猪场舍内环境中葡萄球菌的分离鉴定及耐药分析

Isolation,identification and drug resistance analysis of Staphylococcus in pig farm environment

为了明确重庆市某规模猪场舍内环境中葡萄球菌的病原特性及耐药情况,试验采用空气细菌培养自然沉降法和物体表面采样法采集猪场舍内环境样本20份,进行葡萄球菌的分离纯化,提取分离株基因组DNA,扩增16S rRNA基因序列,构建系统进化树,检测其生化指标,确定敏感药物,并分析病原菌耐药基因的携带情况。结果表明:分离纯化得到2种革兰氏阳性球菌,分别与头状葡萄球菌和沃氏葡萄球菌相似性较高(均为99%),在系统进化树中也分别与其聚为一簇,两种菌的硝酸盐还原、MR-VP和甘露醇等7种指标检测结果均为阳性,确定为头状葡萄球菌和沃氏葡萄球菌,均对多西环素、复方新诺明和阿奇霉素耐药;头状葡萄球菌携带vanA、rpoB、ermB和tetM 4种耐药基因,沃氏葡萄球菌携带ileS、rpoB、blaZ、ermC、aacA-aphD、tetK和tetM 7种耐药基因,两种菌均含有rpoB和tetM基因,并证实头状葡萄球菌和沃氏葡萄球菌的耐药类型与耐药基因具有相关性。说明该猪场舍内环境中存在头状葡萄球菌和沃氏葡萄球菌,在生猪养殖和人畜共患病防控方面应加强对环境中病原微生物的监测。

In order to clarify the pathogenic characteristics and drug resistance of staphylococcus in the environment of a pig farm in Chongqing,the natural sedimentation method and object surface sampling method were used to collect environmental samples in the farm for isolation and purification of staphylococcus.A phylogenetic tree was constructed by 16S rRNA gene sequence which were amplified with the help of bacterial universal primers after the genomic DNA of the isolated strain was extracted.The isolated strain was identified by the biochemical tests and sensitive drugs.And the carrying status of drug resistance genes of pathogenic bacteria was tested by the specific primers.The results showed that two gram-positive cocci were isolated and purified and they had high homology(both 99%)with Staphylococcus capitis and Staphylococcus warneri,and were also clustered with them in the phylogenetic tree.The biochemical tests results of 7 indicators such as nitrate(reduction),MR-VP and mannitol of the two bacteria were all positive so they were identified as Staphylococcus capitis and Staphylococcus warnerii.The drug allergy test showed that the two pathogenic bacterias were resistant to doxycycline,cotrimoxazole and azithromycin.Drug resistance gene analysis found that Staphylococcus capitis carried 4 kinds of drug resistance genes including vanA,rpoB and ermB;Staphylococcus warneri carried 7 kinds of drug resistance genes including ileS,blaZ and ermC,and both bacteria contained rpoB and tetM genes.And it was confirmed that the resistance types of Staphylococcus capitis and Staphylococcus warneri were related to the resistance genes.It suggested that Staphylococcus cephalus and Staphylococcus wornerii existed in the environment of the pig farm,suggesting that the monitoring of pathogenic microorganisms in the environment should be strengthened in terms of pig breeding and zoonotic disease prevention and control.