尿路感染大肠埃希菌生物膜的表型与基因型鉴定和耐药性的关联

Relationship between phenotype,genotype and drug resistance of Escherichia coli biofilm in urinary tract infection

目的探讨尿路感染大肠埃希菌生物膜的表型与基因型鉴定和耐药性的关联。方法2019年1月至2020年1月尿路感染患者分为2组:大肠埃希菌组(UPEC患者,n=210)和非大肠埃希菌组(年龄和性别匹配的其它尿路感染患者,n=64)。对尿液样品进行处理,并按照标准规程通过标准的微生物学程序鉴定细菌分离物。将分离物在BHI肉汤中于37℃下培养24h,通过分光光度法检测两组分离物生物膜形成。使用琼脂扩散技术进行了抗生素敏感性试验。通过PCR检测毒力因子(papC,fimH,kpsMTII和fliC)的存在与否。通过多重PCR检测bla_(TEM),bla_(CTX-M),bla_(SHV)和bla_(OXA)基因。通过对大肠埃希菌菌株进行脉冲场凝胶电泳(PFGE)分析来评估菌株的遗传多样性。结果本研究共涉及210例UPEC患者,平均年龄42.16±5.37,男性患者71例(33.81%),女性患者139例(66.19%)。这些病例更常见于妇产科107(50.95%),其次是泌尿外科71(33.81%)和肾内科32(15.24%)。非大肠埃希菌组与大肠埃希菌组性别、年龄、BMI、吸烟、饮酒和科室均没有差异性统计(P>0.05)。女性的UPEC发病率较高,女性占66.19%,男性占33.81%,21至40岁年龄段的女性更常见,共67人,其次是41至60岁年龄段的女性,共37人(17.62%)。生物膜形成试验阳性率为40.51%(111),大肠埃希菌的试验阳性率为48.57%(102),其他的大肠菌群生物膜试验阳性率为14.06%(9)。210株UPEC菌株对6种抗菌药物表现出不同程度耐药性,其中对氨苄西林耐药率最高(50.48%),对庆大霉素耐药率最低(8.57%)。生物膜阳性组对氨苄西林(71.57%)、庆大霉素(13.73%)和环丙沙星(40.20%)的耐药率高于生物膜阴性组(30.56%,3.70%和10.19%)(P<0.05)。生物膜阳性组β-内酰胺酶(ESBL)、fimH、fliC、papC、kpsMTII、bla_(CTX-M)、bla_(TEM)、bla_(SHV)和bla_(OXA)阳性率均较物膜阴性组高(P<0.05)。bla_(TEM)阳性组对氨苄西林(80.87%)、头孢噻吩(40.87%)、庆大霉素(13.04%)、左氧氟沙星(40.87%)、环丙沙星(37.39%)和头孢他啶(21.74%)的耐药率高于bla_(TEM)阴性组(13.68%,7.37%,3.16%,13.68%,20.00%和3.16%)(P<0.05)。结论UPEC生物膜形成对抗生素(氨苄西林、庆大霉素和环丙沙星)的耐药率高于生物膜阴性,生物膜阳性基因型阳性率较高。blaTEM阳性对抗生素的耐药率高于bla_(TEM)阴性。

Objective To explore the relationship between phenotype,genotype and drug resistance of Escherichia coli biofilm in urinary tract infection.Methods Patients with urinary tract infection from January 2019 to January 2020 were divided into two groups:Escherichia coli group(patients with E.coli infection,n=210)and non-E.Coli group(age and gender matching patients with other urinary tract infections,n=64).Urine samples were processed,and bacterial isolates were identified by standard microbiological procedures in accordance with standard procedures.The isolates were cultured in BHI broth at 37℃for 24 hours,and the biofilm formation of the two isolates was detected spectrophotometrically.Antibiotic sensitivity tests were performed using the agar diffusion technology.The presence or absence of virulence factors(papC,fimH,kpsMTII,and fliC)was detected by PCR.The bla_(TEM),bla_(CTX-M),bla_(SHV) and bla_(OXA) genes were detected by multiplex PCR.Pulsed field gel electrophoresis(PFGE)analysis of Escherichia coli strains was used to assess the genetic diversity of the strains.Results A total of 210 patients with Escherichia coli with urinary tract infection were involved in this study,with an average age of 42.16±5.37,71 male patients(33.81%),and 139 female patients(66.19%).These cases were more common in obstetrics and gynecology 107(50.95%),followed by Urology 71(33.81%)and Nephrology 32(15.24%).There were no statistical differences in gender,age,BMI,smoking,drinking,and departments between the non-E.Coli group and the E.coli group(P>0.05).Women have a higher incidence of urinary tract infections with E.coli,66.19%in women and 33.81%in men.Women in the 21 to 40 age group are more common,67 in total,followed by women in the 41 to 60 age group including a total of 37 people(17.62%).The positive rate of biofilm formation test was 40.51%(111),the positive rate of Escherichia coli was 48.57%(102),and the positive rate of other coliform biofilm tests was 14.06%(9).The 210 UPEC strains showed different degrees of resistance to six antibacterial drugs,of which the rate of resistance to ampicillin was the highest(50.48%),and the rate of resistance to gentamicin was the lowest(8.57%).The biofilm-positive group was more resistant to ampicillin(71.57%),gentamicin(13.73%),and ciprofloxacin(40.20%)than the biofilm-negative group(30.56%,3.70%,and 10.19%)(P<0.05).The positive rates of ESBL,fimH,fliC,papC,kpsMTII,bla_(CTX-M),bla_(TEM),bla_(SHV) and bla_(OXA )in the biofilm-positive group were higher than those in the film-negative group(P<0.05).The blaTEM-positive group was resistant to ampicillin(80.87%),ceftiophene(40.87%),gentamicin(13.04%),levofloxacin(40.87%),ciprofloxacin(37.39%),and ceftazidime(21.74%)The rates were higher than those in the bla_(TEM)-negative group(13.68%,7.37%,3.16%,13.68%,20.00%,and 3.16%)(P<0.05).Conclusion The biofilm forming Escherichia coliin urinary tract infectionswas more resistant to antibiotics(ampicillin,gentamicin,and ciprofloxacin)than the biofilm-negative ones,and the biofilm-positive genotype-positive rate was higher.The resistance rate of bla_(TEM) positive to antibiotics was higher than that of bla_(TEM) negative ones.