青藏高原牦牛携带屎肠球菌及其毒力基因和耐药性检测

目的 了解青藏高原牦牛携带屎肠球菌(Enterococcus faecium)情况,以及耐药性和毒力基因。方法 使用链球菌培养基分离细菌;使用生化反应和16s rDNA序列分析方法鉴定;采用PCR方法检测从牦牛粪便中分离的屎肠球菌携带细胞溶血素(cytolysin,cylA)、明胶酶E(gelatinase, gelE)、表面蛋白(enterococcal surface protein, esp)、胶质蛋白粘附素collagen-binding-adhesin of Enterococcal faecium,acm)、聚集物质(aggregation substance, asa1)及透明质酸酶(hyalronidase,hyl)6种毒力基因的情况;应用K-B纸片法对屎肠球菌分离株进行16种抗生素的敏感性分析;参照PulseNet 脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)实验方法对屎肠球菌分离株进行PFGE分型分析。结果 我们从320份牦牛粪便分离到33株屎肠球菌。这些菌株中毒力基因asa1的阳性率最高,为6.1%,acm和hyl次之,均为3.0%,其他毒力基因皆未检出。33株牦牛屎肠球菌中,有19株菌株具有抗生素耐药性,包括4株多重耐药菌株和15株单耐菌株。牦牛屎肠球菌对利福平耐药率最高,为48.5%;对其他抗生素的耐药率从高到低依次为青霉素G15.2%、四环素12.1%、强力霉素12.1%、红霉素9.1%、环丙沙星6.1%、氨苄西林6.1%、高浓度庆大霉素6.1%、磷霉素6.1%、左氧氟沙星6.1%。所有菌株对万古霉素、替拉考宁和氯霉素均敏感。细菌染色体DNA酶切片段的PFGE分析发现,33株屎肠球菌分离株共产生30种PFGE带型,可分为A-H 8个聚类群,耐药菌株分布在6个聚类群中。结论 青藏高原牦牛携带屎肠球菌,呈现高度遗传多态性,部分菌株携带毒力基因,对多种抗生素耐药。研究提示,屎肠球菌可能会通过牦牛相关食品传播。

Virulence genes and antibiotic resistance of Enterococcus faecium isolated from yaks in Qinghai-Tibet Plateau,China

To isolate Enterococcus faecium from yaks in Qinghai-Tibet Plateau, China and detect its virulence genes and antibiotic resistance, the strains were isolated by Streptococcal medium and were identified by biochemical and 16s rDNA methods. The strains were then analyzed by pulsed-field gel-electrophoresis of digested chromosomal DNA fragments. The virulence genes were detected by PCR and confirmed by sequencing. Antibiotic resistance was performed by the K-B disk method with 16 antibiotics that were widely used in E. faecium. Results showed that 33 E. faecium strains were isolated from 320 fecal samples of yaks. A total of six virulence genes were detected by PCR method, including cytolysin (cylA), gelatinase (gelE), enterococcal surface protein (esp), collagen-binding-adhesin (acm), aggregation substance (asa1) and hyalronidase (hyl). The gene asa1 was found in 6.1% strains tested, followed by acm and hyl (3.0%). None of isolates was tested positive for the rest three virulence genes. Nineteen of 33 isolates were resistant to antibiotics, including 4 isolates having multidrug-resistance and 15 isolates being resistant to only one antibiotic. Of the 16 antibiotics tested, rifampicin resistance was found to be the most prevalent, accounting for 48.5% of isolates, followed by penicillin G (15.2%), tetracycline (12.1%), doxycycline (12.1%), erythromycin (9.1%), ampicillin (6.1%), high-level gentamicin (6.1%), ciprofloxacin (6.1%), fosfomycin (6.1%), and levofloxacin (6.1%). All of the isolates were 100% susceptible to vancomycin, teicoplanin and chloromycetin. Thirty-three E. faecium strains displayed 30 PFGE patterns and then were divided into 8 clusters from A to H, 6 of which included antibiotic-resistant strains. The yaks in the Qinghai-Tibetan Plateau are releasing E. faecium through feces. Those isolates carried some virulence genes and were resistant to various antibiotics, indicating potential hazard to human health.