产志贺毒素大肠埃希菌的分子生物学鉴定和耐药性分析

牛肉样品中产志贺毒素大肠埃希氏菌和肠致病性大肠埃希氏菌的分离鉴定朱应飞，聂　翔，熊丽霞，吴学平，占忠旭，匡佩琳（江西省检验检测认证总院食品检验检测研究院，江西南昌 330000）摘　要：于2018年9月—2019年3月，每个月从超市和农贸市场中抽取20个样本，共抽取140个牛肉及制品样本，在qPCR初筛时stx阳性率为45.8%，eae的阳性率为52.2%。

每月的stx初筛阳性率分别为65%、60%、80%、5%、25%、45%和40%；eae初筛阳性率分别为70%、70%、65%、0%、40%、65%和55%。

检出携带stx基因的产类志贺毒素大肠埃希氏菌样本4份，样本阳性菌株检出率为2.9%；检出携带eae基因的肠致病性大肠埃希氏菌样本12份，样本阳性菌株检出率为8.6%。

关键词：产志贺毒素大肠埃希氏菌；肠致病性大肠埃希氏菌；分离；鉴定Isolation and Identification of Shiga Toxin Producing Escherichia coli and Enteropathogenic Escherichia coli in BeefSamplesZHU Yingfei, NIE Xiang, XIONG Lixia, WU Xueping, ZHAN Zhongxu, KUANG Peilin (Jiangxi General Institute of Testing and Certification Food Testing Institute, Nanchang 330000, China)Abstract: From September 2018 to March 2019, 20 samples were taken from supermarkets and farmers’ markets every month, with a total of 140 beef and product samples taken. the positive rate of stx was 45.8%, and the positive rate of eae was 52.2%. The monthly positive rates of stx initial screening positive rates of 65%, 60%, 80%, 5%, 25%, 45%, and 40%, respectively; The positive rates of eae initial screening were 70%, 70%, 65%, 0%, 40%, 65%, and 55%, respectively. Four Shiga toxin producing Escherichia coli samples carrying the stx gene were detected, with a positive strain detection rate of 2.9%; From September 2018 to March 2019, 12 samples of enteropathogenic Escherichia coli carrying the eae gene were detected, with a positive strain detection rate of 8.6%.Keywords: Shiga toxin producing Escherichia coli; enterogenic Escherichi coli; separation; appraisal产志贺菌毒素的大肠杆菌（Shiga Toxin Producing Escherichia coli，STEC）和肠致病性埃希氏菌（Enterogenic Escherichi coli，EPEC）是世界上最重要的食源性病原体之一。

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１ ， ２方法 ：药敏纸片及 培养基 ：哌拉西林 、头抱他 啶、哌拉西林 一他 唑 巴坦 、头孢噻肟 、氨曲南 、头孢 曲松 、头孢吡肟 、亚胺培 南、庆大 霉 素、美罗培南 、阿米卡星 、头孢 西丁、环丙沙星 、氯霉 素、阿奠西 林 一舒 巴坦和复 方磺胺 甲恶唑纸片以及Ｍｕ ｅ ｌ ｌ ｅ ｒ － Ｈ ｉ ｎ ｔ ｏ ｍＭＨ）培养基等
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的筛选 。
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生 予 以严格 的控制 ，对耐 药株 的流 行和 爆 发予 以预 防 。
【 关 键词 】 大肠埃 希杆 菌 ；耐 药性 ；临床 分 离 ；分析
中图分类号 ：Ｒ ４ ４ ６ ．— ８ １ ９ ４（ ２ ０ １ ６ ）３ ２ — ０ ０ ７ ６ — ０ ２
为６ ０ ． ５ ％， 出现 亚胺 培 南、美 罗培 南敏 感 率 约 ９ ９ ． ５ ％， 出现 头孢 哌 酮或舒 巴坦敏 感率 约 为 １ ０ ０ ． ０ ％。检 出 超广谱 Ｄ ・ 内酰胺 酶 （ Ｅ Ｓ Ｂ Ｌ ｓ ） 大

Multidrug-resistant Escherichia coli producing AmpC enzyme genotypes study and drug resistance analysis Abstract: Objective: Explore the region more resistant drug Escherichia coli (Escherichia coli, E.c oli) production plasmid AmpC beta-lactamase (AmpC enzyme) status and genotype distribution, yield analysis AmpC enzyme Escherichia coli antibiotic resistance to commonly used the characteristics for clinical rational use of antibiotics and prevent resistance genes to spread to provide the theory basis. Methods: Screening test early：screening test：Under the Clinical Laboratory Standards Agency (Clinical labora-tory Standards Insitute, CLSI) disk diffusion method recommended (kerby-Bauer, disk diffusion method) detected 71 Escherichia coli cefoxitin resistance, ie, the inhibition zone≤ 18mm suspicious of AmpC enzyme-positive strains.2.cefoxitin three-dimensional extract test：According to cefoxitinthree-dimensional extract test,whether by extracts of Escherichia coli beta-lactamase hydrolysis of cefoxitin (FOX) to determine whether AmpC enzymes to detect produced AmpC enzymes strains. Kerby-Bauer disk diffusion method : 71strains of Escherichia coli antibiotic susceptibility test to 10 antibiotics were performed by Kerby-Bauer disk diffusion method with the standard of NCCLS. A TCC25922 was used for quality control.The experiment method points:On a flat plate LB doing (37 ℃ incubation 24 hours) for the preparation of bacteria into and 0.5McIntosh standard pipe turbidity the same bacterium fluid, and the bacterium fluid in the uniform coating sterilization Mueller-Hinton culture medium surface, dry for several minutes, by sterile tweezers will drug susceptibility were flat piece of paper stick in the M-H culture medium surface, the distance between each piece of paper > 24 mm, pieces of > 15 mm from the flat. Will the culture dish with a piece of paper in 37 ℃incubation after 24 hours, with mm feet measurement bacteriostatic annulus diameters (bacteriostatic annulus to the edge of the naked eye can't see bacteria obvious growth limit),According to the size of the bacteriostatic annulus diameters reflect the bacteria on the determination of the drug test sensitive degree .Results: 2006 NCCLS standard to judge, and each test all use escherichia coli bacteria strains of the standard quality A TCC is 25922. 4.PCR technology:AmpC gene detection of three-dimensional test positive strains.References to specific design 6 primers, using common plasmid small mention kit extraction escherichia coli plasmid DNA for polymerase chain reaction (PCR) template .Each specimen use to design good respectively six primer amplification, positive amplification fragment length were 520 bp, 462 bp, 405 bp, 346 bp, 302 bp, 190 bp .PCR products were agarose gel electrophoresis, UV detector observations.5. Gene sequencing:Plasmid AmpC enzyme gene amplification-positive strains, respectively, to take the PCR amplification products the nucleic quantitative instrumentmeasured the DNA concentration> 300ug/ml, and sent to Shanghai Sangon biotechnology companies were sequenced. Results: 1. 11 strains harboring AmpC were screened, and 8(11.27%) strains were found by cefoxitin three-dimensional extract test. 2.Antimicrobial susceptibility test results:71 Escherichia coli susceptibility results show the whole sensitive to imipenem, amikacin, cefoxitin, aztreonam, cefepime, sensitive rate is higher are more than 59.15%, lower cephalosporincefotaxime, streptomycin, gentamicin, ciprofloxacin, levofloxacin. Multi-drug resistant strains, 24 (33.80%) and 35 double-resistant strains dominated (33.80%), including the mode of multi-drug resistance phenotype of GEN + CTX + LEV (16 strains, 22.54%)-based. Resistant strains producing AmpC enzyme and non-producing AmpC strain rate: AmpC producing strains of a variety of commonly used antibiotics resistance rates significantly higher than non-AmpC producing strains (χ 2 test, P <0.05).4.PCR detected by plasmid AmpC enzyme gene situation: three-dimensional experimental 8-positive strains for PCR amplification, two positive, sequencing results showed that the two are DHA-1 type.Conclusions: 1. In the region of clinical isolates of Escherichia coli producing ESBLs situation is more serious, the AmpC enzyme is relatively small, and the phenomenon of multiple drug resistance has emerged. 2 in the region of the Escherichia coli plasmid-mediated AmpCenzyme genotypes of DHA-1, resistant strains producing AmpC enzyme was significantly higher than non-AmpC producing strains. (3) in the region of Escherichia coli detected the two suspected SSBLs, the genotype combination of both DHA-1 AmpC and CTX-M type of ESBLs.Key words: Escherichia coli; AmpC gene; multi-drug resistant前言大肠埃希菌为革兰氏阴性杆菌，属于肠杆菌科，是寄居于人和动物肠道中的正常菌群之一。

产志贺毒素大肠埃希菌感染治疗的研究进展
郭沁园;方芳;刘伟佳;薛云新;王岱
【期刊名称】《中国抗生素杂志》
【年(卷),期】2022(47)12
【摘要】产志贺毒素大肠埃希菌是一种常见的食源性病原菌,近年来世界各地都有产志贺毒素大肠埃希菌的疫情暴发,但是目前临床上却没有可以有效治疗这种细菌感染的方法。

产志贺毒素大肠埃希菌产生的志贺毒素容易使患者出现溶血性尿毒综合征等并发症,大大增加了病原菌感染治疗的难度。

本文主要就近年来产志贺毒素大肠埃希菌感染治疗方面的研究进展进行综述,以期为研究有效的治疗方法提供新思路。

【总页数】7页(P1242-1248)
【作者】郭沁园;方芳;刘伟佳;薛云新;王岱
【作者单位】分子疫苗学和分子诊断学国家重点实验室
【正文语种】中文
【中图分类】R978.1
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