尿道致病性大肠杆菌临床分离株种系分型、毒力基因型及生物被膜表型分析

Analysis of phylogenetic typing,virulence genes and biofilm phenotype of uropathogenic Escherichia coli isolates

目的分析尿道致病性大肠杆菌(UPEC)临床分离菌株的种系分型、毒力基因型及生物被膜形成能力。方法采集临床尿路感染患者尿样进行细菌培养,对菌落计数大于10^(5)CFU/mL的样品再进行挑选,将选出的疑似大肠杆菌的菌落用微生物及蛋白快速鉴定质谱仪(MALDI Biotyper)鉴定出UPEC分离株。利用多重聚合酶链反应(PCR)检测UPEC分离株种系分型基因chuA、yjaA以及DNA片段TSPE4.C2;利用PCR检测UPEC分离株中9种毒力基因(fimH、tonB、ireA、fyuA、cnf-1、vat、hlyD、ompR和phoU)的分布情况;通过结晶紫染色法定量检测UPEC分离株生物被膜形成能力。结果共选取UPEC分离株42株,其种系分型以B2型和D型为主,分别占40.5%和31.0%。对9种毒力基因进行检测发现,Ⅰ型菌毛编码基因fimH检出率最高,为92.9%;摄铁相关基因tonB、ireA和fyuA的检出率分别为90.5%、66.7%和76.2%;受检的3种毒素相关基因中,编码细胞坏死因子的cnf-1基因检出率最高(52.4%),溶血素基因hlyD和空泡化毒素基因vat的检出率分别为21.4%和28.6%;其余毒力基因ompR和phoU的检出率均低于30.0%。所有受检菌株均能形成生物被膜,其中16株(占38.1%)具有强生物被膜形成能力,15株(占35.7%)具有中等生物被膜形成能力,其余11株菌株具有弱生物被膜形成能力。进一步分析发现,强生物被膜形成菌株大多属于B2型,且其携带毒力基因数量高于弱生物被膜菌株,差异有统计学意义(P<0.05)。结论UPEC临床分离株生物被膜形成能力与其种系分型和毒力基因分布具有一定关联性,进一步了解UPEC临床分离株的生物学特征尤其是生物被膜形成能力和毒力基因分布情况,可为尿路感染防控提供重要参考依据。

Objective To analyze phylogenetic typing,virulence genes and biofilm phenotype of isolated uropathogenic Escherichia coli(UPEC)strains.Methods Urine samples from patients with clinical urinary tract infection were collected for bacterial culture.The colonies with a colony number greater than 10^(5)CFU/mL after bacterial culture in urine were collected and then subjected to bacterial isolation.The UPEC isolates from suspected Escherichia coli strains were identified by Matrix Assisted Laser Desorption/Ionization technique(MALDI-Biotyper).Multiplex Polymerase Chain Reaction(PCR)was used to detect the phylogeny genes chuA,yjaA and DNA fragment TspE4.C2 of UPEC isolates.The distribution of nine virulence genes(fimH,tonB,ireA,fyuA,cnf-1,vat,hlyD,ompR and phoU)was detected by PCR.The biofilm formationability of UPEC isolates was quantitatively determined by crystal violet staining.Results A total of 42 UPEC isolates were selected,and the main phylogeny typing was B2 and D,accounting for 40.5%and 31.0%,respectively.Detection of nine virulence genes found that the fimH gene encoding typeⅠfimbriae had the highest detection rate(92.9%);the detection rate of iron-uptake related genes tonB,ireA and fyuA were 90.5%,66.7%and 76.2%,respectively;among the three toxin-related genes,the detection rate of cnf-1 gene encoding cytotoxic necrosis factor was the highest(52.4%),was 21.4%for hemolysin encoding gene hlyD and 28.6%for vacuolating toxin gene vat;the detection rates of the other virulence genes such as ompR and phoU were lower than 30.0%.Our results showed that all the tested isolates could form biofilm,among which 16 isolates had strong biofilm formation ability(38.1%),15 isolates had moderate biofilm formation ability(35.7%),and the remaining 11 strains had weak biofilm forming ability.Further analysis showed that the strong biofilm-forming strains mainly belonged to phylogenetic type B2,which possessed the higher numbers of virulence genes than that of the weak biofilm-forming strains(P<0.05).Conclusion The biofilm formation ability of UPEC clinical isolates is correlated with the phylogenetic types and the distribution virulence genes.This study will help to understand the biological characteristics of UPEC clinical isolates,especially the biofilm formation ability and virulence gene distribution,and could provide important references for the prevention and control of urinary tract infection.