt, Bart; Verhaegen, Jan; Masschelein, Joleen; Volckaert, Guido; Lavigne, Rob

2010-02-26

Kal/bat, a polyketide, isolated to high purity (>95%) is characterized by strong and selective antibacterial activity against Staphylococcus species (minimum inhibitory concentration, 0.05 microg/mL), and no resistance was observed in strains already resistant to commonly used antibiotics. The kal/bat biosynthesis gene cluster was determined to a 62 kb genomic region of Pseudomonas fluorescens BCCM_ID9359. The kal/bat gene cluster consists of 16 open reading frames (ORF), encoding a hybrid PKS-NRPS system, extended with trans-acting tailoring functions. A full model for kal/bat biosynthesis is postulated and experimentally tested by gene inactivation, structural confirmation (using NMR spectroscopy), and complementation. The structural and microbiological study of biosynthetic kal/bat analogs revealed the importance of the carbamoyl group and 17-keto group for antibacterial activity. The mechanism of self-resistance lies within the production of an inactive intermediate, which is activated in a one-step enzymatic oxidation upon export. The genetic basis and biochemical elucidation of the biosynthesis pathway of this antibiotic will facilitate rational engineering for the design of novel structures with improved activities. This makes it a promising new therapeutic option to cope with multidrug-resistant clinical infections. Copyright 2010 Elsevier Ltd. All rights reserved.

Role of the Twin-Arginine Translocation Pathway in Staphylococcus▿ †

PubMed Central

Biswas, Lalitha; Biswas, Raja; Nerz, Christiane; Ohlsen, Knut; Schlag, Martin; Schäfer, Tina; Lamkemeyer, Tobias; Ziebandt, Anne-Kathrin; Hantke, Klaus; Rosenstein, Ralf; Götz, Friedrich

2009-01-01

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB. PMID:19633084

Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

PubMed

Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

2013-03-01

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

The Frequency of Staphylococcus aureus Isolated from Endocervix of Infertile Women in Northwest Iran

PubMed Central

Akhi, Mohammad Taghi; Esmailkhani, Aylin; Sadeghi, Javid; Niknafs, Behrooz; Farzadi, Laya; Akhi, Aydin; Nasab, Elmira Najafi

2017-01-01

Background Infertility is one of the major social issues. Due to the asymptomatic cervical infection associated with Staphylococcus aureus (S. aureus), the majority of patients remain undiagnosed. The present study intended to assess the frequency of S. aureus isolated from infertile women’s endocervix in northwest Iran. Materials and Methods In a descriptive cross sectional study, specimens were randomly collected during vagina examination using a sterile speculum and swabbing. After performance of antibiotic susceptibility testing, polymerase chain reaction (PCR) was used to identify methicillin-resistance S. aureus (MRSA) and toxic shock syndrome toxin-1 (TSST-1). Results About 26 (26%) and 9 (9%) women’s urogenital tracts were colonized by S. aureus and Candida spp., respectively, of which three (11.5%) patients were infected with fungi and S. aureus, simultaneously. Antibiotic susceptibility results showed high activity of vancomycin and co-trimoxazole on isolates. Regarding PCR results, mecA sequences were detected in 7 (26.9%) strains, whilst the tst gene encoding TSST-1 was not detected in any of clinical strains. Conclusion The prevalence of S. aureus was very high in infertile women. Therefore, it demands all patients undergoing infertility treatment to be investigated thoroughly for this type of infection. PMID:28367302

A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors.

PubMed

Wladyka, Benedykt; Piejko, Marcin; Bzowska, Monika; Pieta, Piotr; Krzysik, Monika; Mazurek, Łukasz; Guevara-Lora, Ibeth; Bukowski, Michał; Sabat, Artur J; Friedrich, Alexander W; Bonar, Emilia; Międzobrodzki, Jacek; Dubin, Adam; Mak, Paweł

2015-09-28

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.

Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614.

PubMed

Chaitanyakumar, Amballa; Anbalagan, M

2016-12-01

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn 2+ , Fe 2+ , Fe 3+ and Mn 2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes*

PubMed Central

Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

2014-01-01

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256

Structural insights of Staphylococcus aureus FtsZ inhibitors through molecular docking, 3D-QSAR and molecular dynamics simulations.

PubMed

Ballu, Srilata; Itteboina, Ramesh; Sivan, Sree Kanth; Manga, Vijjulatha

2018-02-01

Filamentous temperature-sensitive protein Z (FtsZ) is a protein encoded by the FtsZ gene that assembles into a Z-ring at the future site of the septum of bacterial cell division. Structurally, FtsZ is a homolog of eukaryotic tubulin but has low sequence similarity; this makes it possible to obtain FtsZ inhibitors without affecting the eukaryotic cell division. Computational studies were performed on a series of substituted 3-arylalkoxybenzamide derivatives reported as inhibitors of FtsZ activity in Staphylococcus aureus. Quantitative structure-activity relationship models (QSAR) models generated showed good statistical reliability, which is evident from r 2 ncv and r 2 loo values. The predictive ability of these models was determined and an acceptable predictive correlation (r 2 Pred ) values were obtained. Finally, we performed molecular dynamics simulations in order to examine the stability of protein-ligand interactions. This facilitated us to compare free binding energies of cocrystal ligand and newly designed molecule B1. The good concordance between the docking results and comparative molecular field analysis (CoMFA)/comparative molecular similarity indices analysis (CoMSIA) contour maps afforded obliging clues for the rational modification of molecules to design more potent FtsZ inhibitors.

A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors

PubMed Central

Wladyka, Benedykt; Piejko, Marcin; Bzowska, Monika; Pieta, Piotr; Krzysik, Monika; Mazurek, Łukasz; Guevara-Lora, Ibeth; Bukowski, Michał; Sabat, Artur J.; Friedrich, Alexander W.; Bonar, Emilia; Międzobrodzki, Jacek; Dubin, Adam; Mak, Paweł

2015-01-01

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups. PMID:26411997

Quantitative Proteomic Analysis of Staphylococcus aureus Treated With Punicalagin, a Natural Antibiotic From Pomegranate That Disrupts Iron Homeostasis and Induces SOS.

PubMed

Cooper, Bret; Islam, Nazrul; Xu, Yunfeng; Beard, Hunter S; Garrett, Wesley M; Gu, Ganyu; Nou, Xiangwu

2018-05-01

Staphylococcus aureus, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. While physical and chemical methods are available to control S. aureus, scientists are searching for inhibitory phytochemicals from plants. One promising compound from pomegranate is punicalagin, a natural antibiotic. To get a broader understanding of the inhibitory effect of punicalagin on S. aureus growth, high-throughput mass spectrometry and quantitative isobaric labeling was used to investigate the proteome of S. aureus after exposure to a sublethal dose of punicalagin. Nearly half of the proteins encoded by the small genome were interrogated, and nearly half of those exhibited significant changes in accumulation. Punicalagin treatment altered the accumulation of proteins and enzymes needed for iron acquisition, and it altered amounts of enzymes for glycolysis, citric acid cycling, protein biosynthesis, and purine and pyrimidine biosynthesis. Punicalagin treatment also induced an SOS cellular response to damaged DNA. Transcriptional comparison of marker genes shows that the punicalagin-induced iron starvation and SOS responses resembles those produced by EDTA and ciprofloxacin. These results show that punicalagin adversely alters bacterial growth by disrupting iron homeostasis and that it induces SOS, possibly through DNA biosynthesis inhibition. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and characterization of human pancreatic polypeptide expressed in E. coli.

PubMed

Griko, Y V; Kapanadze, M D

1995-08-04

The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.

Molecular Analysis of the Locus Responsible for Production of Plantaricin S, a Two-Peptide Bacteriocin Produced by Lactobacillus plantarum LPCO10

PubMed Central

Stephens, Sarah K.; Floriano, Belén; Cathcart, Declan P.; Bayley, Susan A.; Witt, Valerie F.; Jiménez-Díaz, Rufino; Warner, Philip J.; Ruiz-Barba, José Luis

1998-01-01

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Díaz, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459–4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides α and β, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated. PMID:9572965

Biochemical and Molecular Analysis of Staphylococcus aureus Clinical Isolates from Hospitalized Patients.

PubMed

Karmakar, Amit; Dua, Parimal; Ghosh, Chandradipa

2016-01-01

Staphylococcus aureus is opportunistic human as well as animal pathogen that causes a variety of diseases. A total of 100 Staphylococcus aureus isolates were obtained from clinical samples derived from hospitalized patients. The presumptive Staphylococcus aureus clinical isolates were identified phenotypically by different biochemical tests. Molecular identification was done by PCR using species specific 16S rRNA primer pairs and finally 100 isolates were found to be positive as Staphylococcus aureus. Screened isolates were further analyzed by several microbiological diagnostics tests including gelatin hydrolysis, protease, and lipase tests. It was found that 78%, 81%, and 51% isolates were positive for gelatin hydrolysis, protease, and lipase activities, respectively. Antibiogram analysis of isolated Staphylococcus aureus strains with respect to different antimicrobial agents revealed resistance pattern ranging from 57 to 96%. Our study also shows 70% strains to be MRSA, 54.3% as VRSA, and 54.3% as both MRSA and VRSA. All the identified isolates were subjected to detection of mecA, nuc, and hlb genes and 70%, 84%, and 40% were found to harbour mecA, nuc, and hlb genes, respectively. The current investigation is highly important and informative for the high level multidrug resistant Staphylococcus aureus infections inclusive also of methicillin and vancomycin.

The Evaluation of Methicillin Resistance in Staphylococcus aboard the International Space Station

NASA Technical Reports Server (NTRS)

Ott, C. M.; Bassinger, V. J.; Fontenot, S. L.; Castro, V. A.; Pierson, D. L.

2005-01-01

The International Space Station (ISS) represents a semi-closed environment with a high level of crewmember interaction. As community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a health concern in environments with susceptible hosts in close proximity, an evaluation of isolates of clinical and environmental Staphylococcus aureus and coagulase negative Staphylococcus was performed to determine if this trend was also present in astronauts aboard ISS or the space station itself. Rep-PCR fingerprinting analysis of archived ISS isolates confirmed our earlier studies indicating a transfer of S. aureus between crewmembers. In addition, this fingerprinting also indicated a transfer between crewmembers and their environment. While a variety of S. aureus were identified from both the crewmembers and the environment, phenotypic evaluations indicated minimal methicillin resistance. However, positive results for the Penicillin Binding Protein, indicative of the presence of the mecA gene, were detected in multiple isolates of archived Staphylococcus epidermidis and Staphylococcus haemolyticus. Phenotypic analysis of these isolates confirmed their resistance to methicillin. While MRSA has not been isolated aboard ISS, the potential exists for the transfer of the gene, mecA, from coagulase negative environmental Staphylococcus to S. aureus creating MRSA strains. This study suggests the need to expand environmental monitoring aboard long duration exploration spacecraft to include antibiotic resistance profiling.

Staphylococcus haemolyticus as an important hospital pathogen and carrier of methicillin resistance genes.

PubMed

Barros, E M; Ceotto, H; Bastos, M C F; Dos Santos, K R N; Giambiagi-Demarval, M

2012-01-01

Phenotypic and molecular methods were used to characterize the antibiotic resistance of 64 clinical isolates of Staphylococcus haemolyticus. By PCR of the mecA gene, 87% were found to be methicillin resistant. Approximately 55% harbored staphylococcal cassette chromosome mec element (SCCmec) type V, and only one SCCmec type IV. Many isolates (75%) displayed multiresistance, and pulsotype analysis showed a high diversity.

Species determination within Staphylococcus genus by extended PCR-restriction fragment length polymorphism of saoC gene.

PubMed

Bukowski, Michal; Polakowska, Klaudia; Ilczyszyn, Weronika M; Sitarska, Agnieszka; Nytko, Kinga; Kosecka, Maja; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

2015-01-01

Genetic methods based on PCR-restriction fragment length polymorphism (RFLP) are widely used for microbial species determination. In this study, we present the application of saoC gene as an effective tool for species determination and within-species diversity analysis for Staphylococcus genus. The unique sequence diversity of saoC allows us to apply four restriction enzymes to obtain RFLP patterns, which appear highly distinctive even among closely related species as well as atypical isolates of environmental origin. Such patterns were successfully obtained for 26 species belonging to Staphylococcus genus. What is more, tracing polymorphisms detected by different restriction enzymes allowed for basic phylogeny analysis for Staphylococcus aureus, which is potentially applicable for other staphylococcal species. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Staphylococcus xylosus Isolate with a New mecC Allotype

PubMed Central

Harrison, Ewan M.; Paterson, Gavin K.; Holden, Matthew T. G.; Morgan, Fiona J. E.; Larsen, Anders Rhod; Petersen, Andreas; Leroy, Sabine; De Vliegher, Sarne; Perreten, Vincent; Fox, Lawrence K.; Lam, Theo J. G. M.; Sampimon, Otlis C.; Zadoks, Ruth N.; Peacock, Sharon J.; Parkhill, Julian

2013-01-01

Recently, a novel variant of mecA known as mecC (mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec. PMID:23274660

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus.

PubMed

Laczkovich, Irina; Teoh, Wei Ping; Flury, Sarah; Grayczyk, James P; Zorzoli, Azul; Alonzo, Francis

2018-04-16

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH-L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α-ketoacid dehydrogenase E2 subunits, while the function of GcvH-L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain-containing proteins; LplA1 targets H proteins and LplA2 targets α-ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH-L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation. © 2018 John Wiley & Sons Ltd.

Enterotoxigenic coagulase positive Staphylococcus in milk and milk products, lben and jben, in northern Morocco.

PubMed

Bendahou, Abdrezzak; Abid, Mohammed; Bouteldoun, Nadine; Catelejine, Dierick; Lebbadi, Mariam

2009-04-30

The aim of this research was to determine the prevalence of enterotoxin genes (sea-seo) in Coagulase Positive Staphylococcus (CPS) isolated from unpasteurized milk and milk products. These results were compared with the results obtained by using the detection kit SET-RPLA for the specific detection of staphylococcal enterotoxins (SEA-SED). Eighty-one samples of milk and milk products were analyzed for the presence of Staphylococcus strains. Forty-six coagulase positive Staphylococcus isolates were tested for the production of staphylococcal enterotoxins (SEA-SED) by using the reversed passive latex agglutination method. The strains were also tested for the presence of se genes (sea-seo) by polymerase chain reaction. One or more classical enterotoxin products (SEA-SED) were observed in 39% of the strains tested, while se genes were detected in 56.5%. SEA and sea were most commonly detected. For newly discovered se genes among CPS isolates tested in this study, except the seh gene which was revealed in four isolates (8.7 %), none of the strains harbored any of the other se genes (see, seg, sei, sej, sek, sel, sem, seo and sen). The finding of a pathogen such as staphylococci-producing SEs and containing se genes in milk and milk products in northern Morocco may indicate a problem for public health in this region. The presence of enterotoxigenic strains in food does not always necessarily mean that the toxin will be produced. For that reason, the combination of both methods (RPLA and PCR) is a guarantee for success in diagnostic analysis tests.

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage

PubMed Central

Hau, Samantha J.; Frana, Timothy; Sun, Jisun; Davies, Peter R.

2017-01-01

ABSTRACT Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCCmec) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants. IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is more associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance markers. This information is important for public health professionals, veterinarians, producers, and consumers. PMID:28526788

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage.

PubMed

Hau, Samantha J; Frana, Timothy; Sun, Jisun; Davies, Peter R; Nicholson, Tracy L

2017-08-01

Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCC mec ) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCC mec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants. IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is more associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance markers. This information is important for public health professionals, veterinarians, producers, and consumers. Copyright © 2017 American Society for Microbiology.

Occurrence of cfr-mediated multiresistance in staphylococci from veal calves and pigs, from humans at the corresponding farms, and from veterinarians and their family members.

PubMed

Cuny, Christiane; Arnold, Phillippe; Hermes, Julia; Eckmanns, Tim; Mehraj, Jaishri; Schoenfelder, Sonja; Ziebuhr, Wilma; Zhao, Qin; Wang, Yang; Feßler, Andrea T; Krause, Gérard; Schwarz, Stefan; Witte, Wolfgang

2017-02-01

This study reports on the emergence of linezolid-resistant coagulase-negative staphylococci (CoNS) containing the multiresistance gene cfr in veal calves and pigs, as well as in humans exposed to these animals. CoNS (Staphylococcus auricularis, Staphylococcus cohnii, Staphylococcus lentus, Staphylococcus kloosii, Staphylococcus sciuri, Staphylococcus simulans), but not Staphylococcus aureus, carrying the gene cfr were detected in samples of 12 out of 52 calves at three farms which had a history of florfenicol use. Nasal swabs from 10 humans living on these farms were negative for cfr-carrying staphylococci. Nasal swabs taken from 142 calves at 16 farms in the same area that did not use florfenicol were also negative for cfr-carrying staphylococci. 14 cfr-carrying CoNS (S. kloosii, S. saprophyticus, S. simulans) were detected in three of eight conventional pig farms investigated. One of 12 humans living on these farms harboured a cfr-carrying S. cohnii. Among the nasal swabs taken from 169 veterinarians from all over Germany, four (2.3%) were positive for cfr-carrying CoNS (three S. epidermidis, one S. saprophyticus), and three (1.1%) of 263 contact persons of this group also harboured cfr-carrying CoNS (one S. epidermidis, two S. saprophyticus). In vitro conjugation of cfr by filter mating to S. aureus 8325-4 was possible for 10 of 34CoNS and the cfr gene was associated with plasmids of 38-40kb. Moreover, a total of 363 humans of a German municipal community were investigated for nasal carriage of cfr-carrying staphylococci to get an idea whether such isolates are disseminated as nasal colonizers in non-hospitalized humans in the community, were all negative. Copyright © 2016 Elsevier B.V. All rights reserved.

Phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus isolates from clinical specimens, an Atomic Force Microscopic (AFM) study.

PubMed

Bazari, Pelin Aslani Menareh; Honarmand Jahromy, Sahar; Zare Karizi, Shohreh

2017-09-01

Staphylococcus aureus is a major cause of nosocomial infections. Biofilm formation is an important factor for bacterial pathogenesis. Its mechanisms are complex and include of many genes depends on expression of icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. The aim of study was to investigate biofilm forming ability of Staphylococcus aureus strains by phenotypic and genotypic methods. Also Atomic Force microscope (AFM) was used to visualize biofilm formation. 140 Isolates were collected from clinical specimens of patients in Milad Hospital, Tehran and diagnosed by biochemical tests. The ability of strains to produce slime was evaluated by CRA method. For diagnosing of bacterial EPS, Indian ink staining were used and finally biofilm surface of 3 isolates observed by AFM. The prevalence of icaA and icaD genes was determined by PCR. By CRA method 15% of samples considered as positive slime producers, 44.28% as intermediate and 40.71% indicative as negative slime producers. 118 staphylococcus aureus strains showed a distinct halo transparent zone but 22 strains showed no halo zone. AFM analysis of Slime positive isolates showed a distinct and complete biofilm formation. In slime negative strain, there was not observed biofilm. The prevalence of icaA, icaD genes was 44.2% and 10% of the isolates had both genes simultaneously. There is a relationship between exopolysaccharide layer and biofilm formation of Staphylococcus aureus isolates. The presence of icaAD genes among isolates is not associated with in vitro formation of biofilm. AFM is a useful tool for observation of bacterial biofilm formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

PubMed Central

Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

2013-01-01

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

PubMed

Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

2014-01-03

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Complete Genome Sequence of the MRSA Isolate HC1335 from ST239 Lineage Displaying a Truncated AgrC Histidine Kinase Receptor

PubMed Central

Botelho, Ana M. N.; Costa, Maiana O. C.; Beltrame, Cristiana O.; Ferreira, Fabienne A.; Lima, Nicholas C. B.; Costa, Bruno S. S.; de Morais, Guilherme L.; Souza, Rangel C.; Almeida, Luiz G. P.; Vasconcelos, Ana T. R.; Nicolás, Marisa F.; Figueiredo, Agnes M. S.

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is still one of the most important hospital pathogen globally. The multiresistant isolates of the ST239-SCCmecIII lineage are spread over large geographic regions, colonizing and infecting hospital patients in virtually all continents. The balance between fitness (adaptability) and virulence potential is likely to represent an important issue in the clonal shift dynamics leading the success of some specific MRSA clones over another. The accessory gene regulator (agr) is the master quorum sensing system of staphylococci playing a role in the global regulation of key virulence factors. Consequently, agr inactivation in S. aureus may represent a significant mechanism of genetic variability in the adaptation of this healthcare-associated pathogen. We report here the complete genome sequence of the methicillin-resistant S. aureus, isolate HC1335, a variant of the ST239 lineage, which presents a natural insertion of an IS256 transposase element in the agrC gene encoding AgrC histidine kinase receptor. PMID:27635055

MRSA virulence and spread

PubMed Central

Otto, Michael

2012-01-01

Summary Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital- and community-associated infections. Resistance to the entire class of β-lactam antibiotics, such as methicillin and penicillin, makes MRSA infections difficult to treat. Hospital-associated MRSA strains are often multi-drug resistant, leaving only lower efficiency drugs such as vancomycin as treatments options. Like many other S. aureus strains, MRSA strains produce a series of virulence factors, such as toxins and adhesion proteins. Recent findings have shed some new light on the molecular events that underlie MRSA epidemic waves. Newly emerging MRSA clones appear to have acquired phenotypic traits that render them more virulent or able to colonize better, either via mobile genetic elements or adaptation of gene expression. Acquisition of Panton-Valentine leukocidin genes and increased expression of core genome-encoded toxins are being discussed as potentially contributing to the success of the recently emerged community-associated MRSA strains. However, the molecular factors underlying the spread of hospital- and community-associated MRSA strains are still far from being completely understood, a situation calling for enhanced research efforts in that area. PMID:22747834

Staphylococcus haemolyticus as an Important Hospital Pathogen and Carrier of Methicillin Resistance Genes

PubMed Central

Barros, E. M.; Ceotto, H.; Bastos, M. C. F.; dos Santos, K. R. N.

2012-01-01

Phenotypic and molecular methods were used to characterize the antibiotic resistance of 64 clinical isolates of Staphylococcus haemolyticus. By PCR of the mecA gene, 87% were found to be methicillin resistant. Approximately 55% harbored staphylococcal cassette chromosome mec element (SCCmec) type V, and only one SCCmec type IV. Many isolates (75%) displayed multiresistance, and pulsotype analysis showed a high diversity. PMID:21976766

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

PubMed Central

2005-01-01

Abstract A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate. PMID:16479723

Phenotypic and Molecular Aspects of Staphylococcus spp. Isolated from Hospitalized Patients and Beef in the Brazilian Amazon.

PubMed

Pieri, Fabio A; Vargas, Taise F; Galvão, Newton N; Nogueira, Paulo A; Orlandi, Patrícia P

2016-03-01

The aim of this study was to characterize and compare Staphylococcus spp. isolated from hospitalized patients and beef marketed in the city of Porto Velho-RO, Brazil. The isolates were subjected to antibiogram tests, adherence capacity tests, detection of the mecA gene, and epidemiological investigation by the random amplified polymorphic DNA (RAPD) technique, using the primers M13 and H12. Among the 123 Staphylococcus spp. isolates, 50 were identified as S. aureus and 73 as coagulase-negative Staphylococcus; among the latter, 7 species were identified. It was observed that the coagulase-negative Staphylococcus isolates showed greater adhesion ability than S. aureus. The profile of antimicrobial susceptibility was different among isolates, all of which were susceptible to vancomycin and linezolid, and had high penicillin resistance rates, varying according to the bacterial class and the source. In this study, all strains were negative for mecA gene detection; however, 36% of S. aureus and 17% of coagulase-negative Staphylococcus were resistant to oxacillin. The genetic relationship of these bacteria, analyzed by RAPD, was able to discriminate the species of coagulase-negative Staphylococcus strains of S. aureus along its origin. It was concluded that the isolates of Staphylococcus spp. derived from beef and human infections differ genetically. Thus, it is suggested that isolates from beef, which were grouped within hospital isolates, were probably carried via contact with beef in hospital professionals or patients.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Short communication: β-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis.

PubMed

Mello, Priscila Luiza; Pinheiro, Luiza; Martins, Lisiane de Almeida; Brito, Maria Aparecida Vasconcelos Paiva; Ribeiro de Souza da Cunha, Maria de Lourdes

2017-08-01

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce β-lactamase, which explains the survival of these strains in a focus of infection despite the use of a β-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated β-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of β-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants.

PubMed

Rodrigues, Marjory Xavier; Silva, Nathália Cristina Cirone; Trevilin, Júlia Hellmeister; Cruzado, Melina Mary Bravo; Mui, Tsai Siu; Duarte, Fábio Rodrigo Sanches; Castillo, Carmen J Contreras; Canniatti-Brazaca, Solange Guidolin; Porto, Ernani

2017-07-01

The aim of this research paper was to characterize coagulase-positive and coagulase-negative staphylococci from raw milk, Minas cheese, and production lines of Minas cheese processing. One hundred isolates from 3 different cheese producers were characterized using molecular approaches, such as PCR, molecular typing, and DNA sequencing. Staphylococcus aureus (88% of the isolates) was the most abundant followed by Staphylococcus epidermidis, Staphylococcus hyicus, and Staphylococcus warneri. Among the 22 enterotoxin genes tested, the most frequent was seh (62% of the isolates), followed by selx and ser. Hemolysin genes were widely distributed across isolates, and Panton-Valentine leukocidin and toxic shock syndrome toxin genes were also identified. Methicillin-resistant S. aureus were staphylococcal cassette chromosome mec III, IVa, IVd, and others nontypeable. In the phenotypic antibiotic resistance, multiresistant isolates were detected and resistance to penicillin was the most observed. Using spa typing, we identified several types and described a new one, t14969, isolated from cheese. These findings suggest that antibiotic resistance and potentially virulent strains from different sources can be found in the Brazilian dairy processing environment. Further research should be conducted with collaboration from regulatory agencies to develop programs of prevention of virulent and resistant strain dissemination in dairy products and the processing environment. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cloning and occurrence of czrC, a gene conferring cadmium and zinc resistance in methicillin-resistant Staphylococcus aureus CC398 isolates.

PubMed

Cavaco, L M; Hasman, H; Stegger, M; Andersen, P S; Skov, R; Fluit, A C; Ito, T; Aarestrup, F M

2010-09-01

We recently reported a phenotypic association between reduced susceptibility to zinc and methicillin resistance in Staphylococcus aureus CC398 isolates from Danish swine (F. M. Aarestrup, L. M. Cavaco, and H. Hasman, Vet. Microbiol. 142:455-457, 2009). The aim of this study was to identify the genetic determinant causing zinc resistance in CC398 and examine its prevalence in isolates of animal and human origin. Based on the sequence of the staphylococcal cassette chromosome mec (SCCmec) element from methicillin-resistant S. aureus (MRSA) CC398 strain SO385, a putative metal resistance gene was identified in strain 171 and cloned in S. aureus RN4220. Furthermore, 81 MRSA and 48 methicillin-susceptible S. aureus (MSSA) strains, isolated from pigs (31 and 28) and from humans (50 and 20) in Denmark, were tested for susceptibility to zinc chloride and for the presence of a putative resistance determinant, czrC, by PCR. The cloning of czrC confirmed that the zinc chloride and cadmium acetate MICs for isogenic constructs carrying this gene were increased compared to those for S. aureus RN4220. No difference in susceptibility to sodium arsenate, copper sulfate, or silver nitrate was observed. Seventy-four percent (n = 23) of the animal isolates and 48% (n = 24) of the human MRSA isolates of CC398 were resistant to zinc chloride and positive for czrC. All 48 MSSA strains from both human and pig origins were found to be susceptible to zinc chloride and negative for czrC. Our findings showed that czrC is encoding zinc and cadmium resistance in CC398 MRSA isolates, and that it is widespread both in humans and animals. Thus, resistance to heavy metals such as zinc and cadmium may play a role in the coselection of methicillin resistance in S. aureus.

Emergence of Hospital- and Community-Associated Panton-Valentine Leukocidin-Positive Methicillin-Resistant Staphylococcus aureus Genotype ST772-MRSA-V in Ireland and Detailed Investigation of an ST772-MRSA-V Cluster in a Neonatal Intensive Care Unit

PubMed Central

Shore, Anna C.; Corcoran, Suzanne; Tecklenborg, Sarah; Coleman, David C.; O'Connell, Brian

2012-01-01

Sequence type 22 (ST22) methicillin-resistant Staphylococcus aureus (MRSA) harboring staphylococcal cassette chromosome mec (SCCmec) IV (ST22-MRSA-IV) has predominated in Irish hospitals since the late 1990s. Six distinct clones of community-associated MRSA (CA-MRSA) have also been identified in Ireland. A new strain of CA-MRSA, ST772-MRSA-V, has recently emerged and become widespread in India and has spread into hospitals. In the present study, highly similar MRSA isolates were recovered from seven colonized neonates in a neonatal intensive care unit (NICU) in a maternity hospital in Ireland during 2010 and 2011, two colonized NICU staff, one of their colonized children, and a NICU environmental site. The isolates exhibited multiantibiotic resistance, spa type t657, and were assigned to ST772-MRSA-V by DNA microarray profiling. All isolates encoded resistance to macrolides [msr(A) and mpb(BM)] and aminoglycosides (aacA-aphD and aphA3) and harbored the Panton-Valentine leukocidin toxin genes (lukF-PV and lukS-PV), enterotoxin genes (sea, sec, sel, and egc), and one of the immune evasion complex genes (scn). One of the NICU staff colonized by ST772-MRSA-V was identified as the probable index case, based on recent travel to India. Seven additional hospital and CA-ST772-MRSA-V isolates recovered from skin and soft tissue infections in Ireland between 2009 and 2011 exhibiting highly similar phenotypic and genotypic characteristics to the NICU isolates were also identified. The clinical details of four of these patients revealed connections with India through ethnic background or travel. Our study indicates that hospital-acquired and CA-ST772-MRSA-V is currently emerging in Ireland and may have been imported from India on several occasions. PMID:22189119

The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential.

PubMed

Rossi, Ciro César; da Silva Dias, Ingrid; Muniz, Igor Mansur; Lilenbaum, Walter; Giambiagi-deMarval, Marcia

2017-03-01

This study aimed to characterize the species, antimicrobial resistance and dispersion of CRISPR systems in staphylococci isolated from the oropharynx of domestic cats in Brazil. Staphylococcus strains (n=75) were identified by MALDI-TOF and sequencing of rpoB and tuf genes. Antimicrobial susceptibility was assessed by disk diffusion method and PCR to investigate the presence of antimicrobial-resistance genes usually present in mobile genetic elements (plasmids), in addition to plasmid extraction. CRISPR - genetic arrangements that give the bacteria the ability to resist the entry of exogenous DNA - were investigated by the presence of the essential protein Cas1 gene. A great diversity of Staphylococcus species (n=13) was identified. The presence of understudied species, like S. nepalensis and S. pettenkoferi reveals that more than one identification method may be necessary to achieve conclusive results. At least 56% of the strains contain plamids, being 99% resistant to at least one of the eight tested antimicrobials and 12% multidrug resistant. CRISPR were rare among the studied strains, consistent with their putative role as gene reservoirs. Moreover, herein we describe for the first time their existence in Staphylococcus lentus, to which the system must confer additional adaptive advantage. Prevalence of resistance among staphylococci against antimicrobials used in veterinary and human clinical practice and the zoonotic risk highlight the need of better antimicrobial management practices, as staphylococci may transfer resistance genes among themselves, including to virulent species, like S. aureus. Copyright © 2017 Elsevier B.V. All rights reserved.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

PubMed

Sansevere, Emily A; Luo, Xiao; Park, Joo Youn; Yoon, Sunghyun; Seo, Keun Seok; Robinson, D Ashley

2017-04-15

ICE 6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE 6013 and further characterized the diversity of this element. ICE 6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS 30 -like DDE transposase (Tpase; encoded by orf1 and orf2 ) of ICE 6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE 6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE 6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE 6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE 6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS 30 -like Tpase functions as the ICE 6013 recombinase and that ICE 6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE 6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus , but its core functions of excision and conjugation are not well studied. Here, we show that ICE 6013 depends on its IS 30 -like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE 6013 A sequence analysis revealed that ICE 6013 has diverged into seven subfamilies that are dispersed among staphylococci. Copyright © 2017 American Society for Microbiology.

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

PubMed

de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

2016-01-01

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A 3-year long study of Staphylococcus aureus isolates from subclinical mastitis in three Azawak zebu herds at the Sahelian experimental farm of Toukounous, Niger.

PubMed

Issa, Abdoulkarim Ibrahim; Duprez, Jean-Noël; Bada-Alambedji, Rianatou; Djika, Mamane; Mainil, Jacques Georges; Bardiau, Marjorie

2016-02-01

Staphylococcus (S.) aureus is one of the most important pathogens causing bovine mastitis. The aim of the present work was to follow in three herds and during the 3 years the clonality of S. aureus isolated from California Mastitis Test (CMT)-positive cows at the experimental station of Toukounous (Niger) by (i) comparing their pulsed field gel electrophoresis (PFGE) fingerprints, (ii) identifying their virulotypes by PCR amplification and (iii) assessing the production of capsule and the formation of biofilm. The 88 S. aureus isolates belonged to 14 different pulsotypes, 3 of them being predominant: A (30 %), D (27 %), B (15 %). A and B pulsotypes had the highest profile similarity coefficient (94 %), while others had similarity coefficients under 60 %. Seventy-five S. aureus isolates were further studied for their virulotypes, capsular antigens and biofilm production. Most surface factor-, leukocidin- and haemolysin-, but not the enterotoxin-encoding genes were detected in the majority (>75 %) of the isolates and were evenly distributed between the A, B and D pulsotype isolates. The majority of the 72 S. aureus positive with the cap5H or cap8H PCR produced the CP5 (82 %) or the CP8 (88 %) capsular antigen, respectively. Biofilm production by the 57 icaA-positive isolates was strong for 8 isolates, moderate for 31 isolates but weak for 18 isolates, implying that the icaA gene may not be expressed in vitro by one third of the positive isolates. Similar to other studies, those results confirm that a restricted number of S. aureus clones circulate within the three herds at Toukounous and that their specific virulence-associated properties must still be further studied.

Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

PubMed

Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

2014-05-01

During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis.

Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

PubMed Central

White, Mark J.; Boyd, Jeffrey M.

2014-01-01

Staphylococcus aureus is an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains of S. aureus possess a plc gene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity of S. aureus to cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context of S. aureus biology. Among a collection of genetically diverse clinical isolates of S. aureus, community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants of S. aureus, we identified both the agr quorum-sensing system and the SrrAB TCS to be positive regulators of plc gene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner. A Δplc mutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections. PMID:24452683

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

PubMed Central

Yin, Shouhui; Jo, Dae Sun; Montgomery, Christopher P.; Daum, Robert S.

2013-01-01

Staphylococcus aureus infections caused by strains that are resistant to all forms of penicillin, so-called methicillin-resistant S. aureus (MRSA) strains, have become common. One strategy to counter MRSA infections is to use compounds that resensitize MRSA to methicillin. S. aureus responds to diverse classes of cell wall-inhibitory antibiotics, like methicillin, using the two-component regulatory system VraSR (vra) to up- or downregulate a set of genes (the cell wall stimulon) that presumably facilitates resistance to these antibiotics. Accordingly, VraS and VraR mutations decrease resistance to methicillin, vancomycin, and daptomycin cell wall antimicrobials. vraS and vraR are encoded together on a transcript downstream of two other genes, which we call vraU and vraT (previously called yvqF). By producing nonpolar deletions in vraU and vraT in a USA300 MRSA clinical isolate, we demonstrate that vraT is essential for optimal expression of methicillin resistance in vitro, whereas vraU is not required for this phenotype. The deletion of vraT also improved the outcomes of oxacillin therapy in mouse models of lung and skin infection. Since vraT expressed in trans did not complement a vra operon deletion, we conclude that VraT does not inactivate the antimicrobial. Genome-wide transcriptional microarray experiments reveal that VraT facilitates resistance by playing a necessary regulatory role in the VraSR-mediated cell wall stimulon. Our data prove that VraTSR comprise a novel three-component regulatory system required to facilitate resistance to cell wall agents in S. aureus. We also provide the first in vivo proof of principle for using VraT as a sole target to resensitize MRSA to β-lactams. PMID:23070169

Dissecting the regulation of bile-induced biofilm formation in Staphylococcus aureus.

PubMed

Ulluwishewa, Dulantha; Wang, Liang; Pereira, Callen; Flynn, Stephanie; Cain, Elizabeth; Stick, Stephen; Reen, F Jerry; Ramsay, Joshua P; O'Gara, Fergal

2016-08-01

Aspiration of bile into the cystic fibrosis (CF) lung has emerged as a prognostic factor for reduced microbial lung biodiversity and the establishment of often fatal, chronic pathogen infections. Staphylococcus aureus is one of the earliest pathogens detected in the lungs of children with CF, and once established as a chronic infection, strategies for its eradication become limited. Several lung pathogens are stimulated to produce biofilms in vitro in the presence of bile. In this study, we further investigated the effects of bile on S. aureus biofilm formation. Most clinical S. aureus strains and the laboratory strain RN4220 were stimulated to form biofilms with sub-inhibitory concentrations of bovine bile. Additionally, we observed bile-induced sensitivity to aminoglycosides, which we exploited in a bursa aurealis transposon screen to isolate mutants reduced in aminoglycoside sensitivity and augmented in bile-induced biofilm formation. We identified five mutants that exhibited hypersensitivity to bile with respect to bile-induced biofilm formation, three of which carried transposon insertions within gene clusters involved in wall teichoic acid (WTA) biosynthesis or transport. Strain TM4 carried an insertion between the divergently oriented tagH and tagG genes, which encode the putative WTA membrane translocation apparatus. Ectopic expression of tagG in TM4 restored a wild-type bile-induced biofilm response, suggesting that reduced translocation of WTA in TM4 induced sensitivity to bile and enhanced the bile-induced biofilm formation response. We propose that WTA may be important for protecting S. aureus against exposure to bile and that bile-induced biofilm formation may be an evolved response to protect cells from bile-induced cell lysis.

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains.

PubMed

Bückle Née Müller, Anne; Kranz, Markus; Schmidt, Herbert; Weiss, Agnes

2017-01-01

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures. The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure. Copyright © 2016 Elsevier GmbH. All rights reserved.

Methicillin resistant S. aureus in human and bovine mastitis.

PubMed

Holmes, Mark A; Zadoks, Ruth N

2011-12-01

Staphylococcus aureus is a ubiquitous organism that causes a variety of diseases including mastitis in cattle and humans. High-level resistance of S. aureus to β-lactams conferred by a mecA gene encoding a modified penicillin binding protein (PBP2a) was first observed in the early 1960's. These methicillin resistant S. aureus (MRSA) have been responsible for both hospital acquired infections (HA-MRSA) and, more recently, community acquired MRSA (CA-MRSA). A small number of human MRSA mastitis cases and outbreaks in maternity or neonatal units have been reported which are generally the result of CA-MRSA. The establishment of the sequence type 398 (ST398) in farm animals, primarily pigs, in the early 2000's has provided a reservoir of infection for humans and dairy cattle, particularly in continental Europe, described as livestock-associated MRSA (LA-MRSA). Prior to the emergence of ST398 there were sporadic reports of MRSA in bovine milk and cases of mastitis, often caused by strains from human associated lineages. Subsequently, there have been several reports describing bovine udder infections caused by ST-398 MRSA. Recently, another group of LA-MRSA strains was discovered in humans and dairy cattle in Europe. This group carries a divergent mecA gene and includes a number of S. aureus lineages (CC130, ST425, and CC1943) that were hitherto thought to be bovine-specific but are now also found as carriage or clinical isolates in humans. The emergence of MRSA in dairy cattle may be associated with contact with other host species, as in the case of ST398, or with the exchange of genetic material between S. aureus and coagulase negative Staphylococcus species, which are the most common species associated with bovine intramammary infections and commonly carry antimicrobial resistance determinants.

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

PubMed

Rosignoli, Carine; Thibaut de Ménonville, Séverine; Orfila, Danielle; Béal, Méline; Bertino, Béatrice; Aubert, Jérôme; Mercenier, Annick; Piwnica, David

2018-04-01

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of Reduced Susceptibility to Quaternary Ammonium Compounds and Bisbiguanides in Clinical Isolates and Laboratory-Generated Mutants of Staphylococcus aureus

PubMed Central

Furi, Leonardo; Ciusa, Maria Laura; Knight, Daniel; Di Lorenzo, Valeria; Tocci, Nadia; Cirasola, Daniela; Aragones, Lluis; Coelho, Joana Rosado; Freitas, Ana Teresa; Marchi, Emmanuela; Moce, Laura; Visa, Pilar; Northwood, John Blackman; Viti, Carlo; Borghi, Elisa; Orefici, Graziella

2013-01-01

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant. PMID:23669380

Identification of Staphylococcus spp. using (GTG)₅-PCR fingerprinting.

PubMed

Svec, Pavel; Pantůček, Roman; Petráš, Petr; Sedláček, Ivo; Nováková, Dana

2010-12-01

A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp. Copyright © 2010 Elsevier GmbH. All rights reserved.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date. Our study evaluates an often-underappreciated aspect of resistance-the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date. Our study evaluates an often-underappreciated aspect of resistance—the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections. PMID:29719870

DNA Microarray for Detection of Macrolide Resistance Genes

PubMed Central

Cassone, Marco; D'Andrea, Marco M.; Iannelli, Francesco; Oggioni, Marco R.; Rossolini, Gian Maria; Pozzi, Gianni

2006-01-01

A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria. PMID:16723563

Molecular Characterization of Staphylococcus aureus Isolated from Bovine Mastitis and Close Human Contacts in South African Dairy Herds: Genetic Diversity and Inter-Species Host Transmission

PubMed Central

Schmidt, Tracy; Kock, Marleen M.; Ehlers, Marthie M.

2017-01-01

Staphylococcus aureus is one of the most common etiological agents of contagious bovine mastitis worldwide. The purpose of this study was to genetically characterize a collection of S. aureus isolates (bovine = 146, human = 12) recovered from cases of bovine mastitis and nasal swabs of close human contacts in the dairy environment. Isolates were screened for a combination of clinically significant antimicrobial and virulence gene markers whilst the molecular epidemiology of these isolates and possible inter-species host transmission was investigated using a combination of genotyping techniques. None of the isolates under evaluation tested positive for methicillin or vancomycin resistance encoding genes. Twenty seven percent of the bovine S. aureus isolates tested positive for one or more of the pyrogenic toxin superantigen (PTSAg) genes with the sec and sell genes predominating. Comparatively, 83% of the human S. aureus isolates tested positive for one or more PTSAg genes with a greater variety of genes being detected. Genomic DNA macrorestriction followed by pulsed-field gel electrophoresis (PFGE) of the bovine isolates generated 58 electrophoretic patterns which grouped into 10 pulsotypes at an 80% similarity level. The majority of the bovine isolates, 93.2% (136/146), clustered into four major pulsotypes. Seven sequence types (ST) were identified among the representative bovine S. aureus isolates genotyped, including: ST8 (CC8), ST97 (CC97), ST351 (CC705), ST352 (CC97), ST508 (CC45), ST2992 (CC97) and a novel sequence type, ST3538 (CC97). Based on PFGE analysis, greater genetic diversity was observed among the human S. aureus isolates. Bovine and human isolates from three sampling sites clustered together and were genotypically indistinguishable. Two of the isolates, ST97 and ST352 belong to the common bovine lineage CC97, and their isolation from close human contacts suggests zoonotic transfer. In the context of this study, the third isolate, ST8 (CC8), is believed to be a human clone which has transferred to a dairy cow and has subsequently caused mastitis. The detection of indistinguishable S. aureus isolates from bovine and human hosts at three of the sampling sites is suggestive of bacterial transmission and supports the need for vigilant monitoring of staphylococcal populations at the human-animal interface. PMID:28428772

Panton-Valentine leukocidin and some exotoxins of Staphylococcus aureus and antimicrobial susceptibility profiles of staphylococci isolated from milks of small ruminants.

PubMed

Ünal, Nilgün; Askar, Şinasi; Macun, Hasan Ceyhun; Sakarya, Fatma; Altun, Belgin; Yıldırım, Murat

2012-03-01

The aims of this study were to determine the existence of pvl gene, some toxin genes, and mecA gene in Staphylococcus aureus strains isolated from sheep milk and to examine antimicrobial resistance profiles in staphylococci from sheep and goats' milk. The milk samples were collected from 13 different small ruminant farms in Kirikkale province from February to August 2009. A total of 1,604 half-udder milk samples from 857 ewes and 66 half-udder milk samples from 33 goats were collected. Staphylococcus spp. were isolated and identified from the samples. Toxin genes and mecA gene among S. aureus strains were determined by PCR. Antimicrobial susceptibility of staphylococci was examined by the disk diffusion method on Mueller-Hinton agar, and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The prevalence of subclinical intramammary infection in both ewes and goats was 5.2%. The most prevalent subclinical mastitis agents were coagulase-negative staphylococci and S. aureus with prevalences 2.8% (n:46) and 1.3% (n = 21), respectively. The prevalence of resistances in isolated Staphylococcus spp. to penicilin G, tetracycline, erythromycin, gentamicin, and enrofloxacin were found as 26.9% (18), 7.5% (5), 6.0% (4), 3.0% (2), and 1.5% (1), respectively. Only 3 of the 21 S. aureus ewe isolates (13.4%) were shown to harbor enterotoxin genes being either seh, sej or sec. However, fourteen (66.6%) of the 21 S. aureus isolates had pvl gene while none of the isolates harbored mecA gene. In conclusion, Staphylococci were shown to be the most prevalent bacteria isolated from subclinical mastitis of ewes and goats and these isolates were susceptible to most of the antibiotics. In addition, S. aureus strains isolated from ewes were harboring few staphylococcal enterotoxin genes. However, Panton-Valentine leukocidin produced by S. aureus could be an important virulence factor and contribute to subclinical mastitis pathogenicity.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

PubMed

Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2018-04-01

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo -derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes , Staphylococcus aureus , and Streptococcus mutans In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins. Copyright © 2018 American Society for Microbiology.

Epigenetic Tailoring for the Production of Anti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii

PubMed Central

Beau, Jeremy; Mahid, Nida; Burda, Whittney N.; Harrington, Lacey; Shaw, Lindsey N.; Mutka, Tina; Kyle, Dennis E.; Barisic, Betty; van Olphen, Alberto; Baker, Bill J.

2012-01-01

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC90 of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC90 of 437 µM, representing a 90% inhibition therapeutic index (TI90 = IC90 A459/IC90 P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM). PMID:22690142

Epigenetic tailoring for the production of anti-infective cytosporones from the marine fungus Leucostoma persoonii.

PubMed

Beau, Jeremy; Mahid, Nida; Burda, Whittney N; Harrington, Lacey; Shaw, Lindsey N; Mutka, Tina; Kyle, Dennis E; Barisic, Betty; van Olphen, Alberto; Baker, Bill J

2012-04-01

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC(90) of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC(90) of 437 µM, representing a 90% inhibition therapeutic index (TI(90) = IC(90) A459/IC(90)P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM).

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

PubMed

Kurucz, Eva; Márkus, Róbert; Zsámboki, János; Folkl-Medzihradszky, Katalin; Darula, Zsuzsanna; Vilmos, Péter; Udvardy, Andor; Krausz, Ildikó; Lukacsovich, Tamás; Gateff, Elisabeth; Zettervall, Carl-Johan; Hultmark, Dan; Andó, István

2007-04-03

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

Emergence of Nasal Carriage of ST80 and ST152 PVL+ Staphylococcus aureus Isolates from Livestock in Algeria

PubMed Central

Agabou, Amir; Ouchenane, Zouleikha; Ngba Essebe, Christelle; Khemissi, Salim; Chehboub, Mohamed Tedj Eddine; Chehboub, Ilyes Bey; Dunyach-Remy, Catherine

2017-01-01

The spread of toxinogenic Staphylococcus aureus is a public health problem in Africa. The objectives of the study were to investigate the rate of S. aureus nasal carriage and molecular characteristics of these strains in livestock and humans in three Algerian provinces. Nasal samples were collected from camels, horses, cattle, sheep and monkeys, as well as humans in contact with them. S. aureus isolates were genotyped using DNA microarray. The rate of S. aureus nasal carriage varied between species: camels (53%), humans and monkeys (50%), sheep (44.2%), horses (15.2%) and cattle (15%). Nine methicillin-resistant S. aureus (MRSA) isolates (7.6%) were identified, isolated from camels and sheep. The S. aureus isolates belonged to 15 different clonal complexes. Among them, PVL+ (Panton–Valentine Leukocidin) isolates belonging to ST80-MRSA-IV and ST152-MSSA were identified in camels (n = 3, 13%) and sheep (n = 4, 21.1%). A high prevalence of toxinogenic animal strains was noted containing TSST-1- (22.2%), EDINB- (29.6%) and EtD- (11.1%) encoding genes. This study showed the dispersal of the highly human pathogenic clones ST152-MSSA and ST-80-MRSA in animals. It suggests the ability of some clones to cross the species barrier and jump between humans and several animal species. PMID:28946704

Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.

PubMed

Wladyka, Benedykt; Wielebska, Katarzyna; Wloka, Marcin; Bochenska, Oliwia; Dubin, Grzegorz; Dubin, Adam; Mak, Pawel

2013-08-01

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3-6.0 μM for staphylococcal strains up to more than 100 μM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 μM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II.

Staphylococcus aureus isolated from wastewater treatment plants in Tunisia: occurrence of human and animal associated lineages.

PubMed

Ben Said, Meriam; Abbassi, Mohamed Salah; Gómez, Paula; Ruiz-Ripa, Laura; Sghaier, Senda; Ibrahim, Chourouk; Torres, Carmen; Hassen, Abdennaceur

2017-08-01

The objective was to characterize Staphylococcus aureus isolated from two wastewater treatment plants (WWTPs) located in Tunis City (Tunisia), during the period 2014-2015. Genetic lineages, antibiotic resistance mechanisms and virulence factors were determined for the recovered isolates. S. aureus isolates were recovered from 12 of the 62 wastewater samples tested (19.35%), and one isolate/sample was characterized, all of them being methicillin-susceptible (MSSA). Six spa types (t587, t674, t224, t127, t701 and t1534) were found among the 12 isolates, and the spa-t587, associated with the new sequence type ST3245, was the most predominant one (7 isolates). The remaining isolates were assigned to five clonal complexes (CC5, CC97, CC1, CC6 and CC522) according to the sequence-type determined and/or the spa-type detected. S. aureus isolates were ascribed to agrI (n = 3), agrII (n = 7) and agrIII (n = 1); however, one isolate was non-typeable. S. aureus showed resistance to (number of isolates): penicillin (12), erythromycin (7), tetracycline (one) and clindamycin (one). Among the virulence factors investigated, only one isolate harboured the tst gene, encoding the TSST-1 (toxic shock syndrome toxin 1). Despite the low number of studied isolates, the present study reports the occurrence of both human- and animal-associated S. aureus clonal complexes in WWTPs in Tunisia.

Staphylococcus aureus from the German general population is highly diverse.

PubMed

Becker, Karsten; Schaumburg, Frieder; Fegeler, Christian; Friedrich, Alexander W; Köck, Robin

2017-01-01

This prospective cohort study evaluates colonization dynamics and molecular characteristics of methicillin-susceptible and - resistant Staphylococcus aureus (MSSA/MRSA) in a German general population. Nasal swabs of 1878 non-hospitalized adults were screened for S. aureus. Participants were screened thrice in intervals of 6-8 months. Isolates were characterized by spa and agr typing, mecA and mecC possession, respectively, and PCRs targeting virulence factors. 40.9% of all participants carried S. aureus at least once while 0.7% of the participants carried MRSA (mainly spa t011). MSSA isolates (n=1359) were associated with 331 different spa types; t084 (7.7%), t091 (6.1%) and t012 (71, 5.2%) were predominant. Of 206 participants carrying S. aureus at all three sampling time points, 14.1% carried the same spa type continuously; 5.3% carried different spa types with similar repeat patterns, but 80.6% carried S. aureus with unrelated spa types. MSSA isolates frequently harboured genes encoding enterotoxins (sec: 16.6%, seg: 63.1%, sei: 64.5%) and toxic shock syndrome toxin (tst: 17.5%), but rarely Panton-Valentine leukocidin (lukS-PV/lukF-PV: 0.2%). MSSA colonizing human nares in the community are clonally highly diverse. Among those constantly carrying S. aureus, clonal lineages changed over time. The proportion of persistent S. aureus carriers was lower than reported elsewhere. Copyright © 2016 Elsevier GmbH. All rights reserved.

Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage.

PubMed

Zeaki, Nikoleta; Cao, Rong; Skandamis, Panagiotis N; Rådström, Peter; Schelin, Jenny

2014-07-16

Three Staphylococcus aureus strains representing different alleles of the Siphoviridae prophage-encoded enterotoxin A (SEA) gene, including two high-SEA-producing strains and one low-SEA-producing strain were studied to investigate sea expression and SEA formation on a frankfurter type of sausage. The effect of lactic acid, an antimicrobial compound used as a preservative in food, was also investigated on the same product. All three strains were grown on pork sausages at 15°C for 14days in the presence or absence of lactic acid (1 or 2% v/v). Growth, sea mRNA expression and SEA formation were regularly monitored and compared between non-treated and treated sausages. For all experiments performed, the extracellular SEA formation significantly differed between the high- and low-SEA-producing strains, although growth and viability were overall the same. For the low producer (Sa51), the accumulated amount of extracellular SEA formed after 14days was close to the detection limit (less than 1ng/g) in all conditions; while Sa21 and Sa17, the two high-producing strains, formed 250±25.37ng/g and 750±82.65ng/g in non-treated sausage and 150±75.75ng/g and 300±83.89ng/g when treated with 1% lactic acid, respectively, after 14days. Sausages treated with 2% lactic acid followed the same pattern as above, but with an extended lag phase to 4days and reduced levels of enterotoxin formed for all strains. The difference in the level of SEA between the two high-producing strains is most likely due to the different clonal lineages of the sea-encoded Siphoviridae phages where induction of the prophage potentially could be the reason for higher production of SEA in one of the lines. Furthermore, a prolonged expression of sea gene in the two high-producing strains was observed during the entire incubation period, while the sea expression was under the detection limit in the low-producing strain. This study indicates that the high-SEA-producing strains, especially the strains with the putative capacity of prophage induction, could be more relevant in food safety aspects than low-producing type of strains on pork sausage. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of Staphylococcus aureus Isolated from Food Products in Western Algeria.

PubMed

Chaalal, Wafaa; Chaalal, Nadia; Bourafa, Nadjette; Kihal, Mebrouk; Diene, Seydina M; Rolain, Jean-Marc

2018-03-15

The current study aimed to characterize Staphylococcus aureus isolates from foodstuffs collected from western Algeria. A total of 153 S. aureus isolates from various raw and processed foods were obtained and identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Isolates were characterized by antimicrobial susceptibility testing and toxin gene detection. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were identified by detection of the mecA gene and characterized by staphylococcal cassette chromosome mec (SCCmec) typing. We found that 30.9% (153/495) of food samples were contaminated with S. aureus. Thirty-three (21.5%) S. aureus isolates were identified as MRSA, and 16.9% (26/153) carried the mecA gene. Three SCCmec types were identified of which type IV was the most common (69.2%) followed by type V (15.3%) and type II (7.6%). Two MRSA isolates were not typable with SCCmec typing. None of the examined isolates harbored mecC. Furthermore, 14.3% (22/153) of the isolates were toxigenic S. aureus. The cytotoxin gene pvl was detected in 11.1% of the S. aureus isolates. This gene was more commonly detected (76.4%) in MRSA isolates than in methicillin-suceptible Staphylococcus aureus (MSSA) isolates. The tsst-1 gene coding for toxic shock syndrome toxin was isolated rarely (3.2%) and only in MSSA isolates. According to disk diffusion test results, 70 isolates were resistant to only one antimicrobial drug, and 51 (33.3%) isolates were multidrug resistant. Other 32 isolates were susceptible to all antibiotics. Our study highlights, for the first time, a high prevalence of multidrug-resistant S. aureus isolates carrying pvl or tsst-1 found in food products in Algeria. The risk of MRSA transmission through the food chain cannot be disregarded, particularly in uncooked foods.

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

PubMed

Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

2015-11-11

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

Resistance to antimicrobials and biofilm formation in Staphylococcus spp. isolated from bovine mastitis in the Northeast of Brazil.

PubMed

da Costa Krewer, Carina; Santos Amanso, Evandro; Veneroni Gouveia, Gisele; de Lima Souza, Renata; da Costa, Mateus Matiuzzi; Aparecido Mota, Rinaldo

2015-03-01

Mastitis is the principal disease affecting dairy herds worldwide. The aim of the present study was to characterize phenotypic and genotypic features associated with resistance to antimicrobials in Staphylococcus spp. isolated from 2064 milk samples of 525 lactating cows in the Northeast of Brazil. Of the 218 isolates analyzed, 57.8% were characterized as Staphylococcus aureus, 28% as coagulase-positive staphylococci other than S. aureus (oCPS), and 14.2% as coagulase-negative staphylococci (CNS). The test for susceptibility to antimicrobials showed amoxicillin (32.6%) to be the less effective drug in vitro, and the multi-drug resistance (MDR) rate for beta-lactams varied from 0 to 0.75. The genotypic characterization showed that 93.1% of the samples were tested positive for the blaZ gene, while none amplified mecA. The antibiotic efflux mechanism was observed in 0.9% of isolates. The biofilm formation was found in 3.7 and 96.3% of samples, respectively, on Congo red agar and on the microplate adhesion test, while the icaD gene was present in 92.2% of Staphylococcus spp. The high frequency of blaZ gene observed in this study was associated with the resistance of most Staphylococcus spp. to one or more of the beta-lactams tested, which are routinely used in Brazilian herds for mastitis treatment. The biofilm formation was also detected in the isolates analyzed being an important characteristic for pathogenicity and antimicrobial resistance of bacteria.

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

PubMed Central

Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

2015-01-01

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China

PubMed Central

Zhu, Kui; Hölzel, Christina S.; Cui, Yifang; Mayer, Ricarda; Wang, Yang; Dietrich, Richard; Didier, Andrea; Bassitta, Rupert; Märtlbauer, Erwin; Ding, Shuangyang

2016-01-01

Bacillus cereus is an important cause of foodborne infectious disease and food poisoning. However, B. cereus has also been used as a probiotic in human medicine and livestock production, with low standards of safety assessment. In this study, we evaluated the safety of 15 commercial probiotic B. cereus preparations from China in terms of mislabeling, toxin production, and transferable antimicrobial resistance. Most preparations were incorrectly labeled, as they contained additional bacterial species; one product did not contain viable B. cereus at all. In total, 18 B. cereus group strains—specifically B. cereus and Bacillus thuringiensis—were isolated. Enterotoxin genes nhe, hbl, and cytK1, as well as the ces-gene were assessed by PCR. Enterotoxin production and cytotoxicity were confirmed by ELISA and cell culture assays, respectively. All isolated B. cereus group strains produced the enterotoxin Nhe; 15 strains additionally produced Hbl. Antimicrobial resistance was assessed by microdilution; resistance genes were detected by PCR and further characterized by sequencing, transformation and conjugation assays. Nearly half of the strains harbored the antimicrobial resistance gene tet(45). In one strain, tet(45) was situated on a mobile genetic element—encoding a site-specific recombination mechanism—and was transferable to Staphylococcus aureus and Bacillus subtilis by electro-transformation. In view of the wide and uncontrolled use of these products, stricter regulations for safety assessment, including determination of virulence factors and transferable antimicrobial resistance genes, are urgently needed. PMID:27242738

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China.

PubMed

Wang, Dong; Zhang, Limei; Zhou, Xuezhang; He, Yulong; Yong, Changfu; Shen, Mingliang; Szenci, Otto; Han, Bo

2016-12-01

Staphylococcus aureusis the leading pathogen involved inbovine mastitis, but knowledgeabout antimicrobial resistance, virulence factors, and genotypes of Staphylococcus aureus resulting in bovine mastitis in Ningxia, China, is limited. Therefore, antimicrobial susceptibility, virulence gene, and randomly amplified polymorphic DNA (RAPD) analyses of Staph. aureus were carried out. A total of 327 milk samples from cows with clinical and subclinical mastitis in 4 regions of Ningxia were used for the isolation and identification of pathogens according to phenotypic and molecular characteristics. Antimicrobial susceptibility against 22 antimicrobial agents was determined by disk diffusion. The presence of 8 virulence genes in Staph. aureus isolates was tested by PCR. Genotypes of isolates were investigated based on RAPD. Results showed that 35 isolates obtained from mastitis milk samples were identified as Staph. aureus. The isolates were resistant to sulfamethoxazole (100%), penicillin G (94.3%), ampicillin (94.3%), erythromycin (68.6%), azithromycin (68.6%), clindamycin (25.7%), amoxicillin (11.4%), and tetracycline (5.7%). All of the isolates contained one or more virulence genes with average (standard deviation) of 6.6±1.6. The most prevalent virulence genes were hlb (97.1%), followed by fnbpA, hla, coa (94.3% each), nuc (85.7%), fnbpB (80%), clfA (77.1%), and tsst-1 (40%). Nine different gene patterns were found and 3 of them were the dominant gene combinations (77.1%). Staphylococcus aureus isolates (n=35) were divided into 6 genotypes by RAPD tying, the genotypes III and VI were the most prevalent genotypes. There was greatvariation in genotypes of Staph. aureus isolates, not only among different farms, but also within the same herd in Ningxia province. The study showed a high incidence of Staph. aureus with genomic variation of resistance genes, which is matter of great concern in public and animal health in Ningxia province of China. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

High Frequency and Diversity of Antimicrobial Activities Produced by Nasal Staphylococcus Strains against Bacterial Competitors

PubMed Central

Janek, Daniela; Zipperer, Alexander; Kulik, Andreas; Krismer, Bernhard; Peschel, Andreas

2016-01-01

The human nasal microbiota is highly variable and dynamic often enclosing major pathogens such as Staphylococcus aureus. The potential roles of bacteriocins or other mechanisms allowing certain bacterial clones to prevail in this nutrient-poor habitat have hardly been studied. Of 89 nasal Staphylococcus isolates, unexpectedly, the vast majority (84%) was found to produce antimicrobial substances in particular under habitat-specific stress conditions, such as iron limitation or exposure to hydrogen peroxide. Activity spectra were generally narrow but highly variable with activities against certain nasal members of the Actinobacteria, Proteobacteria, Firmicutes, or several groups of bacteria. Staphylococcus species and many other Firmicutes were insusceptible to most of the compounds. A representative bacteriocin was identified as a nukacin-related peptide whose inactivation reduced the capacity of the producer Staphylococcus epidermidis IVK45 to limit growth of other nasal bacteria. Of note, the bacteriocin genes were found on mobile genetic elements exhibiting signs of extensive horizontal gene transfer and rearrangements. Thus, continuously evolving bacteriocins appear to govern bacterial competition in the human nose and specific bacteriocins may become important agents for eradication of notorious opportunistic pathogens from human microbiota. PMID:27490492

Comparison of BD GeneOhm Methicillin-Resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA Assay for Screening Patients for the Presence of MRSA Strains▿

PubMed Central

Boyce, John M.; Havill, Nancy L.

2008-01-01

We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) real-time PCR assay with the CHROMagar MRSA assay for the detection of MRSA in 286 nasal surveillance specimens. Compared with the CHROMagar MRSA assay, PCR had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 98.6%, 95.8%, and 100%, respectively. The mean PCR turnaround time was 14.5 h. PMID:18032616

Biological Detection System Technologies Technology and Industrial Base Study. A Primer on Biological Detection Technologies

DTIC Science & Technology

2001-02-01

a novel method for the detection of the mecA gene that confers the principle mechanism of methicillin resistance in Staphylococcus aureus . CPT is a...with the mecA gene was used to develop a culture confirmation F-33 assay for methicillin resistant Staphylococcus aureus . The CPT assay was used to...compounds. Examples of signatures would include peptides, aptamers and phage. These are being sought to serve as adjuncts and/or replacements for antibody

Drug-Encoded Biomarkers for Monitoring Biological Therapies

PubMed Central

Bedenk, Kristina; Zhang, Qian; Frentzen, Alexa; Cappello, Joseph; Fischer, Utz; Szalay, Aladar A.

2015-01-01

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers. PMID:26348361

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2016-08-01

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Purification and characterization of an antifungal protein, C-FKBP, from Chinese cabbage.

PubMed

Park, Seong-Cheol; Lee, Jung Ro; Shin, Sun-Oh; Jung, Ji Hyun; Lee, Young Mee; Son, Hyosuk; Park, Yoonkyung; Lee, Sang Yeol; Hahm, Kyung-Soo

2007-06-27

An antifungal protein was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by buffer-soluble extraction and two chromatographic procedures. The results of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the isolated Chinese cabbage protein was identical to human FK506-binding protein (FKBP). A cDNA encoding FKBP was isolated from a Chinese cabbage leaf cDNA library and named C-FKBP. The open reading frame of the gene encoded a 154-amino acid polypeptide. The amino acid sequence of C-FKBP exhibits striking degrees of identity with the corresponding mouse (61%), human (60%), and yeast (56%) proteins. Genomic Southern blot analyses using the full-length C-FKBP cDNA probe revealed a multigene family in the Chinese cabbage genome. The C-FKBP mRNA was highly expressed in vegetative tissues. We also analyzed the antifungal and peptidyl-prolyl cis-trans isomerase activity of recombinant C-FKBP protein expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, including Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride, whereas it exhibited no activity against E. coli and Staphylococcus aureus. These results suggest that recombinant C-FKBP is an excellent candidate as a lead compound for the development of antifungal agents.

Short communication: In vivo screening platform for bacteriocins using Caenorhabditis elegans to control mastitis-causing pathogens.

PubMed

Son, S J; Park, M R; Ryu, S D; Maburutse, B E; Oh, N S; Park, J; Oh, S; Kim, Y

2016-11-01

This study aimed to develop an in vivo screening platform using Caenorhabditis elegans to identify a novel bacteriocin for controlling the mastitis-causing pathogen Staphylococcus aureus strain RF122 in dairy cows. Using Bacillus spp. isolated from traditional Korean foods, we developed a direct in vivo screening platform that uses 96-well plates and fluorescence image analysis. We identified a novel bacteriocin produced by Bacillus licheniformis strain 146 (lichenicin 146) with a high in vivo antimicrobial activity using our liquid C. elegans-Staph. aureus assay. We also determined the characteristics of lichenicin 146 using liquid chromatography-mass spectrometry and confirmed that it shared homologous sequences with bacteriocin family proteins. In addition, RNA-sequencing analysis revealed genes encoding cell surface or membrane proteins (SAB0993c, SAB0150, SAB0994c, and SAB2375c) that are involved in the bactericidal activity of lichenicin 146 against Staph. aureus strain RF122 infection as well as those encoding transcriptional regulators (SAB0844c and SAB0133). Thus, our direct in vivo screening platform facilitates simple, convenient, cost-effective, and reliable screening of potential antimicrobial compounds with applications in the dairy field. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

PubMed Central

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-01-01

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

PubMed

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-09-15

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Antibiotic resistance in Staphylococcus aureus. Current status and future prospects.

PubMed

Foster, Timothy J

2017-05-01

The major targets for antibiotics in staphylococci are (i) the cell envelope, (ii) the ribosome and (iii) nucleic acids. Several novel targets emerged from recent targeted drug discovery programmes including the ClpP protease and FtsZ from the cell division machinery. Resistance can either develop by horizontal transfer of resistance determinants encoded by mobile genetic elements viz plasmids, transposons and the staphylococcal cassette chromosome or by mutations in chromosomal genes. Horizontally acquired resistance can occur by one of the following mechanisms: (i) enzymatic drug modification and inactivation, (ii) enzymatic modification of the drug binding site, (iii) drug efflux, (iv) bypass mechanisms involving acquisition of a novel drug-resistant target, (v) displacement of the drug to protect the target. Acquisition of resistance by mutation can result from (i) alteration of the drug target that prevents the inhibitor from binding, (ii) derepression of chromosomally encoded multidrug resistance efflux pumps and (iii) multiple stepwise mutations that alter the structure and composition of the cell wall and/or membrane to reduce drug access to its target. This review focuses on development of resistance to currently used antibiotics and examines future prospects for new antibiotics and informed use of drug combinations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Silver-nanoparticles-modified biomaterial surface resistant to staphylococcus: new insight into the antimicrobial action of silver

PubMed Central

Wang, Jiaxing; Li, Jinhua; Guo, Geyong; Wang, Qiaojie; Tang, Jin; Zhao, Yaochao; Qin, Hui; Wahafu, Tuerhongjiang; Shen, Hao; Liu, Xuanyong; Zhang, Xianlong

2016-01-01

Titanium implants are widely used clinically, but postoperative implant infection remains a potential severe complication. The purpose of this study was to investigate the antibacterial activity of nano-silver(Ag)-functionalized Ti surfaces against epidemic Staphylococcus from the perspective of the regulation of biofilm-related genes and based on a bacteria-cell co-culture study. To achieve this goal, two representative epidemic Staphylococcus strains, Staphylococcus epidermidis (S. epidermidis, RP62A) and Staphylococcus aureus (S. aureus, USA 300), were used, and it was found that an Ag-nanoparticle-modified Ti surface could regulate the expression levels of biofilm-related genes (icaA and icaR for S. epidermidis; fnbA and fnbB for S. aureus) to inhibit bacterial adhesion and biofilm formation. Moreover, a novel bacteria-fibroblast co-culture study revealed that the incorporation of Ag nanoparticles on such a surface can help mammalian cells to survive, adhere and spread more successfully than Staphylococcus. Therefore, the modified surface was demonstrated to possess a good anti-infective capability against both sessile bacteria and planktonic bacteria through synergy between the effects of Ag nanoparticles and ion release. This work provides new insight into the antimicrobial action and mechanism of Ag-nanoparticle-functionalized Ti surfaces with bacteria-killing and cell-assisting capabilities and paves the way towards better satisfying the clinical needs. PMID:27599568

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

PubMed Central

Grohmann, L; Brennicke, A; Schuster, W

1992-01-01

The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed. Images PMID:1454526

Determining the prevalence of SCCmec polymorphism, virulence and antibiotic resistance genes among methicillin-resistant Staphylococcus aureus (MRSA) isolates collected from selected hospitals in west of Iran.

PubMed

Taherikalani, Morovat; Mohammadzad, Mohammad Reza; Soroush, Setareh; Maleki, Mohammad Hossein; Azizi-Jalilian, Farid; Pakzad, Iraj; Sadeghifard, Nourkhoda; Asadollahi, Parisa; Emaneini, Mohammad; Monjezi, Aazam; Alikhani, Mohammad Yousef

2016-04-01

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens worldwide and compared to other staphylococcal species that are associated with higher mortality rate. A total of 500 Staphylococcus spp. was collected from selected hospitals in Ilam, Kermanshah, Khorram Abad and Hamadan cities and, via phenotypic and genotypic methods, was assessed to find MRSA. The presence or absence of prevalent antibiotic resistance genes and virulence genes was evaluated among MRSA isolates, using polymerase chain reaction (PCR) method, and then the SCCmec typing of these isolates was assayed by multiplex PCR. A total of 372 (74.4%) Stapylococcus spp. isolates were identified as S. aureus, among which 200 (53.8%) possessed the mecA gene and were distinguished as MRSA. All of MRSA isolates contained blaZ gene. The frequency of ermA and ermC genes among erythromycin-resistant MRSA isolates was 21.6% and 66.7%, respectively. The frequency of the virulence genes eta, hla and sea among MRSA isolates was 10%, 80.5% and 100%, respectively. SCCmec type IV accounted for 30.6% of the MRSA isolates and SCCmec type III, SCCmec type II and SCCmec type I accounted for 30%, 22% and 17.5% of the isolates, respectively. The antibiotic resistance genes and the virulence genes of blaZ, hla, sea, eta and ermC had high frequencies among the MRSA isolates. This study showed that the antibiotic resistance genes had higher frequencies among SCCmec types I and IV, which confirms the previous reports in this field.

Modeling antibiotic and cytotoxic effects of the dimeric isoquinoline IQ-143 on metabolism and its regulation in Staphylococcus aureus, Staphylococcus epidermidis and human cells

PubMed Central

2011-01-01

Background Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. Results Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. Conclusions The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics. PMID:21418624

A mutation of RNA polymerase β' subunit (RpoC) converts heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) into "slow VISA".

PubMed

Matsuo, Miki; Hishinuma, Tomomi; Katayama, Yuki; Hiramatsu, Keiichi

2015-07-01

Various mutations in the rpoB gene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found "slow VISA" (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024-5035, 2014, http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of the rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, in rpoC. Introduction of the wild-type (WT) rpoC gene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that the rpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: the rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ, which is involved in the stringent response. It appears that the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.

PubMed

Hatoum-Aslan, Asma; Maniv, Inbal; Marraffini, Luciano A

2011-12-27

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.

Isolation of methicillin-resistant Staphylococcus spp. from ready-to-eat fish products.

PubMed

Sergelidis, D; Abrahim, A; Papadopoulos, T; Soultos, N; Martziou, E; Koulourida, V; Govaris, A; Pexara, A; Zdragas, A; Papa, A

2014-11-01

A hundred samples from ready-to-eat (RTE) fish products were examined for the presence and antimicrobial susceptibility of Staphylococcus spp. Staphylococci were isolated from 43% of these samples (n = 100). The identified species in the samples were Staphylococcus aureus (7%), Staphylococcus epidermidis (13%), Staphylococcus xylosus (12%), Staphylococcus sciuri (4%), Staphylococcus warneri (3%), Staphylococcus saprophyticus (2%), Staphylococcus schleiferi (1%) and Staphylococcus auricularis (1%). Two Staph. aureus (MRSA) isolates, three Staph. epidermidis (MRSE), five Staph. xylosus, four Staph. sciuri, one Staph. schleiferi and one Staph. saprophyticus isolates were resistant to oxacillin and all of them carried the mecA gene. The two MRSA isolates belonged to the spa types t316 (ST359) and t548 (ST5) and none of them was able to produce enterotoxins. Pulsed field gel electrophoresis for Staph. aureus and Staph. epidermidis isolates revealed 6 and 11 distinct PFGE types, respectively, reflecting diversity. The presence of methicillin-resistant staphylococci, especially MRSA and MRSE, in RTE fish products may constitute a potential health risk for consumers. This study provides the first data on the occurrence of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci in salted and smoked fish products in Greece. These results are important and useful for Staphylococcus spp. risk assessment and management programmes for ready-to-eat fish products. © 2014 The Society for Applied Microbiology.

Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

USDA-ARS?s Scientific Manuscript database

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

Growth promotion of the opportunistic human pathogen, Staphylococcus lugdunensis, by heme, hemoglobin, and coculture with Staphylococcus aureus

PubMed Central

Brozyna, Jeremy R; Sheldon, Jessica R; Heinrichs, David E

2014-01-01

Staphylococcus lugdunensis is both a commensal of humans and an opportunistic pathogen. Little is currently known about the molecular mechanisms underpinning the virulence of this bacterium. Here, we demonstrate that in contrast to S. aureus,S. lugdunensis makes neither staphyloferrin A (SA) nor staphyloferrin B (SB) in response to iron deprivation, owing to the absence of the SB gene cluster, and a large deletion in the SA biosynthetic gene cluster. As a result, the species grows poorly in serum-containing media, and this defect was complemented by introduction of the S. aureusSA gene cluster into S. lugdunensis. S. lugdunensis expresses the HtsABC and SirABC transporters for SA and SB, respectively; the latter gene set is found within the isd (heme acquisition) gene cluster. An isd deletion strain was significantly debilitated for iron acquisition from both heme and hemoglobin, and was also incapable of utilizing ferric-SB as an iron source, while an hts mutant could not grow on ferric-SA as an iron source. In iron-restricted coculture experiments, S. aureus significantly enhanced the growth of S. lugdunensis, in a manner dependent on staphyloferrin production by S. aureus, and the expression of the cognate transporters by S. lugdunensis. PMID:24515974

A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing

PubMed Central

Ao, Wanyuan; Clifford, Adrianne; Corpuz, Maylene; Jenison, Robert

2017-01-01

We describe here a strategy that can distinguish between Staphylococcus species truly present in a clinical sample from contaminating Staphylococcus species introduced during the testing process. Contaminating Staphylococcus species are present at low levels in PCR reagents and colonize lab personnel. To eliminate detection of contaminants, we describe an approach that utilizes addition of sufficient quantities of either non-target Staphylococcal cells (Staphylococcus succinus or Staphylococcus muscae) or synthetic oligonucleotide templates to helicase dependent isothermal amplification reactions to consume Staphylococcus-specific tuf and mecA gene primers such that contaminating Staphylococcus amplification is suppressed to below assay limits of detection. The suppressor template DNA is designed with perfect homology to the primers used in the assay but an internal sequence that is unrelated to the Staphylococcal species targeted for detection. Input amount of the suppressor is determined by a mathematical model described herein and is demonstrated to completely suppress contaminating levels of Staphylococcus while not negatively impacting the appropriate clinical assay limit of detection. We have applied this approach to improve the specificity of detection of Staphylococcus species present in positive blood cultures using a chip-based array that produces results visible to the unaided eye. PMID:28225823

Staph ID/R: a Rapid Method for Determining Staphylococcus Species Identity and Detecting the mecA Gene Directly from Positive Blood Culture

PubMed Central

Pasko, Chris; Dunn, John; Jaeckel, Heidi; Nieuwlandt, Dan; Weed, Diane; Woodruff, Evelyn; Zheng, Xiaotian

2012-01-01

Rapid diagnosis of staphylococcal bacteremia directs appropriate antimicrobial therapy, leading to improved patient outcome. We describe herein a rapid test (<75 min) that can identify the major pathogenic strains of Staphylococcus to the species level as well as the presence or absence of the methicillin resistance determinant gene, mecA. The test, Staph ID/R, combines a rapid isothermal nucleic acid amplification method, helicase-dependent amplification (HDA), with a chip-based array that produces unambiguous visible results. The analytic sensitivity was 1 CFU per reaction for the mecA gene and was 1 to 250 CFU per reaction depending on the staphylococcal species present in the positive blood culture. Staph ID/R has excellent specificity as well, with no cross-reactivity observed. We validated the performance of Staph ID/R by testing 104 frozen clinical positive blood cultures and comparing the results with rpoB gene or 16S rRNA gene sequencing for species identity determinations and mecA gene PCR to confirm mecA gene results. Staph ID/R agreed with mecA gene PCR for all samples and agreed with rpoB/16S rRNA gene sequencing in all cases except for one sample that contained a mixture of two staphylococcal species, one of which Staph ID/R correctly identified, for an overall agreement of 99.0% (P < 0.01). Staph ID/R could potentially be used to positively affect patient management for Staphylococcus-mediated bacteremia. PMID:22170912

Identification of ORF636 in phage phiSLT carrying Panton-Valentine leukocidin genes, acting as an adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of Staphylococcus aureus.

PubMed

Kaneko, Jun; Narita-Yamada, Sachiko; Wakabayashi, Yukari; Kamio, Yoshiyuki

2009-07-01

The temperate phage phiSLT of Staphylococcus aureus carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of S. aureus. ORF636 bound specifically to S. aureus; it did not bind to any other staphylococcal species or to several gram-positive bacteria.

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

PubMed

Schuster, Dominik; Rickmeyer, Jasmin; Gajdiss, Mike; Thye, Thorsten; Lorenzen, Stephan; Reif, Marion; Josten, Michaele; Szekat, Christiane; Melo, Luís D R; Schmithausen, Ricarda M; Liégeois, Florian; Sahl, Hans-Georg; Gonzalez, Jean-Paul J; Nagel, Michael; Bierbaum, Gabriele

2017-01-01

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132 T . Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice. Copyright © 2016 Elsevier GmbH. All rights reserved.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

PubMed

Feng, Zhiyang; Kallifidas, Dimitris; Brady, Sean F

2011-08-02

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

PubMed

Averina, O V; Nezametdinova, V Z; Alekseeva, M G; Danilenko, V N

2012-11-01

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

Complete Genome Sequence of Biofilm-Forming Strain Staphylococcus haemolyticus S167.

PubMed

Hong, Jisoo; Kim, Jonguk; Kim, Byung-Yong; Park, Jin-Woo; Ryu, Jae-Gee; Roh, Eunjung

2016-06-16

Staphylococcus haemolyticus S167 has the ability to produce biofilms in large quantities. Genomic analyses revealed information on the biofilm-related genes of S. haemolyticus S167. Detailed studies of biofilm formation at the molecular level could provide a foundation for biofilm control research. Copyright © 2016 Hong et al.

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Human Genomic Signatures of Brain Oscillations During Memory Encoding.

PubMed

Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

2018-05-01

Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

Identification of Structural and Immunity Genes of a Class IIb Bacteriocin Encoded in the Enterocin A Operon of Enterococcus faecium Strain MXVK29.

PubMed

Escamilla-Martínez, E E; Cisneros, Y M Álvarez; Fernández, F J; Quirasco-Baruch, M; Ponce-Alquicira, E

2017-10-09

The Enterococcus faecium strain MXVK29, isolated from fermented sausages, produces a bacteriocin with a molecular mass of 3.5 kDa that belongs to the class of enterocins II.1, according to the terminal amino acid sequence, and has been identified as enterocin A. This bacteriocin is active against selected strains of Listeria, Staphylococcus, Pediococcus, and Enterococcus. In this study, we identified the genes adjacent to the structural gene for this bacteriocin, such as the immunity gene (entI) and the inducer gene (entF). Accessory genes for this bacteriocin, such as entK, entR, and entT, were identified as well, in addition to the orf2 and orf3, showing a high identity with class IIb peptides bacteriocins. The orf2 shows the consensus motif GxxxG, similar to those shown by bacteriocins such as PlnNC8α, EntCα, and Ent1071A, whereas orf3 shows a consensus motif SxxxS similar to that present in PlnNC8β (AxxxA). PlnNC8 is expressed only in bacterial cocultures, so there is the possibility that the expression of this two-peptide bacteriocin can be induced by a similar mechanism. So far, only the expression of enterocin A has been found in this strain; however, the presence of the genes ent29α and ent29β opens the possibility for further research on its induction, functionality, and origin. Although there are reports on this type of bacteriocin (EntX, EntC, and Ent1071) in other strains of E. faecium, no report exists yet on an Enterococcus strain producing two different classes of bacteriocin.

Improving monitoring of erythromycin ribosome methylase genes in swine and cattle manures with gene targeted approaches

USDA-ARS?s Scientific Manuscript database

Macrolide antibiotics are often used in feed for animal industry to prevent diseases. Resistance to these antibiotics is associated with erythromycin ribosome methylase genes (erm genes), which were first discovered in Staphylococcus aureus. The erm gene confers resistance by methylating rRNA at the...

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

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

'Prevalence and antimicrobial susceptibility of Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains from raw meat and meat products in Zaria, Nigeria.

PubMed

Ndahi, M D; Kwaga, J K P; Bello, M; Kabir, J; Umoh, V J; Yakubu, S E; Nok, A J

2014-03-01

The bacterial genera Listeria and Staphylococcus have been frequently isolated from food products and are responsible for a number of animal and human diseases. The aim of the study was to simultaneously isolate and characterize L. monocytogenes and Staphylococcus species from 300 samples of raw meat and meat products, to determine the susceptibility of the organisms to commonly used antimicrobial agents and to determine the presence of haemolysin A (hyl) virulence gene in L. monocytogenes and staphylococcal cassette chromosome mecA (SCCmec) gene in the Staph. aureus isolates using PCR. Of the 85 Listeria isolates tested, 12 L. monocytogenes were identified and tested for their sensitivity to 14 antimicrobial agents. All the 12 isolates (100%) were resistant to nine antimicrobial agents, but however sensitive to gentamicin. Only one isolate was found to harbour the hylA gene. Twenty-nine isolates were confirmed as Staph. aureus by the Microbact 12S identification system and were all presumptively identified as methicillin-resistant Staph. aureus species using oxacillin-resistant Staph. aureus basal medium (ORSAB). The 29 Staph. aureus isolates were tested for their sensitivity to 16 antimicrobial agents, and 11 were resistant to methicillin. None of the 11 Staph. aureus isolates harboured the methicillin resistance, mecA gene. Listeria monocytogenes and Staphylococcus aureus are important agents of foodborne diseases. Occurrence of these infectious agents was established in meat and meat products in Zaria, Nigeria. Majority of isolates obtained from this study, displayed multidrug resistance to commonly used antimicrobial agents, including methicillin resistance among the Staph. aureus isolates. The potential virulence of L. monocytogenes found in ready-to-eat food was documented by the carriage of hly A gene by one of the isolates. A different mechanism of methicillin resistance or different homologue of mec A gene may be circulating among Nigerian isolates. © 2013 The Society for Applied Microbiology.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

PubMed

Furukawa, Mutsumi; Yoneyama, Hiroshi; Hata, Eiji; Iwano, Hidetomo; Higuchi, Hidetoshi; Ando, Tasuke; Sato, Mika; Hayashi, Tomohito; Kiku, Yoshio; Nagasawa, Yuya; Niimi, Kanae; Usami, Katsuki; Ito, Kumiko; Watanabe, Kouichi; Nochi, Tomonori; Aso, Hisashi

2018-02-26

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

PubMed

Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

2014-01-01

Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo. © 2014 S. Karger AG, Basel.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

PubMed Central

Gagnaire, Julie; Dauwalder, Olivier; Boisset, Sandrine; Khau, David; Freydière, Anne-Marie; Ader, Florence; Bes, Michèle; Lina, Gerard; Tristan, Anne; Reverdy, Marie-Elisabeth; Marchand, Adrienne; Geissmann, Thomas; Benito, Yvonne; Durand, Géraldine; Charrier, Jean-Philippe; Etienne, Jerome; Welker, Martin; Van Belkum, Alex; Vandenesch, François

2012-01-01

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01–10.24; p = 0.023; CI 95%: 1.20–12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies. PMID:22792394

Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.

PubMed

Kurokawa, Kenji; Nishida, Satoshi; Ishibashi, Mihoko; Mizumura, Hikaru; Ueno, Kohji; Yutsudo, Takashi; Maki, Hideki; Murakami, Kazuhisa; Sekimizu, Kazuhisa

2008-03-01

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Escherichia coli arabinose isomerase and Staphylococcus aureus tagatose-6-phosphate isomerase: which is a better template for directed evolution of non-natural substrate isomerization?

PubMed

Kim, Hye Jung; Uhm, Tae Guk; Kim, Seong Bo; Kim, Pil

2010-06-01

Metallic and non-metallic isomerases can be used to produce commercially important monosaccharides. To determine which category of isomerase is more suitable as a template for directed evolution to improve enzymes for galactose isomerization, L-arabinose isomerase from Escherichia coli (ECAI; E.C. 5.3.1.4) and tagatose-6-phosphate isomerase from Staphylococcus aureus (SATI; E.C. 5.3.1.26) were chosen as models of a metallic and non-metallic isomerase, respectively. Random mutations were introduced into the genes encoding ECAI and SATI at the same rate, resulting in the generation of 515 mutants of each isomerase. The isomerization activity of each of the mutants toward a non-natural substrate (galactose) was then measured. With an average mutation rate of 0.2 mutations/kb, 47.5% of the mutated ECAIs showed an increase in activity compared with wild-type ECAI, and the remaining 52.5% showed a decrease in activity. Among the mutated SATIs, 58.6% showed an increase in activity, whereas 41.4% showed a decrease in activity. Mutant clones showing a significant change in relative activity were sequenced and specific increases in activity were measured. The maximum increase in activity achieved by mutation of ECAI was 130%, and that for SATI was 190%. Based on these results, the characteristics of the different isomerases are discussed in terms of their usefulness for directed evolution of non-natural substrate isomerization.

Identification and characterization of a cyclosporin binding cyclophilin from Staphylococcus aureus Newman

PubMed Central

Polley, Soumitra; Seal, Soham; Mahapa, Avisek; Jana, Biswanath; Biswas, Anindya; Mandal, Sukhendu; Sinha, Debabrata; Sau, Keya; Sau, Subrata

2017-01-01

Cyclophilins, a class of peptidyl-prolyl cis-trans isomerase (PPIase) enzymes, are inhibited by cyclosporin A (CsA), an immunosuppressive drug. Staphylococcus aureus Newman, a pathogenic bacterium, carries a gene for encoding a putative cyclophilin (SaCyp). SaCyp shows significant homology with other cyclophilins at the sequence level. A three-dimensional model structure of SaCyp harbors a binding site for CsA. To verify whether SaCyp possesses both the PPIase activity and the CsA binding ability, we have purified and investigated a recombinant SaCyp (rCyp) using various in vitro tools. Our RNase T1 refolding assay indicates that rCyp has a substantial extent of PPIase activity. rCyp that exists as a monomer in the aqueous solution is truly a cyclophilin as its catalytic activity specifically shows sensitivity to CsA. rCyp appears to bind CsA with a reasonably high affinity. Additional investigations reveal that binding of CsA to rCyp alters its structure and shape to some extent. Both rCyp and rCyp-CsA are unfolded via the formation of at least one intermediate in the presence of guanidine hydrochloride. Unfolding study also indicates that there is substantial extent of thermodynamic stabilization of rCyp in the presence of CsA as well. The data suggest that rCyp may be exploited to screen the new antimicrobial agents in the future. PMID:28584448

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

PubMed Central

2013-01-01

Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. Conclusions A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification. PMID:24188142

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Coagulase-Negative Staphylococci Favor Conversion of Arginine into Ornithine despite a Widespread Genetic Potential for Nitric Oxide Synthase Activity

PubMed Central

Sánchez Mainar, María; Weckx, Stefan

2014-01-01

Within ecosystems that are poor in carbohydrates, alternative substrates such as arginine may be of importance to coagulase-negative staphylococci (CNS). However, the versatility of arginine conversion in CNS remains largely uncharted. Therefore, a set of 86 strains belonging to 17 CNS species was screened for arginine deiminase (ADI), arginase, and nitric oxide synthase (NOS) activities, in view of their ecological relevance. In fermented meats, for instance, ADI could improve bacterial competitiveness, whereas NOS may serve as an alternative nitrosomyoglobin generator to nitrate and nitrite curing. About 80% of the strains were able to convert arginine, but considerable inter- and intraspecies heterogeneity regarding the extent and mechanism of conversion was found. Overall, ADI was the most commonly employed pathway, resulting in mixtures of ornithine and small amounts of citrulline. Under aerobic conditions, which are more relevant for skin-associated CNS communities, several strains shifted toward arginase activity, leading to the production of ornithine and urea. The obtained data indeed suggest that arginase occurs relatively more in CNS isolates from a dairy environment, whereas ADI seems to be more abundant in strains from a fermented meat background. With some exceptions, a reasonable match between phenotypic ADI and arginase activity and the presence of the encoding genes (arcA and arg) was found. With respect to the NOS pathway, however, only one strain (Staphylococcus haemolyticus G110) displayed phenotypic NOS-like activity under aerobic conditions, despite a wide prevalence of the NOS-encoding gene (nos) among CNS. Hence, the group of CNS displays a strain- and condition-dependent toolbox of arginine-converting mechanisms with potential implications for competitiveness and functionality. PMID:25281381

A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay.

PubMed

Bartels, Mette Damkjaer; Boye, Kit; Rohde, Susanne Mie; Larsen, Anders Rhod; Torfs, Herbert; Bouchy, Peggy; Skov, Robert; Westh, Henrik

2009-05-01

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade

PubMed Central

Rouillon, Christophe; Zhou, Min; Zhang, Jing; Politis, Argyris; Beilsten-Edmands, Victoria; Cannone, Giuseppe; Graham, Shirley; Robinson, Carol V.; Spagnolo, Laura; White, Malcolm F.

2013-01-01

Summary The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family. PMID:24119402

Targeting agr- and agr-Like Quorum Sensing Systems for Development of Common Therapeutics to Treat Multiple Gram-Positive Bacterial Infections

PubMed Central

Gray, Brian; Hall, Pamela; Gresham, Hattie

2013-01-01

Invasive infection by the Gram-positive pathogen Staphylococcus aureus is controlled by a four gene operon, agr that encodes a quorum sensing system for the regulation of virulence. While agr has been well studied in S. aureus, the contribution of agr homologues and analogues in other Gram-positive pathogens is just beginning to be understood. Intriguingly, other significant human pathogens, including Clostridium perfringens, Listeria monocytogenes, and Enterococcus faecalis contain agr or analogues linked to virulence. Moreover, other significant human Gram-positive pathogens use peptide based quorum sensing systems to establish or maintain infection. The potential for commonality in aspects of these signaling systems across different species raises the prospect of identifying therapeutics that could target multiple pathogens. Here, we review the status of research into these agr homologues, analogues, and other peptide based quorum sensing systems in Gram-positive pathogens as well as the potential for identifying common pathways and signaling mechanisms for therapeutic discovery. PMID:23598501

Distribution of the Multidrug Resistance Gene cfr in Staphylococcus Isolates from Pigs, Workers, and the Environment of a Hog Market and a Slaughterhouse in Guangzhou, China.

PubMed

Wang, Jing; Lin, Da-Chuan; Guo, Xiao-Mu; Wei, Hong-Kun; Liu, Xiao-Qin; Chen, Xiao-Jie; Guo, Jian-Ying; Zeng, Zhen-Ling; Liu, Jian-Hua

2015-07-01

Bacteria harboring cfr, a multidrug resistance gene, have high prevalence in livestock in China and might be transmitted to humans through direct contact or via contaminated food products. To better understand the epidemiology of cfr producers in the food chain, the prevalence and genetic analysis of Staphylococcus isolates recovered from pigs, workers, and meat-handling facilities (a slaughterhouse and a hog market in Guangzhou, China) were examined. Twenty (4.5%) cfr-positive Staphylococcus isolates (18 Staphylococcus simulans, 1 S. cohnii, and 1 S. aureus) were derived from pigs (16/312), the environment (2/52), and workers (2/80). SmaI pulsed-field gel electrophoresis of 26 staphylococcal strains (22 S. simulans and 4 S. cohnii), including previously reported cfr-carrying staphylococci of animal food origin, exhibited 19 major pulsed-field gel electrophoresis patterns (A-S). Clonal spread of cfr-carrying staphylococci among pigs, workers, and meat products was detected. The genetic contexts of cfr in plasmids (pHNKF3, pHNZT2, and pHNCR35) obtained from S. simulans of swine or human origin were similar to that of Staphylococcus species isolated from human clinics and animal-derived food. The cfr-carrying S. aureus strain isolated from floor swabs of the hog market was spa-type t889 and belonged to the ST9 clonal lineage. In summary, both clonal spread and horizontal transmission via mobile elements contributed to cfr dissemination among staphylococcal isolates obtained from different sources. To monitor potential outbreaks of cfr-positive bacteria, continued surveillance of this gene in animals at slaughter and in animal-derived food is warranted.

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Global Transcriptome Analysis of Staphylococcus aureus Response to Hydrogen Peroxide†

PubMed Central

Chang, Wook; Small, David A.; Toghrol, Freshteh; Bentley, William E.

2006-01-01

Staphylococcus aureus responds with protective strategies against phagocyte-derived reactive oxidants to infect humans. Herein, we report the transcriptome analysis of the cellular response of S. aureus to hydrogen peroxide-induced oxidative stress. The data indicate that the oxidative response includes the induction of genes involved in virulence, DNA repair, and notably, anaerobic metabolism. PMID:16452450

Introduction of plasmid DNA into an ST398 livestock-associated methicillin-resistant Staphylococcus aureus strain

USDA-ARS?s Scientific Manuscript database

MRS926 is a livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) strain of sequence type (ST) 398. In order to facilitate in vitro and in vivo studies of this strain, we sought to tag it with a fluorescent marker. We cloned a codon-optimized gene for TurboGFP into a shuttle vector...

Pneumonia and New Methicillin-resistant Staphylococcus aureus Clone

PubMed Central

Tristan, Anne; François, Bruno; Etienne, Jerome; Delage-Corre, Manuella; Martin, Christian; Liassine, Nadia; Wannet, Wim; Denis, François; Ploy, Marie-Cécile

2006-01-01

Necrotizing pneumonia caused by Staphylococcus aureus strains carrying the Panton-Valentin leukocidin gene is a newly described disease entity. We report a new fatal case of necrotizing pneumonia. An S. aureus strain with an agr1 allele and of a new sequence type 377 was recovered, representing a new, emerging, community-acquired methicillin-resistant clone. PMID:16704793

[Molecular study of methicillin-resistant Staphylococcus haemolyticus in a Mexican hospital].

PubMed

Castro, Natividad; Loaiza-Loeza, María Salomé; Calderón-Navarro, Amparo; Sánchez, Alejandro; Silva-Sánchez, Jesús

2006-01-01

To perform the molecular characterization of methicillin-resistant Staphylococcus haemolyticus (MRSH) clinical isolates from patients in a Mexican hospital. Sixty three Staphylococcus ssp. isolates collected from September 2000 to October 2002 were analyzed. Antimicrobial susceptibility was determined by disk diffusion method and the presence of the mecA gene was detected by PCR technique. Isolates characterization was carried out by pulsed field gel electrophoresis (PFGE). The frequency of S. haemolyticus was 25.5% (18 of 63 clinical isolates), all S. haemolyticus isolates were methicillin-resistant and they were positive for the mecA gene. A major pattern (A) with 8 subtypes was identified. This clone was distributed during the 20 months period. Most of them were isolated from the surgery (55%) and pediatric services (27.5%). The methicillin-resistant S. haemolyticus permanence as pathogen in this hospital, suggest the implementation of control programs in order to decrease the prevalence of this multiresistant pathogen.

Attenuating Staphylococcus aureus Virulence Gene Regulation: A Medicinal Chemistry Perspective

PubMed Central

2013-01-01

Virulence gene expression in Staphylococcus aureus is tightly regulated by intricate networks of transcriptional regulators and two-component signal transduction systems. There is now an emerging body of evidence to suggest that the blockade of S. aureus virulence gene expression significantly attenuates infection in experimental models. In this Perspective, we will provide insights into medicinal chemistry strategies for the development of chemical reagents that have the capacity to inhibit staphylococcal virulence expression. These reagents can be broadly grouped into four categories: (1) competitive inhibitors of the accessory gene regulator (agr) quorum sensing system, (2) inhibitors of AgrA–DNA interactions, (3) RNAIII transcription inhibitors, and (4) inhibitors of the SarA family of transcriptional regulators. We discuss the potential of specific examples of antivirulence agents for the management and treatment of staphylococcal infections. PMID:23294220

Molecular detection of Staphylococcus aureus resistant to temperature in milk and its products

NASA Astrophysics Data System (ADS)

Sutejo, Stephani Valentina Harda; Amarantini, Charis; Budiarso, Tri Yahya

2017-11-01

Contamination of Staphylococcus aureus on milk can cause intoxication and infection by Staphylococcal enterotoxin. It has nuc gene, coding thermonuclease enzyme (TNase) that is responsible for nature of resistance in the heating process. This study was conducted to identify nuc gene of as S. aureus isolated from milk and its products like ultra-high temperature, sterile milk, sweetened condensed milk, formula milk, café/milk street traders and fresh milk. Biochemical identification was conducted by using carbohydrate fermentation tests and confirmed by API Staph. Molecular confirmation by amplification of nuc gene using PCR. Based on the results of confirmation using API Staph, all isolates were confirmed as S. aureus with index determinant percentage of 97%. An amplicon product of 270 bp was gained in all isolates. It is concluded that isolate of S. aureus has nuc gene.

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis

PubMed Central

Ciftci, Alper; Findik, Arzu; Onuk, Ertan Emek; Savasan, Serap

2009-01-01

This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains. PMID:24031354

Isolation, pathogenicity and disinfection of Staphylococcus aureus carried by insects in two public hospitals of Vitória da Conquista, Bahia, Brazil.

PubMed

Oliveira, Pollianna S; Souza, Simone G; Campos, Guilherme B; da Silva, Danilo C C; Sousa, Daniel S; Araújo, Suerda P F; Ferreira, Laiziane P; Santos, Verena M; Amorim, Aline T; Santos, Angelita M O G; Timenetsky, Jorge; Cruz, Mariluze P; Yatsuda, Regiane; Marques, Lucas M

2014-01-01

Currently, hospital infection is a serious public health problem, and several factors may influence the occurrence of these infections, including the presence of insects, which are carriers of multidrug-resistant bacterial species. The aim of this study was to isolate staphylococci carried by insects in two public hospitals of Vitoria da Conquista, Bahia and to identify the resistance profile, pathogenicity and efficacy of disinfection of the premises. A total of 91 insects were collected in 21 strategic points of these hospitals, and 32 isolated strains of Staphylococcus aureus were isolated. Based on antibiogram and Minimum Inhibitory Concentration results, 95% of these strains were susceptible to oxacillin. These strains were also evaluated for the presence of resistance genes encoding resistance to oxacillin/methicillin by polymerase chain reaction, but the sample was negative for this gene. Pathogenicity tests were performed in vitro biofilm formation induced by glucose, where it was found that eight (27.58%) strains were classified as biofilm producers and 21 (72.4%) as stronger producers. In addition, we performed PCR for their virulence genes: Sea (enterotoxin A), SEB (B), Sec (C), PVL (Panton-Valentine Leukocidin), ClfA (clumping factor A) and Spa (protein A). Of these, Sea, Spa PVL were positive in 7 (21.8%), 2 (6.3%) and 1 (3.1%) samples, respectively. The analysis of cytokine induction in the inflammatory response of J774 macrophages by isolates from the two hospitals did not show statistical difference at the levels of IL-6, TNF-α, IL-1 and IL-10 production. In addition, we verified the antimicrobial activity of disinfecting agents on these strains, quaternary ammonium, 0.5% sodium hypochlorite, 1% sodium hypochlorite, 2% sodium hypochlorite, 2% glutaraldehyde, Lysoform(®), 70% alcohol solution of chlorhexidine digluconate, 2% peracetic acid, and 100% vinegar. Resistance was seen in only for the following two disinfectants: 70% alcohol in 31 (96.8%) samples tested and vinegar in 30 (93.8%) samples. The study demonstrated the presence of resistant and pathogenic organisms conveyed by insects, thus suggesting improvement in efforts to control these vectors. Copyright © 2013 Elsevier Editora Ltda. All rights reserved.

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

Extensive Genetic Diversity Identified among Sporadic Methicillin-Resistant Staphylococcus aureus Isolates Recovered in Irish Hospitals between 2000 and 2012

PubMed Central

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan

2014-01-01

Clonal replacement of predominant nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains has occurred several times in Ireland during the last 4 decades. However, little is known about sporadically occurring MRSA in Irish hospitals or in other countries. Eighty-eight representative pvl-negative sporadic MRSA isolates recovered in Irish hospitals between 2000 and 2012 were investigated. These yielded unusual pulsed-field gel electrophoresis and antibiogram-resistogram typing patterns distinct from those of the predominant nosocomial MRSA clone, ST22-MRSA-IV, during the study period. Isolates were characterized by spa typing and DNA microarray profiling for multilocus sequence type (MLST) clonal complex (CC) and/or sequence type (ST) and SCCmec type assignment, as well as for detection of virulence and antimicrobial resistance genes. Conventional PCR-based SCCmec subtyping was undertaken when necessary. Extensive diversity was detected, including 38 spa types, 13 MLST-CCs (including 18 STs among 62 isolates assigned to STs), and 25 SCCmec types (including 2 possible novel SCCmec elements and 7 possible novel SCCmec subtypes). Fifty-four MLST-spa-SCCmec type combinations were identified. Overall, 68.5% of isolates were assigned to nosocomial lineages, with ST8-t190-MRSA-IID/IIE ± SCCM1 predominating (17.4%), followed by CC779/ST779-t878-MRSA-ψSCCmec-SCC-SCCCRISPR (7.6%) and CC22/ST22-t032-MRSA-IVh (5.4%). Community-associated clones, including CC1-t127/t386/t2279-MRSA-IV, CC59-t216-MRSA-V, CC8-t008-MRSA-IVa, and CC5-t002/t242-MRSA-IV/V, and putative animal-associated clones, including CC130-t12399-MRSA-XI, ST8-t064-MRSA-IVa, ST398-t011-MRSA-IVa, and CC6-t701-MRSA-V, were also identified. In total, 53.3% and 47.8% of isolates harbored genes for resistance to two or more classes of antimicrobial agents and two or more mobile genetic element-encoded virulence-associated factors, respectively. Effective ongoing surveillance of sporadic nosocomial MRSA is warranted for early detection of emerging clones and reservoirs of virulence, resistance, and SCCmec genes. PMID:24395241

Vaccine potential of poly-1-6 beta-D-N-succinylglucosamine, an immunoprotective surface polysaccharide of Staphylococcus aureus and Staphylococcus epidermidis.

PubMed

Mckenney, D; Pouliot, K; Wang, Y; Murthy, V; Ulrich, M; Döring, G; Lee, J C; Goldmann, D A; Pier, G B

2000-09-29

Staphylococcus aureus and S. epidermidis are among the most common causes of nosocomial infection, and S. aureus is also of major concern to human health due to its occurrence in community-acquired infections. These staphylococcal species are also major pathogens for domesticated animals. We have previously identified poly-N-succinyl beta-1-6 glucosamine (PNSG) as the chemical form of the S. epidermidis capsular polysaccharide/adhesin (PS/A) which mediates adherence of coagulase-negative staphylococci (CoNS) to biomaterials, serves as the capsule for strains of CoNS that express PS/A, and is a target for protective antibodies. We have recently found that PNSG is made by S. aureus as well, where it is an environmentally regulated, in vivo-expressed surface polysaccharide and similarly serves as a target for protective immunity. Only a minority of fresh human clinical isolates of S. aureus elaborate PNSG in vitro but most could be induced to do so under specific in vitro growth conditions. However, by immunofluorescence microscopy, S. aureus cells in infected human sputa and lung elaborated PNSG. The ica genes, previously shown to encode proteins in CoNS that synthesize PNSG, were found by PCR in all S. aureus strains examined, and immunogenic and protective PNSG could be isolated from S. aureus. Active and passive immunization of mice with PNSG protected them against metastatic kidney infections after intravenous inoculation with eight phenotypically PNSG-negative S. aureus. Isolates recovered from kidneys expressed PNSG, but expression was lost with in vitro culture. Strong antibody responses to PNSG were elicited in S. aureus infected mice, and a PNSG-capsule was observed by electron microscopy on isolates directly plated from infected kidneys. PNSG represents a previously unidentified surface polysaccharide of S. aureus that is elaborated during human and animal infection and is a prominent target for protective antibodies.

Comparison of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus (MRSA) PCR Assay to Culture by Use of BBL CHROMagar MRSA for Detection of MRSA in Nasal Surveillance Cultures from an At-Risk Community Population▿

PubMed Central

Farley, Jason E.; Stamper, Paul D.; Ross, Tracy; Cai, Mian; Speser, Sharon; Carroll, Karen C.

2008-01-01

We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture with BBL CHROMagar MRSA for nasal surveillance among 602 arrestees from the Baltimore City Jail. The sensitivity and specificity were 88.5% and 91.0%, respectively, and after secondary analysis using enrichment broth, they were 89.0% and 91.7%, respectively. Twenty-three of 42 false-positive PCR lysates contained methicillin-susceptible S. aureus. PMID:18057129

Identification of ORF636 in Phage φSLT Carrying Panton-Valentine Leukocidin Genes, Acting as an Adhesion Protein for a Poly(Glycerophosphate) Chain of Lipoteichoic Acid on the Cell Surface of Staphylococcus aureus▿

PubMed Central

Kaneko, Jun; Narita-Yamada, Sachiko; Wakabayashi, Yukari; Kamio, Yoshiyuki

2009-01-01

The temperate phage φSLT of Staphylococcus aureus carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of S. aureus. ORF636 bound specifically to S. aureus; it did not bind to any other staphylococcal species or to several gram-positive bacteria. PMID:19429614

rRNA gene restriction patterns as an epidemiological marker in nosocomial outbreaks of Staphylococcus aureus infections.

PubMed

Meugnier, H; Fernandez, M P; Bes, M; Brun, Y; Bornstein, N; Freney, J; Fleurette, J

1993-01-01

rRNA gene restriction patterns (ribotyping) were compared with phage typing, serotyping, enterotoxins and exfoliatin production in the analysis of 26 Staphylococcus aureus strains isolated from two different nosocomial outbreaks. Total DNA was cleaved by EcoRI restriction endonuclease. After agarose gel electrophoresis and Southern transfer, the hybridization of the membranes was done with radiolabelled 16S rRNA gene from Bacillus subtilis inserted into a plasmid vector. Six to 13 fragments were visualized. A core of common fragments was discerned for all strains tested. A full correlation between ribotyping and conventional markers was observed in only one of the outbreaks studied. In both outbreaks, ribotyping proved helpful in characterizing otherwise untypable strains.

Detection of methicillin resistant Staphylococcus aureus (MRSA) from recreational beach using the mecA gene

NASA Astrophysics Data System (ADS)

Zulkifli, Aisya; Ahmad, Asmat

2015-09-01

Water samples were collected in triplicates from three different locations choosen from the recreational beach of Teluk Kemang, Port Dickson as sampling station including main area of recreation activity for the public. Bacteria were isolated from the water and cultured. Out of 286 presumptive Staphylococcus aureus enumerated by using culture method, only 4 (1.4 %) confirmed as Meticillin Resistant S. aureus (MRSA) based on PCR detection of mecA gene. Interestingly, all of MRSA detections were found at the main area of recreational activity. Our results suggested that public beaches may be reservoir for transmission of MRSA to beach visitors and PCR using the mecA gene is the fastest way to detect this pathogenic bacteria.

Study of Staphylococcus aureus N315 Pathogenic Genes by Text Mining and Enrichment Analysis of Pathways and Operons.

PubMed

Yang, Chun-Feng; Gou, Wei-Hui; Dai, Xin-Lun; Li, Yu-Mei

2018-06-01

Staphylococcus aureus (S. aureus) is a versatile pathogen found in many environments and can cause nosocomial infections in the community and hospitals. S. aureus infection is an increasingly serious threat to global public health that requires action across many government bodies, medical and health sectors, and scientific research institutions. In the present study, S. aureus N315 genes that have been shown in the literature to be pathogenic were extracted using a bibliometric method for functional enrichment analysis of pathways and operons to statistically discover novel pathogenic genes associated with S. aureus N315. A total of 383 pathogenic genes were mined from the literature using bibliometrics, and subsequently a few new pathogenic genes of S. aureus N315 were identified by functional enrichment analysis of pathways and operons. The discovery of these novel S. aureus N315 pathogenic genes is of great significance to treat S. aureus induced diseases and identify potential diagnostic markers, thus providing theoretical fundamentals for epidemiological prevention.

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.

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

PubMed

Frey, Amy B; Wilson, Deborah A; LaSalvia, Margaret M; Tan, Carmela D; Rodriguez, E Rene; Shrestha, Nabin K; Hall, Gerri S; Procop, Gary W

2011-11-01

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

Staphylococcus spp. isolated from wild birds apprehended in the local illegal trade in Rio de Janeiro, Brazil and relevance in Public Health.

PubMed

Matias, C A R; Pereira, I A; Rodrigues, D P; Siciliano, S

2018-06-20

This work aimed to investigate the prevalence of Staphylococcus in wild birds seized in illegal trade and their antimicrobial resistance patterns. Cloacal samples were obtained from 109 wild birds apprehended in the street markets in Rio de Janeiro, Brazil. Staphylococcus spp. were pheno and genotypically identified and resistance profile were evaluated by CLSI guidelines and by Polymerase Chain Reaction of mecA and blaZ genes. Staphylococcus was detected in 45,9% (50/109) of the cloacal swab samples and thirty-nine (78,0%) isolates were resistant to one or more of the nine antimicrobials tested, and were also positive to mecA (12/39) or blaZ genes (14/39). High percentage of resistance was detected to ampicillin, oxacillin, cefoxitin, clindamycin and tetracycline, with absence of resistance to vancomycin. Wild birds captured and submitted to captive stress conditions of illegal trade market of Brazil may have an important role as reservoirs of Staphylococcus spp. and its antimicrobial resistance mechanisms. The significance of the present study is revealed by the zoonotic and pathogenic potential of staphylococci and that impact to public health and requires monitoring polices of wild birds health in tropical areas. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Cow teat skin, a potential source of diverse microbial populations for cheese production.

PubMed

Verdier-Metz, Isabelle; Gagne, Geneviève; Bornes, Stéphanie; Monsallier, Françoise; Veisseire, Philippe; Delbès-Paus, Céline; Montel, Marie-Christine

2012-01-01

The diversity of the microbial community on cow teat skin was evaluated using a culture-dependent method based on the use of different dairy-specific media, followed by the identification of isolates by 16S rRNA gene sequencing. This was combined with a direct molecular approach by cloning and 16S rRNA gene sequencing. This study highlighted the large diversity of the bacterial community that may be found on teat skin, where 79.8% of clones corresponded to various unidentified species as well as 66 identified species, mainly belonging to those commonly found in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea, Aerococcus, and Staphylococcus). Several of them, such as nonstarter lactic acid bacteria (NSLAB), Staphylococcus, and Actinobacteria, may contribute to the development of the sensory characteristics of cheese during ripening. Therefore, teat skin could be an interesting source or vector of biodiversity for milk. Variations of microbial counts and diversity between the farms studied have been observed. Moreover, Staphylococcus auricularis, Staphylococcus devriesei, Staphylococcus arlettae, Streptococcus bovis, Streptococcus equinus, Clavibacter michiganensis, Coprococcus catus, or Arthrobacter gandavensis commensal bacteria of teat skin and teat canal, as well as human skin, are not common in milk, suggesting that there is a breakdown of microbial flow from animal to milk. It would then be interesting to thoroughly study this microbial flow from teat to milk.

Staphylococcus epidermidis and Staphylococcus haemolyticus: detection of biofilm genes and biofilm formation in blood culture isolates from patients in a Brazilian teaching hospital.

PubMed

Pinheiro, Luiza; Brito, Carla Ivo; Oliveira, Adilson de; Pereira, Valéria Cataneli; Cunha, Maria de Lourdes Ribeiro de Souza da

2016-09-01

Infections with coagulase-negative staphylococci are often related to biofilm formation. This study aimed to detect biofilm formation and biofilm-associated genes in blood culture isolates of Staphylococcus epidermidis and S. haemolyticus. Half (50.6%) of the 85 S. epidermidis isolates carried the icaAD genes and 15.3% the bhp gene, while these numbers were 42.9% and 0 for S. haemolyticus, respectively. According to the plate test, 30 S. epidermidis isolates were biofilm producers and 40% of them were strongly adherent, while only one (6%) of the 17 S. haemolyticus biofilm-producing isolates exhibited a strongly adherent biofilm. The concomitant presence of icaA and icaD was significantly associated with the plate and tube test results (P ≤ 0.0004). The higher frequency of icaA in S. epidermidis and of icaD in S. haemolyticus is correlated with the higher biofilm-producing capacity of the former since, in contrast to IcaD, IcaA activity is sufficient to produce small amounts of polysaccharide. Although this study emphasizes the importance of icaAD and bhp for biofilm formation in S. epidermidis, other mechanisms seem to be involved in S. haemolyticus. Copyright © 2016 Elsevier Inc. All rights reserved.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Inhibition of Exotoxin Production by Mobile Genetic Element SCCmec-Encoded psm-mec RNA Is Conserved in Staphylococcal Species

PubMed Central

Saito, Yuki; Mao, Han; Sekimizu, Kazuhisa; Kaito, Chikara

2014-01-01

Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved. PMID:24926994

Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli.

PubMed

Wladyka, Benedykt; Puzia, Katarzyna; Dubin, Adam

2005-01-01

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.

Whole Genome Sequencing of Danish Staphylococcus argenteus Reveals a Genetically Diverse Collection with Clear Separation from Staphylococcus aureus.

PubMed

Hansen, Thomas A; Bartels, Mette D; Høgh, Silje V; Dons, Lone E; Pedersen, Michael; Jensen, Thøger G; Kemp, Michael; Skov, Marianne N; Gumpert, Heidi; Worning, Peder; Westh, Henrik

2017-01-01

Staphylococcus argenteus ( S. argenteus ) is a newly identified Staphylococcus species that has been misidentified as Staphylococcus aureus ( S. aureus ) and is clinically relevant. We identified 25 S. argenteus genomes in our collection of whole genome sequenced S. aureus . These genomes were compared to publicly available genomes and a phylogeny revealed seven clusters corresponding to seven clonal complexes. The genome of S. argenteus was found to be different from the genome of S. aureus and a core genome analysis showed that ~33% of the total gene pool was shared between the two species, at 90% homology level. An assessment of mobile elements shows flow of SCC mec cassettes, plasmids, phages, and pathogenicity islands, between S. argenteus and S. aureus . This dataset emphasizes that S. argenteus and S. aureus are two separate species that share genetic material.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with low within-herd prevalence of intra-mammary infections in dairy cows: Genotyping of isolates.

PubMed

Luini, M; Cremonesi, P; Magro, G; Bianchini, V; Minozzi, G; Castiglioni, B; Piccinini, R

2015-08-05

Staphylococcus aureus is one of the most common mastitis-causing pathogens worldwide. In the last decade, livestock-associated methicillin-resistant S. aureus (LA-MRSA) infections have been described in several species, included the bovines. Hence, this paper investigates the diffusion of MRSA within Italian dairy herds; the strains were further characterized using a DNA microarray, which detects 330 different sequences, including the methicillin-resistance genes mecA and mecC and SCCmec typing. The analysis of overall patterns allows the assignment to Clonal Complexes (CC). Overall 163 S. aureus isolates, collected from quarter milk samples in 61 herds, were tested. MRSA strains were further processed using spa typing. Fifteen strains (9.2%), isolated in 9 herds (14.75%), carried mecA, but none harboured mecC. MRSA detection was significantly associated (P<0.011) with a within-herd prevalence of S. aureus intra-mammary infections (IMI) ≤5%. Ten MRSA strains were assigned to CC398, the remaining ones to CC97 (n=2), CC1 (n=2) or CC8 (n=1). In 3 herds, MRSA and MSSA co-existed: CC97-MRSA with CC398-MSSA, CC1-MRSA with CC8-MSSA and CC398-MRSA with CC126-MSSA. The results of spa typing showed an overall similar profile of the strains belonging to the same CC: t127-CC1, t1730-CC97, t899 in 8 out of 10 CC398. In the remaining 2 isolates a new spa type, t14644, was identified. The single CC8 was a t3092. The SCCmec cassettes were classified as type IV, type V or type IV/V composite. All or most strains harboured the genes encoding the β-lactamase operon and the tetracycline resistance. Streptogramin resistance gene was related to CC398. Enterotoxin and leukocidin genes were carried only by CC1, CC8 and CC97-MRSA. The persistence of MRSA clones characterized by broader host range, in epidemiologically unrelated areas and in dairy herds with low prevalence of S. aureus IMI, might enhance the risk for adaptation to human species. Copyright © 2015 Elsevier B.V. All rights reserved.

Nasal Carriage of Staphylococcus aureus among Children in the Ashanti Region of Ghana.

PubMed

Eibach, Daniel; Nagel, Michael; Hogan, Benedikt; Azuure, Clinton; Krumkamp, Ralf; Dekker, Denise; Gajdiss, Mike; Brunke, Melanie; Sarpong, Nimako; Owusu-Dabo, Ellis; May, Jürgen

2017-01-01

Nasal carriage with Staphylococcus aureus is a common risk factor for invasive infections, indicating the necessity to monitor prevalent strains, particularly in the vulnerable paediatric population. This surveillance study aims to identify carriage rates, subtypes, antimicrobial susceptibilities and virulence markers of nasal S. aureus isolates collected from children living in the Ashanti region of Ghana. Nasal swabs were obtained from children < 15 years of age on admission to the Agogo Presbyterian Hospital between April 2014 and January 2015. S. aureus isolates were characterized by their antimicrobial susceptibility, the presence of genes encoding for Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) and further differentiated by spa-typing and multi-locus-sequence-typing. Out of 544 children 120 (22.1%) were colonized with S. aureus, with highest carriage rates during the rainy seasons (27.2%; p = 0.007), in females aged 6-8 years (43.7%) and males aged 8-10 years (35.2%). The 123 isolates belonged to 35 different spa-types and 19 sequence types (ST) with the three most prevalent spa-types being t355 (n = 25), t84 (n = 18), t939 (n = 13), corresponding to ST152, ST15 and ST45. Two (2%) isolates were methicillin-resistant S. aureus (MRSA), classified as t1096 (ST152) and t4454 (ST45), and 16 (13%) were resistant to three or more different antimicrobial classes. PVL and TSST-1 were detected in 71 (58%) and 17 (14%) isolates respectively. S. aureus carriage among Ghanaian children seems to depend on age, sex and seasonality. While MRSA rates are low, the high prevalence of PVL is of serious concern as these strains might serve not only as a source for severe invasive infections but may also transfer genes, leading to highly virulent MRSA clones.

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus - A case of gene duplication and lateral transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebulsky, M. Tom; Speziali, Craig D.; Shilton, Brian H.

Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us tomore » examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.« less

Thioridazine Induces Major Changes in Global Gene Expression and Cell Wall Composition in Methicillin-Resistant Staphylococcus aureus USA300

PubMed Central

Thorsing, Mette; Klitgaard, Janne K.; Atilano, Magda L.; Skov, Marianne N.; Kolmos, Hans Jørn; Filipe, Sérgio R.; Kallipolitis, Birgitte H.

2013-01-01

Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics. PMID:23691239

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Selenium Pretreatment Alleviated LPS-Induced Immunological Stress Via Upregulation of Several Selenoprotein Encoding Genes in Murine RAW264.7 Cells.

PubMed

Wang, Longqiong; Jing, Jinzhong; Yan, Hui; Tang, Jiayong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Tian, Gang; Cai, Jingyi; Shang, Haiying; Zhao, Hua

2018-04-18

This study was conducted to profile selenoprotein encoding genes in mouse RAW264.7 cells upon lipopolysaccharide (LPS) challenge and integrate their roles into immunological regulation in response to selenium (Se) pretreatment. LPS was used to develop immunological stress in macrophages. Cells were pretreated with different levels of Se (0, 0.5, 1.0, 1.5, 2.0 μmol Se/L) for 2 h, followed by LPS (100 ng/mL) stimulation for another 3 h. The mRNA expression of 24 selenoprotein encoding genes and 9 inflammation-related genes were investigated. The results showed that LPS (100 ng/mL) effectively induced immunological stress in RAW264.7 cells with induced inflammation cytokines, IL-6 and TNF-α, mRNA expression, and cellular secretion. LPS increased (P < 0.05) mRNA profiles of 9 inflammation-related genes in cells, while short-time Se pretreatment modestly reversed (P < 0.05) the LPS-induced upregulation of 7 genes (COX-2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, and MCP-1) and further increased (P < 0.05) expression of IFN-β and TNF-α in stressed cells. Meanwhile, LPS decreased (P < 0.05) mRNA levels of 18 selenoprotein encoding genes and upregulated mRNA levels of TXNRD1 and TXNRD3 in cells. Se pretreatment recovered (P < 0.05) expression of 3 selenoprotein encoding genes (GPX1, SELENOH, and SELENOW) in a dose-dependent manner and increased (P < 0.05) expression of another 5 selenoprotein encoding genes (SELENOK, SELENOM, SELENOS, SELENOT, and TXNRD2) only at a high level (2.0 μmol Se/L). Taken together, LPS-induced immunological stress in RAW264.7 cells accompanied with the global downregulation of selenoprotein encoding genes and Se pretreatment alleviated immunological stress via upregulation of a subset of selenoprotein encoding genes.

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

PubMed

Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

2007-05-01

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

Characterization of a novel composite staphylococcal cassette chromosome mec (SCCmec-SCCcad/ars/cop) in the neonatal sepsis-associated Staphylococcus capitis pulsotype NRCS-A.

PubMed

Martins Simões, P; Rasigade, J-P; Lemriss, H; Butin, M; Ginevra, C; Lemriss, S; Goering, R V; Ibrahimi, A; Picaud, J C; El Kabbaj, S; Vandenesch, F; Laurent, F

2013-12-01

Multiresistant Staphylococcus capitis pulsotype NRCS-A has been reported to be a major pathogen causing nosocomial bacteremia in preterm infants. We report that the NRCS-A strain CR01 harbors a novel 60.9-kb composite staphylococcal cassette chromosome mec (SCCmec) element, composed of an SCCmec with strong homologies to Staphylococcus aureus ST398 SCCmec and of an SCCcad/ars/cop harboring resistance genes for cadmium, arsenic, and copper. Whole-genome-based comparisons of published S. capitis strains suggest that strain CR01 acquired the two elements independently.

Vancomycin-Resistant Gram-Positive Cocci Isolated from the Saliva of Wild Songbirds

PubMed Central

Ishihara, Shingo; Bitner, Jessica J.; Farley, Greg H.

2014-01-01

We analyzed highly vancomycin-resistant Gram-positive bacteria isolated from the saliva of migratory songbirds captured, sampled, and released from a birdbanding station in western Kansas. Individual bacterial isolates were identified by partial 16S rRNA sequencing. Most of the bacteria in this study were shown to be Staphylococcus succinus with the majority being isolated from the American Robin. Some of these bacteria were shown to carry vanA, vanB, and vanC vancomycin-resistance genes and have the ability to form biofilms. One of the van gene-carrying isolates is also coagulase positive, which is normally considered a virulence factor. Other organisms isolated included Staphylococcus saprophyticus as well as Enterococcus gallinarum. Given the wide range of the American Robin and ease of horizontal gene transfer between Gram-positive cocci, we postulate that these organisms could serve as a reservoir of vancomycin-resistance genes capable of transferring to human pathogens. PMID:23224296

Vancomycin-resistant gram-positive cocci isolated from the saliva of wild songbirds.

PubMed

Ishihara, Shingo; Bitner, Jessica J; Farley, Greg H; Gillock, Eric T

2013-04-01

We analyzed highly vancomycin-resistant Gram-positive bacteria isolated from the saliva of migratory songbirds captured, sampled, and released from a bird-banding station in western Kansas. Individual bacterial isolates were identified by partial 16S rRNA sequencing. Most of the bacteria in this study were shown to be Staphylococcus succinus with the majority being isolated from the American Robin. Some of these bacteria were shown to carry vanA, vanB, and vanC vancomycin-resistance genes and have the ability to form biofilms. One of the van gene-carrying isolates is also coagulase positive, which is normally considered a virulence factor. Other organisms isolated included Staphylococcus saprophyticus as well as Enterococcus gallinarum. Given the wide range of the American Robin and ease of horizontal gene transfer between Gram-positive cocci, we postulate that these organisms could serve as a reservoir of vancomycin-resistance genes capable of transferring to human pathogens.

Molecular Characterization of Staphylococcus aureus Isolates Transmitted between Patients with Buruli Ulcer.

PubMed

Amissah, Nana Ama; Chlebowicz, Monika A; Ablordey, Anthony; Sabat, Artur J; Tetteh, Caitlin S; Prah, Isaac; van der Werf, Tjip S; Friedrich, Alex W; van Dijl, Jan Maarten; Rossen, John W; Stienstra, Ymkje

2015-01-01

Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. This study investigated possible patient-to-patient transmission events of S. aureus during wound care in a health care center. S. aureus isolates from different BU patients with overlapping visits to the clinic were whole-genome sequenced and analyzed by a gene-by-gene approach using SeqSphere(+) software. In addition, sequence data were screened for the presence of genes that conferred antibiotic resistance. SeqSphere(+) analysis of whole-genome sequence data confirmed transmission of methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus among patients that took place during wound care. Interestingly, our sequence data show that the investigated MRSA isolates carry a novel allele of the fexB gene conferring chloramphenicol resistance, which had thus far not been observed in S. aureus.

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

Isolation and identification of Staphylococcus sp. in powdered infant milk

NASA Astrophysics Data System (ADS)

Palilu, Prayolga Toban; Budiarso, Tri Yahya

2017-05-01

Staphylococcus sp. is one of the most dangerous bacteria that could cause food poisoning. It is a pathogenic bacterium which is able to produce enterotoxin in foods. Milk is an ideal growth medium for Staphylococcus sp., that may cause problem if it is to be consumed, especially by infant. It is the objective of this research to detect the presence of Staphylococcus sp. in powdered infant milk. As many as 14 samples obtained from market were used as samples for bacterial isolation. The isolation were done by employing enrichment step on BHI-broth, continued with Baird-Parker Agar which will produce a typical colony. It is then picked and grown on Mannitol Salt Agar, and gram staining, coagulase assay, and fermentation tests. The confirmation step was done by using API-Staph which gives the identification of Staphylococcus hemoliticus, Staphylococcus aureus and Staphylococcus epidermidis, with a percentage of identity ranging from 65.9-97.7%. Two isolates with the highest identification similarity values were then picked for molecular detection. A PCR primer pair targeting gene coding for enterotoxin A was used, and it gives positive result for the two isolates being tested. It is then concluded that the two isolates belong to Staphylococcus sp., and further research need to be done to correctly identify these isolates.

Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries.

PubMed

Ruppé, Etienne; Barbier, François; Mesli, Yasmine; Maiga, Aminata; Cojocaru, Radu; Benkhalfat, Mokhtar; Benchouk, Samia; Hassaine, Hafida; Maiga, Ibrahim; Diallo, Amadou; Koumaré, Abdel Karim; Ouattara, Kalilou; Soumaré, Sambou; Dufourcq, Jean-Baptiste; Nareth, Chhor; Sarthou, Jean-Louis; Andremont, Antoine; Ruimy, Raymond

2009-02-01

In staphylococci, methicillin (meticillin) resistance (MR) is mediated by the acquisition of the mecA gene, which is carried on the size and composition variable staphylococcal cassette chromosome mec (SCCmec). MR has been extensively studied in Staphylococcus aureus, but little is known about MR coagulase-negative staphylococci (MR-CoNS). Here, we describe the diversity of SCCmec structures in MR-CoNS from outpatients living in countries with contrasting environments: Algeria, Mali, Moldova, and Cambodia. Their MR-CoNS nasal carriage rates were 29, 17, 11, and 31%, respectively. Ninety-six MR-CoNS strains, comprising 75 (78%) Staphylococcus epidermidis strains, 19 (20%) Staphylococcus haemolyticus strains, 1 (1%) Staphylococcus hominis strain, and 1 (1%) Staphylococcus cohnii strain, were analyzed. Eighteen different SCCmec types were observed, with 28 identified as type IV (29%), 25 as type V (26%), and 1 as type III (1%). Fifteen strains (44%) were untypeable for their SCCmec. Thirty-four percent of MR-CoNS strains contained multiple ccr copies. Type IV and V SCCmec were preferentially associated with S. epidermidis and S. haemolyticus, respectively. MR-CoNS constitute a widespread and highly diversified MR reservoir in the community.

Novel staphylococcal species that form part of a Staphylococcus aureus-related complex: the non-pigmented Staphylococcus argenteus sp. nov. and the non-human primate-associated Staphylococcus schweitzeri sp. nov.

PubMed

Tong, Steven Y C; Schaumburg, Frieder; Ellington, Matthew J; Corander, Jukka; Pichon, Bruno; Leendertz, Fabian; Bentley, Stephen D; Parkhill, Julian; Holt, Deborah C; Peters, Georg; Giffard, Philip M

2015-01-01

We define two novel species of the genus Staphylococcus that are phenotypically similar to and have near identical 16S rRNA gene sequences to Staphylococcus aureus. However, compared to S. aureus and each other, the two species, Staphylococcus argenteus sp. nov. (type strain MSHR1132(T) = DSM 28299(T) = SSI 89.005(T)) and Staphylococcus schweitzeri sp. nov. (type strain FSA084(T) = DSM 28300(T) = SSI 89.004(T)), demonstrate: 1) at a whole-genome level considerable phylogenetic distance, lack of admixture, average nucleotide identity <95 %, and inferred DNA-DNA hybridization <70 %; 2) different profiles as determined by MALDI-TOF MS; 3) a non-pigmented phenotype for S. argenteus sp. nov.; 4) S. schweitzeri sp. nov. is not detected by standard nucA PCR; 5) distinct peptidoglycan types compared to S. aureus; 6) a separate ecological niche for S. schweitzeri sp. nov.; and 7) a distinct clinical disease profile for S. argenteus sp. nov. compared to S. aureus. © 2015 IUMS.

Regulation of Expression of abcA and Its Response to Environmental Conditions

PubMed Central

Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

2014-01-01

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters.

PubMed

Kennedy, Jonathan; Baker, Paul; Piper, Clare; Cotter, Paul D; Walsh, Marcella; Mooij, Marlies J; Bourke, Marie B; Rea, Mary C; O'Connor, Paula M; Ross, R Paul; Hill, Colin; O'Gara, Fergal; Marchesi, Julian R; Dobson, Alan D W

2009-01-01

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.

Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

PubMed Central

Zeaki, Nikoleta; Budi Susilo, Yusak; Pregiel, Anna; Rådström, Peter; Schelin, Jenny

2015-01-01

The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards. PMID:26690218

Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification

PubMed Central

Shen, Jiangchuan; Keithly, Mary E.; Armstrong, Richard N.; Higgins, Khadine A.; Edmonds, Katherine A.; Giedroc, David P.

2016-01-01

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus (S. aureus) and is under the transcriptional control of the persulfide sensor CstR and H2S. Here we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (Ethylmalonic Encephalopathy Protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both activities require Cys408. CstB protects S. aureus against H2S toxicity with C201S and C408S cstB genes unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts the cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon the elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S stress, in concert with the other enzymes encoded by the cst operon. PMID:26177047

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

PubMed

Nydam, Marie L; Netuschil, Nikolai; Sanders, Erin; Langenbacher, Adam; Lewis, Daniel D; Taketa, Daryl A; Marimuthu, Arumugapradeep; Gracey, Andrew Y; De Tomaso, Anthony W

2013-01-01

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

PubMed

Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

2016-12-01

Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Slime-producing Staphylococcus epidermidis and S. aureus in acute bacterial conjunctivitis in soft contact lens wearers.

PubMed

Catalanotti, Piergiorgio; Lanza, Michele; Del Prete, Antonio; Lucido, Maria; Catania, Maria Rosaria; Gallè, Francesca; Boggia, Daniela; Perfetto, Brunella; Rossano, Fabio

2005-10-01

In recent years, an increase in ocular pathologies related to soft contact lens has been observed. The most common infectious agents were Staphylococcus spp. Some strains produce an extracellular polysaccharidic slime that can cause severe infections. Polysaccharide synthesis is under genetic control and involves a specific intercellular adhesion (ica) locus, in particular, icaA and icaD genes. Conjunctival swabs from 97 patients with presumably bacterial bilateral conjunctivitis, wearers of soft contact lenses were examined. We determined the ability of staphylococci to produce slime, relating it to the presence of icaA and icaD genes. We also investigated the antibiotic susceptibility and Pulsed Field Gel Electrophoresis (PFGE) patterns of the clinical isolates. We found that 74.1% of the S. epidermidis strains and 61.1% of the S. aureus strains isolated were slime producers and showed icaA and icaD genes. Both S. epidermidis and S. aureus slime-producing strains exhibited more surface hydrophobicity than non-producing slime strains. The PFGE patterns overlapped in S. epidermidis strains with high hydrophobicity. The similar PFGE patterns were not related to biofilm production. We found scarce matching among the Staphylococcus spp. studied, slime production, surface hydrophobicity and antibiotic susceptibility.

Fitness and competitive growth comparison of methicillin resistant and methicillin susceptible Staphylococcus aureus colonies.

PubMed

Durhan, Emine; Korcan, Safiye Elif; Altindis, Mustafa; Konuk, Muhsin

2017-05-01

Exponential developments of both Methicillin resistant Staphylococcus aureus (MRSA) 3R ve 36R and methicillin susceptible Staphylococcus aureus (MSSA) 27S were evaluated in the presence and absence of oxacillin. The strains were isolated from the specimens collected in microbiology department. It was also determined the transfer of mecA gene from 3R to 27S strain by using the replica plate technique. It was observed that the presence of antibiotics in the preliminary culture had a positive impact on the growth of the secondary culture of MRSA isolates. Comparison results of Rt bacteria in three different mixed cultures, assessed with Tukey's HSD test, showed a significant statistical difference among the groups. The values were as following; on the first day; Df: 2, F: 60.90, P: 0.0001, second day; Df:2, F:90.56, P: 0.0000, and third day; Df:2, F:4.86, P:0.0557. As a result of the study, we can suggest that the gene expression levels of the transferred antibiotic resistance genes could help us in both controlling hospital originated sickness and developing new strategies to prevent the spread of resistant bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat[W][OA

PubMed Central

Mugford, Sam T.; Louveau, Thomas; Melton, Rachel; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Tsurushima, Tetsu; Honkanen, Suvi; Rosser, Susan J.; Lomonossoff, George P.; Osbourn, Anne

2013-01-01

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis. PMID:23532069

Detection of oxacillin-susceptible mecA-positive Staphylococcus aureus isolates by use of chromogenic medium MRSA ID.

PubMed

Kumar, V Anil; Steffy, Katherin; Chatterjee, Maitrayee; Sugumar, Madhan; Dinesh, Kavitha R; Manoharan, Anand; Karim, Shamsul; Biswas, Raja

2013-01-01

Reports of oxacillin-susceptible mecA-positive Staphylococcus aureus strains are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using routine phenotypic methods. We describe two such isolates that were detected by chromogenic medium and confirmed by characterization of the mecA gene element.

Presence of the optrA Gene in Methicillin-Resistant Staphylococcus sciuri of Porcine Origin.

PubMed

Fan, Run; Li, Dexi; Wang, Yang; He, Tao; Feßler, Andrea T; Schwarz, Stefan; Wu, Congming

2016-12-01

A total of 57 methicillin-resistant Staphylococcus aureus (MRSA) isolates and 475 methicillin-resistant coagulase-negative staphylococci (MRCoNS) collected from pigs in the Guangdong province of China in 2014 were investigated for the presence of the novel oxazolidinone-phenicol resistance gene optrA The optrA gene was detected in 6.9% (n = 33) of the MRCoNS, all of which were Staphylococcus sciuri isolates, but in none of the MRSA isolates. Five optrA-carrying methicillin-resistant (MR) S. sciuri isolates also harbored the multiresistance gene cfr Pulsed-field gel electrophoresis (PFGE) and dru typing of the 33 optrA-carrying MR S. sciuri isolates revealed 25 patterns and 5 sequence types, respectively. S1 nuclease PFGE and Southern blotting confirmed that optrA was located in the chromosomal DNAs of 29 isolates, including 1 cfr-positive isolate. The remaining four isolates harbored a ∼35-kb pWo28-3-like plasmid on which optrA and cfr were located together with other resistance genes, as confirmed by sequence analysis. Six different types of genetic environments (types I to VI) of the chromosome-borne optrA genes were identified; these types had the optrA gene and its transcriptional regulator araC in common. Tn558 was found to be associated with araC-optrA in types II to VI. The optrA gene in types II and III was found in close proximity to the ccr gene complex of the respective staphylococcal cassette chromosome mec element (SCCmec). Since oxazolidinones are last-resort antimicrobial agents for the control of serious infections caused by methicillin-resistant staphylococci in humans, the location of the optrA gene close to the ccr complex is an alarming observation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

PubMed Central

Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

1989-01-01

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae

PubMed Central

Choury, Danièle; Aubert, Gérald; Szajnert, Marie-France; Azibi, Kemal; Delpech, Marc; Paul, Gérard

1999-01-01

A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new β-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB β-lactamases and to SAR-1, another carbenicillin-hydrolyzing β-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This β-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of the Staphylococcus aureus PC-1 β-lactamase. PMID:9925522

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Genome-Wide Architecture of Disease Resistance Genes in Lettuce

PubMed Central

Christopoulou, Marilena; Wo, Sebastian Reyes-Chin; Kozik, Alex; McHale, Leah K.; Truco, Maria-Jose; Wroblewski, Tadeusz; Michelmore, Richard W.

2015-01-01

Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes. PMID:26449254

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

Staphylococcus argensis sp. nov., a novel staphylococcal species isolated from an aquatic environment.

PubMed

Heß, Stefanie; Gallert, Claudia

2015-08-01

A staphylocoagulase-negative, novobiocin-susceptible strain (M4S-6T) of a species of the genus Staphylococcus was isolated from the river Argen in Southern Germany. It was assigned to the genus Staphylococcus due to the presence of the fatty acids, ai-C15 : 0, i-C15 : 0, i-C17 : 0, ai-C17 : 0, and of menaquinone (MK-7) in the cytoplasmic membrane, which are typical of coagulase-negative staphylococci. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. Although the 16S gene sequence of strain M4S-6T revealed a 98% similarity with its closest relative, Staphylococcus pettenkoferi, it could be distinguished by several phenotypical and physiological markers. In contrast to S. pettenkoferi, M4S-6T was ornithine decarboxylase-positive, urease-negative and could use formiate and l-histidine as carbon-sources; nitrate was not reduced. Whereas S. pettenkoferi could grow with d(-)-mannitol, d-sorbitol, gluconic acid, l-proline, carboxymethylcellulose and lignosulfonate, M4S-6T was not able to grow with these substances. The results of 16S rRNA gene sequence analysis and of phenotypic testing indicated that M4S-6T was a representative of a novel species for which the name Staphylococcus argensis sp. nov., is proposed with the type strain M4S-6T (DSM 29875T = CIP 110904T).

Characterization of Staphylococcus species isolated from raw milk and milk products (lben and jben) in North Morocco.

PubMed

Bendahou, Abdrezzak; Lebbadi, Mariam; Ennanei, Latifa; Essadqui, Fatima Z; Abid, Mohammed

2008-06-01

To investigate the incidence and antibiotic resistance of staphylococcal strains isolated from milk and milk products and to trace the ecological origin of the Staphylococcus aureus isolated. Eighty-one samples of raw milk, lben (whey) and jben (cheese) were analyzed for the presence of staphylococcal strains. Isolates were identified by Gram stains, tests for coagulase, the API staph system and the WalkAway 40/96, which also determines the antimicrobial susceptibility profiles. The S. aureus strains were biotyped, and variable regions of the coagulase gene were amplified using the polymerase chain reaction. The identification results showed a predominance of coagulase-negative staphylococci (54 %). Coagulase-positive staphylococci that were identified were divided into 3 groups comprising S. aureus (40%), Staphylococcus intermedius (2 %) and Staphylococcus hyicus (4%). Among the S. aureus that was isolated, biotype C was the predominant biotype. Among 40 coagulase gene PCR-amplification products, 37 produced a single band, while 3 isolates produced two bands. The antimicrobial susceptibility-profile of the staphylococcal strains revealed a high incidence of S. aureus to penicillin G. In addition, Staphylococcus lentus presented considerable resistance to the oxacillin, erythromycin and lincomycin. The presence of staphylococci in raw milk, lben and jben in areas of northern Morocco poses a health hazard, so it is necessary for the public health inspectors to properly examine the conditions during production, storage and commercialization of all products made with unpasteurized milk.

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Comparative analysis of the virulence characteristics of epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from Chinese children: ST59 MRSA highly expresses core gene-encoded toxin.

PubMed

Li, Shipeng; Sun, Jing; Zhang, Jianzhong; Li, Xiangmei; Tao, Xiaoxia; Wang, Lijuan; Sun, Mingjiao; Liu, Yingchao; Li, Juan; Qiao, Yanhong; Yu, Sangjie; Yao, Kaihu; Yang, Yonghong; Shen, Xuzhuang

2014-02-01

This study aims to investigate the prevalence of a novel cell wall-anchored protein gene, sasX, and to obtain information on the genetic basis for the pathogenic potential of the MRSA strains isolated from Chinese children. The molecular and virulence characteristics of the clinical strains were analyzed. Twenty-two sequence types (STs) were obtained, with six epidemic clones ST59, ST239, ST1, ST910, ST88, and ST338 accounting for 35.8, 22, 6.6, 6.6, 5.3, and 4.1% respectively. The expression levels of hla, psmα, and RNAIII were higher in ST59 than in other STs (p < 0.05). The sasX gene was detected in 26 (10.7%) MRSA isolates. ST239-MRSA-SCCmecIII-t037 (61.5%) was the predominant sasX-positive MRSA clone. The expressions of PSMα and RNAIII were higher in sasX-positive ST239 isolates than in sasX-negative ST239 ones (p < 0.01). Notably, the percentage of invasive infection in infections caused by sasX-positive ST239 MRSA was higher than that by sasX-negative ST239 MRSA (p = 0.008). This study indicated that ST59 was the predominant clone in the MRSA isolates obtained from Chinese children and might have stronger pathogenic potential. The prevalence of the sasX gene in the MRSA isolates from children was relatively low. Furthermore, the sasX gene might be related to the expressions of PSMα and RNAIII and infection invasiveness. © 2013 APMIS Published by John Wiley & Sons Ltd.

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus

PubMed Central

Luong, Thanh T.; Dunman, Paul M.; Murphy, Ellen; Projan, Steven J.; Lee, Chia Y.

2006-01-01

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus. PMID:16484201

Study on Prevalence, Antibiotic Susceptibility, and tuf Gene Sequence-Based Genotyping of Species-Level of Coagulase-Negative Staphylococcus Isolated From Keratitis Caused by Using Soft Contact Lenses.

PubMed

Faghri, Jamshid; Zandi, Alireza; Peiman, Alireza; Fazeli, Hossein; Esfahani, Bahram Nasr; Safaei, Hajieh Ghasemian; Hosseini, Nafiseh Sadat; Mobasherizadeh, Sina; Sedighi, Mansour; Burbur, Samaneh; Oryan, Golfam

2016-03-01

To study on antibiotic susceptibility and identify coagulase-negative Staphylococcus (CoNS) species based on tuf gene sequencing from keratitis followed by using soft contact lenses in Isfahan, Iran, 2013. This study examined 77 keratitis cases. The samples were cultured and the isolation of CoNS was done by phenotypic tests, and in vitro sensitivity testing was done by Kirby-Bauer disk diffusion susceptibility method. Thirty-eight of isolates were conveniently identified as CoNS. In this study, 27 (71.1%), 21 (55.3%), and 16 (42.1%) were resistant to penicillin, erythromycin, and tetracycline, respectively. One hundred percent of isolates were sensitive to gentamicin, and 36 (94.7%) and 33 (86.8%) of isolates were sensitive to chloramphenicol and ciprofloxacin, respectively. Also, resistances to cefoxitin were 7 (18.4%). Analysis of tuf gene proved to be discriminative and sensitive in which all the isolates were identified with 99.0% similarity to reference strains, and Staphylococcus epidermidis had the highest prevalence among other species. Results of this study showed that CoNS are the most common agents causing contact lens-associated microbial keratitis, and the tuf gene sequencing analysis is a reliable method for distinguishing CoNS species. Also gentamycin, chloramphenicol, and ciprofloxacin are more effective than the other antibacterial agents against these types of bacteria.

Rapid Detection of Bacterial Antibiotic Resistance: Preliminary Evaluation of PCR Assays Targeting Tetracycline Resistance Genes

DTIC Science & Technology

2007-08-01

gonorrheae strain 2309 plasmid pOZ101; AF440277, Lactobacillus plantarum plasmid pMD5057; X75073, Neisseria meningitidis plasmid DNA for tet(M...tetracycline resistance tet(M) gene; AY057892, Staphylococcus aureus strain 1802 tetracycline resistance protein tet(M) gene; AY149596, Lactobacillus sakei

Transfer of mupirocin resistance from Staphylococcus haemolyticus clinical strains to Staphylococcus aureus through conjugative and mobilizable plasmids.

PubMed

Rossi, Ciro C; Ferreira, Natália C; Coelho, Marcus L V; Schuenck, Ricardo P; Bastos, Maria do Carmo de F; Giambiagi-deMarval, Marcia

2016-07-01

Coagulase-negative staphylococci are thought to act as reservoirs of antibiotic resistance genes that can be transferred to Staphylococcus aureus, thus hindering the combat of this bacterium. In this work, we analyzed the presence of plasmids conferring resistance to the antibiotic mupirocin-widely used to treat and prevent S. aureus infections in hospital environments-in nosocomial S. haemolyticus strains. About 12% of the 75 strains tested were resistant to mupirocin, and this phenotype was correlated with the presence of plasmids. These plasmids were shown to be diverse, being either conjugative or mobilizable, and capable of transferring mupirocin resistance to S. aureus Our findings reinforce that S. haemolyticus, historically and mistakenly considered as a less important pathogen, is a reservoir of resistance genes which can be transferred to other bacteria, such as S. aureus, emphasizing the necessity of more effective strategies to detect and combat this emergent opportunistic pathogen. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.