猪肉源金黄色葡萄球菌生物膜能力与MLST分型检测

Detection of Biofilm Capacity and MLST Typing of Staphylococcus aureus from Pork

金黄色葡萄球菌在生鲜猪肉中有较高的检出率,是污染生鲜猪肉的主要食源性致病菌之一,对食品安全和人体健康造成巨大威胁。采用刚果红琼脂法定性,96孔微量板定量结合扫描电镜检测其成膜能力,用PCR法检测其成膜基因和黏附基因,最后对分离株进行多位点序列分型分析(MLST)。结果表明,刚果红琼脂法定性试验显示84.93%(62/73)的菌株成生物膜阳性,96孔微量板定量试验显示94.52%(69/73)的菌株成生物膜阳性,扫描电镜观察到不同黏附能力的菌株在钢板上梯度富集,PCR试验发现分离株均含有多重黏附基因,其中同时携带icaD、clfA、clfB、fnbA、fnbB;icaD、clfA、clfB、fnbA、fnbB、cna;icaA、icaD、clfA、clfB、fnbA、fnbB、cna多重黏附基因的菌株数最多,分别为30.14%(22/73),20.55%(15/73),15.07%(11/73)。MLST试验中73株菌分为26个ST型,夏季20个、冬季12个,夏季以CC1、CC5为优势克隆群,冬季以CC15、CC1、CC5为优势克隆群。系统发育树中,分离株与国内食源性、医源性和动物源性分离株有一定的相关性,呈现遗传多样性。研究结果可为生鲜猪肉的安全监管和金黄色葡萄球菌的传播及溯源调查提供一定的科学依据。

S.aureus has a high detection rate in fresh pork,which is one of the main foodborne pathogens that pollute fresh pork,and poses a great threat to food safety and human health.In this study,Congo red Agar method was used to determine the quality,96-well microplate quantitative analysis combined with scanning electron microscope to detect the film-forming ability,PCR method was used to detect the film-forming genes and adhesion genes.Finally,the isolates were analyzed by multi-site sequence typing(MLST).The results showed that the qualitative test of Congo red Agar method showed that 84.93%(62/73)of the strains were biofilm positive,and the 96-well microplate quantitative tes showed that 94.52%(69/73)of the strains were biofilm positive.Scanning electron microscope showed that the strains with different adhesion ability were enriched in gradient on the steel plate.PCR test found that all the strains contained multiple adhesion genes,which carried icaD,clfA,clfB,fnbA and fnbB at the same time.The strains of icaD,clfA,clfB,fnbA,fnbB,cna;icaA,icaD,clfA,clfB,fnbA,fnbB and cna had the largest number of multiple adhesion genes,which were 30.14%(22/73),20.55%(15/73)and 15.07%(11/73),respectively.In MLST test,73 strains were divided into 26 ST types,20 in summer and 12 in winter.CC1 and CC5 were dominant clones in summer and CC15,CC1 and CC5 were dominant in winter.In the phylogenetic tree,the isolates were correlated with domestic food,iatrogenic and animal isolates,showing genetic diversity.The results can provide a scientific basis for the safety supervision of fresh pork,the transmission and the traceability investigation of S.aureus.