Distribution Of Spuerantigen Genes And Antibiotic Resistance And Molecular Typing Of Pathogenic Staphylococcal Strains Isolated From Dairy Cows With Clinical Endometritis In Inner Mongolia

Clinical endometritis is one of the common obstetrics diseases in post partum dairy cows resulting in the degradation of reproductive performance and even infertility. Inner Mongolia autonomous region is one of the major dairy farming regions of China and the amount of dairy cows in stock stands in the forefront amongst all of the provinces in China. However, clinical endometritis produces huge economic losses in dairy cow husbandry. Staphylococci are rendered as one of the potential pathogens causing bovine clinical endometritis. The superantigenic toxins produced by Staphylococci are always associated with the development of inflammatory disease. The antibiotic therapy is known as an effective treatment protocol for infectious diseases caused by Staphylococci in veterinary clinic, unfortunately, due to the widespread existence of unreasonable employment of antimicrobial agents in dairy farms, the pathogenic Staphylococci isolated from bovine express serious resistance to antibiotics commonly used in veterinary clinic. Accordingly, it is of significance to investigate and monitor the antibiotic resistance of pathogenic Staphylococci isolated from dairy cows for the better understanding and interpretation of the development mechanisms and current situation of epidemic spread of their resistance. Furthermore, the choice and employment of antibiotics in the treatment of bacterial infectious diseases in dairy cows may varied in different regions and dairy farms which results in diverse characteristic of antibiotic resistance of Staphylococci isolated from dairy cows of different geographic regions. Therefore, scientific basis of the choice of antibiotics for the treatment of infectious diseases in dairy cows caused by pathogenic Staphylococci in dairy farms of different regions may be provided based on the current investigation of the antimicrobial resistance of pathogenic Staphylococci from dairy cows in different geographic locations. The aim of the present study was to investigate the species distribution of the staphylococcal isolates associated with clinical endometritis of dairy cows in Inner Mongolia and the prevalence of superantigenic toxins genes (SAgs), and try to make a speculation on the potential function of staphylococcal superantigens in the development of clinical endometritis. Furthermore, the profiles of phonotypical antimicrobial resistance and the molecular antibiotic resistance mechanisms of the clinically isolated staphylococcal strains were subsequently investigated to inspect the current situation of antibiotic resistance in clinical staphylococcal isolates and the reasons of the development of resistance to certain antibiotics and to provide scientific therapy protocols for the treatment of clinical endometritis. Moreover, the molecular typing of S.aureus isolated from dairy cows with clinical endometritis targeting at spa gene was also carried out to clarify the prevalence and distribution of spa type and the epidemiological correlation of the isolates in S.aureus of bovine origin in Inner Mongolia render rationale basement for the screening of candidate S.aureus vaccine strains and the control of the diseases caused by infection of S.aureus.(1) The staphylococcal strains were isolated by traditional method and subsequently species level confirmation was made by PCR and sequencing of gap gene. The result revealed that a total of127isolates (48.8%) which belonged to ten different staphylococcal species from260uterine samples in dairy cows with clinical endometritis were isolated. Of the isolates, S.aureus (n=53,41.7%) and coagulase-negative staphylococci (CNS)(n=74,58.3%) belonging to nine varied species were observed. No significant differences of the incidence of staphylococci, S.aureus, and coagulase-negative staphylococci in dairy cows with clinical endometritis in different regions of Inner Mongolia were observed (p>0.05). In CNS, the predominant species was S. saprophyticus (n=38,29.9%) followed by S. chromogenes (n=22,17.3%).(2) The distribution of15staphylococcal SAgs in Staphylococci were investigated by PCR. Except for enterotoxin gene see and seg, all the other genes were found in both S.aureus and CNS. The carrier rate of superantigen genes in coagulase-negative staphylococci isolated from dairy cows with clinical endometritis in western region was significantly higher than that of central region (p<0.05). Isolates positive for sej gene (38.6%) were most commonly observed followed by those positive for the sen (30.7%) and sec genes (21.3%). A total of17genotypes of SAg gene were also found in SAg gene-positive staphylococcal isolates. The sec+sej+sen was the most widespread pattern of SAg genes (n=22,16.5%)(3) The MICs of19antibiotics and combinations frequently used in veterinary clinic to the isolates were determined by broth micro-dilution method. The incidence of resistance to β-lactam antimicrobial agents ranged from57.6%to79.5%, and incidence of resistance to macrolides and tetracyclines were from40%to50%. The isolates showed susceptibility to gentamicin and all of the combinations of certain antibiotics with a lower resistance incidence of15%. A total of43isolates were multi-resistant. Significant differences (p<0.05or p<0.01) of resistance to fluoroquinolone, clindamycin and gentamicin were observed in isolates from different regiongs of Inner Mongolia. Furthermore, the resistance of the isolates to erythromycin was found mainly due to the target methylation contributed by erm gene, the reduced susceptibility to tetracycline was ascribed to the excretion mechanism mediated by the tetK gene and the tolerance to penicillin was contributed by hydrolysis mediated by blaZ gene.(4) The disk diffusion of cefoxitin and amplification of mecA gene by PCR were used to detect the presence of methicillin resistant staphylococci (MRS). A total of22CNS isolates were confirmed as MRS. whilst no methicillin-resistant S. aureus (MRSA) were found. The SCCmec of the MRS strains were molecularly typed by multiplex PCR. Of the MRS isolates,20strains were successfully typed as SCCmec type Ⅱ (n=6,13.6%),type Ⅲ(n=12,54.5%) and Ⅳa(n=2,9.1%), however2isolates were failed to type by the method. The MRS with type Ⅲ SCCmec were found mainly prevailed in the central and western regions of Inner Mongolia, and MRS with type IVa SCCmec were found mainly prevailed in the eastern regions. The MRS with type II and III SCCmec were distributed in the the central, western and eastern regions of Inner Mongolia. All of the MRS isolates were multi-resistant, and the resistance incidence of MRS isolates to penicillin (p<0.01), ampicillin (p<0.01), clarithromycin (p<0.01), erythromycin (p<0.01) and cefotaxime (p<0.05) were significantly higher than MSS.(5) The53S. aureus isolates obtained in present study were molecularly typed by spa typing. A total of51isolates from7different geographical regions were typed to10gene type, whereas2isolates were failed to type. Of the10gene type, t518(n=18,34.0%) was found to be the most frequently observed type followed by t189(n=11,20.8%). The t518type was also found in5geographically different sampling sites with the widest geographical distribution amongst all types, and other types prevailed in1-3sampling sites.The main conclusions of the present study can be drawn as followed:(1) The high incidence of staphylococcal isolates in was uterine samples of dairy cows with clinical endometritis in different regions of Inner Mongolia was observed, and the species of staphylococci were widely distributed. Staphylococci were proved to be one of the main pathogens associated with bovine clinical endometritis in this region.(2)Different superantigens were widely distributed in diverse species staphylococcal isolates from dairy cows with clinical endometritis in Inner Mongolia, and the distributing partten of superantigens in different regiongs of Inner Mongolia varied.(3) The staphylococcal isolates associated with bovine clinical endometritis in Inner Mongolia expressed high resistance to the antibiotics frequently used in veterinary clinic which may serve as a crucial reason causing the failure of treatment for bovine clinical endomitritis in Inner Mongolia.(4) The appearance of MRS isolates were found in staphylococci associated with bovine clinical endometritis in Inner Mongolia, and the type of SCCmec in MRS prevailed in this region were type Ⅱ, type Ⅲ and Ⅳa.(5) The polymorphism of the distribution of spa gene type in S. aureus associated with bovine clinical endometritis in Inner Mongolia were observed with the prevailed gene types of t518and t189.