Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

PubMed

Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

2018-05-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi?

PubMed

Kiraz, Nuri; Oz, Yasemin; Aslan, Huseyin; Erturan, Zayre; Ener, Beyza; Akdagli, Sevtap Arikan; Muslumanoglu, Hamza; Cetinkaya, Zafer

2015-10-01

Although conventional identification of pathogenic fungi is based on the combination of tests evaluating their morphological and biochemical characteristics, they can fail to identify the less common species or the differentiation of closely related species. In addition these tests are time consuming, labour-intensive and require experienced personnel. We evaluated the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeast and 160 moulds in our clinical mycology laboratory. While the yeast isolates were identified at species level with 100% homology, 102 (63.75%) clinically important mould isolates were identified at species level, 56 (35%) isolates at genus level against fungal sequences existing in DNA databases and two (1.25%) isolates could not be identified. Consequently, Whatman FTA filter matrix technology was a useful method for extraction of fungal DNA; extremely rapid, practical and successful. Sequence analysis strategy of D1-D2 region of the large ribosomal subunit gene was found considerably sufficient in identification to genus level for the most clinical fungi. However, the identification to species level and especially discrimination of closely related species may require additional analysis. © 2015 Blackwell Verlag GmbH.

Draft Genome Sequences of Two Species of "Difficult-to-Identify" Human-Pathogenic Corynebacteria: Implications for Better Identification Tests.

PubMed

Pacheco, Luis G C; Mattos-Guaraldi, Ana L; Santos, Carolina S; Veras, Adonney A O; Guimarães, Luis C; Abreu, Vinícius; Pereira, Felipe L; Soares, Siomar C; Dorella, Fernanda A; Carvalho, Alex F; Leal, Carlos G; Figueiredo, Henrique C P; Ramos, Juliana N; Vieira, Veronica V; Farfour, Eric; Guiso, Nicole; Hirata, Raphael; Azevedo, Vasco; Silva, Artur; Ramos, Rommel T J

2015-01-01

Non-diphtheriae Corynebacterium species have been increasingly recognized as the causative agents of infections in humans. Differential identification of these bacteria in the clinical microbiology laboratory by the most commonly used biochemical tests is challenging, and normally requires additional molecular methods. Herein, we present the annotated draft genome sequences of two isolates of "difficult-to-identify" human-pathogenic corynebacterial species: C. xerosis and C. minutissimum. The genome sequences of ca. 2.7 Mbp, with a mean number of 2,580 protein encoding genes, were also compared with the publicly available genome sequences of strains of C. amycolatum and C. striatum. These results will aid the exploration of novel biochemical reactions to improve existing identification tests as well as the development of more accurate molecular identification methods through detection of species-specific target genes for isolate's identification or drug susceptibility profiling.

Use of the MicroSeq 500 16S rRNA Gene-Based Sequencing for Identification of Bacterial Isolates That Commercial Automated Systems Failed To Identify Correctly

PubMed Central

Fontana, Carla; Favaro, Marco; Pelliccioni, Marco; Pistoia, Enrico Salvatore; Favalli, Cartesio

2005-01-01

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux). However, more and more frequently, microbiologists must isolate “difficult” strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 “difficult” clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems—VITEK 2 and Phoenix—while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory. PMID:15695654

Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Wieme, Anneleen D; Spitaels, Freek; Aerts, Maarten; De Bruyne, Katrien; Van Landschoot, Anita; Vandamme, Peter

2014-08-18

Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS identification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical analysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The remaining isolates clustered separately and were assigned through sequence analysis of protein coding genes either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB species. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combining the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were considered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI-TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry. Copyright © 2014 Elsevier B.V. All rights reserved.

Species-Level Identification of Actinomyces Isolates Causing Invasive Infections: Multiyear Comparison of Vitek MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) to Partial Sequencing of the 16S rRNA Gene.

PubMed

Lynch, T; Gregson, D; Church, D L

2016-03-01

Actinomyces species are uncommon but important causes of invasive infections. The ability of our regional clinical microbiology laboratory to report species-level identification of Actinomyces relied on molecular identification by partial sequencing of the 16S ribosomal gene prior to the implementation of the Vitek MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) system. We compared the use of the Vitek MS to that of 16S rRNA gene sequencing for reliable species-level identification of invasive infections caused by Actinomyces spp. because limited data had been published for this important genera. A total of 115 cases of Actinomyces spp., either alone or as part of a polymicrobial infection, were diagnosed between 2011 and 2014. Actinomyces spp. were considered the principal pathogen in bloodstream infections (n = 17, 15%), in skin and soft tissue abscesses (n = 25, 22%), and in pulmonary (n = 26, 23%), bone (n = 27, 23%), intraabdominal (n = 16, 14%), and central nervous system (n = 4, 3%) infections. Compared to sequencing and identification from the SmartGene Integrated Database Network System (IDNS), Vitek MS identified 47/115 (41%) isolates to the correct species and 10 (9%) isolates to the correct genus. However, the Vitek MS was unable to provide identification for 43 (37%) isolates while 15 (13%) had discordant results. Phylogenetic analyses of the 16S rRNA sequences demonstrate high diversity in recovered Actinomyces spp. and provide additional information to compare/confirm discordant identifications between MALDI-TOF and 16S rRNA gene sequences. This study highlights the diversity of clinically relevant Actinomyces spp. and provides an important typing comparison. Based on our analysis, 16S rRNA gene sequencing should be used to rapidly identify Actinomyces spp. until MALDI-TOF databases are optimized. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Species-Level Identification of Actinomyces Isolates Causing Invasive Infections: Multiyear Comparison of Vitek MS (Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry) to Partial Sequencing of the 16S rRNA Gene

PubMed Central

Gregson, D.; Church, D. L.

2016-01-01

Actinomyces species are uncommon but important causes of invasive infections. The ability of our regional clinical microbiology laboratory to report species-level identification of Actinomyces relied on molecular identification by partial sequencing of the 16S ribosomal gene prior to the implementation of the Vitek MS (matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS]) system. We compared the use of the Vitek MS to that of 16S rRNA gene sequencing for reliable species-level identification of invasive infections caused by Actinomyces spp. because limited data had been published for this important genera. A total of 115 cases of Actinomyces spp., either alone or as part of a polymicrobial infection, were diagnosed between 2011 and 2014. Actinomyces spp. were considered the principal pathogen in bloodstream infections (n = 17, 15%), in skin and soft tissue abscesses (n = 25, 22%), and in pulmonary (n = 26, 23%), bone (n = 27, 23%), intraabdominal (n = 16, 14%), and central nervous system (n = 4, 3%) infections. Compared to sequencing and identification from the SmartGene Integrated Database Network System (IDNS), Vitek MS identified 47/115 (41%) isolates to the correct species and 10 (9%) isolates to the correct genus. However, the Vitek MS was unable to provide identification for 43 (37%) isolates while 15 (13%) had discordant results. Phylogenetic analyses of the 16S rRNA sequences demonstrate high diversity in recovered Actinomyces spp. and provide additional information to compare/confirm discordant identifications between MALDI-TOF and 16S rRNA gene sequences. This study highlights the diversity of clinically relevant Actinomyces spp. and provides an important typing comparison. Based on our analysis, 16S rRNA gene sequencing should be used to rapidly identify Actinomyces spp. until MALDI-TOF databases are optimized. PMID:26739153

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

PubMed

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

2015-10-22

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology

PubMed Central

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K. P.; Woo, Patrick C. Y.

2015-01-01

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10–49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n = 2), Pichia (Candida) norvegensis (n = 2), Candida tropicalis (n = 1) and Saccharomyces cerevisiae (n = 1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study. PMID:26506340

Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil▿

PubMed Central

Uhlik, Ondrej; Strejcek, Michal; Junkova, Petra; Sanda, Miloslav; Hroudova, Miluse; Vlcek, Cestmir; Mackova, Martina; Macek, Tomas

2011-01-01

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases. PMID:21821747

A Systematic Approach for Discovering Novel, Clinically Relevant Bacteria

PubMed Central

Simmon, Keith E.; Fisher, Mark A.

2012-01-01

Sequencing of the 16S rRNA gene (16S) is a reference method for bacterial identification. Its expanded use has led to increased recognition of novel bacterial species. In most clinical laboratories, novel species are infrequently encountered, and their pathogenic potential is often difficult to assess. We reviewed partial 16S sequences from >26,000 clinical isolates, analyzed during February 2006–June 2010, and identified 673 that have <99% sequence identity with valid reference sequences and are thus possibly novel species. Of these 673 isolates, 111 may represent novel genera (<95% identity). Isolates from 95 novel taxa were recovered from multiple patients, indicating possible clinical relevance. Most repeatedly encountered novel taxa belonged to the genera Nocardia (14 novel taxa, 42 isolates) and Actinomyces (12 novel taxa, 52 isolates). This systematic approach for recognition of novel species with potential diagnostic or therapeutic relevance provides a basis for epidemiologic surveys and improvement of sequence databases and may lead to identification of new clinical entities. PMID:22377371

A systematic approach for discovering novel, clinically relevant bacteria.

PubMed

Schlaberg, Robert; Simmon, Keith E; Fisher, Mark A

2012-03-01

Sequencing of the 16S rRNA gene (16S) is a reference method for bacterial identification. Its expanded use has led to increased recognition of novel bacterial species. In most clinical laboratories, novel species are infrequently encountered, and their pathogenic potential is often difficult to assess. We reviewed partial 16S sequences from >26,000 clinical isolates, analyzed during February 2006-June 2010, and identified 673 that have <99% sequence identity with valid reference sequences and are thus possibly novel species. Of these 673 isolates, 111 may represent novel genera (<95% identity). Isolates from 95 novel taxa were recovered from multiple patients, indicating possible clinical relevance. Most repeatedly encountered novel taxa belonged to the genera Nocardia (14 novel taxa, 42 isolates) and Actinomyces (12 novel taxa, 52 isolates). This systematic approach for recognition of novel species with potential diagnostic or therapeutic relevance provides a basis for epidemiologic surveys and improvement of sequence databases and may lead to identification of new clinical entities.

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

PubMed Central

França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

2015-01-01

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

PubMed

França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

2015-01-01

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.

Isolation and identification of a bovine viral diarrhea virus from sika deer in china.

PubMed

Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; Zhang, Pengju; Zhang, Lianxue

2011-02-25

Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China) using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE), indirect immunoperoxidase test (IPX) and electron microscopy(EM). The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV) and 3 strains of border disease virus(BDV) in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

PubMed

de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

First molecular identification and characterization of classical swine fever virus isolates from Nepal.

PubMed

Postel, Alexander; Jha, Vijay C; Schmeiser, Stefanie; Becher, Paul

2013-01-01

Classical swine fever (CSF) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. Here, for the first time, molecular identification and characterization of CSFV isolates from two recent outbreaks in Nepal are presented. Analysis of full-length E2-encoding sequences revealed that these isolates belonged to CSFV subgenotype 2.2 and had highest genetic similarity to isolates from India. Hence, for CSFV, Nepal and India should be regarded as one epidemiological unit. Both Nepalese isolates exhibited significant sequence differences, excluding a direct epidemiological connection and suggesting that CSFV is endemic in that country.

Yeast identification: reassessment of assimilation tests as sole universal identifiers.

PubMed

Spencer, J; Rawling, S; Stratford, M; Steels, H; Novodvorska, M; Archer, D B; Chandra, S

2011-11-01

To assess whether assimilation tests in isolation remain a valid method of identification of yeasts, when applied to a wide range of environmental and spoilage isolates. Seventy-one yeast strains were isolated from a soft drinks factory. These were identified using assimilation tests and by D1/D2 rDNA sequencing. When compared to sequencing, assimilation test identifications (MicroLog™) were 18·3% correct, a further 14·1% correct within the genus and 67·6% were incorrectly identified. The majority of the latter could be attributed to the rise in newly reported yeast species. Assimilation tests alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profiles. Assimilation tests still have a useful role in the identification of common species, such as the majority of clinical isolates. It is probable, based on these results, that many yeast identifications reported in older literature are incorrect. This emphasizes the crucial need for accurate identification in present and future publications. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory.

PubMed

Rodríguez-Sánchez, Belén; Marín, Mercedes; Sánchez-Carrillo, Carlos; Cercenado, Emilia; Ruiz, Adrián; Rodríguez-Créixems, Marta; Bouza, Emilio

2014-05-01

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and identification of a bovine viral diarrhea virus from sika deer in china

PubMed Central

2011-01-01

Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China) using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE), indirect immunoperoxidase test (IPX) and electron microscopy(EM). The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV) and 3 strains of border disease virus(BDV) in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer. PMID:21352530

Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems.

PubMed

Isaksson, Jenny; Rasmussen, Magnus; Nilson, Bo; Stadler, Liselott Svensson; Kurland, Siri; Olaison, Lars; Ek, Elisabeth; Herrmann, Björn

2015-04-01

Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid Identification of Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and C. gattii by Use of Rapid Biochemical Tests, Differential Media, and DNA Sequencing ▿

PubMed Central

McTaggart, Lisa; Richardson, Susan E.; Seah, Christine; Hoang, Linda; Fothergill, Annette; Zhang, Sean X.

2011-01-01

Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp. PMID:21593254

Rapid identification of Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and C. gattii by use of rapid biochemical tests, differential media, and DNA sequencing.

PubMed

McTaggart, Lisa; Richardson, Susan E; Seah, Christine; Hoang, Linda; Fothergill, Annette; Zhang, Sean X

2011-07-01

Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp.

Evaluation of MALDI-TOF MS (Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) for routine identification of anaerobic bacteria.

PubMed

Rodríguez-Sánchez, Belén; Alcalá, Luis; Marín, Mercedes; Ruiz, Adrián; Alonso, Elena; Bouza, Emilio

2016-12-01

Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Peptide de novo sequencing of mixture tandem mass spectra

PubMed Central

Hotta, Stéphanie Yuki Kolbeck; Verano‐Braga, Thiago; Kjeldsen, Frank

2016-01-01

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co‐isolation and thus prone to false identifications. The deconvolution approach matched complementary b‐, y‐ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co‐isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. PMID:27329701

Identification of Group G Streptococcal Isolates from Companion Animals in Japan and Their Antimicrobial Resistance Patterns.

PubMed

Tsuyuki, Yuzo; Kurita, Goro; Murata, Yoshiteru; Goto, Mieko; Takahashi, Takashi

2017-07-24

In this study, we conducted a species-level identification of group G streptococcal (GGS) isolates from companion animals in Japan and analyzed antimicrobial resistance (AMR) patterns. Strains were isolated from sterile and non-sterile specimens collected from 72 animals with clinical signs or symptoms in April-May, 2015. We identified the strain by 16S rRNA sequencing, mass spectrometry (MS), and an automated method based on their biochemical properties. Antimicrobial susceptibility was determined using the broth microdilution method and E-test. AMR determinants (erm(A), erm(B), mef(A), tet(M), tet(O), tet(K), tet(L), and tet(S)) in corresponding resistant isolates were amplified by PCR. The 16S rRNA sequencing identified the GGS species as Streptococcus canis (n = 68), Streptococcus dysgalactiae subsp. equisimilis (n = 3), and S. dysgalactiae subsp. dysgalactiae (n = 1). However, there were discrepancies between the sequencing data and both the MS and automated identification data. MS and the automated biochemical technique identified 18 and 37 of the 68 sequencing-identified S. canis strains, respectively. The AMR rates were 20.8% for tetracycline and 5.6% for clarithromycin, with minimum inhibitory concentrations (MIC) 50 -MIC 90 of 2-64 and ≤ 0.12-0.25μg/mL, respectively. AMR genotyping showed single or combined genotypes: erm(B) or tet(M)-tet(O)-tet(S). Our findings show the unique characteristics of GGS isolates from companion animals in Japan in terms of species-level identification and AMR patterns.

Identification of Clinical Coryneform Bacterial Isolates: Comparison of Biochemical Methods and Sequence Analysis of 16S rRNA and rpoB Genes▿

PubMed Central

Adderson, Elisabeth E.; Boudreaux, Jan W.; Cummings, Jessica R.; Pounds, Stanley; Wilson, Deborah A.; Procop, Gary W.; Hayden, Randall T.

2008-01-01

We compared the relative levels of effectiveness of three commercial identification kits and three nucleic acid amplification tests for the identification of coryneform bacteria by testing 50 diverse isolates, including 12 well-characterized control strains and 38 organisms obtained from pediatric oncology patients at our institution. Between 33.3 and 75.0% of control strains were correctly identified to the species level by phenotypic systems or nucleic acid amplification assays. The most sensitive tests were the API Coryne system and amplification and sequencing of the 16S rRNA gene using primers optimized for coryneform bacteria, which correctly identified 9 of 12 control isolates to the species level, and all strains with a high-confidence call were correctly identified. Organisms not correctly identified were species not included in the test kit databases or not producing a pattern of reactions included in kit databases or which could not be differentiated among several genospecies based on reaction patterns. Nucleic acid amplification assays had limited abilities to identify some bacteria to the species level, and comparison of sequence homologies was complicated by the inclusion of allele sequences obtained from uncultivated and uncharacterized strains in databases. The utility of rpoB genotyping was limited by the small number of representative gene sequences that are currently available for comparison. The correlation between identifications produced by different classification systems was poor, particularly for clinical isolates. PMID:18160450

Performance evaluation of three automated identification systems in detecting carbapenem-resistant Enterobacteriaceae.

PubMed

He, Qingwen; Chen, Weiyuan; Huang, Liya; Lin, Qili; Zhang, Jingling; Liu, Rui; Li, Bin

2016-06-21

Carbapenem-resistant Enterobacteriaceae (CRE) is prevalent around the world. Rapid and accurate detection of CRE is urgently needed to provide effective treatment. Automated identification systems have been widely used in clinical microbiology laboratories for rapid and high-efficient identification of pathogenic bacteria. However, critical evaluation and comparison are needed to determine the specificity and accuracy of different systems. The aim of this study was to evaluate the performance of three commonly used automated identification systems on the detection of CRE. A total of 81 non-repetitive clinical CRE isolates were collected from August 2011 to August 2012 in a Chinese university hospital, and all the isolates were confirmed to be resistant to carbapenems by the agar dilution method. The potential presence of carbapenemase genotypes of the 81 isolates was detected by PCR and sequencing. Using 81 clinical CRE isolates, we evaluated and compared the performance of three automated identification systems, MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, which are commonly used in China. To identify CRE, the comparator methodology was agar dilution method, while the PCR and sequencing was the comparator one to identify CPE. PCR and sequencing analysis showed that 48 of the 81 CRE isolates carried carbapenemase genes, including 23 (28.4 %) IMP-4, 14 (17.3 %) IMP-8, 5 (6.2 %) NDM-1, and 8 (9.9 %) KPC-2. Notably, one Klebsiella pneumoniae isolate produced both IMP-4 and NDM-1. One Klebsiella oxytoca isolate produced both KPC-2 and IMP-8. Of the 81 clinical CRE isolates, 56 (69.1 %), 33 (40.7 %) and 77 (95.1 %) were identified as CRE by MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, respectively. The sensitivities/specificities of MicroScan WalkAway, Phoenix 100 and Vitek 2 were 93.8/42.4 %, 54.2/66.7 %, and 75.0/36.4 %, respectively. The MicroScan WalkAway and Viteck2 systems are more reliable in clinical identification of CRE, whereas additional tests are required for the Pheonix 100 system. Our study provides a useful guideline for using automated identification systems for CRE identification.

Targeting Conserved Genes in Penicillium Species.

PubMed

Peterson, Stephen W

2017-01-01

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of dideoxynucleotide-labeled fragments or NGS. The sequences are compared to a database of validated isolates. Identification of species indicates the potential of the fungus to make particular mycotoxins.

Conventional Morphology Versus PCR Sequencing, rep-PCR, and MALDI-TOF-MS for Identification of Clinical Aspergillus Isolates Collected Over a 2-Year Period in a University Hospital at Kayseri, Turkey.

PubMed

Atalay, Altay; Koc, Ayse Nedret; Suel, Ahmet; Sav, Hafize; Demir, Gonca; Elmali, Ferhan; Cakir, Nuri; Seyedmousavi, Seyedmojtaba

2016-09-01

Aspergillus species cause a wide range of diseases in humans, including allergies, localized infections, or fatal disseminated diseases. Rapid detection and identification of Aspergillus spp. facilitate effective patient management. In the current study we compared conventional morphological methods with PCR sequencing, rep-PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification of Aspergillus strains. A total of 24 consecutive clinical isolates of Aspergillus were collected during 2012-2014. Conventional morphology and rep-PCR were performed in our Mycology Laboratory. The identification, evaluation, and reporting of strains using MALDI-TOF-MS were performed by BioMérieux Diagnostic, Inc. in Istanbul. DNA sequence analysis of the clinical isolates was performed by the BMLabosis laboratory in Ankara. Samples consisted of 18 (75%) lower respiratory tract specimens, 3 otomycosis (12.5%) ear tissues, 1 sample from keratitis, and 1 sample from a cutaneous wound. According to DNA sequence analysis, 12 (50%) specimens were identified as A. fumigatus, 8 (33.3%) as A. flavus, 3 (12.5%) as A. niger, and 1 (4.2%) as A. terreus. Statistically, there was good agreement between the conventional morphology and rep-PCR and MALDI-TOF methods; kappa values were κ = 0.869, 0.871, and 0.916, respectively (P < 0.001). The good level of agreement between the methods included in the present study and sequence method could be due to the identification of Aspergillus strains that were commonly encountered. Therefore, it was concluded that studies conducted with a higher number of isolates, which include other Aspergillus strains, are required. © 2016 Wiley Periodicals, Inc.

Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

PubMed

Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

2015-06-01

Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

PubMed

Pryce, Todd M; Palladino, Silvano; Price, Diane M; Gardam, Dianne J; Campbell, Peter B; Christiansen, Keryn J; Murray, Ronan J

2006-04-01

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

Peptide de novo sequencing of mixture tandem mass spectra.

PubMed

Gorshkov, Vladimir; Hotta, Stéphanie Yuki Kolbeck; Verano-Braga, Thiago; Kjeldsen, Frank

2016-09-01

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20-35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions

PubMed Central

Zhao, Jun; Kong, Fanrong; Li, Ruoyu; Wang, Xiaohong; Wan, Zhe; Wang, Duanli

2001-01-01

Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens. PMID:11376067

Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS.

PubMed

Pavlovic, Melanie; Wudy, Corinna; Zeller-Peronnet, Veronique; Maggipinto, Marzena; Zimmermann, Pia; Straubinger, Alix; Iwobi, Azuka; Märtlbauer, Erwin; Busch, Ulrich; Huber, Ingrid

2015-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.

Molecular identification of unusual Mycetoma agents isolated from patients in Venezuela.

PubMed

Rojas, Olga C; León-Cachón, Rafael B R; Moreno-Treviño, Maria; González, Gloria M

2017-02-01

Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques. © 2016 Blackwell Verlag GmbH.

Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2 ▿

PubMed Central

Borman, Andrew M.; Linton, Christopher J.; Oliver, Debra; Palmer, Michael D.; Szekely, Adrien; Johnson, Elizabeth M.

2010-01-01

Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures. PMID:20702674

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish

PubMed Central

Ryan, Sean; Willer, Jason; Marjoram, Lindsay; Bagwell, Jennifer; Mankiewicz, Jamie; Leshchiner, Ignaty; Goessling, Wolfram; Bagnat, Michel; Katsanis, Nicholas

2013-01-01

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans. PMID:24130329

Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China*

PubMed Central

Xie, Liu; Zhang, Jing-ze; Wan, Yao; Hu, Dong-wei

2010-01-01

Strawberry anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry. This study identifies 31 isolates of Colletotrichum spp. which cause strawberry anthracnose in Zhejiang Province and Shanghai City, China. Eleven isolates were identified as C. acutatum, 10 as C. gloeosporioides and 10 as C. fragariae based on morphological characteristics, phylogenetic and sequence analyses. Species-specific polymerase chain reaction (PCR) and enzyme digestion further confirmed the identification of the Colletotrichum spp., demonstrating that these three species are currently the causal agents of strawberry anthracnose in the studied regions. Based on analysis of rDNA internal transcribed spacers (ITS) sequences, sequences of all C. acutatum were identical, and little genetic variability was observed between C. fragariae and C. gloeosporioides. However, the conservative nature of the MvnI specific site from isolates of C. gloeosporioides was confirmed, and this site could be used to differentiate C. gloeosporioides from C. fragariae. PMID:20043353

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability.

PubMed

Yazdani, D; Zainal Abidin, M A; Tan, Y H; Kamaruzaman, S

2011-01-01

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

PubMed

Fadda, M E; Pisano, M B; Scaccabarozzi, L; Mossa, V; Deplano, M; Moroni, P; Liciardi, M; Cosentino, S

2013-01-01

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genomics dataset of unidentified disclosed isolates.

PubMed

Rekadwad, Bhagwan N

2016-09-01

Analysis of DNA sequences is necessary for higher hierarchical classification of the organisms. It gives clues about the characteristics of organisms and their taxonomic position. This dataset is chosen to find complexities in the unidentified DNA in the disclosed patents. A total of 17 unidentified DNA sequences were thoroughly analyzed. The quick response codes were generated. AT/GC content of the DNA sequences analysis was carried out. The QR is helpful for quick identification of isolates. AT/GC content is helpful for studying their stability at different temperatures. Additionally, a dataset on cleavage code and enzyme code studied under the restriction digestion study, which helpful for performing studies using short DNA sequences was reported. The dataset disclosed here is the new revelatory data for exploration of unique DNA sequences for evaluation, identification, comparison and analysis.

Comparison of 16S rRNA sequencing with biochemical testing for species-level identification of clinical isolates of Neisseria spp.

PubMed

Mechergui, Arij; Achour, Wafa; Ben Hassen, Assia

2014-08-01

We aimed to compare accuracy of genus and species level identification of Neisseria spp. using biochemical testing and 16S rRNA sequence analysis. These methods were evaluated using 85 Neisseria spp. clinical isolates initially identified to the genus level by conventional biochemical tests and API NH system (Bio-Mérieux(®)). In 34 % (29/85), more than one possibility was given by 16S rRNA sequence analysis. In 6 % (5/85), one of the possibilities offered by 16S rRNA gene sequencing, agreed with the result given by biochemical testing. In 4 % (3/85), the same species was given by both methods. 16S rRNA gene sequencing results did not correlate well with biochemical tests.

Complete Genome Sequence of Enterococcus faecalis Strain W11 Isolated from an Algal Food Product

PubMed Central

Takizawa, Noboru

2016-01-01

Here, we report the complete genome sequence of Enterococcus faecalis strain W11 isolated from an algal food product in Japan. This study should facilitate the identification of a novel mechanism of glycerol metabolic control in lactic acid bacteria. PMID:27688337

Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

PubMed Central

Garbaj, Aboubaker M.; Awad, Enas M.; Azwai, Salah M.; Abolghait, Said K.; Naas, Hesham T.; Moawad, Ashraf A.; Gammoudi, Fatim T.; Barbieri, Ilaria; Eldaghayes, Ibrahim M.

2016-01-01

Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow’s milk (11%), 3 isolates from she-camel’s milk (11%), two isolates from goat’s milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya. PMID:27956766

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

PubMed

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

2013-01-01

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

Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

PubMed

Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W

2005-04-01

Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

Diagnostic accuracy of a standardized scheme for identification of Streptococcus uberis in quarter milk samples: A comparison between conventional bacteriological examination, modified Rambach agar medium culturing, and 16S rRNA gene sequencing.

PubMed

Wald, Regina; Baumgartner, Martina; Urbantke, Verena; Stessl, Beatrix; Wittek, Thomas

2017-02-01

Bacteriological examination of milk samples is a prerequisite for pathogen-specific therapy and aids in limiting antimicrobial resistance. The aims of this study were to establish a standardized scheme for reliable Streptococcus uberis identification in routine diagnosis and to evaluate the accuracy of conventional tests and growing patterns of Strep. uberis on a selective medium (modified Rambach agar medium, MRAM) using 16S rRNA gene sequencing analysis as a reference method. We obtained isolates of presumptive Strep. uberis (n = 336) from quarter milk samples of dairy cows with intramammary infections and classified the isolates into 2 clusters using biochemical characterization. In cluster 1 (n = 280), cocci grew as non-hemolytic colonies, hydrolyzing esculin, carrying no Lancefield antigen (A/B/C/D/G) or Christie Atkins Munch-Petersen factor, and their growth was inhibited on an Enterococcus agar. Production of β-d-galactosidase on MRAM was shown by 257 of the cluster 1 isolates (91.79%), and 16S rRNA gene sequencing verified 271 (96.79%) of the isolates to be Strep. uberis. In 264 isolates (94.29%), MRAM agreed with the sequencing results. In cluster 2 (n = 56), isolates showed different characteristics: 37 (66.07%) were β-d-galactosidase-positive, and based on 16S sequencing results, 36 (64.29%) were identified correctly as Strep. uberis using biochemical methods. Identification success in this group differed significantly between routine diagnosis and MRAM application: MRAM agreed with sequencing results in 47 isolates (83.93%). To identify Strep. uberis and differentiate it from other lactic acid bacteria in routine diagnosis, we suggest using catalase reaction, hemolysis, esculin hydrolysis, and growth on enterococci agar. Isolates that show a typical biochemical profile can be identified satisfactorily with these tests. For Strep. uberis isolates with divergent patterns, application of MRAM as a follow-up test increased the diagnostic accuracy to 94.64%. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2012-09-01

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

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

PubMed

Vidal-Acuña, M Reyes; Ruiz-Pérez de Pipaón, Maite; Torres-Sánchez, María José; Aznar, Javier

2017-12-08

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation of an insertion sequence (IS1051) from Xanthomonas campestris pv. dieffenbachiae with potential use for strain identification and characterization.

PubMed Central

Berthier, Y; Thierry, D; Lemattre, M; Guesdon, J L

1994-01-01

A new insertion sequence was isolated from Xanthomonas campestris pv. dieffenbachiae. Sequence analysis showed that this element is 1,158 bp long and has 15-bp inverted repeat ends containing two mismatches. Comparison of this sequence with sequences in data bases revealed significant homology with Escherichia coli IS5. IS1051, which detected multiple restriction fragment length polymorphisms, was used as a probe to characterize strains from the pathovar dieffenbachiae. Images PMID:7906933

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance.

PubMed

Hsieh, Ying-Hsin; Wang, Yun F; Moura, Hercules; Miranda, Nancy; Simpson, Steven; Gowrishankar, Ramnath; Barr, John; Kerdahi, Khalil; Sulaiman, Irshad M

2018-05-01

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Genotypic and Phenotypic Characterization of “Streptococcus milleri” Group Isolates from a Veterans Administration Hospital Population

PubMed Central

Clarridge, Jill E.; Osting, Cheryl; Jalali, Mehri; Osborne, Janet; Waddington, Michael

1999-01-01

Because identification of the species within the “Streptococcus milleri” group is difficult for the clinical laboratory as the species share overlapping phenotypic characteristics, we wished to confirm biochemical identification with identification by 16S rRNA gene sequence analysis. Ninety-four clinical isolates previously identified as the “Streptococcus milleri” group were reclassified as S. anginosus, S. constellatus, or S. intermedius with the API 20 Strep system (bioMerieux Vikek, Hazelton, Mo.) and the Fluo-card (Key Scientific, Round Rock, Tex.). In addition, we determined the Lancefield group, hemolysis, colony size, colony texture, repetitive extragenic palindromic PCR (rep-PCR) pattern, and cellular fatty acid (CFA) profile (MIDI, Newark, Del.). 16S rRNA gene sequence analysis with 40 selected representative strains showed three distinct groups, with S. constellatus and S. intermedius found to be more closely related to each other than to S. anginosus, and further distinguished a biochemically distinct group of urogenital isolates within the S. anginosus group of isolates. Except for strains unreactive with the Fluo-card (8%), all S. anginosus and S. intermedius strains identified by sequencing were similarly identified by biochemical testing. However, 23% of the selected S. constellatus isolates identified by sequencing (9% of all S. constellatus isolates) would have been identified as S. anginosus or S. intermedius by biochemical tests. Although most S. anginosus strains formed one unique cluster by CFA analysis and most S. constellatus strains showed similar rep-PCR patterns, neither method was sufficiently dependable for identification. Whereas Lancefield group or lactose fermentation did not correspond to sequence or biochemical type, S. constellatus was most likely to be beta-hemolytic and S. intermedius was most likely to have a dry colony type. The most frequent isolate in our population was S. constellatus, followed by S. anginosus. There was an association of S. anginosus with a gastrointestinal or urogenital source, and there was an association of S. constellatus and S. intermedius with both the respiratory tract and upper-body abscesses. PMID:10523574

High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

PubMed Central

Tani, Akio; Sahin, Nurettin; Matsuyama, Yumiko; Enomoto, Takashi; Nishimura, Naoki; Yokota, Akira; Kimbara, Kazuhide

2012-01-01

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing. PMID:22808262

Genomic and Proteomic Characterization of Bacteriocin-Producing Leuconostoc mesenteroides Strains Isolated from Raw Camel Milk in Two Southwest Algerian Arid Zones

PubMed Central

Benmechernene, Zineb; Fernández-No, Inmaculada; Quintela-Baluja, Marcos; Kihal, Mebrouk; Calo-Mata, Pilar; Barros-Velázquez, Jorge

2014-01-01

Information on the microbiology of camel milk is very limited. In this work, the genetic characterization and proteomic identification of 13 putative producing bacteriocin Leuconostoc strains exhibiting antilisterial activity and isolated from camel milk were performed. DNA sequencing of the 13 selected strains revealed high homology among the 16S rRNA genes for all strains. In addition, 99% homology with Leuconostoc mesenteroides was observed when these sequences were analysed by the BLAST tool against other sequences from reference strains deposited in the Genbank. Furthermore, the isolates were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF MS) which allowed for the identification of 2 mass peaks 6242 m/z and 5118 m/z that resulted to be specific to the species L. mesenteroides. Remarkably, the phyloproteomic tree provided more intraspecific information of L. mesenteroides than phylogenetic analysis. Accordingly, phyloproteomic analysis grouped L. mesenteroides strains into different subbranches, while all L. mesenteroides isolates were grouped in the same branch according to phylogenetic analysis. This study represents, to our knowledge, the first report on the use of MALDI-TOF MS on the identification of LAB isolated from camel milk. PMID:24809059

Evaluation of protein spectra cluster analysis for Streptococcus spp. identification from various swine clinical samples.

PubMed

Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Doto, Daniela S; Calderaro, Franco F; de Souza, Fernando N; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

2017-03-01

Traditional microbiological methods enable genus-level identification of Streptococcus spp. isolates. However, as the species of this genus show broad phenotypic variation, species-level identification or even differentiation within the genus is difficult. Herein we report the evaluation of protein spectra cluster analysis for the identification of Streptococcus species associated with disease in swine by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 250 S. suis-like isolates obtained from pigs with clinical signs of encephalitis, arthritis, pneumonia, metritis, and urinary or septicemic infection were studied. The isolates came from pigs in different Brazilian states from 2001 to 2014. The MALDI-TOF MS analysis identified 86% (215 of 250) as S. suis and 14% (35 of 250) as S. alactolyticus, S. dysgalactiae, S. gallinaceus, S. gallolyticus, S. gordonii, S. henryi, S. hyointestinalis, S. hyovaginalis, S. mitis, S. oralis, S. pluranimalium, and S. sanguinis. The MALDI-TOF MS identification was confirmed in 99.2% of the isolates by 16S rDNA sequencing, with MALDI-TOF MS misidentifying 2 S. pluranimalium as S. hyovaginalis. Isolates were also tested by a biochemical automated system that correctly identified all isolates of 8 of the 10 species in the database. Neither the isolates of the 3 species not in the database ( S. gallinaceus, S. henryi, and S. hyovaginalis) nor the isolates of 2 species that were in the database ( S. oralis and S. pluranimalium) could be identified. The topology of the protein spectra cluster analysis appears to sustain the species phylogenetic similarities, further supporting identification by MALDI-TOF MS examination as a rapid and accurate alternative to 16S rDNA sequencing.

Yeast species associated with orange juice: evaluation of different identification methods.

PubMed

Arias, Covadonga R; Burns, Jacqueline K; Friedrich, Lorrie M; Goodrich, Renee M; Parish, Mickey E

2002-04-01

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus

PubMed Central

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.

2014-01-01

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. PMID:24972080

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

PubMed

Dourado, Ana Catarina; Alves, Paula I L; Tenreiro, Tania; Ferreira, Eugénio M; Tenreiro, Rogério; Fareleira, Paula; Crespo, M Teresa Barreto

2009-12-01

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

Genome Sequence of Janthinobacterium sp. Strain PAMC 25724, Isolated from Alpine Glacier Cryoconite

PubMed Central

Kim, Su Jin; Shin, Seung Chul; Hong, Soon Gyu; Lee, Yung Mi; Lee, Hyoungseok; Lee, Jungeun

2012-01-01

The draft genome of Janthinobacterium sp. strain PAMC 25724, which is a violacein-producing psychrotolerant bacterium, was determined. The strain was isolated from glacier cryoconite of the Alps mountain permafrost region. The sequence will allow identification and characterization of the genetic determination of its cold-adaptive properties. PMID:22461541

Draft Genome Sequence of Lactobacillus johnsonii Strain 16, Isolated from Mice.

PubMed

Buhnik-Rosenblau, Keren; Danin-Poleg, Yael; Elgavish, Sharona; Kashi, Yechezkel

2015-10-08

Here, we report the genome sequence of Lactobacillus johnsonii, a member of the gut lactobacilli. This draft genome of L. johnsonii strain 16 isolated from C57BL/6J mice enables the identification of bacterial genes responsible for host-specific gut persistence. Copyright © 2015 Buhnik-Rosenblau et al.

Isolating a functionally relevant guild of fungi from the root microbiome of Populus

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

Bonito, Gregory; Hameed, Khalid; Ventura, Rafael

Plant roots interact with a bewilderingly complex community of microbes, including root-associated fungi that are essential for maintaining plant health. To improve understanding of the diversity of fungi in the rhizobiome of Populus deltoides, Populus trichocarpa and co-occurring plant hosts Quercus alba and Pinus taeda, we conducted field and greenhouse studies and sampled, isolated, and characterized the diversity of culturable root-associated fungi on these hosts. Using both general and selective isolation media we obtained more than 1800 fungal isolates from individual surface sterilized root tips. Sequences from the ITS and/or D1– D2 regions of the LSU rDNA were obtained frommore » 1042 of the >1800 pure culture isolates and were compared to accessions in the NCBI nucleotide database and analyzed through phylogenetics for preliminary taxonomic identification. Sequences from these isolates were also compared to 454 sequence datasets obtained directly from the Populus rhizosphere and endosphere. Although most of the ectomycorrhizal taxa known to associate with Populus evaded isolation, many of the abundant sequence types from rhizosphere and endosphere 454 datasets were isolated, including novel species belonging to the Atractiellales. Isolation and identification of key endorrhizal fungi will enable more targeted study of plant-fungal interactions. Genome sequencing is currently underway for a subset of our culture library with the aim of understanding the mechanisms involved in host-endophyte establishment and function. As a result, this diverse culture library of fungal root associates will be a valuable resource for metagenomic research, experimentation and further studies on plant-fungal interactions.« less

Isolating a functionally relevant guild of fungi from the root microbiome of Populus

DOE PAGES

Bonito, Gregory; Hameed, Khalid; Ventura, Rafael; ...

2016-05-27

Plant roots interact with a bewilderingly complex community of microbes, including root-associated fungi that are essential for maintaining plant health. To improve understanding of the diversity of fungi in the rhizobiome of Populus deltoides, Populus trichocarpa and co-occurring plant hosts Quercus alba and Pinus taeda, we conducted field and greenhouse studies and sampled, isolated, and characterized the diversity of culturable root-associated fungi on these hosts. Using both general and selective isolation media we obtained more than 1800 fungal isolates from individual surface sterilized root tips. Sequences from the ITS and/or D1– D2 regions of the LSU rDNA were obtained frommore » 1042 of the >1800 pure culture isolates and were compared to accessions in the NCBI nucleotide database and analyzed through phylogenetics for preliminary taxonomic identification. Sequences from these isolates were also compared to 454 sequence datasets obtained directly from the Populus rhizosphere and endosphere. Although most of the ectomycorrhizal taxa known to associate with Populus evaded isolation, many of the abundant sequence types from rhizosphere and endosphere 454 datasets were isolated, including novel species belonging to the Atractiellales. Isolation and identification of key endorrhizal fungi will enable more targeted study of plant-fungal interactions. Genome sequencing is currently underway for a subset of our culture library with the aim of understanding the mechanisms involved in host-endophyte establishment and function. As a result, this diverse culture library of fungal root associates will be a valuable resource for metagenomic research, experimentation and further studies on plant-fungal interactions.« less

Genetic Characterization of Fasciola Isolates from West Azerbaijan Province Iran Based on ITS1 and ITS2 Sequence of Ribosomal DNA

PubMed Central

GALAVANI, Hossein; GHOLIZADEH, Saber; HAZRATI TAPPEH, Khosrow

2016-01-01

Background: Fascioliasis, caused by Fasciola hepatica and F. gigantica, has medical and economic importance in the world. Molecular approaches comparing traditional methods using for identification and characterization of Fasciola spp. are precise and reliable. The aims of current study were molecular characterization of Fasciola spp. in West Azerbaijan Province, Iran and then comparative analysis of them using GenBank sequences. Methods: A total number of 580 isolates were collected from different hosts in five cities of West Azerbaijan Province, in 2014 from 90 slaughtered cattle (n=50) and sheep (n=40). After morphological identification and DNA extraction, designing specific primer were used to amplification of ITS1, 5.8s and ITS2 regions, 50 samples were conducted to sequence, randomly. Result: Using morphometric characters 99.14% and 0.86% of isolates identified as F. hepatica and F. gigantica, respectively. PCR amplification of 1081 bp fragment and sequencing result showed 100% similarity with F. hepatica in ITS1 (428 bp), 5.8s (158 bp), and ITS2 (366 bp) regions. Sequence comparison among current study sequences and GenBank data showed 98% identity with 11 nucleotide mismatches. However, in phylogenetic tree F. hepatica sequences of West Azerbaijan Province, Iran, were in a close relationship with Iranian, Asian, and African isolates. Conclusions: Only F. hepatica species is distributed among sheep and cattle in West Azerbaijan Province Iran. However, 5 and 6 bp variation in ITS1 and ITS2 regions, respectively, is not enough to separate of Fasciola spp. Therefore, more studies are essential for designing new molecular markers to correct species identification. PMID:27095969

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level.

PubMed

Cherkaoui, Abdessalam; Hibbs, Jonathan; Emonet, Stéphane; Tangomo, Manuela; Girard, Myriam; Francois, Patrice; Schrenzel, Jacques

2010-04-01

Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.

Identification and characterization of Citrus tristeza virus isolates breaking resistance in trifoliate orange in California

USDA-ARS?s Scientific Manuscript database

Most Citrus tristeza virus (CTV) isolates in California are biologically mild and symptomless in commercial cultivars on CTV tolerant rootstocks. However, to better define California CTV isolates showing divergent serological and genetic profiles, selected isolates were subjected to deep sequencing ...

Identification of Lactobacillus from the Saliva of Adult Patients with Caries Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

PubMed Central

Ma, Qingwei; Song, Yeqing; Zhang, Qian; Wang, Xiaoyan; Chen, Feng

2014-01-01

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification. PMID:25166027

Partial sequencing of sodA gene and its application to identification of Streptococcus dysgalactiae subsp. dysgalactiae isolated from farmed fish.

PubMed

Nomoto, R; Kagawa, H; Yoshida, T

2008-01-01

To investigate the difference between Lancefield group C Streptococcus dysgalactiae (GCSD) strains isolated from diseased fish and animals by sequencing and phylogenetic analysis of the sodA gene. The sodA gene of Strep. dysgalactiae strains isolated from fish and animals were amplified and its nucleotide sequences were determined. Although 100% sequence identity was observed among fish GCSD strains, the determined sequences from animal isolates showed variations against fish isolate sequences. Thus, all fish GCSD strains were clearly separated from the GCSD strains of other origin by using phylogenetic tree analysis. In addition, the original primer set was designed based on the determined sequences for specifically amplify the sodA gene of fish GCSD strains. The primer set yield amplification products from only fish GCSD strains. By sequencing analysis of the sodA gene, the genetic divergence between Strep. dysgalactiae strains isolated from fish and mammals was demonstrated. Moreover, an original oligonucletide primer set, which could simply detect the genotype of fish GCSD strains was designed. This study shows that Strep. dysgalactiae isolated from diseased fish could be distinguished from conventional GCSD strains by the difference in the sequence of the sodA gene.

Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species.

PubMed

Ferreira-Paim, Kennio; Ferreira, Thatiana Bragine; Andrade-Silva, Leonardo; Mora, Delio Jose; Springer, Deborah J; Heitman, Joseph; Fonseca, Fernanda Machado; Matos, Dulcilena; Melhem, Márcia Souza Carvalho; Silva-Vergara, Mario León

2014-01-01

Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States. In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region. BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified. Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

USDA-ARS?s Scientific Manuscript database

This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India.

PubMed

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal; Singh, Durg Vijai

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates.

Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India

PubMed Central

Panda, Sasmita; Jena, Smrutiti; Sharma, Savitri; Dhawan, Benu; Nath, Gopal

2016-01-01

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates. PMID:27824930

Isolation and identification of methylobacterium species from the tap water in hospitals in Japan and their antibiotic susceptibility.

PubMed

Furuhata, Katsunori; Kato, Yuko; Goto, Keiichi; Hara, Motonobu; Yoshida, Shin-ichi; Fukuyama, Masafumi

2006-01-01

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90 = 1 microg/ml) and tetracycline (MIC90 = 8 microg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.

PubMed

Hoshino, Tomonori; Fujiwara, Taku; Kilian, Mogens

2005-12-01

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.

Complex multifractal nature in Mycobacterium tuberculosis genome

PubMed Central

Mandal, Saurav; Roychowdhury, Tanmoy; Chirom, Keilash; Bhattacharya, Alok; Brojen Singh, R. K.

2017-01-01

The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences. PMID:28440326

Complex multifractal nature in Mycobacterium tuberculosis genome

NASA Astrophysics Data System (ADS)

Mandal, Saurav; Roychowdhury, Tanmoy; Chirom, Keilash; Bhattacharya, Alok; Brojen Singh, R. K.

2017-04-01

The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences.

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

PubMed

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

2005-07-01

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

Draft Genome Sequence of Pseudomonas sp. Strain Ep R1 Isolated from Echinacea purpurea Roots and Effective in the Growth Inhibition of Human Opportunistic Pathogens Belonging to the Burkholderia cepacia Complex.

PubMed

Maggini, Valentina; Presta, Luana; Miceli, Elisangela; Fondi, Marco; Bosi, Emanuele; Chiellini, Carolina; Fagorzi, Camilla; Bogani, Patrizia; Di Pilato, Vincenzo; Rossolini, Gian Maria; Mengoni, Alessio; Firenzuoli, Fabio; Perrin, Elena; Fani, Renato

2017-05-18

In this announcement, we detail the draft genome sequence of the Pseudomonas sp. strain Ep R1, isolated from the roots of the medicinal plant Echinacea purpurea The elucidation of this genome sequence may allow the identification of genes associated with the production of antimicrobial compounds. Copyright © 2017 Maggini et al.

Draft Genome Sequence of Pseudomonas sp. Strain Ep R1 Isolated from Echinacea purpurea Roots and Effective in the Growth Inhibition of Human Opportunistic Pathogens Belonging to the Burkholderia cepacia Complex

PubMed Central

Maggini, Valentina; Presta, Luana; Miceli, Elisangela; Fondi, Marco; Bosi, Emanuele; Chiellini, Carolina; Fagorzi, Camilla; Bogani, Patrizia; Di Pilato, Vincenzo; Rossolini, Gian Maria; Mengoni, Alessio; Firenzuoli, Fabio; Perrin, Elena

2017-01-01

ABSTRACT In this announcement, we detail the draft genome sequence of the Pseudomonas sp. strain Ep R1, isolated from the roots of the medicinal plant Echinacea purpurea. The elucidation of this genome sequence may allow the identification of genes associated with the production of antimicrobial compounds. PMID:28522712

MALDI-TOF MS for identification of Tsukamurella species: Tsukamurella tyrosinosolvens as the predominant species associated with ocular infections.

PubMed

Teng, Jade L L; Tang, Ying; Wong, Samson S Y; Fong, Jordan Y H; Zhao, Zhe; Wong, Chun-Pong; Chen, Jonathan H K; Ngan, Antonio H Y; Wu, Alan K L; Fung, Kitty S C; Que, Tak-Lun; Lau, Susanna K P; Woo, Patrick C Y

2018-05-09

Although Tsukamurella infections have been increasingly reported in Europe, Asia, America, and Africa, indicating that diseases caused by this group of bacteria are emerging in a global scale, species identification within this genus is difficult in most clinical microbiology laboratories. Recently, we showed that groEL gene sequencing is useful for identification of all existing Tsukamurella species. Nevertheless, PCR sequencing is still considered expensive, time-consuming, and technically demanding, and therefore is yet to be incorporated as a routine identification method in clinical laboratories. Using groEL gene sequencing as the reference method, 60 Tsukamurella isolates were identified as five different Tsukamurella species [T. tyrosinosolvens (n = 31), T. pulmonis (n = 25), T. hongkongensis (n = 2), T. strandjordii (n = 1), and T. sinensis (n = 1)]. The most common source of the patient isolates were the eye (n = 18), sputum (n = 6), and blood (n = 6). None of the 60 isolates were identified correctly to species level by MALDI-TOF MS with the original Bruker database V.6.0.0.0. Using the Bruker database extended with 15 type and reference strains which covered all the currently recognized 11 Tsukamurella species, 59 of the 60 isolates were correctly identified to the species level with score ≥2.0. MALDI-TOF MS should be useful for routine species identification of Tsukamurella in clinical microbiology laboratories after optimization of the database. T. tyrosinosolvens was the most common species observed in patients with Tsukamurella infections and the predominant species associated with ocular infections.

Identification of Acinetobacter seifertii isolated from Bolivian hospitals.

PubMed

Cerezales, Mónica; Xanthopoulou, Kyriaki; Ertel, Julia; Nemec, Alexandr; Bustamante, Zulema; Seifert, Harald; Gallego, Lucia; Higgins, Paul G

2018-06-01

Acinetobacter seifertii is a recently described species that belongs to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. It has been recovered from clinical samples and is sometimes associated with antimicrobial resistance determinants. We present here the case of three A. seifertii clinical isolates which were initially identified as Acinetobacter sp. by phenotypic methods but no identification at the species level was achieved using semi-automated identification methods. The isolates were further analysed by whole genome sequencing and identified as A. seifertii. Due to the fact that A. seifertii has been isolated from serious infections such as respiratory tract and bloodstream infections, we emphasize the importance of correctly identifying isolates of the genus Acinetobacter at the species level to gain a deeper knowledge of their prevalence and clinical impact.

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients

PubMed Central

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; de Freitas, Roseli Santos; Nishikaku, Angela; de Souza, Ana Clara; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-01-01

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species. PMID:28423089

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients.

PubMed

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; Freitas, Roseli Santos de; Nishikaku, Angela; Souza, Ana Clara de; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-04-13

The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

StrainSeeker: fast identification of bacterial strains from raw sequencing reads using user-provided guide trees.

PubMed

Roosaare, Märt; Vaher, Mihkel; Kaplinski, Lauris; Möls, Märt; Andreson, Reidar; Lepamets, Maarja; Kõressaar, Triinu; Naaber, Paul; Kõljalg, Siiri; Remm, Maido

2017-01-01

Fast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees. A tool named StrainSeeker was developed that constructs a list of specific k -mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1-2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k -mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain. StrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker's web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker.

Comparison of internal transcribed spacers and intergenic spacer regions of five common Iranian sheep bursate nematodes.

PubMed

Nabavi, Reza; Conneely, Brendan; McCarthy, Elaine; Good, Barbara; Shayan, Parviz; DE Waal, Theo

2014-09-01

Accurate identification of sheep nematodes is a critical point in epidemiological studies and monitoring of drug resistance in flocks. However, due to a close morphological similarity between the eggs and larval stages of many of these nematodes, such identification is not a trivial task. There are a number of studies showing that molecular targets in ribosomal DNA (Internal transcribed spacer 1, 2 and Intergenic spacer) are suitable for accurate identification of sheep bursate nematodes. The objective of present study was to compare the ITS1, ITS2 and IGS regions of Iranian common bursate nematodes in order to choose best target for specific identification methods. The first and second internal transcribed spacers (ITS1and ITS2) and intergenic spacer (IGS) of the ribosomal DNA (rDNA) of 5 common Iranian bursate nematodes of sheep were sequenced. The sequences of some non-Iranian isolates were used for comparison in order to evaluate the variation in sequence homology between geographically different nematode populations. Comparison of the ITS1 and ITS2 sequences of Iranian nematodes showed greatest similarity among Teladorsagia circumcincta and Marshallagia marshalli of 94% and 88%, respectively. While Trichostrongylus colubriformis and M. marshalli showed the highest homology (99%) in the IGS sequences. Comparison of the spacer sequences of Iranian with non-Iranian isolates showed significantly higher variation in Haemonchus contortus compared to the other species. Both the ITS1 and ITS2 sequences are convenient targets to have species-specific identification of Iranian bursate nematodes. On the other hand the IGS region may be a less suitable molecular target.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

16S rRNA Gene Sequencing, Multilocus Sequence Analysis, and Mass Spectrometry Identification of the Proposed New Species “Clostridium neonatale”

PubMed Central

Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose

2014-01-01

In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, “Clostridium neonatale.” To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies. PMID:25232167

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis in comparison to three other methods for identification of Mycobacterium species.

PubMed

Sajduda, Anna; Martin, Anandi; Portaels, Françoise; Palomino, Juan Carlos

2010-02-01

We developed a scheme for rapid identification of Mycobacterium species using an automated fluorescence capillary electrophoresis instrument. A 441-bp region of the hsp65 gene was examined using PCR-restriction analysis (PRA). The assay was initially evaluated on 38 reference strains. The observed sizes of restriction fragments were consistently smaller than the real sizes for each of the species as deduced from the sequence analysis (mean variance=7bp). Nevertheless, the obtained PRA patterns were highly reproducible and resulted in correct species identifications. A blind test was then successfully performed on 64 test isolates previously characterized by conventional biochemical methods, a commercial INNO-LiPA Mycobacteria assay and/or sequence determination of the 5' end of 16S rRNA gene. A total of 14 of 64 isolates were erroneously identified by conventional methods (78% accuracy). In contrast, PRA performed very well in comparison with the LiPA (89% concordance) and especially with DNA sequencing (93.3% of concordant results). Also, PRA identified seven isolates representing five previously unreported hsp65 alleles. We conclude that hsp65 PRA based on automated capillary electrophoresis is a rapid, simple and reliable method for identification of mycobacteria. Copyright 2010 Elsevier B.V. All rights reserved.

Sequence-Based Identification of Aspergillus, Fusarium, and Mucorales in the Clinical Laboratory: Where Are We and Where Should We Go From Here?

USDA-ARS?s Scientific Manuscript database

Accurate identification of fungal species helps establish or exclude a fungal cause of disease. In the past, clinical microbiology labs were restricted to a limited array of phenotypic criteria for categorizing isolates to the species level. This scenario is shifting in favour of DNA sequence-base...

Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii

PubMed Central

Chen, Ying-Sheng; Liu, Yen-Hung; Teng, Shih-Hua; Liao, Chun-Hsing; Hung, Chien-Ching; Sheng, Wang-Huei; Teng, Lee-Jene; Hsueh, Po-Ren

2015-01-01

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the MALDI Bruker Biotyper system (microflex LT; Bruker Daltonik GmbH, Bremen, Germany), on the identification of 50 isolates of clinically encountered molds, including Penicillium marneffei (n = 28), Paecilomyces species (n = 12), Fusarium solani (n = 6), Rhizopus species (n = 3), and Pseudallescheria boydii (n = 1). The isolates were identified to species levels by sequence analysis of the internal transcribed spacer (ITS) regions using primers ITS1 and ITS4. None of the 28 genetically well characterized isolates of P. marneffei were identified as P. marneffei by MALDI-TOF MS, because P. marneffei was not present in either Bruker general library (DB 5627) or Bruker filamentous fungi library V1.0. However, the rate of accurate identification as P. marneffei (score value ≥ 2.000) was 85.7% based on newly created database from one P. marneffei strain (NTUH-3370) by MALDI Biotyper system. Sequencing analysis of these 22 non-P. marneffei isolates of molds revealed seven Paecilomyces variotii, six F. solani, four Paecilomyces lilacinus, and one each of Paecilomyces sinensis, Rhizopus arrhizus, R. oryzae, R. microspores, and P. boydii. Although all the seven P. variotii isolates, four of the six F. solani, two of the four P. lilacinus, and two of the three isolates of Rhizopus species, and the P. boydii isolate had concordant identification results between MALDI-TOF MS and sequencing analysis, the score values of these isolates were all of <1.700. This study indicated that the MALDI Bruker Biotyper is ineffective for identifying P. marneffei and other unusual molds because of the current database limitations. Therefore, it is necessary to continuously update the MALDI-TOF MS databases. PMID:26217315

Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India

PubMed Central

Peddayelachagiri, Bhavani V.; Paul, Soumya; Nagaraj, Sowmya; Gogoi, Madhurjya; Sripathy, Murali H.; Batra, Harsh V.

2016-01-01

Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/μl of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species. PMID:27632353

First description of atypical furunculosis in freshwater farmed Atlantic salmon, Salmo salar L., in Chile.

PubMed

Godoy, M; Gherardelli, V; Heisinger, A; Fernández, J; Olmos, P; Ovalle, L; Ilardi, P; Avendaño-Herrera, R

2010-05-01

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

PubMed

Yadav, Pragya D; Shete, Anita M; Nyayanit, Dimpal A; Albarino, Cesar G; Jain, Shilpi; Guerrero, Lisa W; Kumar, Sandeep; Patil, Deepak Y; Nichol, Stuart T; Mourya, Devendra T

2018-06-25

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.

Identification of Non-diphtheriae Corynebacterium by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Alatoom, Adnan A.; Cazanave, Charles J.; Cunningham, Scott A.; Ihde, Sherry M.

2012-01-01

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum. PMID:22075579

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

PubMed

Ruppitsch, W; Stöger, A; Indra, A; Grif, K; Schabereiter-Gurtner, C; Hirschl, A; Allerberger, F

2007-03-01

In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

PubMed

Cheng, Keding; Chui, Huixia; Domish, Larissa; Sloan, Angela; Hernandez, Drexler; McCorrister, Stuart; Robinson, Alyssia; Walker, Matthew; Peterson, Lorea A M; Majcher, Miles; Ratnam, Sam; Haldane, David J M; Bekal, Sadjia; Wylie, John; Chui, Linda; Tyler, Shaun; Xu, Bianli; Reimer, Aleisha; Nadon, Celine; Knox, J David; Wang, Gehua

2016-08-01

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. Copyright © 2016 Cheng et al.

Isolation and identification of lactic acid bacteria from koumiss in Eastern Inner Mongolia of China

NASA Astrophysics Data System (ADS)

Bai, Lijuan; Ji, Shujuan

2017-01-01

Koumiss is a traditional fermented dairy product and known as its unique physiological actions. Isolation and identification of LAB in it will yield valuable knowledge. In total, 55 LAB strains were isolated and identified of 12 koumiss samples collected in limited regions of Eastern Inner Mongolia. 16S rRNA sequence analysis results showed that were Lactobacillus helveticus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Enterococcus. durans, Leuconostoc lactis and Leuconostoc mesenteroides. It is benefit to further research on koumiss.

Multicenter study evaluating the Vitek MS system for identification of medically important yeasts.

PubMed

Westblade, Lars F; Jennemann, Rebecca; Branda, John A; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B; Ginocchio, Christine C; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Rychert, Jenna A; Sercia, Linda; Burnham, Carey-Ann D

2013-07-01

The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

Phylogeny of isolates of Prunus necrotic ringspot virus from the Ilarvirus Ringtest and identification of group-specific features.

PubMed

Hammond, R W

2003-06-01

Isolates of Prunus necrotic ringspot virus (PNRSV) were examined to establish the level of naturally occurring sequence variation in the coat protein (CP) gene and to identify group-specific genome features that may prove valuable for the generation of diagnostic reagents. Phylogenetic analysis of a 452 bp sequence of 68 virus isolates, 20 obtained from the European Union Ilarvirus Ringtest held in October 1998, confirmed the clustering of the isolates into three distinct groups. Although no correlation was found between the sequence and host or geographic origin, there was a general trend for severe isolates to cluster into one group. Group-specific features have been identified for discrimination between virus strains.

Short communication: Evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci.

PubMed

Cameron, M; Perry, J; Middleton, J R; Chaffer, M; Lewis, J; Keefe, G P

2018-01-01

This study evaluated MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci (CNS). A total of 861 CNS isolates were used in the study, covering 21 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 804) and the remainder were identified by sequencing of hsp60 (n = 56) and tuf (n = 1). The genotypic identification was considered the gold standard identification. Using a direct transfer protocol and the existing commercial database, MALDI-TOF mass spectrometry showed a typeability of 96.5% (831/861) and an accuracy of 99.2% (824/831). Using a custom reference spectra expanded database, which included an additional 13 in-house created reference spectra, isolates were identified by MALDI-TOF mass spectrometry with 99.2% (854/861) typeability and 99.4% (849/854) accuracy. Overall, MALDI-TOF mass spectrometry using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Utility of MALDI-TOF MS for the identification of anaerobic bacteria].

PubMed

Zárate, Mariela S; Romano, Vanesa; Nievas, Jimena; Smayevsky, Jorgelina

2014-01-01

The analysis by MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) has become a reference method for the identification of microorganisms in Clinical Microbiology. However, data on some groups of microorganisms are still controversial. The aim of this study is to determine the utility of MALDI-TOF MS for the identification of clinical isolates of anaerobic bacteria. One-hundred and six anaerobic bacteria isolates were analyzed by MALDI-TOF MS and by conventional biochemical tests. In those cases where identification by conventional methodology was not applicable or in the face of discordance between sequencing methodologies, 16 S rRNA gene sequence analysis was performed. The conventional method and MALDI-TOF MS agreed at genus and species level by 95.3 %. Concordance in gram-negative bacilli was 91.4% and 100% among gram-positive bacilli; there was also concordance both in the 8 isolates studied in gram-positive cocci and in the single gram-negative cocci included. The data obtained in this study demonstrate that MALDI-TOF MS offers the possibility of adequate identification of anaerobic bacteria. Copyright © 2014 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

Evaluation of the Vitek 2 ANC card for identification of clinical isolates of anaerobic bacteria.

PubMed

Lee, E H L; Degener, J E; Welling, G W; Veloo, A C M

2011-05-01

An evaluation of the Vitek 2 ANC card (bioMérieux, Marcy l'Etoile, France) was performed with 301 anaerobic isolates. Each strain was identified by 16S rRNA gene sequencing, which is considered to be the reference method. The Vitek 2 ANC card correctly identified 239 (79.4%) of the 301 clinical isolates to the genus level, including 100 species that were not represented in the database. Correct species identification was obtained for 60.1% (181/301) of the clinical isolates. For the isolates not identified to the species level, a correct genus identification was obtained for 47.0% of them (47/100), and 16 were accurately designated not identified. Although the Vitek 2 ANC card allows the rapid and acceptable identification of the most common clinically important anaerobic bacteria within 6 h, improvement is required for the identification of members of the genera Fusobacterium, Prevotella, and Actinomyces and certain Gram-positive anaerobic cocci (GPAC).

Performance of culture media for the isolation and identification of Staphylococcus aureus from bovine mastitis.

PubMed

Bautista-Trujillo, G U; Solorio-Rivera, J L; Rentería-Solórzano, I; Carranza-Germán, S I; Bustos-Martínez, J A; Arteaga-Garibay, R I; Baizabal-Aguirre, V M; Cajero-Juárez, M; Bravo-Patiño, A; Valdez-Alarcón, J J

2013-03-01

Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt-mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep's blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and β-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95%, respectively) compared with S110 (70.00, 23.73 and 90.91%, respectively), CSA (69.23, 28.13 and 95.74%, respectively) and SBA (68.75, 37.93 and 89.58%, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.

Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation.

PubMed

Schmitt, Bryan H; Cunningham, Scott A; Dailey, Aaron L; Gustafson, Daniel R; Patel, Robin

2013-03-01

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.

Identification of the infectious source of an unusual outbreak of histoplasmosis, in a hotel in Acapulco, state of Guerrero, Mexico.

PubMed

Taylor, Maria Lucia; Ruíz-Palacios, Guillermo M; del Rocío Reyes-Montes, María; Rodríguez-Arellanes, Gabriela; Carreto-Binaghi, Laura E; Duarte-Escalante, Esperanza; Hernández-Ramírez, Aurora; Pérez, Armando; Suárez-Alvarez, Roberto O; Roldán-Aragón, Yuri A; Romero-Martínez, Rafael; Sahaza-Cardona, Jorge H; Sifuentes-Osornio, José; Soto-Ramírez, Luis E; Peña-Sandoval, Gabriela R

2005-09-01

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel's ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92-99%) between them.

Identification by 16S rRNA gene sequencing of an Actinomyces hongkongensis isolate recovered from a patient with pelvic actinomycosis.

PubMed

Flynn, A N; Lyndon, C A; Church, D L

2013-08-01

A case of Actinomyces hongkongensis pelvic actinomycosis in an adult woman is described. Conventional phenotypic tests failed to identify the Gram-positive bacillus isolated from a fluid aspirate of a pelvic abscess. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

PubMed Central

Bossé, Janine T.; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M.; Rogers, Jon; Chaudhuri, Roy R.; Weinert, Lucy A.; Oshota, Olusegun; Holden, Matt T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.

2015-01-01

Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. PMID:25957382

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

De Carolis, E; Posteraro, B; Lass-Flörl, C; Vella, A; Florio, A R; Torelli, R; Girmenia, C; Colozza, C; Tortorano, A M; Sanguinetti, M; Fadda, G

2012-05-01

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

(GTG)5-PCR fingerprinting for the classification and identification of coagulase-negative Staphylococcus species from bovine milk and teat apices: a comparison of type strains and field isolates.

PubMed

Braem, G; De Vliegher, S; Supré, K; Haesebrouck, F; Leroy, F; De Vuyst, L

2011-01-10

Due to significant financial losses in the dairy cattle farming industry caused by mastitis and the possible influence of coagulase-negative staphylococci (CNS) in the development of this disease, accurate identification methods are needed that untangle the different species of the diverse CNS group. In this study, 39 Staphylococcus type strains and 253 field isolates were subjected to (GTG)(5)-PCR fingerprinting to construct a reference framework for the classification and identification of different CNS from (sub)clinical milk samples and teat apices swabs. Validation of the reference framework was performed by dividing the field isolates in two separate groups and testing whether one group of field isolates, in combination with type strains, could be used for a correct classification and identification of a second group of field isolates. (GTG)(5)-PCR fingerprinting achieved a typeability of 94.7% and an accuracy of 94.3% compared to identifications based on gene sequencing. The study shows the usefulness of the method to determine the identity of bovine Staphylococcus species, provided an identification framework updated with field isolates is available. Copyright © 2010 Elsevier B.V. All rights reserved.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in Viking-age settlement.

PubMed

Søe, Martin Jensen; Nejsum, Peter; Fredensborg, Brian Lund; Kapel, Christian Moliin Outzen

2015-02-01

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura , using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and, hence, that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species-specific identification, soil samples offer excellent opportunities for studies of human parasite infections and of human and animal interactions of the past.

Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal.

PubMed

Shahid, M S; Yoshida, S; Khatri-Chhetri, G B; Briddon, R W; Natsuaki, K T

2013-06-01

Carica papaya (papaya) is a fruit crop that is cultivated mostly in kitchen gardens throughout Nepal. Leaf samples of C. papaya plants with leaf curling, vein darkening, vein thickening, and a reduction in leaf size were collected from a garden in Darai village, Rampur, Nepal in 2010. Full-length clones of a monopartite Begomovirus, a betasatellite and an alphasatellite were isolated. The complete nucleotide sequence of the Begomovirus showed the arrangement of genes typical of Old World begomoviruses with the highest nucleotide sequence identity (>99 %) to an isolate of Ageratum yellow vein virus (AYVV), confirming it as an isolate of AYVV. The complete nucleotide sequence of betasatellite showed greater than 89 % nucleotide sequence identity to an isolate of Tomato leaf curl Java betasatellite originating from Indonesian. The sequence of the alphasatellite displayed 92 % nucleotide sequence identity to Sida yellow vein China alphasatellite. This is the first identification of these components in Nepal and the first time they have been identified in papaya.

Rapid Identification and Subtyping of Helicobacter cinaedi Strains by Intact-Cell Mass Spectrometry Profiling with the Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Taniguchi, Takako; Sekiya, Ayumi; Higa, Mariko; Saeki, Yuji; Umeki, Kazumi; Okayama, Akihiko; Hayashi, Tetsuya

2014-01-01

Helicobacter cinaedi infection is recognized as an increasingly important emerging disease in humans. Although H. cinaedi-like strains have been isolated from a variety of animals, it is difficult to identify particular isolates due to their unusual phenotypic profiles and the limited number of biochemical tests for detecting helicobacters. Moreover, analyses of the 16S rRNA gene sequences are also limited due to the high levels of similarity among closely related helicobacters. This study was conducted to evaluate intact-cell mass spectrometry (ICMS) profiling using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a tool for the identification of H. cinaedi. A total of 68 strains of H. cinaedi isolated from humans, dogs, a cat, and hamsters were examined in addition to other Helicobacter species. The major ICMS profiles of H. cinaedi were identical and differed from those of Helicobacter bilis, which show >98% sequence similarity at the 16S rRNA sequence level. A phyloproteomic analysis of the H. cinaedi strains examined in this work revealed that human isolates formed a single cluster that was distinct from that of the animal isolates, with the exception of two strains from dogs. These phyloproteomic results agreed with those of the phylogenetic analysis based on the nucleotide sequences of the hsp60 gene. Because they formed a distinct cluster in both analyses, our data suggest that animal strains may not be a major source of infection in humans. In conclusion, the ICMS profiles obtained using a MALDI-TOF MS approach may be useful for the identification and subtyping of H. cinaedi. PMID:24153128

Characterization of a genetically distinct subpopulation of Staphylococcus haemolyticus isolated from milk of cows with intramammary infections.

PubMed

Wanecka, Anna; Król, Jarosław; Twardoń, Jan; Mrowiec, Jacek; Bania, Jacek; Korzeniowska-Kowal, Agnieszka; Tobiasz, Anna

2018-02-01

The aim of this paper is to describe a novel subpopulation of Staphylococcus haemolyticus isolated from intramammary gland infections (IMI) in cattle. In total, eight isolates originating from milk samples from two unrelated dairy farms were examined phenotypically (using the ID 32 STAPH system) and genotypically. These isolates had almost identical sequences of each of the housekeeping genes examined (dnaJ, rpoB and sodA) but these sequences displayed similarity of only ∼92.5%, 95.0% and 96.8%, respectively, with known S. haemolyticus sequences. The atypical isolates could also be distinguished biochemically by the positive β-galactosidase test (with 2-naphthyl-β-d-galactopyranoside as the substrate). All the isolates were identified as S. haemolyticus upon MALDI-TOF analysis but half of them, that achieved scores 1.7-1.999 (not reliable species identification), required expanding the commercial database for secure identification. Our study has shown that IMI in cattle may be caused by two distinct subpopulations of S. haemolyticus, differing clearly by some genotypic and phenotypic properties. The first of these subpopulations seems to be common to many hosts (including humans), whereas the second (possibly at the subspecies rank) is, so far, found only in cattle. Copyright © 2017 Elsevier B.V. All rights reserved.

Phylogenetic Analysis of Phenotypically Characterized Cryptococcus laurentii Isolates Reveals High Frequency of Cryptic Species

PubMed Central

Ferreira-Paim, Kennio; Ferreira, Thatiana Bragine; Andrade-Silva, Leonardo; Mora, Delio Jose; Springer, Deborah J.; Heitman, Joseph; Fonseca, Fernanda Machado; Matos, Dulcilena; Melhem, Márcia Souza Carvalho; Silva-Vergara, Mario León

2014-01-01

Background Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States. Methods In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region. Results BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99–100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified. Conclusions Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode. PMID:25251413

Species identification and molecular typing of human Brucella isolates from Kuwait.

PubMed

Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Species identification and molecular typing of human Brucella isolates from Kuwait

PubMed Central

Osman, Amr; Shaheed, Faraz; Khan, Mohd W.

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90–99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region. PMID:28800594

A cultivation-independent PCR-RFLP assay targeting oprF gene for detection and identification of Pseudomonas spp. in samples from fibrocystic pediatric patients.

PubMed

Lagares, Antonio; Agaras, Betina; Bettiol, Marisa P; Gatti, Blanca M; Valverde, Claudio

2015-07-01

Species-specific genetic markers are crucial to develop faithful and sensitive molecular methods for the detection and identification of Pseudomonas aeruginosa (Pa). We have previously set up a PCR-RFLP protocol targeting oprF, the gene encoding the genus-specific outer membrane porin F, whose strong conservation and marked sequence diversity allowed detection and differentiation of environmental isolates (Agaras et al., 2012). Here, we evaluated the ability of the PCR-RFLP assay to genotype clinical isolates previously identified as Pa by conventional microbiological methods within a collection of 62 presumptive Pa isolates from different pediatric clinical samples and different sections of the Hospital de Niños "Sor María Ludovica" from La Plata, Argentina. All isolates, but one, gave an oprF amplicon consistent with that from reference Pa strains. The sequence of the smaller-sized amplicon revealed that the isolate was in fact a mendocina Pseudomonas strain. The oprF RFLP pattern generated with TaqI or HaeIII nucleases matched those of reference Pa strains for 59 isolates (96%). The other two Pa isolates (4%) revealed a different RFLP pattern based on HaeIII digestion, although oprF sequencing confirmed that Pa identification was correct. We next tested the effectiveness of the PCR-RFLP to detect pseudomonads on clinical samples of pediatric fibrocystic patients directly without sample cultivation. The expected amplicon and its cognate RFLP profile were obtained for all samples in which Pa was previously detected by cultivation-dependent methods. Altogether, these results provide the basis for the application of the oprF PCR-RFLP protocol to directly detect and identify Pa and other non-Pa pseudomonads in fibrocystic clinical samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Identification and Databases in Fusarium

USDA-ARS?s Scientific Manuscript database

DNA sequence-based methods for identifying pathogenic and mycotoxigenic Fusarium isolates have become the gold standard worldwide. Moreover, fusarial DNA sequence data are increasing rapidly in several web-accessible databases for comparative purposes. Unfortunately, the use of Basic Alignment Sea...

Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

PubMed Central

Westblade, Lars F.; Jennemann, Rebecca; Branda, John A.; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B.; Ginocchio, Christine C.; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Rychert, Jenna A.; Sercia, Linda

2013-01-01

The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories. PMID:23658267

Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Seng, Piseth; Drancourt, Michel; Gouriet, Frédérique; La Scola, Bernard; Fournier, Pierre-Edouard; Rolain, Jean Marc; Raoult, Didier

2009-08-15

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising > or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

Evaluation of VITEK mass spectrometry (MS), a matrix-assisted laser desorption ionization time-of-flight MS system for identification of anaerobic bacteria.

PubMed

Lee, Wonmok; Kim, Myungsook; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Yunsop

2015-01-01

By conventional methods, the identification of anaerobic bacteria is more time consuming and requires more expertise than the identification of aerobic bacteria. Although the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are relatively less studied, they have been reported to be a promising method for the identification of anaerobes. We evaluated the performance of the VITEK MS in vitro diagnostic (IVD; 1.1 database; bioMérieux, France) in the identification of anaerobes. We used 274 anaerobic bacteria isolated from various clinical specimens. The results for the identification of the bacteria by VITEK MS were compared to those obtained by phenotypic methods and 16S rRNA gene sequencing. Among the 249 isolates included in the IVD database, the VITEK MS correctly identified 209 (83.9%) isolates to the species level and an additional 18 (7.2%) at the genus level. In particular, the VITEK MS correctly identified clinically relevant and frequently isolated anaerobic bacteria to the species level. The remaining 22 isolates (8.8%) were either not identified or misidentified. The VITEK MS could not identify the 25 isolates absent from the IVD database to the species level. The VITEK MS showed reliable identifications for clinically relevant anaerobic bacteria.

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

PubMed Central

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

2016-01-01

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

Characterization of Blue Mold Penicillium Species Isolated from Stored Fruits Using Multiple Highly Conserved Loci

PubMed Central

Yin, Guohua; Zhang, Yuliang; Pennerman, Kayla K.; Wu, Guangxi; Hua, Sui Sheng T.; Yu, Jiujiang; Jurick, Wayne M.; Guo, Anping; Bennett, Joan W.

2017-01-01

Penicillium is a large genus of common molds with over 400 described species; however, identification of individual species is difficult, including for those species that cause postharvest rots. In this study, blue rot fungi from stored apples and pears were isolated from a variety of hosts, locations, and years. Based on morphological and cultural characteristics and partial amplification of the β-tubulin locus, the isolates were provisionally identified as several different species of Penicillium. These isolates were investigated further using a suite of molecular DNA markers and compared to sequences of the ex-type for cognate species in GenBank, and were identified as P. expansum (3 isolates), P. solitum (3 isolates), P. carneum (1 isolate), and P. paneum (1 isolate). Three of the markers we used (ITS, internal transcribed spacer rDNA sequence; benA, β-tubulin; CaM, calmodulin) were suitable for distinguishing most of our isolates from one another at the species level. In contrast, we were unable to amplify RPB2 sequences from four of the isolates. Comparison of our sequences with cognate sequences in GenBank from isolates with the same species names did not always give coherent data, reinforcing earlier studies that have shown large intraspecific variability in many Penicillium species, as well as possible errors in some sequence data deposited in GenBank. PMID:29371531

Identification by 16S rRNA Gene Sequencing of an Actinomyces hongkongensis Isolate Recovered from a Patient with Pelvic Actinomycosis

PubMed Central

Flynn, A. N.; Lyndon, C. A.

2013-01-01

A case of Actinomyces hongkongensis pelvic actinomycosis in an adult woman is described. Conventional phenotypic tests failed to identify the Gram-positive bacillus isolated from a fluid aspirate of a pelvic abscess. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software. PMID:23698532

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

PubMed

Cloud, Joann L; Conville, Patricia S; Croft, Ann; Harmsen, Dag; Witebsky, Frank G; Carroll, Karen C

2004-02-01

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5' 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i). conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii). molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii). when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species.

PubMed

Alanio, A; Beretti, J-L; Dauphin, B; Mellado, E; Quesne, G; Lacroix, C; Amara, A; Berche, P; Nassif, X; Bougnoux, M-E

2011-05-01

New Aspergillus species have recently been described with the use of multilocus sequencing in refractory cases of invasive aspergillosis. The classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific patterns of susceptibility to antifungal agents, and misidentification may lead to inappropriate therapy. We developed a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based strategy to adequately identify Aspergillus species to the species level. A database including the reference spectra of 28 clinically relevant species from seven Aspergillus sections (five common and 23 unusual species) was engineered. The profiles of young and mature colonies were analysed for each reference strain, and species-specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized by partial sequencing of the β-tubulin and calmodulin genes. One hundred and thirty-eight isolates of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified, but no isolate was misidentified (specificity: 100%). The database, including species-specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI-TOF MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can give better results than morphological identification in clinical laboratories. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Molecular identification of environmental bacteria in indoor air in the domestic home: description of a new species of Exiguobacterium.

PubMed

Yuan, Ivan; Xu, Jiru; Millar, B Cherie; Dooley, James S G; Rooney, Paul J; Alexander, H Denis; Moore, John E

2007-02-01

The quality of indoor air in terms of its bioaerosol composition with microorganisms is important due to its potential aetiological role in development of conditions such as Sick Building Syndrome. Hence, laboratory identification of bacteriological components in any bioaerosol from buildings may help elucidate the role of such organisms in disease states, particularly allergy-related conditions. A molecular method was developed employing universal or "broad-range" eubacterial PCR to help identify environmental culturable bacteria from domestic household air. In a "proof of concept" experiment, 16S rDNA PCR was performed on a collection of bacterial isolates originating from indoor air in the domestic home. 16S rDNA PCR was performed using a set of universal primers to successfully generate an amplicon of approximately 1400 bp, which was sequenced to obtain each isolate's identity. Sequence analysis was able to identify 12/13 of the isolates, whereby the majority were Gram-positive (12/13). Nine different genera were identified from the 13 isolates examined, of which, 12/13 were Gram-positive, with the exception being Moraxella osloensis, which was Gram-negative, as well as a novel species of Exiguobacterium. The closest phylogenetic neighbour of the wildtype isolate to a named species within this genus was E. aestuarii (1364/1384 bases; 98.4% homology), followed by E. marinum (97.5%) and with E. acetylicum being the most distantly related of all the described species. On account of this divergence within the 16S rDNA gene operon of the unknown Exiguobacterium isolate, we believe this isolate to represent a novel species of Exiguobacterium, which we have tentatively named Exiguobacterium belfastensis. Although from this study, these organisms are usually unlikely to be clinically significant to healthy individuals with a competent immune system, we recommend that molecular identification methods are used, if considered necessary, as an adjunct to first line phenotypic identification schemes, where a definitive identification is required. When the use of molecular identification methods is justified, employment of partial 16S rDNA PCR and sequencing provides a valuable and reliable method of identification of environmental bacteria in the home. This study demonstrates the usefulness of such methods and a full and comprehensive study is now required to examine the diversity of bacteria in indoor air in the home, with particular emphasis on the risk of such environmental organisms to immunosurpressed patients, such as those with haematological malignancies and who are neutropenic.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

PubMed

Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

2012-09-11

Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

Isolation and identification of gold nanoparticles synthesizing fungi from Indian Kolar Gold Field mine soil.

PubMed

Lakshmi, V Jhansi; Kannan, K P

2016-07-01

An indigenous fungal strain was isolated from Indian Kolar Gold Field mine soil. The isolate was heterothallic, branched septate, deeply floccose, fast-growing, dull green with white background conidial columnar mycelium from Aspergillus section Fumigati. Diverse metabolic patterns of the isolate exhibit high metal, thermal resistance which grews well from 28 ± 1 degrees C to 37 degrees C and pH concentration was significant on the growth of isolate. Phylogenetic analysis of 16srRNA β-Tubulin gene sequence established relationship among isolate and other taxa. Molecular identification and morphological features of fungal isolate were consistent with those of Neosartorya udagawae. Heterothallic N. udagawae FJ830683 strain was closely related to homothallic N. aureola EF661890. Fungal isolate extract synthesized narrow sized stable Gold nanoparticles (AuNPs).

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

PubMed Central

Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

2013-01-01

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038

Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections.

PubMed

Ng, L S Y; Sim, J H C; Eng, L C; Menon, S; Tan, T Y

2012-08-01

Aero-tolerant Actinomyces spp. are an under-recognised cause of cutaneous infections, in part because identification using conventional phenotypic methods is difficult and may be inaccurate. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a promising new technique for bacterial identification, but with limited data on the identification of aero-tolerant Actinomyces spp. This study evaluated the accuracy of a phenotypic biochemical kit, MALDI-TOF MS and genotypic identification methods for the identification of this problematic group of organisms. Thirty aero-tolerant Actinomyces spp. were isolated from soft-tissue infections over a 2-year period. Species identification was performed by 16 s rRNA sequencing and genotypic results were compared with results obtained by API Coryne and MALDI-TOF MS. There was poor agreement between API Coryne and genotypic identification, with only 33% of isolates correctly identified to the species level. MALDI-TOF MS correctly identified 97% of isolates to the species level, with 33% of identifications achieved with high confidence scores. MALDI-TOF MS is a promising new tool for the identification of aero-tolerant Actinomyces spp., but improvement of the database is required in order to increase the confidence level of identification.

Molecular Microbial Analysis of Lactobacillus Strains Isolated from the Gut of Calves for Potential Probiotic Use

PubMed Central

Soto, Lorena P.; Frizzo, Laureano S.; Bertozzi, Ezequiel; Avataneo, Elizabeth; Sequeira, Gabriel J.; Rosmini, Marcelo R.

2010-01-01

The intestinal microbiota has an influence on the growth and health status of the hosts. This is of particular interest in animals reared using intensive farming practices. Hence, it is necessary to know more about complexity of the beneficial intestinal microbiota. The use of molecular methods has revolutionized microbial identification by improving its quality and effectiveness. The specific aim of the study was to analyze predominant species of Lactobacillus in intestinal microbial ecosystem of young calves. Forty-two lactic acid bacteria (LAB) isolated from intestinal tract of young calves were characterized by: Amplified Ribosomal DNA Restriction Analysis (ARDRA), by using Hae III, Msp I, and Hinf I restriction enzymes, and 16S rDNA gene sequencing. ARDRA screening revealed nine unique patterns among 42 isolates, with the same pattern for 29 of the isolates. Gene fragments of 16S rDNA of 19 strains representing different patterns were sequenced to confirm the identification of these species. These results confirmed that ARDRA is a good tool for identification and discrimination of bacterial species isolated from complex ecosystem and between closely related groups. This paper provides information about the LAB species predominant in intestinal tract of young calves that could provide beneficial effects when administered as probiotic. PMID:20445780

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

PubMed

Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

2015-08-01

The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Molecular characterization of freshwater snails in the genus Bulinus: a role for barcodes?

PubMed Central

Kane, Richard A; Stothard, J Russell; Emery, Aidan M; Rollinson, David

2008-01-01

Background Reliable and consistent methods are required for the identification and classification of freshwater snails belonging to the genus Bulinus (Gastropoda, Planorbidae) which act as intermediate hosts for schistosomes of both medical and veterinary importance. The current project worked towards two main objectives, the development of a cost effective, simple screening method for the routine identification of Bulinus isolates and the use of resultant sequencing data to produce a model of relationships within the group. Results Phylogenetic analysis of the DNA sequence for a large section (1009 bp) of the mitochondrial gene cytochrome oxidase subunit 1 (cox1) for isolates of Bulinus demonstrated superior resolution over that employing the second internal transcribed spacer (its2) of the ribosomal gene complex. Removal of transitional substitutions within cox1 because of saturation effects still allowed identification of snails at species group level. Within groups, some species could be identified with ease but there were regions where the high degree of molecular diversity meant that clear identification of species was problematic, this was particularly so within the B. africanus group. Conclusion The sequence diversity within cox1 is such that a barcoding approach may offer the best method for characterization of populations and species within the genus from different geographical locations. The study has confirmed the definition of some accepted species within the species groups but additionally has revealed some unrecognized isolates which underlines the need to use molecular markers in addition to more traditional methods of identification. A barcoding approach based on part of the cox1 gene as defined by the Folmer primers is proposed. PMID:18544153

Single-cell isolation by a modular single-cell pipette for RNA-sequencing.

PubMed

Zhang, Kai; Gao, Min; Chong, Zechen; Li, Ying; Han, Xin; Chen, Rui; Qin, Lidong

2016-11-29

Single-cell transcriptome sequencing highly requires a convenient and reliable method to rapidly isolate a live cell into a specific container such as a PCR tube. Here, we report a modular single-cell pipette (mSCP) consisting of three modular components, a SCP-Tip, an air-displacement pipette (ADP), and ADP-Tips, that can be easily assembled, disassembled, and reassembled. By assembling the SCP-Tip containing a hydrodynamic trap, the mSCP can isolate single cells from 5-10 cells per μL of cell suspension. The mSCP is compatible with microscopic identification of captured single cells to finally achieve 100% single-cell isolation efficiency. The isolated live single cells are in submicroliter volumes and well suitable for single-cell PCR analysis and RNA-sequencing. The mSCP possesses merits of convenience, rapidness, and high efficiency, making it a powerful tool to isolate single cells for transcriptome analysis.

Rapid Identification of Bacteria in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿

PubMed Central

Stevenson, Lindsay G.; Drake, Steven K.; Murray, Patrick R.

2010-01-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of <1.7) was obtained for 42 (19.8%) of the isolates, due most commonly to insufficient numbers of bacteria in the blood culture broth. Of the bacteria with a spectral score of ≥1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test. PMID:19955282

Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Stevenson, Lindsay G; Drake, Steven K; Murray, Patrick R

2010-02-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of < 1.7) was obtained for 42 (19.8%) of the isolates, due most commonly to insufficient numbers of bacteria in the blood culture broth. Of the bacteria with a spectral score of > or = 1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test.

Genomic epidemiology of a protracted hospital outbreak caused by multidrug-resistant Acinetobacter baumannii in Birmingham, England.

PubMed

Halachev, Mihail R; Chan, Jacqueline Z-M; Constantinidou, Chrystala I; Cumley, Nicola; Bradley, Craig; Smith-Banks, Matthew; Oppenheim, Beryl; Pallen, Mark J

2014-01-01

Multidrug-resistant Acinetobacter baumannii commonly causes hospital outbreaks. However, within an outbreak, it can be difficult to identify the routes of cross-infection rapidly and accurately enough to inform infection control. Here, we describe a protracted hospital outbreak of multidrug-resistant A. baumannii, in which whole-genome sequencing (WGS) was used to obtain a high-resolution view of the relationships between isolates. To delineate and investigate the outbreak, we attempted to genome-sequence 114 isolates that had been assigned to the A. baumannii complex by the Vitek2 system and obtained informative draft genome sequences from 102 of them. Genomes were mapped against an outbreak reference sequence to identify single nucleotide variants (SNVs). We found that the pulsotype 27 outbreak strain was distinct from all other genome-sequenced strains. Seventy-four isolates from 49 patients could be assigned to the pulsotype 27 outbreak on the basis of genomic similarity, while WGS allowed 18 isolates to be ruled out of the outbreak. Among the pulsotype 27 outbreak isolates, we identified 31 SNVs and seven major genotypic clusters. In two patients, we documented within-host diversity, including mixtures of unrelated strains and within-strain clouds of SNV diversity. By combining WGS and epidemiological data, we reconstructed potential transmission events that linked all but 10 of the patients and confirmed links between clinical and environmental isolates. Identification of a contaminated bed and a burns theatre as sources of transmission led to enhanced environmental decontamination procedures. WGS is now poised to make an impact on hospital infection prevention and control, delivering cost-effective identification of routes of infection within a clinically relevant timeframe and allowing infection control teams to track, and even prevent, the spread of drug-resistant hospital pathogens.

Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

PubMed Central

Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

2011-01-01

Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

Streptococcal Diversity of Human Milk and Comparison of Different Methods for the Taxonomic Identification of Streptococci.

PubMed

Martín, Virginia; Mediano, Pilar; Del Campo, Rosa; Rodríguez, Juan M; Marín, María

2016-11-01

The genus Streptococcus is 1 of the dominant bacterial groups in human milk, but the taxonomic identification of some species remains difficult. The objective of this study was to investigate the discriminatory ability of different methods to identify streptococcal species in order to perform an assessment of the streptococcal diversity of human milk microbiota as accurately as possible. The identification of 105 streptococcal strains from human milk was performed by 16S rRNA, tuf, and sodA gene sequencing, phylogenetic analysis, and Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Streptococcus salivarius, Streptococcus mitis, and Streptococcus parasanguinis were the streptococcal dominant species in the human milk microbiota. Sequencing of housekeeping genes allowed the classification of 96.2% (16S rRNA), 84.8% ( sodA), and 88.6% ( tuf) of the isolates. Phylogenetic analysis showed 3 main streptococcal clusters corresponding with the mitis (73 isolates), salivarius (29), mutans (1)-pyogenic (2) groups, but many of the mitis group isolates (36) could not be assigned to any species. The application of the MALDI-TOF Bruker Biotyper system resulted in the identification of 56 isolates (53.33%) at the species level, but it could not discriminate between S pneumoniae and S mitis isolates, in contrast to the Vitek-MS system. There was a good agreement among the different methods assessed in this study to identify those isolates of the salivarius, mutans, and pyogenic groups, whereas unambiguous discrimination could not be achieved concerning some species of the mitis group ( S mitis, S pneumoniae, S pseudopneumoniae, S oralis).

Characteristics of invasive Acinetobacter species isolates recovered in a pediatric academic center.

PubMed

Jain, Avish L; Harding, Christian M; Assani, Kaivon; Shrestha, Chandra L; Haga, Mercedees; Leber, Amy; Munson, Robert S; Kopp, Benjamin T

2016-07-22

Acinetobacter species are associated with increasing mortality due to emerging drug-resistance. Pediatric Acinetobacter infections are largely undefined in developed countries and clinical laboratory identification methods do not reliably differentiate between members of the Acinetobacter calcoaceticus-baumannii complex, leading to improper identification. Therefore we aimed to determine risk factors for invasive Acinetobacter infections within an academic, pediatric setting as well as defining microbiologic characteristics of predominant strains. Twenty-four invasive Acinetobacter isolates were collected from 2009-2013. Comparative sequence analysis of the rpoB gene was performed coupled with phenotypic characterization of antibiotic resistance, motility, biofilm production and clinical correlation. Affected patients had a median age of 3.5 years, and 71 % had a central catheter infection source. rpoB gene sequencing revealed a predominance of A. pittii (45.8 %) and A. baumannii (33.3 %) strains. There was increasing incidence of A. pittii over the study. Two fatalities occurred in the A. pittii group. Seventeen percent of isolates were multi-drug resistant. A pittii and A. baumannii strains were similar in motility, but A pittii strains had significantly more biofilm production (P value = 0.018). A. pittii was the most isolated species highlighting the need for proper species identification. The isolated strains had limited acute mortality in children, but the occurrence of more multi-drug resistant strains in the future is a distinct possibility, justifying continued research and accurate species identification.

Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

PubMed

Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

2016-03-01

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs.

PubMed

Campbell, A J; Gasser, R B; Chilton, N B

1995-03-01

In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.

Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier

2013-07-01

During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard

2013-01-01

During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories. PMID:23637301

Prospective evaluation of the VITEK MS for the routine identification of bacteria and yeast in the clinical microbiology laboratory: assessment of accuracy of identification and turnaround time.

PubMed

Charnot-Katsikas, Angella; Tesic, Vera; Boonlayangoor, Sue; Bethel, Cindy; Frank, Karen M

2014-02-01

This study assessed the accuracy of bacterial and yeast identification using the VITEK MS, and the time to reporting of isolates before and after its implementation in routine clinical practice. Three hundred and sixty-two isolates of bacteria and yeast, consisting of a variety of clinical isolates and American Type Culture Collection strains, were tested. Results were compared with reference identifications from the VITEK 2 system and with 16S rRNA sequence analysis. The VITEK MS provided an acceptable identification to species level for 283 (78 %) isolates. Considering organisms for which genus-level identification is acceptable for routine clinical care, 315 isolates (87 %) had an acceptable identification. Six isolates (2 %) were identified incorrectly, five of which were Shigella species. Finally, the time for reporting the identifications was decreased significantly after implementation of the VITEK MS for a total mean reduction in time of 10.52 h (P<0.0001). Overall, accuracy of the VITEK MS was comparable or superior to that from the VITEK 2. The findings were also comparable to other studies examining the accuracy of the VITEK MS, although differences exist, depending on the diversity of species represented as well as on the versions of the databases used. The VITEK MS can be incorporated effectively into routine use in a clinical microbiology laboratory and future expansion of the database should provide improved accuracy for the identification of micro-organisms.

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

PubMed

Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

2008-10-01

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However, studies on the accuracy of the software packages have given highly varied results, and interpretation of results remains difficult for most technicians, and even for clinical microbiologists. To fully utilize 16S rDNA sequencing in clinical microbiology, better guidelines are needed for interpretation of the identification results, and additional/supplementary methods are necessary for bacterial species that cannot be identified confidently by 16S rDNA sequencing alone.

MALDI-TOF Mass Spectrometry as a Useful Tool for Identification of Enterococcus spp. from Wild Birds and Differentiation of Closely Related Species.

PubMed

Stępień-Pyśniak, Dagmara; Hauschild, Tomasz; Różański, Paweł; Marek, Agnieszka

2017-06-28

The aim of this study was to explore the accuracy and feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared with rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic ( rpoA gene sequencing) methods were used to identify all the isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level (log(score) > or =2.0), and three isolates (5.6%) were identified at a level of probable genus identification (log(score) 1.88-1.927). Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%), and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus .

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Culture.

PubMed

Noor Uddin, Gazi Md; Larsen, Marianne Halberg; Christensen, Henrik; Aarestrup, Frank M; Phu, Tran Minh; Dalsgaard, Anders

2015-01-01

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

[Enterovirus sequencing as a new approach to the laboratory diagnosis for clinical and epidemiological purposes].

PubMed

Rainetová, P; Jiřincová, H; Musílek, M; Nováková, L; Vodičková, I; Štruncová, V; Švecová, M; Pazdiora, P; Piskunová, N; Trubač, P; Zajíc, T; Havlíčková, M

2015-06-01

Introducing enterovirus sequencing as an advanced approach to classify the viruses isolated according to the novel nomenclature and to characterize isolates in detail. Seventy-five specimens collected from 64 patients in two hospitals, Liberec Regional Hospital, and Plzeň University Hospital, were analyzed. The study patients' age ranged from four to 54 years, with a median of 15 years in males and 16 years in females. In most patients, the reasons for admission were intense headache, fever, vomiting, tiredness, meningeal symptoms, intestinal symptoms (in two patients), and skin symptoms (in one patient). The specimens collected were rectal and throat swabs, cerebrospinal fluid (CSF) and stool specimens. Molecular detection and typing were performed using the RT-PCR method. A segment of the 5´non-coding RNA was selected for typing. Specimens were amplified using single-step PCR with external primers and with the same primers extended to include M13 sequences (Generi-Biotech). The LASERGENE software (DIASTAR) was used in sequence editing, alignment, and quality check. The sequences obtained were checked against the central GenBank sequence database using the BLAST algorithm. The identification of the study isolates resulted in 61 ECHO viruses 30, three coxsackie viruses B1, one coxsackie virus B3, one coxsackie virus A9, one enterovirus 86, one enterovirus 71, Two ECHO viruses 13/coxsackie virus B5, one ECHO virus 7/30/coxsackie virus B4, one coxsackie virus B4/enterovirus B, one enterovirus 87/ECHO virus 30/enterovirus B, and one ECHO virus 3. All viruses isolated, except enterovirus 71 classified into group A, were of group B. The enteroviruses were identified unambigously, although the sequencing only targeted a short, conserved segment that showed considerable variability. The sequencing was an effective alternative to enterovirus identification by the neutralisation test and allowed for detailed characterization of the isolates. The predominance of ECHO 30 as the cause of aseptic meningitis is in accordance with the literature data.

Isolation and characterization of a novel chlorpyrifos degrading flavobacterium species EMBS0145 by 16S rRNA gene sequencing.

PubMed

Amareshwari, P; Bhatia, Mayuri; Venkatesh, K; Roja Rani, A; Ravi, G V; Bhakt, Priyanka; Bandaru, Srinivas; Yadav, Mukesh; Nayarisseri, Anuraj; Nair, Achuthsankar S

2015-03-01

Indiscriminate application of pesticides like chlorpyrifos, diazinon, or malathion contaminate the soil in addition has being unsafe often it has raised severe health concerns. Conversely, microorganisms like Trichoderma, Aspergillus and Bacteria like Rhizobium Bacillus, Azotobacter, Flavobacterium etc have evolved that are endowed with degradation of pesticides aforementioned to non-toxic products. The current study pitches into identification of a novel species of Flavobacterium bacteria capable to degrade the Organophosphorous pesticides. The bacterium was isolated from agricultural soil collected from Guntur District, Andhra Pradesh, India. The samples were serially diluted and the aliquots were incubated for a suitable time following which the suspected colony was subjected to 16S rDNA sequencing. The sequence thus obtained was aligned pairwise against Flavobacterium species, which resulted in identification of novel specie of Flavobacterium later named as EMBS0145, the sequence of which was deposited in in GenBank with accession number JN794045.

Species Identification of Clinical Prevotella Isolates by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Soetens, Oriane; De Bel, Annelies; Echahidi, Fedoua; Vancutsem, Ellen; Vandoorslaer, Kristof; Piérard, Denis

2012-01-01

The performance of matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database. PMID:22301022

(GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

PubMed

Ramírez-Castrillón, Mauricio; Mendes, Sandra Denise Camargo; Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

(GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?

PubMed Central

Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure. PMID:25171185

Identification of bacteria pathogenic to or associated with onion (Allium cepa) based on sequence differences in a portion of the conserved gyrase B gene.

PubMed

Bonasera, Jean M; Asselin, Jo Ann E; Beer, Steven V

2014-08-01

We have developed a method for the identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae, based on sequence variation in a portion of the gyrB gene. Thus, we identified, in most cases to species level, over 1000 isolates from onion bulbs and leaves and soil in which onions were grown. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular characterization of chikungunya virus from Andhra Pradesh, India & phylogenetic relationship with Central African isolates.

PubMed

M Naresh Kumar, C V; Anthony Johnson, A M; R Sai Gopal, D V

2007-12-01

Chikungunya virus has caused numerous large outbreaks in India. Suspected blood samples from the epidemic were collected and characterized for the identification of the responsible causative from Rayalaseema region of Andhra Pradesh. RT-PCR was used for screening of suspected blood samples. Primers were designed to amplify partial E1 gene and the amplified fragment was cloned and sequenced. The sequence was analyzed and compared with other geographical isolates to find the phylogenetic relationship. The sequence was submitted to the Gen bank DNA database (accession DQ888620). Comparative nucleotide homology analysis of the AP Ra-CTR isolate with the other isolates revealed 94.7+/-3.6 per cent of homology of CHIKAPRa-CTR with other isolates of Chikungunya virus at nucleotide level and 96.8+/-3.2 per cent of homology at amino acid level. The current epidemic was caused by the Central African genotype of CHIKV, grouped in Central Africa cluster in phylogenetic trees generated based on nucleotide and amino acid sequences.

Species of Phaeoacremonium Associated with Infections in Humans and Environmental Reservoirs in Infected Woody Plants

PubMed Central

Mostert, Lizel; Groenewald, Johannes Z.; Summerbell, Richard C.; Robert, Vincent; Sutton, Deanna A.; Padhye, Arvind A.; Crous, Pedro W.

2005-01-01

To date, three species of Phaeoacremonium have been associated with phaeohyphomycosis. These are P. parasiticum (formerly Phialophora parasitica), P. inflatipes, and P. rubrigenum. Numerous unknown isolates resembling Phaeoacremonium spp. have in recent years been isolated from human patients as well as from woody plants that appear to be the main environmental source of these fungi. Nine new Phaeoacremonium species, of which six were obtained as etiologic agents of human opportunistic infection, are reported. They can be identified based on their cultural and morphological characters, and the identifications are strongly supported in phylogenetic analyses of partial sequences of the actin, β-tubulin, and calmodulin genes. A multiple-entry electronic key based on morphological, cultural, and β-tubulin sequence data was developed to facilitate routine species identification. Reexamination of all isolates of P. inflatipes associated with human disease showed them to be misidentified and to belong to the new taxa described here. PMID:15814996

Comparison of Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometer to BD Phoenix Automated Microbiology System for Identification of Gram-Negative Bacilli▿

PubMed Central

Saffert, Ryan T.; Cunningham, Scott A.; Ihde, Sherry M.; Monson Jobe, Kristine E.; Mandrekar, Jayawant; Patel, Robin

2011-01-01

We compared the BD Phoenix automated microbiology system to the Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) system for identification of Gram-negative bacilli, using biochemical testing and/or genetic sequencing to resolve discordant results. The BD Phoenix correctly identified 363 (83%) and 330 (75%) isolates to the genus and species level, respectively. The Bruker Biotyper correctly identified 408 (93%) and 360 (82%) isolates to the genus and species level, respectively. The 440 isolates were grouped into common (308) and infrequent (132) isolates in the clinical laboratory. For the 308 common isolates, the BD Phoenix and Bruker Biotyper correctly identified 294 (95%) and 296 (96%) of the isolates to the genus level, respectively. For species identification, the BD Phoenix and Bruker Biotyper correctly identified 93% of the common isolates (285 and 286, respectively). In contrast, for the 132 infrequent isolates, the Bruker Biotyper correctly identified 112 (85%) and 74 (56%) isolates to the genus and species level, respectively, compared to the BD Phoenix, which identified only 69 (52%) and 45 (34%) isolates to the genus and species level, respectively. Statistically, the Bruker Biotyper overall outperformed the BD Phoenix for identification of Gram-negative bacilli to the genus (P < 0.0001) and species (P = 0.0005) level in this sample set. When isolates were categorized as common or infrequent isolates, there was statistically no difference between the instruments for identification of common Gram-negative bacilli (P > 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated Gram-negative bacilli (P < 0.0001). PMID:21209160

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

PubMed Central

Burnham, Carey-Ann D.; Bythrow, Maureen; Garner, Omai B.; Ginocchio, Christine C.; Jennemann, Rebecca; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Sercia, Linda; Westblade, Lars F.; Ferraro, Mary Jane; Branda, John A.

2013-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting. PMID:23658261

Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria.

PubMed

Rychert, Jenna; Burnham, Carey-Ann D; Bythrow, Maureen; Garner, Omai B; Ginocchio, Christine C; Jennemann, Rebecca; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Sercia, Linda; Westblade, Lars F; Ferraro, Mary Jane; Branda, John A

2013-07-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

DOEpatents

Bavykin, Sergei G.; Mirzabekov, Andrei D.

2007-10-30

The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing.

PubMed

Pereira, Leonel; Dias, Nicolina; Santos, Cledir; Lima, Nelson

2014-01-01

In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%. MALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Genetic variation in Pythium myriotylum based on SNP typing and development of a PCR-RFLP detection of isolates recovered from Pythium soft rot ginger.

PubMed

Le, D P; Smith, M K; Aitken, E A B

2017-10-01

Pythium myriotylum is responsible for severe losses in both capsicum and ginger crops in Australia under different regimes. Intraspecific genomic variation within the pathogen might explain the differences in aggressiveness and pathogenicity on diverse hosts. In this study, whole genome data of four P. myriotylum isolates recovered from three hosts and one Pythium zingiberis isolate were derived and analysed for sequence diversity based on single nucleotide polymorphisms (SNPs). A higher number of true and unique SNPs occurred in P. myriotylum isolates obtained from ginger with symptoms of Pythium soft rot (PSR) in Australia compared to other P. myriotylum isolates. Overall, SNPs were discovered more in the mitochondrial genome than those in the nuclear genome. Among the SNPs, a single substitution from the cytosine (C) to the thymine (T) in the partially sequenced CoxII gene of 14 representatives of PSR P. myriotylum isolates was within a restriction site of HinP1I enzyme which was used in the PCR-RFLP for detection and identification of the isolates without sequencing. The PCR-RFLP was also sensitive to detect PSR P. myriotylum strains from artificially infected ginger without the need for isolation for pure cultures. This is the first study of intraspecific variants of Pythium myriotylum isolates recovered from different hosts and origins based on single nucleotide polymorphism (SNP) genotyping of multiple genes. The SNPs discovered provide valuable makers for detection and identification of P. myriotylum strains initially isolated from Pythium soft rot (PSR) ginger by using PCR-RFLP of the CoxII locus. The PCR-RFLP was also sensitive to detect P. myriotylum directly from PSR ginger sampled from pot trials without the need of isolation for pure cultures. © 2017 The Society for Applied Microbiology.

ISOLATION AND IDENTIFICATION OF FRESHWATER BACTERIA ANTAGONISTIC TO GIARDIA INTESTINALIS CYSTS

EPA Science Inventory

We have isolated three freshwater bacterial strains that demonstrate the ability to degrade Giardia intestinalis cysts. These strains have been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the Flavobacterium columnare clade of the ...

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

PubMed

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification by 16S rRNA Gene Sequencing of Lactobacillus salivarius Bacteremic Cholecystitis

PubMed Central

Woo, Patrick C. Y.; Fung, Ami M. Y.; Lau, Susanna K. P.; Yuen, Kwok-Yung

2002-01-01

An anaerobic, nonsporulating, gram-positive bacterium was isolated from blood and bile pus cultures of a 70-year-old man with bacteremic acute cholecystitis. The API 20A system showed that it was 70% Actinomyces naeslundii and 30% Bifidobacterium species, whereas the Vitek ANI system and the ATB ID32A Expression system showed that it was “unidentified.” The 16S rRNA gene of the strain was amplified and sequenced. There were 3 base differences between the nucleotide sequence of the isolate and that of Lactobacillus salivarius subsp. salivarius or L. salivarius subsp. salicinius, indicating that the isolate was a strain of L. salivarius. The patient responded to cholecystectomy and a 2-week course of antibiotic treatment. Identification of the organism in the present study was important because the duration of antibiotic therapy would have been entirely different depending on the organism. If the bacterium had been identified as Actinomyces, penicillin for 6 months would have been the regimen of choice. However, it was Lactobacillus, and a 2-week course of antibiotic was sufficient. PMID:11773128

Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

PubMed

Deshpande, Ashutosh; Inkster, Teresa; Hamilton, Kate; Litt, David; Fry, Norman; Kennedy, Iain T R; Shookhye-Dickson, Jacqueline; Hill, Robert L R

2015-01-01

On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

The usefulness of DNA sequencing after extraction by Whatman FTA filter matrix technology and phenotypic tests for differentiation of Candida albicans and Candida dubliniensis.

PubMed

Kiraz, Nuri; Oz, Yasemin; Aslan, Huseyin; Muslumanoglu, Hamza

2014-02-01

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.

Isolation and identification of multidrug-resistant Staphylococcus haemolyticus from a laboratory-breeding mouse.

PubMed

Huang, Fengying; Meng, Qiuping; Tan, Guanghong; Huang, Yonghao; Wang, Hua; Mei, Wenli; Dai, Haofu

2011-06-01

To analysis and identify a bacterium strain isolated from laboratory breeding mouse far away from a hospital. Phenotype of the isolate was investigated by conventional microbiological methods, including Gram-staining, colony morphology, tests for haemolysis, catalase, coagulase, and antimicrobial susceptibility test. The mecA and 16S rRNA genes were amplified by the polymerase chain reaction (PCR) and sequenced. The base sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by phylogenetic analysis and multiple sequence alignment. The isolate in this study was a gram positive, coagulase negative, and catalase positive coccus. The isolate was resistant to oxacillin, methicillin, penicillin, ampicillin, cefazolin, ciprofloxacin erythromycin, et al. PCR results indicated that the isolate was mecA gene positive and its 16S rRNA was 1 465 bp. Phylogenetic analysis of the resultant 16S rRNA indicated the isolate belonged to genus Saphylococcus, and multiple sequence alignment showed that the isolate was Saphylococcus haemolyticus with only one base difference from the corresponding 16S rRNA deposited in the GenBank. 16S rRNA gene sequencing is a suitable technique for non-specialist researchers. Laboratory animals are possible sources of lethal pathogens, and researchers must adapt protective measures when they manipulate animals. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Intra- and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus: implications for molecular identification and phylogeny on fungal groups.

PubMed

He, Xiao-Lan; Li, Qian; Peng, Wei-Hong; Zhou, Jie; Cao, Xue-Lian; Wang, Di; Huang, Zhong-Qian; Tan, Wei; Li, Yu; Gan, Bing-Cheng

2017-06-26

The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and elongation factor 1-alpha (EF1α) are often used in fungal taxonomy and phylogenetic analysis. As we know, an ideal molecular marker used in molecular identification and phylogenetic studies is homogeneous within species, and interspecific variation exceeds intraspecific variation. However, during our process of performing ITS, RPB2, and EF1α sequencing on the Pleurotus spp., we found that intra-isolate sequence polymorphism might be present in these genes because direct sequencing of PCR products failed in some isolates. Therefore, we detected intra- and inter-isolate variation of the three genes in Pleurotus by polymerase chain reaction amplification and cloning in this study. Results showed that intra-isolate variation of ITS was not uncommon but the polymorphic level in each isolate was relatively low in Pleurotus; intra-isolate variations of EF1α and RPB2 sequences were present in an unexpectedly high amount. The polymorphism level differed significantly between ITS, RPB2, and EF1α in the same individual, and the intra-isolate heterogeneity level of each gene varied between isolates within the same species. Intra-isolate and intraspecific variation of ITS in the tested isolates was less than interspecific variation, and intra-isolate and intraspecific variation of RPB2 was probably equal with interspecific divergence. Meanwhile, intra-isolate and intraspecific variation of EF1α could exceed interspecific divergence. These findings suggested that RPB2 and EF1α are not desirable barcoding candidates for Pleurotus. We also discussed the reason why rDNA and protein-coding genes showed variants within a single isolate in Pleurotus, but must be addressed in further research. Our study demonstrated that intra-isolate variation of ribosomal and protein-coding genes are likely widespread in fungi. This has implications for studies on fungal evolution, taxonomy, phylogenetics, and population genetics. More extensive sampling of these genes and other candidates will be required to ensure reliability as phylogenetic markers and DNA barcodes.

Oligonucleotide Array for Identification and Detection of Pythium Species†

PubMed Central

Tambong, J. T.; de Cock, A. W. A. M.; Tinker, N. A.; Lévesque, C. A.

2006-01-01

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies. PMID:16597974

Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world

PubMed Central

Visagie, C.M.; Hirooka, Y.; Tanney, J.B.; Whitfield, E.; Mwange, K.; Meijer, M.; Amend, A.S.; Seifert, K.A.; Samson, R.A.

2014-01-01

As part of a worldwide survey of the indoor mycobiota, dust was collected from nine countries. Analyses of dust samples included the culture-dependent dilution-to-extinction method and the culture-independent 454-pyrosequencing. Of the 7 904 isolates, 2 717 isolates were identified as belonging to Aspergillus, Penicillium and Talaromyces. The aim of this study was to identify isolates to species level and describe the new species found. Secondly, we wanted to create a reliable reference sequence database to be used for next-generation sequencing projects. Isolates represented 59 Aspergillus species, including eight undescribed species, 49 Penicillium species of which seven were undescribed and 18 Talaromyces species including three described here as new. In total, 568 ITS barcodes were generated, and 391 β-tubulin and 507 calmodulin sequences, which serve as alternative identification markers. PMID:25492981

Molecular application for identification of polycyclic aromatic hydrocarbons degrading bacteria (PAHD) species isolated from oil polluted soil in Dammam, Saud Arabia.

PubMed

Ibrahim, Mohamed M; Al-Turki, Ameena; Al-Sewedi, Dona; Arif, Ibrahim A; El-Gaaly, Gehan A

2015-09-01

Soil contamination with petroleum hydrocarbon products such as diesel and engine oil is becoming one of the major environmental problems. This study describes hydrocarbons degrading bacteria (PHAD) isolated from long-standing petrol polluted soil from the eastern region, Dammam, Saudi Arabia. The isolated strains were firstly categorized by accessible shape detection, physiological and biochemistry tests. Thereafter, a technique established on the sequence analysis of a 16S rDNA gene was used. Isolation of DNA from the bacterial strains was performed, on which the PCR reaction was carried out. Strains were identified based on 16S rDNA sequence analysis, As follows amplified samples were spontaneously sequenced automatically and the attained results were matched to open databases. Among the isolated bacterial strains, S1 was identified as Staphylococcus aureus and strain S1 as Corynebacterium amycolatum.

Digital data for quick response (QR) codes of alkalophilic Bacillus pumilus to identify and to compare bacilli isolated from Lonar Crator Lake, India.

PubMed

Rekadwad, Bhagwan N; Khobragade, Chandrahasya N

2016-06-01

Microbiologists are routinely engaged isolation, identification and comparison of isolated bacteria for their novelty. 16S rRNA sequences of Bacillus pumilus were retrieved from NCBI repository and generated QR codes for sequences (FASTA format and full Gene Bank information). 16SrRNA were used to generate quick response (QR) codes of Bacillus pumilus isolated from Lonar Crator Lake (19° 58' N; 76° 31' E), India. Bacillus pumilus 16S rRNA gene sequences were used to generate CGR, FCGR and PCA. These can be used for visual comparison and evaluation respectively. The hyperlinked QR codes, CGR, FCGR and PCA of all the isolates are made available to the users on a portal https://sites.google.com/site/bhagwanrekadwad/. This generated digital data helps to evaluate and compare any Bacillus pumilus strain, minimizes laboratory efforts and avoid misinterpretation of the species.

Isolation of a variant Porphyromonas sp. from polymicrobial infections in central bearded dragons (Pogona vitticeps).

PubMed

Bemis, David A; Greenacre, Cheryl B; Bryant, Mary Jean; Jones, Rebekah D; Kania, Stephen A

2011-01-01

Isolates of gram-negative anaerobic bacteria from reptiles have only occasionally been identified to the genus and species level in the veterinary medical literature. In particular, reports identifying Porphyromonas spp. from infections in reptiles are scarce. The present report describes unique Porphyromonas isolates obtained from necrosuppurative infections in central bearded dragons (Pogona vitticeps). The isolates grew in the presence of oxygen, were strongly hemolytic, and did not produce detectable black, iron porphyrin pigment. Biochemical identification kit numeric biocodes gave high but unreliable probabilities (>99.9%) for identification as Porphyromonas gingivalis. Partial 16S ribosomal RNA gene sequences of the isolates were identical to each other and shared 91% identity with those of Porphyromonas gulae. The isolates may represent a new reptile-associated Porphyromonas species.

Identification of a novel endophytic Bacillus sp. from Capsicum annuum with highly efficient and broad spectrum plant probiotic effect.

PubMed

Jasim, B; Mathew, J; Radhakrishnan, E K

2016-10-01

The study mainly aimed the isolation and characterization of plant probiotic endophytic bacteria from Capsicum annuum to explore its multipotent agricultural applications. Endophytic bacteria were isolated from the surface sterilized fruit tissue. The isolates were then subjected to PCR-based screening for the presence of potential biosynthetic gene clusters. The PCR positive isolate was then analysed for its inhibitory effect towards fungal and bacterial pathogens. The compounds responsible for the antimicrobial activity was purified from large scale culture and subjected to identification by LC-MS/MS. The ability of the selected isolate in plant growth enhancement was also done using Vigna radiata seedlings. In this study, an endophytic bacterium isolated from C. annuum was found to have the phenotypic and genetic basis for broad antimicrobial property. PCR-based sequence analysis has resulted in the identification of nonribosomal peptide synthases, PKS Type I, Iturin, surfactin, DAPG and gacA genes in the selected isolate CaB 5. The bioactivity-guided fractionation using column and HPLC purification of active fraction followed by LC-MS/MS analysis has proved the presence of surfactin derivatives (M+H(+) - 1008 & 1036) and iturin (M+H(+) - 1058) as the basis of antimicrobial activity of CaB 5. The isolate was identified as a novel Bacillus sp. because of its low (76%) identity to the reported sequences. Endophytes are considered to have the genetic basis for a diverse array of bioactive metabolites which can have significant applications in both pharmaceutical industry and agriculture. The identification of CaB 5 with broad bioactivity and excellent plant growth enhancement on taxonomically distinct plant species as explained in current study and our previous reports highlights its plant probiotic applicability. This proves the potential of the isolate obtained in the study to be an excellent plant probiotic. © 2016 The Society for Applied Microbiology.

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

PubMed

Mulet, M; Gomila, M; Ramírez, A; Cardew, S; Moore, E R B; Lalucat, J; García-Valdés, E

2017-02-01

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identification of M. tuberculosis and non-tuberculosis Mycobacterium species.

PubMed

Şamlı, Asuman; İlki, Arzu

2016-10-01

Mycobacteria are an important cause of morbidity in humans. Rapid and accurate mycobacterial identification is important for improving patient outcomes. However, identification of Mycobacterium species is not easy, due to the slow and fastidious growth of mycobacteria. Recently, biochemical, sequencing, and probing methods have come to be used for identification. This study compared the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of M.tuberculosis and non-tuberculosis Mycobacteria (NTM) to those of nucleic acid hybridization (NAH) and the MPT64 immunochromatographic test. A total of 69 isolates from Marmara University Hospital, Microbiology Laboratory obtained between 2012 and 2013 were included in our study. All strains were grown on Lowenstein-Jensen and Middlebrook 7H9 medium. Among the 69 isolates, 56 (81%) were isolated as Mycobacterium tuberculosis complex (MTC), and 13 (19%) were isolated as NTM by the MPT64 ICT. NAH was able to identify all isolates to the species level. The isolated NTM included M. intracellulare (n:5), M. lentiflavum (n:3), M. xenopi (n:2), M. malmoense (n:1), M. abscessus (n:1), and M. avium (n:1). MALDI-TOF MS identified 88% of the mycobacterial isolates. All M. tuberculosis strains were identified correctly, but the ratio was 38.5% for NTM. Mycobacterial identification using MALDI-TOF MS takes 45 minutes and costs 3 Euro/test, whereas mycobacterial identification using NAH takes 6-7 hours and costs 30 Euro/test. In conclusion, MALDI-TOF MS has the potential to identify mycobacteria in the clinical laboratory setting by reducing identification turnaround time and laboratory costs for isolate referral.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

PubMed

Richter, S S; Sercia, L; Branda, J A; Burnham, C-A D; Bythrow, M; Ferraro, M J; Garner, O B; Ginocchio, C C; Jennemann, R; Lewinski, M A; Manji, R; Mochon, A B; Rychert, J A; Westblade, L F; Procop, G W

2013-12-01

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l'Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer's instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7% of the 965 isolates tested, with 83.8% correct to the species level and 12.8% limited to a genus-level identification. There was no identification for 1.7% of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster).

PubMed

Roriz, Mariana; Santos, Carla; Vasconcelos, Marta W

2011-08-01

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera. The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA. The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines. In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate. Copyright © 2011 Elsevier Inc. All rights reserved.

The population structure of Vibrio cholerae from the Chandigarh Region of Northern India.

PubMed

Abd El Ghany, Moataz; Chander, Jagadish; Mutreja, Ankur; Rashid, Mamoon; Hill-Cawthorne, Grant A; Ali, Shahjahan; Naeem, Raeece; Thomson, Nicholas R; Dougan, Gordon; Pain, Arnab

2014-07-01

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century. Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters. The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.

Multicenter Evaluation of the Vitek MS v3.0 System for the Identification of Filamentous Fungi.

PubMed

Rychert, Jenna; Slechta, E Sue; Barker, Adam P; Miranda, Edwin; Babady, N Esther; Tang, Yi-Wei; Gibas, Connie; Wiederhold, Nathan; Sutton, DeAnna; Hanson, Kimberly E

2018-02-01

Invasive fungal infections are an important cause of morbidity and mortality affecting primarily immunocompromised patients. While fungal identification to the species level is critical to providing appropriate therapy, it can be slow and laborious and often relies on subjective morphological criteria. The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has the potential to speed up and improve the accuracy of identification. In this multicenter study, we evaluated the accuracy of the Vitek MS v3.0 system in identifying 1,601 clinical mold isolates compared to identification by DNA sequence analysis and supported by morphological and phenotypic testing. Among the 1,519 isolates representing organisms in the v3.0 database, 91% ( n = 1,387) were correctly identified to the species level. An additional 27 isolates (2%) were correctly identified to the genus level. Fifteen isolates were incorrectly identified, due to either a single incorrect identification ( n = 13) or multiple identifications from different genera ( n = 2). In those cases, when a single identification was provided that was not correct, the misidentification was within the same genus. The Vitek MS v3.0 was unable to identify 91 (6%) isolates, despite repeat testing. These isolates were distributed among all the genera. When considering all isolates tested, even those that were not represented in the database, the Vitek MS v3.0 provided a single correct identification 98% of the time. These findings demonstrate that the Vitek MS v3.0 system is highly accurate for the identification of common molds encountered in the clinical mycology laboratory. Copyright © 2018 American Society for Microbiology.

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

PubMed

Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

2013-08-01

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of novel bacteriophage peptides using a combination of gene sequence LC-MS-MS analysis and BLASTP

USDA-ARS?s Scientific Manuscript database

Introduction: In an effort to characterize novel bacteriophage with lytic activity against pathogenic E.coli associated with foodborne illness, gene sequencing and mass spectrometry have been used to identify expressed peptides which differentiate isolated bacteriophage from other known phage. Here,...

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.

PubMed

Garcia, Virginie; Bres, Cécile; Just, Daniel; Fernandez, Lucie; Tai, Fabienne Wong Jun; Mauxion, Jean-Philippe; Le Paslier, Marie-Christine; Bérard, Aurélie; Brunel, Dominique; Aoki, Koh; Alseekh, Saleh; Fernie, Alisdair R; Fraser, Paul D; Rothan, Christophe

2016-12-01

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F 2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F 2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

PubMed

Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

2017-06-28

The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

Use of 16S rRNA Sequencing for Identification of Actinobacillus ureae Isolated from a Cerebrospinal Fluid Sample

PubMed Central

Whitelaw, A. C.; Shankland, I. M.; Elisha, B. G.

2002-01-01

Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene. PMID:11825992

Identification and characterization of Highlands J virus from a Mississippi sandhill crane using unbiased next-generation sequencing

USGS Publications Warehouse

Ip, Hon S.; Wiley, Michael R.; Long, Renee; Gustavo, Palacios; Shearn-Bochsler, Valerie; Whitehouse, Chris A.

2014-01-01

Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequencing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus, NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the application of NGS for the rapid identification and characterization of a virus isolated from the brain of an endangered Mississippi sandhill crane. This bird was part of a population restoration effort and was found in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012. Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arboviral etiology was strongly suspected. Because individual molecular assays for several known arboviruses were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the causative viral agent. Whole genome sequencing and phylogenetic analysis revealed the viral isolate to be the Highlands J virus, a known avian pathogen. This study demonstrates the use of unbiased NGS for the rapid detection and characterization of an unidentified viral pathogen and the application of this technology to wildlife disease diagnostics and conservation medicine.

Isolation, identification and screening of antimicrobial thermophilic Streptomyces sp. Al-Dhabi-1 isolated from Tharban hot spring, Saudi Arabia.

PubMed

Al-Dhabi, Naif Abdullah; Esmail, Galal Ali; Duraipandiyan, Veeramuthu; Valan Arasu, Mariadhas; Salem-Bekhit, Mounir M

2016-01-01

The strain Streptomyces sp. Al-Dhabi-1 was isolated from soil sediments collected from Tharban hot spring in the southern west of Saudi Arabia using actinomycetes isolation agar and starch casein agar at 55 °C. Identification of the isolate was done according to morphological, physiological and biochemical characteristics and 16S rRNA sequence similarity as well. 16S rRNA sequence and blast analyses confirmed that the isolate belonging to the genus Streptomyces. The sequence was submitted to GenBank with accession number (KF815080). Ethyl acetate extract of Streptomyces sp. Al-Dhabi-1 showed good antimicrobial activities against tested pathogenic microbes. Minimum inhibitory concentration results showed that the best values were observed against S. agalactiae (<0.039 mg/ml) and Klebsiella pneumonia (0.125 mg/ml). Minimum inhibitory concentration of Al-Dhabi-1 against fungi; Cryptococcus neoformans (0.078 mg/ml), C. albicans (0.156 mg/ml), A. niger (0.625 mg/ml), and T. mentagrophytes (0.156 mg/ml). GC-MS analysis was used for the chemical profile of ethyl acetate extract. Benzeneacetic acid (16.02 %) and acetic acid 2-phenylethyl ester (10.35 %) were the major compounds among 31 substances found the ethyl acetate extract. According to the results of antimicrobial activity against pathogenic microbes, it is clear that the actinomycetes from hot springs with extreme environments are promising source for antimicrobial compounds.

[Morphological and molecular characterization of isolates of Macrophomina phaseolina associated with sugarcane in Mexico].

PubMed

Leyva-Mir, Santos G; Velázquez-Martínez, Guadalupe C; Tlapal-Bolaños, Bertha; Tovar-Pedraza, Juan M; Rosas-Saito, Greta H; Alvarado-Gómez, Omar G

2015-01-01

Charcoal rot caused by Macrophomina phaseolina is an important disease of sugarcane in Mexico. This study was carried out to characterize isolates of M. phaseolina obtained from sugarcane by the combination of morphological and molecular analyses. The morphological characterization of 10 isolates was performed using scanning electron microscopy and light microscopy. To confirm the morphological identification, rDNA from two representative isolates was extracted, and the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction and sequenced using specific primers MpKF1 and MpKR1. Based on their morphological characteristics, all isolates were identified as M. phaseolina. Moreover, the analysis of two ITS sequences showed 100% similarity with the M. phaseolina sequences deposited in the GenBank. To our knowledge, this is the first study in the world aimed at characterizing isolates of M. phaseolina obtained from sugarcane. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria

PubMed Central

Srinivasan, Sujatha; Munch, Matthew M.; Sizova, Maria V.; Fiedler, Tina L.; Kohler, Christina M.; Hoffman, Noah G.; Liu, Congzhou; Agnew, Kathy J.; Marrazzo, Jeanne M.; Epstein, Slava S.; Fredricks, David N.

2016-01-01

Background. Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. Methods. Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. Results. We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. Conclusions. We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously “uncultivated” bacteria are amenable to conventional cultivation. PMID:27449870

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium.

PubMed

Eom, Keeseon S; Chai, Jong-Yil; Yong, Tai-Soon; Min, Duk-Young; Rim, Han-Jong; Kihamia, Charles; Jeon, Hyeong-Kyu

2011-12-01

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).

Mimosa caesalpiniifolia rhizobial isolates from different origins of the Brazilian Northeast.

PubMed

Martins, Paulo Geovani Silva; Junior, Mario Andrade Lira; Fracetto, Giselle Gomes Monteiro; da Silva, Maria Luiza Ribeiro Bastos; Vincentin, Rayssa Pereira; de Lyra, Maria do Carmo Catanho Pereira

2015-04-01

Biological nitrogen fixation from the legume-rhizobia symbiosis is one of the main sources of fixed nitrogen on land environments. Diazotrophic bacteria taxonomy has been substantially modified by the joint use of phenotypic, physiological and molecular aspects. Among these molecular tools, sequencing and genotyping of genomic regions such as 16S rDNA and repetitive conserved DNA regions have boosted the accuracy of species identification. This research is a phylogenetic study of diazotrophic bacteria from sabiá (Mimosa caesalpiniifolia Benth.), inoculated with soils from five municipalities of the Brazilian Northeast. After bacterial isolation and morphophysiological characterization, genotyping was performed using REP, ERIC and BOX oligonucleotides and 16S rDNA sequencing for genetic diversity identification. A 1.5b Kb fragment of the 16S rDNA was amplified from each isolate. Morphophysiological characterization of the 47 isolates created a dendrogram, where isolate PE-GR02 formed a monophyletic branch. The fingerprinting conducted with BOX, ERIC and REP shows distinct patterns, and their compilation created a dendrogram with diverse groups and, after blasting in GenBank, resulted in genetic identities ranging from 77 to 99 % with Burkholderia strains. The 16S rDNA phylogenetic tree constructed with these isolates and GenBank deposits of strains recommended for inoculant production confirm these isolates are distinct from the previously deposited strains, whereas isolates PE-CR02, PE-CR4, PE-CR07, PE-CR09 and PE-GE06 were the most distinct within the group. Morphophysiological characterization and BOX, ERIC and REP compilation enhanced the discrimination of the isolates, and the 16S rDNA sequences compared with GenBank confirmed the preference of Mimosa for Burkholderia diazotrophic bacteria.

Molecular identification and amphotericin B susceptibility testing of clinical isolates of Aspergillus from 11 hospitals in Korea.

PubMed

Heo, Min Seok; Shin, Jong Hee; Choi, Min Ji; Park, Yeon Joon; Lee, Hye Soo; Koo, Sun Hoe; Lee, Won Gil; Kim, Soo Hyun; Shin, Myung Geun; Suh, Soon Pal; Ryang, Dong Wook

2015-11-01

We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and β-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by β-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of ≥2 μg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was ≤75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.

Mistaken identity of a PCR target proposed for identification of Mycoplasma bovis and the effect of sequence variation on assay performance

USDA-ARS?s Scientific Manuscript database

Background. Mycoplasma bovis is an important cause of disease in cattle and has recently emerged as a primary disease agent in bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories use PCR to replace or complement traditional isolation and identification ...

Diagnostics of Neisseriaceae and Moraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms

PubMed Central

Harmsen, Dag; Singer, Christian; Rothgänger, Jörg; Tønjum, Tone; Sybren de Hoog, Gerrit; Shah, Haroun; Albert, Jürgen; Frosch, Matthias

2001-01-01

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes. PMID:11230407

Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus

PubMed Central

2011-01-01

Background Streptococcus is an economically important genus as a number of species belonging to this genus are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene sequence similarity. The variability observed among the members of these groups is low and it is difficult to distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that can be used for preliminary identification and can supplement the existing methods for identification of clinically-relevant isolates of the genus Streptococcus. Methods 16S rRNA gene sequences belonging to the isolates of S. dysgalactiae, S. equi, S. pyogenes, S. agalactiae, S. bovis, S. gallolyticus, S. mutans, S. sobrinus, S. mitis, S. pneumoniae, S. thermophilus and S. anginosus were analyzed with the purpose to define genetic variability within each species to generate a phylogenetic framework, to identify species-specific signatures and in-silico restriction enzyme analysis. Results The framework based analysis was used to segregate Streptococcus spp. previously identified upto genus level. This segregation was validated using species-specific signatures and in-silico restriction enzyme analysis. 43 uncharacterized Streptococcus spp. could be identified using this approach. Conclusions The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus Streptococcus. PMID:21702978

Laboratory Diagnosis and Susceptibility Testing for Mycobacterium tuberculosis.

PubMed

Procop, Gary W

2016-12-01

The laboratory, which utilizes some of the most sophisticated and rapidly changing technologies, plays a critical role in the diagnosis of tuberculosis. Some of these tools are being employed in resource-challenged countries for the rapid detection and characterization of Mycobacterium tuberculosis. Foremost, the laboratory defines appropriate specimen criteria for optimal test performance. The direct detection of mycobacteria in the clinical specimen, predominantly done by acid-fast staining, may eventually be replaced by rapid-cycle PCR. The widespread use of the Xpert MTB/RIF (Cepheid) assay, which detects both M. tuberculosis and key genetic determinants of rifampin resistance, is important for the early detection of multidrug-resistant strains. Culture, using both broth and solid media, remains the standard for establishing the laboratory-based diagnosis of tuberculosis. Cultured isolates are identified far less commonly by traditional biochemical profiling and more commonly by molecular methods, such as DNA probes and broad-range PCR with DNA sequencing. Non-nucleic acid-based methods of identification, such as high-performance liquid chromatography and, more recently, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, may also be used for identification. Cultured isolates of M. tuberculosis should be submitted for susceptibility testing according to standard guidelines. The use of broth-based susceptibility testing is recommended to significantly decrease the time to result. Cultured isolates may also be submitted for strain typing for epidemiologic purposes. The use of massive parallel sequencing, also known as next-generation sequencing, promises to continue to this molecular revolution in mycobacteriology, as whole-genome sequencing provides identification, susceptibility, and typing information simultaneously.

Pseudomonas sp. strain CA5 (a selenite-reducing bacterium) 16S rRNA gene complete sequence. National Institute of Health, National Center for Biotechnology Information, GenBank sequence. Accession FJ422810.1.

USDA-ARS?s Scientific Manuscript database

This study used 1321 base pair 16S rRNA gene sequence methods to confirm the phylogenetic position of a soil isolate as a bacterium belonging to the genus Pesudomonas sp. Morphological, biochemical characteristics, and fatty acid profiles are consistent with the 16S rRNA gene sequence identification...

Genomic characterization of two new enterovirus types, EV-A114 and EV-A121.

PubMed

Deshpande, Jagadish M; Sharma, Deepa K; Saxena, Vinay K; Shetty, Sushmitha A; Qureshi, Tarique Husain I H; Nalavade, Uma P

2016-12-01

Enteroviruses cause a variety of illnesses of the gastrointestinal tract, central nervous system and cardiovascular system. Phylogenetic analysis of VP1 sequences has identified 106 different human enteroviruses classified into four enterovirus species within the genus Enterovirus of the family Picornaviridae. It is likely that not all enterovirus types have been discovered. Between September 2013 and October 2014, stool samples of 6274 apparently healthy children of up to 5 years of age residing in Gorakhpur district, Uttar Pradesh, India were screened for enteroviruses. Virus isolates obtained in RD and Hep-2c cells were identified by complete VP1 sequencing. Enteroviruses were isolated from 3042 samples. A total of 87 different enterovirus types were identified. Two isolates with 71 and 74 % nucleotide sequence similarity to all other known enteroviruses were recognized as novel types. In this paper we report identification and complete genome sequence analysis of these two isolates classified as EV-A114 and EV-A121.

Detection and identification of cutaneous leishmaniasis isolates by culture, Polymerase chain reaction and sequence analyses in Syrian and Central Anatolia patients.

PubMed

Beyhan, Yunus E; Karakus, Mehmet; Karagoz, Alper; Mungan, Mesut; Ozkan, Aysegul T; Hokelek, Murat

2017-09-01

To characterize the cutaneous leishmaniasis (CL) isolates of Syrian and Central Anatolia patients at species levels. Methods: Skin scrapings of 3 patients (2 Syrian, 1 Turkish) were taken and examined by direct examination, culture in Novy-MacNeal-Nicole (NNN) medium, internal transcribed spacer polymerase chain reaction and sequence analysis (PCR). Results:According to microscopic examination, culture and PCR methods, 3 samples were detected positive. The sequencing results of all isolates in the study were identified as Leishmania tropica. The same genotypes were detected in the 3 isolates and nucleotide sequence submitted into GenBank with the accession number: KP689599. Conclusion: This finding could give information about the transmission of CL between Turkey and Syria. Because of the Syrian civil war, most of the Syrian citizens circulating in Turkey and different part of Europe, this can be increase the risk of spreading the disease. So, prevention measurements must be taken urgently.

Identification of risk factors and causes of persistence of Salmonella Gallinarum in laying hens farms from Colombia

USDA-ARS?s Scientific Manuscript database

The presence of Salmonella Gallinarum (SG) was recently identified in brown egg layers in Colombia. During 2013, twenty isolates were analyzed using Intergenic Sequence Ribotyping. Eighteen (90%) were SG and two (10%) were Salmonella Enteritidis (SE). SG was isolated mainly from organs of sick birds...

Clinical Significance and Characterization of Streptococcus tigurinus Isolates in an Adult Population.

PubMed

Bourassa, Lori; Clarridge, J E

2015-11-01

Streptococcus tigurinus is a newly described member of the Streptococcus mitis group. Due to the difficulty in distinguishing viridans group streptococci (VGS) by phenotype, analysis of 16S rRNA sequences is necessary for the accurate identification of most species. Through a laboratory policy of analyzing all clinically significant isolates from the VGS group by16S rRNA gene sequencing, we identified 14 S. tigurinus isolates from 11 patients. The Vitek 2 system most commonly gave an excellent rating to an incorrect identification (e.g., Streptococcus mitis), as did matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (e.g., Streptococcus oralis). S. tigurinus strains were recovered from numerous body sites, including the blood, peritoneal fluid, bone, synovial fluid, a perianal abscess, and an arm wound. Retrospective chart review indicated that most isolates were clinically significant, with bacteremia (n = 5), soft tissue infections (n = 3) osteomyelitis (n = 2), infected joint prosthesis (n = 2), and peritonitis (n = 2) being the most common, thus expanding the spectrum of disease associated with S. tigurinus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Molecular epidemiological analysis of rubella virus isolates from 2001 to 2011 in Shanghai, China].

PubMed

Li, Chong-Shan; Yang, Yu-Ying; Wang, Jian-Guo; Zhu, Zhen; Tang, Wei; Li, Zhi; Sun, Xiao-Dong; Xu, Wen-Bo

2012-03-01

Throat swabs collected from patients whose serum was measles IgM negative and rubella IgM positive during 2001-2011 were used to conduct cell culture for rubella virus. After identification of cell culture with RT-PCR, nucleotide of gene E1 of rubella virus was amplified and sequenced, followed by molecular epidemiological analysis. A total of 31 rubella viruses were isolated from 60 throat swabs. Compared 27 isolates with the WHO reference strains of all genotypes, phylogenetic tree was constructed based on the amplified 739 nucleotide fragment. These isolates belonged to two different genotypes respectively. Isolates 11009, 11052 and 11106 in 2011 belonged to genotype 2B, and others belonged to genotype 1E. Most of mutations were nonsense mutation, and sequence of amino acid was highly conserved. Amino acid sequence of most isolates of genotype 1E was identical, which suggested rubella viruses from same transmission chain might be transmitted continually since 2001. Rubella virus genotype 2B was found to be popular for the first time in Shanghai in 2011. The nucleotide sequences of these genotype 2B isolates showed 99% identity compared with that of isolates recently from Vietnam, Japan and Argentina. The resources of these strains were not confirmed due to the absence of rubella virus surveillance before.

Potential for biocontrol of melanized fungi by actinobacteria isolated from intertidal region of Ilha Do Mel, Paraná, Brazil.

PubMed

Dalitz, Camila de Araújo; Porsani, Mariana Vieira; Figel, Izabel Cristina; Pimentel, Ida C; Dalzoto, Patrícia R

Actinobacteria occur in many environments and have the capacity to produce secondary metabolites with antibiotic potential. Identification and taxonomy of actinobacteria that produce antimicrobial substances is essential for the screening of new compounds, and sequencing of the 16S region of ribosomal DNA (rDNA), which is conserved and present in all bacteria, is an important method of identification. Melanized fungi are free-living organisms, which can also be pathogens of clinical importance. This work aimed to evaluate growth inhibition of melanized fungi by actinobacteria and to identify the latter to the species level. In this study, antimicrobial activity of 13 actinobacterial isolates from the genus Streptomyces was evaluated against seven melanized fungi of the genera Exophiala, Cladosporium, and Rhinocladiella. In all tests, all actinobacterial isolates showed inhibitory activity against all isolates of melanized fungi, and only one actinobacterial isolate had less efficient inhibitory activity. The 16S rDNA region of five previously unidentified actinobacterial isolates from Ilha do Mel, Paraná, Brazil, was sequenced; four of the isolates were identified as Streptomyces globisporus subsp. globisporus, and one isolate was identified as Streptomyces aureus. This work highlights the potential of actinobacteria with antifungal activity and their role in the pursuit of novel antimicrobial substances. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Molecular Identification of Malassezia Species in Patients with Malassezia folliculitis in Sfax, Tunisia.

PubMed

Cheikhrouhou, F; Guidara, R; Masmoudi, A; Trabelsi, H; Neji, S; Sellami, H; Makni, F; Ayadi, A

2017-06-01

Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome. Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm. Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes. There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

Impact of cultivation on characterisation of species composition of soil bacterial communities.

PubMed

McCaig, A E.; Grayston, S J.; Prosser, J I.; Glover, L A.

2001-03-01

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Biodiversity of Environmental Leptospira: Improving Identification and Revisiting the Diagnosis.

PubMed

Thibeaux, Roman; Girault, Dominique; Bierque, Emilie; Soupé-Gilbert, Marie-Estelle; Rettinger, Anna; Douyère, Anthony; Meyer, Michael; Iraola, Gregorio; Picardeau, Mathieu; Goarant, Cyrille

2018-01-01

Leptospirosis is an important environmental disease and a major threat to human health causing at least 1 million clinical infections annually. There has recently been a growing interest in understanding the environmental lifestyle of Leptospira . However, Leptospira isolation from complex environmental samples is difficult and time-consuming and few tools are available to identify Leptospira isolates at the species level. Here, we propose a polyphasic isolation and identification scheme, which might prove useful to recover and identify environmental isolates and select those to be submitted to whole-genome sequencing. Using this approach, we recently described 12 novel Leptospira species for which we propose names. We also show that MALDI-ToF MS allows rapid and reliable identification and provide an extensive database of Leptospira MALDI-ToF mass spectra, which will be valuable to researchers in the leptospirosis community for species identification. Lastly, we also re-evaluate some of the current techniques for the molecular diagnosis of leptospirosis taking into account the extensive and recently revealed biodiversity of Leptospira in the environment. In conclusion, we describe our method for isolating Leptospira from the environment, confirm the usefulness of mass spectrometry for species identification and propose names for 12 novel species. This also offers the opportunity to refine current molecular diagnostic tools.

Occurrence and characterization of hitherto unknown Streptomyces species in semi-arid soils.

PubMed

Kumar, Surendra; Priya, E; Singh Solanki, Dilip; Sharma, Ruchika; Gehlot, Praveen; Pathak, Rakesh; Singh, S K

2016-09-01

Streptomyces the predominant genus of Actinobacteria and plays an important role in the recycling of soil organic matter and production of important secondary metabolites. The occurrence and diversity assessment of Streptomyces species revealed alkaline and poor nutrient status of soils of semi-arid region of Jodhpur, Rajasthan. The morphological and biochemical characterization of 21 Streptomyces isolates facilitated Genus level identification but were insufficient to designate species. Species designation based on 16S rRNA gene delineated 21 isolates into 14 Streptomyces species. Upon BLAST search, the test isolates exhibited 98 to 100% identities with that of the best aligned sequences of the NCBI database. The GC content of 16S rRNA gene sequences of all the Streptomyces isolates tested ranged from 59.03% to 60.94%. The multiple sequence alignment of all the 21 Streptomyces isolates generated a phylogram with high bootstrap values indicating reliable grouping of isolates based on nucleotide sequence variations by way of insertion, deletion and substitutions and 16S rRNA length polymorphism. Some of the Streptomyces species molecularly identified under present study are reported for the first time from semi-arid region of Jodhpur.

Using msa-2b as a molecular marker for genotyping Mexican isolates of Babesia bovis.

PubMed

Genis, Alma D; Perez, Jocelin; Mosqueda, Juan J; Alvarez, Antonio; Camacho, Minerva; Muñoz, Maria de Lourdes; Rojas, Carmen; Figueroa, Julio V

2009-12-01

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2) and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR-RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.

Comparative analysis of the L, M, and S RNA segments of Crimean-Congo haemorrhagic fever virus isolates from southern Africa.

PubMed

Goedhals, Dominique; Bester, Phillip A; Paweska, Janusz T; Swanepoel, Robert; Burt, Felicity J

2015-05-01

Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family with a tripartite, negative sense RNA genome. This study used predictive software to analyse the L (large), M (medium), and S (small) segments of 14 southern African CCHFV isolates. The OTU-like cysteine protease domain and the RdRp domain of the L segment are highly conserved among southern African CCHFV isolates. The M segment encodes the structural glycoproteins, GN and GC, and the non-structural glycoproteins which are post-translationally cleaved at highly conserved furin and subtilase SKI-1 cleavage sites. All of the sites previously identified were shown to be conserved among southern African CCHFV isolates. The heavily O-glycosylated N-terminal variable mucin-like domain of the M segment shows the highest sequence variability of the CCHFV proteins. Five transmembrane domains are predicted in the M segment polyprotein resulting in three regions internal to and three regions external to the membrane across the G(N), NS(M) and G(C) glycoproteins. The corroboration of conserved genome domains and sequence identity among geographically diverse isolates may assist in the identification of protein function and pathogenic mechanisms, as well as the identification of potential targets for antiviral therapy and vaccine design. As detailed functional studies are lacking for many of the CCHFV proteins, identification of functional domains by prediction of protein structure, and identification of amino acid level similarity to functionally characterised proteins of related viruses or viruses with similar pathogenic mechanisms are a necessary step for selection of areas for further study. © 2015 Wiley Periodicals, Inc.

Digital data for quick response (QR) codes of alkalophilic Bacillus pumilus to identify and to compare bacilli isolated from Lonar Crator Lake, India

PubMed Central

Rekadwad, Bhagwan N.; Khobragade, Chandrahasya N.

2016-01-01

Microbiologists are routinely engaged isolation, identification and comparison of isolated bacteria for their novelty. 16S rRNA sequences of Bacillus pumilus were retrieved from NCBI repository and generated QR codes for sequences (FASTA format and full Gene Bank information). 16SrRNA were used to generate quick response (QR) codes of Bacillus pumilus isolated from Lonar Crator Lake (19° 58′ N; 76° 31′ E), India. Bacillus pumilus 16S rRNA gene sequences were used to generate CGR, FCGR and PCA. These can be used for visual comparison and evaluation respectively. The hyperlinked QR codes, CGR, FCGR and PCA of all the isolates are made available to the users on a portal https://sites.google.com/site/bhagwanrekadwad/. This generated digital data helps to evaluate and compare any Bacillus pumilus strain, minimizes laboratory efforts and avoid misinterpretation of the species. PMID:27141529

PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis▿

PubMed Central

Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Van Vinh Chau, Nguyen; Chinh, Nguyen Tran; Farrar, Jeremy

2007-01-01

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939

Evaluation of the Biotyper MALDI-TOF MS system for identification of Staphylococcus species.

PubMed

Zhu, Wenming; Sieradzki, Krzysztof; Albrecht, Valerie; McAllister, Sigrid; Lin, Wen; Stuchlik, Olga; Limbago, Brandi; Pohl, Jan; Kamile Rasheed, J

2015-10-01

The Bruker Biotyper MALDI-TOF MS (Biotyper) system, with a modified 30 minute formic acid extraction method, was evaluated by its ability to identify 216 clinical Staphylococcus isolates from the CDC reference collection comprising 23 species previously identified by conventional biochemical tests. 16S rDNA sequence analysis was used to resolve discrepancies. Of these, 209 (96.8%) isolates were correctly identified: 177 (84.7%) isolates had scores ≥2.0, while 32 (15.3%) had scores between 1.70 and 1.99. The Biotyper identification was inconsistent with the biochemical identification for seven (3.2%) isolates, but the Biotyper identifications were confirmed by 16S rDNA analysis. The distribution of low scores was strongly species-dependent, e.g. only 5% of Staphylococcus epidermidis and 4.8% of Staphylococcus aureus isolates scored below 2.0, while 100% of Staphylococcus cohnii, 75% of Staphylococcus sciuri, and 60% of Staphylococcus caprae produced low but accurate Biotyper scores. Our results demonstrate that the Biotyper can reliably identify Staphylococcus species with greater accuracy than conventional biochemicals. Broadening of the reference database by inclusion of additional examples of under-represented species could further optimize Biotyper results. Published by Elsevier B.V.

Molecular identification of Fasciola spp. (Digenea: Platyhelminthes) in cattle from Vietnam

PubMed Central

Nguyen, S.; Amer, S.; Ichikawa, M.; Itagaki, T.; Fukuda, Y.; Nakai, Y.

2012-01-01

Fasciola spp. were collected from naturally infected cattle at a local abattoir of Khanh Hoa province, Vietnam, for morphological and genetic investigations. Microscopic examination detected no sperm cells in the seminal vesicles, suggesting a parthenogenetic reproduction of the flukes. Analyses of sequences from the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA revealed that 13 out of 16 isolates were of Fasciola gigantica type, whereas three isolates presented a hybrid sequence from F. gigantica and Fasciola hepatica. Interestingly, all the mitochondrial sequences (partial COI and NDI) were of F. gigantica type, suggesting that the maternal lineage of the hybrid form is from F. gigantica. No intra-sequence variation was detected. PMID:22314245

New Genome Sequence of an Echinacea purpurea Endophyte, Arthrobacter sp. Strain EpSL27, Able To Inhibit Human-Opportunistic Pathogens

PubMed Central

Miceli, Elisangela; Presta, Luana; Maggini, Valentina; Fondi, Marco; Bosi, Emanuele; Chiellini, Carolina; Fagorzi, Camilla; Bogani, Patrizia; Di Pilato, Vincenzo; Rossolini, Gian Maria; Mengoni, Alessio; Firenzuoli, Fabio; Perrin, Elena

2017-01-01

ABSTRACT We announce here the draft genome sequence of Arthrobacter sp. strain EpSL27, isolated from the stem and leaves of the medicinal plant Echinacea purpurea and able to inhibit human-pathogenic bacterial strains. The genome sequencing of this strain may lead to the identification of genes involved in the production of antimicrobial molecules. PMID:28642378

New Genome Sequence of an Echinaceapurpurea Endophyte, Arthrobacter sp. Strain EpSL27, Able To Inhibit Human-Opportunistic Pathogens.

PubMed

Miceli, Elisangela; Presta, Luana; Maggini, Valentina; Fondi, Marco; Bosi, Emanuele; Chiellini, Carolina; Fagorzi, Camilla; Bogani, Patrizia; Di Pilato, Vincenzo; Rossolini, Gian Maria; Mengoni, Alessio; Firenzuoli, Fabio; Perrin, Elena; Fani, Renato

2017-06-22

We announce here the draft genome sequence of Arthrobacter sp. strain EpSL27, isolated from the stem and leaves of the medicinal plant Echinacea purpurea and able to inhibit human-pathogenic bacterial strains. The genome sequencing of this strain may lead to the identification of genes involved in the production of antimicrobial molecules. Copyright © 2017 Miceli et al.

Isolation and identification of a thermophilic strain producing trehalose synthase from geothermal water in China.

PubMed

Zhu, Yueming; Zhang, Jun; Wei, Dongsheng; Wang, Yufan; Chen, Xiaoyun; Xing, Laijun; Li, Mingchun

2008-08-01

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.

Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Alatoom, Adnan A; Cunningham, Scott A; Ihde, Sherry M; Mandrekar, Jayawant; Patel, Robin

2011-08-01

We evaluated Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 305 clinical isolates of staphylococci, streptococci, and related genera by comparing direct colony testing with preparatory extraction. Isolates were previously identified by use of phenotypic testing and/or 16S rRNA gene sequencing. Manufacturer-specified score cutoffs for genus- and species-level identification were used. After excluding 7 isolates not present in the Biotyper library, the Biotyper correctly identified 284 (95%) and 207 (69%) isolates to the genus and species levels, respectively, using extraction. By using direct colony testing, the Biotyper identified 168 (56%) and 60 (20%) isolates to the genus and species levels, respectively. Overall, more isolates were identified to the genus and species levels with preparatory extraction than with direct colony testing (P < 0.0001). The analysis was repeated after dividing the isolates into two subgroups, staphylococci, streptococci, and enterococci (n = 217) and "related genera" (n = 81). For the former subgroup, the extraction method resulted in the identification of 213 (98%) and 171 (79%) isolates to the genus and species levels, respectively, whereas the direct colony method identified 136 (63%) and 56 (26%) isolates to the genus and species levels, respectively. In contrast, for the subgroup of related genera, the extraction method identified 71 (88%) and 36 (44%) isolates to the genus and species levels, respectively, while the direct colony method identified 32 (40%) and 4 (5%) isolates to the genus and species levels, respectively. For both subgroups, preparatory extraction was superior to direct colony testing for the identification of isolates to the genus and species levels (P < 0.0001). Preparatory extraction is needed for the identification of a substantial proportion of Gram-positive cocci using the Biotyper method according to manufacturer-specified score cutoffs.

Integrated proteomics, genomics, metabolomics approaches reveal oxalic acid as pathogenicity factor in Tilletia indica inciting Karnal bunt disease of wheat.

PubMed

Pandey, Vishakha; Singh, Manoj; Pandey, Dinesh; Kumar, Anil

2018-05-18

Tilletia indica incites Karnal bunt (KB) disease in wheat. To date, no KB resistant wheat cultivar could be developed due to non-availability of potential biomarkers related to pathogenicity/virulence for screening of resistant wheat genotypes. The present study was carried out to compare the proteomes of T. indica highly (TiK) and low (TiP) virulent isolates. Twenty one protein spots consistently observed as up-regulated/differential in the TiK proteome were selected for identification by MALDI-TOF/TOF. Identified sequences showed homology with fungal proteins playing essential role in plant infection and pathogen survival, including stress response, adhesion, fungal penetration, invasion, colonization, degradation of host cell wall, signal transduction pathway. These results were integrated with T. indica genome sequence for identification of homologs of candidate pathogenicity/virulence related proteins. Protein identified in TiK isolate as malate dehydrogenase that converts malate to oxaloacetate which is precursor of oxalic acid. Oxalic acid is key pathogenicity factor in phytopathogenic fungi. These results were validated by GC-MS based metabolic profiling of T. indica isolates indicating that oxalic acid was exclusively identified in TiK isolate. Thus, integrated omics approaches leads to identification of pathogenicity/virulence factor(s) that would provide insights into pathogenic mechanisms of fungi and aid in devising effective disease management strategies.

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

NASA Astrophysics Data System (ADS)

Knolhoff, Ann M.; Zheng, Jie; McFarland, Melinda A.; Luo, Yan; Callahan, John H.; Brown, Eric W.; Croley, Timothy R.

2015-08-01

The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MSn spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows.

PubMed

Marques, S; Huss, V A R; Pfisterer, K; Grosse, C; Thompson, G

2015-05-01

The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

PCR Followed by Electrospray Ionization Mass Spectrometry for Broad-Range Identification of Fungal Pathogens

PubMed Central

Massire, Christian; Buelow, Daelynn R.; Zhang, Sean X.; Lovari, Robert; Matthews, Heather E.; Toleno, Donna M.; Ranken, Raymond R.; Hall, Thomas A.; Metzgar, David; Sampath, Rangarajan; Blyn, Lawrence B.; Ecker, David J.; Gu, Zhengming; Walsh, Thomas J.

2013-01-01

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses. PMID:23303501

Characterization of Klebsiella isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determination of antimicrobial resistance with VITEK 2 advanced expert system (AES).

PubMed

Karagöz, Alper; Acar, Sümeyra; Körkoca, Hanifi

2015-01-01

The purpose of the study was to evaluate the performance of the VITEK mass spectrometry (MS) (bioMérieux, France) system for the identification of Klebsiella spp. isolated from different sources. Moreover, while assessing the ability of the VITEK 2 automated expert system (AES) to recognize antimicrobial resistance patterns, the researchers have extended the study to compare VITEK 2 with the routine antimicrobial susceptibility testing method. This study tested 51 Klebsiella spp. isolates that were isolated from environmental examples and clinical examples. Results of conventional methods and the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS were compared. Then, any differing results were compared against a reference 16S rRNA gene sequence, and when indicated, a recA sequencing analysis was done. VITEK MS correctly identified 100% of the Klebsiella spp. isolates. There were two K. oxytoca isolates incorrectly identified to the species level with conventional methods according to the 16S rRNA gene sequencing analysis. In addition, a VITEK 2 AST-N261 card was used for the detection of extended spectrum beta-lactamases (ESBL). Using the VITEK 2 AES, ESBL positivity was found at the rate of 16.3% whereas this rate was 4.08% using the disk diffusion method. MALDI-TOF MS is a rapid and accurate method for the identification of Klebsiella spp. Moreover, the bioMérieux AES provides a useful laboratory tool for the interpretation of susceptibility results.

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

PubMed Central

Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

2011-01-01

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

Identification and phylogenetic analysis of a sheep pox virus isolated from the Ningxia Hui Autonomous Region of China.

PubMed

Zhu, X L; Yang, F; Li, H X; Dou, Y X; Meng, X L; Li, H; Luo, X N; Cai, X P

2013-05-14

An outbreak of sheep pox was investigated in the Ningxia Hui Autonomous Region in China. Through immunofluorescence testing, isolated viruses, polymerase chain reaction identification, and electron microscopic examination, the isolated strain was identified as a sheep pox virus. The virus was identified through sequence and phylogenetic analysis of the P32 gene, open reading frame (ORF) 095, and ORF 103 genes. This study is the first to use the ORF 095 and ORF 103 genes as candidate genes for the analysis of sheep pox. The results showed that the ORF 095 and ORF 103 genes could be used for the genotyping of the sheep pox virus.

Rapid identification of pathogenic streptococci isolated from moribund red tilapia (Oreochromis spp.).

PubMed

Abdelsalam, Mohamed; Elgendy, Mamdouh Y; Shaalan, Mohamed; Moustafa, Mohamed; Fujino, Masayuki

2017-03-01

Accurate and rapid identification of bacterial pathogens of fish is essential for the effective treatment and speedy control of infections. Massive mortalities in market-sized red tilapia (Oreochromis spp.) were noticed in mariculture concrete ponds in northern Egypt. Histopathological examination revealed marked congestion in the central vein of the liver with the presence of bacterial aggregates inside the lumen and in the vicinity of the central vein. A total of 12 isolates of streptococci were obtained from the moribund fish. This study documented the ability of the MicroSeq 500 16S bacterial sequencing method to accurately identify Streptococcus agalactiae and S. dysgalactiae mixed infections from moribund red tilapia that were difficult to be recognised by the commercial biochemical systems. The continuously decreasing cost of the sequencing technique should encourage its application in routine diagnostic procedures.

Evaluation of GenoType NTM-DR Assay for Identification of Mycobacterium chimaera.

PubMed

Mok, Simone; Rogers, Thomas R; Fitzgibbon, Margaret

2017-06-01

Identification of species within the Mycobacterium avium complex (MAC) is difficult, and most current diagnostic laboratory tests cannot distinguish between species included in the complex. Differentiation of species within the MAC is important, as Mycobacterium chimaera has recently emerged as a major cause of invasive cardiovascular infections following open heart surgery. A new commercial diagnostic assay, GenoType NTM-DR ver. 1.0, is intended to differentiate between three species within the MAC, namely, Mycobacterium avium , Mycobacterium intracellulare , and Mycobacterium chimaera In this study, we investigated an archival collection of 173 MAC isolates using 16S rRNA and 16S-23S internal transcribed spacer (ITS) gene sequencing, and GenoType NTM-DR was evaluated for identifying M. chimaera and other species belonging to the MAC. Species identification of 157/173 (91%) isolates with the GenoType NTM-DR assay was in agreement with 16S rRNA and 16S-23S ITS gene sequencing results. Misidentification occurred with 16 isolates which belonged to four species included in the MAC that are rarely encountered in clinical specimens. Despite some limitations of this assay, GenoType NTM-DR had 100% specificity for identifying M. chimaera This novel assay will enable diagnostic laboratories to differentiate species belonging to the Mycobacterium avium complex and to accurately identify M. chimaera It can produce rapid results and is also more cost efficient than gene sequencing methods. Copyright © 2017 American Society for Microbiology.

Reads2Type: a web application for rapid microbial taxonomy identification.

PubMed

Saputra, Dhany; Rasmussen, Simon; Larsen, Mette V; Haddad, Nizar; Sperotto, Maria Maddalena; Aarestrup, Frank M; Lund, Ole; Sicheritz-Pontén, Thomas

2015-11-25

Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale. In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

Identification and Characterization of Citrus tristeza virus Isolates Breaking Resistance in Trifoliate Orange in California.

PubMed

Yokomi, Raymond K; Selvaraj, Vijayanandraj; Maheshwari, Yogita; Saponari, Maria; Giampetruzzi, Annalisa; Chiumenti, Michela; Hajeri, Subhas

2017-07-01

Most Citrus tristeza virus (CTV) isolates in California are biologically mild and symptomless in commercial cultivars on CTV tolerant rootstocks. However, to better define California CTV isolates showing divergent serological and genetic profiles, selected isolates were subjected to deep sequencing of small RNAs. Full-length sequences were assembled, annotated and trifoliate orange resistance-breaking (RB) isolates of CTV were identified. Phylogenetic relationships based on their full genomes placed three isolates in the RB clade: CA-RB-115, CA-RB-AT25, and CA-RB-AT35. The latter two isolates were obtained by aphid transmission from Murcott and Dekopon trees, respectively, containing CTV mixtures. The California RB isolates were further distinguished into two subclades. Group I included CA-RB-115 and CA-RB-AT25 with 99% nucleotide sequence identity with RB type strain NZRB-G90; and group II included CA-RB-AT35 with 99 and 96% sequence identity with Taiwan Pumelo/SP/T1 and HA18-9, respectively. The RB phenotype was confirmed by detecting CTV replication in graft-inoculated Poncirus trifoliata and transmission from P. trifoliata to sweet orange. The California RB isolates induced mild symptoms compared with severe isolates in greenhouse indexing tests. Further examination of 570 CTV accessions, acquired from approximately 1960 and maintained in planta at the Central California Tristeza Eradication Agency, revealed 16 RB positive isolates based on partial p65 sequences. Six isolates collected from 1992 to 2011 from Tulare and Kern counties were CA-RB-115-like; and 10 isolates collected from 1968 to 2010 from Riverside, Fresno, and Kern counties were CA-RB-AT35-like. The presence of the RB genotype is relevant because P. trifoliata and its hybrids are the most popular rootstocks in California.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

PubMed

Srinivasan, Sujatha; Munch, Matthew M; Sizova, Maria V; Fiedler, Tina L; Kohler, Christina M; Hoffman, Noah G; Liu, Congzhou; Agnew, Kathy J; Marrazzo, Jeanne M; Epstein, Slava S; Fredricks, David N

2016-08-15

Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Novel Mutations in pncA Gene of Pyrazinamide Resistant Clinical Isolates of Mycobacterium tuberculosis.

PubMed

Kahbazi, Manijeh; Sarmadian, Hossein; Ahmadi, Azam; Didgar, Farshideh; Sadrnia, Maryam; Poolad, Toktam; Arjomandzadegan, Mohammad

2018-04-16

In clinical isolates of Mycobacterium tuberculosis (MTB), resistance to pyrazinamide occurs by mutations in any positions of the pncA gene (NC_000962.3) especially in nucleotides 359 and 374. In this study we examined the pncA gene sequence in clinical isolates of MTB. Genomic DNA of 33 clinical isolates of MTB was extracted by the Chelex100 method. The polymerase chain reactions (PCR) were performed using specific primers for amplification of 744 bp amplicon comprising the coding sequences (CDS) of the pncA gene. PCR products were sequenced by an automated sequencing Bioscience system. Additionally, semi Nested-allele specific (sNASP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were carried out for verification of probable mutations in nucleotides 359 and 374. Sequencing results showed that from 33 MTB clinical isolates, nine pyrazinamide-resistant isolates have mutations. Furthermore, no mutation was detected in 24 susceptible strains in the entire 561 bp of the pncA gene. Moreover, new mutations of G→A at position 3 of the pncA gene were identified in some of the resistant isolates. Results showed that the sNASP method could detect mutations in nucleotide 359 and 374 of the pncA gene, but the PCR-RFLP method by the SacII enzyme could not detect these mutations. In conclusion, the identification of new mutations in the pncA gene confirmed the probable occurrence of mutations in any nucleotides of the pncA gene sequence in resistant isolates of MTB.

Morphologic and Genetic Identification of Taenia Tapeworms in Tanzania and DNA Genotyping of Taenia solium

PubMed Central

Eom, Keeseon S.; Chai, Jong-Yil; Yong, Tai-Soon; Min, Duk-Young; Rim, Han-Jong; Kihamia, Charles

2011-01-01

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n=1) and T. saginata (n=3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1). PMID:22355207

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu.

PubMed

Chitra, M Ananda; Jayalakshmi, K; Ponnusamy, P; Manickam, R; Ronald, B S M

2017-11-01

The study was conducted to isolate and identify Dermatophilus congolensis (DC) using conventional and molecular diagnostic techniques in scab materials collected from skin infections of sheep and goats in the Delta region of Tamil Nadu. A total of 20 scab samples collected from 18 goats and 2 sheep from Nagapattinam, Thanjavur, and Tiruvarur districts of Tamil Nadu. Smears were made from softened scab materials and stained by either Gram's or Giemsa staining. Isolation was attempted on blood agar plates, and colonies were stained by Gram's staining for morphological identification. Identification was also done by biochemical tests and confirmed by 16S rRNA polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the amplified product. The peculiar laddering arrangement of coccoid forms in stained smears prepared from scab materials revealed the presence of DC. Isolated colonies from scab materials of sheep and goats on bovine blood agar plate were small, hemolytic, rough, adherent, and bright orange-yellow in color, but some colonies were white to cream color. Gram-staining of cultured organisms revealed Gram-positive branching filaments with various disintegration stages of organisms. 16S rRNA PCR yielded 500 bp amplicon specific for DC. Sequence analysis of a sheep DC isolate showed 99-100% sequence homology with other DC isolates available in NCBI database, and phylogenetic tree showed a close cluster with DC isolates of Congo, Nigeria, and Angola of Africa. Genes for virulence factors such as serine protease and alkaline ceramidase could not be detected by PCR in any of the DC strains isolated of this study. The presence of dermatophilosis in Tamil Nadu was established from this study.

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu

PubMed Central

Chitra, M. Ananda; Jayalakshmi, K.; Ponnusamy, P.; Manickam, R.; Ronald, B. S. M.

2017-01-01

Aim: The study was conducted to isolate and identify Dermatophilus congolensis (DC) using conventional and molecular diagnostic techniques in scab materials collected from skin infections of sheep and goats in the Delta region of Tamil Nadu. Materials and Methods: A total of 20 scab samples collected from 18 goats and 2 sheep from Nagapattinam, Thanjavur, and Tiruvarur districts of Tamil Nadu. Smears were made from softened scab materials and stained by either Gram’s or Giemsa staining. Isolation was attempted on blood agar plates, and colonies were stained by Gram’s staining for morphological identification. Identification was also done by biochemical tests and confirmed by 16S rRNA polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the amplified product. Results: The peculiar laddering arrangement of coccoid forms in stained smears prepared from scab materials revealed the presence of DC. Isolated colonies from scab materials of sheep and goats on bovine blood agar plate were small, hemolytic, rough, adherent, and bright orange-yellow in color, but some colonies were white to cream color. Gram-staining of cultured organisms revealed Gram-positive branching filaments with various disintegration stages of organisms. 16S rRNA PCR yielded 500 bp amplicon specific for DC. Sequence analysis of a sheep DC isolate showed 99-100% sequence homology with other DC isolates available in NCBI database, and phylogenetic tree showed a close cluster with DC isolates of Congo, Nigeria, and Angola of Africa. Genes for virulence factors such as serine protease and alkaline ceramidase could not be detected by PCR in any of the DC strains isolated of this study. Conclusion: The presence of dermatophilosis in Tamil Nadu was established from this study. PMID:29263591

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

PubMed Central

Vágány, Viktória; Jackson, Alison C.; Harrison, Richard J.; Rainoni, Alessandro; Clarkson, John P.

2016-01-01

Summary Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific. PMID:26609905

Identification of pathogenicity-related genes in Fusarium oxysporum f. sp. cepae.

PubMed

Taylor, Andrew; Vágány, Viktória; Jackson, Alison C; Harrison, Richard J; Rainoni, Alessandro; Clarkson, John P

2016-09-01

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non-pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non-pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific. © 2015 The Authors Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.

Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

PubMed

Hajjaran, Homa; Mohebali, Mehdi; Teimouri, Aref; Oshaghi, Mohammad Ali; Mirjalali, Hamed; Kazemi-Rad, Elham; Shiee, Mohammad Reza; Naddaf, Saied Reza

2014-08-01

The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates using nagt gene allowed unambiguous identification of five prevalent species with a high-resolution phylogeny. Copyright © 2014 Elsevier B.V. All rights reserved.

Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences*

PubMed Central

Zhao, Ya-e; Ma, Jun-xian; Hu, Li; Wu, Li-ping; De Rojas, Manuel

2013-01-01

For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum. Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids. PMID:24009203

Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish.

PubMed

Takahashi, Hajime; Kimura, Bon; Yoshikawa, Miwako; Fujii, Tateo

2003-05-01

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

PubMed Central

Kelly, Shannan; Yamamoto, Hideki

2008-01-01

Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

PubMed

Paul, Catherine J; Twine, Susan M; Tam, Kevin J; Mullen, James A; Kelly, John F; Austin, John W; Logan, Susan M

2007-05-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

Evaluation of MALDI-TOF-MS for the Identification of Yeast Isolates Causing Bloodstream Infection.

PubMed

Turhan, Ozge; Ozhak-Baysan, Betil; Zaragoza, Oscar; Er, Halil; Sarıtas, Zubeyde Eres; Ongut, Gozde; Ogunc, Dilara; Colak, Dilek; Cuenca-Estrella, Manuel

2017-04-01

Infections due to Candida species are major causes of morbidity and mortality in humans, causing a diverse spectrum of clinical disease ranging from superficial and mucosal infections to invasive disease. Several authors have demonstrated that mortality is closely linked to both timing of therapy and/or source control. The rapid identification of pathogenic species is helpful to start timely and effective antifungal therapy. The aim of this study was to assess the performance of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the correct and rapid identification of yeast isolates causing bloodstream infection. Between January 2014 and January 2015, a total of 117 yeast like organisms isolated from blood culture samples of 117 episodes from 102 patients who had blood stream infections were included in the study. The isolates were identified by MALDI-TOF MS. The results were compared with those obtained by the standard mycological methods and/or sequence analysis. One hundred and seventeen yeast isolates including 115 Candida spp and two non-Candida yeasts were analysed. The Biotyper correctly identified 115 (98.3%) isolates to the genus level and 102 (87.2%) isolates to the species level using the manufacturer's recommended cutoff scores. The Bruker Biotyper is a rapid, easy, inexpensive, and highly reliable system for the identification of yeast isolates. Early identification with MALDI-TOF MS would save time for determination of antifungal susceptibility and proper treatment strategy. The expansion of the database of the library by addition of less common species will improve the performance of the system.

Evaluation of T2-weighted versus short-tau inversion recovery sagittal sequences in the identification and localization of canine intervertebral disc extrusion with low-field magnetic resonance imaging.

PubMed

Housley, Daniel; Caine, Abby; Cherubini, Giunio; Taeymans, Olivier

2017-07-01

Sagittal T2-weighted sequences (T2-SAG) are the foundation of spinal protocols when screening for the presence of intervertebral disc extrusion. We often utilize sagittal short-tau inversion recovery sequences (STIR-SAG) as an adjunctive screening series, and experience suggests that this combined approach provides superior detection rates. We hypothesized that STIR-SAG would provide higher sensitivity than T2-SAG in the identification and localization of intervertebral disc extrusion. We further hypothesized that the parallel evaluation of paired T2-SAG and STIR-SAG series would provide a higher sensitivity than could be achieved with either independent sagittal series when viewed in isolation. This retrospective diagnostic accuracy study blindly reviewed T2-SAG and STIR-SAG sequences from dogs (n = 110) with surgically confirmed intervertebral disc extrusion. A consensus between two radiologists found no significant difference in sensitivity between T2-SAG and STIR-SAG during the identification of intervertebral disc extrusion (T2-SAG: 92.7%, STIR-SAG: 94.5%, P = 0.752). Nevertheless, STIR-SAG accurately identified intervertebral disc extrusion in 66.7% of cases where the evaluation of T2-SAG in isolation had provided a false negative diagnosis. Additionally, one radiologist found that the parallel evaluation of paired T2-SAG and STIR-SAG series provided a significantly higher sensitivity than T2-SAG in isolation, during the identification of intervertebral disc extrusion (T2-SAG: 78.2%, paired T2-SAG, and STIR-SAG: 90.9%, P = 0.017). A similar nonsignificant trend was observed when the consensus of both radiologists was taken into consideration (T2-SAG: 92.7%, paired T2-SAG, and STIR-SAG = 97.3%, P = 0.392). We therefore conclude that STIR-SAG is capable of identifying intervertebral disc extrusion that is inconspicuous in T2-SAG, and that STIR-SAG should be considered a useful adjunctive sequence during preliminary sagittal screening for intervertebral disc extrusion in low-field magnetic resonance. © 2017 American College of Veterinary Radiology.

MALDI-TOF Mass Spectrometry Is a Fast and Reliable Platform for Identification and Ecological Studies of Species from Family Rhizobiaceae

PubMed Central

Ferreira, Laura; Sánchez-Juanes, Fernando; García-Fraile, Paula; Rivas, Raúl; Mateos, Pedro F.; Martínez-Molina, Eustoquio; González-Buitrago, José Manuel; Velázquez, Encarna

2011-01-01

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies. PMID:21655291

Genotypic analysis of Mucor from the platypus in Australia.

PubMed

Connolly, J H; Stodart, B J; Ash, G J

2010-01-01

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Uukuniemi virus, Czech Republic.

PubMed

Papa, Anna; Zelená, Hana; Papadopoulou, Elpida; Mrázek, Jakub

2018-04-20

Following the identification of severe fever with thrombocytopenia syndrome and Heartland viruses, the interest on tick-borne phleboviruses has increased rapidly. Uukuniemi virus has been proposed as a model for tick-borne phleboviruses. However, the number of available sequences is limited. In the current study we performed whole-genome sequencing on two Uukuniemi viral strains isolated in 2000 and 2004 from Ixodes ricinus ticks in the Czech Republic. Both strains cluster together with Potepli63 strain isolated in the country in 1963. Although the Czech strains were isolated many years apart, a high identity was seen at the nucleotide and amino acid levels, suggesting that UUKV has a relatively stable genome. Copyright © 2018 Elsevier GmbH. All rights reserved.

16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry.

PubMed

Dec, Marta; Puchalski, Andrzej; Urban-Chmiel, Renata; Wernicki, Andrzej

2016-06-13

The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70-1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats.

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination

PubMed Central

Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-01-01

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. PMID:26712553

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Molecular identification and antifungal susceptibility of Curvularia australiensis, C. hawaiiensis and C. spicifera isolated from human eye infections.

PubMed

Krizsán, Krisztina; Tóth, Eszter; Nagy, László G; Galgóczy, László; Manikandan, Palanisamy; Chandrasekaran, Muthusamy; Kadaikunnan, Shine; Alharbi, Naiyf S; Vágvölgyi, Csaba; Papp, Tamás

2015-10-01

A reliable identification method was developed for three closely related Curvularia species, which are frequently isolated from human keratomycoses. Since the traditionally used morphological method and the increasingly used internal transcribed spacer (ITS)-based molecular method proved to be insufficient to discern C. australiensis, C. hawaiiensis and C. spicifera, other molecular targets, such as β-tubulin, translation elongation factor 1-α and the nuclear ribosomal intergenic spacer (IGS), were tested. Among them, the use of the highly divergent IGS sequence is suggested and the species-specific discriminating characters were determined in appropriate reference strains. It was also concluded that C. hawaiiensis and C. spicifera can be predominantly isolated from eye infections among the three species. The in vitro antifungal susceptibility of 10 currently used antifungal agents against 32 Curvularia isolates was also investigated. MICs were determined in each case. Isolates of C. spicifera proved to be less susceptible to the tested antifungals than those of C. hawaiiensis, which underline the importance of the correct identification of these species. © 2015 Blackwell Verlag GmbH.

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system.

PubMed

Garner, O; Mochon, A; Branda, J; Burnham, C-A; Bythrow, M; Ferraro, M; Ginocchio, C; Jennemann, R; Manji, R; Procop, G W; Richter, S; Rychert, J; Sercia, L; Westblade, L; Lewinski, M

2014-04-01

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M.

PubMed

Enzmann, P J; Kurath, G; Fichtner, D; Bergmann, S M

2005-09-23

Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

PubMed

Shahinyan, Grigor; Margaryan, Armine; Panosyan, Hovik; Trchounian, Armen

2017-05-02

Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Catheter-related bacteraemia and infective endocarditis caused by Kocuria species.

PubMed

Lai, C C; Wang, J Y; Lin, S H; Tan, C K; Wang, C Y; Liao, C H; Chou, C H; Huang, Y T; Lin, H I; Hsueh, P R

2011-02-01

We describe five patients with positive blood culture for Kocuria species. Three patients had catheter-related bacteraemia and one had infective endocarditis caused by Kocuria kristinae, and one had a K. marina isolate, which was considered to be a contaminant. Identification of the isolates was further confirmed by 16S rRNA gene sequence analysis. In conclusion, Kocuria species are an unusual cause of infection in immunocompromised patients. Accurate identification with molecular methods is imperative for the diagnosis of these unusual pathogens. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

PubMed

Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

2015-01-01

Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities were relatively low, ranging from 68.7-97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella.

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

PubMed Central

Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

2015-01-01

Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella. PMID:26103050

Molecular identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein

2011-01-01

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric...

Eubacterium callanderi Bacteremia: Report of the First Case

PubMed Central

Thiolas, Aurélie; Bollet, Claude; Gasmi, Mohammed; Drancourt, Michel; Raoult, Didier

2003-01-01

Eubacterium callanderi is an environmental anaerobic rod-shaped bacterium first isolated in 1998 from an industrial anaerobic digester. We report on the first clinical isolate of E. callanderi, which was recovered from the blood of a patient with a bladder carcinoma. Identification of the organism was made by cell fatty acid chromatographic analysis and 16S rRNA gene sequencing. PMID:12734289

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting.

PubMed

Romi, Wahengbam; Keisam, Santosh; Ahmed, Giasuddin; Jeyaram, Kumaraswamy

2014-02-28

Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.

Candida guilliermondii and Other Species of Candida Misidentified as Candida famata: Assessment by Vitek 2, DNA Sequencing Analysis, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Two Global Antifungal Surveillance Programs

PubMed Central

Woosley, Leah N.; Diekema, Daniel J.; Jones, Ronald N.; Pfaller, Michael A.

2013-01-01

Candida famata (teleomorph Debaryomyces hansenii) has been described as a medically relevant yeast, and this species has been included in many commercial identification systems that are currently used in clinical laboratories. Among 53 strains collected during the SENTRY and ARTEMIS surveillance programs and previously identified as C. famata (includes all submitted strains with this identification) by a variety of commercial methods (Vitek, MicroScan, API, and AuxaColor), DNA sequencing methods demonstrated that 19 strains were C. guilliermondii, 14 were C. parapsilosis, 5 were C. lusitaniae, 4 were C. albicans, and 3 were C. tropicalis, and five isolates belonged to other Candida species (two C. fermentati and one each C. intermedia, C. pelliculosa, and Pichia fabianni). Additionally, three misidentified C. famata strains were correctly identified as Kodomaea ohmeri, Debaryomyces nepalensis, and Debaryomyces fabryi using intergenic transcribed spacer (ITS) and/or intergenic spacer (IGS) sequencing. The Vitek 2 system identified three isolates with high confidence to be C. famata and another 15 with low confidence between C. famata and C. guilliermondii or C. parapsilosis, displaying only 56.6% agreement with DNA sequencing results. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) results displayed 81.1% agreement with DNA sequencing. One strain each of C. metapsilosis, C. fermentati, and C. intermedia demonstrated a low score for identification (<2.0) in the MALDI Biotyper. K. ohmeri, D. nepalensis, and D. fabryi identified by DNA sequencing in this study were not in the current database for the MALDI Biotyper. These results suggest that the occurrence of C. famata in fungal infections is much lower than previously appreciated and that commercial systems do not produce accurate identifications except for the newly introduced MALDI-TOF instruments. PMID:23100350

Identification and complete genome sequence analysis of a genotype XIV Newcastle disease virus from Nigeria

USDA-ARS?s Scientific Manuscript database

The first complete genome sequence of a strain of Newcastle disease virus from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a m...

Draft Genome Sequence of the Fish Pathogen Flavobacterium columnare Genomovar III Strain PH-97028 (=CIP 109753).

PubMed

Criscuolo, Alexis; Chesneau, Olivier; Clermont, Dominique; Bizet, Chantal

2018-04-05

Flavobacterium columnare strain PH-97028 (=CIP 109753) is a genomovar III reference strain that was isolated from a diseased Ayu fish in Japan. We report here the analysis of the first available genomovar III sequence of this species to aid in identification, epidemiological tracking, and virulence studies. Copyright © 2018 Criscuolo et al.

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

PubMed Central

Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia

2016-01-01

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance. PMID:27031639

Polymerase chain reaction-based identification of clinically relevant Pasteurellaceae isolated from cats and dogs in Poland.

PubMed

Król, Jaroslaw; Bania, Jacek; Florek, Magdalena; Pliszczak-Król, Aleksandra; Staroniewicz, Zdzislaw

2011-05-01

A set of polymerase chain reaction (PCR) assays for identification of the most important Pasteurellaceae species encountered in cats and dogs were developed. Primers for Pasteurella multocida were designed to detect a fragment of the kmt, a gene encoding the outer-membrane protein. Primers specific to Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis were based on the manganese-dependent superoxide dismutase gene (sodA) and those specific to [Haemophilus] haemoglobinophilus on species-specific sequences of the 16S ribosomal RNA gene. All the primers were tested on respective reference and control strains and applied to the identification of 47 canine and feline field isolates of Pasteurellaceae. The PCR assays were shown to be species specific, providing a valuable supplement to phenotypic identification of species within this group of bacteria. © 2011 The Author(s)

Identification and characterization of a NBS–LRR class resistance gene analog in Pistacia atlantica subsp. Kurdica

PubMed Central

Bahramnejad, Bahman

2014-01-01

P. atlantica subsp. Kurdica, with the local name of Baneh, is a wild medicinal plant which grows in Kurdistan, Iran. The identification of resistance gene analogs holds great promise for the development of resistant cultivars. A PCR approach with degenerate primers designed according to conserved NBS-LRR (nucleotide binding site-leucine rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from P. atlantica subsp. Kurdica. A DNA fragment of the expected 500-bp size was amplified. The nucleotide sequence of this amplicon was obtained through sequencing and the predicted amino acid sequence compared to the amino acid sequences of known R-genes revealed significant sequence similarity. Alignment of the deduced amino acid sequence of P. atlantica subsp. Kurdica resistance gene analog (RGA) showed strong identity, ranging from 68% to 77%, to the non-toll interleukin receptor (non-TIR) R-gene subfamily from other plants. A P-loop motif (GMMGGEGKTT), a conserved and hydrophobic motif GLPLAL, a kinase-2a motif (LLVLDDV), when replaced by IAVFDDI in PAKRGA1 and a kinase-3a (FGPGSRIII) were presented in all RGA. A phylogenetic tree, based on the deduced amino-acid sequences of PAKRGA1 and RGAs from different species indicated that they were separated in two clusters, PAKRGA1 being on cluster II. The isolated NBS analogs can be eventually used as guidelines to isolate numerous R-genes in Pistachio. PMID:27843981

Incidence of Keratinophilic Fungi from the Selected Soils of Kaziranga National Park, Assam (India).

PubMed

Deshmukh, Sunil Kumar; Verekar, Shilpa Amit; Chavan, Yashwant G

2017-04-01

Seventy-eight soil samples were collected from the various locations in the vicinity of Kaziranga National Park (Assam), India, during April to October 2009 and screened for the presence of keratinophilic fungi using the hair baiting techniques for isolation. Thirty-nine isolates were recovered and identified by recognition of their macro- and micromorphological features. Their identification was also confirmed by the BLAST search of sequences of the ITS1-5.8S-ITS2 rDNA region against the NCBI/GenBank data and compared with deposited sequences for identification purpose. Eleven species related to seven genera were recorded viz. Aphanoascus durus (1.28%), Arthroderma tuberculatum (3.84%), Arthroderma corniculatum (1.28%), Chrysosporium indicum (16.66%), C. tropicum (3.84%), Ctenomyces serratus (5.12%), Keratinophyton punsolae (1.28%), Microsporum appendiculatum (1.28%), Microsporum gypseum complex (11.53%), Trichophyton mentagrophytes (11.28%) and T. terrestre (2.56%).

Bacteremia due to Moraxella atlantae in a cancer patient.

PubMed

De Baere, Thierry; Muylaert, An; Everaert, Els; Wauters, Georges; Claeys, Geert; Verschraegen, Gerda; Vaneechoutte, Mario

2002-07-01

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.

Bacteremia Due to Moraxella atlantae in a Cancer Patient

PubMed Central

De Baere, Thierry; Muylaert, An; Everaert, Els; Wauters, Georges; Claeys, Geert; Verschraegen, Gerda; Vaneechoutte, Mario

2002-01-01

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the Moraxellaceae. After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed. PMID:12089312

Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-03-24

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1998-01-01

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

A report on extensive lateral genetic reciprocation between arsenic resistant Bacillus subtilis and Bacillus pumilus strains analyzed using RAPD-PCR.

PubMed

Khowal, Sapna; Siddiqui, Md Zulquarnain; Ali, Shadab; Khan, Mohd Taha; Khan, Mather Ali; Naqvi, Samar Husain; Wajid, Saima

2017-02-01

The study involves isolation of arsenic resistant bacteria from soil samples. The characterization of bacteria isolates was based on 16S rRNA gene sequences. The phylogenetic consanguinity among isolates was studied employing rpoB and gltX gene sequence. RAPD-PCR technique was used to analyze genetic similarity between arsenic resistant isolates. In accordance with the results Bacillus subtilis and Bacillus pumilus strains may exhibit extensive horizontal gene transfer. Arsenic resistant potency in Bacillus sonorensis and high arsenite tolerance in Bacillus pumilus strains was identified. The RAPD-PCR primer OPO-02 amplified a 0.5kb DNA band specific to B. pumilus 3ZZZ strain and 0.75kb DNA band specific to B. subtilis 3PP. These unique DNA bands may have potential use as SCAR (Sequenced Characterized Amplified Region) molecular markers for identification of arsenic resistant B. pumilus and B. subtilis strains. Copyright © 2016 Elsevier Inc. All rights reserved.

(GTG)5 microsatellite regions in citrinin-producing Penicillium.

PubMed

Di Conza, José Alejandro; Nepote, Andrea Fabiana; González, Ana María; Lurá, María Cristina

2007-03-01

Morphological and cultural characteristics, as well as biochemical properties, are the main criteria used in fungal taxonomy and in the standard description of fungi species. Sometimes, however, this criterion is difficult to apply due to fungal phenotypic variations. This is particularly true in the genus Penicillium. The aims of this work were to determine (GTG)5 microsatellite sequence in potentially citrinin-producing Penicillium strains and to investigate if this sequence could be useful to characterize such fungi. Penicillium citrinum Thom and Penicillium chrysogenum Thom were isolated from different foods. The identification of the isolates at species level was carried out according to classical taxonomy. The production of citrinin was determined by thin layer chromatography. This study proved that microsatellite regions exist as short repeated sequences in all tested strains. The patterns were very similar for all P. citrinum isolates and it was possible to group them in function of the quantity of citrinin produced. Yet, not similar clusters were obtained when P. chrysogenum isolates were analyzed.

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions.

PubMed

Mikaeili, Fattaneh; Mathis, Alexander; Deplazes, Peter; Mirhendi, Hossein; Barazesh, Afshin; Ebrahimi, Sepideh; Kia, Eshrat Beigom

2017-09-26

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

Identification of species and genetic variation in Taenia isolates from human and swine of North India.

PubMed

Singh, Satyendra K; Prasad, Kashi N; Singh, Aloukick K; Gupta, Kamlesh K; Chauhan, Ranjeet S; Singh, Amrita; Singh, Avinash; Rai, Ravi P; Pati, Binod K

2016-10-01

Taenia solium is the major cause of taeniasis and cysticercosis/neurocysticercosis (NCC) in the developing countries including India, but the existence of other Taenia species and genetic variation have not been studied in India. So, we studied the existence of different Taenia species, and sequence variation in Taenia isolates from human (proglottids and cysticerci) and swine (cysticerci) in North India. Amplification of cytochrome c oxidase subunit 1 gene (cox1) was done by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. We identified two species of Taenia i.e. T. solium and Taenia asiatica in our isolates. T. solium isolates showed similarity with Asian genotype and nucleotide variations from 0.25 to 1.01 %, whereas T. asiatica displayed nucleotide variations ranged from 0.25 to 0.5 %. These findings displayed the minimal genetic variations in North Indian isolates of T. solium and T. asiatica.

Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

PubMed

den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

2013-09-01

Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii.

Isolation and molecular identification of endophytic diazotrophs from seeds and stems of three cereal crops.

PubMed

Liu, Huawei; Zhang, Lei; Meng, Aihua; Zhang, Junbiao; Xie, Miaomiao; Qin, Yaohong; Faulk, Dylan Chase; Zhang, Baohong; Yang, Shushen; Qiu, Li

2017-01-01

Ten strains of endophytic diazotroph were isolated and identified from the plants collected from three different agricultural crop species, wheat, rice and maize, using the nitrogen-free selective isolation conditions. The nitrogen-fixing ability of endophytic diazotroph was verified by the nifH-PCR assay that showed positive nitrogen fixation ability. These identified strains were classified by 879F-RAPD and 16S rRNA sequence analysis. RAPD analyses revealed that the 10 strains were clustered into seven 879F-RAPD groups, suggesting a clonal origin. 16S rRNA sequencing analyses allowed the assignment of the 10 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Paenibacillus, Enterobacter, Klebsiella and Pantoea. These representative genus are not endophytic diazotrophs in the conventional sense. They may have obtained nitrogen fixation ability through lateral gene transfer, however, the evolutionary forces of lateral gene transfer are not well known. Molecular identification results from 16S rRNA analyses were also confirmed by morphological and biochemical data. The test strains SH6A and MZB showed positive effect on the growth of plants.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties.

PubMed

Ulbegi-Mohyla, H; Hijazin, M; Alber, J; Lämmler, C; Hassan, A A; Abdulmawjood, A; Prenger-Berninghoff, E; Weiss, R; Zschöck, M

2010-09-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Approaches for monitoring the release of Pochonia chlamydosporia var. catenulata, a biocontrol agent of root-knot nematodes.

PubMed

Atkins, Simon D; Hidalgo-Diaz, Leopoldo; Clark, Ian M; Morton, C Oliver; de Oca, Nivian Montes; Gray, Paul A; Kerry, Brian R

2003-02-01

Pochonia chlamydosporia var. catenulata is a potential biocontrol agent against root-knot nematodes. Diagnosis of isolates has relied on morphological identification, and is both time-consuming and difficult. beta-tubulin primers have been developed for the identification of this fungus that were specific enough to distinguish between varieties of the fungus within the same species. Separate primers have been developed for the specific detection of P. chlamydosporia var. catenulata based on ITS sequences, which were able to detect the fungus in soil from various sites in Cuba where the biocontrol agent had been added. When the PCR diagnosis was combined with serial dilution of soil samples on selective medium, colonies were rapidly identified. The fungus was still present, albeit at low densities, in soils inoculated five years previously. The development of a baiting method allowed quick in situ screening of the isolates' ability to infect nematode eggs, and when combined with PCR diagnosis both varieties of the fungus could be detected in infected eggs. RFLP analysis of ITS sequences from P. chlamydosporia provided an extra level of discrimination between isolates.

Identification of non-streptococcal organisms from human dental plaque grown on the Streptococcus-selective medium mitis-salivarius agar.

PubMed

Kim, Yeon-Hee; Lee, Si Young

2015-02-01

Mitis-salivarius (MS) agar has been used widely in microbial epidemiological studies because oral viridans streptococci can be selectively grown on this medium. Even though the previous findings reported the limited selecting power of MS agar for streptococcus strains, the identities of non-streptococcal strains from human oral samples which can grow on this medium are not clear yet. In this study, we identified non-streptococcal organisms grown on MS agar plates by polymerase chain reaction (PCR) amplification and sequencing of the 16S ribosomal RNA (rRNA) gene. Eighty bacterial colonies on MS plates were isolated from plaque samples, and bacterial identification was achieved with the rapid ID 32 Strep system and mini API reader. The bacterial colonies identified as non-streptococci by the API system were selected for further identification. The 16S rRNA gene was amplified by PCR and verified using DNA sequencing analysis for identification. Sequences were compared with those of reference organisms in the genome database of the National Center for Biotechnology Information using the Basic Local Alignment Search Tool (BLAST). Among the 11 isolated non-streptococcal strains on MS plates, 3 strains were identified as Actinomyces naeslundii, 7 strains were identified as Actinomyces oris and 1 strain were identified as Actinomyces sp. using Blastn. In this study, we showed that some oral Actinomyces species can grow on Streptococcus-selective MS agar plates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis

PubMed Central

Miotto, Olivo; Heiny, AT; Tan, Tin Wee; August, J Thomas; Brusic, Vladimir

2008-01-01

Background The identification of mutations that confer unique properties to a pathogen, such as host range, is of fundamental importance in the fight against disease. This paper describes a novel method for identifying amino acid sites that distinguish specific sets of protein sequences, by comparative analysis of matched alignments. The use of mutual information to identify distinctive residues responsible for functional variants makes this approach highly suitable for analyzing large sets of sequences. To support mutual information analysis, we developed the AVANA software, which utilizes sequence annotations to select sets for comparison, according to user-specified criteria. The method presented was applied to an analysis of influenza A PB2 protein sequences, with the objective of identifying the components of adaptation to human-to-human transmission, and reconstructing the mutation history of these components. Results We compared over 3,000 PB2 protein sequences of human-transmissible and avian isolates, to produce a catalogue of sites involved in adaptation to human-to-human transmission. This analysis identified 17 characteristic sites, five of which have been present in human-transmissible strains since the 1918 Spanish flu pandemic. Sixteen of these sites are located in functional domains, suggesting they may play functional roles in host-range specificity. The catalogue of characteristic sites was used to derive sequence signatures from historical isolates. These signatures, arranged in chronological order, reveal an evolutionary timeline for the adaptation of the PB2 protein to human hosts. Conclusion By providing the most complete elucidation to date of the functional components participating in PB2 protein adaptation to humans, this study demonstrates that mutual information is a powerful tool for comparative characterization of sequence sets. In addition to confirming previously reported findings, several novel characteristic sites within PB2 are reported. Sequence signatures generated using the characteristic sites catalogue characterize concisely the adaptation characteristics of individual isolates. Evolutionary timelines derived from signatures of early human influenza isolates suggest that characteristic variants emerged rapidly, and remained remarkably stable through subsequent pandemics. In addition, the signatures of human-infecting H5N1 isolates suggest that this avian subtype has low pandemic potential at present, although it presents more human adaptation components than most avian subtypes. PMID:18315849

Prevalence and genotypic characterization of bovine Echinococcus granulosus isolates by using cytochrome oxidase 1 (Co1) gene in Hyderabad, Pakistan.

PubMed

Ehsan, Muhammad; Akhter, Nasreen; Bhutto, Bachal; Arijo, Abdullah; Ali Gadahi, Javaid

2017-05-30

Cystic echinococcosis is an important zoonotic disease; it has serious impacts on animals as well as human health throughout the world. Genotypic characterization of Echinocossus granulosus (E. granulosus) in buffaloes has not been addressed in Pakistan. Therefore, the present study was conducted to evaluate the incidence and genotypic characterization of bovine E. granulosus. Out of 832 buffaloes examined, 112 (13.46%) were found infected. The favorable site for hydatid cyst development was liver (8.65%) followed by lungs (4.80%). The rate of cystic echinococcosis was found higher in females 14.43% than males 9.77%. The females above seven years aged were more infected as compared to the young ones. The partial sequence of mitochondrial cytochrome oxidase 1 (CO1) gene was used for identification and molecular analysis of buffalo's E. granulosus isolates. The alignment of redundant sequences were compared with already identified 10 genotypes available at National Centre for Biotechnology Information (NCBI) GenBank. The sequencing and phylogenetic analysis of all randomly selected buffalo isolates were belong to the G1- G3 complex (E. granulosus sensu stricto). All sequences were diverse from the reference sequence. No one showed complete identity to the buffalo strain (G3), representing substantial microsequence variability in G1, G2 and G3 genotypes. We evaluated the echinococcal infectivity and first time identification of genotypes in buffaloes in Sindh, Pakistan. This study will lead to determine accurate source of this zoonotic disease to humans in Pakistan. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment of a Pan-Dermatophyte Nested-PCR Compared with Conventional Methods for Direct Detection and Identification of Dermatophytosis Agents in Animals.

PubMed

Piri, Fahimeh; Zarei Mahmoudabadi, Ali; Ronagh, Ali; Ahmadi, Bahram; Makimura, Koichi; Rezaei-Matehkolaei, Ali

2018-06-26

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002.

PubMed

Cheon, Doo-Sung; Lee, Jiwon; Lee, Kangbum; Lee, Sunhwa; Park, Kwisung; Ahn, Jungbae; Jee, Youngmee; Yoon, Jaedeuk; Cho, Haewol

2004-07-01

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula. Copyright 2004 Wiley-Liss, Inc.

Molecular identification of Giardia and Cryptosporidium from dogs and cats

PubMed Central

Sotiriadou, Isaia; Pantchev, Nikola; Gassmann, Doreen; Karanis, Panagiotis

2013-01-01

The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR) and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU) rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH) locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans. PMID:23477297

Isolation and sequence analysis of a canine distemper virus from a raccoon dog in Jilin Province, China.

PubMed

Cheng, Yuening; Wang, Jianke; Zhang, Miao; Zhao, Jianjun; Shao, Xiqun; Ma, Zengjun; Zhao, Hang; Lin, Peng; Wu, Hua

2015-10-01

Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10(4.6) TCID50/mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China.

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

PubMed

Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

2008-01-01

Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella

PubMed Central

Drancourt, Michel; Roux, Véronique; Fournier, Pierre-Edouard; Raoult, Didier

2004-01-01

We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair. PMID:14766807

Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

PubMed

Röder, Christoph; König, Helmut; Fröhlich, Jürgen

2007-09-01

Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.

Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua

DOE PAGES

Thorell, Kaisa; Hosseini, Shaghayegh; Palacios Gonzales, Reyna Victoria Palacios; ...

2016-02-29

In this study, Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. The complete genomes ofmore » fifty-two Nicaraguan H. pylorii isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South-and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. In conclusion, the discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence properties.« less

Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use.

PubMed

Games, Patrícia Dias; daSilva, Elói Quintas Gonçalves; Barbosa, Meire de Oliveira; Almeida-Souza, Hebréia Oliveira; Fontes, Patrícia Pereira; deMagalhães, Marcos Jorge; Pereira, Paulo Roberto Gomes; Prates, Maura Vianna; Franco, Gloria Regina; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Baracat-Pereira, Maria Cristina

2016-12-15

Antimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves ('Magali R') are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses. Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature. Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessary.

Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

PubMed Central

Ginther, Jennifer L.; Mayo, Mark; Warrington, Stephanie D.; Kaestli, Mirjam; Mullins, Travis; Wagner, David M.; Currie, Bart J.; Tuanyok, Apichai; Keim, Paul

2015-01-01

Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area. PMID:26121041

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination.

PubMed

Jacques, Marie-Agnès; Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-12-28

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

PubMed

Smail, D A; Bain, N; Bruno, D W; King, J A; Thompson, F; Pendrey, D J; Morrice, S; Cunningham, C O

2006-01-01

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

PubMed

Félix-Valenzuela, L; Guardiola-Avila, I; Burgara-Estrella, A; Ibarra-Zavala, M; Mata-Haro, V

2015-10-01

The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A. acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A. acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A. acidocaldarius isolates analysed in this study. Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species. © 2015 The Society for Applied Microbiology.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds

PubMed Central

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa

2015-01-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. PMID:25809972

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

PubMed

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa; Hirai, Itaru

2015-06-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae

PubMed Central

Hussain, Fatma; Ahmed, Nisar; Ghorbani, Abdolbaset

2018-01-01

In pursuit of developing fast and accurate species-level molecular identification methods, we tested six DNA barcodes, namely ITS2, matK, rbcLa, ITS2+matK, ITS2+rbcLa, matK+rbcLa and ITS2+matK+rbcLa, for their capacity to identify frequently consumed but geographically isolated medicinal species of Fabaceae and Poaceae indigenous to the desert of Cholistan. Data were analysed by BLASTn sequence similarity, pairwise sequence divergence in TAXONDNA, and phylogenetic (neighbour-joining and maximum-likelihood trees) methods. Comparison of six barcode regions showed that ITS2 has the highest number of variable sites (209/360) for tested Fabaceae and (106/365) Poaceae species, the highest species-level identification (40%) in BLASTn procedure, distinct DNA barcoding gap, 100% correct species identification in BM and BCM functions of TAXONDNA, and clear cladding pattern with high nodal support in phylogenetic trees in both families. ITS2+matK+rbcLa followed ITS2 in its species-level identification capacity. The study was concluded with advocating the DNA barcoding as an effective tool for species identification and ITS2 as the best barcode region in identifying medicinal species of Fabaceae and Poaceae. Current research has practical implementation potential in the fields of pharmaco-vigilance, trade of medicinal plants and biodiversity conservation. PMID:29576968

New Paenibacillus larvae bacterial isolates from honey bee colonies infected with American foulbrood disease in Egypt.

PubMed

Masry, Saad Hamdy Daif; Kabeil, Sanaa Soliman; Hafez, Elsayed Elsayed

2014-03-04

The American foulbrood disease is widely distributed all over the world and causes a serious problem for the honeybee industry. Different infected larvae were collected from different apiaries, ground in phosphate saline buffer (PSB) and bacterial isolation was carried out on nutrient agar medium. Different colonies were observed and were characterized biologically. Two bacterial isolates (SH11 and SH33) were subjected to molecular identification using 16S rRNA gene and the sequence analysis revealed that the two isolates are Paenibacillus larvae with identity not exceeding 83%. The DNA sequence alignment between the other P. larvae bacterial strains and the two identified bacterial isolates showed that all the examined bacterial strains have the same ancestor, i.e. they have the same origin. The SH33 isolate was closely related to the P. larvae isolated from Germany, whereas the isolate SH11 was close to the P. larvae isolated from India. The phylogenetic tree constructed for 20 different Bacillus sp. and the two isolates SH11 and SH33 demonstrated that the two isolates are Bacillus sp. and they are new isolates. The bacterial isolates will be subjected to more tests for more confirmations.

Performance Assessment of the CapitalBio Mycobacterium Identification Array System for Identification of Mycobacteria

PubMed Central

Liu, Jingbo; Yan, Zihe; Han, Min; Han, Zhijun; Jin, Lingjie; Zhao, Yanlin

2012-01-01

The CapitalBio Mycobacterium identification microarray system is a rapid system for the detection of Mycobacterium tuberculosis. The performance of this system was assessed with 24 reference strains, 486 Mycobacterium tuberculosis clinical isolates, and 40 clinical samples and then compared to the “gold standard” of DNA sequencing. The CapitalBio Mycobacterium identification microarray system showed highly concordant identification results of 100% and 98.4% for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), respectively. The sensitivity and specificity of the CapitalBio Mycobacterium identification array for identification of Mycobacterium tuberculosis isolates were 99.6% and 100%, respectively, for direct detection and identification of clinical samples, and the overall sensitivity was 52.5%. It was 100% for sputum, 16.7% for pleural fluid, and 10% for bronchoalveolar lavage fluid, respectively. The total assay was completed in 6 h, including DNA extraction, PCR, and hybridization. The results of this study confirm the utility of this system for the rapid identification of mycobacteria and suggest that the CapitalBio Mycobacterium identification array is a molecular diagnostic technique with high sensitivity and specificity that has the capacity to quickly identify most mycobacteria. PMID:22090408

Infectious hematopoietic necrosis virus: Monophyletic origin of European isolates from North American Genogroup M

USGS Publications Warehouse

Enzmann, P.-J.; Kurath, G.; Fichtner, D.; Bergmann, S.M.

2005-01-01

Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe. ?? Inter-Research 2005.

Identification of Aeromonas isolates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Lamy, Brigitte; Kodjo, Angeli; Laurent, Frédéric

2011-09-01

We evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying aeromonads with an extraction procedure. Genus-level accuracy was 100%. Compared to rpoB gene sequencing, species-level accuracy was 90.6% (29/32) for type and reference strains and 91.4% for a collection of 139 clinical and environmental isolates, making this system one of the most accurate and rapid methods for phenotypic identification. The reliability of this technique was very promising, although some improvements in database composition, taxonomy, and discriminatory power are needed. Copyright © 2011 Elsevier Inc. All rights reserved.

Isolation and Identification of Active Compounds from Papaya Plants and Activities as Antimicrobial

NASA Astrophysics Data System (ADS)

Prasetya, A. T.; Mursiti, S.; Maryan, S.; Jati, N. K.

2018-04-01

Extraction and isolation of papaya seeds and leaves (Carica papaya L) has been performed using n-hexane and ethanol solvents. Further isolation of the extract obtained using ethyl acetate and diethyl ether solvents. The result of the phytochemical test of papaya extract obtained by mixture of an active compound of flavonoids, alkaloids, tannins, steroids, and saponins. Ethyl acetate isolates containing only flavonoids and diethyl ether isolates contain only alkaloids. Extracts and isolates from papaya plants had gram-positive antibacterial activity greater than the gram-negative bacteria, but both did not have antifungal activity. Papaya extracts have greater antibacterial activity than flavonoid isolates and alkaloid isolates. Strong antibacterial inhibitory sequences are extracts of papaya plants, flavonoid isolates, and alkaloid isolates.

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings

PubMed Central

Iatta, Roberta; Nuccio, Federica; Immediato, Davide; Mosca, Adriana; De Carlo, Carmela; Miragliotta, Giuseppe; Parisi, Antonio; Crescenzo, Giuseppe; Otranto, Domenico

2016-01-01

Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of β-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena. PMID:27413191

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia.

PubMed

Song, Yuli; Liu, Chengxu; Molitoris, Denise; Tomzynski, Thomas J; Mc Teague, Maureen; Read, Erik; Finegold, Sydney M

2002-12-01

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.

Extended-Spectrum β-lactamase (ESBL) producing Enterobacter aerogenes phenotypically misidentified as Klebsiella pneumoniae or K. terrigena

PubMed Central

Claeys, Geert; De Baere, Thierry; Wauters, Georges; Vandecandelaere, Patricia; Verschraegen, Gerda; Muylaert, An; Vaneechoutte, Mario

2004-01-01

Background Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates. Results Ten clinical isolates, identified as K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes. Conclusions Clinical E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification. PMID:15619329

Utilization of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry for identification of infantile seborrheic dermatitis-causing Malassezia and incidence of culture-based cutaneous Malassezia microbiota of 1-month-old infants.

PubMed

Yamamoto, Mikachi; Umeda, Yoshiko; Yo, Ayaka; Yamaura, Mariko; Makimura, Koichi

2014-02-01

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI-TOF-MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1-month-old infants using this technique. A Malassezia species-specific MALDI-TOF-MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1-month-old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI-TOF-MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI-TOF-MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia. © 2014 Japanese Dermatological Association.

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis.

PubMed

Adkins, P R F; Middleton, J R; Calcutt, M J; Stewart, G C; Fox, L K

2017-06-01

Staphylococcus hyicus and Staphylococcus agnetis are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus , S. agnetis , and Staphylococcus aureus Isolates ( n = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus , S. agnetis , and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis ( n = 43) or S. hyicus ( n = 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections. Copyright © 2017 American Society for Microbiology.

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

PubMed Central

Schelfaut, Jacqueline J. G.; Bernards, Alexandra T.; Claas, Eric C. J.

2012-01-01

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated. PMID:22278834

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates

PubMed Central

Faron, Matthew L.; Buchan, Blake W.; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L.; Granato, Paul A.; Wilson, Deborah A.; Procop, Gary W.; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A.

2015-01-01

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria. PMID:26529504

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

PubMed

Faron, Matthew L; Buchan, Blake W; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L; Granato, Paul A; Wilson, Deborah A; Procop, Gary W; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A

2015-01-01

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

[Analysis of 4 clustered high risk acute flaccid paralysis cases in Shanxi Province in 2006].

PubMed

Yan, Dong-mei; Zhang, Yong; Wang, Dong-yan

2010-04-01

Analysis of epidemiology of 4 clustered high risk acute flaccid paralysis(AFP) cases reported by Shanxi province in 2006 and VP1 gene characteristic for type III poliovirus isolated from the four AFP cases. Virus isolation and identification were conducted according to the 4th edition of WHO polio laboratory manual. The sequence of VP1 region were amplified and sequenced. The phylogenetic trees based on VP1 region were constructed. Three of four high risk AFP cases were suspected as vaccine associated paralysis poliomyelitis (VAPP), the onset date of them were close. VP1 sequencing of the four type III isolates revealed that the identity were 99.7%, 99.9%, 99.4% and 99.9% respectively compared with vaccine reference strain-BJOPV3. According to WHO criteria, the four isolates were identified as type III vaccine-related poliovirus. Phylogenetic analysis based on VP1 coding sequence showed that the four type III poliovirus were not related significantly. The type III poliovirus isolated from 3 suspected VAPP cases shared one nucleotide mutation at 2637 (C-->U), which result in the amino acid mutation from Val into Ala. The improvement of laboratory surveillance for clustered high risk AFP cases should be strengthened so as to detect and prevent poliovirus circulation timely.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

PubMed

G Junior, Daniel S; Araújo, Flábio R; Almeida Junior, Nalvo F; Adi, Said S; Cheung, Luciana M; Fragoso, Stenio P; Ramos, Carlos A N; Oliveira, Renato Henrique M de; Santos, Caroline S; Bacanelli, Gisele; Soares, Cleber O; Rosinha, Grácia M S; Fonseca, Adivaldo H

2010-11-01

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Comparing genome versus proteome-based identification of clinical bacterial isolates.

PubMed

Galata, Valentina; Backes, Christina; Laczny, Cédric Christian; Hemmrich-Stanisak, Georg; Li, Howard; Smoot, Laura; Posch, Andreas Emanuel; Schmolke, Susanne; Bischoff, Markus; von Müller, Lutz; Plum, Achim; Franke, Andre; Keller, Andreas

2018-05-01

Whole-genome sequencing (WGS) is gaining importance in the analysis of bacterial cultures derived from patients with infectious diseases. Existing computational tools for WGS-based identification have, however, been evaluated on previously defined data relying thereby unwarily on the available taxonomic information.Here, we newly sequenced 846 clinical gram-negative bacterial isolates representing multiple distinct genera and compared the performance of five tools (CLARK, Kaiju, Kraken, DIAMOND/MEGAN and TUIT). To establish a faithful 'gold standard', the expert-driven taxonomy was compared with identifications based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis. Additionally, the tools were also evaluated using a data set of 200 Staphylococcus aureus isolates.CLARK and Kraken (with k =31) performed best with 626 (100%) and 193 (99.5%) correct species classifications for the gram-negative and S. aureus isolates, respectively. Moreover, CLARK and Kraken demonstrated highest mean F-measure values (85.5/87.9% and 94.4/94.7% for the two data sets, respectively) in comparison with DIAMOND/MEGAN (71 and 85.3%), Kaiju (41.8 and 18.9%) and TUIT (34.5 and 86.5%). Finally, CLARK, Kaiju and Kraken outperformed the other tools by a factor of 30 to 170 fold in terms of runtime.We conclude that the application of nucleotide-based tools using k-mers-e.g. CLARK or Kraken-allows for accurate and fast taxonomic characterization of bacterial isolates from WGS data. Hence, our results suggest WGS-based genotyping to be a promising alternative to the MS-based biotyping in clinical settings. Moreover, we suggest that complementary information should be used for the evaluation of taxonomic classification tools, as public databases may suffer from suboptimal annotations.

Isolation and identification of a new strain of hirame rhabdovirus (HIRRV) from Japanese flounder Paralichthys olivaceus in China.

PubMed

Zhang, Jialin; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2017-04-07

Hirame rhabdovirus virus (HIRRV) is a rhabdovirus that causes acute hemorrhage disease in fish culture, resulting in a great economic loss in parts of Asia and Europe. In this study, we isolated a virus strain named as CNPo2015 from cultured Japanese flounder in Shandong province, China. Cell isolation, electron microscopic observation, RT-PCR detection and phylogenetic analysis were used for virus identification. Further, artificial infection experiment was conducted for virulence testing. The gross signs included abdominal distension, fin reddening and yellow ascitic fluid in the abdominal cavity. Histopathological examination revealed marked cell degeneration and necrosis in the kidney. The tissue homogenates induced obvious cytopathic effects in EPC, FHM and FG cell lines. Electron microscopic observation showed the virus had a bullet-like shape with a capsule membrane. RT-PCR and sequencing analysis revealed that CNPo2015 belonged to the HIRRV with high sequence identity to HIRRV isolates. Infection experiment confirmed that the HIRRV CNPo2015 strain was virulent to flounder juveniles with a LD 50 value of 1.0 × 10 5.9 TCID 50 /fish. In conclusion, we described the first isolation and characterization of a HIRRV from Japanese flounder in China. This will provide a candidate material for further research on the infection mechanism and preventive strategies of HIRRV.

Virus surveys of Capsicum spp. in the Republic of Benin reveal the prevalence of pepper vein yellows virus and the identification of a previously uncharacterised polerovirus species.

PubMed

Afouda, Leonard; Kone, Daouda; Zinsou, Valerien; Dossou, Laurence; Kenyon, Lawrence; Winter, Stephan; Knierim, Dennis

2017-06-01

Surveys were conducted in 2014 and 2015 in Southern and Northern Benin, respectively, to identify the viruses infecting peppers (Capsicum spp.). The samples were screened by ELISA for cucumber mosaic virus (CMV), pepper veinal mottle virus (PVMV), potato virus Y (PVY) and tomato yellow leaf curl virus (TYLCV). A generic reverse transcription PCR (RT-PCR) was used to test for the presence of poleroviruses. ELISA tests confirmed the prevalence of all viruses, while the RT-PCR detected pepper vein yellows virus (PeVYV) which is reported for the first time in Benin. A further, divergent polerovirus isolate was detected from a single pepper sample originating from southern Benin. Screening of samples collected from solanaceous plants during virus surveys in Mali (conducted in 2009) also detected this divergent polerovirus isolate in two samples from African eggplants. The complete genome sequence was obtained from the Mali isolate using transcriptome sequencing and by conventional Sanger sequencing of overlapping RT-PCR products. Based on the sequence characteristics of this isolate we propose a new polerovirus species, African eggplant yellowing virus (AeYV).

Corynebacterium tuberculostearicum: a Potentially Misidentified and Multiresistant Corynebacterium Species Isolated from Clinical Specimens

PubMed Central

Hinić, V.; Lang, C.; Weisser, M.; Straub, C.; Frei, R.

2012-01-01

Corynebacterium tuberculostearicum is a lipophilic corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. The majority of the patients were hospitalized and had a history of prolonged treatment with broad-spectrum antimicrobials. In 7 (38.9%) of the 18 cases, the isolates were found to be clinically relevant. The present report also includes detailed data on the biochemical and molecular identification of C. tuberculostearicum, as well as its identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our data demonstrate that routine biochemical tests do not provide reliable identification of C. tuberculostearicum. MALDI-TOF MS represents a helpful tool for the identification of this species, since all of the strains matched C. tuberculostearicum as the first choice and 58.3% (7/12) of the strains processed with the full extraction protocol generated scores of >2.000. Nevertheless, partial 16S rRNA gene sequencing still represents the gold standard for the identification of this species. Due to the challenging identification of C. tuberculostearicum, we presume that this organism is often misidentified and its clinical relevance is underestimated. The antimicrobial susceptibility profile of C. tuberculostearicum presented here reveals that 14 (87.5%) of the 16 strains analyzed exhibited multidrug resistance. PMID:22593594

Beta-haemolytic group A streptococci emm75 carrying altered pyrogenic exotoxin A linked to scarlet fever in adults.

PubMed

Dong, Hongjun; Xu, Guozhang; Li, Shuhua; Song, Qifa; Liu, Shijian; Lin, Hui; Chai, Yibiao; Zhou, Aimin; Fang, Ting; Zhang, Hongwei; Jin, Chunguang; Lu, Wei; Cao, Guangwen

2008-04-01

To determine the etiological cause of a food-borne outbreak of scarlet fever in adults. Swabs from the throats of the patients and asymptomatic control were cultured on blood agar plates individually. Biochemical identification of all isolates was performed with a VITEX automated system. Antibiotic susceptibility was examined by using the Kirby-Bauer disc diffusion method. emm gene and extracellular pyrogenic exotoxins of each isolate were amplified by using polymerase chain reaction and subjected to DNA sequencing. Sequence differences between the isolated and the highly similar reference sequences were compared on BLAST. Bioinformatics was used to predict protein structures. Beta-haemolytic group A streptococci (GAS) emm75 were identified from 10 of 13 available patients. The isolates were susceptible to penicillin, ampicillin, vancomycin, cefatriaxone, ofloxacin, linezolid and quinupristin. All of the isolates carried pyrogenic exotoxin A (speA) and cysteine protease (speB). Isolated speA was phylogenetically different from 30 highly similar references on BLAST. Differences in the primary sequence of the deduced protein were 14.37-20.12% between the speA and each of 11 references. Secondary protein structure of the speA was different from the references at the N-terminal. GAS emm75 encoding altered speA was responsible for the food-borne outbreak of scarlet fever in adults.

Analysis of the nucleoprotein gene identifies three distinct lineages of viral haemorrhagic septicemia virus (VHSV) within the European marine environment

USGS Publications Warehouse

Snow, M.; Cunningham, C.O.; Melvin, W.T.; Kurath, G.

1999-01-01

A ribonuclease (RNase) protection assay (RPA) has been used to detect nucleotide sequence variation within the nucleoprotein gene of 39 viral haemorrhagic septicaemia virus (VHSV) isolates of European marine origin. The classification of VHSV isolates based on RPA cleavage patterns permitted the identification of ten distinct groups of viruses based on differences at the molecular level. The nucleotide sequence of representatives of each of these groupings was determined and subjected to phylogenetic analysis. This revealed grouping of the European marine isolates of VHSV into three genotypes circulating within distinct geographic areas. A fourth genotype was identified comprising isolates originating from North America. Phylogenetic analyses indicated that VHSV isolates recovered from wild caught fish around the British Isles were genetically related to isolates responsible for losses in farmed turbot. Furthermore, a relationship between naturally occurring marine isolates and VHSV isolates causing mortality among rainbow trout in continental Europe was demonstrated. Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res. 63, 35-44. Available from: 

Identification of Two Novel Mycobacterium avium Allelic Variants in Pig and Human Isolates from Brazil by PCR-Restriction Enzyme Analysis

PubMed Central

Leão, Sylvia Cardoso; Briones, Marcelo R. S.; Sircili, Marcelo Palma; Balian, Simone Carvalho; Mores, Nelson; Ferreira-Neto, José Soares

1999-01-01

Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. avium PCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in the HaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria. PMID:10405407

Harnessing Whole Genome Sequencing in Medical Mycology.

PubMed

Cuomo, Christina A

2017-01-01

Comparative genome sequencing studies of human fungal pathogens enable identification of genes and variants associated with virulence and drug resistance. This review describes current approaches, resources, and advances in applying whole genome sequencing to study clinically important fungal pathogens. Genomes for some important fungal pathogens were only recently assembled, revealing gene family expansions in many species and extreme gene loss in one obligate species. The scale and scope of species sequenced is rapidly expanding, leveraging technological advances to assemble and annotate genomes with higher precision. By using iteratively improved reference assemblies or those generated de novo for new species, recent studies have compared the sequence of isolates representing populations or clinical cohorts. Whole genome approaches provide the resolution necessary for comparison of closely related isolates, for example, in the analysis of outbreaks or sampled across time within a single host. Genomic analysis of fungal pathogens has enabled both basic research and diagnostic studies. The increased scale of sequencing can be applied across populations, and new metagenomic methods allow direct analysis of complex samples.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

Isolation and molecular typing of Naegleria fowleri from the brain of a cow that died of primary amebic meningoencephalitis.

PubMed

Visvesvara, Govinda S; De Jonckheere, Johan F; Sriram, Rama; Daft, Barbara

2005-08-01

Naegleria fowleri causes an acute and rapidly fatal central nervous system infection called primary amebic meningoencephalitis (PAM) in healthy children and young adults. We describe here the identification of N. fowleri isolated from the brain of one of several cows that died of PAM based on sequencing of the internal transcribed spacers, including the 5.8S rRNA genes.

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18.

PubMed

Paulus, Constanze; Rebets, Yuriy; Tokovenko, Bogdan; Nadmid, Suvd; Terekhova, Larisa P; Myronovskyi, Maksym; Zotchev, Sergey B; Rückert, Christian; Braig, Simone; Zahler, Stefan; Kalinowski, Jörn; Luzhetskyy, Andriy

2017-02-10

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family - lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18

PubMed Central

Paulus, Constanze; Rebets, Yuriy; Tokovenko, Bogdan; Nadmid, Suvd; Terekhova, Larisa P.; Myronovskyi, Maksym; Zotchev, Sergey B.; Rückert, Christian; Braig, Simone; Zahler, Stefan; Kalinowski, Jörn; Luzhetskyy, Andriy

2017-01-01

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family – lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites. PMID:28186197

Application of next generation sequencing toward sensitive detection of enteric viruses isolated from celery samples as an example of produce.

PubMed

Yang, Zhihui; Mammel, Mark; Papafragkou, Efstathia; Hida, Kaoru; Elkins, Christopher A; Kulka, Michael

2017-11-16

Next generation sequencing (NGS) holds promise as a single application for both detection and sequence identification of foodborne viruses; however, technical challenges remain due to anticipated low quantities of virus in contaminated food. In this study, with a focus on data analysis using several bioinformatics tools, we applied NGS toward amplification-independent detection and identification of norovirus at low copy (<10 3 copies) or within multiple strains from produce. Celery samples were inoculated with human norovirus (stool suspension) either as a single norovirus strain, a mixture of strains (GII.4 and GII.6), or a mixture of different species (hepatitis A virus and norovirus). Viral RNA isolation and recovery was confirmed by RT-qPCR, and optimized for library generation and sequencing without amplification using the Illumina MiSeq platform. Extracts containing either a single virus or a two-virus mixture were analyzed using two different analytic approaches to achieve virus detection and identification. First an overall assessment of viral genome coverage for samples varying in copy numbers (1.1×10 3 to 1.7×10 7 ) and genomic content (single or multiple strains in various ratios) was completed by reference-guided mapping. Not unexpectedly, this targeted approach to identification was successful in correctly mapping reads, thus identifying each virus contained in the inoculums even at low copy (estimated at 12 copies). For the second (metagenomic) approach, samples were treated as "unknowns" for data analyses using (i) a sequence-based alignment with a local database, (ii) an "in-house" k-mer tool, (iii) a commercially available metagenomics bioinformatic analysis platform cosmosID, and (iv) an open-source program Kraken. Of the four metagenomics tools applied in this study, only the local database alignment and in-house k-mer tool were successful in detecting norovirus (as well as HAV) at low copy (down to <10 3 copies) and within a mixture of virus strains or species. The results of this investigation provide support for continued investigation into the development and integration of these analytical tools for identification and detection of foodborne viruses. Published by Elsevier B.V.

Phylogenetic analyses of the genus Aeromonas based on housekeeping gene sequencing and its influence on systematics.

PubMed

Navarro, Aaron; Martínez-Murcia, Antonio

2018-04-19

The phylogenies derived from housekeeping gene sequence alignments, although mere evolutionary hypotheses, have increased our knowledge about the Aeromonas genetic diversity, providing a robust species delineation framework invaluable for reliable, easy and fast species identification. Previous classifications of Aeromonas, have been fully surpassed by recently developed phylogenetic (natural) classification obtained from the analysis of so-called "molecular chronometers". Despite ribosomal RNAs cannot split all known Aeromonas species, the conserved nature of 16S rRNA offers reliable alignments containing mosaics of sequence signatures which may serve as targets of genus-specific oligonucleotides for subsequent identification/detection tests in samples without culturing. On the contrary, some housekeeping genes coding for proteins show a much better chronometric capacity to discriminate highly related strains. Although both, species and loci, do not all evolve at exactly the same rate, published Aeromonas phylogenies were congruent to each other, indicating that, phylogenetic markers are synchronized and a concatenated multi-gene phylogeny, may be "the mirror" of the entire genomic relationships. Thanks to MLPA approaches, the discovery of new Aeromonas species and strains of rarely isolated species is today more frequent and, consequently, should be extensively promoted for isolate screening and species identification. Although, accumulated data still should be carefully catalogued to inherit a reliable database. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Structural studies of O-polysaccharide isolated from Cronobacter sakazakii Sequence Type 12 from a case of neonatal necrotizing enterocolitis.

PubMed

Marszewska, Kinga; Czerwicka, Małgorzata; Forsythe, Stephen J; Ossowska, Karolina; Dziadziuszko, Halina; Kaczyński, Zbigniew

2015-04-30

The O-polysaccharide (OPS) of Cronobacter sakazakii NTU 696 (Sequence Type 12) from a case of neonatal necrotizing enterocolitis was isolated from the polysaccharide fraction obtained after lipopolysaccharide (LPS) hydrolysis. Purified OPS was analyzed by NMR spectroscopy ((1)H, COSY, TOCSY, NOESY, HSQC, HSQC-TOCSY and HMBC experiments) and chemical methods. Obtained monosaccharide derivatives analyzed by gas chromatography and gas chromatography-mass spectrometry allowed the identification of six sugar components. Performed experiments enabled to establish a structure of the OPS repeating unit of C. sakazakii NTU 696, as: [structure: see text]. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification, characterization and phylogenetic analysis of antifungal Trichoderma from tomato rhizosphere.

PubMed

Rai, Shalini; Kashyap, Prem Lal; Kumar, Sudheer; Srivastava, Alok Kumar; Ramteke, Pramod W

2016-01-01

The use of Trichoderma isolates with efficient antagonistic activity represents a potentially effective and alternative disease management strategy to replace health hazardous chemical control. In this context, twenty isolates were obtained from tomato rhizosphere and evaluated by their antagonistic activity against four fungal pathogens ( Fusarium oxysporum f. sp. lycopersici , Alternaria alternata , Colletotrichum gloeosporoides and Rhizoctonia solani ). The production of extracellular cell wall degrading enzymes of tested isolates was also measured. All the isolates significantly reduced the mycelial growth of tested pathogens but the amount of growth reduction varied significantly as well. There was a positive correlation between the antagonistic capacity of Trichoderma isolates towards fungal pathogens and their lytic enzyme production. The Trichoderma isolates were initially sorted according to morphology and based on the translation elongation factor 1-α gene sequence similarity, the isolates were designated as Trichoderma harzianum , T. koningii , T. asperellum , T. virens and T. viride . PCA analysis explained 31.53, 61.95, 62.22 and 60.25% genetic variation among Trichoderma isolates based on RAPD, REP-, ERIC- and BOX element analysis, respectively. ERG - 1 gene, encoding a squalene epoxidase has been used for the first time for diversity analysis of antagonistic Trichoderma from tomato rhizosphere. Phylogenetic analysis of ERG -1 gene sequences revealed close relatedness of ERG -1sequences with earlier reported sequences of Hypocrea lixii , T. arundinaceum and T. reesei. However, ERG -1 gene also showed heterogeneity among some antagonistic isolates and indicated the possibility of occurrence of squalene epoxidase driven triterpene biosynthesis as an alternative biocontrol mechanism in Trichoderma species.

Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant.

PubMed

Palma, Federica; Pasquali, Frédérique; Lucchi, Alex; Cesare, Alessandra De; Manfreda, Gerardo

2017-08-16

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates.

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes.

PubMed

Nong, Guang; Chow, Virginia; Schmidt, Liesbeth M; Dickson, Don W; Preston, James F

2007-08-01

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.

Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

PubMed

Roy, Avijit; Ananthakrishnan, G; Hartung, John S; Brlansky, R H

2010-10-01

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Characterization of Staphylococcus caprae Clinical Isolates Involved in Human Bone and Joint Infections, Compared with Goat Mastitis Isolates.

PubMed

d'Ersu, J; Aubin, G G; Mercier, P; Nicollet, P; Bémer, P; Corvec, S

2016-01-01

Staphylococcus caprae is an emerging microorganism in human bone and joint infections (BJI). The aim of this study is to describe the features of S. caprae isolates involved in BJI (H for human) compared with those of isolates recovered in goat mastitis (A for animal). Fourteen isolates of each origin were included. Identifications were performed using a Vitek 2 GP ID card, tuf gene sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Vitek MS. Molecular typing was carried out using pulsed-field gel electrophoresis (PFGE) and DiversiLab technology. The crystal violet method was used to determine biofilm-forming ability. Virulence factors were searched by PCR. Vitek MS technology provides an accurate identification for the two types of isolates compared to that of gold-standard sequencing (sensitivity, 96.4%), whereas the Vitek 2 GP ID card was more effective for H isolates. Molecular typing methods revealed two distinct lineages corresponding to the origin despite few overlaps: H and A. In our experimental conditions, no significant difference was observed in biofilm production ability between H and A isolates. Nine isolates (5 H isolates and 4 A isolates) behaved as weak producers while one A isolate was a strong producer. Concerning virulence factors, the autolysin atlC and the serine aspartate adhesin (sdrZ) genes were detected in 24 isolates (86%), whereas the lipase gene was always detected, except in one H isolate (96%). The ica operon was present in 23 isolates (82%). Fibrinogen-binding (fbe) or collagen-binding (cna) genes were not detected by using primers designed for Staphylococcus aureus or Staphylococcus epidermidis, even in low stringency conditions. Although S. caprae probably remains underestimated in human infections, further studies are needed to better understand the evolution and the adaptation of this species to its host. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

PubMed

Dunn, Glynis

2016-01-01

The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence.

Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

PubMed Central

Shittu, Ismaila; Sharma, Poonam; Volkening, Jeremy D.; Solomon, Ponman; Sulaiman, Lanre K.; Joannis, Tony M.; Williams-Coplin, Dawn; Miller, Patti J.; Dimitrov, Kiril M.

2016-01-01

The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II. PMID:26823576

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

PubMed

Naveed, Muhammad; Mubeen, Samavia; Khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

2014-01-01

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

PubMed Central

Naveed, Muhammad; Mubeen, Samavia; khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

2014-01-01

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

Identification and Characterization of the Genes and Enzymes Belonging to the Bile Acid Catabolic Pathway in Pseudomonas.

PubMed

Luengo, José M; Olivera, Elías R

2017-01-01

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and other steroids (e.g., testosterone and 4-androsten-3,17-dione). From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemical defined media containing some cyclopentane-perhydro-phenantrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the 9,10-seco pathway, the route involved in the aerobic assimilation of steroids. In this manuscript, we describe the most relevant methods required for (1) isolation and characterization of these species; (2) determining the chromosomal location, nucleotide sequence, and functional analysis of the catabolic genes (or gene clusters) encoding the enzymes from this pathway; and (3) the tools employed to establish the role of some of the proteins that participate in this route.

Isolation and molecular identification of Bartonellae from wild rats (Rattus species) in Malaysia.

PubMed

Tay, Sun Tee; Mokhtar, Aida Syafinaz; Zain, Siti Nursheena Mohd; Low, Kiat Cheong

2014-06-01

This study describes our investigation on the prevalence and molecular identification of bartonellae from Rattus diardii and R. norvegicus in the urban areas of Malaysia. Of 95 rats investigated, Bartonella tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis were isolated from kidney and spleen homogenates of four rats. Bartonellae DNA was amplified from the rat organ tissues by using primers specific for the bartonellae RNA polymerase beta subunit (rpoB) gene in nine other rats. Sequence analysis of the rpoB gene fragments shows the identification of B. queenslandensis in five rats, B. elizabethae in three rats, and B. tribocorum in one rat. Combining the results of isolation and molecular detection of bartonellae, we found that the prevalence of Bartonella infection in the Rattus spp. investigated in this study was 13.7%. Implementation of effective rat control program in the urban areas is necessary to prevent the spillover of bartonellosis from rats to humans. © The American Society of Tropical Medicine and Hygiene.

Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system.

PubMed

Kämpfer, Peter; Nienhüser, Anita; Packroff, Gabriele; Wernicke, Frank; Mehling, Arnd; Nixdorf, Katja; Fiedler, Stefanie; Kolauch, Claudia; Esser, Michael

2008-07-01

The accuracy of a traditional method (lactose utilization with acid and gas production) for the detection of coliform bacteria and E. coli was tested in comparison with method ISO 9308-1 (based on acid formation from lactose) and the Colilert-18 system (detection of beta-galactosidase). A total of 345 isolates were identified after isolation from water samples using API 20E strips. The Colilert-18 led to the highest number of positive findings (95% of the isolates were assigned to coliforms), whereas the ISO-9308-1 method resulted only in 29% coliform findings. With the traditional method only 15% were rated positive. Most of the isolates were identified by the API 20E system as Enterobacter spp. (species of the Enterobacter cloacae complex), Serratia spp., Citrobacter spp.and Klebsiella spp.; but species identification remained vague in several cases. A more detailed identification of 126 pure cultures by using 16S rRNA gene sequence analysis and analysis of the hsp60 gene resulted in the identification of Enterobacter nimipressuralis, E. amnigenus, E. asburiae, E. hormaechei, and Serratia fonticola as predominat coliforms. These species are beta-galactosidase positive, but show acid formation from lactose often after a prolonged incubation time. They are often not of fecal origin and may interfere with the ability to accurately detect coliforms of fecal origin.

Secretome Analysis Identifies Potential Pathogenicity/Virulence Factors of Tilletia indica, a Quarantined Fungal Pathogen Inciting Karnal Bunt Disease in Wheat.

PubMed

Pandey, Vishakha; Singh, Manoj; Pandey, Dinesh; Marla, Soma; Kumar, Anil

2018-04-01

Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity/virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI-TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host-derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity/virulence factors homologs that were further subjected to sequence- and structure-based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity/virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and characterization of the dominant lactic acid bacteria isolated from traditional fermented milk in Mongolia.

PubMed

Sun, Z H; Liu, W J; Zhang, J C; Yu, J; Gao, W; Jiri, M; Menghe, B; Sun, T S; Zhang, H P

2010-05-01

Five samples of Airag and 20 of Tarag (both in Mongolia) were collected from scattered households. One hundred strains of lactic acid bacteria (LAB) were isolated and identified from these samples according to phenotypic characterization and 16S rRNA gene sequence analysis. Eighty-five isolates belonged to the genus Lactobacillus, 15 being classified as coccoid LAB. All isolates belonged to 5 genera and 11 to different species and subspecies. Lactobacillus (Lb.) helveticus was predominant population in Airag samples, Lb. fermentum and Lb. helveticus were the major LAB microflora in Tarag.

Anaerococcus degenerii sp. nov., isolated from human clinical specimens.

PubMed

Veloo, A C M; Elgersma, P E; van Winkelhoff, A J

2015-06-01

Four clinical isolates of gram-positive strict anaerobic cocci were isolated from four different human mixed anaerobic infections. The taxonomical status of the four strains could not be established using standard identification techniques. The biochemical features of the strains were established and their taxonomic position was determined using 16S rRNA sequencing. The four strains form a homogeneous phenotypical and genotypical cluster. A new Anaerococcus species is proposed for these isolates: Anaerococcus degenerii sp. nov. The type strain is UMCG-104(T) = DSM29674(T) (accession number AM176528). Copyright © 2015 Elsevier Ltd. All rights reserved.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

PubMed Central

Owen, Joseph R.; Noyes, Noelle; Young, Amy E.; Prince, Daniel J.; Blanchard, Patricia C.; Lehenbauer, Terry W.; Aly, Sharif S.; Davis, Jessica H.; O’Rourke, Sean M.; Abdo, Zaid; Belk, Keith; Miller, Michael R.; Morley, Paul; Van Eenennaam, Alison L.

2017-01-01

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. PMID:28739600

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

PubMed

Owen, Joseph R; Noyes, Noelle; Young, Amy E; Prince, Daniel J; Blanchard, Patricia C; Lehenbauer, Terry W; Aly, Sharif S; Davis, Jessica H; O'Rourke, Sean M; Abdo, Zaid; Belk, Keith; Miller, Michael R; Morley, Paul; Van Eenennaam, Alison L

2017-09-07

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates ( Histophilus somni , Mycoplasma bovis , Mannheimia haemolytica , and Pasteurella multocida ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni , M. haemolytica , and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. Copyright © 2017 Owen et al.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

PubMed

El-Gazzar, Mohamed; Ghanem, Mostafa; McDonald, Kristina; Ferguson-Noel, Naola; Raviv, Ziv; Slemons, Richard D

2017-03-01

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

PubMed

Williams, Angela H; Sharma, Mamta; Thatcher, Louise F; Azam, Sarwar; Hane, James K; Sperschneider, Jana; Kidd, Brendan N; Anderson, Jonathan P; Ghosh, Raju; Garg, Gagan; Lichtenzveig, Judith; Kistler, H Corby; Shea, Terrance; Young, Sarah; Buck, Sally-Anne G; Kamphuis, Lars G; Saxena, Rachit; Pande, Suresh; Ma, Li-Jun; Varshney, Rajeev K; Singh, Karam B

2016-03-05

Soil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world's second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula. Focusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp. We demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn't share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Comparison of methods for the identification of microorganisms isolated from blood cultures.

PubMed

Monteiro, Aydir Cecília Marinho; Fortaleza, Carlos Magno Castelo Branco; Ferreira, Adriano Martison; Cavalcante, Ricardo de Souza; Mondelli, Alessandro Lia; Bagagli, Eduardo; da Cunha, Maria de Lourdes Ribeiro de Souza

2016-08-05

Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.

Identification of Escherichia coli and Shigella Species from Whole-Genome Sequences.

PubMed

Chattaway, Marie A; Schaefer, Ulf; Tewolde, Rediat; Dallman, Timothy J; Jenkins, Claire

2017-02-01

Escherichia coli and Shigella species are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species of Shigella are therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982 Escherichia coli and Shigella sp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasive E. coli isolates that were misidentified as Shigella flexneri or S. boydii by the kmer ID, and 8 were S. flexneri isolates misidentified by TB&S as S. boydii due to nonfunctional S. flexneri O antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising both S. boydii and S. dysenteriae strains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data. Shigella can be differentiated from E. coli and accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species of Shigella, and identified emerging pathoadapted lineages. © Crown copyright 2017.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

PubMed

de la Bastide, Paul Y; Leung, Wai Lam; Hintz, William E

2015-01-01

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

PubMed Central

Reuter, Sandra; Ellington, Matthew J.; Cartwright, Edward J. P.; Köser, Claudio U.; Török, M. Estée; Gouliouris, Theodore; Harris, Simon R.; Brown, Nicholas M.; Holden, Matthew T. G.; Quail, Mike; Parkhill, Julian; Smith, Geoffrey P.; Bentley, Stephen D.; Peacock, Sharon J.

2014-01-01

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance. PMID:23857503

Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

PubMed Central

2010-01-01

Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 μmol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days. PMID:20377865

DNA Microarray Profiling of a Diverse Collection of Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates Assigns the Majority to the Correct Sequence Type and Staphylococcal Cassette Chromosome mec (SCCmec) Type and Results in the Subsequent Identification and Characterization of Novel SCCmec-SCCM1 Composite Islands

PubMed Central

Brennan, Orla M.; Deasy, Emily C.; Rossney, Angela S.; Kinnevey, Peter M.; Ehricht, Ralf; Monecke, Stefan; Coleman, David C.

2012-01-01

One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCCM1 from ST8/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmec types and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate. PMID:22869569

Isolation of Cryptococcus laurentii from Canada Goose guano in rural upstate New York.

PubMed

Filion, Tera; Kidd, Sarah; Aguirre, Karen

2006-11-01

Cryptococcus neoformans and Cryptococcus gattii are etiologic agents of cryptococcal pneumonia and meningitis, potentially lethal syndromes associated with AIDS. A related species, Cryptococcus laurentii, has recently been implicated in several cases of human disease. Guano from Canada Goose (Branta canadensis), an organism that lives closely beside man and inhabits recreational space in rural and suburban areas, might be a significant environmental reservoir of Cryptococcus organisms in non-urban areas. Cryptococcal organisms were isolated from Canada Goose guano from a site in rural northern New York, with identification based upon colony and microscopic morphology, ability to metabolize L: -Dopa to melanin, and positive reaction with a commercial anti-cryptococcal capsular polysaccharide latex bead agglutination test. DNA sequences from five positive isolates were identical to each other, and identical to the ITS1-5.8S-ITS2 sequences of C. laurentii strain CBS7140 (Accession AY315665) across a 511 bp sequence. All five isolates of C. laurentii possess three of the known virulence factors common to cryptococcal organisms that cause human disease: capsule, ability to grow at 37 degrees C, and laccase activity.

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

PubMed

Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

2006-08-01

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

PubMed

Branda, John A; Rychert, Jenna; Burnham, Carey-Ann D; Bythrow, Maureen; Garner, Omai B; Ginocchio, Christine C; Jennemann, Rebecca; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Sercia, Linda F; Westblade, Lars F; Ferraro, Mary Jane

2014-02-01

The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level. © 2013.

Molecular Identification of Nocardia Isolates from Clinical Samples and an Overview of Human Nocardiosis in Brazil

PubMed Central

Baio, Paulo Victor Pereira; Ramos, Juliana Nunes; dos Santos, Louisy Sanches; Soriano, Morgana Fonseca; Ladeira, Elisa Martins; Souza, Mônica Cristina; Camello, Thereza Cristina Ferreira; Ribeiro, Marcio Garcia; Hirata Junior, Raphael; Vieira, Verônica Viana; Mattos-Guaraldi, Ana Luíza

2013-01-01

Background Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA) and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease. Methodology/Principal Findings The majority of Brazilian human isolates in cases reported in literature were identified as Nocardia sp. Molecular characterization was used for species identification of Nocardia nova, Nocardia cyriacigeorgica, Nocardia asiatica and Nocardia exalbida/gamkensis. Data indicated that molecular analysis provided a different Nocardia speciation than the initial biochemical identification for most Brazilian isolates. All Nocardia isolates showed susceptibility to trimethoprim-sulfamethoxazole, the antimicrobial of choice in the treatment nocardiosis. N. nova isolated from different clinical specimens from one patient showed identical antimicrobial susceptibility patterns and two distinct clones. Conclusions/Significance Although Brazil is the world's fifth-largest country in terms of land mass and population, pulmonary, extrapulmonary and systemic forms of nocardiosis were reported in only 6 of the 26 Brazilian states from 1970 to 2013. A least 33.8% of these 46 cases of nocardiosis proved fatal. Interestingly, coinfection by two clones may occur in patients presenting nocardiosis. Nocardia infection may be more common throughout the Brazilian territory and in other developing tropical countries than is currently recognized and MLSA should be used more extensively as an effective method for Nocardia identification. PMID:24340116

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates.

PubMed

Fang, H; Ohlsson, A-K; Ullberg, M; Ozenci, V

2012-11-01

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3 % (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Identification, cloning and sequencing of Escherichia coli strain chi1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran.

PubMed

Derakhshandeh, A; Zahraei Salehi, T; Tadjbakhsh, H; Karimi, V

2009-09-01

To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain chi1378 isolated from Iranian poultry and to predict its protein product, Iss. The iss gene from E. coli strain chi1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain chi1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains. The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC). Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.

Molecular analysis of Toxoplasma gondii Surface Antigen 1 (SAG1) gene cloned from Toxoplasma gondii DNA isolated from Javanese acute toxoplasmosis

NASA Astrophysics Data System (ADS)

Haryati, Sri; Agung Prasetyo, Afiono; Sari, Yulia; Dharmawan, Ruben

2018-05-01

Toxoplasma gondii Surface Antigen 1 (SAG1) is often used as a diagnostic tool due to its immunodominant-specific as antigen. However, data of the Toxoplasma gondii SAG1 protein from Indonesian isolate is limited. To study the protein, genomic DNA was isolated from a Javanese acute toxoplasmosis blood samples patient. A complete coding sequence of Toxoplasma gondii SAG1 was cloned and inserted into an Escherichia coli expression plasmid and sequenced. The sequencing results were subjected to bioinformatics analysis. The Toxoplasma gondii SAG1 complete coding sequences were successfully cloned. Physicochemical analysis revealed the 336 aa of SAG1 had 34.7 kDa of weight. The isoelectric point and aliphatic index were 8.4 and 78.4, respectively. The N-terminal methionine half-life in Escherichia coli was more than 10 hours. The antigenicity, secondary structure, and identification of the HLA binding motifs also had been discussed. The results of this study would contribute information about Toxoplasma gondii SAG1 and benefits for further works willing to develop diagnostic and therapeutic strategies against the parasite.

RNA polymerase beta-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp.

PubMed

Ko, K S; Kuwahara, T; Haehwa, L; Yoon, Y-J; Kim, B-J; Lee, K-H; Ohnishi, Y; Kook, Y-H

2007-01-01

Partial rpoB sequences (317 bp) of 11 species of Bacteroides, two Porphyromonas spp. and two Prevotella spp. were compared to delineate the genetic relationships among Bacteroides and closely related anaerobic species. The high level of inter-species sequence dissimilarities (7.6-20.8%) allowed the various Bacteroides spp. to be distinguished. The position of the Bacteroides distasonis and Bacteriodes merdae cluster in the rpoB tree was different from the position in the 16S rRNA gene tree. Based on rpoB sequence similarity and clustering in the rpoB tree, it was possible to correctly re-identify 80 clinical isolates of Bacteroides. In addition to two subgroups, cfiA-negative (division I) and cfiA-positive (division II), of Bacteroides fragilis isolates, two distinct subgroups were also found among Bacteroides ovatus and Bacteroides thetaiotaomicron isolates. Bacteroides genus-specific rpoB PCR and B. fragilis species-specific rpoB PCR allowed Bacteroides spp. to be differentiated from Porphyromonas and Prevotella spp., and also allowed B. fragilis to be differentiated from other non-fragilisBacteroides spp. included in the present study.

Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Cassagne, Carole; Ranque, Stéphane; Normand, Anne-Cécile; Fourquet, Patrick; Thiebault, Sandrine; Planard, Chantal; Hendrickx, Marijke; Piarroux, Renaud

2011-01-01

MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

PubMed

Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

2017-08-16

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina.

PubMed

Scialfa, Exequiel; Grune, Sylvia; Brihuega, Bibiana; Aguirre, Pablo; Rivero, Marina

2017-11-29

Ten Leptospira spp. strains were isolated from water samples from Nievas stream, Olavarría, Buenos Aires province (Argentina). The isolates showed the typical motility and morphology of the genus Leptospira under dark field microscopy, developing in liquid EMJH medium after eight days of incubation at 13°C and 30°C. All isolates were negative by the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Molecular identification by 16S rRNA gene sequencing identified all isolates as nonpathogenic leptospires. Four isolates showed a genetic profile identical to that of the reference strain Leptospira biflexa serovar Patoc, and six isolates revealed sequence similarities within the 97-98% range, closely related to Leptospira yanagawae and Leptospira meyeri, respectively. Strains ScialfaASA42, ScialfaASA45, ScialfaASA44, ScialfaASA47, ScialfaASA49, ScialfaASA50 and ScialfaASA51 possibly represent a novel species of the genus Leptospira. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

[A rarely isolated bacterium in microbiology laboratories: Streptococcus uberis].

PubMed

Eryıldız, Canan; Bukavaz, Şebnem; Gürcan, Şaban; Hatipoğlu, Osman

2017-04-01

Streptococcus uberis is a gram-positive bacterium that is mostly responsible for mastitis in cattle. The bacterium rarely has been associated with human infections. Conventional phenotyphic methods can be inadequate for the identification of S.uberis; and in microbiology laboratories S.uberis is confused with the other streptococci and enterococci isolates. Recently, molecular methods are recommended for the accurate identification of S.uberis isolates. The aim of this report is to present a lower respiratory tract infection case caused by S.uberis and the microbiological methods for identification of this bacterium. A 66-year-old male patient with squamous cell lung cancer who received radiotherapy was admitted in our hospital for the control. According to the chest X-Ray, patient was hospitalized with the prediagnosis of ''cavitary tumor, pulmonary abscess''. In the first day of the hospitalization, blood and sputum cultures were drawn. Blood culture was negative, however, Candida albicans was isolated in the sputum culture and it was estimated to be due to oral lesions. After two weeks from the hospitalization, sputum sample was taken from the patient since he had abnormal respiratory sounds and cough complaint. In the Gram stained smear of the sputum there were abundant leucocytes and gram-positive cocci, and S.uberis was isolated in both 5% sheep blood and chocolate agar media. Bacterial identification and antibiotic susceptibility tests were performed by VITEK 2 (Biomerieux, France) and also, the bacterium was identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) based VITEK MS system as S.uberis. The isolate was determined susceptible to ampicillin, erythromycin, clindamycin, levofloxacin, linezolid, penicillin, cefotaxime, ceftriaxone, tetracycline and vancomycin. 16S, 23S ribosomal RNA and 16S-23S intergenic spacer gene regions were amplified with specific primers and partial DNA sequence analysis of 16S rRNA polymerase chain reaction (PCR) products were performed by 3500xL Genetic Analyzer (Applied Biosystems, USA). According to the partial 16S rRNA gene sequencing results, bacterium was confirmed as S.uberis. This report makes a significant contribution to the number of case reports of human infections caused by S.uberis as the identification was performed by current microbiological methods in our case. In conclusion, S.uberis should be evaluated as an opportunistic pathogen among the immunosuppressed patients and in addition to phenotypic bacteriological methods, the other recent microbiological methods should also be utilized for the identification.

[Identification of lactic acid bacteria in commercial yogurt and their antibiotic resistance].

PubMed

Qin, Yuxuan; Li, Jing; Wang, Qiuya; Gao, Kexin; Zhu, Baoli; Lv, Na

2013-08-04

To identify lactic acid bacteria (LAB) in commercial yogurts and investigate their antibiotic resistance. LABs were cultured from 5 yogurt brands and the isolates were identified at the species level by 16S rRNA sequence. Genotyping was performed by repetitive extragenic palindromic PCR (rep-PCR). The sensitivity to 7 antibiotics was tested for all LAB isolates by Kirby-Bauer paper diffusion (K-B method). Meanwhile, 9 antibiotic resistance genes (ARGs), including erythromycin resistance genes (ermA and ermB) and tetracycline resistance genes (tetM, tetK, tetS, tetQ, tetO, tetL and tetW), were detected by PCR amplification in the identified LAB isolates. The PCR products were confirmed by sequencing. Total 100 LABs were isolated, including 23 Lactobacillus delbrueckii ssp. bulgaricus, 26 Lactobacillus casei, 30 Streptococcus thermophilus, 5 Lactobacillus acidophilus, 6 Lactobacillus plantarum, and 10 Lactobacillus paracasei. The drug susceptibility test shows that all 100 isolates were resistant to gentamicin and streptomycin, 42 isolates were resistant to vancomycin, and on the contrary all were sensitive to cefalexin, erythromycin, tetracycline and oxytetracycline. Moreover, 5 ARGs were found in the 28 sequencing confirmed isolates, ermB gene was detected in 8 isolates, tet K in 4 isolates, tetL in 2 isolates, tetM in 4 isolates, tetO in 2 isolates. erm A, tet S, tet Q and tet W genes were not detected in the isolates. Antibiotic resistance genes were found in 53.57% (15/28) sequenced isolates, 2 -3 antibiotic resistance genes were detected in 4 isolates of L. delbrueckii ssp. bulgaricus. Some LABs were not labeled in commercial yogurt products. Antibiotic resistance genes tend to be found in the starter culture of L. delbrueckii ssp. Bulgaricus and S. thermophilus. All the LAB isolates were sensitive to erythromycin and tetracycline, even though some carried erythromycin and/or tetracycline resistance genes. We proved again that LAB could carry antibiotic resistance gene(s) though it is sensitive to antibiotics.

Early Sex Identification in Date Palm by Male-Specific Sequence-Characterized Amplified Region (SCAR) Markers.

PubMed

Kharb, Pushpa; Mitra, Charu

2017-01-01

Date palm (Phoenix dactylifera L.) is a dioecious plant, and sex of the seedlings can be determined only at the time of first flowering which takes 4-5 years. Female date palm plants are of economic importance as they bear the fruit. Therefore, sex identification at an early stage is highly desirable. DNA-based markers are useful for early sex detection. In this chapter, we describe male-specific sequence-characterized amplified region (SCAR) markers to identify sex in date palm at the seedling stage. Genomic DNA is isolated separately from both male and female date palm genotypes. Amplification of this genomic DNA isolated from male and female plants using the SCAR primers results in an amplicon of 406 bp in both female and male samples and a unique amplicon of 354 bp only in male samples. Based on this amplification pattern, the sex of date palm seedlings can be predicted.

Multilocus sequence identification of Penicillium species in cork bark during plank preparation for the manufacture of stoppers.

PubMed

Serra, Rita; Peterson, Stephen; Venâncio, Armando

2008-04-01

Despite several studies reporting Penicillium as one of the most frequent fungal genera in cork planks, the isolates were rarely identified to species level. We conducted a detailed study to identify Penicillium species from the field to the factory environment prior to and after boiling the cork planks. A total of 84 samples were analyzed. Of the 486 Penicillium isolates phenotypically identified, 32 representative or unusual strains were selected for identification by multilocus DNA sequence type. Cork proved to be a rich source of Penicillium biodiversity. A total of 30 taxa were recognized from cork including rarely seen species and 6 phylogenetically unique groups. Spores of some species lodged deep in cork can survive the boiling process. P. glabrum, P. glandicola and P. toxicarium, species with high CFU numbers in the field, are still frequently present in cork after boiling. Other species are killed by the boiling treatment and replaced by Penicillium species originating from the factory environment. Species known to contribute to cork taint were isolated at all stages. Good manufacturing practices are necessary at all stages in the preparation of cork planks to minimize the load of Penicillium species that produce cork taint.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

PubMed Central

Ülbegi-Mohyla, H.; Hijazin, M.; Alber, J.; Hassan, A. A.; Abdulmawjood, A.; Prenger-Berninghoff, E.; Weiß, R.; Zschöck, M.

2010-01-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens. PMID:20706035

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

PubMed

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

2016-06-23

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

Detection and characterization of carbapenemase-producing Enterobacteriaceae in wounded Syrian patients admitted to hospitals in northern Israel.

PubMed

Lerner, A; Solter, E; Rachi, E; Adler, A; Rechnitzer, H; Miron, D; Krupnick, L; Sela, S; Aga, E; Ziv, Y; Peretz, A; Labay, K; Rahav, G; Geffen, Y; Hussein, K; Eluk, O; Carmeli, Y; Schwaber, M J

2016-01-01

Since 2013, four hospitals in northern Israel have been providing care for Syrian nationals, primarily those wounded in the ongoing civil war. We analyzed carbapenemase-producing Enterobacteriaceae (CPE) isolates obtained from these patients. Isolate identification was performed using the VITEK 2 system. Polymerase chain reaction (PCR) was performed for the presence of bla KPC, bla NDM, and bla OXA-48. Susceptibility testing and genotyping were performed on selected isolates. During the study period, 595 Syrian patients were hospitalized, most of them young men. Thirty-two confirmed CPE isolates were grown from cultures taken from 30 patients. All but five isolates were identified as Klebsiella pneumoniae and Escherichia coli. Nineteen isolates produced NDM and 13 produced OXA-48. Among a further 29 isolates tested, multilocus sequence typing (MLST) showed that ST278 and ST38 were the major sequence types among the NDM-producing K. pneumoniae and OXA-48-producing E. coli isolates, respectively. Most were resistant to all three carbapenems in use in Israel and to gentamicin, but susceptible to colistin and fosfomycin. The source for bacterial acquisition could not be determined; however, some patients admitted to different medical centers were found to carry the same sequence type. CPE containing bla NDM and bla OXA-48 were prevalent among Syrian wounded hospitalized patients in northern Israel. The finding of the same sequence type among patients at different medical centers implies a common, prehospital source for these patients. These findings have implications for public health throughout the region.

Identification of species of viridans group streptococci in clinical blood culture isolates by sequence analysis of the RNase P RNA gene, rnpB.

PubMed

Westling, Katarina; Julander, Inger; Ljungman, Per; Vondracek, Martin; Wretlind, Bengt; Jalal, Shah

2008-03-01

Viridans group streptococci (VGS) cause severe diseases such as infective endocarditis and septicaemia. Genetically, VGS species are very close to each other and it is difficult to identify them to species level with conventional methods. The aims of the present study were to use sequence analysis of the RNase P RNA gene (rnpB) to identify VGS species in clinical blood culture isolates, and to compare the results with the API 20 Strep system that is based on phenotypical characteristics. Strains from patients with septicaemia or endocarditis were analysed with PCR amplification and sequence analysis of the rnpB gene. Clinical data were registered as well. One hundred and thirty two VGS clinical blood culture isolates from patients with septicaemia (n=95) or infective endocarditis (n=36) were analysed; all but one were identified by rnpB. Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii strains were most common in the patients with infective endocarditis. In the isolates from patients with haematological diseases, Streptococcus mitis and S. oralis dominated. In addition in 76 of the isolates it was possible to compare the results from rnpB analysis and the API 20 Strep system. In 39/76 (51%) of the isolates the results were concordant to species level; in 55 isolates there were no results from API 20 Strep. Sequence analysis of the RNase P RNA gene (rnpB) showed that almost all isolates could be identified. This could be of importance for evaluation of the portal of entry in patients with septicaemia or infective endocarditis.

Isolation and molecular characterization of Fikirini rhabdovirus, a novel virus from a Kenyan bat.

PubMed

Kading, Rebekah C; Gilbert, Amy T; Mossel, Eric C; Crabtree, Mary B; Kuzmin, Ivan V; Niezgoda, Michael; Agwanda, Bernard; Markotter, Wanda; Weil, M Ryan; Montgomery, Joel M; Rupprecht, Charles E; Miller, Barry R

2013-11-01

Zoonotic and vector-borne pathogens have comprised a significant component of emerging human infections in recent decades, and bats are increasingly recognized as reservoirs for many of these disease agents. To identify novel pathogens associated with bats, we screened tissues of bats collected in Kenya. Virus isolates were identified by next generation sequencing of viral nucleic acid preparations from the infected cell culture supernatant and characterized. Here we report the identification of Fikirini rhabdovirus, a novel rhabdovirus isolated from a bat, Hipposideros vittatus, captured along the Kenyan coast.

Identification of novel Theileria genotypes from Grant's gazelle

PubMed Central

Hooge, Janis; Howe, Laryssa; Ezenwa, Vanessa O.

2015-01-01

Blood samples collected from Grant's gazelles (Nanger granti) in Kenya were screened for hemoparasites using a combination of microscopic and molecular techniques. All 69 blood smears examined by microscopy were positive for hemoparasites. In addition, Theileria/Babesia DNA was detected in all 65 samples screened by PCR for a ~450-base pair fragment of the V4 hypervariable region of the 18S rRNA gene. Sequencing and BLAST analysis of a subset of PCR amplicons revealed widespread co-infection (25/39) and the existence of two distinct Grant's gazelle Theileria subgroups. One group of 11 isolates clustered as a subgroup with previously identified Theileria ovis isolates from small ruminants from Europe, Asia and Africa; another group of 3 isolates clustered with previously identified Theileria spp. isolates from other African antelope. Based on extensive levels of sequence divergence (1.2–2%) from previously reported Theileria species within Kenya and worldwide, the Theileria isolates detected in Grant's gazelles appear to represent at least two novel Theileria genotypes. PMID:25973394

Identification of novel Theileria genotypes from Grant's gazelle.

PubMed

Hooge, Janis; Howe, Laryssa; Ezenwa, Vanessa O

2015-08-01

Blood samples collected from Grant's gazelles (Nanger granti) in Kenya were screened for hemoparasites using a combination of microscopic and molecular techniques. All 69 blood smears examined by microscopy were positive for hemoparasites. In addition, Theileria/Babesia DNA was detected in all 65 samples screened by PCR for a ~450-base pair fragment of the V4 hypervariable region of the 18S rRNA gene. Sequencing and BLAST analysis of a subset of PCR amplicons revealed widespread co-infection (25/39) and the existence of two distinct Grant's gazelle Theileria subgroups. One group of 11 isolates clustered as a subgroup with previously identified Theileria ovis isolates from small ruminants from Europe, Asia and Africa; another group of 3 isolates clustered with previously identified Theileria spp. isolates from other African antelope. Based on extensive levels of sequence divergence (1.2-2%) from previously reported Theileria species within Kenya and worldwide, the Theileria isolates detected in Grant's gazelles appear to represent at least two novel Theileria genotypes.

Isolation and identification of efficient Egyptian malathion-degrading bacterial isolates.

PubMed

Hamouda, S A; Marzouk, M A; Abbassy, M A; Abd-El-Haleem, D A; Shamseldin, Abdelaal

2015-03-01

Bacterial isolates degrading malathion were isolated from the soil and agricultural waste water due to their ability to grow on minimal salt media amended with malathion as a sole carbon source. Efficiencies of native Egyptian bacterial malathion-degrading isolates were investigated and the study generated nine highly effective malathion-degrading bacterial strains among 40. Strains were identified by partial sequencing of 16S rDNA analysis. Comparative analysis of 16S rDNA sequences revealed that these bacteria are similar with the genus Acinetobacter and Bacillus spp. and RFLP based PCR of 16S rDNA gave four different RFLP patterns among strains with enzyme HinfI while with enzyme HaeI they gave two RFLP profiles. The degradation rate of malathion in liquid culture was estimated using gas chromatography. Bacterial strains could degrade more than 90% of the initial malathion concentration (1000 ppm) within 4 days. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reclassification of Borrelia spp. isolated in South Korea using Multilocus Sequence Typing.

PubMed

Park, Kyung-Hee; Choi, Yeon-Joo; Kim, Jeoungyeon; Park, Hye-Jin; Song, Dayoung; Jang, Won-Jong

2018-05-31

Using Borrelia isolated from South Korea, we evaluated by MLST and three intergenic genes (16S rRNA, ospA, and 5S-23S IGS) typing to analyze the relationship between host and vector and molecular background. Using the MLST analysis, we identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study was first report of the identification of B. yangtzensis using the MLST in South Korea.

Isolation and Molecular Typing of Naegleria fowleri from the Brain of a Cow That Died of Primary Amebic Meningoencephalitis

PubMed Central

Visvesvara, Govinda S.; De Jonckheere, Johan F.; Sriram, Rama; Daft, Barbara

2005-01-01

Naegleria fowleri causes an acute and rapidly fatal central nervous system infection called primary amebic meningoencephalitis (PAM) in healthy children and young adults. We describe here the identification of N. fowleri isolated from the brain of one of several cows that died of PAM based on sequencing of the internal transcribed spacers, including the 5.8S rRNA genes. PMID:16081978

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization.

PubMed

Ouoba, L I I; Parkouda, C; Diawara, B; Scotti, C; Varnam, A H

2008-01-01

To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Direct identification of pathogens from positive blood cultures using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

PubMed

Rodríguez-Sánchez, B; Sánchez-Carrillo, C; Ruiz, A; Marín, M; Cercenado, E; Rodríguez-Créixems, M; Bouza, E

2014-07-01

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Viral forensic genomics reveals the relatedness of classic herpes simplex virus strains KOS, KOS63, and KOS79

PubMed Central

Bowen, Christopher D.; Renner, Daniel W.; Shreve, Jacob T.; Tafuri, Yolanda; Payne, Kimberly M.; Dix, Richard D.; Kinchington, Paul R.; Gatherer, Derek; Szpara, Moriah L.

2016-01-01

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains. PMID:26950505

Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

PubMed Central

2014-01-01

Background This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing. Results Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues. Conclusion Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family. PMID:24410890

Viral forensic genomics reveals the relatedness of classic herpes simplex virus strains KOS, KOS63, and KOS79.

PubMed

Bowen, Christopher D; Renner, Daniel W; Shreve, Jacob T; Tafuri, Yolanda; Payne, Kimberly M; Dix, Richard D; Kinchington, Paul R; Gatherer, Derek; Szpara, Moriah L

2016-05-01

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation.

PubMed

Neill, John D; Dubovi, Edward J; Ridpath, Julia F

2015-09-30

Bovine viral diarrhea viruses (BVDV) are most commonly associated with infections of cattle. However, BVDV are often isolated from closely related ruminants with a number of BVDV-1b viruses being isolated from alpacas that were both acutely and persistently infected. The complete nucleotide sequence of the open reading frame of eleven alpaca-adapted BVDV isolates and the region encoding the envelope glycoproteins of an additional three isolates were determined. With the exception of one, all alpaca isolates were >99.2% similar at the nucleotide level. The Hercules isolate was more divergent, with 95.7% sequence identity to the other viruses. Sequence similarity of the 14 viruses indicated they were isolates of a single BVDV strain that had adapted to and were circulating through alpaca herds. Hercules was a more distantly related strain that has been isolated only once in Canada and represented a separate adaptation event that possessed the same adaptive changes. Comparison of amino acid sequences of alpaca and bovine-derived BVDV strains revealed three regions with amino acid sequences unique to all alpaca isolates. The first contained two small in-frame deletions near the N-terminus of the E2 glycoprotein. The second was found near the C-terminus of the E2 protein where four altered amino acids were located within a 30 amino acid domain that participates in E2 homodimerization. The third region contained three variable amino acids in the C-terminus of the E(rns) within the amphipathic helix membrane anchor. These changes were found in the polar side of the amphipathic helix and resulted in an increased charge within the polar face. Titration of bovine and alpaca viruses in both bovine and alpaca cells indicated that with increased charge in the amphipathic helix, the ability to infect alpaca cells also increased. Published by Elsevier B.V.

Clinical characteristics of bacteraemia caused by Lactobacillus spp. and antimicrobial susceptibilities of the isolates at a medical centre in Taiwan, 2000-2014.

PubMed

Lee, Meng-Rui; Tsai, Chia-Jung; Liang, Sheng-Kai; Lin, Ching-Kai; Huang, Yu-Tsung; Hsueh, Po-Ren

2015-10-01

The clinical characteristics of 89 patients with Lactobacillus bacteraemia treated at a university-affiliated hospital in northern Taiwan during 2000-2014 were retrospectively evaluated. Lactobacillus spp. were identified by 16S rRNA sequencing analysis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibilities of the isolates were determined by broth microdilution. The most commonly isolated species was Lactobacillus salivarius (n = 21), followed by Lactobacillus paracasei (n = 16) and Lactobacillus fermentum (n = 13). Excluding three isolates with lower 16S rRNA sequence similarity, MALDI-TOF/MS provided correct identification for 84.9% (73/86) of Lactobacillus isolates. Concordant identification was lowest for Lactobacillus casei (11%). The main infection foci were intra-abdominal infection (49%) and catheter-related bloodstream infection (17%). Only one-half of the patients received adequate antibiotic treatment during the bacteraemic episode. The majority of patients with Lactobacillus bacteraemia were immunocompromised. The 7-day and in-hospital mortality rates were 21% and 62%, respectively, and underlying malignancy was associated with a higher in-hospital mortality rate (odds ratio = 2.666). There were no significant differences in mortality (7-day, 14-day, 30-day and in-hospital) among patients with bacteraemia due to different Lactobacillus spp. Minimum inhibitory concentrations were highest for glycopeptides, cephalosporins and fluoroquinolones and were lowest for carbapenems and aminopenicillins. Lactobacillus bacteraemia was associated with a high mortality rate, and patient outcome was associated with underlying malignancy. MALDI-TOF/MS was able to accurately identify 84.9% of the Lactobacillus isolates, and L. salivarius was the predominant pathogen. The accuracy rate for identification of Lactobacillus spp. by MALDI-TOF/MS was lowest for L. casei. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

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

Kamb, A.; Weir, M.; Rudy, B.

1989-06-01

The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this methodmore » to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.« less

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

PubMed

Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

2011-07-01

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

Etiological agents causing leptospirosis in Sri Lanka: A review.

PubMed

Naotunna, Chamidri; Agampodi, Suneth Buddhika; Agampodi, Thilini Chanchala

2016-04-01

To systematically review the etiological agent causing human leptospirosis in Sri Lanka. Published articles on leptospirosis and Leptospira in Sri Lanka were all reviewed to determine serovar, strain and species level identification of Leptospira. After screening process, 74 full text articles/reports were reviewed and among of them, 12 published papers describing isolation of Leptospira from Sri Lankan patients/animals, 5 molecular epidemiology papers on newer typing methods citing Sri Lanka isolates, with a descriptions of the isolates and 6 published papers reporting PCR based species level identification were identified. Published literature showed that more than 40 strains classified under at least 20 serovars and 10 serogroups have been isolated from Sri Lanka. These isolates belong to four species, namely, Leptospira interrogans, Leptospira kirschneri, Leptospira borgpetersenii, and Leptospira santarosai. In addition, recent studies on direct patient samples without culture and isolation showed Leptospira from Leptospira weilli is also circulating in Sri Lanka. Multi locus sequence typing showed 13 genotypes of Leptospira from Sri Lankan isolates. This review shows the diversity of Leptospira in Sri Lanka, but culture isolation data has not been published in Sri Lanka during last 30 years. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Identification of viral and non-viral reverse transcribing elements in pineapple (Ananas comosus), including members of two new badnavirus species.

PubMed

Gambley, C F; Geering, A D W; Steele, V; Thomas, J E

2008-01-01

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

PubMed Central

Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

PubMed

Govan, V A; Brözel, V; Allsopp, M H; Davison, S

1998-05-01

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

PubMed Central

Govan, V. A.; Brözel, V.; Allsopp, M. H.; Davison, S.

1998-01-01

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae. PMID:9572987

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients.

PubMed

Banko, Ana; Lazarevic, Ivana; Cupic, Maja; Stevanovic, Goran; Boricic, Ivan; Jovanovic, Tanja

2012-04-01

Seven strains of Epstein-Barr virus (EBV) are defined based on C-terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30-bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C-terminal region of LMP1 in Serbian isolates. This study included 53 EBV-DNA-positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506-bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non-deleted (66%), and the rest had 30-bp, rare 69-bp, or yet unknown 27-bp deletions, which were not related to malignant or non-malignant isolate origin. However, the majority of 69-bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33-bp repeats were found in the majority of non-deleted isolates (68.6%), whereas most 69-bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95-8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical-associated characteristics in the LMP1 C terminus of investigated isolates. Copyright © 2012 Wiley Periodicals, Inc.

Development of chemiluminescent probe hybridization, RT-PCR and nucleic acid cycle sequencing assays of Sabin type 3 isolates to identify base pair 472 Sabin type 3 mutants associated with vaccine associated paralytic poliomyelitis.

PubMed

Old, M O; Logan, L H; Maldonado, Y A

1997-11-01

Sabin type 3 polio vaccine virus is the most common cause of poliovaccine associated paralytic poliomyelitis. Vaccine associated paralytic poliomyelitis cases have been associated with Sabin type 3 revertants containing a single U to C substitution at bp 472 of Sabin type 3. A rapid method of identification of Sabin type 3 bp 472 mutants is described. An enterovirus group-specific probe for use in a chemiluminescent dot blot hybridization assay was developed to identify enterovirus positive viral lysates. A reverse transcription-polymerase chain reaction (RT-PCR) assay producing a 319 bp PCR product containing the Sabin type 3 bp 472 mutation site was then employed to identify Sabin type 3 isolates. Chemiluminescent nucleic acid cycle sequencing of the purified 319 bp PCR product was then employed to identify nucleic acid sequences at bp 472. The enterovirus group probe hybridization procedure and isolation of the Sabin type 3 PCR product were highly sensitive and specific; nucleic acid cycle sequencing corresponded to the known sequence of stock Sabin type 3 isolates. These methods will be used to identify the Sabin type 3 reversion rate from sequential stool samples of infants obtained after the first and second doses of oral poliovirus vaccine.

Sequence analysis of porcine kobuvirus VP1 region detected in pigs in Japan and Thailand.

PubMed

Okitsu, Shoko; Khamrin, Pattara; Thongprachum, Aksara; Hidaka, Satoshi; Kongkaew, Sompreeya; Kongkaew, Apisek; Maneekarn, Niwat; Mizuguchi, Masashi; Hayakawa, Satoshi; Ushijima, Hiroshi

2012-04-01

Porcine kobuvirus is a new candidate species of the genus Kobuvirus in the family Picornaviridae, and information is still limited. The identification of porcine kobuvirus has been performed by the sequence analyses of the 3D region of the viruses. Therefore, the purpose of this study was to characterize the molecular properties of VP1 nucleotide sequences of the porcine kobuviruses isolated from porcine stool samples in Japan during 2009 and Thailand between 2006 and 2008. In addition, previous identification of a unique porcine kobuvirus; Japanese H023/2009/JP, which is a bovine kobuvirus-like strain based on sequence analysis of the 3D region, was also included in this study. All of the strains were amplified by the VP1-specific primer pair: the amplicons were subjected to direct sequencing and compared with the VP1 nucleotide sequences of reference strains. The VP1 sequences of strains from the GenBank database revealed high nucleotide sequence identity at 84.3-100%. On the other hand, the nucleotide identities among the 15 porcine kobuvirus strains analyzed in this study ranged from 78.8 to 99.8%. The results revealed that diversity of the strains in this study were higher than those of the strains in previous studies. Furthermore, it was found that the VP1 region of the bovine kobuvirus-like strain, H023/2009/JP, clustered with nine porcine kobuvirus strains that were isolated in Thailand and Japan. Since this strain was previously found to be closely related to bovine kobuviruses in the 3D gene region, it may be a natural recombinant.

Identification and Genetic Characterization of Ralstonia solanacearum Species Complex Isolates from Cucurbita maxima in China

PubMed Central

She, Xiaoman; Yu, Lin; Lan, Guobing; Tang, Yafei; He, Zifu

2017-01-01

Ralstonia solanacearum species complex is a devastating phytopathogen with an unusually wide host range, and new host plants are continuously being discovered. In June 2016, a new bacterial wilt on Cucurbita maxima was observed in Guangdong province, China. Initially, in the adult plant stage, several leaves of each plant withered suddenly and drooped; the plant then wilted completely, and the color of their vasculature changed to dark brown, ultimately causing the entire plant to die. Creamy-whitish bacterial masses were observed to ooze from crosscut stems of these diseased plants. To develop control strategies for C. maxima bacterial wilt, the causative pathogenic isolates were identified and characterized. Twenty-four bacterial isolates were obtained from diseased C. maxima plants, and 16S rRNA gene sequencing and pathogenicity analysis results indicated that the pathogen of C. maxima bacterial wilt was Ralstonia solanacearum. The results from DNA-based analysis, host range determination and bacteriological identification confirmed that the 24 isolates belonged to R. solanacearum phylotype I, race 1, and eight of these isolates belonged to biovar 3, while 16 belonged to biovar 4. Based on the results of partial egl gene sequence analysis, the 24 isolates clustered into three egl- sequence type groups, sequevars 17, 45, and 56. Sequevar 56 is a new sequevar which is described for the first time in this paper. An assessment of the resistance of 21 pumpkin cultivars revealed that C. moschata cv. Xiangyu1 is resistant to strain RS378, C. moschata cv. Xiangmi is moderately resistant to strain RS378, and 19 other pumpkin cultivars, including four C. maxima cultivars and 15 C. moschata cultivars, are susceptible to strain RS378. To the best of our knowledge, this is the first report of C. maxima bacterial wilt caused by R. solanacearum race 1 in the world. Our results provide valuable information for the further development of control strategies for C. maxima wilt disease. PMID:29093727

Identification and Genetic Characterization of Ralstonia solanacearum Species Complex Isolates from Cucurbita maxima in China.

PubMed

She, Xiaoman; Yu, Lin; Lan, Guobing; Tang, Yafei; He, Zifu

2017-01-01

Ralstonia solanacearum species complex is a devastating phytopathogen with an unusually wide host range, and new host plants are continuously being discovered. In June 2016, a new bacterial wilt on Cucurbita maxima was observed in Guangdong province, China. Initially, in the adult plant stage, several leaves of each plant withered suddenly and drooped; the plant then wilted completely, and the color of their vasculature changed to dark brown, ultimately causing the entire plant to die. Creamy-whitish bacterial masses were observed to ooze from crosscut stems of these diseased plants. To develop control strategies for C. maxima bacterial wilt, the causative pathogenic isolates were identified and characterized. Twenty-four bacterial isolates were obtained from diseased C. maxima plants, and 16S rRNA gene sequencing and pathogenicity analysis results indicated that the pathogen of C. maxima bacterial wilt was Ralstonia solanacearum . The results from DNA-based analysis, host range determination and bacteriological identification confirmed that the 24 isolates belonged to R. solanacearum phylotype I, race 1, and eight of these isolates belonged to biovar 3, while 16 belonged to biovar 4. Based on the results of partial egl gene sequence analysis, the 24 isolates clustered into three egl- sequence type groups, sequevars 17, 45, and 56. Sequevar 56 is a new sequevar which is described for the first time in this paper. An assessment of the resistance of 21 pumpkin cultivars revealed that C. moschata cv. Xiangyu1 is resistant to strain RS378, C. moschata cv. Xiangmi is moderately resistant to strain RS378, and 19 other pumpkin cultivars, including four C. maxima cultivars and 15 C. moschata cultivars, are susceptible to strain RS378. To the best of our knowledge, this is the first report of C. maxima bacterial wilt caused by R. solanacearum race 1 in the world. Our results provide valuable information for the further development of control strategies for C. maxima wilt disease.

Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review.

PubMed

Ponce-Alonso, M; Rodríguez-Rojas, L; Del Campo, R; Cantón, R; Morosini, M-I

2016-03-01

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011-14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A β-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoβ sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoβ correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory.

PubMed

Fatania, Nita; Fraser, Mark; Savage, Mike; Hart, Jason; Abdolrasouli, Alireza

2015-12-01

Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Phenotypic and genotypic identification of Aeromonas spp. isolated from a chlorinated intermittent water distribution system in Lebanon.

PubMed

Tokajian, Sima; Hashwa, Fuad

2004-06-01

Aeromonas spp. were detected in samples collected from both untreated groundwater and treated drinking water in Lebanon. Aeromonas spp. levels ranged between 2 and 1,100 colonies per 100 ml in the intake underground well and between 3 and 43 colonies per 100 ml in samples from the distribution system. Samples positive for Aeromonas spp. from the network had a free chlorine level ranging between 0 and 0.4 mg l(-1). Multiple antibiotic-resistance was common among the isolated aeromonads; all were resistant to amoxycillin while 92% showed resistance to cephalexin. Haemolysis on blood agar was detected in 52% of the isolates recovered from the distribution network and 81% of isolates from the untreated underground source. The Biolog microbial identification system assigned identities to all of the isolated presumptive aeromonads (at least at the genus level), which was not the case with the API 20NE strips. Differences at the species level were observed when results from the Biolog system were compared with identification based on the MicroSeq 500 16S rDNA sequence analysis. The presence of Aeromonas spp. in drinking water can be an important threat to public health, thus greater awareness of Aeromonas strains as potential enteropathogens is warranted.

Reemergence of rabies in the southern Han river region, Korea.

PubMed

Oem, Jae-Ku; Kim, Seong-Hee; Kim, Yeon-Hee; Lee, Myoung-Heon; Lee, Kyoung-Ki

2014-07-01

Recently, 11 cases of animal rabies were reported in the southern region (Suwon and Hwaseong cities) of Gyeonggi Province, South Korea. The cases were temporally separated into two cases in dogs (Canis lupus familiaris) in spring 2012 and nine cases in domestic animals and wildlife in winter 2012-13. All carcasses were submitted for histopathologic examination and viral antigen identification. Sequences of the glycoprotein, nucleoprotein, and glycoprotein-large polymerase protein intergenic noncoding loci of the 11 strains were determined and compared with published reference sequences. All rabies strains were closely related to the Gangwon strains isolated in 2008-09, suggesting that the rabies virus strains isolated in Gyeonggi were introduced from Gangwon Province.

Draft Genome Sequence of Mycobacterium chimaera Type ...

EPA Pesticide Factsheets

We report the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169T, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, though Fl-0169T possesses unique virulence genes. Evidence suggests that M. avium, M. intracellulare, and M. chimaera are differently virulent and a comparative genomic analysis is critically needed to identify diagnostic targets that reliably differentiate species of MAC. With treatment costs for Mycobacterium infections estimated to be >$1.8 B annually in the U.S., correct species identification will result in improved treatment selection, lower costs, and improved patient outcomes.

Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

PubMed

Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

2010-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

PubMed

Cheng, Jiujun; Nordeste, Ricardo; Trainer, Maria A; Charles, Trevor C

2017-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Isolation of two new retrotransposon sequences and development of molecular and cytological markers for Dasypyrum villosum (L.).

PubMed

Zhang, Jie; Jiang, Yun; Xuan, Pu; Guo, Yuanlin; Deng, Guangbing; Yu, Maoqun; Long, Hai

2017-10-01

Dasypyrum villosum is a valuable genetic resource for wheat improvement. With the aim to efficiently monitor the D. villosum chromatin introduced into common wheat, two novel retrotransposon sequences were isolated by RAPD, and were successfully converted to D. villosum-specific SCAR markers. In addition, we constructed a chromosomal karyotype of D. villosum. Our results revealed that different accessions of D. villosum showed slightly different signal patterns, indicating that distribution of repeats did not diverge significantly among D. villosum accessions. The two SCAR markers and FISH karyotype of D. villosum could be used for efficient and precise identification of D. villosum chromatin in wheat breeding.

Identification of Lactobacillus strains with probiotic features from the bottlenose dolphin (Tursiops truncatus)

PubMed Central

Diaz, MA; Bik, EM; Carlin, KP; Venn-Watson, SK; Jensen, ED; Jones, SE; Gaston, EP; Relman, DA; Versalovic, J

2013-01-01

Aims In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus. Methods and Results We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti. Conclusions Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals. Significance and Impact of the Study This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals. PMID:23855505

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species.

PubMed

Wyllie, Anne L; Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A M; Trzciński, Krzysztof; van der Ende, Arie

2017-09-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease ( n = 101) and from carriage ( n = 103), and on non-typeable pneumococci from asymptomatic individuals ( n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae , targeting cpsA , lytA , piaB , ply , Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae , whereas assays targeting cpsA , ply , Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. © 2017 The Authors.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species

PubMed Central

Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A. M.; Trzciński, Krzysztof; van der Ende, Arie

2017-01-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae. Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. PMID:28931649

[A new herbs traceability method based on DNA barcoding-origin-morphology analysis--an example from an adulterant of 'Heiguogouqi'].

PubMed

Gu, Xuan; Zhang, Xiao-qin; Song, Xiao-na; Zang, Yi-mei; Li Yan-peng; Ma, Chang-hua; Zhao, Bai-xiao; Liu, Chun-sheng

2014-12-01

The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adulterant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced; DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows: in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp cells of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains.

PubMed

Hamet, Maria Fernanda; Londero, Alejandra; Medrano, Micaela; Vercammen, Elisabeth; Van Hoorde, Koenraad; Garrote, Graciela L; Huys, Geert; Vandamme, Peter; Abraham, Analía G

2013-12-01

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Streptococcus ovuberis sp. nov., isolated from a subcutaneous abscess in the udder of a sheep.

PubMed

Zamora, Leydis; Pérez-Sancho, Marta; Fernández-Garayzábal, Jose Francisco; Orden, Jose Antonio; Domínguez-Bernal, Gustavo; de la Fuente, Ricardo; Domínguez, Lucas; Vela, Ana Isabel

2017-11-01

One unidentified, Gram-stain-positive, catalase-negative coccus-shaped organism was recovered from a subcutaneous abscess of the udder of a sheep and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolate was tentatively assigned to the genus Streptococcus, although the organism did not appear to match any recognized species. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus and showed that the nearest phylogenetic relatives of the unknown coccus corresponded to Streptococcus moroccensis and Streptococcus cameli (95.9 % 16S rRNA gene sequence similarity). The sodA sequence analysis showed less than 89.3 % sequence similarity with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from close relatives of the genus Streptococcusby using biochemical tests. A mass spectrometry profile was also obtained for the novel isolate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a representative of a novel species of the genus Streptococcus, Streptococcus ovuberis sp. nov. The type strain of Streptococcus ovuberissp. nov. is VB15-00779 T (=CECT 9179 T =CCUG 69612 T ).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

PubMed Central

Suttisunhakul, Vichaya; Pumpuang, Apinya; Ekchariyawat, Peeraya; Wuthiekanun, Vanaporn; Elrod, Mindy G.; Turner, Paul; Currie, Bart J.; Phetsouvanh, Rattanaphone; Dance, David A. B.; Limmathurotsakul, Direk; Peacock, Sharon J.

2017-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species. PMID:28384252

Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

PubMed

Canchignia, Hayron; Altimira, Fabiola; Montes, Christian; Sánchez, Evelyn; Tapia, Eduardo; Miccono, María; Espinoza, Daniel; Aguirre, Carlos; Seeger, Michael; Prieto, Humberto

2017-03-17

The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

Biological and phylogenetic characteristics of yellow fever virus lineages from West Africa.

PubMed

Stock, Nina K; Laraway, Hewád; Faye, Ousmane; Diallo, Mawlouth; Niedrig, Matthias; Sall, Amadou A

2013-03-01

The yellow fever virus (YFV), the first proven human-pathogenic virus, although isolated in 1927, is still a major public health problem, especially in West Africa where it causes outbreaks every year. Nevertheless, little is known about its genetic diversity and evolutionary dynamics, mainly due to a limited number of genomic sequences from wild virus isolates. In this study, we analyzed the phylogenetic relationships of 24 full-length genomes from YFV strains isolated between 1973 and 2005 in a sylvatic context of West Africa, including 14 isolates that had previously not been sequenced. By this, we confirmed genetic variability within one genotype by the identification of various YF lineages circulating in West Africa. Further analyses of the biological properties of these lineages revealed differential growth behavior in human liver and insect cells, correlating with the source of isolation and suggesting host adaptation. For one lineage, repeatedly isolated in a context of vertical transmission, specific characteristics in the growth behavior and unique mutations of the viral genome were observed and deserve further investigation to gain insight into mechanisms involved in YFV emergence and maintenance in nature.

Biological and Phylogenetic Characteristics of Yellow Fever Virus Lineages from West Africa

PubMed Central

Laraway, Hewád; Faye, Ousmane; Diallo, Mawlouth; Niedrig, Matthias

2013-01-01

The yellow fever virus (YFV), the first proven human-pathogenic virus, although isolated in 1927, is still a major public health problem, especially in West Africa where it causes outbreaks every year. Nevertheless, little is known about its genetic diversity and evolutionary dynamics, mainly due to a limited number of genomic sequences from wild virus isolates. In this study, we analyzed the phylogenetic relationships of 24 full-length genomes from YFV strains isolated between 1973 and 2005 in a sylvatic context of West Africa, including 14 isolates that had previously not been sequenced. By this, we confirmed genetic variability within one genotype by the identification of various YF lineages circulating in West Africa. Further analyses of the biological properties of these lineages revealed differential growth behavior in human liver and insect cells, correlating with the source of isolation and suggesting host adaptation. For one lineage, repeatedly isolated in a context of vertical transmission, specific characteristics in the growth behavior and unique mutations of the viral genome were observed and deserve further investigation to gain insight into mechanisms involved in YFV emergence and maintenance in nature. PMID:23269797

Molecular characterization of Trichinella species from wild animals in Israel.

PubMed

Erster, Oran; Roth, Asael; King, Roni; Markovics, Alex

2016-11-15

Trichinellosis is a worldwide disease caused by nematode worms of the genus Trichinella, frequently diagnosed in Israel. However, the identity of the Israeli isolates have not been studied. Here we describe the molecular characterization of 58 isolates collected from jackals (Canis aureus), wild boar (Sus scrofa), foxes (Vulpes vulpes) and a wolf (Canis lupus) in central and northern Israel. Isolates were analyzed using the multiplex PCR analysis encompassing expansion segment V (ESV) and internal sequence 1 (ITS-1) markers, which identified 52 of the 58 samples. Out of the six unidentified samples, four were successfully identified using extended PCR assays for ESV and ITS-1, developed in this study. Our analysis identified 44 isolates as T. britovi, 8 as T. spiralis, four mixed infections, and two isolates were not identified. Clonal analysis of the ITS-1 sequences from six isolates confirmed the initial identification of four mixed infections. These results show that the prevalent species in Israel are T. britovi and T. spiralis, with nearly 7% (4 of 58) incidence of mixed infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Toxocara nematodes in stray cats from shiraz, southern iran: intensity of infection and molecular identification of the isolates.

PubMed

Mikaeili, Fattaneh; Mirhendi, Hossein; Hosseini, Mostafa; Asgari, Qasem; Kia, Eshrat Beigom

2013-10-01

Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection. This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites. Eight out of 30 cats (26.7%) were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes. The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.

Is Malassezia nana the main species in horses' ear canal microbiome?

PubMed

Aldrovandi, Ana Lúcia; Osugui, Lika; Acqua Coutinho, Selene Dall'

2016-01-01

The objective of this study was to characterize genotypically Malassezia spp. isolated from the external ear canal of healthy horses. Fifty-five horses, 39 (70.9%) males and 16 (29.1%) females, from different breeds and adults were studied. External ear canals were cleaned and a sterile cotton swab was introduced to collect cerumen. A total of 110 samples were cultured into Dixon medium and were incubated at 32°C for up to 15 days. Macro- and micromorphology and phenotypic identification were performed. DNA was extracted, strains were submitted to polymerase chain reaction technique, and the products obtained were submitted to Restriction Fragment Length Polymorphism using the restriction enzymes BstCI and HhaI. Strains were sent off to genetic sequencing of the regions 26S rDNA D1/D2 and ITS1-5.8S-ITS2 rDNA. Malassezia spp. were isolated from 33/55 (60%) animals and 52/110 (47%) ear canals. No growth on Sabouraud dextrose agar was observed, confirming the lipid dependence of all strains. Polymerase chain reaction-Restriction fragment length polymorphism permitted the molecular identification of Malassezia nana - 42/52 (81%) and Malassezia slooffiae - 10/52 (19%). Sequencing confirmed RFLP identification. It was surprising that M. nana represented over 80% of the strains and no Malassezia equina was isolated in this study, differing from what was expected. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

PubMed

Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

2016-03-01

The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

PubMed Central

Gianecini, R.; Oviedo, C.; Littvik, A.; Mendez, E.; Piccoli, L.; Montibello, S.

2014-01-01

One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin. PMID:25367903

Genetic diversity of Echinococcus granulosus in center of Iran.

PubMed

Pestechian, Nader; Hosseini Safa, Ahmad; Tajedini, Mohammadhasan; Rostami-Nejad, Mohammad; Mousavi, Mohammad; Yousofi, Hosseinali; Haghjooy Javanmard, Shaghayegh

2014-08-01

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon.

PubMed Central

Fuerst, J A; Gwilliam, H G; Lindsay, M; Lichanska, A; Belcher, C; Vickers, J E; Hugenholtz, P

1997-01-01

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology. PMID:8979353

Genetic, phenotypic and matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based identification of anaerobic bacteria and determination of their antimicrobial susceptibility at a University Hospital in Japan.

PubMed

Yunoki, Tomoyuki; Matsumura, Yasufumi; Nakano, Satoshi; Kato, Karin; Hotta, Go; Noguchi, Taro; Yamamoto, Masaki; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

2016-05-01

The accuracies of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the phenotypic method using VITEK 2 were compared to the accuracy of 16S rRNA sequence analysis for the identification of 170 clinically isolated anaerobes. The antimicrobial susceptibility of the isolates was also evaluated. Genetic analysis identified 21 Gram-positive species in 14 genera and 29 Gram-negative species in 11 genera. The most frequently isolated genera were Prevotella spp. (n = 46), Bacteroides spp. (n = 25) and Clostridium spp. (n = 25). MALDI-TOF MS correctly identified more isolates compared with VITEK 2 at the species (80 vs. 58%, respectively; p < 0.01) and genus (85 vs. 71%, respectively; p < 0.01) levels. More than 90% of the isolates of the three major genera identified (Prevotella, Bacteroides, and Clostridium species other than Clostridium difficile) were susceptible to beta-lactam/beta-lactamase inhibitor combinations, carbapenems, metronidazole and chloramphenicol. MALDI-TOF MS provided better identification results than VITEK2. Commonly used anti-anaerobic agents indicated that the isolates of the three most frequently identified anaerobic genera exhibited good antimicrobial susceptibility. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Molecular and antimicrobial susceptibility characterization of Globicatella sulfidifaciens isolated from sow's urinary tract infection.

PubMed

Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Amigo, Cristina R; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

2017-12-01

The Globicatella genus comprises Gram-positive, facultative anaerobic, α-hemolytic and catalase negative cocci morphologically and phenotypically very similar to Streptococcus and Aerococcus genus which can lead to misidentification and underestimation of this pathogen. Globicatella species have already been isolated from human and animals with heart and brain disorders. Their clinical relevance in animals, and its zoonotic potential, remains unknown due to the difficulty in their identification. To present the isolation, phenotypic and molecular characterization of G. sulfidifaciens from urinary tract infection in sows. Urine samples from 140 sows of two swine herds located in São Paulo State (Brazil) yielded the isolation of three presumptive G. sulfidifaciens strains. Identification and species confirmation were done by MALDI-TOF MS and 16S rRNA sequencing. Strains were further characterized by single enzyme amplified fragments length polymorphism (SE-AFLP) and broth microdilution techniques. All three isolates were confirmed as G. sulfidifaciens. The SE-AFLP genotyping resulted in distinct fingerprint patterns for each strain. All isolates presented high MIC values to tetracycline, sulphonamides, aminoglycosides and tylosin tartrate, which present high usage in human and animal medicine. Globicatella sulfidifaciens could be related to sporadic urinary tract infections in swine and appear to present alarming antimicrobial susceptibility profile. It is necessary to differentiate Streptococcus-like microorganisms in routine laboratory diagnostics for the correct identification of underestimated species potentially pathogenic to animals.

Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

PubMed

Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

2006-09-01

This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

PubMed

Preveena, Jagadesan; Bhore, Subhash J

2013-01-01

In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study.

Genotyping of clinical isolates of Acanthamoeba genus in Venezuela.

PubMed

Wagner, Carolina; Reyes-Batlle, María; Ysea, María Alejandra Vethencourt; Pérez, Mónica V Galindo; de Rondón, Carmen Guzmán; Paduani, Anaibeth J Nessi; Pérez, Angelyseb Dorta; López-Arencibia, Atteneri; Sifaoui, Ines; de Galindo, María Virginia Pérez; de Suárez, Eva Pérez; Martínez-Carretero, Enrique; Valladares, Basilio; Piñero, José E; Lorenzo-Morales, Jacob

2016-12-01

Free-living amoebae of Acanthamoeba genus are opportunistic pathogens distributed worldwide. Strains included in this genus are causative agents of a fatal encephalitis and a sight-threating keratitis in humans and other animals. In this study, 550 clinical samples which were collected between 1984 and 2014 from different patients with suspected infections due to Acanthamoeba were initially screened for the presence of this amoebic genus at the Laboratorio de Amibiasis-Escuela de Bioanálisis at the Universidad Central de Venezuela. Samples were cultured in 2% Non-Nutrient agar plates seeded with a layer of heat killed Escherichia coli. From the 550 clinical samples included in this study, 18 of them were positive for Acanthamoeba genus after culture identification. Moreover, positive samples were confirmed after amplification of the Diagnostic Fragment 3 (DF3) of the Acanthamoeba18S rDNA genus and sequencing was carried out in order to genotype the isolated strains of Acanthamoeba. Furthermore, the pathogenic potential of the strains was checked by performing thermotolerance and osmotolerance assays. Sequencing of the DF3 region resulted in the identification of genotype T4 in all the isolated strains. Moreover, most isolates were thermotolerant or both thermotolerant and osmotolerant and thus were classified as potentially pathogenic strains. To the best of our knowledge, this is the first report on the molecular characterization at the genotype level of Acanthamoeba strains in Venezuela.

Indigenous Halomonas spp., the Potential Nitrifying Bacteria for Saline Ammonium Waste Water Treatment.

PubMed

Sangnoi, Yutthapong; Chankaew, Sunipa; O-Thong, Sompong

2017-01-01

Toxic nitrogen compounds are one cause decreasing of shrimp production and water pollution. Indigenous Halomonas spp., isolated from Pacific white shrimp farm are benefitted for saline ammonium waste water treatment. This study aimed to isolate the heterotrophic-halophilic Halomonas spp. and investigate their ammonium removal efficiency. Halomonas spp., were isolated by culturing of samples collected from shrimp farm into modified Pep-Beef-AOM medium. Ammonium converting ability was tested and monitored by nitrite reagent. Ammonium removal efficiency was measured by the standard colorimetric method. Identification and classification of Halomonas spp., were studied by morphological, physiological and biochemical characteristics as well as molecular information. There were 5 strains of heterotrophic-halophilic nitrifying bacteria including SKNB2, SKNB4, SKNB17, SKNB20 and SKNB22 were isolated. The identification result based on 16S rRNA sequence analysis indicated that all 5 strains were Halomonas spp., with sequence similarity values of 91-99 %. Ammonium removal efficiency of all strains showed a range of 23-71%. The production of nitrite was low detected of 0.01-0.15 mg-N L-1, while the amount of nitrate was almost undetectable. This might suggest that the indigenous Halomonas spp., as nitrifying bacteria involved biological nitrification process for decreasing and transforming of ammonia. Due to being heterotrophic, halophilic and ammonium removing bacteria, these Halomonas spp., could be developed for use in treatment of saline ammonium waste water.

Isolation and characterization of NBS-LRR- resistance gene candidates in turmeric (Curcuma longa cv. surama).

PubMed

Joshi, R K; Mohanty, S; Subudhi, E; Nayak, S

2010-09-08

Turmeric (Curcuma longa), an important asexually reproducing spice crop of the family Zingiberaceae is highly susceptible to bacterial and fungal pathogens. The identification of resistance gene analogs holds great promise for development of resistant turmeric cultivars. Degenerate primers designed based on known resistance genes (R-genes) were used in combinations to elucidate resistance gene analogs from Curcuma longa cultivar surama. The three primers resulted in amplicons with expected sizes of 450-600 bp. The nucleotide sequence of these amplicons was obtained through sequencing; their predicted amino acid sequences compared to each other and to the amino acid sequences of known R-genes revealed significant sequence similarity. The finding of conserved domains, viz., kinase-1a, kinase-2 and hydrophobic motif, provided evidence that the sequences belong to the NBS-LRR class gene family. The presence of tryptophan as the last residue of kinase-2 motif further qualified them to be in the non-TIR-NBS-LRR subfamily of resistance genes. A cluster analysis based on the neighbor-joining method was carried out using Curcuma NBS analogs together with several resistance gene analogs and known R-genes, which classified them into two distinct subclasses, corresponding to clades N3 and N4 of non-TIR-NBS sequences described in plants. The NBS analogs that we isolated can be used as guidelines to eventually isolate numerous R-genes in turmeric.

reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra

PubMed Central

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R.; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W.; Moritz, Robert L.

2016-01-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contributes to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), that enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the following iterations. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website. PMID:26419769

reSpect: software for identification of high and low abundance ion species in chimeric tandem mass spectra.

PubMed

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W; Moritz, Robert L

2015-11-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contribute to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), which enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post-search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the iterations that follow. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website. Graphical Abstract ᅟ.

reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra

NASA Astrophysics Data System (ADS)

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R.; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W.; Moritz, Robert L.

2015-11-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contribute to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), which enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post-search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the iterations that follow. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website.

Internet-accessible DNA sequence database for identifying fusaria from human and animal infections.

PubMed

O'Donnell, Kerry; Sutton, Deanna A; Rinaldi, Michael G; Sarver, Brice A J; Balajee, S Arunmozhi; Schroers, Hans-Josef; Summerbell, Richard C; Robert, Vincent A R G; Crous, Pedro W; Zhang, Ning; Aoki, Takayuki; Jung, Kyongyong; Park, Jongsun; Lee, Yong-Hwan; Kang, Seogchan; Park, Bongsoo; Geiser, David M

2010-10-01

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

Use of an improved atpA amplification and sequencing method to identify members of the Campylobacteraceae and Helicobacteraceae

USDA-ARS?s Scientific Manuscript database

Emerging Campylobacter spp. and arcobacters have been increasingly isolated from human clinical samples, food and the environment. Unambiguous species identification of such organisms is of obvious importance in epidemiological studies, but is also necessary to accurately assess their host range and...

An enrichment, amplification, and sequence-based typing (EAST) approach for foodborne pathogen detection and surveillance

USDA-ARS?s Scientific Manuscript database

Introduction: Detection of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture. This is typically followed by strain typing, which provides information critical to outbreak and source investigations. In the early 1990’s pul...

Variability in secondary structure of 18S ribosomal RNA as topological marker for identification of Paramecium species.

PubMed

Shakoori, Farah R; Tasneem, Fareeda; Al-Ghanim, K; Mahboob, S; Al-Misned, F; Jahan, Nusrat; Shakoori, Abdul Rauf

2014-12-01

Besides cytological and molecular applications, Paramecium is being used in water quality assessment and for determination of saprobic levels. An unambiguous identification of these unicellular eukaryotes is not only essential, but its ecological diversity must also be explored in the local environment. 18SrRNA genes of all the strains of Paramecium species isolated from waste water were amplified, cloned and sequenced. Phylogenetic comparison of the nucleotide sequences of these strains with 23 closely related Paramecium species from GenBank Database enabled identification of Paramecium multimicronucleatum and Paramecium jenningsi. Some isolates did not show significant close association with other Paramecium species, and because of their unique position in the phylogenetic tree, they were considered new to the field. In the present report, these isolates are being designated as Paramecium caudatum pakistanicus. In this article, secondary structure of 18SrRNA has also been analyzed as an additional and perhaps more reliable topological marker for species discrimination and for determining possible phylogenetic relationship between the ciliate species. On the basis of comparison of secondary structure of 18SrRNA of various isolated Paramacium strains, and among Paramecium caudatum pakistanicus, Tetrahymena thermophila, Drosophila melanogaster, and Homo sapiens, it can be deduced that variable regions are more helpful in differentiating the species at interspecific level rather than at intraspecific level. It was concluded that V3 was the least variable region in all the organisms, V2 and V7 were the longest expansion segments of D. melanogaster and there was continuous mutational bias towards G.C base pairing in H. sapiens. © 2014 Wiley Periodicals, Inc.

Isolation, biochemical and molecular identification of Nocardia species among TB suspects in northeastern, Tanzania; a forgotten or neglected threat?

PubMed

Hoza, Abubakar S; Mfinanga, Sayoki G S; Moser, Irmgard; König, Brigitte

2017-06-08

Pulmonary nocardiosis mimic pulmonary tuberculosis in most clinical and radiological manifestations. In Tanzania, where tuberculosis is one of the major public health threat clinical impact of nocardiosis as the cause of the human disease remains unknown. The objective of the present study was to isolate and identify Nocardia isolates recovered from TB suspects in Northeastern, Tanzania by using biochemical and molecular methods. The study involved 744 sputum samples collected from 372 TB suspects from four periphery diagnostic centers in Northeastern, Tanzania. Twenty patients were diagnosed as having presumptively Nocardia infections based on microscopic, cultural characteristics and biomèrieux ID 32C Yeast Identification system and confirmed using 16S rRNA and hsp65 gene specific primers for Nocardia species and sequencing. Biochemically, the majority of the isolates were N. asteroides (n = 8/20, 40%), N. brasiliensis (n = 4/20, 20%), N. farcinica (n = 3/20, 15%), N. nova (n = 1/20, 5%). Other aerobic actinomycetales included Streptomyces cyanescens (n = 2/20, 10%), Streptomyces griseus, Actinomadura madurae each (n = 1/20, 5%). Results of 16S rRNA and hsp65 sequencing were concordant in 15/17 (88. 2%) isolates and discordant in 2/17 (11.8%) isolates. Majority of the isolates belonged to N. cyriacigeorgica and N. farcinica, four (23.5%) each. Our findings suggest that Nocardia species may be an important cause of pulmonary nocardiosis that is underdiagnosed or ignored. This underscores needs to consider pulmonary nocardiosis as a differential diagnosis when there is a failure of anti-TB therapy and as a possible cause of human infections.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory.

PubMed

Wattal, C; Oberoi, J K; Goel, N; Raveendran, R; Khanna, S

2017-05-01

The study evaluates the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Vitek MS for identification of microorganisms in the routine clinical microbiology laboratory. From May 2013 to April 2014, microbial isolates recovered from various clinical samples were identified by Vitek MS. In case of failure to identify by Vitek MS, the isolate was identified using the Vitek 2 system (bioMerieux, France) and serotyping wherever applicable or otherwise by nucleic acid-mediated methods. All the moulds were identified by Lactophenol blue mounts, and mycobacterial isolates were identified by molecular identification systems including AccuProbe (bioMerieux, France) or GenoType Mycobacterium CM (Hain Lifescience, Germany). Out of the 12,003 isolates, the Vitek MS gave a good overall ID at the genus and or species level up to 97.7% for bacterial isolates, 92.8% for yeasts and 80% for filamentous fungi. Of the 26 mycobacteria tested, only 42.3% could be identified using the Saramis RUO (Research Use Only) database. VITEK MS could not identify 34 of the 35 yeast isolates identified as C. haemulonii by Vitek 2. Subsequently, 17 of these isolates were identified as Candida auris (not present in the Vitek MS database) by 18S rRNA sequencing. Using these strains, an in-house superspectrum of C. auris was created in the VITEK MS database. Use of MALDI-TOF MS allows a rapid identification of aerobic bacteria and yeasts in clinical practice. However, improved sample extraction protocols and database upgrades with inclusion of locally representative strains is required, especially for moulds.

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings.

PubMed

Iatta, Roberta; Nuccio, Federica; Immediato, Davide; Mosca, Adriana; De Carlo, Carmela; Miragliotta, Giuseppe; Parisi, Antonio; Crescenzo, Giuseppe; Otranto, Domenico; Cafarchia, Claudia

2016-09-01

Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of β-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5.

PubMed

Alkotaini, Bassam; Anuar, Nurina; Kadhum, Abdul Amir Hassan; Sani, Asmahani Azira Abdu

2014-04-01

A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).

Isolation of Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole goats fed native forage diet.

PubMed

Grilli, D J; Cerón, M E; Paez, S; Egea, V; Schnittger, L; Cravero, S; Escudero, M Sosa; Allegretti, L; Arenas, G N

2013-09-01

We isolated and identified functional groups of bacteria in the rumen of Creole goats involved in ruminal fermentation of native forage shrubs. The functional bacterial groups were evaluated by comparing the total viable, total anaerobic, cellulolytic, hemicellulolytic, and amylolytic bacterial counts in the samples taken from fistulated goats fed native forage diet (Atriplex lampa and Prosopis flexuosa). Alfalfa hay and corn were used as control diet. The roll tubes method increased the possibility of isolating and 16S rDNA gene sequencing allowed definitive identification of bacterial species involved in the ruminal fermentation. The starch and fiber contents of the diets influenced the number of total anaerobic bacteria and fibrolytic and amylolytic functional groups. Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans were the main species isolated and identified. The identification of bacterial strains involved in the rumen fermentation helps to explain the ability of these animals to digest fiber plant cell wall contained in native forage species.

Identification of phylogenetic position in the Chlamydiaceae family for Chlamydia strains released from monkeys and humans with chlamydial pathology.

PubMed

Karaulov, Alexander; Aleshkin, Vladimir; Slobodenyuk, Vladimir; Grechishnikova, Olga; Afanasyev, Stanislav; Lapin, Boris; Dzhikidze, Eteri; Nesvizhsky, Yuriy; Evsegneeva, Irina; Voropayeva, Elena; Afanasyev, Maxim; Aleshkin, Andrei; Metelskaya, Valeria; Yegorova, Ekaterina; Bayrakova, Alexandra

2010-01-01

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S-23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the Gen-Bank electronic database has been demonstrated. Differences in the 16S-23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and nonplasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B-B, Ba, D, Da, E, L1, L2, L2a; intermediate group-F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life development cycle disorder, and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration.

Isolation and identification of local bacteria endophyte and screening of its antimicrobial property against pathogenic bacteria and fungi

NASA Astrophysics Data System (ADS)

Fikri, Ahmad Syairazie Ibrahim; Rahman, Irman Abdul; Nor, Norefrina Shafinaz Md; Hamzah, Ainon

2018-04-01

Endophytes are organisms, often fungi and bacteria that live in living plant cells. These organisms reside in the living tissues of the host plant in a variety of relationships, ranging from symbiotic to slightly pathogenic. The endophytes may produce a plethora of substances that have potential to be used in modern medicine, agriculture and industry. The aims of this study are to isolate, identify and screening antimicrobial activity of bacterial endophytes. The endophytes were isolated using nutrient agar, incubated at 37°C for 48 hours. Identification of the isolates were done based on morphological characteristics, biochemical tests and 16S rDNA molecular analysis. Disk diffusion method was used to screen for antimicrobial activity of metabolites from endophytes against pathogenic bacteria. Screening for antifungal activity of selected endophytes was done using dual culture method againts pathogenic fungi followed by Kirby-Bauer method. Results showed endophytes designated as B2c and B7b have positive antimicrobial activity. The metabolites from isolate B2c showed antimicrobial activity against pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus and Staphylococcus epidermis, while isolate B7b have positive activities againts MRSA, S. aureus and Pseudomonas aeruginosa. Isolates B2c displayed antifungal activity against Fusarium oxysporum, Fusarium solani, Phytophthora palmivora and Colletotrichum gloeosporioides. Identification using biochemical tests and 16S rDNA sequences identified isolate B2c as Pseudomonas resinovorans with 97% homology and isolate B7b as Bacillus subtilis with 98% homology.

[Isolation and identification of cow-origin Cryptosporidium isolates in Hefei].

PubMed

Sun, Tao; Liu, Wei; Wang, Ju-Hua; Xue, Xiu-Heng; Zhao, Chang-Cheng; Li, Pei-Ying

2011-12-01

To isolate cow-origin Cryptosporidium in Hefei, and identify its species. 285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp., the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software. Five fecal samples were positive by morphological methods with an infection rate of 1.8% (5/285). Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 microm x 6.13 microm and 7.58 microm x 6.20 microm, and a shape index of 1.20 and 1.22, respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp, respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni (DQ989573), and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates (AY954892 and DQ989576), and they were placed into the same clade. The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

(GTG)(5)-PCR fingerprinting of lactobacilli isolated from cervix of healthy women.

PubMed

Svec, P; Sedláček, I; Chrápavá, M; Vandamme, P

2011-01-01

A group of lactobacilli isolated from the cervix of 31 healthy women was characterized by (GTG)(5)-polymerase chain reaction (PCR) fingerprinting in order to evaluate this method for identification of vaginal lactobacilli. Obtained fingerprints were compared with profiles available in an in-house database of the CCM bacteria collection covering type and reference strains of multiple lactic acid bacteria including lactobacilli. Selected strains representing individual clusters were further identified by pheS gene sequencing. In total, six lactobacillus species were found among lactobacilli isolated from the cervix of healthy women. The (GTG)(5)-PCR method identified Lactobacillus gasseri (11 strains), Lactobacillus fermentum (one), and some of the Lactobacillus jensenii strains (eight out of 11), but failed to identify the remaining strains, including the Lactobacillus crispatus (18), Lactobacillus mucosae (one), and Lactobacillus vaginalis (one) species. L. jensenii strains were distributed over two fingerprint clusters. The majority of samples was dominated by one (GTG)(5)-PCR type. The rep-PCR fingerprinting using the (GTG)(5) primer allowed straightforward identification of many, but not all, isolates. This method has been shown to be a useful tool for fast screening and grouping of vaginal lactobacilli, but its combination with another identification method is needed to obtain reliable identification results. In addition, Lactobacillus acidophilus was not shown to be the most common inhabitant of the female genital tract as generally assumed.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

PubMed Central

Pérez-Osorio, Ailyn C.; Boyle, David S.; Ingham, Zachary K.; Ostash, Alla; Gautom, Romesh K.; Colombel, Craig; Houze, Yolanda

2012-01-01

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampinr TB identification; 60.6% and 100% for isoniazidr TB identification; and 75.0% and 98.1% for pyrazinamider TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced. PMID:22162548

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

PubMed

Biondi, Enrico; Zamorano, Alan; Vega, Ernesto; Ardizzi, Stefano; Sitta, Davide; De Salvador, Flavio Roberto; Campos-Vargas, Reinaldo; Meneses, Claudio; Perez, Set; Bertaccini, Assunta; Fiore, Nicola

2018-05-01

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

PubMed

Lorenzini, Marilinda; Simonato, Barbara; Zapparoli, Giacomo

2018-05-07

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.

Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

PubMed

Al-Qahtani, Ahmed Ali; Mubin, Muhammad; Dela Cruz, Damian M; Althawadi, Sahar Isa; Ul Rehman, Muhammad Shah Nawaz; Bohol, Marie Fe F; Al-Ahdal, Mohammed N

2017-01-30

In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

Morphological and molecular characterization of Fusarium spp pathogenic to pecan tree in Brazil.

PubMed

Lazarotto, M; Milanesi, P M; Muniz, M F B; Reiniger, L R S; Beltrame, R; Harakava, R; Blume, E

2014-11-11

The occurrence of Fusarium spp associated with pecan tree (Carya illinoinensis) diseases in Brazil has been observed in recent laboratory analyses in Rio Grande do Sul State. Thus, in this study, we i) obtained Fusarium isolates from plants with disease symptoms; ii) tested the pathogenicity of these Fusarium isolates to pecan; iii) characterized and grouped Fusarium isolates that were pathogenic to the pecan tree based on morphological characteristics; iv) identified Fusarium spp to the species complex level through TEF-1α sequencing; and v) compared the identification methods used in the study. Fifteen isolates collected from the inflorescences, roots, and seeds of symptomatic plants (leaf necrosis or root rot) were used for pathogenicity tests. Morphological characterization was conducted using only pathogenic isolates, for a total of 11 isolates, based on the mycelial growth rate, sporulation, colony pigmentation, and conidial length and width variables. Pathogenic isolates were grouped based on morphological characteristics, and molecular characterization was performed by sequencing TEF-1α genes. Pathogenic isolates belonging to the Fusarium chlamydosporum species complex, Fusarium graminearum species complex, Fusarium proliferatum, and Fusarium oxysporum were identified based on the TEF-1α region. Morphological characteristics were used to effectively differentiate isolates and group the isolates according to genetic similarity, particularly conidial width, which emerged as a key morphological descriptor in this study.

Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036) from the faeces of exclusively breast-fed infants.

PubMed

Muñoz-Quezada, Sergio; Chenoll, Empar; Vieites, José María; Genovés, Salvador; Maldonado, José; Bermúdez-Brito, Miriam; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, María José; Romero, Fernando; Suárez, Antonio; Ramón, Daniel; Gil, Angel

2013-01-01

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytes in vitro. The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity with Lactobacillus paracasei, Lactobacillus rhamnosus and Bifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S-23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and named L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibited in vitro the meningitis aetiological agent Listeria monocytogenes and human rotavirus infections. B. breve CNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

Screening for and isolation and identification of malathion-degrading bacteria: cloning and sequencing a gene that potentially encodes the malathion-degrading enzyme, carboxylestrase in soil bacteria.

PubMed

Goda, Sayed K; Elsayed, Iman E; Khodair, Taha A; El-Sayed, Walaa; Mohamed, Mervat E

2010-11-01

Five malathion-degrading bacterial strains were enriched and isolated from soil samples collected from different agricultural sites in Cairo, Egypt. Malathion was used as a sole source of carbon (50 mg/l) to enumerate malathion degraders, which were designated as IS1, IS2, IS3, IS4, and IS5. They were identified, based on their morphological and biochemical characteristics, as Pseudomonas sp., Pseudomonas putida, Micrococcus lylae, Pseudomonas aureofaciens, and Acetobacter liquefaciens, respectively. IS1 and IS2, which showed the highest degrading activity, were selected for further identification by partial sequence analysis of their 16S rRNA genes. The 16S rRNA gene of IS1 shared 99% similarity with that of Alphaprotoebacterium BAL284, while IS2 scored 100% similarity with that of Pseudomonas putida 32zhy. Malathion residues almost completely disappeared within 6 days of incubation in IS2 liquid cultures. LC/ESI-MS analysis confirmed the degradation of malathion to malathion monocarboxylic and dicarboxylic acids, which formed as a result of carboxylesterase activity. A carboxylesterase gene (CE) was amplified from the IS2 genome by using specifically designed PCR primers. The sequence analysis showed a significant similarity to a known CE gene in different Pseudomonas sp. We report here the isolation of a new malathion-degrading bacteria from soils in Egypt that may be very well adapted to the climatic and environmental conditions of the country. We also report the partial cloning of a new CE gene. Due to their high biodegradation activity, the bacteria isolated from this work merit further study as potential biological agents for the remediation of soil, water, or crops contaminated with the pesticide malathion.

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007.

PubMed

Logotheti, Maria; Pogka, Vasiliki; Horefti, Elina; Papadakos, Konstantinos; Giannaki, Maria; Pangalis, Anastasia; Sgouras, Dionyssios; Mentis, Andreas

2009-11-01

Aseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV). Identification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007. In total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains. EV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006. It is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.

Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts.

PubMed

Cutillas, Cristina; de Rojas, Manuel; Ariza, Concepción; Ubeda, José Manuel; Guevara, Diego

2007-01-01

Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica -- swine and Sus scrofa scrofa -- wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica -- swine and S. scrofa scrofa -- wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.

Comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing.

PubMed

O'Flaherty, Brigid M; Li, Yan; Tao, Ying; Paden, Clinton R; Queen, Krista; Zhang, Jing; Dinwiddie, Darrell L; Gross, Stephen M; Schroth, Gary P; Tong, Suxiang

2018-06-01

Next generation sequencing (NGS) technologies have revolutionized the genomics field and are becoming more commonplace for identification of human infectious diseases. However, due to the low abundance of viral nucleic acids (NAs) in relation to host, viral identification using direct NGS technologies often lacks sufficient sensitivity. Here, we describe an approach based on two complementary enrichment strategies that significantly improves the sensitivity of NGS-based virus identification. To start, we developed two sets of DNA probes to enrich virus NAs associated with respiratory diseases. The first set of probes spans the genomes, allowing for identification of known viruses and full genome sequencing, while the second set targets regions conserved among viral families or genera, providing the ability to detect both known and potentially novel members of those virus groups. Efficiency of enrichment was assessed by NGS testing reference virus and clinical samples with known infection. We show significant improvement in viral identification using enriched NGS compared to unenriched NGS. Without enrichment, we observed an average of 0.3% targeted viral reads per sample. However, after enrichment, 50%-99% of the reads per sample were the targeted viral reads for both the reference isolates and clinical specimens using both probe sets. Importantly, dramatic improvements on genome coverage were also observed following virus-specific probe enrichment. The methods described here provide improved sensitivity for virus identification by NGS, allowing for a more comprehensive analysis of disease etiology. © 2018 O'Flaherty et al.; Published by Cold Spring Harbor Laboratory Press.

Modified Primers for the Identification of Nonpathogenic Fusarium oxysporum Isolates That Have Biological Control Potential against Fusarium Wilt of Cucumber in Taiwan

PubMed Central

Wang, Chaojen; Lin, Yisheng; Lin, Yinghong; Chung, Wenhsin

2013-01-01

Previous investigations demonstrated that Fusarium oxysporum (Fo), which is not pathogenic to cucumbers, could serve as a biological control agent for managing Fusarium wilt of cucumber caused by Fo f. sp. cucumerinum (Foc) in Taiwan. However, thus far it has not been possible to separate the populations of pathogenic Fo from the nonpathogenic isolates that have biological control potential through their morphological characteristics. Although these two populations can be distinguished from one another using a bioassay, the work is laborious and time-consuming. In this study, a fragment of the intergenic spacer (IGS) region of ribosomal DNA from an Fo biological control agent, Fo366, was PCR-amplified with published general primers, FIGS11/FIGS12 and sequenced. A new primer, NPIGS-R, which was designed based on the IGS sequence, was paired with the FIGS11 primer. These primers were then evaluated for their specificity to amplify DNA from nonpathogenic Fo isolates that have biological control potential. The results showed that the modified primer pair, FIGS11/NPIGS-R, amplified a 500-bp DNA fragment from five of seven nonpathogenic Fo isolates. These five Fo isolates delayed symptom development of cucumber Fusarium wilt in greenhouse bioassay tests. Seventy-seven Fo isolates were obtained from the soil and plant tissues and then subjected to amplification using the modified primer pair; six samples showed positive amplification. These six isolates did not cause symptoms on cucumber seedlings when grown in peat moss infested with the isolates and delayed disease development when the same plants were subsequently inoculated with a virulent isolate of Foc. Therefore, the modified primer pair may prove useful for the identification of Fo isolates that are nonpathogenic to cucumber which can potentially act as biocontrol agents for Fusarium wilt of cucumber. PMID:23762289

Noncytopathogenic Pestivirus Strains Generated by Nonhomologous RNA Recombination: Alterations in the NS4A/NS4B Coding Region

PubMed Central

Gallei, Andreas; Orlich, Michaela; Thiel, Heinz-Juergen; Becher, Paul

2005-01-01

Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous recombination between identical duplicated viral sequences. To allow the identification of recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous recombination while the other 14 strains were generated by nonhomologous recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses. PMID:16254361

High frequency of pathogenic Aspergillus species among nonsporulating moulds from respiratory tract samples.

PubMed

Dufresne, Philippe J; Moonjely, Soumya S; Ozaki, Koyomi; Tremblay, Cécile; Laverdière, Michel; Dufresne, Simon F

2017-02-01

Nonsporulating moulds (NSM) represent an identification challenge for clinical laboratories. Data on the prevalence of pathogenic species among NSM are lacking. We prospectively investigated consecutive thermotolerant (36°C) clinical NSM isolates from respiratory tract samples. A total of 123 isolates were identified by DNA sequencing and phenotypically characterized. Of those, 13 (11%) were pathogenic species (Aspergillus fumigatus, n = 10; A. flavus, n = 1; A. hiratsukae, n = 1; Schizophyllum commune, n = 1). Presumptive identification of Aspergillus species among NSM can be achieved by simple phenotypic testing. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

PubMed Central

Ferreira, M A; Tooley, P W; Hatziloukas, E; Castro, C; Schaad, N W

1996-01-01

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds. PMID:8572716

Nanobacteria may be linked to calcification in placenta.

PubMed

Lu, He; Guo, Ya-nan; Liu, Sheng-nan; Zhang, De-chun

2012-05-01

Placental calcification is a common pathologic condition in obstetrics. To detect the bacteria infection mechanisms for calcification, an experiment was performed to isolate, culture, and identify the nanobacteria in placental calcification. Sixteen cases of placental calcification of pregnant women were collected for the purpose of the isolation of nanobacteria, cultivation, and identification of 16S rDNA sequence. Under transmission electron microscope, novel oval-shape nanobacteria-like particles (NLP) in extracellular matrix of calcified placenta tissues were found with 50-500 nm in diameter, and among hydroxyapatite crystals aggregation existed. After about 4 weeks of culturing and isolating NLP from these calcified tissues, all calcified placental tissue samples and one adjacent tissue of calcified placental tissue samples showed white granular depositions, which were firmly attached to the bottom of the culture tubes and visible to the naked eyes. In the control group they could not be seen. After PCR was amplified a 1407-bp fragment was obtained and submitted to GenBank after sequencing with accession number JN029830. The 16S rDNA sequence homology between the isolation strain and strain nanobacteria (X98418) was 92% in GenBank. For the first time isolated, cultured, and identified nanobacteria in placental calcification indicated that nanobacteria infection is related to placental calcification.

A combination of PhP typing and β-d-glucuronidase gene sequence variation analysis for differentiation of Escherichia coli from humans and animals.

PubMed

Masters, N; Christie, M; Katouli, M; Stratton, H

2015-06-01

We investigated the usefulness of the β-d-glucuronidase gene variance in Escherichia coli as a microbial source tracking tool using a novel algorithm for comparison of sequences from a prescreened set of host-specific isolates using a high-resolution PhP typing method. A total of 65 common biochemical phenotypes belonging to 318 E. coli strains isolated from humans and domestic and wild animals were analysed for nucleotide variations at 10 loci along a 518 bp fragment of the 1812 bp β-d-glucuronidase gene. Neighbour-joining analysis of loci variations revealed 86 (76.8%) human isolates and 91.2% of animal isolates were correctly identified. Pairwise hierarchical clustering improved assignment; where 92 (82.1%) human and 204 (99%) animal strains were assigned to their respective cluster. Our data show that initial typing of isolates and selection of common types from different hosts prior to analysis of the β-d-glucuronidase gene sequence improves source identification. We also concluded that numerical profiling of the nucleotide variations can be used as a valuable approach to differentiate human from animal E. coli. This study signifies the usefulness of the β-d-glucuronidase gene as a marker for differentiating human faecal pollution from animal sources.

Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species.

PubMed

Arbefeville, S; Harris, A; Ferrieri, P

2017-09-01

Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU rDNA Fungal Identification Kit was equivalent to the in-house developed ITS regions assay to identify fungi at the genus level. The MycoBank database gave a better curated database and thus allowed a better genus and species identification for both D2 region of the LSU rRNA gene and ITS regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and screening of rare actinomycetes isolated from Neesia altissima Bl.

NASA Astrophysics Data System (ADS)

Pratiwi, R. H.; Hidayat, I.; Hanafi, M.; Mangunwardoyo, W.

2017-07-01

Actinomycetes is the main source of antibiotics and endophytic actinomycetes from medicinal plants has considerable potential as like the host. The aim of this research is to identify rare actinomycetes isolated from Neesia altissima and to screen their antagonistic activity against diarrhea-causing bacteria in order to find new potential secondary metabolites. Samples of N. altissima were collected from mount Halimun-Salak National Park. Endophytic actinomycetes were isolated from roots of N. altissima by surface sterilization method. Screening of antagonistic activity was conducted against five diarrhea-causing bacteria such as Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 25241, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 by using diffusion disc methods. The endophytic actinomycete showed in vitro antibacterial activity against four diarrhea-causing bacteria, except the B. cereus ATCC 10876. The phylogenetic tree generated from 16S rRNA sequence showed that sequence of endophytic actinomycetes isolates nested in the clade belonging to the genus Nonomuraea. Sequence of UICC B-94 formed a monophyletic clade with N. jabiensis strain A4036 and N. rubra strain AC 615. Therefore, it is named as Nonomuraea sp. strain UICC B-94.

Cyprinid herpesvirus 2 infection emerged in cultured gibel carp, Carassius auratus gibelio in China.

PubMed

Xu, Jin; Zeng, Lingbing; Zhang, Hui; Zhou, Yong; Ma, Jie; Fan, Yuding

2013-09-27

An epizootic with severe mortality has emerged in cultured gibel carp, Carassius auratus gibelio, in China since 2009, and caused huge economic loss. The signs and epidemiology background of the disease were investigated. Parasite examination, bacteria and virus isolation were carried out for pathogen isolation. The causative pathogen was obtained and identified as Cyprinid herpesvirus 2 (CyHV-2) by experimental infection, electron microscopy, cell culture, PCR assay and sequence alignment, designated as CyHV-2-JSSY. Experimental infection proved the high virulence of CyHV-2-JSSY to healthy gibel carp. Electron microscopy revealed that the viral nucleocapsid was hexagonal in shape measuring 110-120 nm in diameter with a 170-200 nm envelope. The virus caused significant CPE in Koi-Fin cells at the early passages, but not beyond the fifth passages. Sequence alignment of the partial viral helicase gene (JX566884) showed that it shared 99-100% identity to the published sequences of other CyHV-2 isolates. This study represented the first isolation and identification of CyHV-2 in cultured gibel carp in China and laid a foundation for the further studies of the disease. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

PubMed Central

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

1999-01-01

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Identification of chitinolytic bacteria isolated from shrimp pond sediment and characterization of their chitinase encoding gene

NASA Astrophysics Data System (ADS)

Triwijayani, A. U.; Puspita, I. D.; Murwantoko; Ustadi

2018-03-01

Chitinolytic bacteria are a group of bacteria owning enzymes that able to hydrolyze chitin. Previously, we isolated chitinolytic bacteria from shrimp pond sediment in Bantul, Yogyakarta, and obtained five isolates showing high chitinolytic index named as isolate PT1, PT2, PT5, PT6 and PB2. The aims of this study were to identify chitinolytic bacteria isolated from shrimp pond sediment and to characterize the chitinase encoding gene from each isolate. The molecular technique was performed by amplification of 16S rDNA, amplification of chitinase encoding gene and sequence analysis. Two chitinolytic bacteria of PT1 and PT2 were similar to Aeromonas bivalvium strain D15, PT5 to Pseudomonas stutzeri strain BD-2.2.1, PT6 to Serratia marcescens strain FZSF02 and PB2 to Streptomyces misionensis strain OsiRt-1. The comparison of chitinase encoding gene between three isolates with those in Gen Bank shows that PT1 had similar sequences with the chi1 gene in Aeromonas sp. 17m, PT2 with chi1 gene in A. caviae (CB101) and PT6 with chiB gene in S. Marcescens (BJL200).

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

PubMed Central

Gebhardt, J S; Nierzwicki-Bauer, S A

1991-01-01

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla. Images PMID:1685078

Morphological and molecular characterization of fungal pathogen, Magnaphorthe oryzae

NASA Astrophysics Data System (ADS)

Hasan, Nor'Aishah; Rafii, Mohd Y.; Rahim, Harun A.; Ali, Nusaibah Syd; Mazlan, Norida; Abdullah, Shamsiah

2016-02-01

Rice is arguably the most crucial food crops supplying quarter of calories intake. Fungal pathogen, Magnaphorthe oryzae promotes blast disease unconditionally to gramineous host including rice species. This disease spurred an outbreaks and constant threat to cereal production. Global rice yield declining almost 10-30% including Malaysia. As Magnaphorthe oryzae and its host is model in disease plant study, the rice blast pathosystem has been the subject of intense interest to overcome the importance of the disease to world agriculture. Therefore, in this study, our prime objective was to isolate samples of Magnaphorthe oryzae from diseased leaf obtained from MARDI Seberang Perai, Penang, Malaysia. Molecular identification was performed by sequences analysis from internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes. Phylogenetic affiliation of the isolated samples were analyzed by comparing the ITS sequences with those deposited in the GenBank database. The sequence of the isolate demonstrated at least 99% nucleotide identity with the corresponding sequence in GenBank for Magnaphorthe oryzae. Morphological observed under microscope demonstrated that the structure of conidia followed similar characteristic as M. oryzae. Finding in this study provide useful information for breeding programs, epidemiology studies and improved disease management.

Morphological and molecular characterization of fungal pathogen, Magnaphorthe oryzae

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

Hasan, Nor’Aishah, E-mail: aishahnh@ns.uitm.edu.my; Rafii, Mohd Y., E-mail: mrafii@upm.edu.my; Department of Crop Science, Universiti Putra Malaysia

2016-02-01

Rice is arguably the most crucial food crops supplying quarter of calories intake. Fungal pathogen, Magnaphorthe oryzae promotes blast disease unconditionally to gramineous host including rice species. This disease spurred an outbreaks and constant threat to cereal production. Global rice yield declining almost 10-30% including Malaysia. As Magnaphorthe oryzae and its host is model in disease plant study, the rice blast pathosystem has been the subject of intense interest to overcome the importance of the disease to world agriculture. Therefore, in this study, our prime objective was to isolate samples of Magnaphorthe oryzae from diseased leaf obtained from MARDI Seberangmore » Perai, Penang, Malaysia. Molecular identification was performed by sequences analysis from internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes. Phylogenetic affiliation of the isolated samples were analyzed by comparing the ITS sequences with those deposited in the GenBank database. The sequence of the isolate demonstrated at least 99% nucleotide identity with the corresponding sequence in GenBank for Magnaphorthe oryzae. Morphological observed under microscope demonstrated that the structure of conidia followed similar characteristic as M. oryzae. Finding in this study provide useful information for breeding programs, epidemiology studies and improved disease management.« less

Streptococcus caprae sp. nov., isolated from Iberian ibex (Capra pyrenaica hispanica).

PubMed

Vela, A I; Mentaberre, G; Lavín, S; Domínguez, L; Fernández-Garayzábal, J F

2016-01-01

Biochemical and molecular genetic studies were performed on a novel Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from tonsil samples of two Iberian ibexes. The micro-organism was identified as a streptococcal species based on its cellular, morphological and biochemical characteristics. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from ibex was Streptococcus porci 2923-03T (96.6 % 16S rRNA gene sequence similarity). Analysis based on rpoB and sodA gene sequences revealed sequence similarity values lower than 86.0 and 83.8 %, respectively, from the type strains of recognized Streptococcus species. The novel bacterial isolate was distinguished from Streptococcus porci and other Streptococcus species using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as representing a novel species of the genus Streptococcus, for which the name Streptococcus caprae sp. nov. is proposed. The type strain is DICM07-02790-1CT ( = CECT 8872T = CCUG 67170T).

Phenotypic, Genotypic, and Antimicrobial Characteristics of Streptococcus halichoeri Isolates from Humans, Proposal To Rename Streptococcus halichoeri as Streptococcus halichoeri subsp. halichoeri, and Description of Streptococcus halichoeri subsp. hominis subsp. nov., a Bacterium Associated with Human Clinical Infections.

PubMed

Shewmaker, P L; Whitney, A M; Humrighouse, B W

2016-03-01

Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324(T), 97.9% similarity to S. canis ATCC 43496(T), and 97.8% similarity to S. ictaluri ATCC BAA-1300(T). A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844(T) = CCUG 67100(T) = LMG 28801(T). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand.

PubMed

Prachantasena, Sakaoporn; Charununtakorn, Petcharatt; Muangnoicharoen, Suthida; Hankla, Luck; Techawal, Natthaporn; Chaveerach, Prapansak; Tuitemwong, Pravate; Chokesajjawatee, Nipa; Williams, Nicola; Humphrey, Tom; Luangtongkum, Taradon

2016-01-01

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.

Multicenter Study for Defining the Breakpoint for Rifampin Resistance in Neisseria meningitidis by rpoB Sequencing▿

PubMed Central

Taha, Muhamed-Kheir; Thulin Hedberg, Sara; Szatanik, Marek; Hong, Eva; Ruckly, Corinne; Abad, Raquel; Bertrand, Sophie; Carion, Francoise; Claus, Heike; Corso, Alejandra; Enríquez, Rocío; Heuberger, Sigrid; Hryniewicz, Waleria; Jolley, Keith A.; Kriz, Paula; Mollerach, Marta; Musilek, Martin; Neri, Arianna; Olcén, Per; Pana, Marina; Skoczynska, Anna; Sorhouet Pereira, Cecilia; Stefanelli, Paola; Tzanakaki, Georgina; Unemo, Magnus; Vázquez, Julio A.; Vogel, Ulrich; Wasko, Izabela

2010-01-01

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates. PMID:20606072

Previously unknown species of Aspergillus.

PubMed

Gautier, M; Normand, A-C; Ranque, S

2016-08-01

The use of multi-locus DNA sequence analysis has led to the description of previously unknown 'cryptic' Aspergillus species, whereas classical morphology-based identification of Aspergillus remains limited to the section or species-complex level. The current literature highlights two main features concerning these 'cryptic' Aspergillus species. First, the prevalence of such species in clinical samples is relatively high compared with emergent filamentous fungal taxa such as Mucorales, Scedosporium or Fusarium. Second, it is clearly important to identify these species in the clinical laboratory because of the high frequency of antifungal drug-resistant isolates of such Aspergillus species. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been shown to enable the identification of filamentous fungi with an accuracy similar to that of DNA sequence-based methods. As MALDI-TOF MS is well suited to the routine clinical laboratory workflow, it facilitates the identification of these 'cryptic' Aspergillus species at the routine mycology bench. The rapid establishment of enhanced filamentous fungi identification facilities will lead to a better understanding of the epidemiology and clinical importance of these emerging Aspergillus species. Based on routine MALDI-TOF MS-based identification results, we provide original insights into the key interpretation issues of a positive Aspergillus culture from a clinical sample. Which ubiquitous species that are frequently isolated from air samples are rarely involved in human invasive disease? Can both the species and the type of biological sample indicate Aspergillus carriage, colonization or infection in a patient? Highly accurate routine filamentous fungi identification is central to enhance the understanding of these previously unknown Aspergillus species, with a vital impact on further improved patient care. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species.

PubMed

Pattemore, Julie A; Hane, James K; Williams, Angela H; Wilson, Bree A L; Stodart, Ben J; Ash, Gavin J

2014-08-07

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102). Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae. The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

PubMed Central

Skoglund, Pontus; Northoff, Bernd H.; Shunkov, Michael V.; Derevianko, Anatoli P.; Pääbo, Svante; Krause, Johannes; Jakobsson, Mattias

2014-01-01

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation—summarized in a postmortem degradation score—which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material. PMID:24469802