长春地区致儿童腹泻大肠埃希菌的分离鉴定与耐药分析

Isolation,identification,and analysis of the drug resistance of Escherichia coli isolated from children with diarrhea in Changchun,China

目的了解长春地区致儿童细菌性腹泻的病原菌类型、病原学特征和抗生素耐药模式,为临床合理用药提供参考。方法采集2019年长春市儿童医院和长春中医药大学附属医院儿童腹泻样品50份,进行16s rDNA扩增子高通量测序分析和大肠埃希菌的分离鉴定;E-test法测定细菌的耐药谱与抗生素最小抑菌浓度;ERIC-PCR检测细菌的同源性,并测定13种毒力基因,进行系统分群和血清型鉴定。结果通过16S rDNA扩增子组学测序分析,排除志贺菌、沙门菌等感染的可能,确定致儿童腹泻粪便样品中大肠埃希菌为优势病原菌。分离获得110株大肠埃希菌,系统进化分群以A群、B2群和D群为主。检测到O抗原血清型23种,其中优势血清型为O119,检出13株大肠埃希菌EAST1基因PCR阳性,7株eae基因PCR阳性,其中1株同时携带EAST1基因和eae基因。分离株对21种抗生素的耐药率为4.3%~68.1%,多药耐药菌菌株比例高达63.8%。49.3%的菌株为ESBL表型,喹诺酮类抗生素的耐药率约为50%,阿米卡星和碳青霉烯类抗生素耐药率较低(5.8%和4.3%)。结论长春地区致泻性大肠埃希菌是引起儿童腹泻的重要病原菌之一,存在非典型肠致病性大肠埃希菌和肠集聚性大肠埃希菌的流行。目前临床常用的β-内酰胺类和喹诺酮类抗生素已不能达到良好的治疗效果,ESBL菌株对12种抗生素有较高的耐药水平,儿童感染性腹泻大肠埃希菌的耐药形势严峻,应进一步加强耐药性监测与抗生素应用管理。

Objective To study the distribution and patterns of antibiotic resistance of pathogenic Escherichia coli isolated from children with bacterial diarrhea in the Changchun area in order to provide a reference for rational drug use in clinical settings.Methods Fifty stool samples were collected from children with bacterial diarrhea in Changchun Children’s Hospital and the Changchun University of Traditional Chinese Medicine Hospital in 2019.Patients with diarrhea ranged in age from 1 month to 5 years.The V3-V4 region of 16 S rDNA was amplified from the 50 samples and a small fragment library was constructed using the Illumina novaseq sequencing platform.The effective data were clustered into operational taxonomic units,and then the OTUs sequences were analyzed using species annotation.MacConkey agar medium was used to isolate E.coli.The drug resistance spectrum and biochemical properties of the isolates were identified by an automated Phoenix-100 microbiology system.The minimal inhibitory concentrations of 13 antibiotics were determined using an E-test.Serotypes of the isolates were identified using E.coli O antisera.ERIC-PCR was used to determine the homology of the isolates.Systematic grouping and detection of 13 virulence genes were performed using PCR assays.Results Based on sequencing of 16 S rDNA amplicons,E.coli was identified as the dominant pathogen in stool samples from children with diarrhea.No 16 S rDNA sequences of Shigella,Salmonella,Vibrio parahaemolyticus,or Aeromonas were found.A total of 110 strains of E.coli were isolated,and the phylogenetic clusters were mainly A,B2,and D.Among them,29 ERIC fingerprints were found.Twenty-three serotypes of the O antigen were detected,which were mainly O1,O86,O119,O44,O125,O148,O153,O143,and O164.O119 was the dominant serotype.Thirteen isolates of E,coli were positive for the EAST1 gene.Seven isolates were positive for the eae gene,and one isolate carried both the EAST1 gene and the eae gene.The resistance of the isolates to 21 antibiotics ranged from 4.3%to 68.1%,and the proportion of multidrug-resistant strains was as high as 63.8%.The resistance of the isolates to quinolones was about 50%,while their resistance of amikacin and carbapenem antibiotics was low(5.8%and 4.3%).Forty-nine-point-three percent of the isolates were of the ESBL-producing phenotype,and the isolates were highly resistant toβ-lactam antibiotics.Conclusion Diarrhea-causing E.coli was one of the key pathogenic bacteria causing diarrhea in children in the Changchun area.There was an epidemic of atypical enteropathogenic E.coli and intestinal aggregation of E.coli infection.At present,β-lactams and quinolones commonly used in clinical practice are inefficacious.ESBL-producing strains were highly resistant to 12 antibiotics,and E.coli found in children with infectious diarrhea has serious drug resistance.Monitoring of drug resistance and management of antibiotic use should be further enhanced.