2016-2017年宁夏地区牛源金黄色葡萄球菌的全基因组分析

Whole-genome analysis of bovine Staphylococcus aureus strains in Ningxia from 2016 to 2017

摘要:

[目的] 了解宁夏地区奶牛乳腺炎金黄色葡萄球菌（Staphylococcus aureus，SA）代表菌株的基因组序列基本特征，进一步探究其耐药基因型、毒力及进化关系，为兽医临床防治提供理论依据。[方法] 采用纸片法对97株金黄色葡萄球菌临床分离株进行抗菌药物敏感性试验，同时进行葡萄球菌蛋白A（Staphylococcus aureus protein A，spa）分型、多位点序列分型（multilocus sequence typing，MLST），根据分型结果选取16株代表菌株进行全基因组测序，并对获得的测序序列进行处理分析。[结果] 药敏试验结果显示97株分离株对18种抗菌药物存在不同程度的耐药，其中9株耐甲氧西林金黄色葡萄球菌（methicillin-resistantStaphylococcus aureus，MRSA）对青霉素、氨苄西林、苯唑西林、头孢噻呋、磺胺异噁唑、红霉素、庆大霉素和克林霉素等8种抗菌药物完全耐药，甲氧西林敏感金黄色葡萄球菌（methicillin-sensitiveStaphylococcus aureus，MSSA）菌株对青霉素、氨苄西林、磺胺异噁唑耐药率较高。耐药基因数据库（antibiotic resistance genes database，ARDB）注释分析显示16株代表菌株共携带21种耐药基因，其中norA、tet38、bacA、mepA的携带率较高，与药敏试验结果具有一定的相关性。毒力基因数据库（virulence factors of pathogenic bacteria，VFDB）注释分析显示所有菌株携带多种与粘附、宿主免疫逃逸、分泌、胞外酶编码、铁摄取等疾病相关的毒力基因，MRSA菌株均携带较多毒力因子，MSSA菌株携带毒力因子数目不等。基因岛预测结果显示16株代表菌株存在不同数量的基因岛且MRSA菌株携带基因岛数目及毒力基因岛较多，但耐药基因岛数目与MSSA差异不明显。SNP分析结果显示部分分离株同源性较高，同源性较高的两株MRSA的全基因组基本序列特征差异较小，携带的耐药、毒力基因情况相似。[结论] 宁夏地区牛源SA分离株耐药性情况严重且具有较高的毒力水平，本研究为家畜相关MRSA（livestock-associated MRSA，LA-MRSA）与MSSA基因组序列信息的比较分析及宁夏地区SA感染的临床防控提供参考依据。

Abstract:

[Objective] To understand the basic characteristics of the genome sequence of the representative strain of Staphylococcus aureus from dairy cows with mastitis in Ningxia, and to further explore its drug-resistant genotype, virulence and evolutionary relationship, to serve as a theoretical basis for veterinary clinical prevention and treatment. [Methods] In total 97 clinical isolates of S. aureus were tested for antimicrobial susceptibility by the paper disk method. Staphylococcal protein A typing and multi-site sequence typing were done at the same time. According to the typing results, 16 representative strains were sequenced and the obtained sequences were analyzed by network database. [Results] Drug susceptibility test shows that 97 strains were resistant to 18 antibacterial drugs, of which 9 strains of methicillin-resistant Staphylococcus aureus (MRSA) were completely resistant to 8 antibacterial drugs, such as penicillin, ampicillin, oxacillin, ceftiofur, sulfisoxazole, erythromycin, gentamicin, and clindamycin. Methicillin-sensitive Staphylococcus aureus (MSSA) strains were highly resistant to penicillin, ampicillin, and sulfisoxazole. Antibiotic resistance genes database (ARDB) annotation analysis shows that 16 representative strains carried 21 drug resistance genes, among which the carrying rates of norA, tet38, bacA, and mepA were higher, correlating with to the results of drug sensitivity test. Virulence Factors Database (VFDB) annotation analysis shows that all strains carried a variety of virulence genes related to diseases such as adhesion, host immune escape, secretion, extracellular enzyme encoding, and iron uptake. MRSA strains all carried more virulence factors, MSSA strains carried different number of virulence factors. The results of gene island prediction show that 16 representative strains had different number of gene islands, and MRSA strains carried more gene islands and virulence gene islands, but the number of drug resistance gene islands was not significantly different from that of MSSA. The results of SNP analysis show that some isolates had higher homology, and the two MRSA with higher homology had little differences in the basic sequence characteristics of the whole genome, both carried similar resistance and virulence genes. [Conclusion] The drug resistance of S. aureus isolates from cattle in Ningxia is serious and highly virulent. Our findings provide a reference for the comparative analysis of genome sequence information of livestock-associated MRSA (LA-MRSA) and MSSA, and the clinical prevention and control of S. aureus infection in Ningxia.