| Objective:In this study,stool of outpatients with diarrhea in a sentinel hospital in Changping district,Beijing from Jan.to Dec.of 2018 were collected,and multiple common pathogen were detected to maintain the diarrhea pathogen of the hospital.For isolated strains,virulence gene detection,drug resistance test and PFGE analysis were done to learn the characteristics of virulence genes,molecular epidemiology and drug resistance,so as to provide a reliable basis for the formulation of preventive measures and clinical rational drug use.Methods:According to the definition of diarrhea case,379 samples were collected and screened for Diarrheal Escherichia coli(DEC),Salmonella,Shigella,Vibrio parahaemolyticus and Campylobacter.Among them,120 samples were selected randomly,and were extracted by Roche viral nucleic acid kit.Rotavirus,Norovirus,Adenovirus and Astrovirus were detected.Bacteria isolated from positive samples were examined for virulence genes by Polymerase chain reaction(PCR).For DEC,Salmonella,Vibrio parahaemolyticus strains minimal inhibitory concentration(MIC)were measured by Broth microdilution method and PFGE patterns were obtained.Results:Among 379 diarrhea stool samples,68 pathogenic bacteria were detected,with a positive rate of 17.94%,including 25 strains(6.59%)of DEC,mainly enteropathogenic Escherichia coli(13 strains);22 strains(5.80%)of Vibrio parahaemolyticus,mainly O3:K6 serotype(12 strains),19 strains(5.01%)of Salmonella,mainly Salmonella enteritidis(11 strains);1 strain of Campylobacter jejuni and 1 strain of Shigella flexneri(0.26%each).Of the 120 diarrhea samples,40 were positive for viral nucleic acid,with a total detection rate of 33.33%,and the number of Norovirus was the highest(23,19.17%),mainly GII-type Norovirus(18),followed by Rotavirus(group A)(10,8.33%)and Astrovirus(7,5.84%).Seasonal distribution showed that bacterial diarrhea had the highest incidence in summer(63.60%),followed by spring(25.80%)and viral diarrhea had the highest incidence in in winter(34.80%),followed by spring(27.50%).Virulence gene detection results showed that the virulence genotypes of enteropathogenic Escherichia coli(EPEC)were eae+and bfp-,which were atypical EPEC(aEPEC)strains;the virulence genotypes of enteroaggregative Escherichia coli(EAEC)were aggR+ and aaf Ⅱ-.In enterotoxigenic Escherichia coli(ETEC),the carrying rates of st and It were 87.50%(7/8)and 50.00%(4/8)respectively.The carrying rate of tdh virulence gene of Vibrio parahaemolyticus was 86.36%(19/22),and the virulence genotypes were tdh+ and trh-.The carrying rate of trh virulence gene was 4.54%(1/22),and the virulence genotypes were tdh-and trh+.2 strains that did not carry trh and virulence genes were non-pathogenic strains which accounted for 9.10%(2/22).1 Shigella strain carried four virulence genes including ial,ipaH sen and set1.1 Campylobacter jejuni virus carried five virulence genes of CadF,flaA,cdtA,cdtB and cdtC simultaneously.The results of drug sensitivity test showed that the total drug resistance rate of DEC was 76.00%(19/25,number of drug resistant strains/total number of strains),and the highest drug resistance rate was the rate to ampicillin(AMP)(52.00%),followed by that to nalidixic acid(NAL)and compound sulfamethoxazole(SXT),and multidrug-resistant strains accounted for 42.10%(8/19).The total drug resistance rate of Vibrio parahaemolyticus was 81.80%(18/22),which was only resistant to cefazolin.The total drug resistance rate of Salmonella was 89.50%(17/19),and the highest drug resistance rate was the rate to sulfamethoxazole(Sul)and NAL(68.40%),followed by AMP(47.40%),and multidrug-resistant strains accounted for 52.94%(9/17).PFGE clustering results showed that in DEC,4 strains of EAEC produced 4 PFGE band patterns,with similarity coefficients of 62.4%-82.9%;13 strains of EPEC produced 13 band patterns,and the similarity coefficients of genetic fingerprinting were 50.1%～78.1%;8 ETEC strains produced 6 band patterns(of which cp2018DE05 and cp2018DE18 strains had the same band pattern,cp2018DE22 and cp2018DE24 strains had the same band pattern),and the similarity coefficients between band patterns were 63.7～100%.Vibrio parahaemolyticus produced a total of 15 band patterns,and the similarity coefficients of genetic fingerprinting were 60.4%～100%.Predominant serotype O3:K6 strains clustered,O4 serogroup strains clustered,and 2 02 serogroup strains clustered into a small cluster.Salmonella produces 13 band patterns with similarity coefficients of 44.8%～100%.Among them,the predominant serotype Salmonella enteritidis clustered into a large cluster,2 Salmonella Stanley strains were of the same band pattern,and 2 Salmonella London strains were of the same band pattern.Conclusion:1.Epidemiological characteristics of multiple-pathogen diarrhea:Common diarrhea-causing bacterial infections in this hospital in 2018 included Escherichia coli,Vibrio parahaemolyticus,Salmonella,Shigella and Campylobacter jejuni;Viral diarrheal infections include Norovirus,Rotavirus(group A)and Astrovirus.There was no significant difference in gender and age among diarrhea pathogens,and the detection of diarrheal pathogens had characteristics of time distribution.2.Pathogenic characteristics of pathogenic bacteria in diarrhea:Virulence genotypes of EPEC were atypical EPEC,virulence genotypes of EAEC were aggR+and aafⅡ-,st was the main virulence gene carried by ETEC,and tdh was the main virulence gene carried by Vibrio parahaemolyticus.3.Resistance of diarrheal pathogens:The drug resistance spectrum of DEC and Salmonella was extensive,the drug resistance spectrum of Vibrio parahaemolyticus was single,and most strains were only resistant to CFZ.4.Molecular subtyping characteristics of PFGE:The PFGE band patterns of DEC,Salmonella and Vibrio parahaemolyticus were polymorphism. |
PDF Full Text Request