STEC O157 Research Articles

Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy

The aim of this study was to monitor the presence of Shiga toxin (Stx)-producing Escherichia coli in dairy farms authorized to sell raw milk and other farms, located in the same area, which sell milk to industry or use it to produce Parmesan or Grana cheese. Our research was focused on the serogroups O157 and O26, which are the most common in human cases in Italy and genetic markers that characterize the strains that can cause hemorrhagic colitis and hemolytic uremic syndrome (EHEC) in humans. Overall, 255 bulk-milk and 225 milk filter samples were screened for the presence of Shiga toxin genes (stx1 and stx2), O157 and O26 serogroups by using PCR. The samples were collected in 193 bovine dairy farms located in Northern Italy, including 32 farms selling raw milk to consumers. According to the preliminary PCR screening test, 32 out of 255 (12.5%; CI95%, 8.7% to 17.3%) bulk milk samples and 68 out of 225 (30.2%; CI95%, 24.3% to 36.7%) milk filters were positive for stx genes. Of the 32 milk samples that were stx-positive, 4 (1.6%, CI95%, 0.4% to 4%) were also positive by PCR for the rfbEO157 gene and 6 (2.4%, CI95%, 0.9% to 5.1%) were positive for the wzxO26 gene. The culture detection method, which was based on the immunomagnetic separation, achieved isolation rates of E. coli serogroups O157 and O26 in 25–67% of the milk samples that tested positive by PCR for these serogroups. STEC O26 was detected in one milk filter (1.6%) from a farm that sells raw milk to consumers directly and one sample (1.4%) of bulk milk intended for pasteurization. The presence of STEC O157 was also detected in 2 milk filters (1.7%) from farms that use milk to produce Grana cheese. All the STEC stains O157 and O26 isolated carried the genes eae and espK and genes belonging to the pathogenicity island OI-122 (efa1/2, sen, pagC), which are markers suitable for screening the human virulent EHEC strains. These virulence markers were also detected in the three strains of stx-negative E. coli O157 isolated from two filters and one milk sample. These strains could be therefore EHEC strains that have lost the stx genes (EHEC-derivative strains). Concern arise for the presence of EHEC O26 and E. coli O157 isolates that are suspected to be an EHEC-derivative in the milk filters sampled in farms that are used to sell raw milk to consumers and in other dairy farms.

Shiga toxin-producingEscherichia coliin swine: the public health perspective

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that are an important public health concern. STEC infection is associated with severe clinical diseases in human beings, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), which can lead to kidney failure and death. Cattle are the most important STEC reservoir. However, a number of STEC outbreaks and HUS cases have been attributed to pork products. In swine, STEC strains are known to be associated with edema disease. Nevertheless, the relationship between STEC of swine origin and human illness has yet to be determined. This review critically summarizes epidemiologic and biological studies of swine STEC. Several epidemiologic studies conducted in multiple regions of the world have demonstrated that domestic swine can carry and shed STEC. Moreover, animal studies have demonstrated that swine are susceptible to STEC O157:H7 infection and can shed the bacterium for 2 months. A limited number of molecular epidemiologic studies, however, have provided conflicting evidence regarding the relationship between swine STEC and human illness. The role that swine play in STEC transmission to people and the contribution to human disease frequency requires further evaluation.

Molecular Identification of Shiga-Toxin Producing and Enteropathogenic <i>Escherichia coli</i> (STEC and EPEC) in Diarrheic and Healthy Young Alpacas

Isolation and biochemical and molecular identification of 303 strains of Escherichia coli obtained from diarrheic and healthy young alpacas of Puno-Peru, were realized. PCR amplification for 7 virulence factor genes associated with STEC, STEC O157:H7, EPEC: sxt1, sxt2, rfbO157, fliCH7, hlyA, eae y bfp were determined. A total of 39 strains (12.88%) showed amplification for one or more of these genes. Twenty three strains (59%) were classified as STEC and 16 strains (41%) as EPEC. An 88.18% (34/39) of STEC and EPEC strains were obtained from healthy alpacas and only 11.82% (5/39) from diarrheic alpacas considering this specie as potential zoonotic reservoir of STEC and EPEC.

STEC in the beef chain: One health approach

Since the early 1980s, E. coli O157 has emerged as one of the most significant pathogens of public health relevance not because of the incidence of the illness, which is much lower than that of other food borne pathogens such as Campylobacter or Salmonella, but because of the severity of the symptoms, the low infectious dose and potential sequelae. Shiga toxin - producing (STEC) Escherichia coli is a human pathogen that can cause hemorrhagic colitis (bloody diarrhea) and sometimes hemolytic uremic syndrome (HUS), a life-threatening disease that causes kidney damage. Cattle carry mixtures of O157 and non-O157 STEC in their intestines that are not necessarily pathogenic to humans. The O157 STEC serogroup was until recently responsible for the majority of disease outbreaks reported for STECs in North America, however now the non-O157 STECs account for almost 50% of the reported human disease outbreaks in North America and Europe. The STECs are shed at significant levels by healthy/asymptomatic cattle, e.g. cattle with jejunal hemorrhage syndrome. The shedding leads to contamination of the farm environment. This may lead to direct or indirect contamination of cattle hides, which, in turn, can serve as the main source of carcass contamination during slaughter and dressing of cattle at abattoirs or contamination of fresh beef and beef products. A science-based risk assessment is needed to assess the public health impact, the consumer exposure to the pathogen and for design of the most effective risk mitigation strategies regarding prevention and reduction of beef-borne O157 and non-O157 STECs.

The inhibitory effect of colloidal bismuth hydroxide gel on Escherichia coli O157:H7 and on the activity of Shiga toxins.

BackgroundShiga toxin-producing Escherichia coli (STEC) is the causative agent of hemolytic uremic syndrome (HUS). Colloidal bismuth hydroxide gel (CBHG) is an anti-diarrheal and antisecretory compound, which does not inhibit gastrointestinal motility and reaches an in vivo gut concentration of 10.8 mg/ml of bismuth. Its action on bacteria has not been studied. We analyzed its inhibitory effects on STEC, as well as the deactivation of the Shiga toxin (Stx) and its ability to block the spread of genes encoding Stx. We determined a minimum inhibitory concentration and bactericidal concentration for the STEC O157:H7 strain (EDL933), with CBHG and Chobet® bismuth cream with pectin (CBCHP). We analyzed its effect on Stx by means of cytotoxicity assay and ELISA, as well as its effect on the free 933 W Stx phage.ResultsEffect on the EDL933 strain: CBHG: MIC 10 mg/ml of bismuth. CBCHP: MIC 6 mg/ml and MBC 15 mg/ml of bismuth. Effect on EDL933 virulence factors: significant decrease in active Stx and 933 W Stx phage titer. ELISA did not find significant differences with treatment.ConclusionsThe results obtained may be useful in the development of new therapeutic strategies based on the use of CBHG to prevent or improve the prognosis of HUS, as it can be used to control STEC infections.

Stochastic Simulation Model Comparing Distributions of STEC O157 Faecal Shedding Prevalence Between Cattle Vaccinated With Type III Secreted Protein Vaccines and Non‐Vaccinated Cattle

Pens of cattle with high Escherichia coli O157:H7 (STEC O157) prevalence at harvest may present a greater risk to food safety than pens of lower prevalence. Vaccination of live cattle against STEC O157 has been proposed as an approach to reduce STEC O157 prevalence in live cattle. Our objective was to create a stochastic simulation model to evaluate the effectiveness of pre-harvest interventions. We used the model to compare STEC O157 prevalence distributions for summer- and winter-fed cattle to summer-fed cattle immunized with a type III secreted protein (TTSP) vaccine. Model inputs were an estimate of vaccine efficacy, observed frequency distributions for number of animals within a pen, and pen-level faecal shedding prevalence for summer and winter. Uncertainty about vaccine efficacy was simulated using a log-normal distribution (mean = 58%, SE = 0.14). Model outputs were distributions of STEC O157 faecal pen prevalence of summer-fed cattle unvaccinated and vaccinated, and winter-fed cattle unvaccinated. The simulation was performed 5000 times. Summer faecal prevalence ranged from 0% to 80% (average = 30%). Thirty-six per cent of summer-fed pens had STEC O157 prevalence >40%. Winter faecal prevalence ranged from 0% to 60% (average = 10%). Seven per cent of winter-fed pens had STEC O157 prevalence >40%. Faecal prevalence for summer-fed pens vaccinated with a 58% efficacious vaccine product ranged from 0% to 52% (average = 13%). Less than one per cent of vaccinated pens had STEC O157 prevalence >40%. In this simulation, vaccination mitigated the risk of STEC O157 faecal shedding to levels comparable to winter, with the major effects being reduced average shedding prevalence, reduced variability in prevalence distribution, and a reduction in the occurrence of the highest prevalence pens. Food safety decision-makers may find this modelling approach useful for evaluating the value of pre-harvest interventions.

Assessment of Heterogeneity of Efficacy of a Three-Dose Regimen of a Type III Secreted Protein Vaccine for Reducing STEC O157 in Feces of Feedlot Cattle

Preharvest control of Escherichia coli O157:H7 (STEC O157) may prevent human illness by reducing the presence of STEC O157 throughout the beef production chain. Immunization of cattle with a type III secreted protein vaccine inhibits colonization of cattle with STEC O157 and reduces the probability of fecal shedding and hide contamination. Our objectives were to perform a meta-analysis to estimate efficacy of a three-dose regimen of TTSP vaccine at reducing the presence of STEC O157 in the feces of feedlot cattle and to test factors that might modify vaccine efficacy. Pen-level data (n=184 pens, 1462 cattle) from four randomized controlled vaccine trials conducted from 2002 to 2008 at the University of Nebraska-Lincoln were analyzed. Factors explaining a culture-positive fecal sample were tested in generalized estimating equations logistic regression and log-binomial models. An autoregressive correlation structure was defined to account for clustering of repeated test-periods within block. Clustering or potential confounding by study was accounted for by treating study as a fixed effect. STEC O157 was detected from 661 of 5451 postvaccination fecal samples. The probability to detect STEC O157 postvaccination was 8.4% and 15.8% in vaccinated and unvaccinated cattle, respectively. Interactions between vaccination and (1) study; (2) prevalence of control pens within each time-place cluster; and (3) days from vaccination were not significant or fit poorly with observed data. Adjusting for study, cattle in pens receiving three doses of vaccine were less likely to shed STEC O157 (odds ratio=0.46, p<0.0001). Model-adjusted vaccine efficacy was 48% (95% confidence interval: 0.37-0.57). We concluded that a three-dose regimen type III secreted protein vaccine was efficacious at reducing the probability of detecting STEC O157 in the feces of cattle and that vaccine efficacy was not modified by study or level of prevalence observed in control pens.

Impact of Management Practices and Distillers' Grains Feeding on the Prevalence ofEscherichia coliO157 in Feedlot Cattle in Minnesota

Escherichia coli O157 is a foodborne pathogen that can be transmitted by contaminated ground beef and is shed naturally in cattle feces. Recent reports indicated that feeding distillers' grains (DG) to cattle increased fecal shedding and prevalence of E. coli O157. In Minnesota, feeding DG with solubles (DGS) to livestock became widespread within the last 10 years, but there is no report about the prevalence of E. coli O157 in beef cattle in this state. This study was undertaken to survey the fecal prevalence of E. coli O157 in cattle fed diets containing DG and its association with environmental conditions and management practices. Fecal samples were collected from three feedlots during a 1-year period. All animals in those feedlots were fed different DGS levels. E. coli O157 presence was determined using a combination of enrichment, immunomagnetic separation, plating onto sorbitol MacConkey agar, and confirmation of isolates by immunoassay and multiplex virulence genes polymerase chain reaction analysis. Overall, E. coli O157 was confirmed in 9.7% of samples. Prevalence during summer was 30% and declined to less than 10% the rest of the year. In animals grouped by dietary DGS concentration, no significant difference in prevalence (12.0 and 5.5%) was detected between the low and the high average groups (less and more than 20%). Previous feeding of DGS before arriving to the feedlot also had no influence on fecal prevalence. The presence of several interacting variables, uncontrolled in a real-life feedlot environment, was the likely reason for our observation and suggested that at the levels studied, DGS had no effect on the STEC O157 prevalence in cattle populations.

Outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 associated with romaine lettuce consumption, 2011.

BackgroundShiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States.Materials and MethodsWe defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted.ResultsWe identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1–97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5–176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar.ConclusionsThis multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.

Prevalence and Characterization of Shiga-Toxin O157:H7 and Non-O157:H7 Enterohemorrhagic Escherichia Coli Isolated from Different Sources

ABSTRACTShiga-toxin-producing Escherichia coli (STEC) is recognized as an important foodborne pathogen responsible for sporadic cases to serious outbreaks worldwide. The morbidity and mortality associated with several recent outbreaks due to STEC have highlighted the threat this organism poses to public health. This study was conducted to identify, characterize the virulence traits and antibiogram of enterohemorrhagic E. coli (EHEC) from different sources. A total of 384 samples from human, animal and environmental sources were collected from different locations in Ismailia city, Egypt. E. coli isolates (n = 283) were identified by conventional microbiology culture, and phenotypically characterized with biochemical and motility tests. Multiplex PCR (mPCR) was applied for the detection of virulence genes (stx1, stx2, eaeA, and EHEC hlyA). From the overall prevalence of E. coli isolates, 31 % (89/283) were isolated from stools of people with diarrhea; 17.3 % (49/283) were from stools of sheep, cattle and chicken with diarrhea; 16.5 % (47/283) were from urine of peple with a urinary tract infection; 17.3 % (49/283) were from fresh water; 6.4 % (18/283) from seafood; 6.02 % (17/283) from processed meat products; 3.9 % (11/283) from dairy products; and 1.1 % (3/283) from poultry products (liver). The antibiotic sensitivity pattern showed that the isolates carried a multidrug resistance (MDR) phenotype to at least four antibiotics from different classes: erythromycin (E), gentamicin (CN), cefazolin (CZ), thiampinicol (TP), vancomycin (VA), ciprofloxacin (CIP), and ampicillin (AM). Shiga toxin was identified in ten suspected EHEC by mPCR. Serotyping of these 10 E. coli isolates demonstrated the circulation of five serotypes (O157, O158, O114, O125 and O26) (10/283 [4%]): three isolates from people (serotype O157, O158), four isolates from animals (serotype O114, O26), two isolates from meat products (serotype O125, O158), and an isolate from fresh water (serotype O114). This study identified STEC O157 from human cases with diarrhea, and demonstrated that meat and water were contaminated with more than one serotype of non-O157 STEC strains. This is a cause of concern owing to their potential to cause human infections.

Comparison of Three Different Methods for Detection of Shiga Toxin-Producing Escherichia coli in a Tertiary Pediatric Care Center

Shiga toxin-producing Escherichia coli (STEC) is a well-known cause of sporadic and epidemic food-borne gastroenteritis. A low infectious dose, approximately 10 microorganisms, is sufficient to cause disease that may lead to hemolytic-uremic syndrome. The objective of this study was to compare the performances of an in-house real-time PCR, a commercial enzyme immunoassay (EIA) (Premier EHEC; Meridian Bioscience), and culture on sorbitol MacConkey agar for the detection of STEC in a tertiary care pediatric hospital. Of 632 stool samples tested, 21 were positive for STEC. All were detected by PCR, 6 were detected by EIA, and only 5 O157 STEC isolates were identified by culture. Among the 15 specimens falsely negative by EIA, there were 9 Stx1, 2 Stx2, and 4 Stx1 and Stx2 STEC isolates. The latter group included 2 O157 STEC isolates that would have been missed if only EIA had been performed. To our knowledge, this is the first prospective study performed in a pediatric hospital which demonstrates the superiority of PCR over EIA for the detection of STEC. We conclude that PCR is specific and more sensitive than EIA. PCR should be considered for routine use in clinical settings where molecular detection facilities are available. Its lower limit of detection, equivalent to the infectious dose, is an obvious advantage for patient care and public health surveillance.

Escherichia coli O157 in foodstuffs: first year results of monitoring plan in Piedmont region, 2011

A total of 260 food samples were examined for the presence of Shiga toxin-producing Escherichia coli O157 (STEC O157). Samples were collected between May 2011 and September 2011 in Piedmont region and included 120 minced meat and meat preparation (hamburger and meat balls) and 180 soft and semi-soft cheeses made from raw milk with less than 60 days of ripening. Samples were collected at re-tail level. All samples were tested using an Enzyme Linked Fluorescent Assay (ELFA) (AFNOR BIO 12/8 - 07/00), with kit VIDAS&reg; ECO (bioM&eacute;rieux); if positive, the samples have been tested with VIDAS&reg; ICE and spread onto selective media to allow the growth of the strains. If present, all the strains have been tested to detect the genes encoding the pathogen factors (stx1, stx2 and eae). STEC O157 was not detected in any products. This survey on the presence of STEC O157 in foodstuffs provided data, demonstrating a low prevalence of the pathogen in our Region.

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes.

Molecular Characterization Reveals Three Distinct Clonal Groups among Clinical Shiga Toxin-Producing Escherichia coli Strains of Serogroup O103

Shiga toxin-producing Escherichia coli (STEC) is one of the most important groups of food-borne pathogens, and STEC strains belonging to the serotype O103:H2 can cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. STEC O103:non-H2 strains are also sometimes isolated from human patients, but their genetic characteristics and role in significant human enteric disease are not yet understood. Here, we investigated 17 STEC O103:non-H2 strains, including O103:H11, O103:H25, O103:HUT (UT [untypeable]), and O103:H- (nonmotile) isolated in Japan, and their characteristics were compared to those of STEC O103:H2 and other serotype STEC strains. Sequence analyses of fliC and eae genes revealed that strains possessed any of the following combinations: fliC-H2/eae-epsilon, fliC-H11/eae-beta1, and fliC-H25/eae-theta, where fliC-H2, -H11, and -H25 indicate fliC genes encoding H2, H11, and H25 flagella antigens, respectively, and eae-epsilon, -beta1, and -theta indicate eae genes encoding epsilon, beta1, and theta subclass intimins, respectively. Phylogenetic analysis based on the sequences of seven housekeeping genes demonstrated that the O103:H11/[fliC-H11] and O103:H25/[fliC-H25] strains formed two distinct groups, different from that of the O103:H2/[fliC-H2] strains. Interestingly, a group consisting of O103:H11 strains was closely related to STEC O26:H11, which is recognized as a most important non-O157 serotype, suggesting that the STEC O103:H11 and STEC O26:H11 clones evolved from a common ancestor. The multiplex PCR system for the rapid typing of STEC O103 strains described in the present study may aid clinical and epidemiological studies of the STEC O103:H2, O103:H11, and O103:H25 groups. In addition, our data provide further insights into the high variability of STEC stains with emerging new serotypes.

Molecular Characterization of Shiga Toxin-Producing Escherichia coli Isolated from the Environment of a Dairy Farm

Environmental samples were taken from ground, cattle water troughs, and feeders from a dairy farm with different STEC prevalence between animal categories (weaning calves, rearing calves, and dairy cows). Overall, 23 % of samples were positive for stx genes, stx(2) being the most prevalent type. Isolates were analyzed by PCR monoplex to confirm generic E. coli and by two multiplex PCR to investigate the presence of stx(1), stx(2), eae, saa, ehxA, and other putative virulence genes encoded in STEC plasmids: katP, espP, subA, and stcE. The toxin genes were subtyped and the strains were serotyped. The ground and the environment of the rearing calves were the sites with the highest number of STEC-positive samples; however, cattle water troughs and the environment of cows were the places with the greater chance of finding stx(2EDL933) which is a subtype associated with serious disease in humans. Several non-O157 STEC serotypes were detected. The serotypes O8:H19; O26:H11; O26:H-; O118:H2; O141:H-; and O145:H- have been asociated with human illness. Furthermore, the emergent pathogen STEC O157:H- (stx(1)-ehxA-eae) was detected in the environment of the weaning calves. These results emphasize the risk that represents the environment as source of STEC, a potential pathogen for human and suggest the importance of developing control methods designed to prevent contaminations of food products and transmission from animal to person.

Characterization of 4 T1-like lytic bacteriophages that lyse Shiga-toxin Escherichia coli O157:H7

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003-0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ∼44 kb, but EcoRІ or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%-82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%-92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.

Genomic, Proteomic and Physiological Characterization of a T5-like Bacteriophage for Control of Shiga Toxin-Producing Escherichia coli O157:H7

Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31×10−9 ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60%) to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37–41%) to tail fibers of E. coli phage phiEco32 of Podoviridae, a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein (pb5) shared an overall identify of 29–72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip (pb2). The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent.

Phylogeny of Shiga Toxin-Producing Escherichia coli O157 Isolated from Cattle and Clinically Ill Humans

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H− (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.

A new health threat in Europe: Shiga toxin–producing Escherichia coli O104:H4 infections

Since early May 2011, a large outbreak of enterohemorrhagic gastroenteritis and hemolytic uremic syndrome (HUS) related to infections with Shiga toxin-producing Escherichia coli O104:H4 (STEC O104:H4) had been reported in Germany, which marks one of the largest outbreaks ever described of the HUS worldwide. The HUS outbreak was unusual, and there were important differences between this outbreak and previous large outbreaks, primarily those of STEC O157:H7 both clinically and microbiologically. This outbreak highlights the importance of collaboration of all aspects of public health to detect the outbreak, to identify and characterize the causative agent, to find the vehicles of transmission, and to control the infection.

Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food

The aim of the current study was to evaluate a multiplex PCR (mPCR) detection test combined with the evaluation of a previously described isolation method. Minced beef, raw-milk cheese and sprouted seed samples were inoculated with low amounts (7–58 cfu 25 g −1) of non-stressed, cold-stressed or freeze-stressed clinical STEC strains, including serogroups O26, O103, O111, O145, sorbitol fermenting (SF) O157 and non-sorbitol fermenting (NSF) O157. The inoculated pathogen was detected using a 24 h-enrichment followed by an mPCR protocol, and in parallel isolated using an enrichment step of 6 and 24 h, followed by selective plating of the enriched broth and selective plating of the immunomagnetic separation (IMS) product. Recovery results were evaluated and compared. Successful mPCR detection and isolation was obtained for non-stressed and cold-stressed STEC cells in minced beef and raw-milk cheese samples, except for serogroups O111 and SF O157. For freeze-stressed cells and sprouted seed samples, false negatives were often found. Isolation was better after 24 h-enrichment compared to 6 h-enrichment. IMS improved in some cases the isolation of non-stressed and cold-stressed cells belonging to serogroups O111 and O157 from minced beef and raw-milk cheese and freeze-stressed cells of all tested serogroups from minced beef.