OmpR coordinates the expression of virulence factors of Enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans.

Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.

Do changes in STEC diagnostics mislead interpretation of disease surveillance data in Switzerland? Time trends in positivity, 2007 to 2016.

BackgroundLaboratory-confirmed cases of Shiga toxin-producing Escherichia coli (STEC) have been notifiable to the National Notification System for Infectious Diseases in Switzerland since 1999. Since 2015, a large increase in case numbers has been observed. Around the same time, syndromic multiplex PCR started to replace other diagnostic methods in standard laboratory practice for gastrointestinal pathogen testing, suggesting that the increase in notified cases is due to a change in test practices and numbers.AimThis study examined the impact of changes in diagnostic methods, in particular the introduction of multiplex PCR panels, on routine STEC surveillance data in Switzerland.MethodsWe analysed routine laboratory data from 11 laboratories, which reported 61.9% of all STEC cases from 2007 to 2016 to calculate the positivity, i.e. the rate of the number of positive STEC tests divided by the total number of tests performed.ResultsThe introduction of multiplex PCR had a strong impact on STEC test frequency and identified cases, with the number of tests performed increasing sevenfold from 2007 to 2016. Still, age- and sex-standardised positivity increased from 0.8% in 2007 to 1.7% in 2016.ConclusionIncreasing positivity suggests that the increase in case notifications cannot be attributed to an increase in test numbers alone. Therefore, we cannot exclude a real epidemiological trend for the observed increase. Modernising the notification system to address current gaps in information availability, e.g. diagnostic methods, and improved triangulation of clinical presentation, diagnostic and serotype information are needed to deal with emerging disease and technological advances.

Influences of epigallocatechin gallate and citric acid on Escherichia coli O157:H7 toxin gene expression and virulence-associated stress response.

Citric acid and EGCG at their minimum inhibitory concentrations were tested in this study. Logarithmic phase cells of Escherichia coli O157:H7 (ATCC 43895) were exposed to EGCG and citric acid respectively. The results of RT-real time PCR showed that both EGCG and citric acid increased stx2 and oxyR expression and decreased stx1, recA and Q expression. The result of Western blotting for RecA protein further indicated that both EGCG and citric acid decreased RecA production. Both EGCG and citric acid increased the level of intracellular reactive oxygen species and H2 O2 production and decreased superoxide dismutase activity. Therefore, EGCG and citric acid might induce stx2 production by increasing oxidative stress response and inhibit stx1 production by suppressing SOS response. In our study, the differential effects of the two antimicrobials were observed. EGCG reduced ompC and rpoS expression. However, citric acid caused an increase in ompC and rpoS expression. Membrane permeability is associated with toxin release. Citric acid increased the outer membrane permeability of E. coli O157:H7. However, the outer membrane of E. coli O157:H7 remained unaffected by EGCG. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxins are the major virulence factors of Escherichia coli O157:H7. The use of antimicrobials triggering Shiga toxin production is controversial. (-)-epigallocatechin-3-gallate (EGCG) citric acid are often used singly or in combination to prevent micro-organisms in some food products. This study evaluated toxin induction in E. coli O157:H7 in response to EGCG and citric acid and investigated the potential mechanism of action. The findings may contribute to the proper use of EGCG and citric acid as antimicrobials.

Antimicrobial Drug-Resistant Shiga Toxin-Producing Escherichia coli Infections, Michigan, USA.

High frequencies of antimicrobial drug resistance were observed in O157 and non-O157 Shiga toxin-producing E. coli strains recovered from patients in Michigan during 2010-2014. Resistance was more common in non-O157 strains and independently associated with hospitalization, indicating that resistance could contribute to more severe disease outcomes.

Shiga Toxin 1-Producing Shigella sonnei Infections, California, United States, 2014-2015.

Shiga toxins (Stx) are primarily associated with Shiga toxin-producing Escherichia coli and Shigella dysenteriae serotype 1. Stx production by other shigellae is uncommon, but in 2014, Stx1-producing S. sonnei infections were detected in California. Surveillance was enhanced to test S. sonnei isolates for the presence and expression of stx genes, perform DNA subtyping, describe clinical and epidemiologic characteristics of case-patients, and investigate for sources of infection. During June 2014-April 2015, we identified 56 cases of Stx1-producing S. sonnei, in 2 clusters. All isolates encoded stx1 and produced active Stx1. Multiple pulsed-field gel electrophoresis patterns were identified. Bloody diarrhea was reported by 71% of case-patients; none had hemolytic uremic syndrome. Some initial cases were epidemiologically linked to travel to Mexico, but subsequent infections were transmitted domestically. Continued surveillance of Stx1-producing S. sonnei in California is necessary to characterize its features and plan for reduction of its spread in the United States.

A Rapid Immunoassay for Detection of Shiga Toxin-Producing Escherichia coli Directly from Human Fecal Samples and Its Performance in Detection of Toxin Subtypes.

Fecal samples (n = 531) submitted to a regional clinical laboratory during a 6-month period were tested for the presence of Shiga toxin using both a Vero cell cytotoxicity assay and the Shiga Toxin Quik Chek test (STQC), a rapid membrane immunoassay. Testing the samples directly (without culture), 9 positives were identified by the Vero cell assay, all of which were also detected by the STQC. The correlation between the two assays was 100%. Not all of the identified positive samples were detected when fecal broth cultures were tested. By testing broth cultures of characterized isolates representing all described Shiga toxin subtypes, the STQC detected all subtypes. Levels of induction of toxin production by ciprofloxacin differed among the strains tested, with more toxin induction seen in strains harboring Stx2 phages than in those harboring Stx1 phages.

The nitric oxide reductase of enterohaemorrhagic Escherichia coli plays an important role for the survival within macrophages.

In enterohaemorrhagic Escherichia coli (EHEC) O157, there are two types of anaerobic nitric oxide (NO) reductase genes, an intact gene (norV) and a 204 bp deletion gene (norVs). Epidemiological analysis has revealed that norV-type EHEC are more virulent than norVs-type EHEC. Thus, to reveal the role of NO reductase during EHEC infection, we constructed isogenic norV-type and norVs-type EHEC mutant strains. Under anaerobic conditions, the norV-type EHEC was protected from NO-mediated growth inhibition, while the norVs-type EHEC mutant strain was not, suggesting that NorV of EHEC was effective in the anaerobic detoxification. We then investigated the role of NO reductase within macrophages. The norV-type EHEC produced a lower NO level within macrophages compared with the norVs-type EHEC. Moreover, the norV-type EHEC resulted in higher levels of Shiga toxin 2 (Stx2) within macrophages compared with the norVs-type EHEC. Finally, the norV-type EHEC showed a better level of survival than the norVs-type EHEC. These data suggest that the intact norV gene plays an important role for the survival of EHEC within macrophages, and is a direct virulence determinant of EHEC.

Verocytotoxin-producing Escherichia coli O128ab:H2 bacteremia in a 27-year-old male with hemolytic-uremic syndrome.

Verocytotoxin-producing Escherichia coli (VTEC) strains of serotype O128ab:H2 were isolated from blood and stool of a 27-year-old male presenting diarrhea-associated hemolytic-uremic syndrome complicated by bacteremia. This report once again illustrates the pathogenic potential of a non-O157 VTEC strain carrying a virulence profile previously associated with mild disease.

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009.

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.

Investigation of a spatiotemporal cluster of verotoxin-producing Escherichia coli O157 infections in eastern England in 2007.

An outbreak of verotoxin-producing Escherichia coli O157 (VTEC O157) infections linked to an open farm occurred in eastern England in April and May 2007. This paper describes the investigation and highlights the importance of multidisciplinary collaboration for successful control of such outbreaks. There was a temporal cluster of 12 confirmed symptomatic cases of VTEC O157 and one asymptomatic carrier, from five families. The investigation revealed that four of these cases formed part of an outbreak involving two families who visited an open farm. The phenotypic and genotypic characteristics of the isolates from the two families and the putative farm animal contacts were indistinguishable, indicating that the animals were the source of the primary infections. No epidemiological link could be established between the remaining three families affected and the open farm or people having visited the farm. Control measures included improved hand washing facilities on the farm, information for visitors and staff, restricted access and suspended petting and feeding of animals, and thorough cleaning and disinfection of affected areas.

Shiga toxin 2-specific but not shiga toxin 1-specific human monoclonal antibody protects piglets challenged with enterohemorrhagic Escherichia coli producing shiga toxin 1 and shiga toxin 2.

Escherichia coli strains that produce Shiga toxin 2 (Stx2) are isolated from hemolytic-uremic syndrome (HUS) cases more frequently than are strains that produce both Shiga toxin 1 (Stx1) and Stx2, whereas strains that produce only Stx1 are rarely isolated from HUS cases. Studies have implicated Stx2 as the sole contributor to acute kidney failure and other systemic complications in humans. The aim of the present study was to determine whether Stx2-specific antibody would be as effective against Shiga toxin-producing Escherichia coli (STEC) strains that produce both Stx1 and Stx2 as it is against strains that produce only Stx2, compared with Stx1-specific antibody. We found that Stx2-specific and Stx1-specific antibodies protected 100% and 0% of piglets, respectively, against oral challenge with a Stx1- and Stx2-producing STEC strain. We conclude that Stx2-specific antibody is sufficient to protect piglets, and possibly humans, against STEC strains that produce both toxins.

Production of verotoxin and distribution of O islands 122 and 43/48 among verotoxin-producing Escherichia coli O103:H2 isolates from cattle and humans.

This study investigated variations in the occurrence of markers of O islands 122 and 43/48 and in verotoxin 1 production in 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of bovine and human origins. None of the genes that were investigated appear to be virulence indicators for human O103:H2 VTEC.

Escherichia coli O157:H7 strain origin, lineage, and Shiga toxin 2 expression affect colonization of cattle.

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10(3) to 10(5) CFU and maximum colonization at 10(7) CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx(1)) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx(2)). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx(2) expression in modulating the amount of E. coli O157:H7 colonization of cattle.

Genotype, serotype, and antibiotic resistance of sorbitol-negative Escherichia coli isolates from feedlot cattle.

Rectal fecal samples from 80 steers receiving Rumensin, Revalor-S, and Liquamycin alone or in combination for growth promotion and disease prevention were examined for the presence of non-O157:H7 Shiga toxin-producing Escherichia coli. All isolates were identified with the API 20E test, virulence genes were detected with a PCR assay, and antibiotic susceptibilities were determined with the Sensititre system. Of the 153 E. coli isolates recovered 126 (82.3%) were sorbitol negative. Isolates were classified into 14 biochemical E. coli groups; 51.6% were negative for arginine dihydrolase, ornithine decarboxylase, sorbitol, and saccharose reactions but positive for lysine decarboxylase, indole production, and rhamnose reactions. Twenty-one O:H serotypes were detected in the 153 E. coli isolates. The most frequent serotypes were O2:H42 (49.7% of isolates), O49:NM (13.7%), O?:H25 (9.2%), and O10:NM (7.2%). One isolate of E. coli O172:H25 and one of E. coli O157: H39 were found. The stx1 gene was found in the two E. coli O98:H25 isolates. The eaeA and e-hlyA genes were detected in 21, 14, and 10 isolates of serotypes O49:NM, O?:H25, and O10:NM, respectively, and in each isolate of serotype O156:H25 and O172:H25. Four E. coli O132:H18 isolates were multiresistant to ampicillin, chloramphenicol, kanamycin, streptomycin, and sulfisoxazole. Tetracycline resistance due to the tet(B) gene was observed in 74 of the 76 E. coli O2:H42 isolates. Except for one isolate, all tetracycline-resistant isolates were negative for the virulence genes eaeA and e-hlyA or stx1. Pulsed-field gel electrophoresis typing revealed that the tetracycline-resistant serotypes were genetically diverse. Our data illustrate that cattle are a potential source of some atypical antibiotic-resistant E. coli isolates that harbor virulence genes.

Transcriptional and Translational Inhibitors Block SOS Response and Shiga Toxin Expression in Enterohemorrhagic Escherichia coli.

Shiga toxins (Stx) induce the symptoms of the life-threatening hemolytic uremic syndrome (HUS) and are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC). The bacterial SOS response is the essential signal for high level production and release of Stx1/2. To assess the potential effectiveness of different antibiotics in blocking SOS response and Stx1/2 production, we constructed a reporter gene based test system that allows for the time-resolved, simultaneous read-out of the SOS response (recAP-cfp) and Stx1 production (stx1::yfp) in EHEC O157:H7 EDL933. We find that cells exposed to inhibitory or subinhibitory concentrations of ciprofloxacin did induce the SOS response, but not when the cells were exposed to rifaximine, azithromycin, tetracycline, gentamicin or ampicillin. Cell lysis and the peak in Stx1 production were substantially delayed with respect to the peak of the SOS response. We used this feature to show that adding transcriptional or translational inhibitors can block Stx1 production even after the SOS response is fully induced. RT-qPCR based tests with other clinically relevant EHEC isolates showed similar results for both Stx1 and Stx2. These observations suggest that transcriptional and translational inhibitors may be of value in treating EHEC infections.

[Molecular detection of shiga toxin-producing (stx1) Escherichia coli and rotavirus in stools of children with diarrhea]. / Detección molecular de Escherichia coli productor de shiga toxina (Stx1) y rotavirus en heces de niños con diarrea.

The presence of E. coli producer of shiga toxin and rotavirus was investigated in 90 stool samples from children less than 3 years old with diarrhea. Three aliquots were separated from each sample: the first one underwent previous enrichment for E. coli O157, the second one was plated on agar MacConkey-Sorbitol and Red Eosine with MUG, and the last one was frozen at -70 degrees C for the later analysis of rotavirus. The search of the antigen O157 of E. coli was carried out by immunochromatography in vitro of Coris Bioconcept (Belgium). The presence of the antigen O157 (rbf O157) and the genes that code for the shiga toxin (stx1 and stx2) were determined by PCR. Rotavirus were detected by electrophoresis in polyacrylamide gels. The 90 samples analyzed by immunochromatography were negative for the antigen O157. The isolates were STEC strains non O157 and contained the gene sx1, showing a 10% of positivity. The electrophoresis for the viral RNA detected rotavirus in 21 (23.33%) samples. This result confirms the rotavirus prevalence and suggests, that the circulation of STEC strains non O157 is an indication of the involvement of these strains in the ethiology of acute diarrheas.

Characterization of Shiga toxigenic Escherichia coli isolated from foods.

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.

Testing of swine feces obtained through the National Animal Health Monitoring System's Swine 2000 study for the presence of Escherichia coli O157:H7.

Fecal samples collected from healthy pigs from 13 of the top 17 swine-producing states were tested for Escherichia coli O157:H7 as part of the National Animal Health Monitoring System Swine 2000 study. Serogroup O157 strains were isolated from 106 of 2,526 fecal samples. None of the isolates were positive by PCR for the fliCh7 (H7 flagellin) gene or for the hly933 (hemolysin) gene; however, one isolate was positive for the stxl gene (Shiga toxin 1), an additional four isolates were positive for the stx2 gene (Shiga toxin 2), and three isolates possessed the eae gene (intimin).

Epidemiology and characterization of Escherichia coli outbreak on a pig farm in South Africa.

An investigation of mortality of piglets through clinical signs, post-mortem, histopathology and bacteriological analyses revealed the causal organism to be Escherichia coli, mainly O149:K91:K88 which belongs to the enterotoxigenic biotypes. Molecular characterization and epidemiologic analysis elucidated it as shiga-toxin (ST) E. coli resistant to ampicillin, cefotaxime, tetracycline, trimethoprim-sulfamethoxazole, tylosin and neomycin. Conventional PCR results detected genes for ST-2, adhesin involved in diffuse adherence (AIDA-1) and F18 fimbriae virulence factors. Survival analyses and logistic regression of piglet mortality patterns showed that season of weaning, weaning weight and age of dam had significant influence on survival rate of piglets. Factors affecting pathogenicity of bowel edema and survival of affected piglets on a farm with persistent infection were reported for the first time. An association of E. coli O149:K91:K88 (F4) with clinical edema disease was made even though it has been reported in the past that this serotype does not produce ST. It was concluded that more stringent measures to mitigate the impact of the disease need to be targeted for spring and in older sows.