Showing papers on "Typing published in 2012"
TL;DR: This work proposes ribosomal multilocus sequence typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrating microbial genealogy and typing, and employs curated reference sequences to identify gene variants efficiently and rapidly.
Abstract: No single genealogical reconstruction or typing method currently encompasses all levels of bacterial diversity, from domain to strain. We propose ribosomal multilocus sequence typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrating microbial genealogy and typing. As with multilocus sequence typing (MLST), rMLST employs curated reference sequences to identify gene variants efficiently and rapidly. The rps loci are ideal targets for a universal characterization scheme as they are: (i) present in all bacteria; (ii) distributed around the chromosome; and (iii) encode proteins which are under stabilizing selection for functional conservation. Collectively, the rps loci exhibit variation that resolves bacteria into groups at all taxonomic and most typing levels, providing significantly more resolution than 16S small subunit rRNA gene phylogenies. A web-accessible expandable database, comprising whole-genome data from more than 1900 bacterial isolates, including 28 draft genomes assembled de novo from the European Bioinformatics Institute (EBI) sequence read archive, has been assembled. The rps gene variation catalogued in this database permits rapid and computationally non-intensive identification of the phylogenetic position of any bacterial sequence at the domain, phylum, class, order, family, genus, species and strain levels. The groupings generated with rMLST data are consistent with current nomenclature schemes and independent of the clustering algorithm used. This approach is applicable to the other domains of life, potentially providing a rational and universal approach to the classification of life that is based on one of its fundamental features, the translation mechanism.
451 citations
TL;DR: Over a 2-month period, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MR SA isolates.
348 citations
TL;DR: It was found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type, and spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step.
Abstract: Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.
215 citations
TL;DR: Here, the lab's efforts to develop high-resolution and high-throughput HLA DNA typing using the 454 Sequencing System are reviewed, and the potential future developments and applications of HLADNA typing are discussed.
Abstract: The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome, with a highly clustered and patchwork pattern of sequence motifs. In the three decades since the first HLA gene was isolated by molecular cloning (a cDNA clone of HLA-B7), thousands of alleles have been identified and the names and sequences of all known alleles have been curated in the IMGT/HLA database. This extensive allelic diversity made and continues to make high-resolution HLA DNA typing very challenging. The first attempt at HLA DNA typing involved restriction fragment length polymorphism (RFLP) analysis, but this approach had many limitations. The development of PCR in 1985 allowed for the amplification of the polymorphic exons of the HLA class I and class II genes and the analysis of the polymorphic sequence motifs with sequence-specific oligonucleotide (SSO) hybridization probes. The immobilization of these probes on membranes and later on beads, along with primer sets for sequence-specific priming (SSP), gave rise to the current set of HLA typing reagents. Sanger sequencing has provided high-resolution typing but, in many cases, genotyping 'ambiguity' remains an issue. In the past few years, the introduction of next-generation sequencing, with the critical properties of massively parallel and clonal sequencing, has significantly reduced HLA genotyping ambiguity. Here, our lab's efforts to develop high-resolution and high-throughput HLA DNA typing using the 454 Sequencing System are reviewed, and the potential future developments and applications of HLA DNA typing are discussed.
187 citations
TL;DR: This work describes a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST.
Abstract: Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach-which we call CH (fumC/fimH) typing-consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.
171 citations
TL;DR: The most frequently detected MLST sequence types (STs) were ST14 (n = 13), ST11, ST149, ST231, and ST147 as mentioned in this paper, and IncA/C was the most commonly detected plasmid replicon type.
Abstract: Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of bla(NDM-1).
169 citations
TL;DR: The SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.
Abstract: Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.
147 citations
02 Jul 2012
TL;DR: Staphylococcus aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages, and novel and unique sas alleles were identified in the animal-associated strains that may represent animal- associated sAs alleles.
Abstract: Staphylococcus aureus is an important pathogen of man, but is also able to colonize and cause disease in a wide variety of mammals and birds. An extended multilocus sequencing approach, involving multilocus sequence typing (MLST), sas typing, spa typing and agr typing, was used to examine the molecular diversity of 118 S. aureus isolates recovered from a range of host species and to compare these data with the known diversity of human-derived isolates. MLST revealed that the commonest animal-associated MLST types were ST133, ST5, ST71, ST97, ST126 and ST151. ST133 appears to be an ungulate-animal-specific genotype, as no evidence of ST133 associating with humans has yet been found in the literature. Novel and unique sas alleles were identified in the animal-associated strains that may represent animal-associated sas alleles. However, sas typing exhibited a lower typeability than MLST for the animal strains (91.3 %). Phylogenetic analyses using neighbour-joining and maximum-parsimony trees localized ruminant-associated MLST lineages to both previously identified S. aureus subspecies aureus subgroups, thus explaining the finding of all four agr types within the ruminant-associated strains. S. aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages.
115 citations
TL;DR: Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis patterns with congruent subtypes that substantiated a predominant clone that has been causing human illness across Canada for more than 2 decades.
Abstract: Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades.
109 citations
TL;DR: A high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs is introduced.
Abstract: Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.
90 citations
TL;DR: In order to fully explore the possibilities of genotyping in the molecular epidemiology of tuberculosis and to study the phylogeny of the causative bacteria reliably, the application of whole-genome sequencing (WGS) analysis for all M. tuberculosis isolates is the optimal, although currently still a costly solution.
TL;DR: The results of this study confirm that MRSA, including LA-MRSA, is common in herds of swine in Ohio and is shown to persist in the farm to processing and retail continuum.
Abstract: The main goal of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), particularly livestock-associated MRSA (LA-MRSA) in pigs and pork The genotypic relatedness of isolates on the farm, at slaughter, and at the retail level was assessed Paired nasal and perianal swab samples were collected from 10 cohorts of market-age pigs (24 pigs per cohort) and carcasses at slaughterhouse, and pork samples were collected at retail Staphylococci were isolated using selective enrichment method Isolates were tested for antimicrobial resistance by broth microdilution Duplex PCR was used to confirm MRSA using species-specific (nuc) and methicillin resistance (mecA) genes The clonal relatedness of isolates was determined using pulsed-field gel electrophoresis (PFGE), Staphylococcus protein A (spa) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element (SCCmec) typing MRSA was detected in 5 of the 10 cohorts (50%), with the prevalence ranging from 0% to 125% per cohort Of all the pigs sampled on the farm before they went to market, 3% (7/240) were MRSA positive A higher prevalence of MRSA was detected at holding pens at the slaughterhouse (11% [27/240]) MRSA was also detected in 2% (4/235) of the carcasses and 4% (5/135) of the retail pork While the isolates appear predominantly to be highly clonal, PFGE had a relatively higher discriminatory power (discriminatory index [DI] = 0624) Four genotypic clusters were identified by PFGE; of the four clusters, clonal type B was predominant across the farm-to-retail continuum MLST findings revealed that sequence type 5 (ST5) was the most predominant subtype (32/50) The livestock-associated MRSA (clonal complex 398 [CC398] or sequence type 398 [ST398]) was the second common type (12/50) and was detected at all stages from farm to retail Nine of the 50 (18%) MRSA isolates belonged to spa type 539/t034 that were of ST398 based on MLST The results of this study confirm that MRSA, including LA-MRSA, is common in herds of swine in Ohio and hereby shown to persist in the farm to processing and retail continuum
TL;DR: A high rate of associated mortality and great difficulty in controlling the spread of this clone, without permanent behavioral changes in the personnel, were observed.
Abstract: We study the epidemiology, molecular basis, clinical risk factors, and outcome involved in the clonal dissemination of VIM-1-producing Klebsiella pneumoniae isolates in the hospital setting. All patients infected/colonized by carbapenem-nonsusceptible K. pneumoniae (CNSKP) in 2009 were included. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and for incompatibility group by a PCR-based replicon typing scheme. Risk factors associated with CNSKP colonization/infection were assessed by an observational case-control study. All 55 patients studied were infected ( n = 28) or colonized ( n = 27) by VIM-1-producing K. pneumoniae. All but one acquired isolates of a single clone (PFGE cluster 1 [C1], sequence type 15 [ST15]), while another clone (PFGE C2, ST340) was detected in four patients. C1 isolates also produced the new extended-spectrum β-lactamase SHV-134. bla VIM-1 was carried in a class 1 integron and an untypeable plasmid of ∼50 bp. The number of days that the patient received mechanical ventilation, the use of parenteral nutrition, previous treatment with linezolid, and treatment with extended-spectrum cephalosporins for more than 7 days were detected to be independent risk factors for CNSKP acquisition. The VIM-1-producing K. pneumoniae ST15 clone has a high capacity to spread among intensive care unit patients with severe underlying conditions. A high rate of associated mortality and great difficulty in controlling the spread of this clone, without permanent behavioral changes in the personnel, were observed.
TL;DR: There is no consensus regarding the single best method for typing of MRSA strains and a need for harmonisation of typing techniques by following standard protocols is emphasised to establish surveillance networks and facilitate global MRSA control.
TL;DR: A simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing is presented, providing the cornerstone for the development of robust typing strategies.
Abstract: Background
Trypanosoma cruzi is the causative agent of Chagas' Disease The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events Because there are important biological differences between these lineages, strain typing methods are essential to study the T cruzi species Currently, there are a number of typing methods available for T cruzi, each with its own advantages and disadvantages However, most of these methods are based on the amplification of a variable number of loci
Methodology/Principal Findings
We present a simple typing assay for T cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group) Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites
Conclusions/Significance
Based on these findings we propose a simple typing assay for T cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies
TL;DR: Sequence-based typing of bla OXA-51 variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.
Abstract: This study investigated the correlation between blaOXA-51 variants and Acinetobacter baumannii worldwide clonal lineages 1 to 8 (WW1 to -8). The blaOXA-51-like genes of 102 A. baumannii isolates were sequenced. Using DiversiLab repetitive-sequence-based PCR (rep-PCR) typing, 92 of these isolates had previously been assigned to WW1 to -8 and 10 were unclustered. Clustering of DNA sequences was performed using the neighbor-joining method and the Jukes-Cantor phylogenetic correction. blaOXA-51 variants were in good correlation with DiversiLab-defined clonal lineages. Sequence-based typing of blaOXA-51 variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.
TL;DR: The feasibility and accuracy of Ion Torrent Personal Genome Machine (PGM) sequencing for genomic typing of meningococci was explored and the amount of typing information could be substantially increased by the combined use of genome sequencing and BIGSdb compared to conventional methods.
Abstract: Neisseria meningitidis causes invasive meningococcal disease in infants, toddlers and adolescents worldwide. DNA sequence based typing has become the standard for molecular epidemiology of the organism including multilocus sequence typing, analysis of genetic determinants of antibiotic resistance, and sequence typing of vaccine antigens. However, PCR of multiple targets and consecutive Sanger sequencing provides logistic constraints to reference laboratories. Taking advantage of the recent development of benchtop next generation sequencers (NGS) and of BIGSdb, a database accommodating and analyzing genome sequence data, we therefore explored the feasibility and accuracy of Ion Torrent Personal Genome Machine™ (PGM™) sequencing for genomic typing of meningococci. Three strains from a previous meningococcus B community outbreak were selected to compare conventional typing results with data generated by semiconductor chip based sequencing. In addition, sequencing of the meningococcal type strain MC58 provided information about the general performance of the technology. The PGM™ technology generated sequence information for almost all target genes addressed. The results were 100% concordant with conventional typing results with no further editing necessary. In addition, the amount of typing information, i.e. nucleotides and target genes analyzed, could be substantially increased by the combined use of genome sequencing and BIGSdb compared to conventional methods. In a near future, affordable and fast benchtop-NGS machines like the PGM™ might enable reference laboratories to switch to genomic typing on a routine basis. This will reduce workload and rapidly provide information for laboratory surveillance, outbreak investigation, assessment of vaccine preventability and antibiotic resistance gene monitoring.
TL;DR: The sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥1 allele from 43/46 strains.
Abstract: The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires’ disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥1 allele from 43/46 strains.
TL;DR: Comparative genomic fingerprinting appears to be one of the optimal methods for the detection of clusters of cases, and it could be supplemented by the sequencing of the flaA gene short variable region (flaA SVR sequence typing), with or without subsequent multilocus sequence typing (MLST).
Abstract: Campylobacter spp. may be responsible for unreported outbreaks of food-borne disease. The detection of these outbreaks is made more difficult by the fact that appropriate methods for detecting clusters of Campylobacter have not been well defined. We have compared the characteristics of five molecular typing methods on Campylobacter jejuni and C. coli isolates obtained from human and nonhuman sources during sentinel site surveillance during a 3-year period. Comparative genomic fingerprinting (CGF) appears to be one of the optimal methods for the detection of clusters of cases, and it could be supplemented by the sequencing of the flaA gene short variable region (flaA SVR sequence typing), with or without subsequent multilocus sequence typing (MLST). Different methods may be optimal for uncovering different aspects of source attribution. Finally, the use of several different molecular typing or analysis methods for comparing individuals within a population reveals much more about that population than a single method. Similarly, comparing several different typing methods reveals a great deal about differences in how the methods group individuals within the population.
TL;DR: This review describes the design as well as advantages and disadvantages for 3 current phenotypesic techniques and 6 genotypic techniques for typing pathogenic Campylobacter spp.
TL;DR: It is suggested that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent and is an excellent candidate to become the gold standard for T. cruzi genetic typing.
TL;DR: A set of 415 clinical samples isolated from 294 patients suspected of having syphilis collected in the Czech Republic between 2004 and 2010 was tested for the presence of treponemal DNA, and variants of arp and tpr genes appear to combine independently with sequence variants of TP0136, TP0548 and 23S rRNA genes.
Abstract: A set of 415 clinical samples isolated from 294 patients suspected of having syphilis collected in the Czech Republic between 2004 and 2010 was tested for the presence of treponemal DNA. Standard serological tests showed that 197 patients were syphilis-seropositive and 97 patients were syphilis-seronegative. In each sample, PCR tests for polA (TP0105), tmpC (TP0319), TP0136, TP0548 and 23S rRNA genes were performed. Samples taken from 91 patients were PCR-positive. Molecular typing of treponemal DNA was based on the sequencing of TP0136, TP0548 and 23S rRNA genes. Treponemal DNA was typeable in samples taken from 64 PCR-positive patients and 9 different genotypes were found. The proportion of treponemal strains resistant to macrolide antibiotics was 37.3%. In the DNA samples taken from 39 patients, a parallel treponemal typing approved by Centers for Disease Control and Prevention was performed. The variants of arp and tpr genes appear to combine independently with sequence variants of TP0136, TP0548 and 23S rRNA genes.
TL;DR: This first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.
Abstract: Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.
TL;DR: The results show that the proportions of β-lactam-resistant invasive H. influenzae have increased in Sweden in the last decade.
Abstract: The proportions of Haemophilus influenzae resistant to ampicillin and other beta-lactam antibiotics have been low in Sweden compared to other countries in the Western world. However, a near-doubled proportion of nasopharyngeal Swedish H. influenzae isolates with resistance to beta-lactams has been observed in the last decade. In the present study, the epidemiology and mechanisms of antimicrobial resistance of H. influenzae isolates from blood and cerebrospinal fluid in southern Sweden from 1997 to 2010 (n = 465) were studied. Antimicrobial susceptibility testing was performed using disk diffusion, and isolates with resistance to any tested beta-lactam were further analyzed in detail. We identified a significantly increased (P = 0.03) proportion of beta-lactam-resistant invasive H. influenzae during the study period, which was mainly attributed to a significant recent increase of beta-lactamase-negative beta-lactam-resistant isolates (P = 0.04). Furthermore, invasive beta-lactamase-negative beta-lactam-resistant H. influenzae isolates from 2007 and onwards were found in higher proportions than the corresponding proportions of nasopharyngeal isolates in a national survey. Multiple-locus sequence typing (MIST) of this group of isolates did not completely separate isolates with different resistance phenotypes. However, one cluster of beta-lactamase-negative ampicillin-resistant (BLNAR) isolates was identified, and it included isolates from all geographical areas. A truncated variant of a beta-lactamase gene with a promoter deletion, bla(TEM-1)-P Delta dominated among the beta-lactamase-positive H. influenzae isolates. Our results show that the proportions of beta-lactam-resistant invasive H. influenzae have increased in Sweden in the last decade.
TL;DR: Traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates, and whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease.
Abstract: Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system Group A streptococcus (GAS; Streptococcus pyogenes) isolates PS001 to PS011 were recovered from nine patients Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm140, emm750, emm770, emm890, and emm899 were each recovered from a single patient, ruling out a single source of infection However, emm288 GAS were recovered from four different patients To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm284 reference strain, MGAS6180 A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease
TL;DR: The results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide.
Abstract: Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
TL;DR: Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST 93-IVa [2B]-t202-dt10 predominant across Australia.
Abstract: In Australia the PVL - positive ST93-IV [2B], colloquially known as “Queensland CA-MRSA” has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.
TL;DR: MALDI-ToF MS has potential as a rapid and reproducible platform for the identification and typing of Mycobacterium species and was developed and validated and matched completely to classical spoligotyping data.
TL;DR: This study confirmed the importance of the widely disseminated emm1-T1-ST28 clone in invasive infections but also identified other clones linked to either invasive infections or pharyngitis, which may be more limited in their temporal and geographical spread.
Abstract: Background
A few lineages of Group A streptococci (GAS) have been associated with a reemergence of severe invasive streptococcal disease in developed countries. However, the majority of the comparisons between invasive and non-invasive GAS isolates have been performed for collections of reduced genetic diversity or relied on limited typing information to distinguish clones. We characterized by several typing methods and compared a collection of 160 isolates recovered from normally sterile sites with 320 isolates associated with pharyngitis and recovered in the same time period in Portugal.
TL;DR: Antibiotic resistance to macrolides and clindamycin in GAS is prevalent in Shanghai and emm12 GAS strain caused the large 2011 outbreak of scarlet fever in Shanghai.
Abstract: Background An unprecedented, large outbreak of childhood scarlet fever occurred in Shanghai between April and July 2011. Investigation of the epidemiology could enhance our understanding of the factors related to the outbreak. Methods We retrospectively analyzed the demographic and seasonal characteristics of children with scarlet fever and the outcome. During the peak month of the 2011 outbreak, 45 GAS isolates recovered from pediatric patients and 13 (43.3%) GAS isolates recovered from 30 asymptomatic student contacts were characterized by emm typing, superantigen profiles, pulsed-field gel electrophoresis genotypes, mutilocus sequence typing and antimicrobial susceptibility. Results The 2011 outbreak of scarlet fever started in April and peaked in May and June. Boys outnumbered girls (65.1% versus 34.9%). Preschool and primary school children accounted for 96% of cases. No severe outcome was found. emm1, emm12 and emm75 were identified among 58 GAS isolates, and 53 (91.4%) isolates belonged to emm12, st36. Ten pulsed-field gel electrophoresis genotypes were identified among emm12 GAS isolates, 43 (81.1%) shared SPYS16.001 genotype and the remaining 7 genotypes detected were related to SPYS16.001 closely or possibly. No streptococcal pyrogenic exotoxin A and streptococcal pyrogenic exotoxin M were detected in 58 isolates. All emm12 GAS isolates were resistant to azithromycin and clindamycin. Conclusions emm12 GAS strain caused the large 2011 outbreak of scarlet fever in Shanghai. Antibiotic resistance to macrolides and clindamycin in GAS is prevalent in Shanghai.