奶牛乳房炎分离大肠杆菌的耐药性及脉冲场凝胶电泳分型研究

为了解宁夏地区奶牛乳房炎分离大肠杆菌的耐药表型及同源性特征,本试验采用肉汤微量稀释法测定了16种抗菌药物对66株奶牛乳房炎分离大肠杆菌的最低抑菌浓度(MIC),同时进行了脉冲场凝胶电泳(PFGE)分子分型研究.结果显示,66株大肠杆菌对氨苄西林、链霉素、四环素、多西环素、头孢菌素类抗生素和磺胺甲氧嘧啶的耐药率都比较高,而对阿米卡星、卡那霉素、多粘菌素、氟苯尼考及喹诺酮类药物则比较敏感.分离菌株最多耐受15种药,且主要以8耐、5耐和7耐为主.66株大肠杆菌中有54株能被PFGE分型,可分为A-a 27个型.分型结果显示,宁夏地区的奶牛场分离的菌株之间既存在垂直传播也存在水平传播,同一养殖场不同奶牛之间和不同养殖场的奶牛之间存在多重耐药大肠杆菌的克隆传播.     

猪源大肠杆菌耐药性质粒的监测研究

在１１５株猪大肠杆菌耐药性监测的基础上,提取耐药类型最普遍的５株耐药菌的质粒进行质粒指纹图谱分析。结果表明：同一来源、耐药类型相同的菌株有相似的质粒图谱,来源不同者,酶切图谱有提示出它们之间的同源性,说明质粒指纹图谱可作为流行病学调查的工具。     

四川规模化猪场仔猪黄、白痢的分子流行病学研究

在系统鉴定、药敏试验和血甭型鉴定的基础上,对52株致病性大肠杆菌和4株标准血清型大肠杆菌进行了质粒DNA分析,结果表明,菌株的质粒得率为100%,来源相同的菌株具有相同或相似的质粒图谱,来源不同的菌株具有不同的质粒图谱,质粒DNA分析为四川规模化猪场致病性E.coli的分子流行病学调查提供了科学依据。质粒图谱与血清型及耐药谱之间无明显关系。     

某规模化种鸡场沙门菌流行病学调查和耐药性分析

试验旨在调查国内某规模化种鸡场沙门菌流行状况,为种鸡场沙门菌净化提供基础资料。1 744份样品被采集进行沙门菌分离、血清型鉴定、耐药性分析及脉冲场凝胶电泳(PFGE)方法亚分型。结果显示:168株沙门菌被分离鉴定,分离率为9.63%,其中肠炎沙门菌有157株,为优势血清型(93.45%);抗生素耐药性试验表明,多重耐药现象较为严重,耐药种类在三种以上的肠炎沙门菌株有130株(77.38%);随机选取28株肠炎沙门菌进行PFGE亚分型显示,这些菌株属于同一个聚类簇,表明它们可能来自于同一克隆株。     

养殖场中动物源大肠杆菌blaCTX-M-27基因的传播特征探究

从2014年和2016年保存的来自河南、广东、山东和湖北4个省1 100株食品动物肠道大肠杆菌中筛选出58株blaCTX-M-27阳性菌株,采用药敏试验测定产blaCTX-M-27的大肠杆菌对20种抗生素的敏感性,并用PCR的方法检测其所携带的其他耐药基因;通过脉冲场凝胶电泳分析各菌株之间的亲缘关系,通过接合或转化试验、复制子分型和Southern blot对携带blaCTX-M-27的质粒特征进行分析。结果显示,58株blaCTX-M-27阳性大肠杆菌对除了β-内酰胺类外的其他抗生素,尤其氟喹诺酮类呈现高水平耐药;大多数菌株还同时携带2~3种编码其他抗生素的耐药基因。PFGE分型结果表明,来源于同一采样地及不同省的菌株存在克隆现象,但不同省或来自于同一省份的不同采样地之间的菌株亲缘关系较远;菌株携带的blaCTX-M-27基因全部定位在质粒上,其中38株位于可接合的IncFIB质粒,另外20株位于不可分型的质粒。     

猪源耐黏菌素大肠杆菌PFGE分型及耐药性研究

为了解河南省焦作市、许昌市规模化养殖厂耐黏菌素大肠杆菌的流行情况,对分离到的32株耐黏菌素大肠杆菌使用微量肉汤稀释法检测其药物敏感性,并采用脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)对分离株进行分型研究。结果显示,32株耐黏菌素大肠杆菌对四环素、多西环素、磺胺异噁唑、复方新诺明的耐药率均高达90%以上,32株猪源大肠杆菌可分为30种带型。结果表明,两地市养殖场猪源大肠杆菌耐药性普遍存在,且以多重耐药为主,PFGE分型结果显示,32株耐黏菌素大肠杆菌整体上表现出较大的遗传多样性,但不同菌株存在着不同程度的亲缘关系。     

大雁、斑头雁和天鹅源沙门氏菌血清及脉冲场凝胶电泳分型研究

为了解大雁、斑头雁及天鹅源沙门氏菌流行特征和脉冲场凝胶电泳（PFGE）分型,本研究对33株沙门氏菌进行血清型鉴定,采用PFGE对其进行基因分型,并用BioNumerics 6.6软件进行聚类分析。鉴定出丙型副伤寒沙门氏菌6株、汤卜逊沙门氏菌19株、乙型副伤寒沙门氏菌1株、伊鲁木沙门氏菌1株、奥斯陆沙门氏菌1株、5株未定型。PFGE聚类分型共分为16个型别,其相似度为53.7%~100.0%,且大多数菌株与人源菌株相似度超过80%。结果表明,大雁、斑头雁及天鹅源沙门氏菌相同血清型PFGE带型高度相似,可能来自同一克隆株,不同血清型PFGE带型差别较大。     

食品动物源沙门氏菌质粒介导喹诺酮类耐药基因的检测与分析

为了解食品动物源沙门氏菌质粒介导喹诺酮类耐药性(Plasmid-mediated quinolone resistance,PMQR),采用微量肉汤稀释法和PCR方法,检测了316株食品动物源沙门氏菌对20种抗菌药物的敏感性,以及菌株中PMQR基因的携带率.结果显示:316株沙门氏菌对20种抗菌药物呈不同程度的耐药性,95.57％菌株为多重耐药菌;316株菌中未检出qnrA、qnrC、qnrD、qnrS和qepA基因,7.91％菌株检出qnrB基因,15.19％菌株检出aac(6 ′ )-Ib-cr基因,7.91％菌株检出oqxA基因,8.86％菌株检出oqxB基因,这是首次在沙门氏菌中发现oqxAB基因;98.11％PMQR阳性菌同时携带2种及以上的耐药基因,呈8～17耐的多重耐药性,其中以qnrB和aac(6′)-Ibcr基因型为主;53株PMQR阳性菌分属于5种不同的基因型,耐药表型或耐药基因型不同的菌株却有相同的PFGE谱型.本次检测的316株食品动物源沙门氏菌耐药较为严重;菌株主要携带qnrB、aac(6 ′ )-Ib-cr及oqxAB基因;不同来源菌株存在同一耐药克隆株的流行.     

规模羊场环境气载产气荚膜梭菌耐药性及其ERIC-PCR分型

为监测不同规模羊场空气中分离的气载产气荚膜梭菌耐药性变迁及不同菌株间的遗传相似性,从而为临床治疗和控制感染提供依据,收集了2016—2017年来自不同规模羊场分离的气载产气荚膜梭菌30株,采用K-B法进行药敏试验,应用基因间重复序列聚合酶链反应(ERIC-PCR),对菌株进行DNA分型及同源性分析。结果显示:30株气载产气荚膜梭菌在监测的9种药物中,对庆大霉素的耐药性最强(86.7%),其次为青霉素(43.3%),对头孢噻肟耐药率最低(1%),对其他药物耐药率在3.3%~36.7%之间;ERIC-PCR谱型表现为14个基因型(Ⅰ~XIV),其中大部分来源于不同的克隆株,且有2个克隆株的相似度为99.0%。本研究结果表明:规模羊场的气载产气荚膜梭菌多重耐药较严重,分子多样性复杂;气载产气荚膜梭菌来源广泛,不同地区、环境和条件下,同一种病原菌的基因型也存在一定差异,可能存在多个基因型;同一个规模羊场的基因型也不同,不同羊场之间也存在相同的基因型。     

猪源耐药大肠杆菌质粒及酶切图谱分析

为了解河北省猪源耐药大肠杆菌的质粒携带情况,追踪耐药菌株的同源性,试验对已经过系统鉴定及药敏试验的12株猪源大肠杆菌进行质粒提取并使用限制性内切酶HindⅢ酶切,进行质粒谱型分析,探讨大肠杆菌耐药性与质粒及其酶切图谱之间的关系。结果表明:分离菌株质粒的获得率为100%;来源相同的菌株一般有相同或相似的质粒酶切图谱,来源不同的菌株酶切图谱大多不同,但亦有相同的。说明河北省不同地区的大肠杆菌也有同源性菌株存在;大肠杆菌的质粒携带与细菌的耐药性之间并没有明显的对应关系。     

Dissemination of antimicrobial resistant strains of Campylobacter coli and Campylobacter jejuni among cattle in Washington State and California

The purpose of this study was to investigate the genetic similarity of Campylobacter jejuni and Campylobacter coli with similar antimicrobial resistance phenotypes, isolated from cattle on different farms and at different times, in order to evaluate the possible existence of disseminated antimicrobial resistant clones. PFGE after SmaI and KpnI restriction identified 23 and 16 distinct PFGE patterns among 29 C. jejuni and 66 C. coli isolates, respectively. In C. coli, 51 (77%) of the resistant isolates demonstrated one of the four indistinguishable PFGE patterns, whereas only 24% doxycycline resistant C. jejuni shared one of the two indistinguishable PFGE patterns. The genetic mechanisms of resistance were homogeneous within and between these clonal types. Genetically indistinguishable (clonal) groups of C. coli accounted for most Campylobacter sp. with multiple antimicrobial resistance observed in this study, consistent with a role for clonal dissemination in the epidemiology of resistance in this species.     

屠宰前鸡、猪源大肠杆菌耐药性调查

本试验旨在了解屠宰前鸡、猪源食品动物体内大肠杆菌耐药情况,分析潜在的食品安全问题。从广州市畜禽交易市场随机采集待屠宰鸡和猪的粪便样品,分离鉴定大肠杆菌,并采用琼脂稀释法检测大肠杆菌对15种抗菌药物的敏感性。结果显示,从658份猪源样品和133份鸡源样品中共分离鉴定出731株大肠杆菌,其中猪源606株,鸡源125株。药敏试验结果显示,731株大肠杆菌均表现出不同程度的耐药,耐药谱广且多重耐药现象严重。对复方新诺明和四环素的耐药率为90.0%以上,仅对头孢西丁、黏菌素和阿米卡星较敏感(耐药率均低于3%)。鸡源大肠杆菌对头孢噻肟、头孢曲松、新霉素、阿米卡星、萘啶酸和环丙沙星的耐药率显著高于猪源大肠杆菌(P＜0.05)。鸡源大肠杆菌中3耐及3耐以上的菌株占97.60%,猪源大肠杆菌占94.72%。结果表明,屠宰前畜禽体内大肠杆菌对临床常用抗菌药物的耐药性非常严重,以多重耐药为主,且耐药谱丰富多样。提示屠宰前畜禽携带的耐药菌对食品安全和人类健康存在较大的安全隐患。     

3株猪源大肠埃希氏菌耐药基因的初步定位

本试验旨在对临床分离的猪源大肠埃希氏菌耐药基因进行初步定位。采用常规细菌分离培养、16S rRNA PCR扩增和序列测定方法从江西省3个规模化猪场送检的子宫脓液中分离鉴定病原菌,并通过质粒提取、转化大肠埃希氏菌DH5α感受态细胞及药敏试验对临床分离株的耐药基因进行初步定位。结果显示,分离鉴定到3株大肠埃希氏菌,其中JX-22分离株仅对氧氟沙星、大观霉素敏感,JX-26分离株仅对链霉素、氧氟沙星等4种药物敏感,JX-28分离株仅对氧氟沙星等3种药物敏感,均为多重耐药菌;3株大肠埃希氏菌均可纯化到分子质量大小不一的质粒。分离株、质粒转化菌及大肠埃希氏菌DH5α感受态细胞药敏试验对比结果显示,3株大肠埃希氏菌的耐链霉素、林可霉素、甲硝唑、氨苄西林、阿莫西林、大观霉素、丁胺卡那基因,JX-22和JX-26分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐头孢曲松基因,JX-28分离株的耐头孢曲松、头孢噻肟、诺氟沙星基因均定位于细菌质粒上;JX-28分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐诺氟沙星基因和JX-26分离株的耐头孢曲松、头孢噻肟基因均定位于其染色体上;3株分离株均无氧氟沙星耐药基因。本试验初步确定3株多重耐药猪源大肠埃希氏菌的大部分耐药基因定位于质粒上,为进一步研究猪源大肠埃希氏菌的耐药机理和有效控制措施奠定基础。     

Clonal spread of antimicrobial-resistant Escherichia coli isolates among pups in two kennels

Although the dog breeding industry is common in many countries, the presence of antimicrobial resistant bacteria among pups in kennels has been infrequently investigated. This study was conducted to better understand the epidemiology of antimicrobial-resistant Escherichia coli isolates from kennel pups not treated with antimicrobials. We investigated susceptibilities to 11 antimicrobials, and prevalence of extended-spectrum β-lactamase (ESBL) in 86 faecal E. coli isolates from 43 pups in two kennels. Genetic relatedness among all isolates was assessed using pulsed-field gel electrophoresis (PFGE). Susceptibility tests revealed that 76% of the isolates were resistant to one or more of tested antimicrobials, with resistance to dihydrostreptomycin most frequently encountered (66.3%) followed by ampicillin (60.5%), trimethoprim-sulfamethoxazole (41.9%), oxytetracycline (26.7%), and chloramphenicol (26.7%). Multidrug resistance, defined as resistance against two or more classes of antimicrobials, was observed in 52 (60.5%) isolates. Three pups in one kennel harboured SHV-12 ESBL-producing isolates. A comparison between the two kennels showed that frequencies of resistance against seven antimicrobials and the variation in resistant phenotypes differed significantly. Analysis by PFGE revealed that clone sharing rates among pups of the same litters were not significantly different in both kennels (64.0% vs. 88.9%), whereas the rates among pups from different litters were significantly different between the two kennels (72.0% vs. 33.3%, P < 0.05). The pups in the two kennels had antimicrobial-resistant E. coli clones, including multidrug-resistant and ESBL-producing clones. It is likely that resistant and susceptible bacteria can clonally spread among the same and/or different litters thus affecting the resistance prevalence.     

THE ISOLATION OF ANTIBIOTIC RESISTANT COLIFORMS FROM MEAT AND SEWAGE

Beef and pig carcases, meat products, frozen chickens, and sewage were examined in 3 separate surveys for antibiotic resistant coliforms. Escherichia coli was isolated from 18 of 50 beef carcases; the numbers were low and resistance was found only to tetracycline. E. coli was isolated from 45 of 50 pig carcases; the numbers were high and showed a range of patterns of multiple antibiotic resistance. In meat products, the proportion of E. coli in contaminating organisms was low, and most resistance found was to tetracycline and streptomycin. E. coli was isolated from 66 of 75 chickens and these gave 23 patterns of antibiotic resistance, often multiple. Sewage from hospital or domestic origin and abattoir effluent yielded approximately 10⁶ coliforms/ml, most of which were resistant to one or more antibiotics; few of those from hospital or domestic origin however, were classified as E. coli of faecal origin. Twenty-four patterns of resistance were found in coliforms from domestic sewage, 19 from hospital sewage and 11 from abattoir effluent. Transfer of resistance, often multiple, was achieved from 55% of 447 resistant strains to an E. coli K₁₂ recipient. Much more information is required on the prevalence of R-factors in bacteria associated with food producing animals and their products.     

Isolation,Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets

In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.     

Antibiotic resistance among indicator bacteria isolated from healthy pigs in New Zealand

AIM: To determine the resistance to antibiotics among the indictor bacteria, Escherichia coli and Enterococcus spp, isolated from the faeces of healthy pigs on three conventional pig farms and one organic farm in the North Island of New Zealand. METHODS: Faecal samples, collected at intervals between March and October 2001, were plated onto MacConkey agar and Slanetz-Bartley agar and examined after 1-3 days incubation for colonies resembling E. coli and Enterococcus spp, respectively. Typical colonies were subcultured for further identification and storage. The isolates were tested for antibiotic resistance, using disc diffusion, to ampicillin, gentamicin, streptomycin, and tetracycline. Escherichia coli isolates were also tested for resistance to ciprofloxacin, cotrimoxazole and neomycin. Enterococcus spp isolates were also tested for resistance to vancomycin, erythromycin and virginiamycin. RESULTS: A total of 296 E. coli and 273 Enterococcus spp isolates were obtained from the three conventional farms, and 79 E. coli and 80 Enterococcus spp isolates were obtained from the organic farm. All the E. coli isolates from both the conventional and organic pig farms were susceptible to ciprofloxacin, and all the Enterococcus spp isolates were susceptible to ampicillin, gentamicin and vancomycin. Isolates of E. coli from conventional pig farms were resistant to gentamicin (0.7%), neomycin (0.7%), ampicillin (2.7%), cotrimoxazole (11%), streptomycin (25%) and tetracycline (60%). Enterococcus spp isolates from the same farms were resistant to erythromycin (68%), tetracycline (66%), streptomycin (54%) and virginiamycin (49%). By contrast, for the organic pig farm 相似文献   

腹泻仔猪肠道致病性大肠埃希氏菌和沙门氏菌的分离鉴定与耐药性研究

为探明仔猪细菌性腹泻肠道致病性大肠埃希氏菌和沙门氏菌流行的血清型、耐药表型及耐药基因型,本试验采集了贵州省5个地（州）市7个规模化养猪场的128份腹泻仔猪肠道样本,并对采集的样本进行了大肠埃希氏菌和沙门氏菌分离与鉴定,通过动物试验鉴定菌株的致病性,利用血清学方法鉴定其血清型,并通过药敏纸片法对主要致病菌进行耐药性研究,采用PCR技术检测各致病菌株耐药相关基因,分析细菌耐药表型和耐药基因型相关性。结果显示,本研究共分离鉴定到78株致病性大肠埃希氏菌与21株沙门氏菌,致病性大肠埃希氏菌血清型以O138、O87为主,沙门氏菌血清型以鼠伤寒沙门氏菌、肠炎沙门氏菌居多;药敏试验结果表明,本试验分离到的78株致病性大肠埃希氏菌对β-内酰胺类药物耐药率达80%以上,对其他种类的抗菌药耐药率均超过40%,分离鉴定的21株沙门氏菌对氨基糖苷类药物耐药率达50%以上,对其他种类的抗菌药耐药率均达20%以上;本试验分离鉴定的致病性大肠埃希氏菌共检出12种耐药相关基因,沙门氏菌共检出10种耐药相关基因,两种细菌耐药基因型与耐药表型符合率均达60%以上,且均为多重耐药。本研究为仔猪腹泻的综合防控提供了理论依据。     

Study on Effect of Nitrogen and Phosphorous on the Resistance of E.coli to Chloramphenicol and its Mechanism

This study was designed to explore the effect of nitrogen and phosphorous on the resistance of E.coli from environment and the mechanism.Microcosms were established to study the effect of nitrogen and phosphorous on the resistant phenotype of E.coli to chloramphenicol (CHL).cat gene of isolated drug-resistant strains and susceptible strains were detected.The results showed that,different concentration of nitrogen and phosphorous could induce the formation of antibiotics resistance of E.coli to CHL.The rate of cat gene of 46 strains of chloramphenicol resistant E.coli was 89.13%,which was 0 in the 16 strains of chloramphenicol sensitive E.coli.The results indicated that,nitrogen and phosphorous in the microcosms could induce the formation and maintenance of resistance to chloramphenicol in E.coli,which had correlation with cat gene.     

Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources

The aim of this study was to determine the molecular epidemiology of cefoxitin-resistance Escherichia coli identified in cattle entering feedlots and determine if there were any similarities to E. coli causing human infections in Canadian hospitals. A total of 51 E. coli were isolated from a total of 2483 cattle entering four feedlots in southern Alberta, Canada. DNA fingerprinting using pulsed-field gel electrophoresis revealed thirty-two unique patterns with two major clusters observed comprised of Cluster A (11 strains) and Cluster B (7 strains). PCR and sequence analysis revealed 38 isolates (74.5%) harboured bla(CMY-2), whereas the remainder were found to contain mutations in the promoter region of the chromosomal ampC gene, which has been previously associated with cefoxitin resistance. No resistance to nalidixic acid, ciprofloxacin, or amikacin was observed in the clinical isolates. bla(CMY-2) harbouring plasmids were transferred to E. coli DH10B. All of the plasmids carrying bla(CMY-2) contained the A/C replicon and also harboured other resistance genes. Plasmid fingerprinting using BglII revealed 17 unique patterns with all but one clustering within 70% similarity. Comparison of the plasmid fingerprints to those isolated from human clinically significant E. coli in Canada during a similar time period [Mulvey, M.R., Bryce, E., Boyd, D.A., Ofner-Agostini, M., Land, A.M., Simor, A.E, Paton, S., 2005. The Canadian Hospital Epidemiology Committee, and The Canadian Nosocomial Infection Surveillance Program, Health Canada. Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrob. Agents Chemother. 49, 358-365] revealed four strains that harboured bla(CMY-2) A/C replicon type plasmid with fingerprint similarities of greater than 90% to the ones identified in E. coli from the cattle in this study. These findings highlight the potential linkage of multidrug resistant organisms in food producing animals and human infections in Canadian hospitals. The plasmids conferred resistance to multiple antibiotics which could limit options for the treatment of infections caused by these strains.