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A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.Vancomycin-resistant Enterococcus faecium (VREF) has recently emerged as an important threat in U.S. hospitals (5, 24). In Europe, VREF isolates are found relatively frequently in the community and farm animals, while prevalence in hospitals is generally low (14). The latter observation was explained by the use of the glycopeptide avoparcin as an antimicrobial growth promoter in animal feeding operations.Several molecular typing schemes have been developed to study the epidemiology of VREF. Of these, pulsed-field gel electrophoresis analysis of genomic restriction fragments has been considered the "gold standard" for the study of hospital outbreaks because of its high degree of isolate differentiation (15,17,20,23). However, due to this high degree of isolate differentiation, pulsed-field gel electrophoresis typing is less suitable for determining the degree of relatedness among epidemiologically unrelated isolates. Recently, amplified fragment length polymorphism (AFLP) analysis was applied as a new method for the typing of VREF (1, 33). AFLP analysis is a robust and fast typing technique with high intra-and interexperimental reproducibilities and appears to be discriminatory enough for the recognition of hospital outbreaks (1, 32, 33). In addition, AFLP analysis has allowed the detection of associations among different E. faecium genetic lineages and different human and animal hosts (33), suggesting the existence of host-specific VREF lineages. Whether this is also true for vancomycin-sensitive E. faecium (VSEF) is not known, since VSEF isolates were not included in that study. AFLP typing also disclosed two different human-associated lineages. One lineage comprised epidemic-related isolates recovered from hospitalized patients, while isolates of the other lineage were mainly from nonhospitalized persons. Interes...

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BackgroundMolecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.Methodology/Principal FindingsA new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness.ConclusionsThe MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.

A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.Vancomycin-resistant Enterococcus faecium (VREF) has recently emerged as an important threat in U.S. hospitals (5, 24). In Europe, VREF isolates are found relatively frequently in the community and farm animals, while prevalence in hospitals is generally low (14). The latter observation was explained by the use of the glycopeptide avoparcin as an antimicrobial growth promoter in animal feeding operations.Several molecular typing schemes have been developed to study the epidemiology of VREF. Of these, pulsed-field gel electrophoresis analysis of genomic restriction fragments has been considered the "gold standard" for the study of hospital outbreaks because of its high degree of isolate differentiation (15,17,20,23). However, due to this high degree of isolate differentiation, pulsed-field gel electrophoresis typing is less suitable for determining the degree of relatedness among epidemiologically unrelated isolates. Recently, amplified fragment length polymorphism (AFLP) analysis was applied as a new method for the typing of VREF (1, 33). AFLP analysis is a robust and fast typing technique with high intra-and interexperimental reproducibilities and appears to be discriminatory enough for the recognition of hospital outbreaks (1, 32, 33). In addition, AFLP analysis has allowed the detection of associations among different E. faecium genetic lineages and different human and animal hosts (33), suggesting the existence of host-specific VREF lineages. Whether this is also true for vancomycin-sensitive E. faecium (VSEF) is not known, since VSEF isolates were not included in that study. AFLP typing also disclosed two different human-associated lineages. One lineage comprised epidemic-related isolates recovered from hospitalized patients, while isolates of the other lineage were mainly from nonhospitalized persons. Interes...

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra-and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Background: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing.

The epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.