Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Response of Chinese hamster ovary cells to a cytolethal distending toxin (CDT) of Escherichia coli and possible misinterpretation as heat-labile (LT) enterotoxin

Increased incubation of the Chinese hamster ovary cell (CHO) assay up to 96–120 h allowed differentiation of cytotonic and cytotoxic effects. Strains of Escherichia coli O128 found to produce a heat-labile cytolethal CHO toxin were compared with E. coli heat-labile enterotoxin (LT). The cytolethal toxin was unrelated to LT, heat-stable enterotoxin (ST), Verotoxin (VT) or hemolysins. Since CHO elongation induced by either the E. coli LT or E. coli O128 filtrates could not be differentiated after 24 h, continued incubation for 96–120 h was essential for observation of progressive morphological changes and cytolethal events. Comparative responses in at least two cell systems is recommended to prevent misinterpretation of elongation at 24 h in the CHO assay.     

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.     

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.     

Genotypic and Phenotypic Profiles of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Associated with Acute Diarrhea in Tunis,Tunisia

No past studies of acute diarrhea in Tunisia have examined the phenotypic and genotypic profiles of enterotoxigenic Escherichia coli (ETEC) isolates. We determined 65 ETEC isolates derived from a total of 327 E. coli isolates collected from a previous study (acute diarrheal and healthy persons, children and adults n = 214) and 32 E. coli isolates derived from an acute diarrheal outbreak in Kabaria-Ennour city, Tunis. All E. coli isolates were screened by polymerase chain reaction (PCR) for ETEC virulence genes: sta (heat-stable toxin gene) and elt (heat-labile toxin gene). Seventy-two percent (47 of 65) of ETEC strains expressed the sta gene only, 21.5% (14 of 65) expressed the elt gene and 6.1% (4 of 65) expressed both genes. For the outbreak isolates, the elt gene was predominant (10 isolates out of 14). Ganylioside GM1 enzyme-linked immunosorbent assay (GM1-ELISA) was used to validate the PCR results and this was confirmed by dot blot assay. The same results were obtained. The most common colonization factors (CFs) were CFA/I (44.6%) and coli surface antigen 6 (CS6) (11%), and 44.6% of the isolates showed no association with either CFAs. Resistance of ETEC isolates to tetracycline (38.5%), streptomycin (26%), and beta-lactam agents (ticarcillin 26%, amoxicillin 24.6%, cephalotin 21.5%) was common. Regarding serotypes, the majority of ETEC isolates serotyped as O86:H(-) (n = 16), O128:H2 (n = 11), and O127:H21 (n = 10). Other serotypes found were O111:H(-) (n = 6) and O126: H(-) (n = 5). DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) using the XbaI enzyme was conducted to investigate the epidemiological clonal relationship among ETEC isolates. Major patterns were identified among which some of outbreak ETEC isolates belonged. These data suggest that a proportion of acute diarrhea in Tunis represents the confluence of small epidemics by clonality-related ETEC isolates that are transiently introduced or that persist in our community.     

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment

The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.     

Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli.

The genes for a new enterotoxin were cloned from Escherichia coli SA53. The new toxin was heat labile and activated adenylate cyclase but was not neutralized by antisera against cholera toxin or E. coli heat-labile enterotoxin. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on polyacrylamide gels that are similar to those of cholera toxin and E. coli heat-labile enterotoxin subunits. A 0.8-kilobase DNA probe for the new enterotoxin failed to hybridize with the cloned structural genes for E. coli heat-labile enterotoxin.     

Fluorogenic assay for rapid detection of Escherichia coli in food

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.     

Fluorogenic assay for rapid detection of Escherichia coli in food.

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.     

Improved Method for Purification of Human Escherichia Col I Heat-Stable Enterotoxin by Hydrophobic Interaction,Molecular-Sieve and High Performance Ion Exchange Chromatography

Abstract

A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity.     

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11

Abstract Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

弧菌三联融合毒素基因表达及ELISA检测方法建立

采用柔性Linker(GGGGS)和重叠延伸PCR方法将三个毒素基因, 即副溶血性弧菌的耐热型直接溶血素基因tdh、创伤弧菌的溶细胞毒素基因vvhA和拟态弧菌的热不稳定型溶血素基因vmhA的成熟蛋白基因片段进行串联, 得到三联融合毒素基因tdh-vvhA-vmhA(命名为FT)。一致性比对分析与预计融合的基因序列一致性为99.6%。FT的开放阅读框架全长3225 bp, 编码1074个氨基酸残基, 预测分子量为120.4 kDa。将FT亚克隆至表达载体pET-22b(+)中, 再转化至E. coli BL21 (DE3)中进行表达, 经SDS-PAEG分析, 融合蛋白分子量与预测的相符。终浓度为1mmol/L的IPTG, 37 ℃诱导4 h, 融合毒素蛋白表达量最高, 经薄层扫描分析, 此时融合毒素蛋白占全菌体蛋白的11.49%。融合毒素蛋白包涵体经纯化, 免疫豚鼠制备血清抗体, 确定间接ELISA的最佳工作条件, 并进行敏感性、特异性、重复性及模拟样品检测试验, 建立了食物中毒性弧菌广谱、快速、特异的同步免疫学检测方法。     

Enterotoxigenicity of diarrhoeal isolates of Salmonella bareilly

Whole cell cultures, cell-free supernatants, and cell sonicates from ten strains of Salmonella bareilly induced fluid accumulation in ligated rabbit and rat ileal loops. All strains had an intracellular vascular permeability factor, half were suckling mouse positive indicating the presence of a heat-stable type of activity. The toxin(s), however, were Immunologically distinct from the heat-labile toxin of Escherichia coll LT and cholera toxin. Besides enterotoxigenicity, all strains exhibited potential Invasive character.The authors are with the Department of Microbiology, Panjab University, Chandigarh, India 160 014. M. Saxena is the corresponding author.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Pilus antigen 987P produced by strains of Escherichia coli serotypes O141:K--, H- and O8:K85:H--

Escherichia coli strains isolated from the alimentary tract of 68 weaned and 44 unweaned pigs with diarrhoea in various parts of Hungary, were tested for the presence of pilus antigens K88, K99 and 987P. K88 was detected in 30% of the strains from newborn pigs and in 12% of the strains isolated from weaned pigs. One strain carried K99. Based on agglutination test and immunoelectron microscopic studies with specific absorbed antisera, five non-haemolytic E. coli strains isolated from newborn pigs were found to produce so-called 987P pili. Three of these strains were designated serologically as O8:K85:H--,987P+ and two as O141: K--:H--,987P+. The Y1 cell assay, the infant mouse assay, and the ligated intestinal loop assay in less than 3-week-old pigs indicated that none of the strains produced heat-labile enterotoxin but all produced a heat-stable enterotoxin detectable in infant mice and in pig loops (STa). All the strains induced diarrhoea in newborn, colostrum deprived pigs and colonized the lower small intestine by adhesion to the villous epithelium. The results have confirmed earlier findings about adhesive virulence attributes caused by 987P pili.