Showing papers on "Typing published in 2010"
TL;DR: The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.
Abstract: Objectives: IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. Methods: We compared the available IncF replicon sequences, identifying the combination of the different IncF replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. Results: We devised a replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the blaCTX-M-15 gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. Conclusions: The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.
550 citations
TL;DR: These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.
Abstract: A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S–23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.
240 citations
TL;DR: An enhanced T. pallidum strain typing system that shows biological and clinical relevance is described and is believed to be the most discriminating typing system.
Abstract: Background. Strain typing is a tool for determining the diversity and epidemiology of infections. Methods. Treponema pallidum DNA was isolated from 158 patients with syphilis from the United States, China, Ireland, and Madagascar and from 15 T. pallidum isolates. Six typing targets were assessed: (1) the number of 60-bp repeats in the acidic repeat protein gene, (2) restriction fragment length polymorphism (RFLP) analysis of T. pallidum repeat (tpr) subfamily II genes, (3) RFLP analysis of the tprC gene, (4) determination of tprD allele in the tprD gene locus, (5) the presence of a 51-bp insertion between tp0126 and tp0127, and (6) sequence analysis of an 84-bp region of tp0548. The combination of targets 1 and 2 comprises the Centers for Disease Control and Prevention (CDC) T. pallidum subtyping method. Results. Adding sequence analysis of tp0548 to the CDC method yielded the most discriminating typing system. Twenty-five strain types were identified and designated as "CDC subtype/tp0548 sequence type." Type 14d/f was found in samples from 5 of 6 locations. In Seattle, Washington, strain types changed from 1999 through 2008 (P<.001). Twenty-one (50%) of 42 patients infected with type 14d/f had neurosyphilis compared with 10 (24%) of 41 patients infected with any of the other types combined (P = .02). Conclusion. We describe an enhanced T. pallidum strain typing system that shows biological and clinical relevance.
197 citations
TL;DR: It was concluded that the gyrB and gpi genes are not good candidates for use in MLST analysis and that a SBT-bla OXA-51-like gene scheme produced results comparable to those produced by MLST for the identification of the major epidemic lineages.
Abstract: This study used a diverse collection of epidemiologically unrelated Acinetobacter baumannii isolates to compare the robustness of a multilocus sequence typing (MLST) scheme, based on conserved regions of seven housekeeping genes, gltA, gdhB, recA, cpn60, rpoD, gyrB, and gpi, with that of sequence-based typing of bla(OXA-51-like) genes (SBT-bla(OXA-51-like) genes). The data obtained by analysis of MLST and SBT-bla(OXA-51-like) genes were compared to the data generated by pulsed-field gel electrophoresis (PFGE). The topologies of the phylogenetic trees generated for the gyrB and gpi genes showed evidence of recombination and were inconsistent with those of the trees generated for the other five genes. MLST identified 24 sequence types (STs), of which 19 were novel, and 5 novel alleles. Clonality was demonstrated by eBURST analysis and standardized index of association values of >1 (P < 0.001). MLST data revealed that all isolates harboring the major bla(OXA-51-like) alleles OXA-66, OXA-69, and OXA-71 fell within the three major European clonal lineages. However, the MLST data were not always in concordance with the PFGE data, and some isolates containing the same bla(OXA-51-like) allele demonstrated <50% relatedness by PFGE. It was concluded that the gyrB and gpi genes are not good candidates for use in MLST analysis and that a SBT-bla(OXA-51-like) gene scheme produced results comparable to those produced by MLST for the identification of the major epidemic lineages, with the advantage of having a significantly reduced sequencing cost and time. It is proposed that studies of A. baumannii epidemiology could involve initial screening of bla(OXA-51-like) alleles to identify isolates belonging to major epidemic lineages, followed by MLST analysis to categorize isolates from common lineages, with PFGE being reserved for fine-scale typing.
136 citations
TL;DR: The cumulative data indicate a recent emergence of a certain multidrug-resistant MRSP-lineage (ST71) in central and southern European countries during the last few years.
124 citations
TL;DR: PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments.
Abstract: A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHTbeta (n=14) were observed in 83% of the strains, while pS86, pCF10, pAM373, pMBB1 or pEF418 were not detected. Furthermore, 61% of the strains contained the axe-txe (n=42) or/and the omega-epsilon-zeta (n=18) plasmid stabilization loci. Sequence analyses divided the omega-epsilon-zeta operon into two distinct phylogenetic groups. The present typing scheme accounted for about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to >200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and pRUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments.
112 citations
TL;DR: The ability to recognize and subcategorize plasmids by pDLST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful to analyse their distribution in nature and to discover of their evolutionary origin.
Abstract: Objectives: IncHI2 plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance genes. These plasmids are usually .250 kb, and technical difficulties can impair plasmid DNA purification and comparison by restriction fragment length polymorphism. We analysed the available IncHI2 whole DNA plasmid sequences to devise a rapid typing scheme to categorize the members of this plasmid family into homogeneous groups. Methods: We compared the available full IncHI2 plasmid sequences, identifying conserved and variable regions within the backbone of this plasmid family, to devise an IncHI2 typing method based on sequence typing and multiplex PCRs. A collection of IncHI2 plasmids carrying extended-spectrum b-lactamase and quinolone resistance genes, identified in strains from different sources (animals and humans) and geographical origins, was tested by these typing systems. Results: We devised a plasmid double locus sequence typing (pDLST) scheme and a multiplex PCR discriminating IncHI2 plasmid variants. These systems were tested on a collection of IncHI2 plasmids, demonstrating that the plasmids carrying blaCTX-M-2 and blaCTX-M-9 belonged to two major plasmid variants, which were highly conserved among different enterobacterial species disseminated in several European countries. Conclusions: The ability to recognize and subcategorize plasmids by pDLST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful to analyse their distribution in nature and to discover of their evolutionary origin.
106 citations
TL;DR: MIRU-VNTR typing appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the Mycobacterium avium complex and providing new epidemiological knowledge on MAC.
Abstract: Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.
83 citations
TL;DR: High-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences, which reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help understand how M. Ulcerans is transmitted.
Abstract: Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted.
80 citations
TL;DR: DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp.
Abstract: Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMerieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpson's index of diversity and adjusted Rand's and Wallace's coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.
80 citations
TL;DR: The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.
TL;DR: The results obtained were in complete agreement with those obtained by MLEE, suggesting that PCR-RFLP analysis of hsp70 could soon replace MLEe for routine Leishmania typing, and led to the identification of restriction enzymes that could be used for PCR- RFLP-based identification.
TL;DR: Combining PFGE and spa typing with DNA sequencing of the staphylococcal cassette chromosome mec element-associated direct repeat unit (dru typing) would improve isolate discrimination of ST22-MRSA-IV isolates significantly enhances discrimination and could be applied to epidemiological investigations of other highly clonal MRSA strains.
Abstract: ST22-methicillin-resistant Staphylococcus aureus type IV (ST22-MRSA-IV) is endemic in Irish hospitals and is designated antibiogram-resistogram type-pulsed-field group (AR-PFG) 06-01. Isolates of this highly clonal strain exhibit limited numbers of pulsed-field gel electrophoresis (PFGE) patterns and spa types. This study investigated whether combining PFGE and spa typing with DNA sequencing of the staphylococcal cassette chromosome mec element (SCCmec)-associated direct repeat unit (dru typing) would improve isolate discrimination. A total of 173 MRSA isolates recovered in one Irish hospital during periods in 2007 and 2008 were investigated using antibiogram-resistogram (AR), PFGE, spa, dru, and SCCmec typing. Isolates representative of each of the 17 pulsed-field group 01 (PFG-01) spa types identified underwent multilocus sequence typing, and all isolates were ST22. Ninety-seven percent of isolates (168 of 173) exhibited AR-PFG 06-01 or closely related AR patterns, and 163 of these isolates harbored SCCmec type IVh. The combination of PFGE, spa, and dru typing methods significantly improved discrimination of the 168 PFG-01 isolates, yielding 65 type combinations with a Simpson's index of diversity (SID) of 96.53, compared to (i) pairwise combinations of spa and dru typing, spa and PFGE typing, and dru and PFGE typing, which yielded 37, 44, and 43 type combinations with SIDs of 90.84, 91.00, and 93.57, respectively, or (ii) individual spa, dru, and PFGE typing methods, which yielded 17, 17, and 21 types with SIDs of 66.9, 77.83, and 81.34, respectively. Analysis of epidemiological information for a subset of PFG-01 isolates validated the relationships inferred using combined PFGE, spa, and dru typing data. This approach significantly enhances discrimination of ST22-MRSA-IV isolates and could be applied to epidemiological investigations of other highly clonal MRSA strains.
TL;DR: The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.
TL;DR: Pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates and two recent isolates from the authors' collection were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements.
TL;DR: Strain typing based on the presence or absence of distributed genes is able to resolve all completely sequenced genomes of six bacterial species by the development of a clustering method, neighbour grouping, which is completely consistent with the lower-resolution MLST method, but provides far greater resolving power.
Abstract: The most widely used DNA-based method for bacterial strain typing, multi-locus sequence typing (MLST), lacks sufficient resolution to distinguish among many bacterial strains within a species Here, we show that strain typing based on the presence or absence of distributed genes is able to resolve all completely sequenced genomes of six bacterial species This was accomplished by the development of a clustering method, neighbour grouping, which is completely consistent with the lower-resolution MLST method, but provides far greater resolving power Because the presence/absence of distributed genes can be determined by low-cost microarray analyses, it offers a practical, high-resolution alternative to MLST that could provide valuable diagnostic and prognostic information for pathogenic bacterial species
TL;DR: Two standard methods for Clostridium difficile typing are described and the difficulties of inter-laboratory comparability and unification of typing nomenclature are discussed.
Abstract: Molecular typing methods for Clostridium difficile are based on gel electrophoresis of restriction fragments (endonuclease restriction analysis, REA; pulsed field gel electrophoresis PFGE; toxinotyping), PCR amplification (PCR ribotyping, arbitrarily primed PCR, multilocus variable-number tandem-repeat analysis MLVA), and sequence analysis (multilocus sequence typing MLST; slpA typing, tandem repeat sequence typing). We will describe two standard methods (PCR ribotyping predominantly used throughout Europe and PFGE which is predominantly used in North America) and will discuss the difficulties of inter-laboratory comparability and unification of typing nomenclature.
TL;DR: An assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence- based typing is presented.
TL;DR: No correlation could be established between the anatomical site of isolation and the emm or the FCT type, and there was no relationship between biofilm formation and emm type, antibiotic-resistance or PFGE patterns, but a novel association between bioFilm formation and FCTtype became obvious among strains from the authors' collection.
Abstract: Streptococcus pyogenes is an important human pathogen for which
an association between infection site and selected epidemiological or functional
markers has previously been suggested. However, the studies involved often
used strains with an insufficiently defined clinical background and laboratory
history. Thus, the major goal of the present study was to investigate these
relationships in 183 prospectively collected, well-defined, low-passage isolates
from a North-East German centre for tertiary care. For each isolate the clinical
background (91 respiratory, 71 skin and 21 invasive isolates) and
antibiotic-resistance pattern was recorded. All isolates were classified according
to their emm type, antibiotic-resistance and PFGE pattern (SmaI restriction analysis of genomic DNA). As novel discriminatory
methods we performed a PCR-based typing of the pilus-protein-encoding FCT
region (FCT) and biofilm-formation phenotyping in various culture
media. Forty-one isolates were found to be resistant to at least one of the
tested antibiotics. emm typing revealed emm28, emm12, emm1, emm4, emm89 and emm2 as the
most frequent types in our collection. The novel FCT typing showed isolates
encoding FCT types 4 and 2 to be the most common. Overall 113 strains with
unique combinations of emm and FCT types, antibiotic-resistance and
PFGE patterns were identified. The majority of all isolates revealed an association
of biofilm-formation capacity with growth media. Comparing all results for
potential associations, no correlation could be established between the anatomical
site of isolation and the emm or the FCT type. There was no relationship
between biofilm formation and emm type, antibiotic-resistance or
PFGE patterns. However, a novel association between biofilm formation and
FCT type became obvious among strains from our collection.
TL;DR: Fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types associated with dermatological infections, finding that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis.
Abstract: Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.
TL;DR: A high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed and the emergence of the bla CTX-M-14 gene among several clones of Salmonella serovar Choleraesuis was revealed.
Abstract: The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.
TL;DR: Based on the phylogenetic analysis, the Indian isolates could be grouped in to two subgroups, viz., 1.1 and 2.2, which revealed predominance of subgroup 1.2 and involvement of viruses of more than one subgroup in an outbreak.
TL;DR: A typing system for Clostridium difficile using sequencing of the surface-layer protein A encoding gene (slpA) was evaluated and used to analyse clinical isolates in Japan and found to have reliable typability and discriminatory power in comparison with PCR ribotyping.
Abstract: A typing system for Clostridium difficile using sequencing of the surface-layer protein A encoding gene (slpA) was evaluated and used to analyse clinical isolates in Japan. A total of 160 stool specimens from symptomatic patients in Japan was examined and 87 C. difficile isolates were recovered. slpA sequence typing was found to have reliable typability and discriminatory power in comparison with PCR ribotyping, and the typing results were highly reproducible and comparable. slpA sequence typing was used to type C. difficile in DNA extracted directly from stool specimens. Among the 90 stool specimens in which direct typing results were obtained, 77 specimens were positive for C. difficile culture, and typing results from isolated strains agreed with those from direct typing in all 77 specimens. The slpA sequence type smz was dominant at all four hospitals examined, and this endemic type was detected by culture and/or direct typing in 61 (62 %) of 99 stool specimens positive for toxic culture and/or direct slpA sequence typing. Comparison of epidemic strains reported throughout the world revealed one isolate identified as slpA sequence type gc8, which was found to correspond to PCR ribotype 027 (BI/NAP1/027), whereas no isolates were found with the slpA gene identical to that of PCR ribotype 078 strain. slpA sequence typing is valuable for comparison of C. difficile strains epidemic in diverse areas because the typing results are reproducible and can easily be shared. In addition, slpA sequence typing could be applied to direct typing without culture.
TL;DR: Ten penicillinase-producing Neisseria gonorrhoeae (PPNG) strains isolated from 2000 to 2008 were characterized by multilocus sequence typing, multiantigen sequence typed, and plasmid typing, suggesting independent origins of these PPNG strains.
Abstract: Ten penicillinase-producing Neisseria gonorrhoeae (PPNG) strains isolated from 2000 to 2008 were characterized by multilocus sequence typing, multiantigen sequence typing, and plasmid typing. Sequence analysis showed that 8 strains contained a TEM-1 β-lactamase gene. However, two other genetically distinct PPNG strains, isolated in 2004 and 2008, each contained a TEM-135 β-lactamase on different plasmids, a Toronto/Rio type R plasmid and an Asia type R plasmid, suggesting independent origins of these PPNG strains.
TL;DR: 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin and subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak.
Abstract: As an issue of biosecurity, species-specific genetic markers have been well characterized. However, Bacillus anthracis strain-specific information is currently not sufficient for traceability to identify the origin of the strain. By using genome-wide screening using short read mapping, we identified strain-specific single nucleotide polymorphisms (SNPs) among B. anthracis strains including Japanese isolates, and we further developed a simplified 80-tag SNP typing method for the primary investigation of traceability. These 80-tag SNPs were selected from 2,965 SNPs on the chromosome and the pXO1 and pXO2 plasmids from a total of 19 B. anthracis strains, including the available genome sequences of 17 strains in the GenBank database and 2 Japanese isolates that were sequenced in this study. Phylogenetic analysis based on 80-tag SNP typing showed a higher resolution power to discriminate 12 Japanese isolates rather than the 25 loci identified by multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the 80-tag PCR testing enabled the discrimination of B. anthracis from other B. cereus group species, helping to identify whether a suspected sample originates from the intentional release of a bioterrorism agent or environmental contamination with a virulent agent. In conclusion, 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin. Subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak.
TL;DR: The genomic instability of the Liverpool epidemic strain of Pseudomonas aeruginosa suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.
Abstract: The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 “anomalous” CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.
TL;DR: It is suggested that introduction of the DiversiLab system would be useful for rapid exclusion of E. coli isolates during outbreak investigations, and that the approach could be employed for surveillance for pathogenic or antibiotic-resistant clones of this organism.
TL;DR: The Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships, offering a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures.
Abstract: Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of VRE. Two sets of VRE isolates evaluated in this study were collected by active microbial surveillance at a large teaching hospital in Taiwan during 2008. The first set included 90 isolates randomly selected from the surveillance cohort. The first set consisted of 34 pulsotypes and 10 Rep-PCR types. There was good correlation between the two methods (P < 0.001). The second set included 68 VRE isolates collected from eight clusters of colonization. A dominant clone was detected in five out of eight clusters by both methods. Two clusters were characterized by Rep-PCR as being caused by a dominant clone, whereas PFGE showed polyclonal origins. One cluster was shown to be polyclonal by both methods. A single Rep-PCR clone type was detected among 12 of 14 vancomycin-intermediate enterococci, whereas PFGE detected six pulsotypes. In conclusion, the Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships. The ease of use and more rapid turnaround time of Rep-PCR compared to PFGE offers a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures. PFGE remains the preferred method to confirm clonal spread.
TL;DR: An extensive molecular characterization of methicillin-susceptible S. aureus carried by children and a broad diversity in MSSA carriage strains suggests a limited selection pressure in this geographical area.
TL;DR: It is concluded that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.
Abstract: To overcome some of the deficiencies with current molecular typing schema for Campylobacter spp., we developed a prototype PCR binary typing (P-BIT) approach. We investigated the distribution of 68 gene targets in 58 Campylobacter jejuni strains, one Campylobacter lari strain, and two Campylobacter coli strains for this purpose. Gene targets were selected on the basis of distribution in multiple genomes or plasmids, and known or putative status as an epidemicity factor. Strains were examined with Penner serotyping, pulsed-field gel electrophoresis (PFGE; using SmaI and KpnI enzymes), and multilocus sequence typing (MLST) approaches for comparison. P-BIT provided 100% typeability for strains and gave a diversity index of 98.5%, compared with 97.0% for SmaI PFGE, 99.4% for KpnI PFGE, 96.1% for MLST, and 92.8% for serotyping. Numerical analysis of the P-BIT data clearly distinguished strains of the three Campylobacter species examined and correlated somewhat with MLST clonal complex assignations and with previous classifications of "high" and "low" risk. We identified 18 gene targets that conferred the same level of discrimination as the 68 initially examined. We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.