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The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific ( rfbE ), Shiga toxin ( stx 1 and/or stx 2), and intimin ( eae ) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx 1 and/or stx 2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus . Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δ ldh, surpassed AR113Δ 0273&2024 , exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene's pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113Δ tagO and Δ ccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

Escherichia coli O157:H7 ( E. coli O157:H7) and Campylobacter jejuni ( C. jejuni ) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2 , and eaeA . Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Microbial contamination in foods could lead to illnesses and substantial losses in both food industry and public health sectors. Rapid detection of microbial hazards (i.e., pathogens, hygiene indicator microorganisms) can accelerate surveillance and diagnostic processes reducing transmission and minimizing undesirable consequences. This study developed a multiplex PCR (m-PCR) for the detection of six common foodborne pathogens and hygiene indicators using specific primers for uidA of Escherichia coli , stx2 of Escherichia coli O157:H7, invA of Salmonella spp., int of Shigella spp., ntrA of Klebsiella pneumoniae , and ail of Yersinia enterocolitica and Yersinia pseudotuberculosis . Sensitivity of the m-PCR was 100 fg or ∼20 bacterial cells. Each primer set amplified only the targeted strain, and specificity was demonstrated by lack of nonspecific bands with DNA from 12 other bacterial strains. Following ISO 16140-2:2016, the relative limit of detection of the m-PCR was comparable to that of the gold-standard method; however, the processing time was five times faster. The m-PCR was applied to detect the six pathogens in 100 natural samples (50 pork meat and 50 local fermented food samples) and compared to results of the gold-standard method. Positive cultures for Klebsiella , Salmonella , and E. coli were 66%, 82%, and 88%, respectively, of meat samples and 78%, 26%, and 56%, respectively, of fermented food samples. Escherichia coli O157:H7, Shigella , and Yersinia were not detected in any of the samples by both standard and m-PCR methods. The developed m-PCR assay showed comparable results with the traditional culture technique proving its rapid and reliable detection of six foodborne pathogens and hygiene indicators in food.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common E. coli pathotypes reported to cause several outbreaks of foodborne illnesses. EHEC is a zoonotic pathogen, and ruminants, especially cattle, are considered important reservoirs for the most common EHEC serotype, E. coli O157:H7. Humans are infected indirectly through the consumption of food (milk, meat, leafy vegetables, and fruits) and water contaminated by animal feces or direct contact with carrier animals or humans. E. coli O157:H7 is one of the most frequently reported causes of foodborne illnesses in developed countries. It employs two essential virulence mechanisms to trigger damage to the host. These are the development of attaching and effacing (AE) phenotypes on the intestinal mucosa of the host and the production of Shiga toxin (Stx) that causes hemorrhagic colitis and hemolytic uremic syndrome. The AE phenotype is controlled by the pathogenicity island, the locus of enterocyte effacement (LEE). The induction of both AE and Stx is under strict and highly complex regulatory mechanisms. Thus, a good understanding of these mechanisms, major proteins expressed, and environmental cues involved in the regulation of the expression of the virulence genes is vital to finding a method to control the colonization of reservoir hosts, especially cattle, and disease development in humans. This review is a concise account of the current state of knowledge of virulence gene regulation in the LEE-positive EHEC.

The objective of this study was to evaluate the inhibitory effect of lactic acid (LA) and peroxyacetic acid (PAA) on the biofilm formation of Escherichia coli O157:H7 in beef extract (BE). BE medium was used as the growth substrate in this study, to make the control effect closer to the situation of the factory. The biofilm inhibitory efficacy of LA and PAA was tested by using a crystal violet staining assay and microscopic examination. And then, extracellular polymeric substance (EPS) production, metabolic activity, and real-time polymerase chain reaction assay were used to reveal the biofilm inhibition mechanism of LA and PAA. The results showed that both LA and PAA significantly inhibited biofilm formation of E. coli O157:H7 at minimum inhibitory concentrations (MICs) ( p  < 0.05). At MIC, LA and PAA showed different effects on the biofilm metabolic activity and the EPS production of E. coli O157:H7. Supporting these findings, expression analysis showed that LA significantly suppressed quorum sensing genes ( luxS and sdiA ) and adhesion genes ( flhC ), while PAA downregulated the transcription of extracellular polysaccharide synthesis genes ( adrB and adrA ) and the global regulatory factor csgD . This result revealed that LA and PAA had different biofilm inhibitory mechanisms on E. coli O157:H7; LA inhibited the biofilm formation mainly by inhibiting metabolic activity, while PAA inhibited EPS production. This study provided a theoretical basis for the control of E. coli O157:H7 biofilm in the actual production process.

Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.

Rapid detection and timely treatment of diseases caused by foodborne pathogens is important for improving the curative efficiency and preventing the spread of disease. In this study, we developed an assay utilizing a recently introduced ultra-fast convection polymerase chain reaction (cPCR) in conjunction with a simple nucleic acid lateral flow (NALF) immunoassay for ultra-fast on-site molecular detection of foodborne pathogens. Two Salmonella enterica serovars, Salmonella Enteritidis and Salmonella Typhimurium, and Escherichia coli O157:H7 were used as the target pathogens. We confirmed the specific amplification of the target species with specifically designed modified primer sets for cPCR in singleplex and duplex modes. After cPCR amplification, we compared the detection specificity and sensitivity using agarose gel electrophoresis and NALF assays with one or two test lines. The cPCR amplicons were readily and sensitively detected using the NALF assay, and the sensitivity was comparable with that of agarose gel electrophoresis. To confirm the application of the assay in real-life samples, the assay was used to test artificially contaminated milk. Without sample pre-enrichment, the limit of detection (LOD) was 4.5 × 10 3 colony-forming units (CFU)/mL for Salmonella species; 4.5 × 10 4 CFU/mL to differentiate Salmonella Enteritidis and Salmonella Typhimurium; and 2.3 × 10 3 CFU/mL for E. coli O157:H7, in a duplex assay. With a 6 h pre-enrichment, the LOD was 4.5 CFU/mL for Salmonella Enteritidis and Salmonella Typhimurium, and 2.3 CFU/mL for E. coli O157:H7. The cPCR amplification took only 14 min, and the NALF assay took ca. 5 min. The total analysis time was less than 20 min. Based on these observations, we propose that the developed assay is simple, ultra-fast, and applicable for on-site detection of foodborne pathogens.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx 1 , stx 2 , eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and β-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx 1a +stx 2a +λ/γ1-eae, O26: β-eae alone, and O111: stx 1a +θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx 1a and stx 2a ) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 was isolated from 30 (4%) of 744 cattle hide swab samples collected at Estonian slaughterhouses within a 3-year monitoring program of zoonotic pathogens. The isolates were characterized by determining the presence of STEC main virulence factors, the antimicrobial resistance profiles, and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). Thirteen strains carried the stx2 gene alone and 17 both the stx1 and stx2 genes. The most frequently detected stx subtype was stx2c, occurring alone (n = 12) or in combination with subtype stx1a (n = 13). All isolates harbored the intimin-coding eae gene and produced enterohemolysin. Twelve isolates (40%) showed resistance to at least one of the 14 antimicrobials and the isolates were predominantly resistant to streptomycin, sulfamethoxazole, and ampicillin. No extended-spectrum beta-lactamase-producing isolates were detected. PFGE characterization of the isolates showed an overall similarity higher than 75%, and four clusters based on 100% similarity were revealed.

The lack of pattern diversity among pulsed-field gel electrophoresis (PFGE) profiles for Escherichia coli O157:H7 in Canada does not consistently provide optimal discrimination, and therefore, differentiating temporally and/or geographically associated sporadic cases from potential outbreak cases can at times impede investigations. To address this limitation, DNA sequence-based methods such as multilocus variable-number tandem-repeat analysis (MLVA) have been explored. To assess the performance of MLVA as a supplemental method to PFGE from the Canadian perspective, a retrospective analysis of all E. coli O157:H7 isolated in Canada from January 2008 to December 2012 (inclusive) was conducted. A total of 2285 E. coli O157:H7 isolates and 63 clusters of cases (by PFGE) were selected for the study. Based on the qualitative analysis, the addition of MLVA improved the categorization of cases for 60% of clusters and no change was observed for ∼40% of clusters investigated. In such situations, MLVA serves to confirm PFGE results, but may not add further information per se. The findings of this study demonstrate that MLVA data, when used in combination with PFGE-based analyses, provide additional resolution to the detection of clusters lacking PFGE diversity as well as demonstrate good epidemiological concordance. In addition, MLVA is able to identify cluster-associated isolates with variant PFGE pattern combinations that may have been previously missed by PFGE alone. Optimal laboratory surveillance in Canada is achieved with the application of PFGE and MLVA in tandem for routine surveillance, cluster detection, and outbreak response.

Background and Objective: Stable flies (Stomoxys calcitrans L.) are very common around confined and pastured cattle, and due to their painful bites they are very important animal pests. Cattle are asymptomatic reservoirs of foodborne pathogens, Escherichia coli O157:H7 and other Shiga-toxigenic E. coli serotypes (STEC). In the present study, the potential of stable flies to carry STEC in a beef cattle feedlot was assessed., Methods: Stable flies (n = 180) were collected over 3 summer months and processed individually for STEC-8 that included the serotype O157 and seven non-O157 serotypes (O26, O45, O103, O104, O111, O121, and O145). Isolation and detection of STEC was based on direct plating as well as the enrichment/immunomagnetic separation approach. Modified Posse agar (mP) was used for culturing non-O157 serotypes and sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) for E. coli O157. Multiplex polymerase chain reactions were used for differentiation of individual serotypes and detection of virulence genes (stx1, stx2, eae, and ehxA)., Results and Conclusions: Of 180 stable flies, 67 (37.2%) carried enterics on mP (mean: 3.6 ± 1.05 × 10(6) colony-forming units [CFU]/fly) and 55/180 (30.5%) were positive for bacteria on CT-SMAC (mean: 1.2 ± 1.08 × 10(4) CFU/fly). However, stable flies positive for E. coli serotypes of interest were very rare (prevalence: 1.1%). The three serotype-positive isolates, two E. coli O26 and one E. coli O45, were recovered from two flies and neither of them harbored the virulence genes. We conclude that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

Between April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km(2) were analyzed to examine the environmental dissemination of Escherichia coli O157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfb(O157)-positive, and 62 (24.7%) E. coli O157, and 32 (12.7%) Shiga toxin-producing E. coli (STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfb(O157)- positive, and 9 (15.0%) E. coli O157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coli in the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coli O157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coli O157 in the aquatic rural environment.

Escherichia coli O157:H7 has been the causative agent of many outbreaks associated with leafy green produce consumption. Elucidating the mechanism by which contamination occurs requires monitoring interactions between the pathogen and the plant under typical production conditions. Intentional introduction of virulent strains into fields is not an acceptable practice. As an alternative, attenuated strains of natural isolates have been used as surrogates of the virulent strains; however, the attachment properties and environmental stabilities of these attenuated isolates may differ from the unattenuated outbreak strains. In this study, the Shiga toxin (stx1, stx2, and/or stx2c) genes as well as the eae gene encoding intimin of two E. coli O157:H7 outbreak isolates, F4546 (1997 alfalfa sprout) and K4492 (2006 lettuce), were deleted. Individual gene deletions were confirmed by polymerase chain reaction (PCR) and DNA sequencing. The mutant strains did not produce Shiga toxin. The growth kinetics of these mutant strains under nutrient-rich and minimal conditions were identical to those of their wild-type strains. Attachment to the surface of lettuce leaves was comparable between wild-type/mutant pairs F4546/MD46 and K4492/MD47. Adherence to soil particles was also comparable between the virulent and surrogate pairs, although the F4546/MD46 pair exhibited statistically greater attachment than the K4492/MD47 pair (p≤0.05). Wild-type and mutant pairs F4546/MD46 and K4492/MD47 inoculated into wet or dry soils had statistically similar survival rates over the 7-day storage period at 20°C. A plasmid, pGFPuv, containing green fluorescent protein was transformed into each of the mutant strains, allowing for ease of identification and detection of surrogate strains on plant material or soil. These pGFPuv-containing surrogate strains will enable the investigation of pathogen interaction with plants and soil in the farm production environment where the virulent pathogen cannot be used.

Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment to pathogenic E. coli and other manure-borne pathogens. Therefore, there is a need to improve manure management practices in urban and periurban areas to prevent pathogen spread and associated human health risks.

This study was aimed at evaluating the prevalence of Campylobacter spp., Escherichia coli O157, Salmonella spp., and related virulence factors (the cdt, stx, and eae genes) in urban pigeons of the coastal area of the Campania region (southern Italy). To achieve this goal, cloacal swab samples from a total of 1800 urban pigeons were collected and subjected to culture methods, PCR, and serotyping. The results of the present study showed a prevalence of 48.3% (870/1800), 7.8% (141/1800), and 0.9% (16/1800), for C. jejuni, E. coli O157, and S. Typhimurium, respectively. All C. jejuni isolates (870/870) carried cdt genes, whereas all E. coli O157 isolates carried stx genes, and 14.9% (21/141) carried the eae gene. These findings clearly show that urban pigeons in the coastal area of the Campania region may constitute an environmental reservoir of these pathogens, thus representing a source of infection for other birds, livestock, and humans.

The objective of this study was to determine the frequency and duration of super-shedding in cattle by enumerating Escherichia coli O157:H7 in feces and to compare lineage and pulsed-field gel electrophoresis (PFGE) subtypes from super- and low-shedders. E. coli O157:H7 was enumerated from fecal samples obtained from the rectums of 400 feedlot cattle. Super-shedding steers (N=11) were identified, transported, and penned individually. Freshly voided fecal pats were sampled 2 h before and 6 h after feeding for 7 d, then once daily for an additional 19 d. Isolates (N=126) were subtyped using PFGE, and lineage was typed using a lineage-specific polymorphism assay. Of the 11 super-shedders identified at the commercial feedlot, only five were confirmed as super-shedders at the research feedlot, with no super-shedders identified 6 d after sampling at the commercial feedlot. Super-shedding was not consistent in fecal pats collected from the same individual at different times of the day. Isolates exhibited three distinct PFGE subtypes, with most isolates (97.6%) displaying the same subtype, including those obtained from steers that transitioned from super- to low-shedding. The short duration of super-shedding and its lack of continuance suggest that these individuals may not play as great a role in the dissemination of E. coli O157:H7 within the feedlot as previously proposed.

Foodborne disease outbreaks are often caused by one of the major pathogens. Early identification of the causal pathogen is crucial for disease control and prevention. We describe a real-time polymerase chain reaction (rtPCR) assay that can identify, in a single reaction, up to eight common foodborne bacterial pathogens, including Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli O157, Vibrio parahaemolyticus, V. vulnificus, Campylobacter jejuni, Enterobacter sakazakii, and Shigella spp. This multiplex rtPCR assay takes advantage of modified molecular beacons and the multicolor combinational probe coding strategy to discriminate each pathogen and the homo-tag assisted non-dimer (HAND) system to prevent dimer formation. The detection limits of the assay ranged from 1.3×10(3) colony-forming units (CFU)/g stool (L. monocytogenes) to 1.6×10(4) CFU/g stool (Shigella spp.). The target genes were 100% specific as assessed on 986 reference strains covering 41 species since no cross-reactions were observed. The assay was applied to the detection of foodborne pathogens in 11,167 clinical samples and the results were compared with culture methods for further validation. The sensitivity and specificity of the rtPCR were 100% and 99%, respectively. When performed in a 96-well rtPCR system, more than 90 samples could be analyzed within 3 h. Given the high accuracy, sensitivity, specificity, and short turn-around time, the established assay could be used for the rapid and reliable identification of the causative pathogens responsible for a certain foodborne disease outbreak and rapid screening of these major foodborne pathogens in laboratory-based surveillance of outpatient clinical samples or even food samples.

Background: The growth of Escherichia coli O157:H7 in contaminated dairy and other refrigerated food products due to temperature fluctuation poses a major food safety threat. Effective control or inhibition of E. coli O157:H7 growth depends on our understanding of mechanisms that regulate its growth at low temperature. We hypothesized that polynucleotide phosphorylase (PNPase) plays a critical role in E. coli O157:H7 low-temperature growth., Methods: To test this hypothesis, the pnp deletion mutant of E. coli O157:H7 was generated using the λ Red recombinase system, and the growth and survival of wild-type and pnp deletion mutant strains were compared at low temperatures., Results: The growth of pnp deletion mutant strains in Luria Broth (LB) and agar plate at 37°C was similar to their corresponding wild-type strains, while the deletion of pnp impaired E. coli O157:H7 growth in LB at 10°C and 22°C; growth impairment could be partially recovered in the mutant strains by ectopic expression of the pnp complementation plasmid, demonstrating that growth impairment was PNPase-specific. During 14 days of 10°C storage in both LB and milk, wild type strain EDL933 grew and reached >8 log10 colony-forming units per milliliter after 4 days of 10°C storage, while EDL933Δpnp gradually died off with effects more pronounced in milk, which were again mitigated by pnp overexpression. In addition, pnp deletion impaired the motility of E. coli O157:H7 but did not affect its susceptibility to H2O2., Conclusion: PNPase is required for the growth of E. coli O157:H7 at low temperature; PNPase thus provides a molecular target to control the growth of E. coli O157:H7, which may have important practical applications in dairy and other food industry.

To understand the nature of a bacterial strain, it is necessary to be able to identify and measure the proteins expressed by the bacteria. In this research, the entire protein complements produced by Escherichia coli O157:H7 strain 43894OW and its naturally occurring curli producing variant 43894OR were compared to better understand the unique capabilities of these two closely related strains. A nonlabeled proteomic comparison was performed utilizing the spectra counting and peptide fractionation abilities of a quadrupole-time of flight analyzer mass spectrometer to identify and quantitate the proteins produced by the two strains. The process reliably identified and measured the concentration of 419 proteins from strains 43894OW and 43894OR within three separate biological replicates. From these two sets, 59 proteins were identified that were preferentially expressed in strain 43894OW compared to 43894OR and 14 proteins that were conversely preferentially expressed in 43894OR. A subset of the preferentially expressed proteins was assayed to determine whether their levels of gene transcription corresponded with the observed protein expression. From the resulting list of confirmed differentially expressed proteins, it was observed that the proteins contributing to acid survival--GadA and GadB--were overexpressed in 43894OW compared to 43894OR. The predicted enhanced acid resistance phenotype of 43894OW was confirmed by experimentation at pH 2.5. Additionally, a knockout mutation in the csgD genes of the 43894OR strain was constructed and suggested that CsgD had a repressive effect on acid survival in 43894OR.

Enterohemorrhagic Escherichia coli (EHEC) causes a wide spectrum of food- and waterborne infectious diseases, including diarrhea, hemorrhagic colitis, and even hemolytic-uremic syndrome. Porcine attaching and effacing-associated protein (Paa) was first identified in a porcine enteropathogenic E. coli strain. It has been proven essential in the attaching and effacing mechanism of EHEC. However, the immunologic function of the Paa protein has yet to be established. In the present study, recombinant Paa protein was overexpressed successfully in engineered E. coli and effectively purified to homogeneity. Comparative experiments were carried out in mice with a known adhesion factor (intimin) as reference to investigate the immunogenicity of Paa. Intraperitoneal immunization of Paa protein in mice elicited significantly high levels of serum immunoglobulin G antibodies via Th2-mediated humoral immune response. In mice challenged with E. coli O157:H7, Paa protein exhibited immunological effectiveness against pathogenic bacteria colonization and excretion in vivo. Compared with the intimin, Paa showed better protective effect against E. coli O157:H7 infection in mice, particularly those challenged with high lethal doses of the pathogen. Seventy percent of the mice challenged with 50 minimal lethal dose (MLD) in the Paa group survived, whereas only 50% survived in the intimin group. This finding is the first description of the immunologic function of the Paa protein. These attributes provide support for the development of Paa-based vaccine, which can be beneficial in treating infectious diseases caused by E. coli O157:H7.

To better understand the epizootiology of Escherichia coli O157:H7 among cattle, all E. coli O157 isolates recovered on a research feedlot during a single feeding period were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Three distinct MLVA subtypes (A, B, C), accounting for 24%, 15%, and 64% of total isolates, respectively, were identified. Subtypes A and B were isolated at the initiation of sampling, but their prevalence waned and subtype C, first isolated on the third sampling date, became the predominant subtype on the feedlot. Supershedding events, however, occurred with equal frequency for all three MLVA-types. Using a multilevel logistic regression model, we investigated whether the odds of shedding subtype C relative to subtypes A or B were associated with time, diet, or the presence of a penmate shedding high numbers of subtype C. Only time and exposure to an animal shedding MLVA-type C at 10³ colony-forming units or greater in the pen at the time of sampling were significantly associated with increased shedding of subtype C. High-level shedding of those E. coli O157 subtypes better suited for survival in the environment and/or in the host appear to play a significant role in the development of predominant E. coli O157 subtypes. Supershedding events alone are neither required nor sufficient to drive the epidemiology of specific E. coli O157 subtypes. Additional factors are necessary to direct successful on-farm transmission of E. coli O157.

Treatment of Escherichia coli O157:H7 by certain antimicrobial agents often exacerbates the patient's condition by increasing either the release of preformed Shiga toxins (Stx) upon cell lysis or their production through the SOS response-triggered induction of Stx-producing prophages. Recommended subinhibitory concentrations (sub-MICs) of azithromycin (AZI), gentamicin (GEN), imipenem (IMI), and rifampicin (RIF) were evaluated in comparison to norfloxacin (NOR), an SOS-inducer, to assess the role of the SOS response in Stx release. Relative expression of recA (SOS-inducer), Q (late antitermination gene of Stx-producing prophage), stx1, and stx2 genes was assessed at two sub-MICs of the antimicrobials for two different strains of E. coli O157:H7 using reverse transcription-real-time polymerase chain reaction. Both strains at the two sub-MICs were also subjected to Western blotting for LexA protein expression and to reverse passive latex agglutination for Stx detection. For both strains at both sub-MICs, NOR and AZI caused SOS-induced Stx production (high recA, Q, and stx2 gene expression and high Stx2 production), so they should be avoided in E. coli O157:H7 treatment; however, sub-MICs of RIF and IMI induced Stx2 production in an SOS-independent manner except for one strain at the first twofold dilution below MIC of RIF where Stx2 production decreased. Moreover, GEN caused somewhat increased Stx2 production due to its mode of action rather than any effect on gene expression. The choice of antimicrobial therapy should rely on the antimicrobial mode of action, its concentration, and on the nature of the strain.

Enterohemorrhagic Escherichia coli (EHEC) causes diarrhea, often with severe complications. Rapid and discriminatory typing of EHEC using advanced molecular methods is needed for determination of the genetic relatedness of clones responsible for foodborne outbreaks and for finding out the transmission sources of the outbreaks. This study evaluated the potential of DiversiLab, a semiautomated repetitive sequence-based polymerase chain reaction method for the genotyping of EHEC strains. A set of 52 EHEC strains belonging to 15 O:H serotypes was clustered into 10 DiversiLab groups. All of the O157 strains and one O55 strain were classified into the same group based on a 90% similarity threshold. The other serotypes were classified to their own DiversiLab group, with the exception of one R:H(-) strain that was grouped with O5:H(-) strains. In addition, O26 and O111 strains were grouped together but ultimately subdivided according to their O-serotypes based on a 95% similarity threshold. The O104 strain, which was associated with a major outbreak of hemolytic uremic syndrome in Germany in May 2011, was also classified independently. The DiversiLab performed well in identifying isolates, but the discriminatory power of the repetitive sequence-based polymerase chain reaction method was lower than that of pulsed-field gel electrophoresis. Analysis of 15 enteropathogenic E. coli (EPEC) strains revealed that some EPEC strains clustered together with EHEC strains. Therefore, the DiversiLab system cannot be used to discriminate between these pathogroups. In conclusion, DiversiLab is a rapid and easy system for the primary exclusion of unrelated EHEC strains based on their serotypes, but more discriminatory methods, such as pulsed-field gel electrophoresis, are needed for accurate typing of the EHEC strains.

Sheep flocks were tested for Escherichia coli O157 from pooled fecal samples while they grazed on pasture in winter, brassicas in spring, and on pasture during the summer. The winter pasture study reported an average individual prevalence of 3.1% (95% confidence interval [CI]: 0.6-5.6%) and an average farm-level prevalence of 10.4% (95% CI: 2.1-18.8%) over the 3-year study period. The spring brassica study reported a prevalence of 0% and the summer pasture study had an individual prevalence of 6.3% (95% CI: 2.1-12.1%) and a farm prevalence of 36.8% (95% CI: 15.8-57.8%). Analysis showed significant differences between the shedding of E. coli O157 in sheep grazing on brassicas in spring when compared to sheep grazing on pasture in the summer (p<0.01) and in winter (p=0.044; odds ratio [OR]=0.106). Furthermore, sheep excreted a lower prevalence of E. coli O157 in winter while grazing on pasture (p=0.017; OR=0.199). E. coli O157 isolates were characterized using polymerase chain reaction for the presence of known virulence factors; all carried the eae and stx2 gene and 10/11 positive flocks possessed the stx2c gene, suggesting that sheep are a potential source of human infection.

Escherichia coli O157:H7 is frequently isolated from cases of diarrhea in many industrialized countries; however, it is seldom found in developing countries. The present manuscript reports the presence of E. coli O157:H7 in Ecuadorian livestock, a country where enterohemorrhagic E. coli disease in humans has never been reported. The Ecuadorian isolates were genetically related to some strains linked to clinical cases in the United States as assessed by multiple-locus variable number tandem repeat (VNTR) analysis.

While efforts to control foodborne illness associated with the Shiga toxin-producing Escherichia coli (E. coli) O157 through processes and procedures implemented at harvest facilities have been very successful, there is concern about the burden of illness associated with other Shiga toxin-producing E. coli. The U.S. Department of Agriculture Food Safety and Inspection Service announced plans to classify an additional six non-O157 Shiga toxin-producing E. coli as adulterants. Little is known about the prevalence and distribution of these E. coli in the animal production environment. An investigation of the prevalence of O157 and the six major non-O157 E. coli serogroups was conducted in 21 feedlots over the period July 2011 to October 2011. Individual fecal swabs were collected from cattle approximately 60 days after their arrival in the feedlot and were pooled for evaluation using a polymerase chain reaction assay to identify the presence of seven E. coli O-types (O157, O45, O103, O121, O145, O26, and O111) and four virulence genes (stx1, stx2, eaeA, and ehxA). Overall, 1145 fecal pools were evaluated, with 506 (44.2%) being positive for one or more of the E. coli O-serogroups. The pool prevalences for E. coli O157, O45, O26, O103, O121, O145, and O111 were 19.7%, 13.8%, 9.9%, 9.3%, 5.5%, 1.1%, and 0.5%, respectively. Nearly all pools were positive for ehxA (99.7%) or stx2 (98.6%). The pool level prevalence for stx1 and eae was 65.5% and 69.3%, respectively. Pools that were positive for one or more of the other E. coli O-serogroups were 1.37 times more likely to be positive for E. coli O157. Conversely, pools that were positive for E. coli O157 were 1.43 times more likely to be positive for at least one of the other E. coli O-serogroups evaluated. These data will be useful to understand the expected prevalence of potential Shiga toxin-producing E. coli in cattle feedlots.

The use of luminescent plasmids in bacteria may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding patterns of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth characteristics of E. coli O157:H7 in mixed ruminal and fecal microbial fluid cultures would be altered when transformed with one of the two luminescent plasmids: pAK1-lux (PAK) or pXEN-13 (XEN). Transformants harboring the luminescent plasmids were compared to the non-transformed parental strain (wild type [WT]) after incubating in mixed ruminal or fecal microbial fluid media for 6 h in triplicate (n=3). The transformants and WT exhibited similar growth rates. Within mixed ruminal microbial fluid fermentations and mixed fecal microbial fluid, all transformants grew similarly (p=0.28) through the 6-h study. The reflective light unit (RLU; photons/pixel per second) photonic emissions of each plasmid within ruminal fluid differed at 0 h (p=0.002) and 2 h (p=0.02) and within fecal fluid at 0 h (p=0.009) and 2 h (p=0.04). The RLU remained the same within rumen fluid at 4 h (p=0.22) and 6 h (p=0.80) and within fecal fluid at 4 h (p=0.06) and 6 h (p=0.29). Growth of E. coli O157:H7 transformed with the bioluminescent plasmids was not altered in comparison to the WT, suggesting that both plasmids may serve as useful models for in vivo studies.

A robust semiquantitative method for measuring the colonization potential of O157 enterohemorrhagic Escherichia coli (EHEC) strains was developed combining an established ex vivo model infection system, bovine in vitro organ culture, with detection of bacteria attached to tissue sections by immunofluorescent assay (bIVOC-IFA) using Quantum dot(®) nanocrystal technology. The method was tested on ten O157 strains chosen to reflect a diversity of genotypes found in New Zealand based on the novel polymerase chain reaction-binary typing (P-BIT) system. High- and low-colonizing EHEC O157 strains were identified using bIVOC-IFA, with the highest colonizing strain belonging to the pulsed-field gel electrophoresis type most commonly identified from New Zealand beef meat. Furthermore, all of the toxigenic O157 strains exhibiting a low-colonizing phenotype were closely related, belonging to the same P-BIT genotype cluster. Future use of this method to characterize EHEC strains could provide valuable information for risk assessment and risk management interventions aimed at improving food safety along the beef farm to fork continuum.

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

Whereas the association of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 with the hemolytic uremic syndrome (HUS) is well established, the medical importance of many non-O157 serotypes remains unclear. Using polymerase chain reaction (PCR), we have investigated the distribution of the pathogenicity island O island 122 (OI-122) and other virulence genes in VTEC belonging to seropathotypes (SPT) A through D, and assessed their association with human disease. Two hundred sixty-five VTEC isolated from human stools comprising 52 O157 (of which 14 associated with HUS) and 213 non-O157 isolates (of which 19 associated with HUS) were studied. A complete OI-122 (COI-122) was detected in all O157, but in only 35 (16.4%) of non-O157 strains. A progressive decrease in the frequency of COI-122 was observed from SPT A through D, with a concomitant increase in the frequencies of incomplete and absent OI-122. We focused on the variable virulence profiles of the non-O157 serotypes and found that COI-122 was also more frequently present in isolates associated with HUS (p=0.001). The individual genes vtx2, eae, espP, as well as the OI-122-associated genes sen, nleB, nleE, and the efa gene cluster were significantly more often present in non-O157 VTEC associated with HUS. Non-O157 isolates carrying the combined virulence profile vtx2-nleE-efa showed the strongest association with HUS (p<0.0001). Molecular risk assessment by determination of virulence profiles of individual isolates may be useful in the identification of highly virulent non-O157 strains. We showed that the detection of a specific gene combination could assist in identifying non-O157 VTEC isolates that pose a serious public health concern.

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10⁵ colony-forming units (CFU)/g before enrichment and 10² CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.

Raw milk is increasingly appreciated by consumers but can be contaminated by a variety of zoonotic pathogens. Therefore, preventive measures, such as on-farm hazard analysis critical control point (HACCP) programs, must be applied to protect consumers. The aim of the present study was the comparison of a multiplex real-time polymerase chain reaction (PCR) assay with a culture-based approach in an on-farm quality assurance program for the detection of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in bulk tank milk, in-line milk filters, manure, and feces. Results revealed that the real-time PCR was more sensitive in detecting E. coli O157 than the culture method in filters (48% vs. 4% positive), manure (93% vs. 7% positive) and feces (60% vs. 4% positive). The two methods were equally efficient in detecting L. monocytogenes (8% of filters), while Salmonella spp. was not detected in any sample. In conclusion, the real-time PCR, by reducing analysis time to two working days, can be proposed as a useful tool in the raw milk primary production setting as a rapid and user-friendly screening method.

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used-official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)-was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) >300,000/mL and total bacterial count (TBC) >50,000 CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumer's safety, a new approach for the raw milk chain is required.

There is a lack of information about the role of poultry, specifically chicken, in transmission of Escherichia coli (E. coli) O157 and subsequent human illnesses. This study was therefore aimed at investigating the presence of E. coli O157 and its virulence genes in various samples collected from broiler chickens and humans in Eastern Turkey by culture, immunomagnetic separation (IMS), and polymerase chain reaction (PCR). The genetic relationship between broiler and human isolates was also examined by pulsed-field gel electrophoresis (PFGE). In the PCR analysis of sorbitol-negative isolates, E. coli O157 was identified in 0.1% (1/1000) and 0.4% (4/1000) of the liver and cecum samples of broiler chickens, respectively. On the other hand, none of the carcass samples were determined to be positive for E. coli O157. Overall, the results indicated that 12% (3/25) of the flocks were positive for E. coli O157. The differences between the flocks in terms of the positivity were determined to be statistically significant (p<0.001). Ten (2.7%) of 367 human stool samples were also positive for E. coli O157 in the PCR examination. None of the broiler and human E. coli O157 isolates possessed H7, shigatoxins 1-2, or enterohemolysin genes, whereas all the broiler isolates and one of the human isolates were positive for intimin gene. In the PFGE analysis, a total of eight different profiles (four from broiler and four from human isolates) were observed. However, there were no genetic relationships between broiler and human E. coli O157 isolates. It can be concluded that more detailed studies are needed in poultry to better understand the role of these species in the epidemiology of E. coli 0157 infections in humans.

Shiga-toxigenic Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh produce. It is generally recognized that bacterial attachment to vegetal matrices constitutes the first step in contamination of fresh produce. Cellular appendages, such as curli fibers, and cellulose, a constituent of extracellular matrix, have been suggested to be involved in E. coli attachment and persistence in fresh produce. A comparative evaluation was conducted on the ability of Shiga toxin-producing E. coli O157:H7 strains EDL933 and 86-24, linked to two independent foodborne disease outbreaks in humans, and their mutants deficient in curli and/or cellulose expression to colonize and to firmly attach to spinach leaf. Inoculated spinach leaves were incubated at 22°C, and at 0, 24, and 48 h after incubation loosely and strongly attached E. coli O157:H7 populations were determined. Curli-expressing E. coli O157:H7 strains developed stronger association with leaf surface, whereas curli-deficient mutants attached to spinach at significantly (p<0.01) lower numbers. Attachment of cellulose-impaired mutants to spinach leaves was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains. Laser scanning confocal microscopy (LSCM) analysis of inoculated leaves revealed that curli-expressing E. coli O157:H7 were surrounded by extracellular structures strongly immunostained with anti-curli antibodies. Production of cellulose was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment of E. coli O157:H7 to spinach whereas cellulose is dispensable.

Cattle are asymptomatic reservoirs for Escherichia coli O157, a major foodborne pathogen. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations. The objective of this research was to evaluate a multiplex, real-time polymerase chain reaction (mqPCR) assay for quantification of E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. In pure culture, the minimum detection limit of the assay was 1.4×10(3) CFU/mL and 3.6×10(3) CFU/mL for the single and five-strain mixture of E. coli O157, respectively. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. In feces spiked with the pooled mixture of five E. coli O157 strains, the minimum detection limit was 3.6×10(4) CFU/g. We also evaluated the assay with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. For the majority of samples tested, the concentration of E. coli O157 detected by the real-time and culture methods was within one log difference. However, the assay could only be evaluated for cattle shedding high concentrations of E. coli O157. In conclusion, the mqPCR quantifying E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets may have use in detecting and quantifying super shedders, but is not applicable for quantification in animals shedding low concentrations (10(2) to 10(3) CFU/g feces).

We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24 h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.

Objectives: In summer 2009, the Utah Department of Health investigated an outbreak of Shiga-toxigenic Escherichia coli (STEC) O157:H7 (O157) illness associated with attendance at multiple rodeos., Materials and Methods: Patients were interviewed regarding exposures during the week before illness onset. A ground beef traceback investigation was performed. Ground beef samples from patient homes and a grocery store were tested for STEC O157. Rodeo managers were interviewed regarding food vendors present and cattle used at the rodeos. Environmental samples were collected from rodeo grounds. Two-enzyme pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) were performed on isolates., Results: Fourteen patients with primary STEC O157 illness were reported in this outbreak. Isolates from all patients were indistinguishable by PFGE. Isolates from nine patients had identical MLVA patterns (main outbreak strain), and five had minor differences. Thirteen (93%) patients reported ground beef consumption during the week before illness onset. Results of the ground beef traceback investigation and ground beef sampling were negative. Of 12 primary patients asked specifically about rodeo attendance, all reported having attended a rodeo during the week before illness onset; four rodeos were mentioned. All four rodeos had used bulls from the same cattle supplier. An isolate of STEC O157 identified from a dirt sample collected from the bullpens of one of the attended rodeos was indistinguishable by PFGE and MLVA from the main outbreak strain., Discussion: Recommendations were provided to rodeo management to keep livestock and manure separate from rodeo attendees. This is the first reported STEC O157 outbreak associated with attendance at multiple rodeos. Public health officials should be aware of the potential for rodeo-associated STEC illness.

Molecular subtyping methods have previously shown that there is a nonrandom distribution of Escherichia coli O157:H7 strains among clinical and nonclinical isolates. Two examples include the lineage-specific polymorphism assay (LSPA) and clade typing assay. The clade typing method was previously used to identify a phylogenetic group of E. coli O157:H7 designated clade 8, which is believed to be more virulent than non-clade 8 isolates, and the LSPA previously indicated that clade 8 isolates are LSPA genotype 211111. Published screens have suggested that LSPA 211111 comprise anywhere from 3.9% to greater than 46% of clinical isolates. To determine the prevalence of such isolates within Pennsylvania, we applied LSPA and screened 52 clinical isolates. We found that 31% of isolates were LSPA 211111 and that 13/16 of these could be classified as clade 8. A rapid polymerase chain reaction screen for clade 8 isolates was developed and shown to have a specificity and sensitivity of 0.92 and 1.0, respectively. Polymerase chain reaction screens indicated that all isolates carried hlyA and eaeA and that all but one of the isolates carried katP. The most common LSPA genotype seen within our collection was 111111, and 29 of 30 of these carried both stx1 and stx2. Clade 8 isolates were more diverse, with four different Shiga toxin profiles observed. We conclude that E. coli O157:H7 of LSPA 211111 and clade 8 are common to clinical isolates in Pennsylvania and suggest that further studies are needed to determine whether their prevalence is increasing as observed elsewhere.

During a Escherichia coli O157 prevalence study on cattle farms, 324 E. coli O157 isolates were collected from 68 out of 180 cattle farms. All isolates harbored the eaeA gene and the enterohemolysin (ehxA) gene. The majority of the strains only contained vtx2 (245 isolates), the combination of vtx1 and vtx2 was detected in 50 isolates, and in 29 isolates none of the vtx genes was present. Pulsed-field gel electrophoresis (PFGE) revealed that at a similarity level of 98% the isolates grouped into 83 different genotypes, 76 of which were only detected on one farm. Twenty-two out of the 68 positive farms harbored isolates belonging to more than one PFGE type, with a maximum of four different PFGE types. Minimal inhibitory concentrations of 10 antimicrobial agents were determined on a subset of 116 isolates, that is, one isolate per positive age category per farm. Acquired resistance to at least one antimicrobial agent was detected in 18 isolates and within a farm, only one resistance pattern was observed. All these 18 isolates were resistant toward streptomycin, and 16 of them also showed resistance toward sulfisoxazole. Six isolates were resistant to three or more antimicrobial agents.

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

Ten (2.7%) Shiga toxin-producing Escherichia coli (STEC) were isolated from 370 samples of raw minced beef, mutton, pork, and chicken from the Jilin region of China; and additional 10 E. coli O157:H7 isolates were previously isolated from different Jilin regions. Seventeen of the isolates were multiresistant, exhibiting resistance to ampicillin, ciprofloxacin, tetracycline, sulfamethoxazole-trimethoprim, gentamycin, and streptomycin. Class 1 integrons were detected in nine (45.0%) of the STEC isolates and consisted of serogroups O157, O62, O113, O149, and O70. Integrons containing amplicons of a 0.5-1.5 or 1.0 kb gene cassette were found in seven (77.8%) of the integron-containing isolates. Sequencing analysis revealed that these gene cassettes encode genes conferring resistance to trimethoprim (dfrA1) and streptomycin (aadA1). The 0.5 kb cassette described here was found to encode a putative transporter peptide in the STEC. Seventeen isolates contained plasmids with different bands, and transfer by conjugation between strains of E. coli demonstrated that class 1 integrons located on mobile plasmids could contribute to the emergence and dissemination of antimicrobial resistance to ampicillin, gentamycin, streptomycin, and sulfamethoxazole-trimethoprim amongst STEC. These data revealed the high prevalence of antimicrobial-resistant STEC isolates in Jilin's surrounding regions, providing important and useful surveillance information reflecting antimicrobial selection pressure., (© Mary Ann Liebert, Inc.)

In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU/g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak.

Beef organ meat, such as liver, and beef are major food sources contaminated with Escherichia coli O157. This study investigated the detection method of E. coli O157 in beef liver and carcass. In an experiment with beef liver inoculated with E. coli O157, the direct plating method, plating after the immunomagnetic separation (IMS) method, and Shiga toxin (Stx)-producing E. coli detection and E. coli O157 detection loop-mediated isothermal amplification (LAMP) assays were compared for the detection of Stx-producing E. coli O157. Fifty percent and 45% of samples were positive by Stx-producing E. coli detection LAMP assay and E. coli O157 detection LAMP assay, respectively. Thirty-five percent and 10% of samples were positive by the IMS method and direct plating method, respectively. In an examination of beef swab samples, contamination frequencies with E. coli O157 were analyzed by LAMP assays and the IMS method. E. coli O157 was detected in 12 of 230 samples (5.2%). There was no sample positive for E. coli O157 isolation but negative for LAMP assays for Stx gene and O157 antigen gene. Four samples (1.7%) were positive by both LAMP assays but negative by the IMS method. The result that there was no sample positive for the O157 antigen gene, but not the Stx gene, indicated that the IMS method failed to detect E. coli O157. Twenty-nine samples (12.6%) were positive for the Stx gene but not the O157 antigen gene. The results indicated that screening of Stx gene and O157 antigen gene by LAMP assays is effective in saving time and effort to isolate E. coli O157 by the IMS method because the LAMP assay is more sensitive. This suggested that samples positive for Stx gene and O157 antigen gene should be examined by the IMS method to isolate E. coli O157.

This study assessed the distribution of class 1 integrons in commensal bacteria isolated from agricultural and nonfarm environments, and the transferability of class 1 integrons to pathogenic bacteria. A total of 26 class 1 integron-positive isolates were detected in fecal samples from cattle operations and a city park, water samples from a beef ranch and city lakes, and soil, feed (unused), manure, and compost samples from a dairy farm. Antimicrobial susceptibility testing of class 1 integron-positive Enterobacteriaceae isolates from city locations displayed multi-resistance to 12-13 out of the 22 antibiotics tested, whereas class 1 integron-positive Enterobacteriaceae isolates from cattle operations only displayed tetracycline resistance. Most class 1 integrons had one gene cassette belonging to the aadA family that confers resistance to streptomycin and spectinomycin. One isolate from a dog fecal sample collected from a city dog park transferred its class 1 integron to a strain of Escherichia coli O157:H7 at a frequency of 10(-7) transconjugants/donor by in vitro filter mating experiments under the stated laboratory conditions. Due to the numerous factors that may affect the transferability testing, further investigation using different methodologies may be helpful to reveal the transferability of the integrons from other isolates. The presence of class 1 integrons among diverse commensal bacteria from agricultural and nonfarm environments strengthens the possible role of environmental commensals in serving as reservoirs of antibiotic resistance genes.

We investigated the efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved a short enrichment period of 8 h followed by capture of the pathogen on O157-specific immunomagnetic beads. The captured cells were treated with O157-specific lytic bacteriophage, CSLO157. Upon phage-induced lysis, the enzyme adenylate kinase, which was released from the lysed cells, was measured in terms of relative light units using luciferin-luciferase assay. The plaque forming efficiency (e.g., phage susceptibility) and ability to capture cells with immunomagnetic beads were examined using an array of 74 E. coli O157:H7 isolates obtained from various clinical and foodborne samples. Immunmagnetic beads successfully captured all 74 isolates; however, only 53 isolates showed susceptibility toward the bacteriophage. Susceptible isolates were further classified into two broad groups, moderately sensitive isolates, which generated phage titer ∼ 10(7)pfu/mL (group I, n=15), and highly susceptible isolates, which generated high phage titer ∼ 10(9)pfu/mL (group II, n=38). We selected 15 isolates (9 from group I and 6 from group II) and individually spiked beef samples (ca. 3-8 cells/25 g beef) to evaluate the bacteriophage-based detection system. Eight out of nine isolates from group I and all six isolates from group II were successfully detected. Pathogenic E. coli strains belonging to other serogroups (12 serogroups, 67 isolates) as well as nontarget microorganisms (n=18) were not lysed by the bacteriophage and hence were not detected. The method is high-throughput compatible, is rapid, and can provide live culture the following day by streaking an aliquot before phage lysis on conventional selective agar media.

The Shiga-like toxins (Stx) are critical virulence factors for enterohemorrhagic Escherichia coli (EHEC), causing severe human illnesses of bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). A subunit of Stx, Stx1B protein, mediates the interaction between EHEC and the specific receptor of host organs. In this article, the recombinant expression vector pGEX-Stx1B bearing a signal peptide sequence-deleted Stx1B gene was constructed and the expression of protein of interest was achieved in a prokaryotic system. The resulting Stx1B protein was used to immunize BALB/c mice followed by the preparation of corresponding monoclonal antibodies (MAbs). One MAb (1G11) was generated. Western blot analysis showed that the MAb was capable of reacting with EHEC Stx1B protein. It had high affinity binding to the Stx1B protein and it distinguished EHEC from other control bacteria. Therefore, the MAb generated in this study can be used as a specific reagent to investigate the pathogenesis mechanism and to develop a diagnostic method of EHEC infection in both humans and animals.

In a previous study we showed that an Escherichia coli O157:H7 strain that was unable to form biofilm was retained in large numbers in dual-strain biofilms formed with an E. coli O-:H4 companion strain. In this study we tested additional companion strains for their ability to retain E. coli O157:H7 strain 0475s. Companion strains producing biofilm that withstood aggressive washes were able to significantly increase serotype O157:H7 retention. Dual-strain biofilms with certain companion strains retained higher percentages of strain 0475s, and that ability was independent of biofilm total cell numbers. Tests with additional non-biofilm-forming E. coli O157:H7 strains showed that enhancement by companion strains was not unique to strain 0475s. Experiments using an E. coli companion strain with deletions of various curli and cellulose genes indicated that dual-strain biofilm formation was dependent on companion strain properties. Strain 0475s was not able to generate biofilm or persist on plastic when grown in broth with a biofilm-forming companion and separated by a 0.2 microm porous membrane, indicating a requirement for intimate contact with the companion strain. When dual-strain biofilms and planktonic cells were challenged with 5% H(2)O(2), strain 0475 showed greater survival in biofilms with certain companion strains compared to the corresponding planktonic cells. The results of this study indicate that non-biofilm-forming E. coli O157:H7 strains are retained on solid surfaces associated with biofilms generated by companion strains. However, properties other than biofilm mass enable certain companion strains to retain greater numbers of E. coli O157:H7.