Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

stx2vha is the dominant genotype of Shiga toxin-producing Escherichia coli O157:H7 isolated from patients and domestic animals in three regions of China

Shiga toxin-producing Escherichia coli(STEC) O157: H7 strains were isolated from domestic animals and patients from Xuzhou City, Jiangsu Province, China and the bordering Anhui and Henan Provinces and were examined for the stx genotype. Of 390 strains, 277 were identified as genotype stx2vha ; 41, stx2 ; 51, stx2-stx1 ; 1, stx2-stx2vha-stx1 ; 5, stx2-stx2vha ; and 15 were un-typeable. Of the 277 stx2vha-bearing isolates, 116 were isolated from goats; 42, cattle; 38, hens, and 35 from pigs. The study shows stx2vha is the dominant genotype and goats are an important reservoir.     

Identification of shiga toxin-producing Escherichia coli possessing insertionally inactivated Shiga toxin gene

We have investigated the Shiga toxin genes of Shiga toxin-producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Characterization of inducible stx2-positive Escherichia coli O157:H7/H7- strains isolated from cattle in France

Aims:  To quantify the variability of the Shiga toxin 2 (Stx2) production by a panel of stx2 -positive Escherichia coli O157:H7/H7- isolates from healthy cattle before and after induction with enrofloxacin.
Methods and Results:  ProSpecT^® ELISA was used to quantify the Stx2 production by stx2 -positive E. coli O157:H7/H7- isolates in native conditions (basal level) or after induction with enrofloxacin. Whereas only 15·2% of the E. coli O157:H7/H7- strains studied displayed significant amounts of detectable Stx2 without induction, most of them were shown to be inducible, and at various levels, in presence of subinhibitory concentrations of enrofloxacin.
Conclusions:  We demonstrated the capability of a highly elevated proportion of stx2 -positive, but constitutively Stx2 -negative, E. coli O157:H7/H7- isolates from healthy cattle to produce significant levels of Shiga toxin Stx2 in presence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family only licensed for veterinary use.
Significance and Impact of the Study:  This study documents the risk that bovine-associated Shiga toxin producing E. coli isolates may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones in the oral treatment of food animals like cattle or poultry.     

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhoea in Paraná State, Brazil

Aims:  To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC.
Methods and Results:  A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx -positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx ₁ eae ehxA and stx ₁ respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested.
Conclusions:  These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence.
Significance and Impact of Study:  These results indicate that molecular methods are required to diagnosis of STEC infections.     

Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.     

Virulence characteristics of Shiga toxin‐producing Escherichia coli from raw meats and clinical samples

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae‐negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx_2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non‐O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.     

A rapid bioluminescent enzyme immunoassay (BLEIA) for the detection of Shiga toxin types 1 and 2.

In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture-based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin-detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non-Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non-Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stool samples we noted a tendency for the system to give unexpectedly high background level. Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.     

Stx genotypes and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli strains isolated from human infections, cattle and foods in Brazil

A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in S?o Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.     

The characterization of Shiga toxin-non-producing Escherichia coli serotype O157:H7 isolated from carcasses of cattle at a slaughter house.

A bacteriological investigation of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was performed on 298 carcasses of cattle at slaughter houses between July 1996 and January 1997 in Gifu Prefecture, Japan. As a result, four Stx-non-producing Escherichia coli O157:H7 strains were isolated from two slaughtered carcasses of cattle. The purpose of this study was to examine the characterization of isolates. Isolates possessed the E. coli attaching and effacing gene (eaeA), and hemolysin gene (hlyA), and harbored 3.0-MDa and 60-MDa plasmids. The Xba I pulsed-field gel electrophoresis (PFGE) pattern showed three similar patterns. Consequently, a closely related genotype of Stx-non-producing E. coli O157:H7 may widely exist in cattle.     

Shiga toxin-producing and attaching and effacing Escherichia coli in cats and dogs in a high hemolytic uremic syndrome incidence region in Argentina

Shiga toxin-producing Escherichia coli (STEC), responsible for the hemolytic uremic syndrome, is an endemic pathogen in Argentina. We studied the prevalence of STEC in fecal samples from cats and dogs of Buenos Aires city and suburbs. Cultures were used for screening stx1/stx2 and rfbO157 by multiplex PCR. All E. coli-positive colonies for these genes were further characterized for the eae gene and for serotypes. In dogs, 17 (3.7%), 19 (4.2%) and 34 (7.5%) of samples were positive for stx2, stx1 and rfb, respectively. In cats, six (4.0%) of the samples were positive for stx2, three (2.0%) for stx1 and four (2.7%) for rfbO157. In 18 (4.0%) of the dog samples, a bacteriological diagnosis was obtained by isolation. The percentage of positive isolates corresponding to the rfbO157 and to the stx2 genotypes were 2.9% and 1.1%, respectively. In four of the cat samples, the bacteriological diagnosis for stx2 (2.6% prevalence of STEC) was confirmed. Although these data suggest that the high infection index of STEC in children in Argentina does not seem to be due mainly to the role of cats and dogs, there are some strains with virulence genes in common for humans and their domestic animals.     

Prevalence of secreted autotransporter toxin gene among diffusely adhering Escherichia coli isolated from stools of children

In this report, we analyzed the prevalence of the sat gene in 336 Escherichia coli samples collected from stools of children with and without diarrhea in Brazil and in 100 uropathogenic E. coli strains. The results show a high correlation between diffusely adhering E. coli (DAEC) and the presence of sat (44%) in intestinal isolates. DAEC strain FBC114 expresses a 107-kDa protein, which showed 98% homology with Sat.     

Survival and spread of Shiga toxin‐producing Escherichia coli in alpine pasture grasslands

Aims: To determine the fate of Shiga toxin‐producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. Methods and Results: During the summers of 2005 and 2006, the field survival of STEC was monitored in cowpats and underlying soils in four different alpine pasture units. A most probable number (MPN)‐PCR stx assay was used to enumerate STEC populations. STEC levels ranged between 3·9 and 5·4 log₁₀ CFU g^?1 in fresh cowpats and slowly decreased until their complete decay (inactivation rates k < 0·04 day^?1). PFGE typing of STEC strains isolated from faecal and soil samples assessed the persistence of various clonal types for at least 2 months in cowpats and their vertical dispersal down through the soil at a depth up to at least 20 cm. STEC cells counts in soil were always below 2 log₁₀ CFU g^?1, regardless of the pasture unit investigated. The soil became rapidly free of detectable STEC once the cowpat had decomposed. The eight STEC strains isolated during this study belonged to six distinct serotypes and tested positive for the gene(s) stx2, including the stx2g and stx2 NV206 variants. Conclusions: STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. Significance and impact of the Study: This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.