DNA probes for the detection of toxin genes in Escherichia coli isolated from diarrhoeal disease in cattle and pigs

DNA gene probes specific for genes coding for heat labile toxin (LT), heat stable toxins (STpa, STpb) and Vero-cell toxins (VT1, VT2) were used to examine 1031 diarrhoeal disease isolates of E. coli (345 from cattle and 686 from pigs). Of the bovine strains, 60 hybridized with the STpa probe and most possessed the K99 (F5) or F41 adhesin. Five bovine strains possessed STpb genes and five either VT1 or VT2 genes. Of the porcine strains, 245 hybridized with one or more gene probes. Of 160 K88 (F4) positive strains, 133 possessed both LT and STpb genes, whilst 17 possessed LT or STpb or STpa alone or in combination. Ten K88 strains did not possess toxin genes. Isolates bearing the K99 (F5) adhesion possessed either STpa, STpb and VT2 genes alone or in combination; in one isolate only the LT gene was detected. Isolates belonging to serogroup 0138:K81 were more heterogeneous as to their toxin genes; of the 60 strains, fourteen carried only VT2 genes, thirty-two carried VT2, STpa and STpb genes, one carried LT, VT2, STpa and STpb genes, two carried STpb gene, four carried STpa and STpb genes, one carried LT and VT2 genes, two carried LT and STpa genes, whilst four carried none. Twenty-four percent of all toxigenic strains apparently did not possess adhesins.     

Identification of putative adhesin genes in shigatoxigenic Escherichia coli isolated from different sources

Shiga toxin-producing Escherichia coli (STEC) is an important pathogen responsible for severe human intestinal and systemic infections. The bacterial factors required for colonization of the hosts are still not well defined. In this study, the prevalence of seven putative adhesive genes that are not encoded in the locus of enterocyte effacement (LEE) in 74 STEC strains isolated from humans (n=39), food (n=6), cattle (n=11), and pigs (n=18) was investigated by PCR. In addition, Shiga toxin (stx) and intimin (eaeA including alpha, beta, gamma, delta, epsilon, zeta variants) genes were tested. The most prevalent adhesin was that encoded by toxB gene (52 of 74 isolates; 70.3%). This marker was found in all 12 strains of O157:H7 serotype and in 23 of 32 (71.9%) isolates of the O157:NM serogroup. Moreover, this gene was also present in other 17 STEC of the non-O157 serogroup. The second most prevalent adhesin was that encoded by the lpfAO157/OI-154 gene (43 isolates; 58.1%). This marker was detected in LEE-positive strains of the O157 serogroup but also in 9 LEE-negative isolates of porcine origin. Several STEC isolates tested (42 strains; 56.7%) had the efa1 gene of the Efa1 putative adhesive marker. This adhesin was almost exclusively found among eaeA-positive strains recovered from humans, food and cattle. On the other hand, iha marker was detected either in LEE-positive (29 isolates) or LEE-negative (12 strains) STEC. Only two eaeA-negative strains had the saa putative adhesive gene. These results show that STEC strains may be able to express several putative adhesins. However, further studies are needed to evaluate the role of the genes identified in the present study in the pathogenesis of human infections.     

Detection of entero- and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction.

Oligonucleotide primers were designed for the specific polymerase chain reaction (PCR) amplification of the enterotoxins STIa and LTI and of the verocytotoxins VT1 and VT2. All of 184 E. coli isolates from cases of diarrhoea from pigs, cattle and sheep gave identical toxin profiles by PCR and gene probe. Differentiation between VT2 and VT2v was achieved using two oligonucleotide primers pairs in PCR and showed that all of 34 VT2+ porcine isolates, of which 23 were 0138:K1, harboured VT2v whereas 20 VT2+ bovine and ovine isolates harboured VT2. No isolate harboured both VT2 polymorphs. Simplified methods for sample preparation for PCR were examined and showed that PCR was not inhibited by direct addition of broth culture to the reaction mixture.     

Characterization and immuno-detection of AIDA-I adhesin isolated from porcine Escherichia coli

A relatively high percentage of porcine Escherichia coli isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). This gene and its corresponding protein were first identified and characterized in E. coli strain 2787 isolated from human infantile diarrhea. Little is known about the properties of the AIDA-I protein and its immuno-detection on surface of AIDA-I positive porcine E. coli isolates. In this study, we demonstrated that the AIDA-I adhesin isolated from porcine AIDA-I positive E. coli is an acidic protein consisting of five isoforms. It has a similar molecular weight (100 kDa) and relatively high amino acid homology (78-87%) with the AIDA-I adhesin expressed by human AIDA-I positive E. coli strain 2787. Based on limited comparison, it appears that there is a very high homology among AIDA-I proteins expressed by porcine AIDA-I positive E. coli isolates. Sensitivity of detection of surface AIDA-I adhesin of PCR-positive AIDA-I E. coli by immuno-dot-blot and coagglutination tests was 76 and 71%, respectively, whereas specificity was 89 and 84%, respectively. These tests are unlikely to be used for diagnostic detection of AIDA-I positive E. coli due to the relatively low sensitivity; however, they may be potentially useful for identification of false positive reactions generated by other diagnostic tests.     

猪和野生动物源大肠杆菌及沙门菌中磺胺类药物耐药基因的检测

对从四川省10个规模化猪场和16种野生动物的粪样中分离鉴定出的67株大肠杆菌、57株沙门菌进行了药敏试验。结果，猪源和野生动物源分离菌对磺胺类药物的耐药率分别为72．0％（72／100）和29．2％（7／24）。根据GenBank中登录的序列，设计了3对特异性引物，对磺胺类药物的耐药基因Sul1、Sul2、Sul3进行了三重PCR检测。在这124株细菌中，Sul1基因的检出率最高，为47．6％（59／124），Sul2基因的检出率为17．7％（22／124），Sul3基因的检出率为18．5％（23／124）。药敏试验结果与基因检测结果的符合率为89．9％。     

Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from animals and food in Argentina (n=44) and Brazil (n=20) were examined and compared in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. The clonal relatedness of STEC O157 isolates (n=22) was established by phage typing (PT) and pulsed-field gel electrophoresis (PFGE). All O157 strains studied carried eae and enterohemorrhagic E. coli (EHEC)-hly sequences. In Argentina, these strains occurred both in cattle and meat, and 50% of them carried stx2/stx2vh-a genes, whereas in Brazil the O157 strains were isolated from animals, and most harbored the stx2vh-a sequence. At least 13 different O:H serotypes were identified among the non-O157 strains studied, with serotype O113:H21 being found in both countries. All but one non-O157 strains did not carry eae gene, but EHEC-hlyA gene was found in 85.7% of them, and the stx2 genotype was also more prevalent in Argentina than in Brazil (P<0.01), where stx1 alone or in association was most common (68.8%). One STEC strain isolated from a calf in Brazil harbored the new variant referred to as stx2-NV206. PFGE analysis showed that STEC O157 strains were grouped in four clusters. One Brazilian strain was considered possibly related (> or =80%) to Argentinean strains of cluster I. Differences in the pathogenic potential, especially in regard to serotypes and stx genotypes, were observed among the STEC strains recovered from animals and food in both countries.     

合肥地区动物源致病性大肠杆菌ESBLs和PMQR基因流行分布

从安徽省合肥地区多个养殖厂分离鉴定了65株致病性大肠杆菌,用PCR方法检测ESBLs和PMQR基因的流行分布情况,用微量肉汤稀释法测定30株ESBLs和/或PMQR阳性菌对16种抗菌药物的最小抑菌浓度.PCR结果显示:ESBLs阳性率为24.6％(16/65),分别为blaOXA(16株)、blaTEM(15株)和blaCTX-M(14株),未检出blaSHV; PMQR的阳性率为46.2％(30/65),分别为qnrA(9株)、qnrS(18株)、aac(6')-Ib～cr(28株),未检出qnrB、qnrC、qnrD和qepA;且携带ESBLs基因的阳性菌株均携带PMQR基因.首次检测出有7株大肠杆菌同时携带ES-BLs基因型(blaOXA、blaTEM、blaCTX-M)和PMQR基因型(qnrA、qnrS、aac(6')-Ib-cr).药物敏感性测定结果显示:30株携带ESBLs和/或PMQR基因的阳性大肠杆菌对16种抗菌药物的耐药率为16.7％～100％,耐药谱较广,耐药性较严重.16株同时携带ESBLs和PMQR基因的阳性菌株对11种及11种以上药物耐药的菌株占87.5％,14株仅携带PMQR基因的阳性菌株对11种及11种以上药物耐药的菌株占28.5％,表明携带PMQR基因的同时携带ESBLs基因大大增强了菌株的耐药性,携带耐药基因的数量和种类与菌株的多重耐药性相关.     

Detection of toxin genes in Escherichia coli isolated from normal dogs and dogs with diarrhea.

The etiology of acute, nonviral diarrhea in dogs is poorly understood. Enterotoxigenic and verotoxigenic Escherichia coli are causal agents of diarrhea in humans, pigs, and cattle, but the association of these toxigenic E. coli with diarrhea in dogs has not been explored to a significant extent. In this study, DNA hybridization and PCR amplification were used to identify the frequency with which the genes for E. coli enterotoxins (STap, STb, and LTI) and verotoxins (VT1 and VT2) occur in association with diarrhea in dogs. Genes for VT1 (8.9%), VT2 (22.2%), STa (26.7%), and STb (4.4%) were identified in E. coli cultured from feces of 20 of 45 dogs (44.4%) with diarrhea. Genes for VT2, STa, and STb were not identified in feces from normal dogs. Genes for VT1 were observed in similar proportions in fecal samples from diarrheic (8.9%) and normal (12.3%) dogs. Heat labile enterotoxin (LTI) was not detected in fecal samples from either diarrheic or normal dogs. Our results suggest that heat stable enterotoxins and VT2 may be causally associated with diarrhea in dogs. Dogs appear to be able to carry VT1-producing E. coli without showing overt signs of disease.     

Prevalence of eae and shiga toxin genes among Escherichia coli strains isolated from healthy calves

Strains of Escherichia coli (n = 390) isolated from 132 healthy, 4-8-week old calves, were tested by polymerase chain reaction (PCR) for the eae (intimin) gene and shiga toxin genes (stx1 and stx2). All strains were also analysed for F5, F17 and F41 fimbriae and for the heat-labile (LT) and heat-stable (STI and STII) genetic markers. Overall, the eae gene was detected in 84 (21.5%) of the strains tested. Only 21 (5.4%) isolates were positive for stx1 (18 strains) or stx2 (three strains); nine of the stx1-positive isolates also possessed the eae gene. A high percentage (29.2%) of the isolates tested expressed F17 but no enterotoxin genes were detected. None of the eae- or stx-positive strains belonged to the O157 serogroup.     

Examination of Escherichia coli from poultry for selected adhesin genes important in disease caused by mammalian pathogenic E. coli.

A collection of 1601 extraintestinal and intestinal Escherichia coli isolated from chickens, turkeys and ducks, in Belgium, France and Spain, was hybridised with gene probes specific for fimbrial and afimbrial adhesins (F17, F18, S , Bfp, Afa, Cs31A, Intimin , Aida-1) of intestinal, urinary and invasive E. coli of mammals and with a probe specific for the P (Pap/Prs) fimbrial adhesin of urinary and invasive E. coli of mammals and birds. Three hundred and eighty-three strains (23.9%) were P-positive, 76 strains (4.8%) were Afa-positive, 75 strains (4.7%) were F17-positive, 67 strains (4.2%) were S-positive, 23 (1.4%) were Intimin-positive, and all were F18-, Cs31A-, Aida1- and Bfp-negative. The 75 F17-positive strains harboured different major subunit A-encoding gene variants, but the f17Ac variant was the most frequent (52 strains, 69.3%) and seven strains (9.3%) were not typeable. The f17G gene variant coding for the GII adhesin was the most frequent (56 strains, 75.0%), whereas the f17GI gene variant was present in four strains (5%) and 15 strains (20.0%) were not typeable. All Afa-positive strains harboured the afa-8 variant. The 23 Intimin-positive E. coli tested positive for the beta-variant (16 strains; 69.6%) or for the gamma-variant (seven strains; 30.4%) of the eae gene. Chicken and turkey E. coli were more frequently probe-positive (43.6 and 43.1%, respectively) than duck E. coli (31.5%) and extraintestinal E. coli were also more frequently probe-positive (48.4%) than intestinal strains (18.5%). Different combinations of probe positive hybridisation results were observed in 72 of the 540 probe-positive E. coli (13.3%). The most frequent combinations were between AfaE-8 and F17 probes (47 strains; 8.7%) and between P and S probes (13 strains; 2.4%). Although f17- and afa-8-related DNA sequences can be plasmid-located in mammalian E. coli, they were not in avian E. coli. Besides the P fimbrial adhesins, F17 and S fimbrial and Afa-VIII and Intimin afimbrial adhesins may thus represent colonisation factors of avian pathogenic E. coli.     

Characters of Escherichia coli 078 isolated from septicaemic animals

Twenty-one Escherichia coli isolates of serogroup 078 from animal septicaemia were obtained from laboratories in France, England and Canada. The bacteria were compared for outer membrane protein (OMP) patterns, lipopolysaccharide patterns, surface proteins of fimbrial types, biotypes, antibiotypes, colicin production, hydroxamate production and virulence in mice. Sixteen isolates from bovine, ovine, porcine and avian species in France and England had a similar OMP pattern. This characteristic associated with minor properties like surface proteins type, colicin V production and virulence in mice made these 16,078 E. coli isolates from 4 animal species, good candidates for the same clonal grouping. The five other bovine isolates with "Vir" or "31a" phenotypes were heterogeneous for most of the characteristics studied.     

Shiga toxin genes in avian Escherichia coli

The purpose of this study was to determine the presence of stx genes in avian pathogenic Escherichia coli (APEC). We examined 97 APEC isolates: 34 from lesions of avian cellulitis, 31 from avian septicemia, 13 from swollen head syndrome (SHS) in chickens, and 19 from diseased turkeys. We also examined five isolates from the feces of healthy chickens. All 102 E. coli isolates were tested for the presence of stx genes by PCR amplification and by colony blots using probes specific for stx1 and stx2. Fifty-three percent (52) of the 97 APEC carried stx gene sequences: one isolate carried stx2 sequences, two carried both stx1 and stx2 sequences, and the remaining 49 isolates carried only stx1 sequences. Twenty-six isolates were positive by both hybridization and PCR amplification, 10 were positive by PCR only, and 16 were positive by hybridization only. All the stx-positive isolates were negative by PCR for the eae and E-hlyA genes. The five isolates from healthy chickens were all negative for stx. All 13 SHS isolates were positive for the stx1 gene and had low titres for cytotoxicity in the Vero cell assay (VCA). Other stx-positive isolates were negative in the VCA. The stx1 gene from one SHS E. coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of Shigella dysenteriae. The observations indicate that stx1 gene sequences are widespread among APEC but that cytotoxicity on Vero cells is uncommon.     

Penetration of drug resistance in Escherichia coli isolated from laboratory animals

A total of 713 strains of fecal Escherichia coli (E. coli) isolated from laboratory animals in the colonies of 4 research laboratories and 4 commercial breeders in Japan in 1994 were examined in regard to resistance to 8 antibacterial agents. The incidence of resistance to sulfadimethoxine (Su), streptomycin (Sm), ampicillin, cephaloridine, tetracycline, chloramphenicol, kanamycin, and gentamicin was 99.9%, 32.5%, 6.7%, 0.7%, 7.0%, 2.6%, 6.6% and 0.7%, respectively. These results indicated that Su and Sm resistance are penetrating into normal E. coli strains isolated from laboratory animals.     

High technology diagnostics: detection of enterotoxigenic Escherichia coli, using DNA probes

A relatively new technique termed colony blot hybridization appears to be a reliable replacement for the ligated porcine gut loop assay previously used to detect stable toxin-B producing Escherichia coli. The highly reproducible blot assay enables screening of large numbers of isolants for the presence of E coli stable-B toxin genes, thus avoiding variations in phenotypic expression and poor repeatability inherent in the in vivo assays. Availability of this diagnostic test can aid practitioners in identifying enterotoxigenic E coli related disease in swine and in determining the benefits of vaccination programs.     

Virulence genes of Escherichia coli strains isolated from mastitic milk

Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis. Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E. coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e. Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA. The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%). The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E. coli isolates and the systemic clinical condition of the respective infected cows.     

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan

A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced bla_CTX-M-2, while the nine poultry isolates produced bla_CTX-M-25 (4), bla_SHV-2 (3), bla_CTX-M-15 (1) and bla_CTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan.     

仔猪腹泻源大肠杆菌耐药性调查及耐药基因检测

对泰州地区分离的仔猪腹泻源大肠杆菌的耐药性及其携带的相关耐药基因情况进行检测,为养殖场今后合理用药提供科学依据。采用微量肉汤稀释法对分离的49株大肠杆菌进行8种抗菌药物的药敏试验;采用PCR方法对多黏菌素耐药基因mcr-1及超广谱β-内酰胺酶(ESBL)耐药基因blaCTX-M、blaSHV和blaTEM进行检测。药敏试验结果显示:大肠杆菌对氯霉素、氟苯尼考、氨苄西林、四环素、庆大霉素和恩诺沙星等的耐药率分别达到100%、97.96%、95.92%、89.80%、59.18%和51.02%;而对多黏菌素E和头孢噻呋的耐药率也达到44.90%和32.65%。进一步耐药基因检测结果显示:ESBL耐药基因中blaCTX-M的检出率最高,达到12.24%(6/49);其次为blaSHV,8.16%(4/49);有一株大肠杆菌携带blaTEM基因。其中一株同时携带blaCTX-M和blaSHV。多黏菌素耐药基因mcr-1的检出率达到30.61%(15/49),其中4株同时携带blaCTX-M基因。泰州地区分离大肠杆菌耐药情况和多重耐药现象较为严重,多黏菌素耐药基因mcr-1及ESBL耐药基因blaCTX-M、blaSHV和blaTEM的广泛分布表明这些耐药基因存在快速传播的风险。因此,有必要加强猪场细菌耐药性监测。     

多重PCR方法检测鸭源产志贺氏毒素大肠杆菌

为了检测产志贺毒素大肠杆菌(STEC)在鸭源大肠杆菌中的分布情况,本研究建立检测STEC的多重PCR方法,针对STEC特有的毒力基因stx1、stx2、h&A和eaeA筛选了4对引物,通过对PCR反应条件的优化、特异性和灵敏度的检测,建立检测STEC的多重PCR,并应用该方法调查254株鸭源致病性E.coli和115株外表健康鸭的泄殖腔分离的E.coli中STEC的分布情况.在254株鸭源致病性E.coli中检测出6株STEC,从外表健康鸭的泄殖腔分离的115株E.coil中未检测到STEC.检出的STEC的血清型分别为O36、O60、O77、O78、O158和O7 & O92.本实验建立的检测STEC的多重PCR方法特异性好、灵敏度高;对鸭源E.coli的检测结果证实鸭致病性大肠杆菌中存在STEC菌株,分布频率较低,但其血清型具有广泛的宿主源,存在引起人类疾病的可能性.