Staphylococcus aureus caused bovine mastitis
Background: Bovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany.  Methods: Forty Staphylococcus aureus isolates collected from clinical bovine mastitis cases of 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing. Results: Out of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against benzylpenicillin, tetracycline, and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole i.e. 35, 35, 30 and 22.5%, respectively. Whereas resistance against clindamycin and erythromycin were rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid, and rifampicin. The isolates were assigned to five different sequence types: ST398 (n=31), ST1074 (n=4), ST504 (n=3), ST582 (CC15) (n=1) and ST479 (n=1).  These isolates were assigned to seven clusters with up to 100 SNP which facilitated geographic mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST 504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI, and blaR associated with benzylpenicillin resistance were found in 31 (80%) strains, all were belonging to ST398. Methicillin resistance associated mecA, was identified in 30 (96.8%) isolates of ST398.  All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3%  of tetracycline resistant isolates assigned to ST398 were additionally carrying tetK gene.  The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all isolates (n=14) that were phenotypically resistant to ciprofloxacin and moxifloxacin. Sixty-eight virulence genes belonging to different categories were identified among isolates. Both lukD/E and lukM/F-PV-P83 were  identified among in  22.5% of isolates (all except ST398).  Conclusion: In this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Different five sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive medical care (e.g. Nesse-Apfelstädt). On the other hand, some regions had different clusters at the same year or during different years (e.g. Unstrut-Hainich-Kreis). The results of this study showed that different sequence types and accompained different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scope. The detection of LA-MRSA in dairy farms, which is known for cabapility to highly spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses the risk for transmission of such strains from animals to humans on the dairy farms.