| Objective:To investigate the distribution of Shigella serotypes,resistance to antibiotics and virulence genes of children in Anhui,and to analyze the relationship between Shigella virulence genes and drug resistance.Meterials and Methods:Materials:A total of 345 clinical isolates of non-repetitive Shigella were collected from 34 hospitals in Anhui from 2010 to 2015.The quality control strain was Escherichia coli ATCC25922 of drug-sensitive test,preserved by Anhui Provincial Center for Bacterial Drug Resistance.Methods:1.A total of 345 clinical isolates of non-repetitive Shigella were collected from 34 hospitals in Anhui from 2010 to 2015 were identified using conventional biochemistry and the Vit KE-2 Compact automated microbiological analysis system.2.The Minimal inhibitory concentration(MIC)of 345 Shigella isolates from children against 18 antibacterial agents was determined by AGAR dilution method.3.16 Virulence Genes(VRGs)were detected: ial、ipa C、ipa B、ipa D、ipa A、ipa H、set、sen、sat、vir F、vir B、ics A、ics B、sig A、pic、sep A.Sixteen pairs of primers were amplified by Polymerase Chain Reaction(PCR).The primers were divided into five groups for amplification(Group 1: ial,ipa H,set,vir F;Group 2: sen,sig A,pic,sep A;Group 3: ics A,ics B,vir B;Group 4: ipa C,ipa B,i Pa D,ipa A;Group 5: sat).The reaction system was as follows: premixed Taq enzyme polymer 12.5ul,upstream primer 0.5ul,downstream primer 0.5ul,DNA template 2ul,dd H2 O 6.5ul-9.5ul(the total reaction system was 25ul).The reaction conditions were as follows: pre-denaturation at 95℃ for15min;95℃ denaturation 45 s,annealing 45s(annealing temperature: 57℃ for Group 1,58℃ for Group 2,60℃ for Group 3 and 4,59℃ for Group 5),72℃ extension for 1min,after 35 cycles;Extended at 72℃ for 10 min.A sample of 8ul PCR product was placed on a 1% agarose gel containing 0.5ug/ml ethidium bromide,electrophoresis at 150 V for30min,observed and photographed through the gel imaging system,and the experimental results were analyzed.Results::1.Among the 345 clinical isolates of Shigella from children,Shigella flexneri was the most prevalent iserotype(n=298,86.4%),Shigella sonnei is the subpopular serotype(n=44,12.8%).Among Shigella flexneri,296 strains(99.3%)were from northern Anhui,and 2 strains(0.7%)were from central Anhui.Among the Shigella strains,43 strains(97.7%)were from northern Anhui,and 1 strain(2.3%)was from central Anhui.The proportion of male children infected with Shigella flexneri was higher than that of female children(59.4%vs40.6%,P<0.0001).2.Among Shigella flexneri,the resistance to ampicillin was the highest(98.3%),followed by the resistance to nalidixic acid(97%),tetracycline(93.3%),chloramphenicol(88.6%)and ciprofloxacin(80.2%).Amikacin was the most sensitive(98.7%),followed by cefoxitin(94.6%)and piperacillin/tazobactam(93.3%).Among Shigella sonnei,all of them were resistant to azithromycin and ampicillin(100%),followed by naliditic acid(97.7%),gentamicin(93.2%),tetracycline(90.9%)and cotrimoxazole(88.6%).Respectively,Amikacin was the most sensitive(100%),followed by piperacillin/tazobactam(97.7%)and imipenem(97.7%).The resistance of Shigella flexneri to chloramphenicol and ciprofloxacin was significantly higher than that of Shigella sonnei(P<0.0001);The sensitivity of Shigella flexneri to levofloxacin and azithromycin was significantly higher than that of Shigella sonnei(P<0.0001).3.In this study,345 children shigella strains of clinical isolates carry 16 kinds of virulence genes of:(1)in the separation of 298 strains of Shigella flexneri: invasion of related genes in ipa H gene detection frequency is highest,298 strains(100%),ipa B gene was 296strains(99.3%),ipa D gene and ipa C gene were all 295 strains(99.0%),the ipa A gene was295 strains(99.0%),and ial was 288 strains(96.6%).Among the enterotoxin genes,283 strains of set gene(95.0%)and 278 strains of sen gene(93.3%)were found.Among the regulatory genes,there were 296 strains of ics A gene(99.3%),293 strains of ics B and vir B gene(98.3%),and 288 strains of vir F gene(96.6%).Among the SPATEs genes,there were 280 strains of sig A gene(94.0%),279 strains of sat gene(93.6%),269 strains of pic gene(90.3%),and 267 strains of sep A gene(89.6%).(2)Among the 44 isolates of Shigella sonnei,the frequency of ipa H gene was also the highest,with 44 isolates(100%),followed by 43 isolates of ipa B gene(97.7%),42 isolates of ipa C gene(95.5%),41 isolates of ipa D gene(93.2%),41 isolates of ipa A gene(93.2%),and 34 isolates of ial gene(77.3%).Among the enterotoxin genes,39 strains(88.6%)of sen gene were found,while set gene was not detected in all the isolates of Shigella sonnei in this study.Among the regulatory genes,there were 43 strains of ics A gene(97.7%),42 strains of vir B gene and ics B gene(95.5%),and 34 strains of vir F gene(77.3%).Among the SPATEs,there were 43 strains(97.7%)of sig A,but the sat,pic and sep A genes were not detected in the Shigella sonnei isolates in this study.Conclusions:1.Shigella flexneri and Shigella sonnei were the main epidemic subgroup in children of Shigella serotypes in Anhui,followed by Shigella sonnei.The frequency of bacterial dysentery was the highest in children in northern Anhui province.2.The most common multi-drug resistance phenotype of pediatric clinical isolates of Shigella in Anhui was ampicillin-nadiridinic acid-tetracycline.The resistance of Shigella flexneri to fluoroquinolones was significantly higher than that of Shigella sonnei.3.The resistance of VRGS(ial,ipaA,ipaC,ipa D,virB,vir F,set,icsB,pic,sepA)positive Shigella flexneri to CHL and CIP was higher than that of VRG negative Shigella flexneri;The drug-resistance of Shigella flexneri with set positive strains was higher than that of Shigella flexneri with set negative strains to CRO and CAZ.VRGS(ial,vir F,set,ics B,vir B,pic,sep A)negative strains of Shigella flexneri showed more frequent resistance to GEN and AZM.The drug resistance rates of VRG-positive Shigella flexneri and VRGpositive Shigella sonnei were similar,but the drug resistance rates of VRG-positive Shigella flexneri to CRO and CAZ were higher than those of VRG-positive Shigella sonnei.4.Shigella,which carries several virulence genes,is related to drug resistance.Further studies on the regulation and expression mechanism of virulence genes and the genetic mechanism of drug resistance in Shigella will be helpful to guide the empirical antimicrobial therapy for Shigellosis. |
PDF Full Text Request