Verocytotoxigenic Escherichia Coli Research Articles

Verocytotoxigenic (Shiga Toxin–Producing)Escherichia coli: Virulence Factors and Pathogenicity in the Farm to Fork Paradigm

Verocytotoxigenic Escherichia coli (VTEC) are a good example of the evolution and emergence of pathogenic E. coli. Unknown before the late 1970s, these bacteria are a major cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. The production of verocytotoxins is the main virulence feature of VTEC but cannot be solely responsible for full pathogenicity. VTEC associated with severe human disease are usually capable of colonizing the intestinal mucosa with a characteristic attaching-and-effacing mechanism, genetically governed by the locus of enterocyte effacement, and possess other mobile genetic elements carrying additional virulence genes such as plasmids, phages, and pathogenicity islands (e.g., O-I 122). Despite the huge amount of data collected after the sequencing of the full genome of VTEC O157, the virulence and the evolution of the different VTEC serotypes have only been partially unraveled. A greater understanding of the factors governing the development of severe disease in humans and the colonization of animal hosts must be achieved before effective intervention strategies aimed at the reduction of the burden of infection can be developed. Defining all the factors characterizing a fully pathogenic VTEC strain will be crucial to improve the efficacy of the diagnosis of human infections, the surveillance of animal reservoirs, the assessment of public health risks, and the development of control interventions. An overview of the VTEC virulence factors, including their genetic basis and function, would start this process and is the objective of this article.

Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen systems by carvacrol and thymol

The antimicrobial activities of thymol and carvacrol were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains ( n = 11) and other bacterial species and spoilage bacteria ( n = 7) using a model broth system. The effects of pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competing microflora on the activities of thymol and carvacrol against E. coli O157:H7 strain 380-94 were also determined. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) and numbers of surviving E. coli O157:H7 were determined following incubation. The mean numbers of VTEC surviving exposure to thymol or carvacrol at concentrations of ≥ 500 μg/ml were between 2.0 and 7.8 log cfu/ml less than the numbers in the corresponding controls. The susceptibility of E. coli O157:H7 to carvacrol or thymol was found to increase with decreasing storage temperature, water activity, pH and E. coli O157:H7 inoculum size. Sodium chloride (0.5–2.5%) and the presence of a microflora cocktail did not significantly ( p > 0.05) affect the antimicrobial activities of thymol or carvacrol against E. coli O157:H7. The antimicrobial activity of carvacrol against E. coli O157:H7 was also tested in a model rumen system. A MIC of 500 μg/ml carvacrol reduced E. coli O157:H7 inoculated at levels of 10 3 and 10 6 cfu/ml to undetectable levels in the system after 24 h incubation. This concentration of carvacrol significantly ( p < 0.05) decreased the total gas production and volatile fatty acid concentrations in the model rumen assay.

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

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

Aspectos clínicos y patogénicos de las infecciones por Escherichia coli O157:H7 y otros E. coli verotoxigénicos

Verocytotoxigenic Escherichia coli (VTEC), particularly serotype O157:H7, have recently emerged as an important group of food-borne pathogens in humans, causing diseases ranging from mild diarrhea to hemorrhagic colitis (HC), and complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura, typically affecting children, the elderly, and immunocompromised patients. Since its identification as a pathogen in 1982, E. coli O157:H7 has been detected in many sporadic cases and outbreaks of HC and HUS, particularly in North America, the United Kingdom, and Japan. In Spain, VTEC is a relatively common cause of sporadic infection, and a total of 10 outbreaks have been reported to date. Thus, knowledge of the clinical and pathogenic aspects of these infections is of particular interest.

Verocytotoxigenic Escherichia coli O157 in animals on public amenity premises in England and Wales, 1997 to 2007

At the request of the public health authorities, 31 public amenity premises in England and Wales containing animals of various species were investigated for the presence of verocytotoxigenic Escherichia coli...

Factors related to the carriage ofVerocytotoxigenic E. coli,Salmonella, thermophilicCampylobacterandYersinia enterocoliticain cattle, sheep and pigs at slaughter

A 12-month abattoir study was undertaken from January 2003. We collected 7492 intestinal samples from cattle, sheep and pigs at slaughter. Rectal samples were taken from cattle and sheep and caecal samples from pigs. They were examined for verocytotoxigenic E. coli (VTEC) O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. Data were collected on the animal from which the sample came and this information was analysed to look at potential risk factors for carriage of these organisms. Logistic regression models were run where an adequate number of positive results were available. This revealed that VTEC O157 carriage in cattle was associated with the summer period and that age was a protective factor. Salmonella carriage in pigs was associated with lairage times >12 h, the North East and not feeding when there was no bedding available. In cattle, carriage was associated with the summer period, the Eastern region of GB and dairy animals. In sheep a spring seasonal effect was seen, which coincided with the lambing period. The carriage of thermophilic Campylobacter in cattle was associated with single-species abattoirs, with age a protective factor. In sheep, winter was a risk period with lairage management influential. For pigs, lairage times of <12 h were found to be associated with carriage. A seasonal trend for carriage of Y. enterocolitica in all species was demonstrated with the period December-May a risk. For cattle, age was also a risk factor; for sheep feeding in the lairage and for pigs being held overnight were risk factors.

Surveillance of Dairy Production Holdings Supplying Raw Milk to the Farmhouse Cheese Sector for Escherichia coli O157, O26 and O111

Clinically healthy domestic animals can harbour Escherichia coli O157 and other verocytotoxigenic E. coli (VTEC) strains in their faeces. Milk filters can be used to microbiologically monitor direct milk secretion and environmental contamination for these pathogens. The aim of this study was to establish baseline data on the prevalence and characteristics of VTEC organisms in lactating animals (bovine, ovine and caprine) supplying milk to the farmhouse cheese sector, with particular emphasis on serogroups O157, O111 and O26. Fifty-six bovine, 13 caprine and 5 ovine herds/flocks, the majority of which supplying milk for farmhouse cheese production, were surveyed from May 2004 to July 2005. Milk filters were analysed by immunomagnetic separation followed by PCR, on a serogroup-specific basis for E. coli O157, O26 and O111. Positive isolates were examined using a multiplex PCR protocol, for their potential to produce verocytotoxins (vt1/vt2), the haemolysin-encoding gene (hlyA) and the gene encoding attaching and effacement (eae). Five verocytotoxigenic and 22 non-virulent E. coli O157 isolates were detected. Seventeen E. coli O26 isolates were also detected, four of which were verocytotoxigenic, seven isolates contained the eae gene only and six isolates were devoid of any of the virulence factors. The VTEC O157 and O26 isolates contained the hlyA and eae genes along with the verocytotoxin genes. No E. coli O111 isolates were detected. Some of the herds were positive on more than one occasion and multiple E. coli serogroups were isolated from the same milk filter sample. Although all food products tested were VTEC negative, routine surveillance for such pathogens in raw milk/raw milk products is of public health importance. Herd-level surveillance along with subsequent risk management action may be a cost-effective component of risk reduction strategies for food production, drinking water supplies and the protection of public health.

Escherichia coli serogroup O26 ? a new look at an old adversary

Escherichia coli serogroup O26 played an important part in the early work on Verocytotoxin and is an established diarrhoeal pathogen. Recently, Verocytotoxigenic E. coli (VTEC) O26 has been increasingly associated with diarrhoeal disease and frequently linked to outbreaks and cases of haemolytic uraemic syndrome (HUS). This review investigates the pathogenicity, geographical distribution, changing epidemiology, routes of transmission and improved detection of VTEC O26. Laboratory data on VTEC O26 isolates and clinical data on HUS suggest a true difference in the incidence of VTEC O26 in different geographic locations. However, few diagnostic laboratories use molecular methods to detect VTEC and so it is difficult to assess the role of VTEC O26 in causing diarrhoeal disease. VTEC O26 is frequently found in the cattle population but rarely in food. However, the small number of outbreaks analysed to date are thought to be food-borne rather than associated with direct or indirect contact with livestock or their faeces. The increase in awareness of VTEC O26 in the clinical and veterinary setting has coincided with the development of novel techniques that have improved our ability to detect and characterize this pathogen.

Occurrence and Characterization of Verocytotoxigenic Escherichia coli (VTEC) Strains from Dairy Farms in Trinidad

A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P < 0.05; chi(2)). For LT production, the highest frequency was detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P < 0.05; chi(2)). Of the 61 VT-producing isolates by vero cell cytotoxicity assay tested by PCR, the VT, LT and eae genes were detected in 62.3%, 4.9% and 1.6% respectively (P < 0.05; chi(2)). Amongst the 45 E. coli isolates that were VT positive (vero cell) or VT-gene positive by PCR, 2.2%, 2.2%, 4.4% and 6.7% belonged to non-O157 strains O91, O111, O103 and O157, respectively, as determined by the Dry spot test. Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.

Vero‐Cytotoxigenic Escherichia coli O157 in Pasteurized Milk Containers at the Point of Retail: A Qualitative Approach to Exposure Assessment

Dairies within the United Kingdom are classified into two groups, namely, off-farm and on-farm dairies (the latter often being small scale). We propose a model for the probability of milk sold as pasteurized reaching the point of retail contaminated with Vero-cytotoxigenic Escherichia coli (VTEC) O157 from each of these two pathways. We evaluate qualitatively the exposures inherent in each, and compare and contrast the two situations. The model framework is generic, in that it can in principle be used, with the relevant data modifications, to provide a qualitative assessment of the likely exposure from milk sold as pasteurized to any potentially milk-borne pathogenic organism. Furthermore, the methodological approaches presented are widely applicable in the microbial risk assessment field. The specific example presented will be of particular interest to the UK dairy and public health communities: we conclude that the exposure potential per liter consumed from milk processed in off-farm dairies is negligible, whereas the exposure potential per liter consumed from milk processed on-farm is low, but not sufficiently small to be regarded as negligible. We also identify areas of data sparsity, which need to be addressed for quantitative risk assessment to proceed, and highlight the critical points in the pasteurized milk production chain, which, in the event of a breakdown, have the potential to increase the risk to the consumer.

Survival of antibiotic resistant and antibiotic sensitive strains of E. coli O157 and E. coli O26 in food matrices

Escherichia coli O157:H7 or E. coli O26, which were AS (antibiotic sensitive), AR (laboratory created antibiotic resistant mutants), or naturally MAR (multi-antibiotic resistant), were inoculated into laboratory media, yoghurt or orange juice and their growth/survival monitored during enrichment at 37 °C or storage at 4 °C. The strains were also inoculated into minced beef and their thermal inactivation ( D-values) examined at 55 °C, with and without a prior heat shock at 48 °C. The growth kinetics (lag phases, growth rates) of the VTEC (verocytotoxigenic E. coli), incubated over 24 h at 37 °C in laboratory media, were similar regardless of the presence or absence of antibiotic resistance. In yoghurt and orange juice, E. coli O157:H7 MAR died off significantly faster ( P < 0.05) than any of other VTEC strains examined. E. coli O157:H7 MAR was also found to be significantly more heat sensitive ( P < 0.05) than the other VTEC strains tested. The reasons for the observed differences in survival of the different VTEC strains and the link between antibiotic resistance and survival in VTEC organisms are discussed.

Clinical, microbiological and epidemiological aspects of Escherichia coli O157 infection.

In the last decade infections caused by Escherichia coli O157:H7 and other verocytotoxigenic E. coli (VTEC) have emerged as a major public health concern in North America and in Europe, and increasingly in other areas of the world. Although absolute numbers of infections are low in comparison with other enteric pathogens such as Salmonella or Campylobacter, it is well-recognised that E. coli O157 can produce severe, potentially life-threatening, illness. As a consequence of this awareness, there has been a rapid expansion of our knowledge about these organisms and the diseases which they cause. In this article, the clinical, microbiological and epidemiological features of VTEC O157 infection are reviewed.

Laboratory-based surveillance of human verocytotoxigenic Escherichia coli infection in the Republic of Ireland, 2002–2004

The aim of this study was to examine the frequency and distribution of human verocytotoxigenic Escherichia coli (VTEC) O157 and non-O157 in the Republic of Ireland, and also to examine the presence of virulence genes in these isolates. This genetic information combined with phenotypic tests was used to produce a complete laboratory-based surveillance of human clinical VTEC infection in the Republic of Ireland between 2002 and 2004. Between January 2002 and December 2004 a total of 207 VTEC isolates were studied (one isolate per patient), 185 (89 %) of these were E. coli O157. The remaining 22 (11 %) were non-O157 E. coli, made up of 15 (7.2 %) E. coli O26, one (0.5 %) E. coli O103, one (0.5 %) E. coli O146, one (0.5 %) E. coli O145, two (1 %) E. coli O111 and two (1 %) ungroupable VTEC. These isolates originated from the eight health boards in the Republic of Ireland and represented over 90 % of the clinical cases of VTEC in the Republic of Ireland during this period. The results showed that VTEC O157 was the predominant serogroup and had a predominant toxin genotype of VT2 alone. Phage type 32 was the most common phage type of E. coli O157 identified. Non-O157 VTEC was a small proportion of all VTEC (10 % in 2002, 8 % in 2003, 15.5 % in 2004). In 2004 it was noted that there was an increase in the number and variety of non-O157 VTEC strains; however, this requires further monitoring in the future to see if this trend is sustained. It was also noted throughout the study period that the incidence of VTEC was higher in rural areas. Implementation of real-time PCR for the detection and subtyping of VTEC has aided outbreak investigations and is important for enhanced surveillance of VTEC in the Republic of Ireland.

Prevalence and Characterization ofEscherichia coliO26 and O111 in Retail Minced Beef in Ireland

Verocytotoxigenic Escherichia coli (VTEC) O157 are recognized as bacterial pathogens with significant public health impact. However, other serogroups, including O26, O111, O103, and O145, have the potential to cause the same spectrum of illness. In this study, 800 minced (ground) beef samples covering a large geographical region in Ireland were collected and tested for Escherichia coli (E. coli) O26 and E. coli O111 by conventional microbiological protocols. Two minced beef samples (0.25%) tested positive for E. coli O26, indicating fecal contamination. None of these isolates possessed verocytotoxin-encoding genes, (vt1/vt2 also known as stx1/stx2), the hemolysinencoding gene (hlyA), or the E. coli attachment-effacement (eae) gene, as determined by polymerase chain reaction (PCR). None of the beef samples analyzed contained E. coli O111. Although the E. coli O26 isolates were nonvirulent, the presence of these isolates in raw minced beef is an indication of fecal contamination and therefore potentially of public health significance.

Virulence genes in verocytotoxigenic Escherichia coli strains isolated from humans and cattle

Verocytotoxigenic Escherichia coli (VTEC) causing diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome usually have additional traits such as the adhesin intimin and a large plasmid that seems to increase virulence. There are, however, isolates of VTEC causing serious symptoms that do not harbour these traits. In the present study we have used PCR with primers detecting adhesin genes other than eaeA, namely fimA, papC, sfaD/sfaE and daaE. We have also used PCR to detect the genes hlyA and iutA that besides the plasmid-borne gene E-hly possibly support the bacterial access to iron. The aim of the study was to identify and compare the presence of virulence genes in VTEC isolates of human and cattle origin. The main finding was that the absence of E-hly might be compensated for by the gene iutA coding for aerobactin or hlyA coding for alpha-haemolysin as 94% of the human VTEC isolates had at least one of these genes. Interestingly, only 45% of VTEC isolated from cattle had any of these genes. We propose that this might be the reason for the relatively low incidence of symptomatic VTEC infections among humans in relation to the high number of VTEC among cattle.

Evidence of transmission of verocytotoxin-producing Escherichia coli O111 from a cattle stable to a child

Infections with verocytotoxigenic Escherichia coli (VTEC) other than O157 have been assumed to have the same epidemiology as those with VTEC O157, but the source of infection is rarely defined for sporadic cases. This report describes a child with VTEC O111:H- infection who was probably infected by playing in a cattle stable and/or by drinking raw milk from the cows in this stable. E. coli O111 isolates colonising the cattle were indistinguishable from the patient isolate by the use of serotyping, pulsed-field gel electrophoresis, and virulence profiling.

Distribution of saa gene variants in verocytotoxigenic Escherichia coli isolated from cattle and food

The pathogenesis of verocytotoxigenic Escherichia coli (VTEC) infection in humans is multifactorial, given that verocytotoxins are the principal virulence factor. Most strains causing serious diseases possess the eae gene that encodes the adhesin intimin, but its presence is not essential for virulence as some cases are caused by eae-negative strains. An autoagglutinating adhesin designated Saa was found in some eae-negative strains. This protein varies in size as a consequence of variation in the number of copies of a 37-aa repeat unit in the C-terminal region. Based on these findings, we designed PCR primers to amplify the region coding for these differences to detect saa gene variants present in VTEC strains isolated in Argentina from cattle and meat. The gene saa was detected in 36 (31.6%) eae-negative strains and 5 variants were found. Strains isolated from cattle possessed 4 saa variants, whereas 2 variants were present in isolates from meat. Saa variant 1 predominated (18 strains) and was distributed in strains isolated both from cattle and from meat. Our study revealed the existence of two novel saa variants, termed 4 and 5, which have a higher number of 111-bp repeats than saa genes previously studied.

VerotoxigenicEscherichia colifrom animals, humans and foods: who's who?

Verocytotoxigenic (shigatoxigenic) and enterohaemorrhagic Escherichia co/i, VTEC (STEC) and EHEC, produce a toxin active on Vero cells in vitro. VTEC and EHEC have been isolated from humans and different animal species, mainly ruminants and pigs. The verocytotoxins, also named shiga toxins (Stx), are active in vivo on the endothelial cells of the blood vessels of the gastro-intestinal mucosa, the kidneys, the brain, and other tissues of humans and piglets, leading to fluid leakage or haemorrhages. Conversely, their role in diseases of young ruminants remains unclear. Adult ruminants can also act as asymptomatic carriers of VTEC and EHEC strains similar to those causing diseases in humans. And they are incriminated as an important source of direct or indirect contamination of humans by the most famous EHEC strain belonging to the O157:H7 serotype, through faecal contamination of either foods of animal origin, or other foodstuffs (fruit, vegetables, etc.), or the environment. But dozens of non-O157 human and ruminant VTEC and EHEC strains with similar general and virulence-associated properties, have been described, whose epidemiology is much less well understood. The purpose of this review manuscript is to describe and compare the properties of human, ruminant and food VTEC and EHEC strains.

Comparison of a real-time PCR and an IMS/culture method to detect Escherichia coli O26 and O111 in minced beef in the Republic of Ireland

This study compared an immuno-magnetic separation (IMS)/culture method and a real-time PCR method to detect Verocytotoxigenic Escherichia coli (VTEC) serovar O26 and/or O111 in minced beef. A total of 65 samples were examined, 40 of which were frozen beef samples previously established as containing E. coli O157, and 25 were samples of fresh minced beef, purchased from butcher shops in the Dublin area. After selective enrichment, all samples were (a) subjected to IMS, plated on differential media and identified as E. coli O26 or O111 using biochemical and immuno-logical methods; and (b) subjected to DNA extraction and real-time PCR analysis using primers and probes against E. coli O111 and O26 serovar specific genes, and verotoxin genes. Overall, from the 65 minced beef samples collected, three were positive for E. coli O26 by real-time PCR, with only one of these samples positive for E. coli O26 by the culture method. One sample was positive for E. coli O111 by both real-time PCR and the culture method. The two samples found positive for E. coli O26 by real-time PCR method but not by culture method belonged to the group of frozen beef samples, indicating that the previously developed culture method for the detection of E. coli O26 may not be suitable for the detection of freeze injured cells. In conclusion, this study highlights the role of beef meat in the transmission of non-O157 VTEC. The results of the study emphasize that the analyses for emergent pathogens should be included in food safety surveillance systems and that the development of standard methods for the detection of E. coli O26 and O111 in routine food testing is needed in order to reduce the consumer exposure to contaminated food.

Presence and characterisation of verotoxin producing E. coli in fresh Italian pork sausages, and preparation and use of an antibiotic-resistant strain for challenge studies

One hundred and twenty six samples of fresh pork sausages were analysed for the presence of verocytotoxigenic Escherichia coli (VTEC). Selective enrichment followed by DNA extraction and PCR amplification of the stx1 and stx2 genes highlighted the occurrence of the above mentioned genes in 20 out of 126 samples screened. From the stx positive enriched cultures, isolation was performed on CT-SMAC agar plates after immuno-magnetic separation of E. coli O157. Fifty three non-sorbitol fermenting isolates were obtained and further characterised, along with the reference strain E. coli ATCC 35150 T. All the isolates were characterised by PCR assays, assessing the presence of stx1, stx2, rfbE O157:H7, eae and hlyA genes. The overall prevalence of VTEC was found to be 16%. VTEC strains were also characterised by plasmid profiling and REA-PFGE analysis, which allowed strain clustering into 5 and 8 groups, respectively. In addition, an antibiotic resistant E. coli O157:H7 strain was selected and used in challenge tests of raw pork at 4 °C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1 week at 4 °C in the raw food studied.