| Bovine mastitis is one of the most prevalent dairy cow diseases worldwide caused by multiple pathogens, Staphylococci, Streptococci and E. coli in particular. Among them, Staphylococci aureus is the contagious pathogen with public health significance, coagulase-negative Staphylococci (CNS) include about 20 different species, some of which have become the dominant mastitis pathogens in many well-managed dairy herds. More than 30 staphylococcal virulence genes have been identified, but their specific roles in bovine mastitis are largely unclear. Currently, the control of bovine mastitis relies mainly on management practice and anti-microbial reagents, but the antibiotic resistance has become a major concern due to long-term use of antibiotics. The systematic investigation into mastitis staphylococci in our country is limited. In this study, we investigated systematically into the species, virulence genes and antibiotic resistance of Staphylococci from mastitis milk, aiming at providing a scientific rationale for efficient control of bovine mastitis.1. Isolation and speciation of Staphylococci from mastitis milkDuring the period from June,2012 to June,2014,209 milk samples were collected from the cow quarters with mastitis according to clinical examination and somatic cell counting of milk samples. The milk samples were inoculated onto sheep blood agar plates and 104 staphylococcus-positive samples were selected according to colony morphology and Gram’s staining of susceptive bacteria. Among 104 staphylococal isolates,100% were positive for catalase (Cat),27.9% for thermonuclease (Nuc),26.0% for coagulase (Coa),23.1% for Nuc gene and 26.9% for Coa gene. According to these data,28 isolates (26.9%) were identified as S. aureus and the remaining 76 isolates (73.1%) as CNS. The identities of 28 S. aureus isolates were confirmed by partial sequencing of elongation Tu factor (Tuf) and/or 16S RNA genes. The 76 CNS isolates were assigned to 13 different species with S. arlettae, S. sciuri, S. xylosus and S. chromogenes as the dominant species, followed by S. epidermidis, S. simulans, S. equorum, S. haemolyticus, S. warneri, S. hyicus, S. saprophyticus, S. succinus and S. muscae. These data suggest that staphylococci were the major pathogens of bovine mastitis at this dairy farm, but the dominant CNS species were different from previously reported. The partial Tuf gene sequencing was efficient for staphylococcal speciation, but 16S rRNA gene sequence analysis was needed for speciation of some CNS isolates.2. Detection of staphylococcal virulence genes in mastitis milkThirty seven virulence genes were divided into general virulence factors, exotoxins and biofilm formation genes, which were detected with singular, duplex or multiplex PCR. Among 28 S. aureus isolates, the most prevalent general virulence genes were coa and Ig (100%), followed by hla (96.4%), hlb and fib (92.9%), clfA (89.3%), clfB and nuc (85.7%), map (78.6%), cap5 (46.4%), cap8 and ebp (39.3%), and cna (10.7%). All isolates were negative for bbp. The relatively prevalent exotoxin genes were seg (14.3%), sei (10.7%) and sea (7.1%) without other toxin genes detected. The most prevalent biofilm formation gene was eno (100%), followed by spa (96.4%), fnbB (75.0%), icaD (71.4%), agr-1 (64.3%), agr-2 (17.9%). Although 19 different virulence gene combinations were detected, only one was dominant combination (32.1%). Among 76 CNS isolates, the relatively prevalent general virulence genes were Ig (18.4%) and map (11.8%). Among 12 exotoxin genes, only enterotoxin genes seb and sei were detected with low prevalence (2.6% and 5.3%). Among 11 biofilm formation genes, the most prevalent one was eno (53.9%), followed by bap (10.5%), agr-2 (3.9%), fnbA and fnbB (2.6%). The virulence gene combinations were significantly few and simpler than that of S. aureus isolates. The high prevalence of hla gene in S. aureus isolates indicates their high virulence for bovine mastitis. The low prevalence of exotoxin and bofilm formation genes suggests the involvement of other virulence genes in bovine mastitis pathogenesis.3. Detection of antibiotic resistance genes of Staphylococci and their relationships with antibiotic resistanceSeventeen staphylococcal antibiotic resistance genes were detected using singular, duplex or multiplex PCR, including, mecA and blaZ conferring resistance to β-lactam antibiotics, aacA-aphD, aac(6’)/aph(2"), aph(3’)-Ⅲa and ant(4’)-Ⅰa to aminoglycosides, erm to erythromycin, tet to tetracyclines, msr to macrolides, and linA to lincosamide. Among 28 S. aureus isolates,17.9% had no antibiotic resistance gene and the remaining harbored one or more antibiotic resistance genes. Among them,82.1% were positive for blaZ,35.7% for mecA,32.1% for aacA-aphD,28.6% for aac(6’)/aph(2"),10.7% for tetK, and 7.1% for ermC and linA. In most isolates, these resistance genes were present as 2 or more gene combinations. Among 76 CNS isolates,27.6% had no antibiotic resistance gene and the remaining harbored one or more antibiotic resistance genes. Among them,38.2% were positive for linA,34.2% for tetK,30.3% for blaZ,21.1% for aacA-aphD,19.7% for msrB,17.1% for mecA and msrA,13.2% for ermC, 10.5% for aac(6’)/aph(2"),9.2% for ermB,2.6% for tetM. S. sciuri was the species with highest percentage of mecA gene, followed by S. epidermidis and S. haemolyticus. In most CNS isolates, these resistance genes were also present as multi-gene combinations and S. epidermidis was the species with most complex gene combinations. Among 28 S. aureus isolates,100% were resistant to two or more antibiotics,82.1% were resistant to penicillin conferred mainly by blaZ, 46.4% to streptomycin conferred by aacA-aphD, aac(6’)/aph(2") or unknown gene,35.7% to kanamycin or tobramycin and 32.1% to gentamicin conferred by aacA-aphD or aac(6’)/aph(2"), 32.1% to cefoxitin conferred by mecA and/or blaZ,14.3% to erythromycin conferred by ermC or unknown gene, and 10.7% to tetracycline conferred by tetK or unknown gene. Among 76 CNS isolates,79.4% were resistant to two or more antibiotics,86.8% were resistant to penicillin conferred by blaZ or unknown gene,48.7% to erythromycin conferred by erm, msr or unknown gene,11.8-46.1% to streptomycin, kanamycin, gentamicin or tobramycin conferred by aacA-aphD, aac(6’)/aph(2") or unknown gene,39.5% to tetracycline conferred by tet or unknown gene,30.3% to clindamycin conferred by erm, linA or unknown gene,27.6% to cefoxitin conferred by mecA, blaZ and/or unknown gene. These data indicate that the prevalence of mecA and blaZ genes in the staphylococcal isolates was significantly higher than previously reported. The possible reasons could be due to the long-term use of penicillin for mastitis control and intra-farm circulation of resistant staphylococcal isolates. |
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