Analysis Of The Characters Of Resistance Pattern Of Staphylococci Isolated From Bovine Mastitis In Jiangsu Province

Mastitis is the most common and costly disease of dairy cattle, and threaten public hygiene. Mastitis usually occurs in response to intramammary bacterial infection. In this study, we aimed to investigate the resistance patterns of Coagulase-Negative Staphylococci (CNS) and Staphylococcus aureus (SA) in terms of antibiotic resistance and antibiotic resistance genes with statistical analysis.1. Investigating the main pathogens causing dairy bovine mastitis in Jiangsu areaSix-hundred and thirty quarter milk samples that were positive by the California Mastitis Test (CMT) were collected from dairy cows at five dairy farms in Jiangsu Province, China, from March 2012 to March 2014. The quarter milk samples were used to investigate the microbial panorama. The most common isolates of 825 bacteriological diagnoses were Staphylococci (47.63%) followed by Streptococcus (28.73%), Bacillus (23.03%) and Prototheca zopfi (0.6%) which are only susceptible to gentamycin in this study.2. Analysis of drug resistance pattern of Staphylococci isolated from bovine mastitis in Jiangsu Province, ChinaAll staphylococci isolates were tested for susceptibility to 15 drugs using a circular disk diffusion method, as recommended by the Committee for Clinical and Laboratory Standards Institute (CLSI,2010a) and to detect antimicrobial resistance genes (tetK, tetM, tetL, tetO, ermC, blaZ) by polymerase chain reaction methods. In drug susceptibility tests, the majority of staphylococci were sensitive to chloramphenicol, nitrofurantoin and clarithromycin, but showed resistance to penicillin, ceftazidime and norfloxacin. And chloramphenicol was the most effective drug against CNS (86.67%), but the highest proportion of SA was susceptible to clarithromycin (92.26%). Additionally, CNS were mainly double drug-resistant, however, SA were mainly resistant to four classes of antibiotics. Revealing the characters of phenotypic pattern of resistance of staphylococci isolated from Jiangsu area through analyzing the chi-square tests. The results show that SA was significantly better to resist antibiotics interfering in the metabolism of nucleotides than CNS. However, CNS isolates were significantly better to resist antibiotics interfering in bacterial protein synthesis, except chloramphenicol for which there was no significant difference. This is the first study to analyze the drug resistance by using the chi-square test from drug’s mechanism. From the analysis on genetic pattern of resistance mechanisms, resistance genes were detected more frequently in CNS than in SA, so CNS may be more relying on resistance genes. However, the effect of resistance genes in SA reached 100%. It can be found out that 1/3 of CNS can be resistant to β-lactam antibiotics by β-lactamase coded by blaZ and CNS resist tetracycline mainly depends on tetK mediated protein pump mechanism. However,27.2% of SA are resistant tom-lactam antibiotics through β-lactamase coded by blaZ and other 5 resistance genes were rare to be found.3. Detecting the reasons why some of the resistance genes lost their functions in StaphylococciThis study found that some of the resistance genes within staphylococci are not available to resist certain antibiotics. These drug resistant genes were sequenced to explore the reasons. These different genes with different effectiveness were ligated with T4-vector respectively and sequenced. The results showed that the seven blaZ genes detected in staphylococci isolates which were susceptible to penicillin were much different to the blaZ genes detected in those staphylococci isolates which were resistant to penicillin (homology between them less than 50%). And this kind of gene weren’t reported before. Therefore, we argue that the seven sequences should be other kings of genes performing unknown function and had not been reported yet. And this gene are only found in these seven strains, thus this gene should be unnecessary in staphylococci. However, due to these genes(tetK and tetL) are respectively high homology with the real tetK and tetL with effectiveness, these tetK and tetL and the real tetK and tetL are respectively joining together with PGEM-6P-1 in BL21 respectively and then test their susceptible to certain antibiotics-tetracycline. We found that tetK genes which can resist tetracycline in staphylococci (MP0072 and MP00159) can also resist tetracycline in BL21. Equally, the tetK genes which can’t resist tetracycline in staphylococci (MP00101 and MP00109) were also susceptible to tetracycline in BL21. And the BL21 with tetK gene cloned from MP00101, there are many bacteria growth in the inhibition circle in disk diffusion method. It can be accord with the sequence result that the gene is only one mutation in one site but the gene cloned from MP00109 are five sites. The above results about tetK amplified from MP00101 illustrate the site may exist the potency to be back mutation or make up the site mutation by changing other sequence under the pressure of antibiotic. Meanwhile, we found that tetL genes which can resist tetracycline in staphylococci (MP00213 and MP00350) are intermediary resistant to tetracycline in BL21, equally, the tetL gene which can’t resist tetracycline in staphylococci (MP00101 and MP00109) are also susceptible to tetracycline in BL21. The above results show that tetL gene may affect the resistance in BL21.Overall, the main pathogens which caused bovine mastitis in Jiangsu province were Staphylococci (47.63%). Meanwhile, five strains of Prototheca zopfi were also firstly found in bovine mastitis in Jiangsu area and most antibiotics are not available to them except gentamycin. Additionally, the characters of phenotypic pattern of resistance of staphylococci isolated from Jiangsu area are as following:SA was significantly better to resist antibiotics interfering in the metabolism of nucleotides than CNS but CNS was significantly better to resist antibiotics interfering in bacterial protein synthesis, except chloramphenicol which there was no significant difference. Therefore, in therapy for bovine mastitis caused by staphylococci in farms, treatment with florfenicol, or with antibiotics interfering in the metabolism of nucleotides in concert with drugs interfering with bacterial protein synthesis, may be suitable. As for the reasons that some of resistance genes were disabled were mainly contributing to the mutation in few sites of them, and the mutation were also the source for stronger resistance ability within bacteria.