Nationwide molecular epidemiology of methicillin-resistant Staphylococcus aureus responsible for horse infections in France.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in horse infections is not well documented, especially in France. The aim of the study was to evaluate the prevalence of MRSA isolates in horse infections from 2007 to 2013 in France and to characterize phenotypically and genotypically this collection. RESULTS: Out of 1393 S. aureus horse isolates, 85 (6.1%) were confirmed to be MRSA. Interestingly, the prevalence of MRSA significantly increased from 2007-2009 to 2010-2013 (0.7 vs. 9.5%, P <0.0001). Resistance to methicillin was due to the presence of the mecA gene in 84 strains (98.8%) while one strain (1.2%) possessed the mecC gene. The vast majority of the strains (83/85, 97.6%) was resistant to at least three different classes of antibiotics. Multi-locus sequence typing (MLST) showed that MRSA strains belonged mainly since not all belong to two sequence types (STs): ST398 (53/85, 62.4%) and ST8 (28/85, 32.9%). It is worth to note that all ST398 MRSA isolates were detected in the period 2010-2013. Other molecular typing methods were also used, such SCC mec analysis, spa typing and rep-PCR (Diversilab, bioMérieux). All these four techniques were in good agreement, with spa typing and rep-PCR being more discriminative than MLST and SCC mec typing. CONCLUSIONS: This study is the first epidemiological study in France with extensive characterization of MRSA isolates associated with horse infections in stud farms. It shows that there is a significant increase of MRSA prevalence between 2007 and 2013, which mainly results from the spread of ST398 clones. It also highlights the importance of horses as a potential reservoir of important antimicrobial resistance genes.

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium.

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 µg/ml) but susceptible to teicoplanin (MIC, 0.5 µg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

Changing trends in vancomycin-resistant enterococci in French hospitals, 2001-08.

OBJECTIVES: Unprecedented outbreaks of vancomycin-resistant enterococci (VRE) have occurred in French hospitals since 2004. The aim of this study was to provide a picture of the spread and control of VRE in France and to characterize the isolates. METHODS: Notification of VRE cases to Institut de Veille Sanitaire has been mandatory since 2001. Isolates of VRE were sent to the National Reference Centre for species and vancomycin-resistance gene identification. Isolates were tested for antimicrobial susceptibility and typed by PFGE and multilocus sequence typing. RESULTS: Five hundred and four VRE notifications from 195 hospitals were recorded, corresponding to 2475 cases of infection (n=243) or colonization (n=2232) and 74 episodes of clustered cases. Outbreaks were controlled by implementation of infection control measures, although the number of new hospitals reporting isolation of VRE was increasing. The majority of 902 VRE isolated from 2006 to 2008 were Enterococcus faecium (94.8%) with the vanA or vanB gene. No isolate was resistant to linezolid, tigecycline or fusidic acid. PFGE analysis showed 161 different patterns. Generally a few predominant clones and several minor clones spread in a single hospital. In a subset of 46 representatives of PFGE clones, 13 different sequence types were characterized, all belonging to clonal complex CC17, while the esp and hyl genes were inconsistently detected. CONCLUSIONS: The national mandatory notification of unusual nosocomial events allowed rapid identification of VRE outbreaks and early implementation of control measures that have proved effective. However, VRE continue to emerge in a growing number of hospitals.

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium.

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.

Rapid detection of vancomycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/vanB assay.

The detection of vancomycin-resistant enterococci using a novel commercial multiplex real-time polymerase chain reaction assay (Xpert vanA/vanB, Cepheid, Sunnyvale, CA) was evaluated on 804 rectal swab specimens. Compared to enriched culture, sensitivity and negative predictive value of this method were 100%. Many false-positive results were recovered (sensitivity, 85.4%; positive predictive value, 8.7%), especially for the vanB gene.