Showing papers on "Typing published in 2007"
TL;DR: An update to the multiplex PCR strategy for SCCmec typing that was described in 2002 is described so that S CCmec types IV and V may be properly identified.
Abstract: Staphylococcal cassette chromosome mec (SCCmec) typing is important for the identification and definition of methicillin-resistant Staphylococcus aureus clones, and for routine purposes, multiplex PCR assays are the most adequate for SCCmec typing. Here, we describe an update to the multiplex PCR strategy for SCCmec typing that we described in 2002 so that SCCmec types IV and V may be properly identified.
484 citations
TL;DR: The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles to significantly increase the index of discrimination calculated for a panel of unrelated strains.
Abstract: The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.
250 citations
TL;DR: A simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal capsular polysaccharides and well adapted for GBS capsularpolysaccharide typing in large-scale epidemiological studies.
Abstract: We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.
247 citations
TL;DR: The results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission and show a comparable to slightly better predictive value than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg.
Abstract: An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (≥6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.
228 citations
TL;DR: The identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs) are reported, indicating a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.
Abstract: Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Merial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.
188 citations
Health Protection Agency1, Virginia Commonwealth University2, Statens Serum Institut3, Robert Koch Institute4, National and Kapodistrian University of Athens5, Erasmus University Rotterdam6, Carlos III Health Institute7, University of the Basque Country8, Swedish Institute9, University of Ljubljana10, Imperial College London11
TL;DR: It is demonstrated that the integration of such technologies is achievable, although common protocols (such as those developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis.
Abstract: We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.
188 citations
TL;DR: The development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis and results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonello serotypeEnteritidis.
Abstract: Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.
173 citations
TL;DR: Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters, and MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.
Abstract: Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.
171 citations
TL;DR: It is suggested that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection, for long-term, nationwide epidemiological surveillance studies.
Abstract: Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional “violations” of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.
149 citations
TL;DR: Multiple-locus variable-number tandem repeats analysis improves surveillance and outbreak investigations and provides new insights into infectious disease prevention and control.
Abstract: In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all Salmonella enterica serotype Typhimurium isolates are currently subtyped by using phage typing, antimicrobial resistance profiles, and pulsed-field gel electrophoresis (PFGE). We evaluated the value of real-time typing that uses multiple-locus variable-number tandem-repeats analysis (MLVA) of S. Typhimurium to detect possible outbreaks. Because only a few subtypes identified by PFGE and phage typing account for most infections, we included MLVA typing in the routine surveillance in a 2-year period beginning December 2003. The 1,019 typed isolates were separated into 148 PFGE types and 373 MLVA types. Several possible outbreaks were detected and confirmed. MLVA was particularly valuable for discriminating within the most common phage types. MLVA was superior to PFGE for both surveillance and outbreak investigations of S. Typhimurium.
126 citations
TL;DR: A novel assay based on multiplex linear DNA amplification and subsequent microarray hybridisation confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages.
TL;DR: The pattern of susceptibility of MRSA isolates both to gentamicin and to trimethoprim-sulfamethoxazole was shown to be an excellent phenotypic marker for the discrimination of the EMRSA-15 clone from other nonmultiresistant MRSA clones present in the hospital.
Abstract: Two hundred eighty methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered from a tertiary care hospital in Oporto, Portugal, between 2003 and 2005 were studied by a combination of molecular typing techniques in order to investigate the genetic backgrounds associated with the changes in the resistance phenotypes observed since 2001 and compare them to those previously found in the hospital. All MRSA isolates were grouped into resistance profiles for a panel of seven antibiotics and characterized by pulsed-field gel electrophoresis (PFGE) and SCCmec (staphylococcal cassette chromosome mec) typing. Representative isolates of PFGE types were further studied by spa typing and multilocus sequence typing. Our findings clearly document that the increasing isolation of nonmultiresistant MRSA strains was associated with the decline (from 69% in 1996 to 2000 to 12% in 2003 to 2005) and massive replacement of the multiresistant Brazilian clone (ST239-IIIA) by the epidemic EMRSA-15 clone (ST22-IV), in which resistance to antibiotics other than β-lactams is very rare, as the major clone (80% of isolates). The Iberian clone (ST247-IA), a major clone in 1992 to 1993, was represented in the present study by just one isolate. Two other pandemic MRSA clones were detected, as sporadic isolates, for the first time in our hospital: the New York/Japan (ST5-II) and the EMRSA-16 (ST36-II) clones. Furthermore, the pattern of susceptibility of MRSA isolates both to gentamicin and to trimethoprim-sulfamethoxazole was shown to be an excellent phenotypic marker for the discrimination of the EMRSA-15 clone from other nonmultiresistant MRSA clones present in our hospital.
TL;DR: P pulsed-field gel electrophoresis was more discriminatory than MLST for determining genetic differences in P. aeruginosa, and while MLST was better for detecting genetic relatedness, PFGE was more discriminated against.
Abstract: For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa.
TL;DR: In this paper, the authors attribute Panton-Valentine leucocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) to clonal lineages by molecular typing with special reference to isolates exhibiting spa type t008/multilocus sequence type (MLST) ST8 [widely disseminated in the USA as ‘community-associated MRSA (caMRSA), USA300’].
Abstract: Objectives: The aim of this paper is to attribute Panton-Valentine leucocidin (PVL)-positive methicillinresistant Staphylococcus aureus (MRSA) to clonal lineages by molecular typing with special reference to isolates exhibiting spa type t008/multilocus sequence type (MLST) ST8 [widely disseminated in the USA as ‘community-associated MRSA (caMRSA) USA300’]. Methods :P VL-positive MRSA (n 5 117) were detected among 4815 MRSA sent to the German National Reference Laboratory for typing. These isolates were analysed by PFGE, spa typing, multilocus sequence typing, grouping of SCCmec elements and PCR detection of arcA, msr(A), mph(B) and the � 6 AT repeat unit in the SACOL0058 sequence. Results: Among the 117 isolates, 80 exhibited spa type t044 (corresponding to MLST ST80) and 23 exhibited spa type t008/MLST ST8. Other spa types were sporadically represented. Further characterization of isolates exhibiting t008/ST8 by PCR [arcA, msr(A), mph(B), � 6 AT repeat signature] indicates the arrival of caMRSA ‘USA300’ in Central Europe. Conclusions: caMRSA ST80 still predominate; however, caMRSA ST8 exhibiting the characteristics of the ‘USA300’ clone became the second most frequent. Routine detection of this clone in clinical bacteriology can be easily performed by PCR.
TL;DR: The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit-VNTR typing method dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained.
Abstract: The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem repeat (VNTR) typing method was evaluated for its ability to differentiate 181 Mycobacterium tuberculosis Beijing family strains. Compared with the original 12-loci MIRU-VNTR typing method, the 15-loci system dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained. The clonal stability and allelic diversity of a total of 31 VNTR loci were evaluated. VNTRs 3232, 3820, and 4120 were identified as the effective hypervariable VNTR set for the second-line typing of clustered strains following the 15-loci based scheme. Consequently, the discriminatory power of the new scheme (18 loci) equaled that of IS6110 RFLP.
TL;DR: This work compared multilocus sequence typing, pulsed-field gel electrophoresis, and amplified fragment length polymorphism for typing of Staphylococcus aureus and shows that the methods yield similar results, although with differences in resolving power and reproducibility.
TL;DR: The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulent traits transferable to human-adapted clusters.
Abstract: VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.
TL;DR: The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method, and permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica.
Abstract: Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present.
TL;DR: The RM test correctly identified 102 MRSA isolates as belonging to one of the six lineages/CCs and could lead to greater understanding of the carriage, spread and virulence of different MRSA lineages.
Abstract: Multilocus sequence typing (MLST) and multi-strain microarray analysis have shown that most human Staphylococcus aureus strains belong to ten dominant clonal complexes (CCs) or lineages, each with unique surface architecture. Meticillin-resistant S. aureus (MRSA) strains currently belong to six of these lineages (CC1, CC5, CC8, CC22, CC30 and CC45), each of which has independently acquired mobile genetic elements (MGEs) carrying antibiotic resistance genes. MLST and microarrays are expensive and time consuming methods for routine determination of S. aureus lineage. A restriction-modification (RM) test has now been developed that is rapid, simple, inexpensive and accurately determines lineage of hospital-acquired MRSA. The RM test is based on three PCRs for hsdS gene variants, as hsdS genes likely control the independent evolution of S. aureus lineages. The RM test correctly identified 102 MRSA isolates as belonging to one of the six lineages/CCs. Real-time MRSA typing can be used to identify and track changes in local MRSA outbreaks, and provide support for targeting infection control strategies. Simple and accurate typing methods will also support large scale epidemiological studies, and could lead to greater understanding of the carriage, spread and virulence of different MRSA lineages.
TL;DR: An SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing and showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates.
Abstract: Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.
TL;DR: Findings are consistent with multiple recent acquisitions of the more mobile type IV SCCmec by MSSA and suggest that certain genetic backgrounds are conferring a selective advantage, regardless of the resistance profile.
Abstract: Objectives: Surveillance of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) infections has shown the emergence and spread of several epidemic MRSA clones over the past 10 years in Belgium. Whether these clones have been imported from abroad or else have arisen locally via staphylococcal cassette chromosome mec (SCCmec) acquisition by successful methicillin-susceptible S. aureus (MSSA) clones is unknown. Methods: We determined by PFGE, spa typing, multi-locus sequence typing (MLST) and agr group analysis the genetic relatedness of 103 MSSA and 511 MRSA strains from a national survey of patients admitted to 112 Belgian hospitals in 2003. Results: The 103 MSSA strains presented very diverse genetic backgrounds, they were distributed into 40 distinct PFGE types and clustered in 15 distinct MLST CCs. Up to 45% harboured the same genotype as five major epidemic HA-MRSA clones. These MRSA clones all harbour a type IV SCCmec element. Conclusions: These findings are consistent with multiple recent acquisitions of the more mobile type IV SCCmec by MSSA and suggest that certain genetic backgrounds are conferring a selective advantage, regardless of the resistance profile. However, since the predominant MSSA and MRSA lineages identified in Belgium are disseminated worldwide, importation of epidemic MRSA strains remains an alternative hypothesis.
TL;DR: This data indicates that community-onset MRSA with diverse genetic backgrounds is rapidly emerging in this previously low MRSA prevalence area.
Abstract: In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for <15 isolates per year during 1980–2002. However, since 2003 an epidemic increase has been observed, with 33 MRSA cases in 2003 and 110 in 2004. We analyzed these 143 cases epidemiologically and characterized isolates by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had no identified risk factors. We identified 29 spa types (t) and 16 sequence types (STs) belonging to 8 clonal complexes and 3 ST singletons. The most common clonal types were t024/ST8-IV, t019/ST30-IV, t044/ST80-IV, and t008/ST8-IV (USA300). A total of 86% of isolates harbored SCCmec IV, and 44% had PVL. Skin and soft tissue infections dominated. CO-MRSA with diverse genetic backgrounds is rapidly emerging in a low MRSA prevalence area.
TL;DR: In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.
Abstract: Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.
TL;DR: Data for pairs of isolates from seven patients showed that each patient was colonized and/or infected with strain types that were indistinguishable by MLST, making this system ideal for strain typing of C. krusei clinical isolates.
Abstract: A multilocus sequence typing (MLST) scheme for Candida krusei was devised, based on sequencing of six gene fragments of the species. The existence of heterozygous results for each of the six fragments sequenced confirms that C. krusei is diploid for at least part of its genome. The C. krusei MLST scheme had a discriminatory index of 0.998, making this system ideal for strain typing of C. krusei clinical isolates. MLST data for 122 independent C. krusei isolates from a range of geographical sources were analyzed by eBURST, structure, and the unweighted-pair group method using average linkages to derive a population structure comprising four subtype strain clusters. There was no evidence of geographical associations with particular subtypes. Data for pairs of isolates from seven patients showed that each patient was colonized and/or infected with strain types that were indistinguishable by MLST. The C. krusei MLST database can be accessed online at http://pubmlst.org/ckrusei/.
TL;DR: Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis.
Abstract: Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis. Both techniques showed that interpatient isolates belonged to different genotypes and that intrapatient isolates from deep sites were all of the same genotype. By contrast, multiple genotypes were found among isolates originating from respiratory samples. Both techniques have specific advantages and disadvantages. AFLP is more universally applicable, but short tandem repeat analysis offers better discriminatory power and should be the preferred method for standardizing typing of clinical isolates of Aspergillus fumigatus.
TL;DR: High prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity and RT‐PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples.
Abstract: From 2000 to 2005, a total of 1,096 enterovirus infections were diagnosed either by isolation of virus from cell culture or by RT-PCR (5′non-coding region (NCR)). Typing of viruses (n = 674) was carried out by immunofluorescence with monoclonal antibodies, neutralization test or molecular methods. Seasons with high enterovirus activity were characterized by high prevalence of echovirus 30 (62.2% in 2000, 25.5% in 2001) and echovirus 13 (34.5% in 2001). In contrast, in the 2003 season, which had very low enterovirus activity, these types were rare. During this season, cell culture sensitivity (human colonic carcinoma cells and human embryonic lung fibroblasts (HEL)) was exceptionally low. In order to determine the type of “non-cultivable” enteroviruses, purified RNA from selected stool samples was subjected to direct molecular typing. VP1/2A-specific fragments were amplified by RT-PCR, cloned and sequenced. The predominant virus identified was coxsackie A. Consequently, rhabdomyosarcom cells were introduced into the daily routine, which improved the isolation of enteroviruses. Echovirus 30 was again most commonly isolated during seasons 2004 and 2005 with increasing enterovirus activity. In conclusion, high prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity. The type of circulating enteroviruses may influence isolation of enterovirus from cell culture. RT-PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples. In cases of severe enterovirus infection, virological diagnosis should not solely rely on virus isolation from cell culture. J. Med. Virol. 79:956-962, 2007. © 2007 Wiley-Liss, Inc.
TL;DR: Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates and MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic bru cellosis.
Abstract: Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates. A perfect concordance was obtained among these molecular assays. However, MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.
TL;DR: In this article, the performance of the four highly polymorphic S. aureus markers (clfA, clfB, fnbA, and spa) was compared to those of PFGE.
Abstract: Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of these methods showed convincing evidence for performance in epidemiological typing comparable to that of PFGE. By using three sets of 48 MRSA strains comprising isolates that were (i) genetically highly diverse, (ii) genetically related, and (iii) obtained from long-term carriers, we analyzed the performance of the four highly polymorphic S. aureus markers: clfA, clfB, fnbA, and spa. Typeability, discriminatory power, in vivo stability, and evolution of these markers were compared to those of PFGE. Clearly, none of the markers alone could match the discriminatory power of PFGE (63 genotypes; index of discrimination of 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3' partial sequences of approximately 500 bp, the majority of each marker's discriminatory power was displayed, and using the partial sequences, the best performance was obtained with the combination of clfB and spa (57 genotypes; index of discrimination of 0.94). Genetic changes were not observed for any of the sequence markers over a period of 3 years and in the case of partial sequences for a period of more than 4 years. This is in contrast to PFGE where changes occurred after several months. The genetic differences found between isolate pairs of long-term carriers and among highly related isolates indicated clonal evolution. A typing scheme based on 500-bp 3' partial sequences of clfB and spa is proposed.
TL;DR: Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP).
Abstract: Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify “clonal” and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study. Conidia of Aspergillus fumigatus become airborne easily, and subsequent inhalation of these infectious particles is believed to be the route of transmission for invasive aspergillosis. Accordingly, several nosocomial outbreaks of invasive aspergillosis have been reported with strong evidence suggesting that such infections are acquired from the environment of the hospital (10, 31, 35, 43). The Centers for Disease Control and Prevention (CDC) recommends that in the setting of a nosocomial aspergillosis outbreak and in the presence of continuing evidence of Aspergillus infection in the hospital population, an environmental assessment should be undertaken to determine and eliminate the source of infection (51). In such nosocomial outbreak investigations, Aspergillus strain typing methods can
TL;DR: Analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain of M. hyopneumoniae, indicating that specific strains or clones are responsible for local outbreaks.
Abstract: Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia in swine. Various reports indicate that different strains are circulating in the swine population. We investigated the variety of M. hyopneumoniae strains by a newly developed genetic typing method based on the polyserine repeat motif of the LppS homolog P146. PCR amplification using M. hyopneumoniae specific, conserved primers flanking the region encoding the repeat motif, followed by sequencing and cluster analysis was carried out. The study included strains isolated from different geographic regions as well as lysates from lung swabs from a series of pig farms in Switzerland. High diversity of M. hyopneumoniae was observed but farms being in close geographic or operative contact generally seemed to be affected by the same strains. Moreover, analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain. The results indicate that multiple strains of M. hyopneumoniae are found in the swine population but that specific strains or clones are responsible for local outbreaks. The method presented is a highly reproducible epidemiologic tool allowing direct typing of M. hyopneumoniae from clinical material without prior isolation and cultivation of strains.