| Objective(1)To understand the epidemiological characteristics of ExPEC though the analysis of the Escherichia coli isolated from patients with infectious disease and obtain the lineages of pathogenic Escherichia coli by PFGE typing.(2)The antibiotic sensitivity test was conducted to analyze the drug resistance of ExPEC,and the difference of drug resistance between ExPEC strains was compared in different clusters.(3)The ExPEC strains of dominant clone group was sequenced,and the MLST type was blasted.The difference in virulence and drug resistance of ExPEC strains between MLST types and PFGE clusters were analyzed.(4)The sequence data of strains were analyzed to predict the Phylogenetic between different clones of ExPEC.Methods(1)Bacteria stored in the refrigerator at-80℃ was resuscitated,single colony was selected after selective culture,and the pathogen species was identified by autoflex maX MALDI-TOF.(2)The pulsed-field gel electrophoresis(PFGE)typing was conducted in the identified Escherichia coli.The images data were processed by bionumeric software,and the type information of strain was obtained through analysis.(3)According to the results of PFGE typing,the whole genome sequence and assembly of dominant clone strains was conducted,and antibiotic sensitivity test was carried out by referring the manual of Gram Negative strains GNX2F susceptibility plate.(4)Drug resistance gene and virulence genes of sequenced E.coli strains were detected in the Center for Genomic Epidemiology,VFDB and CARD.(5)MUMmer(V3.23)software was used to compare the assembled genomes to the reference genome to obtain SNP(single nucleotide polymorphism)markers,and FastTree(V2.1.11)software was used to map and visualize the evolutionary tree.(6)The data of drug resistance phenotype and the carriers of virulence gene and drug resistance gene in ExPEC were analyzed by spss22.0.Results(1)761 strains were identified as E.coli,A total of 324 strains of Escherichia coli belong to 7 Clusters were identified by PFGE typing.(2)Antibiotics sensitive test was conducted to seven major Clusters include 324 E.coli strains,the drug resistance rates of SXT(67.08%),CIP(58.77%),LEV(57.23%),DOX(52.31%),CTX(51.38%)were detected with the higher frequency and carbapenems resistant strains was detected:ETP(4.31%),DOR(1.54%),MERO(1.23%)and IMI(0.62%).It is noteworthy that four ExPEC strains were resistant to colistin B and colistin,and only tigacycline had no resistance.214 strains were resistant to three or more antibiotics simultaneously,and the rate of multi-drug resistance was 66.05%.(3)The seven Clusters,Cluster 2 to Cluster 7 except Clusterl,are mainly belonging to CC69,CC95,CC14,CC10,CC131 and CC38,respectively.4.The following is the multi-drug resistance rates of the seven Clusters:Cluster1:40.74%,Cluster 2:64.91%,Cluster 3:20.59%,Cluster 4:64.62%,Cluster 5:72.73%,Cluster 6:86.89%,Cluster 7:88.89%.(4)All 324 strains belong to 6 Clades in phylogenetic tree,CC14 and CC131 are the largest Clades of ExPEC in this research,and the β-lactamase gene ampC was present in 94.14%strains.Virulence factors such as entA was detected with the highest frequency in 91.04%strains,and allR was detected in 90.12%strains,and the invasive factors ompA and aslA were 41.36%and 80.86%respectively.All strains of ST1193 showed gyrA,gyrB,parC and parE gene point mutations related to quinolone resistance,except the two strains without gyrA mutation.Conclusions(1)E.coli is an important pathogen of infectious diseases.The patients infected by ExPEC are mainly middle-aged and elderly people between 40 and 90 years old except for newborn patients in this study.(2)There are mainly seven epidemic 7 Clusters of the strains collected in this study,including the pandemic clone ST 131,which has been prevalent all over the world.The multi-drug resistance characteristics of st131 is significant,and it is a great threat to the clinical treatment of infectious disease,the difference of drug resistance characteristics between different Clusters are varied greatly,so the new star ST1193 may become the next popular multi-drug resistant ExPEC lineage.As an important pathogenic agent of zoonotic E.coli,ST95 is a major threat to food safety and human health.(3)The serious multi-drug resistance of ExPEC strain should be taken into account,especially the emergence of pan-drug resistant strains and the prevalence of colistin resistant strains,the virulence factors that resist to human immune system are widely carried in ExPEC strains.The spread of these factors in different strains in the intestinal environment will pose a serious threat to human health. |
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