Identification of a truncated Tn1721-like transposon located on a small plasmid of Escherichia coli isolated from Varanus indicus

The 9.1 kb plasmid pDEWT1 was isolated from an Escherichia coli strain obtained from the faeces of a free-living lizard (Varanus indicus) in Indonesia. This plasmid mediated tetracycline resistance via a tet gene of hybridization class A. Molecular analysis of a 7755 bp segment of plasmid pDEWT1 including the tetR-tet(A) region and its flanking areas suggested that pDEWT1 harboured a truncated copy of the tet(A)-carrying transposon Tn1721 in which the part responsible for chemotaxis and transposition functions was lost. Analysis of the sequences at the integration site revealed the presence of the 5-bp direct repeat TACTT. The sequences upstream and downstream of the integration site showed striking homology to sequences of a non-coding region detectable on a small cryptic plasmid from Yersinia enterocolitica.     

蛋源大肠埃希菌的分离鉴定及毒力基因和耐药基因检测

为了调查鸡蛋中大肠埃希菌的感染情况,为生产实践提供参考,从昆明市区鸡蛋中分离出了3株大肠埃希菌,并对其毒力基因、耐药基因、动物致病性及药物敏感性进行了研究。结果表明,菌株KM1携带的毒力基因检出率75%,耐药基因检出率77.3%;菌株KM2携带的毒力基因和耐药基因检出率居中;菌株KM3毒力基因检出率91.7%,耐药性基因检出率50%。动物毒力检测结果 KM1为不致病菌株,KM2为低致病菌株,KM3为中度致病菌株。药敏试验结果显示,分离菌株对头孢噻肟、头孢曲松钠敏感,对庆大霉素、氨苄西林等具有不同程度的耐药性。中草药对3株大肠埃希菌的抑制效果各有所不同,相比之下连翘与松针的效果较好。本试验为研究昆明市区鸡蛋中大肠埃希菌污染与防控提供了理论依据。     

白鹭大肠埃希菌的分离鉴定与药敏试验

对3份白鹭新鲜粪便进行细菌分离纯化、染色镜检和生化鉴定,并用Kirby-Bauer法对分离细菌用30种抗菌药进行了耐药性试验。结果表明,分离到3株革兰阴性小杆菌为大肠埃希菌,分别编号为E1、E2和E3。3株白鹭大肠埃希菌都对青霉素G、阿莫西林、万古霉素和洁霉素耐药,对常用广谱抗菌药物有2重以上耐药性,但对试验用的氨基苷类抗生素、喹诺酮类药物和复方新诺明高度敏感。     

Non‐O157 Shiga Toxin‐producing Escherichia coli Isolated from Diarrhoeic Calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin‐producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx₁ gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H‐ (n = 4), O26:H11 (n = 4), O26:H‐ (n = 1), O111:H‐ (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp‐2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H‐ showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non‐typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

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。     

黑颈鹤源大肠杆菌的快速鉴定及致病特性的初步研究

对黑颈鹤病原菌进行快速鉴定及毒力因子分析。采用16S rRNA序列分析法鉴定黑颈鹤病原菌,其毒力基因分别用大肠杆菌的irp2、papC、iucD、tsh、iss毒力基因的特异性引物进行PCR检测,并将扩增出来的irp2基因序列与Genbank~（TM）相应序列进行同源性分析。分离菌株鉴定为大肠埃希氏菌,鉴定结果与生化检测结果一致,分离菌携带irp2毒力基因,这从毒力试验得到证实,其序列与与Genbank~（TM）上发表的HPI irp2基因序列同源性高达98%以上。采用16S rRNA序列分析法可以对黑颈鹤大肠埃希氏菌感染进行快速鉴定,黑颈鹤源性大肠杆菌携带有irp2毒力基因。     

扎布耶盐山羊大肠杆菌分离鉴定及药敏试验

为掌握西藏自治区仲巴县帕江乡扎布耶盐山羊的发病情况,采集发病羊的粪便、血液、肝脏、脾脏、胸腔、腹腔和心包腔内蓄积的浆液性纤维性渗出物等进行病原菌分离鉴定及药敏试验。经病原菌分离鉴定,该地区扎布耶盐山羊羔羊感染的病原菌为大肠杆菌,检出率为97.14%(34/35);药敏试验结果表明,该研究分离得到的大肠杆菌对头孢唑林、青霉素、阿莫西林、头孢他啶、氟苯尼考、头孢拉啶6种药物高度敏感。该调查研究为仲巴县帕江乡扎布耶盐山羊大肠杆菌病的防治提供了诊断方法和用药方案。     

Anti‐microbial Susceptibility for east1+Escherichia coli Isolated from Diarrheic Pigs in Korea

The in vitro susceptibilities of 128 isolates of east1⁺Escherichia coli from pre‐weaned and post‐weaned pigs with diarrhoea were tested with nine commonly used anti‐microbial agents by an agar dilution minimal inhibitory concentration (MIC) procedure according to National Committee for Clinical Laboratory Standards guidelines. For the isolates from pre‐weaned and post‐weaned pigs, most of them were susceptible to low concentrations (MIC₉₀) of tetracycline (4 and 2 μg/ml), ceftiofur (2 and 2 μg/ml), and colistin (4 and 2 μg/ml). Marked resistance was found in others.     

Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug‐Resistant Escherichia coli Isolated from Healthy Companion Animals

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug‐resistant E. coli (n = 36; MDR, resistance to ≥2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside‐ and/or trimethoprim‐resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β‐lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim‐sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.     

鸡舍内外环境中气载大肠杆菌同源性的分子鉴定

采用ANDERSEN-6级空气微生物样品收集器和RCS离心式采样器在5个鸡场舍内空气、舍外上风向和下风向不同距离收集气载大肠杆菌;并收集鸡的粪便,分离大肠杆菌。利用大肠杆菌基因间重复一致序列为引物的聚合酶链式反应（ERIC-PCR）分型技术,扩增不同测量点收集的大肠杆菌的DNA图谱。通过每个采样点的大肠杆菌的浓度变化以及大肠杆菌遗传相似性分析确认动物舍微生物气溶胶向舍外环境的传播。结果显示：5个鸡舍内空气中大肠杆菌的浓度远远高于舍外上风和下风向的大肠杆菌浓度（P〈0.05或P〈0.01）,但是舍外不同距离间的大肠杆菌浓度差异并不显著（P〉0.05）。同样,ERIC-PCR结果表明,从鸡的粪便中分离的大肠杆菌与从舍内空气中分离的部分大肠杆菌（34.1%）,以及从鸡场舍外下风方向分离到的多数大肠杆菌（54.5%）与从舍内空气或粪便中分离的大肠杆菌相似性可达100%。而从鸡舍上风向分离到的大肠杆菌与从舍内空气或粪便中分离的大肠杆菌相似性为73%-92%。从而说明来自动物体的大肠杆菌既能污染舍内空气,又能对其周围的环境构成污染。本研究揭示了微生物气溶胶的传播规律,具有公共卫生及流行病学意义。     

鸡源大肠埃希菌的耐药性与分子分型研究

为了解规模化养殖厂鸡源大肠埃希菌的耐药性以及分子流行规律和特征,为该菌的分子溯源及风险评估提供依据,本研究收集2013-2014年上海地区养殖场分离的鸡源大肠埃希菌共计741株,采用微量稀释肉汤法分别对常用的9类13种抗菌药物进行药物敏感性测定,以了解大肠埃希菌的耐药表型。在筛选出多重耐药严重的菌株后,同时采用脉冲场凝胶电泳(PFGE)分型方法及核糖体(RP)分型方法进行分子分型分析。结果显示,2013-2014年所分离的鸡源大肠埃希菌对氨苄西林、阿莫西林/克拉维酸、大观霉素、四环素、多西环素、氟苯尼考、磺胺异噁唑、复方新诺明、氧氟沙星和恩诺沙星的耐药率较高,在50%以上,且主要为多重耐药菌株。用XbaⅠ酶切后进行PFGE分析,结果表明,29株鸡源大肠埃希菌可分为22个带型;EcoRⅠ酶切后的RP聚类分析结果表明,鸡源大肠埃希菌分为14个带型。鸡源大肠埃希菌耐药率较高,同一分型菌株的耐药谱型并非完全一致,菌株基因型呈多态性。     

内蒙古地区羊源大肠杆菌耐药性研究

为确定内蒙古地区羊源大肠杆菌的耐药表型及其耐药基因的流行情况,本研究采用微量稀释法测定了内蒙古地区108株羊源大肠杆菌对13种临床常用抗菌药物的最小抑菌浓度。结果显示,分离菌株对阿莫西林、头孢噻吩、磺胺甲唑、黏菌素的耐药率最高,均达100.0%,对阿莫西林/克拉维酸、四环素、环丙沙星的耐药率在50%~80%之间,对头孢噻肟、美洛培南、新霉素的耐药率均低于10%,较为敏感。108株羊源大肠杆菌中耐7种以上药物的菌株占94.4%,其中15.6%菌株对13种抗菌药物耐药,只有1株菌对所有抗菌药物敏感。采用PCR方法对羊源大肠杆菌分离株所携带的6种相关耐药基因进行检测,结果显示,6种耐药基因中的4种耐药基因bla_TEM、proP-2、sul-Ⅰ、ampG检出率超过50%,耐药基因aph（3'）-Ⅰ携带率达40%,只有耐药基因aac（3）-Ⅱ检出率仅为5.5%。由此可见,内蒙古地区羊源大肠杆菌对13种抗菌药物产生了不同程度的耐药性,且存在严重的多重耐药情况,羊源大肠杆菌分离株携带aph（3'）-Ⅰ、sul-Ⅰ、ampG、bla_TEM、proP-2、aac（3）-Ⅱ耐药基因。     

鸡致病性埃希氏大肠杆菌不同菌株间同源性抗原的研究

通过兔制备了３株引起鸡败血症的埃希氏大肠杆菌（Ｅ．ｃｏｌｉ）高免血清,对分离自新疆不同地区的３０余株鸡致病性Ｅ．ｃｏｌｉ进行了玻板凝集试验和双向免疫扩散试验。结果证明,在琼扩试验中,不同的Ｅ．ｃｏｌｉ菌株间存在着同源性抗原成份,这种同源性抗原在绝大多数Ｅ．ｃｏｌｉ间都有数种之多。但是,这种相互间的同源抗原在玻板凝集试验中有时并不能表现,甚至有沉淀线出现的血清与抗原之间在玻板凝集试验中也不能检测出来。     

Study on the Drug-resistance of Escherichia coli Isolated from Sheep in Inner Mongolia

In order to determine the drug resistance phenotype and prevalence of drug resistance genes in Escherichia coli (E.coli) isolated from sheep in Inner Mongolia, the minimum inhibitory concentrations (MICs) of the isolates to antibiotics commonly used in veterinary clinical were detected by micro-dilution method in vitro. The results showed that the highest resistance rates of the isolates to amoxicillin,cephalothin,sulfamethoxazole and polymyxin were up to 100.0%,respectively.To amoxicillin-clavulanic, tetracycline and ciprofloxacin were 50% to 80%. These isolates were sensitive to cefotaxime, meropenem trihydrate, neomycin, and their resistance rates were all less than 10%. Among the 108 strains of E.coli from sheep, 94.4% of them were resistant to more than 7 antimicrobial agents,15.6% of them were resistant to 13 antimicrobial agents, only one strain was sensitive to all antimicrobial agents. Six kinds of resistance genes among the 108 E.coli isolates were detected by PCR method.The results showed that detection rates of 4 kinds of drug resistance genes including bla_TEM, proP-2, sul-Ⅰ and ampG were all over 50%,the detection rate of resistance gene aph (3')-Ⅰ was up to 40%, only resistance gene aac(3)-Ⅱ detection rate was 5.5%. Thus, the sheep E.coli isolates in Inner Mongolia produced various degrees of resistance to 13 kinds of antibiotics, and their multi-drug resistances were very serious. They carried aph(3')-Ⅰ, sul-Ⅰ, ampG, bla_TEM, proP-2 and aac(3)-Ⅱ resistance genes.     

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.     

Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended‐Spectrum β‐Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended‐spectrum β‐lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K‐:H2) was observed among 17 EPEC strains, the O92:K‐serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K‐serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla_TEM gene was demonstrated in the majority of ampicillin‐resistant isolates. The aac(3)‐II or aac(3)‐IV genes were detected in the four gentamicin‐resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin‐resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline‐resistant isolates. A total of nine EPEC isolates showed the phenotype SXT‐resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.     

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。     

Low Occurrence of Extended‐Spectrum β‐lactamase‐Producing Escherichia coli in Finnish Food‐Producing Animals

ESBL/AmpC‐producing Escherichia coli is increasingly isolated from humans and animals worldwide. The occurrence of ESBL/AmpC‐producing E. coli was studied in food‐producing animals in Finland, a country with a low and controlled use of antimicrobials in meat production chain. A total of 648 cattle, 531 pig, 495 broiler and 35 turkey faecal samples were collected from four Finnish slaughterhouses to determine the presence of extended‐spectrum β‐lactamase (ESBL/AmpC)‐producing E. coli. In addition, 260 broiler and 15 turkey samples were screened for carbapenemase‐producing E. coli. Susceptibility to different class of cephalosporins and meropenem was determined with disc diffusion tests according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Determination of ESBL/AmpC production was performed with a combination disc diffusion test according to the recommendations of the European Food Safety Authority (EFSA). Plasmidic bla_ESBL/AmpC genes were characterized by polymerase chain reaction and sequencing. A collection of isolates producing AmpC enzyme but not carrying plasmidic bla_AmpC was analysed by PCR and sequencing for possible chromosomal ampC promoter area mutations. Altogether ESBL/AmpC‐producing E. coli was recovered from five cattle (0.8%), eight pig (1.5%) and 40 broiler samples (8.1%). No ESBL/AmpC‐producing E. coli was found in turkey samples. Carbapenem resistance was not detected. Altogether ESBL/AmpC‐producing E. coli was found on 4 (2.0%), 3 (4.5%) and 14 (25%) cattle, pig and broiler farms, respectively. From cattle samples 3 (27%) bla_CTX‐M‐1 and from broiler samples 13 (33%) bla_CTX‐M‐1 and 22 (55%) bla_CMY‐2 gene‐carrying isolates were detected. In pigs, no plasmidic bla_ESBL/AmpC gene‐carrying isolates were found. In all analysed isolates, the same mutations in the promoter region of chromosomal ampC were detected. The results showed low occurrence of ESBL/AmpC‐producing E. coli in Finnish food‐producing animals. In pigs, plasmidic bla_ESBL/AmpC‐carrying E. coli was not detected at all.     

一株不发酵乳糖致病性大肠杆菌的分离与鉴定

从广东某珍珠鸡场发病死亡的初产珍珠鸡肝脏中分离到一株细菌,经培养特性与生化特性观察,以致病性试验和血清型鉴定,结合其16SrRNA序列分析,判定其为大肠杆菌。这是一株不发酵乳糖的致病性大肠杆菌。