携带新德里金属β-内酰胺酶的猪源大肠杆菌全基因组测序及植物提取物药物筛选

Whole genome sequencing analysis of Escherichia coli from pigs carrying Delhi metal β-lactamase and drug screening of sensitive plant extracts

关键词:  碳青霉烯酶  β内酰胺酶  新德里金属β-内酰胺酶  NDM5  NDM4  blaTEM  AmpC  大肠埃希氏菌  细菌耐药

Key words:  Carbapenase  β lactamase  New Delhi metallo-β-lactamase  NDM5  NDM4  blaTEM  AmpC  E. coli  Bacterial drug resistance

摘要:

为了解大肠杆菌携带新德里金属β-内酰胺酶（New Delhi metallo-β-lactamase，NDM）NDM耐药基因情况及筛选敏感植物提取物，为携带NDM的超级耐药大肠杆菌的防控和药物研究奠定基础。采用16S rRNA菌种鉴定，PCR筛查菌耐药基因，blaNDM、blaTEM和AmpC酶耐药基因的确认，全基因组测序，BLAST和MEGA软件对序列进行生物信息学分析、系统进化树分析、NDM亚型鉴定和6种植物提取物药敏药物筛选研究。结果表明，从2021年（1-12月）临床猪腹泻病例中分离鉴定的91株大肠杆菌（Escherichia coli，E.coli）中鉴定出1株同时携带新德里金属β-内酰胺酶NDM4、NDM5和β内酰胺酶blaTEM、AmpC酶的大肠杆菌（HN2187）。对其进行11种抗生素和6种植物提取物药物敏感性测试，结果显示HN2187菌株对其均耐药。而HN2187对血根碱、贯叶连翘提取物敏感，对黄芩、姜黄素、千里光和大蒜提取物耐药。HN2187产B类酶，携带NDM4、NDM5、blaTEM、AmpC酶耐药基因，其中blaNDM-4位于质粒pNDM4-HN2187（Plasmid B）上，blaNDM-5位于质粒pNDM5-HN2187（Plasmid A）上，blaTEM1位于质粒pTEM1-HN2187（Plasmid B）上，AmpC酶耐药基因位于染色体上，分别介导blaNDM-4、blaNDM-5、blaTEM和AmpC耐药基因，是耐药菌传播的重要载体。以上研究表明，携带NDM4和NDM5的多重耐药菌已在养殖场传播和流行，应加强该类耐药基因的流行调查、监测和新药物研究，为临床超级细菌和多重耐药菌感染的流行和防治提供理论基础。

Abstract:

To investigate the New Delhi metallo-β-lactamase (NDM) resistant genes in E. coli and to screen sensitive plant extracts, to lay a foundation for the prevention and control of NDM resistant E. coli and drug research. 16S rRNA strain identification, PCR screening, blaNDM, blaTEM and AmpC enzyme resistance genes confirmation, whole genome sequencing, BLAST and MEGA software for bioinformatics analysis, phylogenetic tree analysis, NDM subtype identification and drug susceptibility screening of six plant extracts. The results showed that one strain of E. coli (HN2187) producing carbapenemases NDM4 and NDM5 and β-lactamases blaTEM and AmpC was identified from 91 strains from clinical swine diarrhea cases in 2021 (January-December). The results showed that HN2187 strain was resistant to 11 antibiotics. However, HN2187 was sensitive to the extracts of Macleaya cordata and Perilla frutescens, and resistant to the extracts of Baicalin, Curcumin, Senecio and garlic?extract. HN2187 produces class B enzymes and carries NDM4, NDM5, blaTEM and AmpC enzyme resistance genes, in which blaNDM-4 was located at the plasmid pNDM4-HN2187 (Plasmid B) and blaNDM-5 was located at the plasmid pNDM5-HN2187 (Plasmid A). blaTEM1 was located at the plasmid pTEM1-HN2187 (Plasmid B), the AmpC resistance gene was located on the chromosome, which mediates blaNDM-4, blaNDM-5, blaTEM and AmpC resistance genes, respectively, and is an important vector for the transmission of drug-resistant bacteria. The above studies indicate that multidrug-resistant bacteria carrying NDM4 and NDM5 have spread and become prevalent in livestock farms. It is necessary to strengthen the epidemic investigation, surveillance and new drug research of these resistance genes, so as to provide a theoretical basis for the epidemic and prevention of clinical superbug strains and multidrug-resistant strains.