H7 Serotype Research Articles

The 2011 Shiga toxin-producing Escherichia coli O104:H4 German outbreak: a lesson in genomic plasticity

Escherichia coli is known as a versatile bacterial species encompassing both commensal and intraintestinal and extraintestinal pathogenic strains. An E. coli genome contains between 4200 and 5500 genes, with <2000 genes being conserved among all strains of the species (the core genome). Also, the pan-genome (i.e. all of the non-orthologous genes found in the species) consists of almost 20 000 genes [1]. Continuous gene flux has occurred during E. coli divergence, as a result of horizontal gene transfers and deletions confined to a small number of conserved positions in the chromosome. This chromosomal plasticity accelerates the adaptation of E. coli to varied environments and lifestyles, as it allows multiple gene combinations that, with epistatic interactions, result in phenotypic diversification. The outbreak of bloody diarrhoea and haemolytic–uraemic syndrome in Germany in May and June 2011, caused by an E. coli O104:H4 strain [2], dramatically illustrates this tremendous E. coli genome plasticity. Owing to the advances in high-throughput sequencing technologies, several complete sequences of isolates from the outbreak were determined within a few days in China and in Germany [3]. It appears that the strain, which belongs to the B1 phylogenetic group of E. coli [4], is of multilocus sequence type (ST)678 (http:// mlst.ucc.ie) and exhibits an O104:H4 serotype [5]. It shares 93% of its sequence with another O104:H4 ST678 strain, E. coli 55989, that was isolated in the 1990s in Bangui (Central African Republic) from a human immunodeficiency virusinfected adult with chronic diarrhoea [1,6]. Surprisingly, this 55989 strain is an enteroaggregative E. coli (EAEC) strain, with genes encoding proteins involved in the biogenesis of aggregative adherence fimbriae, which mediate the adherence of EAEC to the intestinal mucosa [7]. The German outbreak strain, which can be considered as an enterohaemorrhagic E. coli (EHEC) strain according to the disease that it causes, has acquired, since its divergence from the 55989 strain, the phage-mediated Stx2 Shiga toxin but not the locus of enterocyte effacement located on a 35-kb pathogenic island usually present in EHEC, allowing the attaching bacteria to efface the microvilli. It also possesses, like the 55989 strain, several characteristics of extraintestinal pathogenic E. coli (ExPEC) (https://www.genoscope.cns.fr/agc/microscope/home/ index.php) [8]. It has the high-pathogenicity island, involved in iron capture, that has been shown to spread within the species by horizontal gene transfer, implicating homologous recombination [9], and the adhesin-coding gene iha in close vicinity to the siderophore-encoding gene aer, both of which present in the virulent emerging clone O25b ST131 [10]. In this regard, the 55989 strain is virulent in a mouse model of septicaemia, as it killed ten mice of ten inoculated [1]. In addition, the German outbreak strain has acquired resistance to numerous antibiotics, including third-generation cephalosporins, owing to several plasmid-borne genes, such as the extended spectrum b-lactamase CTX-M gene [2], which has been spreading dramatically in E. coli across the world in the last 10 years [11]. Fortunately, antibiotics are not indicated for treatment of the human disease, as they release the toxin, worsening the symptoms. In sum, it appears that the outbreak strain is an illustration of the genomic plasticity of E coli, with the acquisition of genes providing from different E coli pathovars (EAEC, EHEC, and ExPEC) and the acquisition of antibiotic resistance. But what is the origin of this strain? Stx2-positive EAEC strains were previously described in Bangui during the same study that allowed isolation of the 55989 strain, although other characteristics of these strains, such as the serotype and ST, were not provided [6]. Sporadic cases of Stx2 O104:H4 ST678 strains causing diarrhoea and haemolytic– uraemic syndrome were reported in 2001 in Germany [12], in 2004 in France, in 2005 in South Korea [13], in 2009 in Georgia, and in 2010 in Finland [5]. Unlike classic EHEC, which has cattle as its reservoir, EAEC seems to have humans as its reservoir. An organic sprout farm near Hamburg has been incriminated [14] in the actual outbreak, but the exact mechanism of contamination needs to be determined. The main lesson from this outbreak is that we should be aware of the capacity of the E. coli species to produce new combinations of genes, leading to the emergence of highly aggressive strains. Furthermore, antibiotic pressure in human

Phosphorylation of the epidermal growth factor receptor (EGFR) is essential for interleukin-8 release from intestinal epithelial cells in response to challenge with Escherichia coli O157 : H7 flagellin

Enterohaemorrhagic Escherichia coli O157 : H7 is a major foodborne and environmental pathogen responsible for both sporadic cases and outbreaks of food poisoning, which can lead to serious sequelae, such as haemolytic uraemic syndrome. The structural subunit of E. coli O157 : H7 flagella is flagellin, which is both the antigenic determinant of the H7 serotype, an important factor in colonization, and an immunomodulatory protein that has been determined to be a major pro-inflammatory component through the instigation of host cell signalling pathways. Flagellin has highly conserved N- and C-terminal regions that are recognized by the host cell pattern recognition receptor Toll-like receptor (TLR) 5. Activation of this receptor triggers cell signalling cascades, which are known to activate host cell kinases and transcription factors that respond with the production of inflammatory mediators such as the chemokine interleukin-8 (IL-8), although the exact components of this pathway are not yet fully characterized. We demonstrate that E. coli O157 : H7-derived flagellin induces rapid phosphorylation of the epidermal growth factor receptor (EGFR), as an early event in intestinal epithelial cell signalling, and that this is required for the release of the pro-inflammatory cytokine IL-8.

OCCURRENCE, VIRULENCE GENES AND ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI O157 IN BOVINE, CAPRINE, OVINE AND PORCINE CARCASSES IN GREECE

ABSTRACTOne thousand and two hundred carcasses (620 bovine, 130 caprine, 230 ovine and 220 porcine) from several slaughterhouses throughout Greece were examined for the presence of Escherichia coli O157 after evisceration and before chilling. Twelve E. coli O157 strains (1.0%) were isolated of which eight were from bovine (1.3%), one from caprine (0.8%) and three from ovine (1.3%) meat. None was isolated from pork meat. Six out of the 12 E. coli O157 isolates (50.0%) could be classified as Shiga‐toxigenic based on polymerase chain reaction (PCR), belonged to the E. coli O157:H7 serotype and were PCR‐positive for the stx1, stx2 and eae genes except one isolate that was stx1‐negative. The remaining six E. coli O157 isolates tested negative for Shiga‐toxin production, both by immunoassay and by PCR, were PCR‐negative for the eae gene, and all except one (strain LFH34, a bovine carcass isolate) were also PCR‐negative for the H7 flagellar gene. Among the 15 antimicrobials tested, the E. coli O157 isolates were the most frequently resistant to ticarcillin (9 out of the 12 isolates, 75%).PRACTICAL APPLICATIONSThe Shiga toxin‐producing Escherichia coli (STEC) strains are severe human pathogens. Although the prevalence of STEC in bovine meat has been investigated in several countries worldwide, no such study has been conducted previously in Greece. In addition, very few studies in the literature have reported on the prevalence and characterization of STEC isolated from animal carcasses other than bovine. The findings of the present study are also useful for epidemiological purposes (monitoring the evolution and spread of STEC worldwide).

Draft Genome Sequences of Six Escherichia coli Isolates from the Stepwise Model of Emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).

Shiga Toxin-Producing Escherichia coli in Children: Diagnosis and Clinical Manifestations of O157:H7 and Non-O157:H7 Infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.

Escherichia coliO157:H7: Animal Reservoir and Sources of Human Infection

This review surveys the literature on carriage and transmission of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the context of virulence factors and sampling/culture technique. EHEC of the O157:H7 serotype are worldwide zoonotic pathogens responsible for the majority of severe cases of human EHEC disease. EHEC O157:H7 strains are carried primarily by healthy cattle and other ruminants, but most of the bovine strains are not transmitted to people, and do not exhibit virulence factors associated with human disease. Prevalence of EHEC O157:H7 is probably underestimated. Carriage of EHEC O157:H7 by individual animals is typically short-lived, but pen and farm prevalence of specific isolates may extend for months or years and some carriers, designated as supershedders, may harbor high intestinal numbers of the pathogen for extended periods. The prevalence of EHEC O157:H7 in cattle peaks in the summer and is higher in postweaned calves and heifers than in younger and older animals. Virulent strains of EHEC O157:H7 are rarely harbored by pigs or chickens, but are found in turkeys. The bacteria rarely occur in wildlife with the exception of deer and are only sporadically carried by domestic animals and synanthropic rodents and birds. EHEC O157:H7 occur in amphibian, fish, and invertebrate carriers, and can colonize plant surfaces and tissues via attachment mechanisms different from those mediating intestinal attachment. Strains of EHEC O157:H7 exhibit high genetic variability but typically a small number of genetic types predominate in groups of cattle and a farm environment. Transmission to people occurs primarily via ingestion of inadequately processed contaminated food or water and less frequently through contact with manure, animals, or infected people.

Virulence gene profiles and intimin subtypes of Shiga toxin-producing Escherichia coli isolated from healthy and diarrhoeic calves

The virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrhoeic and non-diarrhoeic calves were compared. The strains were also tested for O157:H7, O111 and O26 serotypes, using...

Characterization of Shiga toxin-producing Escherichia coli isolated from dairy cows in Argentina

To feno-genotypically characterize the Shiga toxin-producing Escherichia coli (STEC) population in Argentinean dairy cows. From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52% stx2 and 37% stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty-five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29% stx2, saa, ehxA. One hundred and fifty-six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2(+) , eae(+) , ehxA(+) virulence pattern. We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. The non-O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.

Phenotypic diversity of Escherichia coli O157:H7 strains associated with the plasmid O157.

Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

Prevalence, characterization and clonal analysis of Escherichia coli O157: non-H7 serotypes that carry eae alleles

We examined O157:non-H7 strains isolated from various sources and geographical locations and found 15/57 strains to carry eae alleles, including alpha, beta, epsilon and kappa/delta, suggesting that these strains may be prevalent. All strains were serologically and genetically confirmed to be O157, but none were the H7 serotype or carried any trait virulence factors of the Escherichia coli O157:H7 serotype. Genetic H typing of the eae-positive strains showed that the alpha-eae-bearing strain was H45, while the beta- and epsilon-eae strains were H16 and the kappa/delta-eae strains were H39. The beta- and epsilon-eae-bearing O157:H16 strains shared approximately 90% pulsed-field gel electrophoresis (PFGE) similarity and were distinct from the other strains that had other eae alleles. Interestingly, an epsilon-eae O157:H16 strain isolated from meat in France shared PFGE similarity to the O157:H16 strains from water in the United States. Multilocus sequence typing showed that there is clonal diversity within the O157 serogroup, as some O157:non-H7 strains clustered with EPEC clonal groups, while others clustered within the ST-171 group of diverse strains and serotypes that had not previously included any strains from the O157 serogroup. Clonal analysis also showed that none of the eae-positive O157:non-H7 strains we examined were closely related to the pathogenic O157:H7 serotype.

Enrichment and Detection of Escherichia coli O157:H7 from Water Samples Using an Antibody Modified Microfluidic Chip

Low abundant (<100 cells mL(-1)) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using benchtop real-time quantitative polymerase chain reaction (PCR), targeting genes which effectively discriminated the O157:H7 serotype from other nonpathogenic bacteria. The recovery of target cells from water samples was determined to be approximately 72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and wastewater samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies.

English

&nbsp; The incidence of&nbsp;Escherichia coli&nbsp;0157: H7 was assessed in meat samples from slaughtered cattle in Ibadan metropolis by culturing on sorbitol MacConkey agar and confirmed using serological agglutination kits. The&nbsp;isolates were tested for susceptibility to seven commonly used antimicrobial agents. Out of the total of the 116 non-sorbitol fermenting (NSF)&nbsp;E. coli&nbsp;isolated from the samples, 71 (comprising of 18.4, 2.0, 3.2 and 4.8% from Bodija abattoir, Bashorun, Apata and Iwo Road slaughter slabs, respectively) were confirmed as&nbsp;E. coli&nbsp;O157:H7 serotype. Antibiotics susceptibility profile showed that all the isolates were resistant to one or multiple antibiotics, resulting in eight different resistance patterns. Tetracycline resistant isolates were the highest with 91.4% incidence. The results are of public health significance confirming cattle as major reservoir of EHEC and antimicrobial-resistant organisms. The high level of carcass contamination with microorganisms may be due to unhygienic slaughtering and meat processing engaged in these abattoir and slabs. Indiscriminate and misuse of antimicrobials on livestock in Nigeria could also be responsible for the multiple resistance pattern of the organism. Application of food hygiene practices such as HACCP is recommended for high quality farm to fork wholesome and safe meat for public consumption in Nigeria. &nbsp; Key words:&nbsp;EHEC,&nbsp;Escherichia coli, beef, food safety Ibadan, Nigeria.

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in fresh ground beef from butcher shops in Concepción, Tucumán Province

Shiga toxin-producing Escherichia coli is an emerging foodborne pathogen. There are many STEC serotypes associated with human diseases, being the O157:H7 serotype the most prevalent. Ground beef is the main transmission vehicle. In Concepción city, Tucumán Province, between September and December 2004, two hemolytic uremic syndrome (HUS) cases were diagnosed. The main objective of this work was to detect, isolate and characterize STEC O157 and non-O157 strains in fresh ground beef. Between September and December 2004, 53 fresh ground beef samples were collected from butcher shops in Concepción city. The USDA-FSIS (2002) methodology was used for detection, isolation and characterization of STEC O157:H7. Two PCR techniques for E. coli non-O157 detection and a previous intra-laboratory validated methodology for the isolation and characterization of these strains were used. The stx2 gen was identified in seven samples and the rfbO157 gene also in four of them. However, only one E. coli O157:H7 strain, biotype C, carrying the eae, stx2 and ehxA genes, was isolated. The present study shows the importance of implementing techniques for the detection of this emerging pathogen in meat samples.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA)

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additonal research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.

Similarity and Divergence among Adherent-Invasive Escherichia coli and Extraintestinal Pathogenic E. coli Strains

Adherent-invasive Escherichia coli (AIEC) pathovar strains, which are associated with Crohn's disease, share many genetic and phenotypic features with extraintestinal pathogenic E. coli (ExPEC) strains, but little is known about the level of genetic similarity between the two pathovars. We aimed to determine the frequency of strains with the "AIEC phenotype" among a collection of ExPEC strains and to further search for a common phylogenetic origin for the intestinal and extraintestinal AIEC strains. The adhesion, invasion, and intramacrophage replication capabilities (AIEC phenotype) of 63 ExPEC strains were determined. Correlations between virulence genotype and AIEC phenotype and between intestinal/extraintestinal origin, serotype, and phylogroup were evaluated for the 63 ExPEC and 23 intestinal AIEC strains. Phylogenetic relationships between extraintestinal and intestinal AIEC strains were determined using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Only four (6.35%) ExPEC strains, belonging to the O6:H1, O83:H1, and O25:H4 serotypes, were classified as having an AIEC phenotype. These strains were found to be genetically related to some intestinal AIEC strains of the same serotypes as revealed by MLST. No particular virulence gene sets correlated with the intestinal/extraintestinal origin of the strains or with the AIEC phenotype, whereas the gene sets did correlate with the serogroup. We identified two intestinal AIEC strains and one extraintestinal AIEC strain belonging to the O25:H4 serotype that also belonged to the emerging and virulent clonal group ST131. In conclusion, the ExPEC and AIEC pathovars share similar virulence gene sets, and certain strains are phylogenetically related. However, the majority of ExPEC strains did not behave like AIEC strains, thus confirming that the AIEC pathovar possesses virulence-specific features that, to date, are detectable only phenotypically.

Occurrence and characterisation of enterohaemorrhagic Escherichia coli isolates from diarrhoeic calves

The presence of major virulence factors of enterohaemorrhagic Escherichia coli (EHEC; stx1, stx2, eae, Ehly) were determined among isolates from 158 diarrhoeic calves by multiplex polymerase chain reaction (PCR). Strains positive for virulence factors were subjected to serotype specific PCR assays for O157:H7 and O111 antigens. Additionally, serogroups were determined by three monovalent antisera for O26, O111 and O157 somatic antigens and enterohaemolysin production were also shown phenotypically. Thirteen (8.2%) calves carried strains positive for one or more of the virulence factors tested, and eleven (6.9%) calves harboured the shiga toxin-producing strains (stx1 or stx2). stx1 was detected in eight (5%) and stx2 in three (1.9%) calves. eae and Ehly were observed in the same frequency (6.3%) and were detected in parallel. Of the 13 virulence-positive strains, the predominant genotype was (stx1/eae/Ehly) at 53.8%. None of the EHEC in this study belonged to O157:H7 or O111 serotypes, but four strains (30.7%) belonged to the O26 serogroup. The results show the possible role of stx1/eae in calf diarrhoea and the particular importance of O26 EHEC. Calves can also act as a reservoir for EHEC and in the transmission of the disease to humans.

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine ( n = 950), caprine ( n = 460) and ovine ( n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx 2 -positive, five of which were also stx 1 -positive. The remaining isolate was positive only for the stx 1 gene. All stx-positive isolates (whether positive for stx 1 , stx 2 or stx 1 and stx 2 ) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Characterization of Virulence Genes of Non-O157 Shiga Toxin-Producing &lt;i&gt;Escherichia coli&lt;/i&gt; Isolates from Two Provinces of Iran

Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types among non-O157 isolates. PCR-RFLP results for the fliC gene and classical serology examination with H antisera indicated the presence of nine different H types. In this study, three new serotypes of non-O157:H7 (i.e., O25:H3, O85:H32, and O162:H21) were found to be Shiga toxin producers. These findings reconfirm the results of our previously reported studies showing that non-O157:H7 serotypes are more prevalent under the present conditions. More detailed characterization of these isolates will require additional genetic studies.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.