The Study Of Mastitis-inducing Staphylococcal Isolates For Molecular Typing And Antibiotic Resistance Profiling

Healthy development of the dairy industry encompasses improvement of the animal health, improvement of milk quality and enhancement of the market competitiveness. The disease prevention and control play a key role in animal health. Bovine mastitis is a major cause of economic losses to the dairy industry. The occurrence of bovine mastitis also has serious consequences for public health. Staphylococcus aureus(S. aureus) is considered to be one of the most important contagious pathogens due to the fact that they are very difficult to be eradicated from mammary glands and the infection rate is very high. The antibiotic therapy has becoming less effective because most bacteria including S. aureus have developed the antibiotic-resistance. In particular, emergence of multi-drug resistant isolates in bovine milk is a major public health concern. It is important to study mastitis-inducing pathogens. The primary purpose of this research was to determine molecular genetic characteristics and antibiotic resistance profiling of S. aureus and coagulase negative staphylococci(Co NS) isolates recovered from milk of mastitic cows in the Shaanxi Province in Northwestern China. One hundred twenty one S. aureus isolates were also genotyped using the spa typing and the multilocus sequence typing(MLST). Furthermore, the Macrolides-LincosamidesStreptogramins(MLS) antibiotics resistance phenotypes and genotypes were determined among 121 S. aureus and 97 Co NS isolates from mastitic milk. In addition, the integron/gene cassettes of 121 S. aureus isolates were analyzed using PCR and direct sequencing technique. We discovered that:1. Among 121 S. aureus isolates, we found 113 methicillin-susceptible Staphylococcus aureus(MSSA), 1 mec A-positive and phenotype-positive MRSA and 7 mec A- and mec Cnegative but phenotype-positive MRSA. Resistance was common among isolates against ampicillin or penicillin(80.2%), kanamycin(68.6%), gentamicin(67.8%) and tetracycline(43%). Antibiotic susceptibility testing showed resistance to a single antibiotic class in 19 isolates, resistance to two antibiotic classes in 23 isolates, resistance to three antibiotic classes in 27 isolates, resistance to four antibiotic classes in 31 isolates and resistance to five antibiotic classes in 8 isolates. Sixty-six isolates were multidrug resistance(resistance to 3 or more than 3 antibiotics classes). The predominant multidrug resistance profile was penicillin/ampicillin/kanamycin/gentamicin/tetracycline(46 isolates).2. 97 Co NS isolates encompassed 9 Co NS species. Among them, we found two MR-Co NS including one oxacillin-susceptible mec A-positive Staphylococcus haemolyticus(OS-MRSH) and one mec A-positive and methicillin-resistant Staphylococcus epidermidis(MRSE) isolates. Resistance was common among isolates against ampicillin or penicillin(93.8%), kanamycin(72.2%), gentamicin(64.9%) and tetracycline(63.9%). Eleven, 24, 30, 19 and 8 isolates showed resistance to 1, 2, 3, 4 and 5 antibiotic classes, respectively. Fifty-seven Co NS were multidrug resistance isolates(resistance to equal or larger than 3 antibiotics classes). The predominant multidrug resistance profile was penicillin/ampicillin/ kanamycin/gentamicin/tetracycline(44 isolates).3. 121 S. aureus isolates belonged to 6 spa types: t524(n=63), t11772(n=31), t4207(n=15), t521(n=5), t11771(n=3) and t11807(n=4). At the same time, 4 MLST types were characterized in the present study, ST71(n=67) and ST2738(n=45) were identified as dominant sequence types, the rest of 2 MLST types were ST2683(n=4) and ST97(n=5). We found that t11772, t11771 and t11807 were novel spa types, ST2683 and ST2738 were novel MLST types. The 121 S. aureus isolates had 12 different spa-MLST type combinations. The spa-MLST types t11772-ST2738(n=27), t11807-ST2683(n=4) and t11771-ST2738(n=3) were newly identified genotypes of S. aureus.4. The mec A-positive and phenotype-positive MRSA isolate had a composite genotype t524-ST71-SCCmec IVa, while 7 mec A-negative but phenotype-positive MRSA isolates were all t524-ST71. The OS-MRSH isolate contained a type V SCCmec cassette, while the MRSE isolate possessed a non-typeable SCCmec. In our research, 2 types of SCCmec cassette(IVa and V) were identified.5. 40(33.1%) S. aureus and 65(67%) Co NS were resistant to erythromycin. Among 40 erythromycin-resistant(ER-R) S. aureus and 65 ER-R Co NS isolates, 95% of S. aureus and 61.5% of Co NS exhibited the inducible MLS(i MLS) resistance phenotype and 5% of S. aureus and 30.8% of Co NS expressed the constitutive MLS resistance(c MLS) phenotype. At the same time, 7.7% of Co NS exhibited resistance to erythromycin but susceptibility to clindamycin(the MS phenotype). One or more MLS antibiotics resistance genes were detected individually or in combinations of each staphylococci isolates. Resistance genes were also existed in erythromycin-susceptible isolates.6. All 121 S. aureus isolates carried the class 1 integrase gene int I1. Among them, 103 isolates were positive for gene cassettes. However, class 2 integrons and class 3 integrons were not detected in all isolates. Sequencing analyses revealed that 12 antibiotic resistance gene cassettes were encoded resistance to antibiotics including trimethoprim(dhfr V, dfr A1, dfr A12), aminoglycosides(aad A1, aad A5, aad A4, aad A24, aac A4, aad A2, aad B), chloramphenicol(cml A6) and quaternary ammonium compound(qac H). Totally, 11 gene cassette arrays(A-K) were identified. Among them, the gene cassette arrays dfr A1-aad A1(D, 44.6%), aad A2(K, 31.4%), dfr A12-hypothetical protein-aad A2(G, 27.3%) and aad A1(A, 25.6%) were found most prevalent. Our data revealed a high rate of class I integrons in bovine milk associated pathogenic S. aureus isolates in Shaanxi province in the Northwestern China and class 1 integron/cassettes were significantly associated with resistance to aminoglycoside antibiotics. However, the class 1 integron/cassettes examined in this study did not account for the total resistance phenotypes observed(tetracyclines, pencillins and MLS antibiotics) among the pathogenic S. aureus isolates.In conclusion, our work studied the molecular genetic characteristics and antibiotic resistance patterns of staphylococcal isolates recovered from milk of mastitic cows. Our results confirm that integrons play an very important role in acquisition and dissemination of antibiotic resistance genes of bovine mastitis associated staphylococci. Our data support the notion that antibiotics should be used appropriately and judiciously to prevent and treat bovine mastitis caused by staphylococci and provide the basis for further studies.