Identification of bovine methicillin resistant staphylococci from Europe, Africa and North America by colony hybridization, PCR and antibiotic sensitivity.

Mastitis is the costliest pathology in dairy cattle and staphylococci are the most prevalent bacterial mastitis pathogens worldwide. Antimicrobial treatment of mastitis has led to the selection of resistant staphylococci, of which the Methicillin Resistant S. aureus (MRSA) are the most studied ones. Still, MR has also been described for non-aureus staphylococci (MRS) species. Bovine MRS(A) represent not only a problem in the treatment of mastitis, but also a potential hazard in public health via the inter-Staphylococcus transferability of the mobile “Staphylococcal Cassette Chromosome” (SCC) carrying the mec genes encoding MR and the zoonotic potential of some Staphylococcus species. The aim of this study is the comparison of genetic and phenotypic methods for the identification of MRS(A) isolated from bovine mastitis in European, African and North-American countries. A total of 1168 mastitis-associated staphylococci were isolated between 2005 and 2014 in Belgium, Italy, Switzerland, Senegal, Niger and Canada, and kept at -80°C until further use. Out of them, 867 isolates were identified to S. aureus while 301 isolates were non aureus staphylococci. All 1168 staphylococci were tested genetically by the dot blot hybridization assay on positively charged nylon membranes (Roche) after DNA extraction with 32P-radioactively labelled probes derived from the mecA and mecC genes and phenotypically by growth on “Chrom MRSA ID®” agar plates. Isolates positive at both or either tests were further studied by PCR targeting the same two genes and by the disk diffusion assay to oxacillin and cefoxitin. A total of 265 isolates (23%) were positive at both or either tests. Out of them, 27 S. aureus (10%) but no non-aureus (0%) tested positive both for DNA hybridization with the mecA probe and for growth on “Chrom MRSA ID®” plates. No isolate tested positive with the mecC probe. In addition, 32 S. aureus (12%) and 15 non aureus (6%) were positive with the mecA probe only and 169 S. aureus (64%) and 22 non aureus (8%) grew on “Chrom MRSA ID®” plates only. The S. aureus originate from Belgium (105), Italy (6), Canada (31), Senegal (38) and Niger (48) whereas the non-aureus originate from Belgium (25), Italy (1) and Niger (11). All of them are being tested with the PCR targeting the mecA gene and by the disk diffusion assay to oxacillin and cefoxitin. Most isolates (72%) grew on “Chrom MRSA ID®” plates only while few (18%) were positive to the hybridization with the mecA probe only. This high difference between the results of both tests could be explained by the weak specificity of phenotypic tests comparing to genetic tests. The others 10% of the isolates (S. aureus) which are positive with the two methods (dot blot hybridization and “Chrom MRSA ID®”) can be considered as MRSA mediated by the mecA gene. However, results of PCR and disk diffusion assay will confirm respectively the presence of mec genes and which of the two methods is the most suitable for identifying MRS from mastitis cases in cattle. Comparison of the results of phenotypic and genetic assays will indicate whether other variant(s) than mecA and mecC may be present in MRS. Further genetic and phenotypic studies are needed to (i) identify the non-aureus isolates to the species level; (ii) compare the MRS(A) isolated in the different countries by their biotypes, serotypes, lysotypes, and virulotypes, without forgetting their SCCmec and their clonal complex; and (iii) identify the mec gene variant present in hybridization-positive PCR-negative isolates, if any