Study On Antibiotic Resistance Analysis And Molecular Epidemiology Of Methicillin-Resistant Staphylococcus Aureus From Animals

Staphylococcus aureus (SA), one of the most common pathogen in medical and veterinary clinic, causes many diseases such as skin and soft tissue infections, sepsis, endocarditis, pneumonia, enteritis, meningitis, osteomyelitis, toxic shock syndrome and so on. With the extensive and abuse application of antibiotics, the methicillin-resistant or mec gene positive Staphylococcus aureus strains were known as methicillin-resistant Staphylococcus aureus (MRSA) first appeared in1961in UK. MRSA is resistant to all beta-lactam antibiotics and multiple drug resistance, which is serious clinical problem of anti-infective treatment of the veterinary. Therefore, it’s very important to analyze and investigate the epidemiology and antibiotic resistance of MRSA from animals. In order to control the large-scale infection of MRSA between human and animals and to guide the correct use of antimicrobials, a series of experiments were carried out to identify, detect, analyze antibiotic resistance and to investigate molecular epidemiological of MRSA from animals. This study supplied a foundation for further study on mechanisms of resistance of MRSA from animals.1. Isolation and identification of Staphylococcus aureus from animals and establish-ment of PCR detection methodNasal or rectal swabs of healthy animals were collected from Nanjing and surrounding regions. A total of156Staphylococcus aureus strains were isolated by bacteriological methods for further studies. The heat-stable nuclease gene nuc was selected as the target gene of Staphylococcus aureus, specific primers was designed, and appropriate PCR reaction system were optimized to established a rapid detection method with high specificity and sensitivity. The sensitivity can be up to269pg.2. Study on the detection and antibiotic resistance of methicillin-resistant Staphylococ-cus aureus from animals156strains were tested for MRSA by cefoxitin disk diffusion method, oxacillin disk diffusion method and PCR method, meanwhile, the antimicrobial sensitivity were tested by K-B method. The detection rate by cefoxitin, oxacillin disk diffusion methods and PCR method was21.79%,19.87%and16.67%, respectively. Compared with PCR method as a gold standard, the sensitivity of oxacillin disk diffusion method was96.15%, specificity was93.08%and coincidence rate was93.59%, while the sensitivity of cefoxitin method was96.15%, specificity was95.38%and coincidence rate was95.51%. There were no significant difference among three methods by χ2test. Cefoxitin disk diffusion method had high degree of specificity and coincidence rate with the PCR method. In a word, Cefoxitin disk diffusion method was more suitable for clinical diagnosis for MRSA. The resistance rates to antibiotic of26MRSA from animals were from0%to100%, a large proportion (76.92%) being multi-resistance strains and mostly resistant to ampicillin (100%), cefotaxime (96.2%), ciprofloxacin (69.2%) and gentamicin (46.2%). Methicillin-sensitive Staphylococcus aureus (MSSA) showed multiple drug resistance too, but the multi-drug resistance rate (36.15%) of MSSA was significantly lower than that of MRSA. However, teicoplanin resistant strain was not found in this study.3. Study on molecular evolution of MRSA isolates from animalsIn this study MLST was first applied to research the origin and genetic background of animal source MRSA in China. The clones, types and evolution of MRSA from animals can be analyzed by MLST and bio-information technology. The tpi, yqiL, arcC, aroE, glpF, gmk and pta of housekeeping genes of Staphylococcus aureus were amplified by PCR, then the sequence of the amplified DNA fragments were analyzed and the results submitted to the International Database Site (http://www.mlst.net) for the ST type. The results showed that26strains of MRSA consisted of20types of STs.2009S001,2010S011,2010S024were ST-9type,2009M016was ST-88,2010M093belonged to ST-188,2010DC002was ST-133, and other strains were not found their ST type in the database. In these new ST strains, monkey strains accounted for95%(19/20) and pig strains2009S001accounted for5%(1/20). This results provided genetic background and possible evolutionary routes of these26strains, and laid basis for further study on analysis of a large number of MRSA from animals.