Identification And Molecular Relatedness Of Methicillin-resistant Staphylococcus Aureus Associated With Bovine Mastitis

Bovine mastitis caused by bacterial pathogens is a serious problem for dairyindustries with considerable economic consequences. S. aureus is a common andimportant cause of bovine mastitis. Despite this fact, methicillin-resistantStaphylococcus aureus (MRSA) isolates have been infrequently reported with mastitis.There have been a few reports of MRSA colonization and/or infections in dairy cattlesince the very first report of MRSA in mastitis in1972. In China, there have been afew reports of MRSA of bovine origin. The aim of this study was to investigate theantimicrobial resistance of Staphylococcus aureus (S.aureus) isolated from bovinemastitis in Gansu and Shanghai, to determine if methicillin-resistant Staphylococcusaureus (MRSA) is present on dairy farms and to find out the molecular relatedness ofMRSA via various genetic typing methods, to provide credible theory evidence forsurveillance of MRSAin mastitis, prevention and treatment on bovine mastitis.1K-B disk diffusion method was used to test drug sensitivity of69totalS.aureus strains to8commonly used antibiotics, meanwhile, agar screen wasperformed to identify the minimum inhibitory concentration of oxacillin andvancomycin to all strains. The sensitive of all isolates to gentamicin, ciprofloxacin,tetracycline, cefazolin, vancomycin and oxacillin was more than60%. No isolateidentified was resistant to vancomycin. The resistance to penicillin and sulfafurazolewas95.7%,89.9%, respectively.2Cefoxitin disk diffusion and PCR assay was used to detect the presence ofMRSA in bovine mastitis. The genotypic detection result was inconsistent with thephenotypic detection result. The PCR assay generated twenty eight (40.58%)mecA-positive S. aureus isolates, seventeen (24.63%) of which were defined asOS-MRSAand the other eleven isolates belonged to MRSA. Eight OS-MRSAisolateswere collected from Gansu. Eleven MRSA isolates and nine OS-MRSA isolates werecollected from Shanghai. However, fifteen phenotypic MRSA isolates collected fromShanghai were identified by cefoxitin disk diffusion, and five of15phenotypic MRSAisolates had oxacillin MIC lower than1μg/mL.3PCR assay was performed to determine the presence of PVL toxicin gene inmecA-positive isolates. The X region of spa gene of mecA-positive isolates wereamplified by PCR assay, and submitted to the typing database(http://www.ridom.de/spaserver/) after sequencing. Meanwhile, Staphylococcalchromosomal cassette mec (SCCmec) type of mecA-positive isolates were identifiedby multiplex PCR assay. Eight OS-MRSA isolates of Gansu origin, five OS-MRSAand five MRSA isolates of Shanghai origin were determined to be spa type267,SCCmec type V, and PVL negative. Four MRSA and four OS-MRSA of Shanghaiorigin belonged to spa type1234, SCCmec type Ⅱ, and were PVL negative, The resttwo MRSA isolates with untypable spa were SCCmec typeⅤand typeⅡ, respectively,and PVL negative.This study suggested that antimicrobial resistance was serious in tested S. aureusisolates, a high incidence of MRSA and OS-MRSA associated with dairy farms inShanghai and Gansu. There were two main lineage of MRSA, one group of whichwere negative with PVL and belonged to spa t267and SCCmec typeⅤ, and anothergroup of which were also negative with PVL and belonged to spa t1234and SCCmectypeⅡ. These two lineages of MRSA were firstly found in bovine mastitis, and maybe commonly associated with bovine mastitis in China, which awaits for furtherinvestigation. These OS-MRSA isolates were susceptible to oxacillin and could bemistakenly identified as MSSA if testing of the mecA gene were not conducted.Findings from this study and the increasing reports of OS-MRSA in clinical settingsunderscore the need for genetic tests as well as phenotypic assays to accuratelyidentify MRSA.