Virulence gene profiles and phylogeny of Shiga toxin-positive Escherichia coli strains isolated from FDA regulated foods during 2010-2017.

Illnesses caused by Shiga toxin-producing Escherichia coli (STECs) can be life threatening, such as hemolytic uremic syndrome (HUS). The STECs most frequently identified by USDA's Microbiological Data Program (MDP) carried toxin gene subtypes stx1a and/or stx2a. Here we described the genome sequences of 331 STECs isolated from foods regulated by the FDA 2010-2017, and determined their genomic identity, serotype, sequence type, virulence potential, and prevalence of antimicrobial resistance. Isolates were selected from the MDP archive, routine food testing by FDA field labs (ORA), and food testing by a contract company. Only 276 (83%) strains were confirmed as STECs by in silico analysis. Foods from which STECs were recovered included cilantro (6%), spinach (25%), lettuce (11%), and flour (9%). Phylogenetic analysis using core genome MLST revealed these STEC genomes were highly variable, with some clustering associated with ST types and serotypes. We detected 95 different sequence types (ST); several ST were previously associated with HUS: ST21 and ST29 (O26:H11), ST11 (O157:H7), ST33 (O91:H14), ST17 (O103:H2), and ST16 (O111:H-). in silico virulome analyses showed ~ 51% of these strains were potentially pathogenic [besides stx gene they also carried eae (25%) or 26% saa (26%)]. Virulence gene prevalence was also determined: stx1 only (19%); stx2 only (66%); and stx1/sxt2 (15%). Our data form a new WGS dataset that can be used to support food safety investigations and monitor the recurrence/emergence of E. coli in foods.

Pectin-derived neoglycolipids: Tools for differentiation of Shiga toxin subtypes and inhibitors of Shiga toxin-mediated cellular injury.

Gut pathogenic enterohemorrhagic Escherichia coli (EHEC) release Shiga toxins (Stxs) as major virulence factors, which bind to globotriaosylceramide (Gb3Cer, Galα1-4 Galß1-4Glcß1-1Cer) on human target cells. The aim of this study was the production of neoglycolipids (neoGLs) using citrus pectin-derived oligosaccharides and their application as potential inhibitors of Stxs. The preparation of neoGLs starts with the reduction of the carboxylic acid group of the pectic poly(α1-4)GalUA core structure to the corresponding alcohol, followed by hydrolytic cleavage of resulting poly(α1-4)Gal into (α1-4)Galn oligosaccharides and their linkage to phosphatidylethanolamine (PE). Thin-layer chromatography overlay assays of the produced (α1-4)Galn-PE and corresponding Amadori (α1-4)Galn=PE neoGLs revealed distinguishable binding patterns for Stx1a, Stx2a, and Stx2e. Furthermore, prepared neoGLs protected Vero cells against the cytotoxic action of Stxs when applied as multivalent glycovesicles. The produced neoGLs are applicable for differentiation of Stx subtypes and represent a promising approach to combat infections of EHEC by blocking their major toxins.

Genetic characterization of Shiga toxin producing Escherichia coli belonging to the emerging hybrid pathotype O80:H2 isolated from humans 2010-2017 in Switzerland.

Shiga toxin-producing E. coli (STEC) O80:H2 is an uncommon hybrid pathotype that has recently emerged in France. We analysed 18 STEC O80:H2 isolated from humans in Switzerland during 2010-2017. All isolates carried stx2a or stx2d, the rare eae variant eae-ξ and at least seven virulence genes associated with pS88, a plasmid that is found in extraintestinal pathogenic E. coli (ExPEC). Whole genome sequencing (WGS) identified additional chromosomal extraintestinal virulence genes encoding for type 1 fimbria (fimA, fimC and fimH), aerobactin (iuc/iutA) and afimbrial adhesins (afaA/C/D/E-VIII). Core genome multi-locus sequence typing (cgMLST) detected two closely related but distinct subclusters with different stx2 and iuc/iutA genotypes. All isolates were multidrug resistant (MDR), but susceptible to third generation cephalosporins and azithromycin. STEC/ExPEC hybrid pathotypes such as STEC O80:H2 represent a therapeutical challenge in the event of extraintestinal infection.

[Zoonotic significance of Escherichia coli strains isolated from cattle and pigs].

OBJECTIVES: The aim of this study was to monitor characteristics of Shiga toxin-producing Escherichia coli (STEC) obtained from animals according to the serogroup they belonged to, Shiga toxin type and subtype and adhesion factor intimin. Then, based on the results, to evaluate the occurrence of Shiga toxin subtypes and their possible significance for humans. MATERIALS AND METHODS: The study included 131 STEC strains isolated from rectal swabs from cattle (80) and pigs (51) sampled on farms in the Czech Republic from 2000 to 2017. Selected strains differed in origin and serogroup. The presence of Shiga toxins, intimin and the Shiga toxin subtypes stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2g was determined by PCR. RESULTS: The stx1 gene was confirmed in 33 % of the strains (43/131), the stx2 gene in 55 % (72/131) and 16 strains carried the genes for both toxins simultaneously (12 %). Strains harboring the eae gene were detected in 46 (35 %) cases, mostly in rectal swabs from cattle. STEC from cattle belonged to 21 different serogroups. The presence of Shiga toxin 1 (55; 69 %) predominated in these strains, with subtypes stx1a (54) and stx1d (1). Shiga toxin 2 was confirmed in 39 of the bovine strains (49 %), with the following subtypes: stx2a (9), stx2e (6), stx2g (3), stx2a, stx2c (5), stx2a, stx2b (1) and stx2c, stx2d (1). Also combinations of stx1a, stx2a (12) and stx1a, stx2c (2) were detected. STEC from pigs belonged to 5 different serogroups. Shiga toxin 2 was most frequently detected (49; 96 %), with subtypes stx2e (42) and stx2a (7). Shiga toxin 1 was detected in 4 strains (8 %), as subtypes stx1a (1) and stx1c (1) and also in the combination stx1a, stx2a (2). CONCLUSION: STEC strains isolated from cattle, compared to those from pigs, belonged to a larger spectrum of serogroups, they more often carried adherence factor intimin and the diversity of Shiga toxin subtypes was higher, including those associated with serious human diseases. In the set of isolates from pigs, the stx2e gene predominated; its significance for human health has not been fully clarified yet.

Comparative Genomic and Phylogenetic Analysis of a Shiga Toxin Producing Shigella sonnei (STSS) Strain.

Shigella strains are important agents of bacillary dysentery, and in recent years Shigella sonnei has emerged as the leading cause of shigellosis in industrialized and rapidly developing countries. More recently, several S. sonnei and Shigella flexneri strains producing Shiga toxin (Stx) have been reported from sporadic cases and from an outbreak in America. In the present study we aimed to shed light on the evolution of a recently identified Shiga toxin producing S. sonnei (STSS) isolated in Europe. Here we report the first completely assembled whole genome sequence of a multidrug resistant (MDR) Stx-producing S. sonnei (STSS) clinical strain and reveal its phylogenetic relations. STSS 75/02 proved to be resistant to ampicillin, streptomycin, tetracycline, chloramphenicol, thrimetoprim, and sulfomethoxazol. The genome of STSS 75/02 contains a 4,891,717 nt chromosome and seven plasmids including the 214 kb invasion plasmid (pInv) harboring type III secretion system genes and associated effectors. The chromosome harbors 23 prophage regions including the Stx1 converting prophage. The genome carries all virulence determinants necessary for an enteroinvasive lifestyle, as well as the Stx1 encoding gene cluster within an earlier described inducible converting prophage. In silico SNP genotyping of the assembled genome as well as 438 complete or draft S. sonnei genomes downloaded from NCBI GenBank revealed that S. sonnei 75/02 belongs to the more recently diverged global MDR lineage (IIIc). Targeted screening of 1131 next-generation sequencing projects taken from NCBI Short Read Archive of confirms that only a few S. sonnei isolates are Stx positive. Our results suggest that the acquisition of Stx phages could have occurred in different environments as independent events and that multiple horizontal transfers are responsible for the appearance of Stx phages in S. sonnei strains.

Virulence genes, Shiga toxin subtypes, major O-serogroups, and phylogenetic background of Shiga toxin-producing Escherichia coli strains isolated from cattle in Iran.

The aim of this study was to investigate the virulence potential of the isolated bovine STEC for humans in Iran. In this study a collection of STEC strains (n = 50) had been provided via four stages, including sampling from feces of cattle, E. coli isolation, molecular screening of Shiga toxin (stx) genes, and saving the STEC strains from various geographical areas in Iran. The STEC isolates were subjected to stx-subtyping, O-serogrouping, and phylo-grouping by conventional polymerase chain reaction (PCR). Occurrence of stx1 (52%) and stx2 (64%) was not significantly different (p = 0.1), and 16% of isolates carried both stx1 and stx2, simultaneously. In addition, 36% and 80% of the isolates were positive for eae and ehxA, respectively. Molecular subtyping showed that stx1a (52%), stx2a (44%), stx2c (44%), and stx2d (30%) were the most prevalent subtypes; two combinations stx2a/stx2c and stx2c/stx2d coexisted in 18% and 10% of STEC strains, respectively. Three important non-O157 serogroups, including O113 (20%), O26 (12%), and O111 (10%), were predominant, and none of the isolates belonged to O157. Importantly, one O26 isolate carried stx1, stx2, eae and ehxA and revealed highly virulent stx subtypes. Moreover, all the 21 serogrouped strains belonged to the B1 phylo-type. Our study highlights the significance of non-O157 STEC strains carrying highly pathogenic virulence genes in cattle population as the source of this pathogen in Iran. Since non-O157 STEC strains are not routinely tried in most diagnostic laboratories, majority of the STEC-associated human infections appear to be overlooked in the clinical settings.

Shiga Toxin Subtypes of Non-O157 Escherichia coli Serogroups Isolated from Cattle Feces.

Shiga toxin producing Escherichia coli (STEC) are important foodborne pathogens responsible for human illnesses. Cattle are a major reservoir that harbor the organism in the hindgut and shed in the feces. Shiga toxins (Stx) are the primary virulence factors associated with STEC illnesses. The two antigenically distinct Stx types, Stx1 and Stx2, encoded by stx1 and stx2 genes, share approximately 56% amino acid sequence identity. Genetic variants exist within Stx1 and Stx2 based on differences in amino acid composition and in cytotoxicity. The objective of our study was to identify the stx subtypes in strains of STEC serogroups, other than O157, isolated from cattle feces. Shiga toxin gene carrying E. coli strains (n = 192), spanning 27 serogroups originating from cattle (n = 170) and human (n = 22) sources, were utilized in the study. Shiga toxin genes were amplified by PCR, sequenced, and nucleotide sequences were translated into amino acid sequences using CLC main workbench software. Shiga toxin subtypes were identified based on the amino acid motifs that define each subtype. Shiga toxin genotypes were also identified at the nucleotide level by in silico restriction fragment length polymorphism (RFLP). Of the total 192 STEC strains, 93 (48.4%) were positive for stx1 only, 43 (22.4%) for stx2 only, and 56 (29.2%) for both stx1 and stx2. Among the 149 strains positive for stx1, 132 (88.6%) were stx1a and 17 (11.4%) were stx1c. Shiga toxin 1a was the most common subtype of stx1 among cattle (87.9%; 123/140) and human strains (100%; 9/9) of non-O157 serogroups. Of the total 99 strains positive for stx2, 79 were stx2a (79.8%), 11 (11.1%) were stx2c, 12 (12.1%) were stx2d. Of the 170 strains originating from cattle feces, 58 (34.1%) were stx2a subtype, 11 (6.5%) were stx2c subtype, and 11 were of subtype stx2d (6.5%). All but one of the human strains were positive for stx2a. Three strains of cattle origin were positive for both stx2a and stx2d. In conclusion, a number of non-O157 STEC serogroups harbored by cattle possess a wide variety of Shiga toxin subtypes, with stx1a and stx2a being the most predominant stx subtypes occurring individually or in combination. Cattle are a reservoir of a number of non-O157 STEC serogroups and information on the Shiga toxin subtypes is useful in assessing the potential risk as human pathogens.

Shiga Toxin Subtypes and Virulence Genes in Escherichia coli Isolated from Cattle.

Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H- strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfAO113 were detected in more than 12 serotypes.

Serotipos, perfiles de virulencia y subtipos de stx en Escherichia coli productor de toxina Shiga aislados de productos de pollo / Serotypes, virulence profiles and stx subtypes of Shigatoxigenic Escherichia coli isolated from chicken derived products

Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen that causes hemolytic uremic syndrome (HUS) and the consumption of chicken products has been related to some HUS cases. We performed a non-selective isolation and characterization of STEC strains from retail chicken products. STEC isolates were characterized according to the presence of stx1, stx2, eae, saa and ehxA; stx subtypes and serotypes. Most of them carried stx2, showing subtypes associated with severe human disease. Although reported in other avian species, the stx2f subtype was not detected. The isolates corresponded to different serotypes and some of them, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been identified among STEC isolated from patients suffering from diarrhea, hemorrhagic colitis, HUS, as well as from cattle. Considering the virulence profiles and serotypes identified, our results indicate that raw chicken products, especially hamburgers sold at butcheries, can be vehicles for high-risk STEC strains.

Escherichia coli productor de toxina de Shiga (STEC) es un patógeno transmitido por alimentos que causa el síndrome urémico hemolítico (SUH). Algunos casos de SUH están relacionados con el consumo de productos de pollo. Se realizó el aislamiento no selectivo y la caracterización de cepas STEC provenientes de productos de pollo atendiendo a la presencia de stx1, stx2, eae, saa y ehxA, subtipos de stx y serotipos. La mayoría de los aislamientos portaba stx2 y subtipos de stx asociados con enfermedades graves en humanos. Aunque se ha detectado en otras especies aviares, el subtipo stx2f no se encontró. Se detectaron diferentes serotipos, entre ellos O22:H8, O113:H21, O130:H11, O171:H2 y O178:H19, también identificados como STEC aislados de pacientes con diarrea, colitis hemorrágica y SUH, y de ganado bovino. Teniendo en cuenta los perfiles de virulencia y los serotipos identificados, nuestros resultados indican que los productos de pollo crudos, especialmente las hamburguesas que se venden en las carnicerías, pueden ser vehículos de cepas STEC de alto riesgo.

Virulence characterization of non-O157 Shiga toxin-producing Escherichia coli isolates from food, humans and animals.

A total of 359 non-O157 STEC isolates from food, humans and animals were examined for serotypes, Shiga toxin subtypes and intimin subtypes. Isolates solely harboring stx2 from the three sources were selected for Vero cell cytotoxicity test. stx subtypes in eae negative isolates were more diverse than in eae positive isolates primarily carrying stx2a. Four eae subtypes (eaeß,eaeε1,eaeγ1 and eaeγ2/θ) were observed and correlated with serotypes and flagella. Food isolates showed more diverse serotypes, virulence factors and cell cytotoxicities than human isolates. Some isolates from produce belonged to serotypes that have been implicated in human diseases, carried stx2a or/and stx2dact and exhibited high cell cytotoxicity similar to human isolates. This indicates that foods can be contaminated with potentially pathogenic STEC isolates that may cause human diseases. Given the increased produce consumption and growing burden of foodborne outbreaks due to produce, produce safety should be given great importance.

Shiga Toxin (Stx) Classification, Structure, and Function.

Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic ribosome and halts protein synthesis in target cells. The function of the B pentamer is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then to the endoplasmic reticulum. In humans infected with Stx-producing E. coli, the most serious manifestation of the disease, hemolytic-uremic syndrome, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.

Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Swine plays an important role as a carrier of this pathogen. In this study we determined the prevalence and characteristics of STEC from healthy swine collected between May 2011 and August 2012 from 3 cities/provinces in China. RESULTS: A total of 1003 samples, including 326 fecal, 351 small intestinal contents and 326 colon contents samples, was analyzed. Two hundred and fifty five samples were stx-positive by PCR and 93 STEC isolates were recovered from 62 stx-positive samples. Twelve O serogroups and 19 O:H serotypes including 6 serotypes (O100:H20/[H20], O143:H38/[H38], O87:H10, O172:H30/[H30], O159:H16, O9:H30/[H30]) rarely found in swine and ruminants were identified. All 93 STEC isolates harbored stx2 only, all of which were stx2e subtype including 1 isolate being a new variant of stx2e. 53.76%, 15.05% and 2.15% STEC isolates carried astA, hlyA and ehxA respectively. Four STEC isolates harbored the high-pathogenicity island. Of the 15 adherence-associated genes tested, 13 (eae, efa1, iha, lpfAO113, lpfAO157/OI-154, lpfAO157/OI-141, toxB, saa, F4, F5, F6, F17 or F41) were all absent while 2 (paa and F18) were present in 7 and 4 STEC isolates respectively. The majority of the isolates were resistant to tetracycline (79.57%), nalidixic acid (78.49%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin (55.91%). The STEC isolates were divided into 63 pulsed-field gel electrophoresis patterns and 21 sequence types (STs). Isolates of the same STs generally showed the same or similar drug resistance patterns. A higher proportion of STEC isolates from Chongqing showed multidrug resistance with one ST (ST3628) resistant to 14 antimicrobials. CONCLUSIONS: Our results indicate that swine is a significant reservoir of STEC strains in China. Based on comparison by serotypes and sequence types with human strains and presence of virulence genes, the swine STEC may have a low potential to cause human disease.

[Epidemic of EHEC (Escherichia coli O104:H4) in Europe in 2011--clinical and therapeutic problems]. / Epidemia EHEC (Escherichia coli O104:H4) w Europie w 2011 roku--problemy kliniczne i terapeutyczne.

Large outbreak of bloody diarrhoea complicated by haemolytic uraemic sundrome (HUS) has been observed in north Germany since May 2011. Epidemy spreaded throughout Germany and other countries and ceased at the end of July 2011. The WHO and German authorities confirmed that this epidemy was related to infection by new, unusual enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli 014:H4 strain.

Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes.

Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx(1d) and stx(2f), and some also missed stx(2b) and stx(2g). Most of the serological assays examined did not detect Stx2c, Stx2e, Stx2f, and Stx2g, and some strain-to-strain variation in reactivity was observed for Stx2b.

[In silico analysis of the capability of two polimerase chain reaction techniques for stx gene detection]. / Análisis in silico de la capacidad de dos técnicas de PCR para la detección del gen stx.

Shiga toxin-producing Escherichia coli is an emergent pathogen, being the Shiga toxin (Stx) the main virulence factor. These toxins are classified into 6 types (1,2, 2c, 2d, 2e and 2f) and 22 variants. In Argentina, two PCR for stx gene detection, PCR-MK and multiplex-PCR, were validated. The aim of this work was to analyze, by using bioinformatic tools, the stx variants that could be amplified by these PCRs, and to experimentally show the amplification of 8 stx variants. Twenty-five nucleotide sequences were collected from GenBank corresponding to 21 stx variants. The BLAST 2 sequences program was used to analyze the complementarities between the nucleotide sequence of the variants and the primers corresponding to the PCR studied. PCR-MK could detect types stx1, stx2, stx2c, stx2d and stx2f, but not type stx2e and three type stx2c variants. On the other hand, the multiplex-PCR could detect types stx1, stx2, stx2c, stx2d, but not stx2e and stx2f types. It was experimentally determined that both PCRs can detect those variants that cause severe disease in humans.

Análisis in silico de la capacidad de dos técnicas de PCR para la detección del gen stx / In silico analysis of the capability of two polimerase chain reaction techniques for stx gene detection.

Escherichia coli productor de toxina Shiga es un patógeno emergente cuyo principal factor de virulencia son las toxinas Shiga (Stx), codificadas por los genes stx. Estas toxinas se clasifican en 6 tipos (1, 2, 2c, 2d, 2e y 2f) que agrupan a 22 variantes. En Argentina se validaron dos técnicas de PCR para la detección de los genes stx, PCR-MK y PCR múltiple. Los objetivos del trabajo fueron analizar mediante el uso de herramientas bioinformáticas la capacidad de dichas técnicas para detectar las variantes del gen stx y demostrar experimentalmente la amplificación de 8 variantes stx. Se recopilaron 25 secuencias nucleotídicas de la base de datos GenBank correspondientes a 21 variantes de stx. Se utilizó el programa BLAST 2 sequences para analizar la complementariedad de las bases nucleotídicas entre las secuencias de las variantes y las secuencias de los cebadores utilizados en las PCR estudiadas. La técnica de PCR-MK permite detectar los tipos stx1, stx2, stx2c, stx2d y stx2f, aunque no permite detectar el tipo stx2e y tres variantes del tipo stx2c. La PCR múltiple permite detectar los tipos stx1, stx2, stx2c, stx2d, pero no los tipos stx2e y stx2f. Se demostró experimentalmente que ambas técnicas de PCR son apropiadas para la detección de las variantes que están asociadas a enfermedad grave en el hombre.

Shiga toxin-producing Escherichia coli is an emergent pathogen, being the Shiga toxin (Stx) the main virulence factor. These toxins are classified into 6 types (1, 2, 2c, 2d, 2e and 2f) and 22 variants. In Argentina, two PCR for stx gene detection, PCR-MK and multiplex-PCR, were validated. The aim of this work was to analyze, by using bioinformatic tools, the stx variants that could be amplified by these PCRs, and to experimentally show the amplification of 8 stx variants. Twentyfive nucleotide sequences were collected from GenBank corresponding to 21 stx variants. The BLAST 2 sequences program was used to analyze the complementarities between the nucleotide sequence of the variants and the primers corresponding to the PCR studied. PCR-MK could detect types stx1, stx2, stx2c, stx2d and stx2f, but not type stx2e and three type stx2c variants. On the other hand, the multiplex-PCR could detect types stx1, stx2, stx2c, stx2d, but not stx2e and stx2f types. It was experimentally determined that both PCRs can detect those variants that cause severe disease in humans.

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escherichia coli from calves.

AIMS: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. METHODS AND RESULTS: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx(1) exhibited stx(1c) in 13 (31.70%) isolates, while that of stx(2) revealed stx(2c) in eight (24.24%) and stx(2d) in two (6.06%) isolates. Subtyping of eae gene showed the presence of eae-beta, eae-eta and eae-zeta in two, three and four isolates respectively. None of the E. coli isolates possessed stx(2e), stx(2f), eae-alpha, eae-delta, eae-epsilon and eae-xi. All EPEC isolates were atypical. CONCLUSIONS: stx(1), stx(1c), stx(2), stx(2c), stx(2d), eae-beta, eae-eta and eae-zeta subtypes are prevalent in STEC and EPEC isolates in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first subtype analysis of stx(2) and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans.

Racing, ornamental and city pigeons carry shiga toxin producing Escherichia coli (STEC) with different Shiga toxin subtypes, urging further analysis of their epidemiological role in the spread of STEC.

Pigeons are known to shed zoonotic pathogens. Therefore, in this study a total of 366 droppings from pigeons were analysed using PCR and DNA-DNA-hybridization for Shiga toxin producing E. coli (STEC). Specimens were collected from three different groups of pigeons: 247 collective and 3 individual droppings from racing pigeons, 26 collective and 40 individual from ornamental pigeons as well as 50 collective droppings from city pigeons. Initial screening experiments revealed a total 245 (66.9%) droppings to be Shiga toxin gene positive. Of these 36% were positive for stx1, 9% for stx2 and 37% for stx2f. Prevalence significantly (p < 0.001) differed in regard to the pigeon groups examined. Droppings from racing pigeons showed prevalence of 45.6% for stx1, 3.2% for stx2, and 33.2% for stx2f, while the distribution of stx-positive specimens was more even in ornamental pigeons (15% stx1, 27% stx2, and 26% stx2f). In specimens from city pigeons, stx2f was found to be most prevalent with 76% (2% stx1, 16% stx2). In 161 samples, stx genes were detected by PCR as well as DNA-DNA-hybridization. From these 161 samples, 20 were randomly chosen for isolation of STEC. A total of 27 STEC strains were isolated from 13 of these 20 samples. Six of the STEC were positive for stx1, 21 harbored stx2f. Further typing for virulence factor genes revealed the existence of eae in 4 of the 6 stx1-positive strains, as well as in 19 of the 21 stx2f-positive strains. eae is known to be crucially involved in the ability of E. coli strains to cause the "attaching and effacing" lesion in the gut, while stx2fSTEC are assumed to be host specific for pigeons. Here we report the first description of stx1- and eae-positive STEC strains in pigeons from Germany, especially in racing and ornamental pigeons. Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons.

[The serotype and Shiga toxin type of Shiga toxin-producing Escherichia coli from humans and various animals].

To clear the route of STEC (Shiga toxin-producing Escherichia coli) infection to humans, we examined the serotype. Shiga toxin genotype and eae gene of STEC strains from humans and various animals. The most predominant serotype originated from humans was O157:H7, followed by O26:H11, and other serotypes were O91:H21, O103:H2, O111:NM, O121:H19, etc. The eae gene was found in 79 of 93 strains from human origin. The serotypes of STEC from cattle were significant by similar to that of STEC from humans. The eae gene was found in 44 of 87 strains from cattle. Shiga toxin genotypes possessed O157 strains from humans and cattle, were divided into six groups, stx1, stx2, stx2c, stx1 + stx2, stx1 + stx2c and stx2 + stx2c. Moreover, frequency rates of Shiga toxin genotypes of O157 were also similar to both human and cattle origins. The serotypes of STEC from sheep were also a little similar to that of STEC from humans. Seven of 8 strains from deer possessed stx2d gene that the strains from humans seldom possessed, and none of the strains possessed eae gene. In STEC originated from swine, 15 of 25 strains were O139:H1 that Shiga toxin genotype was stx2e. It was thought that the sources of STEC infection to human are cattle and sheep, and deer and swine had little possibility to human STEC infection.

Prevalence and characteristics of shiga toxin-producing Escherichia coli in beef cattle slaughtered on Prince Edward Island.

Fecal swabs obtained from a random sample of 1,000 beef slaughter steers and heifers from 123 Prince Edward Island (P.E.I.) farms were examined for the presence of Shiga toxin-producing Escherichia coli (STEC) using a Vero cell assay (VCA). Multiple isolates from each positive sample were tested similarly. VCA-positive isolates were confirmed as E. coli biochemically, tested for drug resistance, serotyped, and tested by polymerase chain reaction (PCR). Animals were classified as positive when an isolate was positive on VCA and the presence of the gene responsible for toxin production was confirmed by PCR. The prevalence of STEC in beef slaughter steers and heifers on P.E.I. was 4% (40 of 1,000). The total number of isolates was 43, and these comprised 26 serotypes, including 13 isolates belonging to 6 serotypes known to be associated with human illness. The most frequently isolated STEC serotype was E. coli O157 (5 isolates out of 43). Of the five E. coli O157 isolates, four were E. coli O157:H7, a serious human pathogen. The majority of STEC isolates, including all O157:H7, isolates, were susceptible to 16 commonly used antimicrobial drugs. According to PCR, 65% of the STEC isolates had the gene for Stx1. Four of these isolates, including two O157:H7, had genes for Shiga toxin (Stx)1 and Stx2.