Pathogenic Mechanism Of Different Extraintestinal Pathogenic Escherichia Coli Clones

Objective: In this study,Escherichia coli(E.coli)clinical isolates and commensal strains were used to study the molecular and genome characteristics.The aim of the present study was to explore the pathogenic mechanism of different extraintestinal pathogenic Escherichia coli(E.coli)clones.Methods:1.Bacterial Strains: A total of 700 non-repetitive E.coli clinical isolates were collected from Fujian Medical University Union Hospital between August 2014 and August 2015.Fifty E.coli isolates were collected from feces samples of random recruited medical students in Fujian Medical University during January 2016 and February 2016.2.Phylogenetic grouping: The main phylogenetic groups(A,B1,B2,and D)of E.coli isolates were identified by a triplex PCR,and all of the positive PCR products were sequenced.3.O types: Sixteen classic O serotype groups(O1,O2,O4,O6,O7,O8,O12,O15,O16,O18,O21,O22,O25,O75,O83,and O157)were detected by PCR,and all of the positive PCR products were sequenced.4.Virulence factor genes: All the isolates were detected for 26 known virulence factors(VFs)genes using a previous described multiplex PCR method.These genes were included as following: adhesions(pap AH,pap EF,pap C,pap G allele I,pap G II/III,pap G allele II,sfa/foc DE,afa/dra BC,fim H,gaf D,sfa S,foc G,nfa E),toxins(hly A,cnf1,cdt B),siderophores(fyu A,iut A),protections and invasions(kps MT Ⅱ,kps MT Ⅲ,tra T,cva C,kps MT K1/K5),miscellaneous(rfc,PAI),and the positive products were sequenced.5.Comparative genomics: Two clinical isolates and one commensal isolate were genotyped.Two clinical isolates were from urinary,and blood sample.All three strains belonged to the B2-O25-ST131.The bacterial genomic DNA was extracted andsent to Shanghai Zeta Biosciences for second-generation sequencing,genome annotation and analysis.6.Statistical analysis: Categorical variables were analyzed using the c2 or Fisher exact test,data were analyzed using SPSS19.0 and a P value <0.05 was considered statistically significant.The mean of the age is used mean ± standard deviation.Results1.Bacterial Strain:(1)700 non-repetitive E.coli clinical strains were isolated from different types of samples,including 357 urine(51%),103 blood(14.7%),86secretion(12.3%),73 drainage fluid(10.4%),44 bile(6.3%),26 pus(3.7%),11others(1.6%).(2)The 700 clinical strains were from 25 clinical departments,which including 98 urology surgery(14%),71 outpatient service(10.1%),59 emergency(8.4%),61 hepatobiliary surgery(8.7%),50 colorectal surgery(7.1%),49 hematology department(7%),39 neurology department(5.6%),31 endocrinology department(4.4%),28 obstetrics and gynecology department(4%),26 oncology department(3.7%),26 cardiology department(3.7%),98 other medical departments(14%),40 other surgical departments(5.7%).(3)Age distribution: 0 to 20 years old(37,5.35%),21 to 30 years old(44,6.3%),31 to 40 years old(49,7%),41 to 50 years old(88,12.6%),51 to 60 years old(160,22.9%),61 to 70 years old(150,21.4%),71 to 80 years old(114,16.3%),81 to 100 years old(58,8.3%).The average age is(56.4±19.4)years old.(4)Gender distribution: 280 isolate were from men(40%),and420 isolate were from women(60%).2.Phylogenetic grouping: 700 clinical isolates were mainly belonged to group B2(247,35.3%)and group D(232,33.1%),followed by group A(137,19.6%)and group B1(84,12%).Of 50 commensal E.coli strains,group A accouted for 60.0% of the isolates,group B1 accouted for 14.0%,group B2 accouted for 18.0% and group D accouted for 8.0%.3.O types: 700 clinical strains were mainly belonged to O25 group(11.9%),followed by O1 group(9.4%)and O75 group(7.6%).O22 group and O157 group were not detected.In the 50 E.coli isolates from feces,only 8 isolates could be typed,including 1 O6(2%),1 O7(2%),2 O12(4%),1 O16(2%),1 O21(2%),1 O25(2%),and1 O75(2%).4.Virulence factor genes: Among 700 clinical strains,the detection rates of virulence genes fim H,fyu A,iut A,kps MT II and tra T were all higher than 50%.The detection rates of virulence genes pap G allele I,gaf D,nfa E,and rfc were all less than 3%,and cdt B was not detected in all strains.Phylogenetic group B2 and D carried more virulence factors than phylogenetic group A and B1.Analysis of 50 E.coli commensal strains found that all strains of virulence factors carried less than 10,and seven strains(14%)did not detect any virulence genes.5.Comparative genomics: The genomes of the three strains were similar,including the GC content,the number of r RNA and t RNA.According to the results of evolutionary analysis and homologous clustering,the strains were has high homology.The genomic islands and prophage associated genes of three isolates were as follows:EC191(416,105);EC192(340,19);EC193(433,224).There 13 ISs in the urine isolate,9 ISs in the blood isolate,and 8 ISs in the commensal isolate.Conclusion:1.The detection rate of E.coli isolated from the urinary was the highest(14%);The elderly is more susceptible to E.coli than other people,especially among the 51-80 age groups.2.The detection rate of O25 was the highest in E.coli clinical strain.The dominant O type of E.coli was different among urinary system and blood system,O25 and O75 was dominant in UPEC,and O25 and O1 was dominant in SEPEC.3.Ex PEC was mainly belong to group B2,and commensal E.coli strains was maily belong to group A.There were no statistically significant differences in the different infection sites among group A,group B1 and group D(P >0.05).And there were statistically significant differences in the different infection sites among group B2(P<0.05).4.The virulence factors carried by E.coli are related to their infection and colonization ability.Ex PEC isolates often carried virulence genes such as fim H、fyu A、iut A and kps MT II which can help E.coli invade,colonize,and avoid the host’s immune response.5.The insertion sequences,phage-related genes,genomic island,and potential virulence genes contained in the UPEC and SEPEC are different.The insertion sequences in the genome of the UPEC is more than that in the SEPEC.Both of the two clinical isolates are carried a large number of potential virulence factors,and some virulence factors in the UPEC and SEPEC were different,such as toxins and invasion.6.The characteristics,molecular biological characteristics and genomic characteristics of Ex PEC clones were different in different infection sites,which made the difference of pathogenicity among different clones.