Molecular Biological Characteristics And Homology Analysis Of Escherichia Coli Sequence Type 131 Clone

Objectives:The main purpose of this study is to detect the epidemiology of Escherichia coli sequence type 131（E.coli ST131）among unselected E.coli clinical strains isolated from Fujian Medical University Union Hospital and to analyze the molecular biological characteristics of E.coli ST131 clone.The aim of this study was to prevent and control the outbreak of infection and E.coli ST131 clonal strains of infectious diseases to provide experimental basis for the treatment.Methods:1.Bacterial Strains and Screening of E.coli ST131:Strains of non-repetitive E.coli strains were consecutively collected between August 2014 and August 2015 in Clinical Microbiology Laboratory of Fujian Medical University Union Hospital.For the detection of all E.coli ST131 isolates,PCR was used to screen ST131-associated specific nucleotide sequences（SNPs）in gyrB and mdh.All the non-O25b,non-O16and non-phylogenetic group B2 ST131 strains would be confirmed by multilocus sequence typing（MLST）to be ST131 according to the Achtman scheme using seven housekeeping genes（adk,fumC,gyrB,icd,mdh,purA and recA）（http://mlst.ucc.ie/mlst/dbs/Ecoli）.2.Detection of fimH:All E.coli ST131 clones were further examined by PCR using specific primers for the fimH30 allele for identification of the H30 and H30Rx subclones.All the H30-ST131 PCR negative isolates underwent direct sequenceing of fimH as previously described.3.Virulence genes testing:26 known of virulence genes（papAH,papC,papEF,papGⅡ/Ⅲ,papGalleleⅠ,papGalleleⅡ,sfa/focDE,afa/draBC,fimH,gafD,sfaS,focG,hlyA,cnf1,cdtB,fyuA,iutA,KpsMTⅡ,kpsMTⅢ,traT,cvaC,K1/K5,rfc and PAI）were performed on all ST131 isolates by a multiplex PCR,and analyze the virulence factors of E.coli ST131 clones.4.Susceptibility testing:Antimicrobial susceptibility of E.coli ST131 clone was determined by KB method.Drugs tested included cefotaxime,ceftazidime,cefepime,aztreonam,ciprofloxacin,amikacin,cotrimoxazole,imipenem,ertapenem,and levofloxacin.The results were based on the breakpoints of the2016 CLSI criteria.E.coli ATCC25922 was used for routine quality control.5.Detection of Commonβ-Lactamase genes:Commonβ-Lactamases genes(bla_CTX-M,bla_TEM and bla_SHV)of E.coli ST131 clone were detected by PCR and DNA sequencing.6.Quinolone resistance mechanisms:As to CIP-nonsusceptible isolates,the presence of plasmid-mediated quinolone resistance determinants（PMQRs;qnrA,qnrB,qnrC,qnrD,qnrS and aac（6’）-Ib-cr）were detected by PCR.As to CIP-resistant isolates,mutations in quinolone-resistant determining region（QRDR）of gyrA,parC,and parE were detected by PCR and DNA sequencing.7.PFGE analysis:Pulsed-field gel electrophoresis（PFGE）analysis of E.coli ST131clones were performed using XbaI digestion.According to the unweighted pair group method of Dice coefficient,BioNumerics software was used to construct PFGE dendrogram.8.Statistical analysis:Data were analyzed using the SPSS 19.0.Comparisons of proportions were performed usingχ2 or Fisher’s exact test.P<0.05 was considered to be statistically significant.Results:1.E.coli ST131 clone screening:83（11.6%）of the 700 clinical E.coli isolates were identified as E.coli ST131 clones.Phylogenetic analysis of 83 strains of E.coli ST131showed that all strains were belonged to group B2.O serotypes showed that 83 strains of E.coli ST131 clones belonged to two types,O25b（66.3%）and O16（33.7%）.2.H30-ST131 accounted for 63.9%（53/83）of E.coli ST131 isolates,including 13H30 Rx-ST131 and 40 H30 non-Rx-ST131 isolates;the remaining 30 belonged to H41-ST131 subclones.94.5%of the O25b-ST131 clone（52/55）contained the fimH30allele and 96.4%of the O16-ST131 clone（27/28）contained the fimH41 allele.3.Virulence factors testing:The virulence genes with the detection rates higher than60%in E.coli ST131 clone were fimH（98.8%）,fyuA（97.6%）,iutA（92.8%）,PAI（91.6%）,traT（88.0%）,kpsMT II（74.7%）.In contrast,11 virulence genes with a detection rate of less than 10%included cvaC（3.6%）,sfa/focDE（1.2%）,sfaS（8.4%）,afa/draBC（8.4%）.Seven virulence genes were not detected:focG,papG allele I,gafD,cdtB,kpsMTIII,nfaE and rfc.The virulence genes of papAH,papC,papEF,papG allele II,papGⅡ/Ⅲ,afa/draBC and kpsMT K5 were significantly higher in O25b-ST131 clones than in O16-ST131 clones（P<0.05）.The virulence fraction of H30 Rx-ST131 clone was significantly higher than that of H30 non-Rx ST131 and H41-ST131 clones（P<0.05）.The virulence genes of papAH,papC,papEF,papG allele II,papGⅡ/Ⅲ,hlyA and cnf1 were significantly higher in H30 Rx-ST131clones than in H30 non-Rx ST131 and H41-ST131 clones（P<0.001）.The virulence factor kpsMT K5 was more prevalent in H30-ST131 clones than in H41-ST131 clones（P<0.05）.4.Susceptibility testing:Eighty-three strains of E.coli ST131 clones were resistant to11 common antibacterial agents.The highest rates of resistant were cefotaxime（71.7%）,ciprofloxacin（69.9%）,levofloxacin（69.9%）,and cotrimoxazole（67.5%）.In contrast,resistance rates werelow to aztreonam（36.1%）,cefepime（31.3%）,ceftazidime（26.5%）,amikacin（8.4%）and piperacillin/tazobactam（1.2%）.Imipenem and ertapenem were all sensitive.The detection rate of multidrug-resistant bacteria was 55.4%（46/83）.Compared with O25b-ST131 clone,O16-ST131 clone had lower resistance to fluoroquinolones（such as ciprofloxacin and levofloxacin）and cefepime（P<0.05）.In contrast,all H30 non-Rxand H30 Rx-ST131 clones were resistant to fluoroquinolones such as ciprofloxacin and levofloxacin（P<0.001）.Compared with the H30 Rx-ST131 clones,the resistance rates of ceftazidime,cefepime and aztreonam were lower in H41-ST131 and H30 non-Rx ST131 clones（P<0.01）.5.Commonβ-Lactamase genes characterization:bla_TEM genes were harbored by34（41%）E.coli ST131 clones.62（74.7%）E.coli ST131 isolates which were nonsusceptible to cefotaxime or ceftazidime harbored blaCTX-M genes.Of these,37strains carried bla_CTX-M-14,22 strains carried bla_CTX-M-15TX-M-15 and one strain carried blaCTX-M-123.The remaining two strains co-produced blaCTX-M-14 and blaCTX-M-15.No SHV,CTX-M-2,CTX-M-8 and CTX-M-25 were detected in this study.H30Rx-ST131 carried more bla_CTX-M-15TX-M-15 than H30 non-Rx ST131 and H41-ST131（P<0.001）.The bla_CTX-M-14TX-M-14 and bla_TEM-1EM-1 were mainly composed of O16-H41 ST131and O25b-H30 non-Rx ST131 clones（P<0.001）,whereas H30 Rx-ST131 did not carry the bla_CTX-M-14 and bla_TEM-1.The drug resistance rates of cefepime and amikacin in CTX-M-15-H30Rx group were higher than those in CTX-M-14-H30R group（P<0.001）.6.Quinolone resistance mechanisms:Three types of PMQR determinants were found in 17 E.coli ST131 clones,including aac（6’）-Ib-cr（18.1%,15/83）,qnrS1（2.4%,2/83）and qnrB1（2.4%,2/83）.Two strains co-produced qnrB1 and aac（6’）-Ib-cr.All 58ciprofloxacin-resistant E.coli ST131 clones contained 4 or 5 non-synonymous mutations in gyrA,parC and parE.All of these E.coli ST131 clones have a set of 3conserved QRDR amino acid substitutions（GyrA Ser83→Leu,Asp87→Asn and ParC Ser80→Ile）.The positive rates of aac（6’）-Ib-cr of H30 Rx and H41-ST131 were significantly higher than those of H30 non-Rx（P<0.001）.Five site mutations（GyrA S83→L,D87→N,ParC S80→I,E84→V and ParE I529→L）were found to be significantly higher in H30-ST131 clones than in H41-ST131 clones（P<0.001）.7.PFGE analysis:PFGE analysis of the 83 strains of E.coli ST131 clonesshowed that these strains were divided into 68 pulsotypes（named 1-68）.Using 70%similarity cut-off value,these subtypes were divided into 23 different PFGE clusters（A^W）.Two PFGE clusters（C and D）were predominant,grouping 13 and 14 E.coli ST131clones,respectively,while the other 21 PFGE clusters contained as many as 9 E.coli ST131 clones,respectively.Cluster C contained 92.3%（12/13）of H30 Rx-ST131clones,and most of the O16 ST131 clones belonged to two clusters（H and I）with 5and 9 E.coli ST131 clones,respectively.Conclutions:1.This is the first study to reveal the prevalence and molecular characteristic of ST131 clonal group among consecutive clinical E.coli isolates in China.2.The prevalence of E.coli ST131 is relatively low in this study,belonging to type B2.H30 lineage accounts for the majority of E.coli ST131 clones,which is a very important lineage of ST131 subclones.3.O16-B2-ST131 subclone may be an important type of E.coli ST131 in China.4.FimH,fyuA,iutA,kpsMTII and traT are the most common virulence factors of E.coli ST131 clones found in this study.5.E.coli ST131 clone is a major clonal group of multidrug-resistant ExPEC.6.The H30 Rx subtype is strongly associated with multiple drug resistance and the presence of blaCTX-M-15.7.Fluoroquinolone resistance of E.coli ST131 was mainly related to QRDR chromosomal mutation,especially to the mutations of GyrA and ParC subunits.8.PFGE clustering analysis of E.coli ST131 clones showed polymorphic distribution among theisolates,showing a high degree of genetic diversity and no major clones.