Experimental infection in calves with a specific subtype of verocytotoxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 of bovine origin

Background  

In Sweden, a particular subtype of verocytotoxin-producing Escherichia coli (VTEC) O157:H7, originally defined as being of phage type 4, and carrying two vtx ₂ genes, has been found to cause the majority of reported human infections during the past 15 years, including both sporadic cases and outbreaks. One plausible explanation for this could be that this particular subtype is better adapted to colonise cattle, and thereby may be excreted in greater concentrations and for longer periods than other VTEC O157:H7 subtypes.     

Genotypic characterization to identify markers associated with putative hypervirulence in Swedish Escherichia coli O157:H7 cattle strains

Aims: To establish whether investigated subtyping methods could identify any specific characteristics that distinguish Swedish VTEC O157:H7 strains isolated from cattle farms associated with human enterohaemorrhagic Escherichia coli (EHEC) cases from cattle strains isolated in prevalence studies. Methods and Results: Strains (n = 32) isolated in a dairy herd prevalence study and strains isolated from farms associated with human cases (n = 13) were subjected to typing. Partial sequencing of the vtx₂ genes could not identify any unique variants of vtx₂ or vtx_2c in strains associated with human cases. A specific variant of VTEC O157:H7, which was overrepresented among farms associated with human cases (P = 0·01), was by two different single‐nucleotide‐polymorphism (SNP) assays identified as clade 8, a subgroup of VTEC O157:H7 strains considered to be putatively hypervirulent. Multi‐locus variable number tandem repeat analysis (MLVA) typing of all strains produced similar results as pulsed‐field gel electrophoresis (PFGE) typing regarding clustering of the strains, but MLVA distinguished slightly better among strains than PFGE. Conclusion: In Sweden, VTEC O157:H7 strains from the putatively hypervirulent clade 8 are overrepresented among isolates from cattle farms associated with human cases compared with VTEC O157:H7 strains isolated in prevalence studies. Significance and Impact of the Study: Real‐time PCR SNP typing for clade 8 can be used to identify cattle farms that are at higher risk of causing EHEC infections in humans.     

Verotoxinogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>(VTEC) O157:H7 – A Nationwide Swedish Survey of Bovine Faeces

In the autumn of 1995 the first outbreaks of enterohemorrhagic Escherichia coli O157:H7 including ca 100 human cases were reported in Sweden. From outbreaks in other countries it is known that cattle may carry these bacteria and in many cases is the source of infection. Therefore, the present study was performed to survey the Swedish bovine population for the presence of verotoxin-producing E. coli (VTEC) of serotype O157:H7. Individual faecal samples were collected at the 16 main Swedish abattoirs from April 1996 to August 1997. Of 3071 faecal samples, VTEC O157 were found in 37 samples indicating a prevalence of 1.2% (CI_95% 0.8–1.6). All 37 isolates carried genes encoding for verotoxin (VT1 and/or VT2), intimin, EHEC-haemolysin and flagellin H7 as determined by PCR. Another 3 strains were of serotype O157:H7 but did not produce verotoxins. The 37 VTEC O157:H7 strains were further characterised by phage typing and pulsed-field gel electrophoresis. The results clearly show that VTEC O157:H7 is established in the Swedish bovine population and indicate that the prevalence of cattle carrying VTEC O157:H7 is correlated to the overall geographical distribution of cattle in Sweden. Results of this study have formed the basis for specific measures recommended to Swedish cattle farmers, and furthermore, a permanent monitoring programme was launched for VTEC O157:H7 in Swedish cattle at slaughter.     

Multiple‐locus variable‐nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio,USA

Aims: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. Methods and Results: During a three‐year period (2007–2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple‐locus variable‐nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Conclusions: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. Significance and Impact of Study: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on‐farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply.     

Prevalence of verotoxigenic Escherichia coli O157 (VTEC O157) and compliance with microbiological safety standards in bovine carcasses from an industrial beef slaughter plant

The presence of Salmonella spp. and levels of Enterobacteriaceae and aerobic plate count were determined in 300 bovine carcasses randomly collected in an industrial cattle slaughterhouse in Catalonia (Spain) as part of a control programme to validate good slaughter practices according to Commission Regulation No 2073/2005. The verotoxigenic Escherichia coli O157 (VTEC O157), although not currently legislated, was also investigated in the same carcasses due to the importance of bovines as a reservoir for this micro‐organism. Virulence genes (vtx1, vtx2 and eae), the presence of fliC_H7 and antimicrobial susceptibility were studied in E. coli O157 isolates. Levels of Enterobacteriaceae and aerobic colonies and the presence of Salmonella were within the admissible range stipulated by current legislation. However, VTEC O157 was detected in 14·7% of carcasses. Among the VTEC O157 strains tested for antimicrobial susceptibility, 65% were multiresistant. Overall, the results of this study indicate that even with good manufacturing practices, contamination with VTEC O157 can occur and cattle meat can pose a risk to human health. These results confirm the need for a review of the appropriateness of introducing antimicrobial treatments in the processing of cattle carcasses in Europe.

Significance and Impact of the Study

This study describes the prevalence of verotoxigenic and multidrug‐resistant E. coli O157 strains in bovine carcasses. These results suggest that despite the good manufacturing practices used in the slaughterhouse studied (the largest in Catalonia slaughtering over 81 000 cattle per year), the absence of verotoxigenic E. coli O157 in bovine carcasses cannot be guaranteed.     

Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle

Background  

Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle.     

Occurrence of verocytotoxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> in the faeces of free-ranging wild lagomorphs in southwest Spain

Verocytotoxin-producing Escherichia coli (VTEC) is an important group of emerging food-borne pathogens and represents a major public health concern worldwide. The aim of this work was to analyse faecal samples from hunted wild lagomorphs for the presence of E. coli O157:H7 and non-O157 VTEC. During two hunting seasons, faecal samples from 241 animals were collected, including wild rabbit (Oryctolagus cuniculus) and hare (Lepus granatensis) and were examined for VTEC. Overall, VTEC were detected and isolated in four (1.66%) of the 241 animals sampled. E. coli O157:H7 was isolated only from one of 124 (0.81%) wild rabbit faecal samples while non-O157 VTEC were isolated from two of 124 (1.61%) wild rabbit faecal samples and one of 117 (0.85%) hare faecal samples. VTEC isolates obtained in the present study (four in total) belonged to four different O:H serotypes, including two serotypes (O84:H− and O157:H7) previously associated with human infection and in particular with causing the life-threatening haemolytic–uraemic syndrome. Although these results indicate a low prevalence of VTEC infection in free-ranging wild lagomorphs, they may play an important role as a source of exposure to human beings and livestock and as a vehicle for dispersing these pathogens. These findings have implications for the zoonotic risk to hunters, people consuming meat from wild animals and others in contact with wild animal faeces, and also in the development of programmes for controlling VTEC at the farm level.     

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.     

First isolation and further characterization of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) O157:H45 strains from cattle

Background  

Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes. The mechanism of EPEC pathogenesis is based on the ability to introduce the attaching-and-effacing (A/E) lesions and intimate adherence of bacteria to the intestinal epithelium. The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains are not well established. E. coli O157:H45 EPEC strains, however, are described in association with enterocolitis and sporadic diarrhea in human. Moreover, a large outbreak associated with E. coli O157:H45 EPEC was reported in Japan in 1998. During a previous study on the prevalence of E. coli O157 in healthy cattle in Switzerland, E. coli O157:H45 strains originating from 6 fattening cattle and 5 cows were isolated. In this study, phenotypic and genotypic characteristics of these strains are described. Various virulence factors (stx, eae, ehxA, astA, EAF plasmid, bfp) of different categories of pathogenic E. coli were screened by different PCR systems. Moreover, the capability of the strains to adhere to cells was tested on tissue culture cells.     

Detection of Verotoxin-Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin, Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.     

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cu,Zn Superoxide Dismutases

Background  

Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.     

Acid stress damage of DNA is prevented by Dps binding in <Emphasis Type="Italic">Escherichia coli</Emphasis>O157:H7

Background  

Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

Detection and Prevalence of Verotoxin-Producing Escherichia coli O157 and Non-O157 Serotypes in a Canadian Watershed

Verotoxin-producing Escherichia coli (VTEC) strains are the cause of food-borne and waterborne illnesses around the world. Traditionally, surveillance of the human population as well as the environment has focused on the detection of E. coli O157:H7. Recently, increasing recognition of non-O157 VTEC strains as human pathogens and the German O104:H4 food-borne outbreak have illustrated the importance of considering the broader group of VTEC organisms from a public health perspective. This study presents the results of a comparison of three methods for the detection of VTEC in surface water, highlighting the efficacy of a direct VT immunoblotting method without broth enrichment for detection and isolation of O157 and non-O157 VTEC strains. The direct immunoblot method eliminates the need for an enrichment step or the use of immunomagnetic separation. This method was developed after 4 years of detecting low frequencies (1%) of E. coli O157:H7 in surface water in a Canadian watershed, situated within one of the FoodNet Canada integrated surveillance sites. By the direct immunoblot method, VTEC prevalence estimates ranged from 11 to 35% for this watershed, and E. coli O157:H7 prevalence increased to 4% (due to improved method sensitivity). This direct testing method provides an efficient means to enhance our understanding of the prevalence and types of VTEC in the environment. This study employed a rapid evidence assessment (REA) approach to frame the watershed findings with watershed E. coli O157:H7 prevalences reported in the literature since 1990 and the knowledge gap with respect to VTEC detection in surface waters.     

First isolation of the enterohaemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> O145:H- from cattle in feedlot in Argentina

Background  

Enterohaemorrhagic Escherichia coli (EHEC) is considered to be common cause of haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome (HUS) in humans. In a previous paper, we have demonstrated that EHEC are commonly found in the intestines of livestock. Infections in humans are, in part, a consequence of consumption of undercooked meat or raw milk. Argentina has one of the highest records of HUS (300–400 cases/year; 22/100,000 children under 4 years of age). The aim of this work is to communicate the first isolation of O145:H-from cattle in this country and characterize the virulence cassette, providing useful information to evaluate the risk of foodborne transmission of this emergent non-O157:H7 serotype.     

Effect of Sand and Sawdust Bedding Materials on the Fecal Prevalence of Escherichia coli O157:H7 in Dairy Cows

Farm management practices that reduce the prevalence of food-borne pathogens in live animals are predicted to enhance food safety. To ascertain the potential role of livestock bedding in the ecology and epidemiology of Escherichia coli O157:H7 on farms, the survival of this pathogen in used-sand and used-sawdust dairy cow bedding was determined. Additionally, a longitudinal study of mature dairy cattle housed on 20 commercial dairy farms was conducted to compare the prevalence of E. coli O157:H7 in cattle bedded on sand to that in cattle bedded on sawdust. E. coli O157:H7 persisted at higher concentrations in used-sawdust bedding than in used-sand bedding. The overall average herd level prevalence (3.1 versus 1.4%) and the number of sample days yielding any tests of feces positive for E. coli O157:H7 (22 of 60 days versus 13 of 60 days) were higher in sawdust-bedded herds. The choice of bedding material used to house mature dairy cows may impact the prevalence of E. coli O157:H7 on dairy farms.     

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Identifying the mechanism of Escherichia coli O157:H7 survival by the addition of salt in the treatment with organic acids

Aim

In this study, the effects of the addition of salt to treatment with acids (one of several organic acids and salt in various solutions including rich or minimal broth, buffer, or distilled water) on the reduction of Escherichia coli O157:H7 were investigated. The protein expression profiles corresponding to acid stress (acetic acid) with or without salt addition were studied using a comparative proteomic analysis of E. coli O157:H7.

Methods and Results

When acetic, lactic, or propionic acid was combined with 3% NaCl, mutually antagonistic effects of acid and salt on viability of E. coli O157:H7 were observed only in tryptone and yeast extract broth. After exposure to acetic acid alone or in combination with salt, approximately 851 and 916 protein spots were detected, respectively. Analysis of 10 statistically significant differentially expressed proteins revealed that these proteins are mainly related to energy metabolism.

Conclusions

When we compared protein expression of E. coli O157:H7 treated with acetic acid and the combination of the acid and salt, the differentially expressed proteins were not related to acid stress‐ and salt stress‐inducible proteins such as stress shock proteins.

Significance and Impact of the Study

According to these results, the increased resistance of E. coli O157:H7 to acetic acid after the addition of salt may not be the result of synthesis of proteins related to these phenomena; therefore, further research needs to be conducted to identify the mechanism of the mutually antagonistic effect of some organic acids and salt.     

Phylogenetic classification of Escherichia coli O157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms

Background

Cattle are a reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157), and are known to harbor subtypes not typically found in clinically ill humans. Consequently, nucleotide polymorphisms previously discovered via strains originating from human outbreaks may be restricted in their ability to distinguish STEC O157 genetic subtypes present in cattle. The objectives of this study were firstly to identify nucleotide polymorphisms in a diverse sampling of human and bovine STEC O157 strains, secondly to classify strains of either bovine or human origin by polymorphism-derived genotypes, and finally to compare the genotype diversity with pulsed-field gel electrophoresis (PFGE), a method currently used for assessing STEC O157 diversity.

Results

High-throughput 454 sequencing of pooled STEC O157 strain DNAs from human clinical cases (n = 91) and cattle (n = 102) identified 16,218 putative polymorphisms. From those, 178 were selected primarily within genomic regions conserved across E. coli serotypes and genotyped in 261 STEC O157 strains. Forty-two unique genotypes were observed that are tagged by a minimal set of 32 polymorphisms. Phylogenetic trees of the genotypes are divided into clades that represent strains of cattle origin, or cattle and human origin. Although PFGE diversity surpassed genotype diversity overall, ten PFGE patterns each occurred with multiple strains having different genotypes.

Conclusions

Deep sequencing of pooled STEC O157 DNAs proved highly effective in polymorphism discovery. A polymorphism set has been identified that characterizes genetic diversity within STEC O157 strains of bovine origin, and a subset observed in human strains. The set may complement current techniques used to classify strains implicated in disease outbreaks.