猪肉源大肠杆菌分离鉴定及其生物被膜形成

为研究猪肉来源大肠杆菌分离菌株的生物被膜形成及相关基因表达变化,首先从生猪屠宰线、猪胴体表面及生鲜猪肉中采用选择平板和特异性聚合酶链式反应（polymerase chain reaction,PCR）分离鉴定大肠杆菌,采用微孔板结晶紫染色法评价分离菌株15 ℃培养72 h时生物被膜形成能力,进而选取代表菌株研究生物被膜形成过程中被膜量、胞外聚合物（extracellular polymeric substances,EPS）组成,以及基于反转录荧光定量PCR的成膜基因表达的变化。结果表明：共分离到猪肉源大肠杆菌31 株,包括生猪屠宰线11 株、猪胴体表面4 株、生鲜猪肉16 株;微孔板结晶紫染色结果显示,被膜形成能力存在明显菌株差异,64.52%菌株成膜能力较弱,选取其中1 株（D4-18）进行成膜过程研究;微孔板中菌株D4-18 15 ℃培养168 h过程中被膜量持续增加,培养72 h和168 h时,菌株D4-18分泌EPS,培养72 h时,papC、fimH、csgA基因表达量分别为0.095、0.933、0.435 copies/cm2,随着培养时间延长,松散型EPS中蛋白质及多糖含量、紧密型EPS中蛋白质含量极显著增加（P＜0.01）,培养168 h时,papC和fimH基因表达量增加,csgA基因表达量无显著变化,表明上述基因在大肠杆菌生物被膜形成过程中发挥了不同程度的调控作用。

Isolation,Identification and Biofilm Formation Ability of Escherichia coli from Pork

This paper aims to investigate the biofilm formation and related genes expression of Escherichia coli isolated from pork. E. coli was isolated from pig slaughtering line, carcass surfaces and pork meat and identified using selective agar plates and specific polymerase chain reaction (PCR). After culture at 15 ℃ for 72 h, the biofilm forming abilities of the isolates were evaluated by means of microplate crystal violet staining. Furthermore, representative strains were selected to explore the changes of biofilm biomass, the composition of extracellular polymeric substances (EPS), and biofilm related-genes expression by means of reverse transcription real-time PCR in the process of biofilm formation. The results showed that a total of 31 strains of E. coli were isolated and identified in this study, among which 11 were obtained from pig slaughtering line, 4 from the surface of pig carcasses, and 16 from raw pork meat. The results of crystal violet staining showed that there were significant differences in the biofilm-forming ability among the 31 isolates, and 64.52% of them had weak biofilmforming ability, one of which was then selected to study the biofilm formation process. The biofilm biomass of strain D4-18 on the microplate increased continuously during 168 h incubation at 15 ℃. After incubation for 72 and 168 h, EPS were secreted by strain D4-18, and the gene expression levels of papC、fimH、csgA genes were 0.095、0.933、0.435 copies/cm2, respectively. With the increase of culture time, the contents of protein and polysaccharide in loose EPS (L-EPS) and the protein content in bound EPS (B-EPS) increased significantly (P < 0.01). After incubation for 168 h, the expression of the papC and fimH genes increased, while csgA gene expression did not change significantly, indicating that these genes play different regulatory roles in the biofilm formation of E. coli.