一株多重耐药大肠杆菌全基因组测序及其耐药性分析

doi:10.13304/j.nykjdb.2023.0169

摘要/Abstract

摘要：

为了解大肠杆菌携带的粘菌素耐药基因并筛选敏感药物，解决多重耐药和动物临床无药可选的困局，采用16S rRNA菌种全基因组测序，PCR筛查mcr-4、mcr-5、blaTEM和AmpC酶耐药基因，应用BLAST和MEGA软件进行生物信息学和系统进化树分析，并对该菌株进行了78种抗生素抗菌药物敏感性测试及4种天然植物提取物（黄藤素、黄连素、黄芩苷和博落回）的抑菌、杀菌效果试验。结果表明，从2021年（1—12月）和2022年（1—6月）猪临床腹泻病例肠道中分离鉴定的145株大肠埃希菌（Escherichia coli，E.coli）中，鉴定出1株同时携带粘菌素耐药基因（mcr-4，mcr-5）和β内酰胺酶blaTEM、AmpC基因的猪源大肠埃希氏菌临床菌株HN2149。78种抗生素药敏试验结果显示，HN2149菌株对头孢吡肟、头孢地嗪、磷霉素、头孢克肟、头孢唑林、头孢哌酮、美洛培南、头孢西丁、头孢呋辛、头孢曲松、头孢噻肟、头孢他啶、替卡西林/克拉维酸、头孢哌酮/舒巴坦、氨曲南、哌拉西林/他唑巴坦、头孢噻肟/克拉维酸、头孢唑肟、头孢美唑、头孢他啶/克拉维酸和头孢他美敏感，对其余57种抗菌药物表现为耐药。4种植物提取物的药敏结果显示，博落回对菌株HN2149的抑菌、杀菌效果较好，其余3种提取物均对该菌株无抑菌和杀菌效果。以上研究结果为猪大肠杆菌病的防控提供参考。

关键词: 碳青霉烯酶, β-内酰胺酶, mcr-4, mcr-5, blaTEM, AmpC, 大肠埃希氏菌, 细菌耐药

Abstract:

In order to understand colistin resistance genes carried by Escherichia coli， screen sensitive plant drugs， and solve the dilemma of multiple drug resistance and no drug option in animal clinic， the resistance genes of Escherichia coli were identified by 16S rRNA sequencing and PCR. The antimicrobial susceptibility of 78 antibiotics were detected， and the bacteriostatic and bactericidal effects of 4 kinds of natural plant extracts （palmatine， berberine， baicalin and macleaya cordata） were tested. The results showed that 145 strains of E.coli were isolated and identified from intestinal tract of swine clinical diarrhea cases in 2021 （from January to December） and 2022 （from January to June）， and a clinical strain carrying colistin resistance genes （mcr-4，mcr-5） and β-lactamases blaTEM and AmpC was identified， which named HN2149. The antimicrobial susceptibility of 78 antibiotics showed that the HN2149 strains was sensitive to cefepime， cefodizime， fosfomycin， cefixime， meropenem， cefoxitin， cefazolin， cefoperazone， cefotaxime， ceftazidime， cefuroxime， ceftriaxone， aztreonam， piperacillin-tazobactam， ticacillin/clavulanic acid， cefoperazone sulbactam， ceftazidime/clavulanic acid， cefotaxime/clavulanic acid， ceftizoxime， cefmetazole， cefetamet， and was resistant to 57 antibiotics. The drug sensitivity of 4 plant extracts showed that the macleaya cordata had the best bacteriostatic and bactericidal effect on HN2149 strain， while the other 3 extracts had no effects on HN2149 strain. Above results provided reference for the prevention and control of swine colibacillosis.

Key words: carbapenemase, β-lactamase, mcr-4, mcr-5, blaTEM, AmpC, Escherichia coli, bacterial drug resistance

中图分类号:

S852

李海利, 徐引弟, 王治方, 朱文豪, 张立宪, 马春江. 一株多重耐药大肠杆菌全基因组测序及其耐药性分析[J]. 中国农业科技导报, 2024, 26(6): 113-121.

Haili LI, Yindi XU, Zhifang WANG, Wenhao ZHU, Lixian ZHANG, Chunjiang MA. Whole Genome Sequencing of Multi-drug Resistant Escherichia coli and Its Drug Resistance Analysis[J]. Journal of Agricultural Science and Technology, 2024, 26(6): 113-121.

图/表 7

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引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp	退火温度 Annealing temperature/℃
Mcr4-F	TTGCAGACGCCCATGGAATA	207	57
Mcr4-R	GCCGCATGAGCTAGTATCGT	207	57
Mcr5-F	GGACGCGACTCCCTAACTTC	608	58
Mcr5-R	ACAACCAGTACGAGAGCACG	608	58
blaTEM-F	CCAATGCTTAATCAGTGAGG	400	57
blaTEM-R	ATGAGTATTCAACATTTCCG	400	57
AmpC-F	GATCGTTCTGCCGCTGTG	270	57
AmpC-R	GGGCAGCAAATGTGGAGCAA	270	57

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp	退火温度 Annealing temperature/℃
Mcr4-F	TTGCAGACGCCCATGGAATA	207	57
Mcr4-R	GCCGCATGAGCTAGTATCGT	207	57
Mcr5-F	GGACGCGACTCCCTAACTTC	608	58
Mcr5-R	ACAACCAGTACGAGAGCACG	608	58
blaTEM-F	CCAATGCTTAATCAGTGAGG	400	57
blaTEM-R	ATGAGTATTCAACATTTCCG	400	57
AmpC-F	GATCGTTCTGCCGCTGTG	270	57
AmpC-R	GGGCAGCAAATGTGGAGCAA	270	57