Showing papers on "Typing published in 2016"
01 Dec 2016
TL;DR: Kaptive, a novel software tool that automates the process of identifying K-loci based on full locus information extracted from whole genome sequences, is introduced, highlighting the extensive diversity of Klebsiella K- loci and the proteins that they encode.
Abstract: Klebsiella pneumoniae is a growing cause of healthcare-associated infections for which multi-drug resistance is a concern. Its polysaccharide capsule is a major virulence determinant and epidemiological marker. However, little is known about capsule epidemiology since serological typing is not widely accessible and many isolates are serologically non-typeable. Molecular typing techniques provide useful insights, but existing methods fail to take full advantage of the information in whole genome sequences. We investigated the diversity of the capsule synthesis loci (K-loci) among 2503 K. pneumoniae genomes. We incorporated analyses of full-length K-locus nucleotide sequences and also clustered protein-encoding sequences to identify, annotate and compare K-locus structures. We propose a standardized nomenclature for K-loci and present a curated reference database. A total of 134 distinct K-loci were identified, including 31 novel types. Comparative analyses indicated 508 unique protein-encoding gene clusters that appear to reassort via homologous recombination. Extensive intra- and inter-locus nucleotide diversity was detected among the wzi and wzc genes, indicating that current molecular typing schemes based on these genes are inadequate. As a solution, we introduce Kaptive, a novel software tool that automates the process of identifying K-loci based on full locus information extracted from whole genome sequences (https://github.com/katholt/Kaptive). This work highlights the extensive diversity of Klebsiella K-loci and the proteins that they encode. The nomenclature, reference database and novel typing method presented here will become essential resources for genomic surveillance and epidemiological investigations of this pathogen.
325 citations
TL;DR: It is demonstrated that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST),Multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping.
Abstract: Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 L. monocytogenes isolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferred in silico from the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97 L. monocytogenes isolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospective L. monocytogenes surveillance and investigated for other pathogens relevant to public health.
183 citations
TL;DR: Overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed, and Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted.
129 citations
TL;DR: WgMLST opens up possibilities for the creation of a Web-accessible database for the global surveillance of ESBL-producing bacterial clones and may contribute to further elucidation of the complex epidemiology of antimicrobial-resistant Enterobacteriaceae.
Abstract: Molecular typing has become indispensable in the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings, but current methods are labor-intensive, are difficult to standardize, or have limited resolution. Whole-genome multilocus sequence typing (wgMLST) has emerged as a whole-genome sequencing (WGS)-based gene-by-gene typing method that may overcome these limitations and has been applied successfully for several species in outbreak settings. In this study, genus-, genetic-complex-, and species-specific wgMLST schemes were developed for Citrobacter spp., the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae and used to type a national collection of 1,798 extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) isolates obtained from patients in Dutch hospitals. Genus-, genetic-complex-, and species-specific thresholds for genetic distance that accurately distinguish between epidemiologically related and unrelated isolates were defined for Citrobacter spp., the E. cloacae complex, E. coli, and K. pneumoniae wgMLST was shown to have higher discriminatory power and typeability than in silico MLST. In conclusion, the wgMLST schemes developed in this study facilitate high-resolution WGS-based typing of the most prevalent ESBL-producing species in clinical practice and may contribute to further elucidation of the complex epidemiology of antimicrobial-resistant Enterobacteriaceae wgMLST opens up possibilities for the creation of a Web-accessible database for the global surveillance of ESBL-producing bacterial clones.
82 citations
TL;DR: WGS was more discriminatory than the band-based techniques and was used to construct a more accurate transmission map that resolved many of the plausible transmission routes suggested by epidemiologic links.
Abstract: Acinetobacter baumannii frequently causes nosocomial infections and outbreaks. Whole-genome sequencing (WGS) is a promising technique for strain typing and outbreak investigations. We compared the performance of conventional methods with WGS for strain typing clinical Acinetobacter isolates and analyzing a carbapenem-resistant A. baumannii (CRAB) outbreak. We performed two band-based typing techniques (pulsed-field gel electrophoresis and repetitive extragenic palindromic-PCR), multilocus sequence type (MLST) analysis, and WGS on 148 Acinetobacter calcoaceticus-A. baumannii complex bloodstream isolates collected from a single hospital from 2005 to 2012. Phylogenetic trees inferred from core-genome single nucleotide polymorphisms (SNPs) confirmed three Acinetobacter species within this collection. Four major A. baumannii clonal lineages (as defined by MLST) circulated during the study, three of which are globally distributed and one of which is novel. WGS indicated that a threshold of 2,500 core SNPs accurately distinguished A. baumannii isolates from different clonal lineages. The band-based techniques performed poorly in assigning isolates to clonal lineages and exhibited little agreement with sequence-based techniques. After applying WGS to a CRAB outbreak that occurred during the study, we identified a threshold of 2.5 core SNPs that distinguished nonoutbreak from outbreak strains. WGS was more discriminatory than the band-based techniques and was used to construct a more accurate transmission map that resolved many of the plausible transmission routes suggested by epidemiologic links. Our study demonstrates that WGS is superior to conventional techniques for A. baumannii strain typing and outbreak analysis. These findings support the incorporation of WGS into health care infection prevention efforts.
78 citations
TL;DR: Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA, which makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates.
76 citations
TL;DR: The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime, although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime.
Abstract: BACKGROUND As the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is constantly changing globally, determining the prevailing MRSA clones in a local healthcare facility is important for better management of infections. This study investigated clonal composition and distribution of MRSA isolates in Kuwait's hospitals using a combination of molecular typing methods. MATERIALS AND METHODS In total, 400 non-repeat MRSA isolates were obtained between 1992 and 2010 in 13 public hospitals and were characterized using antibiogram, SCCmec typing, spa typing, and multilocus-sequence typing. Clonal assignment and detection of virulence factors and antibiotic resistance genes were performed by DNA microarray. RESULTS The isolates were resistant to kanamycin (74.2%), erythromycin (69.5%), tetracycline (66.7%), gentamicin (61%), ciprofloxacin, (61%), fusidic acid (53.5%), clindamycin (41.5%), high-level mupirocin resistance (5.2%) and carried aphA3, aacA-aphD, ermA, ermC, mupA, tetK, tetM, fusC and far1. Molecular typing revealed 31 different MRSA clones consisting of ST239-MRSA-III (52.2%), ST22-MRSA-IV (9.2%), ST80-MRSA-IV (7.5%), ST5-MRSA-II/IV/V/VI (6.5%), ST30-MRSA-IV (3.5%), ST241-MRSA-III (2.7%), ST6-MRSA-IV (2.2%), ST36-MRSA-II (2%) and ST772-MRSA-V (1.75%). The isolates differed in the carriage of genes for enterotoxins, Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin (tst-1), arginine catabolic mobile element (ACME) and exfoliative toxins. The number of clones increased from one (ST239-III-t037) in 1992 to 30 in 2010 including ST8-IV-t008 [PVL+] [ACME+] (USA300), ST772-V (Bengal Bay clone) and ST2816 identified for the first time in Kuwait. CONCLUSION The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime. Although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime, the newly emerged clones consisted mostly of community-associated.
64 citations
TL;DR: The results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST, and this method appears to be more efficient.
Abstract: A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary stud...
64 citations
TL;DR: PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.
Abstract: Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.
59 citations
TL;DR: Improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance are highlighted.
Abstract: Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and real-time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.
56 citations
TL;DR: The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains and increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.
Abstract: Bovine mastitis caused by multidrug resistant Staphylococcus aureus is a huge problem reported worldwide, resulting in prolonged antibiotic treatment and death of livestock. The current study is focused on surveillance of antibiotic susceptibility along with genotypic and phenotypic characterization of the pathogenic S. aureus strains causing mastitis in India. One hundred and sixty seven milk samples were collected from mastitis-affected cows from different farms in India resulting in thirty nine isolated S. aureus strains. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2μg/ml). All strains were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India. Additionally, 32 strains were positive for the pvl gene, a virulence determinant; of these 17 were also OS-MRSA strains. Molecular characterization based on multilocus sequence typing (MLST), spa typing, agr typing and SCCmec classification revealed strains belonging to different groups. Moreover, strains showed spa types (t2526, t9602) and MLST sequence types, ST-72, ST-88 and ST-239 which have been earlier reported in human infections. The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains. Increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.
TL;DR: It is proposed that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary.
Abstract: Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current “gold standard” typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila. However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard “typing panel,” previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila.
07 May 2016
TL;DR: The theoretical analysis and user study show that IJQwerty improves the accuracy and input speed of gesture typing over Qwerty once a user reaches the expert mode, and is also extremely easy to learn.
Abstract: Despite of a significant body of research in optimizing the virtual keyboard layout, none of them has gained large adoption, primarily due to the steep learning curve. To address this learning problem, we introduced three types of Qwerty constraints, Qwerty1, QwertyH1, and One-Swap bounds in layout optimization, and investigated their effects on layout learnability and performance. This bounded optimization process leads to IJQwerty, which has only one pair of keys different from Qwerty. Our theoretical analysis and user study show that IJQwerty improves the accuracy and input speed of gesture typing over Qwerty once a user reaches the expert mode. IJQwerty is also extremely easy to learn. The initial upon-use text entry speed is the same with Qwerty. Given the high performance and learnability, such a layout will more likely gain large adoption than any of previously obtained layouts. Our research also shows the disparity from Qwerty substantially affects layout learning. To minimize the learning effort, a new layout needs to hold a strong resemblance to Qwerty.
TL;DR: Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.
Abstract: Multi isolate whole genome sequencing and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and longterm evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.
TL;DR: Significant associations were observed between presence of the icaD, icaA, fnbA, clfA and the cna genes as well as biofilm formation, suggesting a possible link between the agr types and certain SE genes.
TL;DR: The results showed that the population of carbapenem-resistant A. baumannii in South America was principally associated with ST79, ST25 and ST15 (92 %) and harboured the blaOXA-23 gene mainly.
Abstract: One hundred and twenty-six epidemiologically sequential, unrelated, carbapenem-resistant Acinetobacter baumannii isolates from nine hospitals in six countries of South America were collected between July 2013 and June 2014. Genes coding for Ambler class D and B carbapenemases were sought by PCR. All isolates were typed using the 3-locus sequence typing and blaOXA-51-like sequence-based typing techniques. The blaOXA-23 gene was recovered in all the participating hospitals and in all the isolates of seven of nine medical centres. The blaOXA-72 gene was only recovered in the two medical centres from Guayaquil city, Ecuador. Trilocus sequence typing revealed the presence of sequence groups SG2, SG4 and SG5. blaOXA-51-like sequence-based typing revealed the presence of blaOXA-132, blaOXA-65, blaOXA-69 and blaOXA-64. Our results showed that the population of carbapenem-resistant A. baumannii in South America was principally associated with ST79, ST25 and ST15 (92 %) and harboured the blaOXA-23 gene mainly. CC2 was not detected.
TL;DR: Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.
Abstract: Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4 PCR amplicons were pooled and sheared using Covaris fragmentation Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 978% of samples were successfully amplified using long-range PCR Among these, 982% were successfully reported by NGS, with an accuracy rate of 9984% in an independent blind Quality Control audit performed by the NDMP In this study, NGS-HLA typing identified 23 null alleles (0023%), 92 rare alleles (0091%) and 42 exon novelties (0042%) Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation
TL;DR: This study is the first report that shows high genetic diversity and remarkable antibiotic resistance of Campylobacter strains isolated from meat in Vietnam which can be considered of high public health significance.
Abstract: Campylobacter species are recognized as the most common cause of foodborne bacterial gastroenteritis in humans. In this study nine Campylobacter strains isolated from chicken meat and pork in Hanoi, Vietnam, were characterized using molecular methods and tested for antibiotic resistance.
The nine isolates (eight C. jejuni and one C. coli) were identified by multiplex PCR, and tested for the presence or absence of 29 gene loci associated with virulence, lipooligosaccharide (LOS) biosynthesis and further functions. flaA typing, multilocus sequence typing and microarray assay investigation showed a high degree of genetic diversity among these isolates. In all isolates motility genes (flaA, flaB, flhA, fliM), colonization associated genes (cadF, docB), toxin production genes (cdtA, cdtB, secD, secF), and the LOS biosynthesis gene pglB were detected. Eight gene loci (fliY, virB11, Cje1278, Cj1434c, Cj1138, Cj1438c, Cj1440c, Cj1136) could not be detected by PCR. A differing presence of the gene loci ciaB (22.2 %), Cje1280 (77.8 %), docC (66.7 %), and cgtB (55.6 %) was found. iamA, cdtC, and the type 6 secretion system were present in all C. jejuni isolates but not in C. coli. flaA typing resulted in five different genotypes within C. jejuni, MLST classified the isolates into seven sequence types (ST-5155, ST-6736, ST-2837, ST-4395, ST-5799, ST-4099 and ST-860). The microarray assay analysis showed a high genetic diversity within Vietnamese Campylobacter isolates which resulted in eight different types for C. jejuni. Antibiotic susceptibility profiles showed that all isolates were sensitive to gentamicin and most isolates (88.8 %) were sensitive to chloramphenicol, erythromycin and streptomycin. Resistance rates to nalidixic acid, tetracycline and ciprofloxacin were 88.9, 77.8 and 66.7 %, respectively. To the best of our knowledge, this study is the first report that shows high genetic diversity and remarkable antibiotic resistance of Campylobacter strains isolated from meat in Vietnam which can be considered of high public health significance. These preliminary data show that large scale screenings are justified to assess the relevance of Campylobacter infections on human health in Vietnam.
25 Aug 2016
TL;DR: NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies.
Abstract: Whole-genome sequencing (WGS) provides the highest resolution analysis for comparison of bacterial isolates in public health microbiology. However, although increasingly being used routinely for some pathogens such as Listeria monocytogenes and Salmonella enterica, the use of WGS is still limited for other organisms, such as Neisseria gonorrhoeae. Multi-antigen sequence typing (NG-MAST) is the most widely performed typing method for epidemiological surveillance of gonorrhoea. Here, we present NGMASTER, a command-line software tool for performing in silico NG-MAST on assembled genome data. NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies. The source code and user documentation are available at https://github.com/MDU-PHL/ngmaster.
TL;DR: The genetic environment of the blaK PC-2 gene was diverse and Tn1721-blaKPC-2-ΔTn3-IS26 was dominant in clinical K. pneumoniae isolates in Huashan Hospital, China, and should foster further studies about the mechanism of theBlaKpc-2 dissemination.
Abstract: Infection caused by carbapenem-resistant Klebsiella pneumoniae has become a major healthcare threat and KPC-2 enzyme is a dominant factor mediating carbapenems resistance in K. pneumoniae. This study was designed to determine the genetic environment of blaKPC-2, which prevailed in clinical K. pneumoniae isolates recovered in Huashan Hospital, Shanghai, China. Forty-two clinical isolates were included in this study by blaKPC-2 screening. After multilocus sequence typing and plasmid analyses of PCR-based replicon typing (PBRT), junction PCR, mapping PCR and crossing PCR assays, primer walking, and amplicon sequencing were used to analyze the genetic environment of the blaKPC-2 gene. ST423, ST65, ST977, and ST11 were all detected in KPC-2-producing K. pneumoniae. Two types of blaKPC-2-bearing genetic structure were found: Tn1721-blaKPC-2-Tn3 and Tn1721-blaKPC-2-ΔTn3-IS26; and were carried in IncX and IncFII plasmids, respectively. In conclusion, the genetic environment of the blaKPC-2 gene was diverse and Tn1721-blaKPC-2-ΔTn3-IS26 was dominant in clinical K. pneumoniae isolates in Huashan Hospital. This study sheds some light on the genetic environment and should foster further studies about the mechanism of the blaKPC-2 dissemination.
TL;DR: This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.
Abstract: Background
Rapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST) data direct from patient specimens while minimising costs for subsequent sequencing.
Methodology and Findings
An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK) and the Lao Peoples Democratic Republic (Lao PDR). Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine) PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50%) and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%). No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25%) and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively). All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14]).
Conclusion
Typing information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.
TL;DR: This study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina, and allowed, for the first time, the characterization of isolates of intermediate pathogenicity species from symptomatic patients.
TL;DR: In this paper, a rapid clonotyping method for extraintestinal E coli based on detection of the presence or absence of 7 single nucleotide polymorphisms (SNPs) within 2 genes (fumC and fimH) was developed.
Abstract: Background. Escherichia coli is a highly clonal pathogen. Extraintestinal isolates belong to a limited number of genetically related groups, which often exhibit characteristic antimicrobial resistance profiles. Methods. We developed a rapid clonotyping method for extraintestinal E coli based on detection of the presence or absence of 7 single nucleotide polymorphisms (SNPs) within 2 genes (fumC and fimH). A reference set of 2559 E coli isolates, primarily of urinary origin, was used to predict the resolving power of the 7-SNP-based typing method, and 582 representative strains from this set were used to evaluate test robustness. Results. Fifty-four unique SNP combinations ("septatypes") were identified in the reference strains. These septatypes yielded a clonal group resolution power on par with that of traditional multilocus sequence typing. In 72% of isolates, septatype identity predicted sequence type identity with at least 90% (mean, 97%) accuracy. Most septatypes exhibited highly distinctive antimicrobial susceptibility profiles. The 7-SNP-based test could be performed with high specificity and sensitivity using single or multiplex conventional polymerase chain reaction (PCR) and quantitative PCR. In the latter format, E coli presence and septatype identity were determined directly in urine specimens within 45 minutes with bacterial loads as low as 10(2) colony-forming units/mL and, at clinically significant bacterial loads, with 100% sensitivity and specificity. Conclusions. 7-SNP-based typing of E coli can be used for both epidemiological studies and clinical diagnostics, which could greatly improve the empirical selection of antimicrobial therapy.
TL;DR: The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass.
TL;DR: This study showed that MRSP isolates from clinical cases in individual dogs and cats in Thailand are multi-resistant with similar resistance genes and characteristics as isolate from Europe and North America.
TL;DR: Molecular typing methods can precisely identify clinically significant variant antigens that cannot be distinguished by serological typing; this capability has been exploited for the resolution of typing discrepancies and shows promise for the improved transfusion management of patients with sickle cell anemia.
Abstract: The clinical importance of blood group antigens relates to their ability to evoke immune antibodies that are capable of causing hemolysis. The most important antigens for safe transfusion are ABO and D (Rh), and typing for these antigens is routinely performed for patients awaiting transfusion, prenatal patients, and blood donors. Typing for other blood group antigens, typically of the Kell, Duffy, Kidd, and MNS blood groups, is sometimes necessary, for patients who have, or are likely to develop antibodies to these antigens. The most commonly used typing method is serological typing, based on hemagglutination reactions against specific antisera. This method is generally reliable and practical for routine use, but it has certain drawbacks. In recent years, molecular typing has emerged as an alternative or supplemental typing method. It is based on detecting the polymorphisms and mutations that control the expression of blood group antigens, and using this information to predict the probable antigen type. Molecular typing methods are useful when traditional serological typing methods cannot be used, as when a patient has been transfused and the sample is contaminated with red blood cells from the transfused blood component. Moreover, molecular typing methods can precisely identify clinically significant variant antigens that cannot be distinguished by serological typing; this capability has been exploited for the resolution of typing discrepancies and shows promise for the improved transfusion management of patients with sickle cell anemia. Despite its advantages, molecular typing has certain limitations, and it should be used in conjunction with serological methods.
Journal Article•
TL;DR: The genetic relatedness of multidrug resistant S. aureus isolates from different sources in El-Sharkia and Assuit governorates - Egypt was studied to detect the highest virulent strain from each group and revealed different sequence types and different sequences of coa and spa genes.
Abstract: In recent years, the increasing incidence of diseases caused by Staphylococcus aureus (S. aureus) has been noted in the university hospitals of El-Sharkia and Assuit governorates - Egypt. Therefore, we studied the genetic relatedness of multidrug resistant S. aureus isolates from different sources in the above mentioned governorates. One hundred and fifty six S. aureus isolates were divided into 5 different groups, 1 non clinical isolates from different food products and 4 different clinical isolates of human and animal sources in the 2 different governorates. Epidemiological characteristics of 156 S. aureus isolates were determined by phenotypic methods including quantitative antibiogram typing and biofilm production. Genetic typing of 35 multidrug resistant (MDR) isolates (7 from each group) based on 16S rRNA gene sequence, virulence and antimicrobial resistance gene profiles was done. The genetic relatedness of the highest virulent strain from each group was detected based on different single locus sequence typing and multi-locus sequence typing (MLST). S. aureus strains isolated from different sources and geographical areas showed high diversity. The genetic typing revealed different sequence types and different sequences of coa and spa genes. S. aureus isolates were found highly diverse in Egypt.
TL;DR: A new typing resolution score (TRS) is presented, based on the likelihood of self-match, that allows the systematic comparison of HLA typings across different methods, data sets and populations and can help define standards for acceptable recruitment HLA typing.
TL;DR: It is shown how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance and how Pdr1 sequencing showed that resistance arose independently in each patient.
Abstract: The opportunistic yeast Candida glabratais increasingly refractory to antifungal treatment or prophylaxis and relatedly is increasingly implicated in health care-associated infections. To elucidate the epidemiology of these infections, strain typing is required. Sequence-based typing provides multiple advantages over length-based methods, such as pulsed-field gel electrophoresis (PFGE); however, conventional multilocus sequence typing (targeting 6 conserved loci) and whole-genome sequencing are impractical for routine use. A commercial sequence-based typing service for C. glabratathat targets polymorphic tandem repeat-containing loci has recently been developed. These CgMT-J and CgMT-M services were evaluated with 56 epidemiologically unrelated isolates, 4 to 7 fluconazole-susceptible or fluconazole-resistant isolates from each of 5 center A patients, 5 matched pairs of fluconazole-susceptible/resistant isolates from center B patients, and 7 isolates from a center C patient who responded to then failed caspofungin therapy. CgMT-J and CgMT-M generated congruent results, resolving isolates into 24 and 20 alleles, respectively. Isolates from all but one of the center A patients shared the same otherwise rare alleles, suggesting nosocomial transmission. Unexpectedly, Pdr1 sequencing showed that resistance arose independently in each patient. Similarly, most isolates from center B also clustered together; however, this may reflect a dominant clone since their alleles were shared by multiple unrelated isolates. Although distinguishable by their echinocandin susceptibilities, all isolates from the center C patient shared alleles, in agreement with the previously reported relatedness of these isolates based on PFGE. Finally, we show how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance.
TL;DR: The development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae was described and proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.
Abstract: Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.