Shiga Toxin-producing Escherichia Coli Bacteria Research Articles

Occurrence, antibiotic resistance and molecular characterisation of Shiga toxin-producing Escherichia coli isolated from broiler chickens in Tunisia

ABSTRACT 1. Shiga toxin-producing Escherichia coli (STEC) strains are associated with disease outbreaks which cause a public health problem. The aim of this study was to determine the frequency of STEC strains, their virulence factors, phylogenetic groups and antimicrobial resistance profiles in broiler chickens. 2. A total of 222 E.coli isolates were collected from the caecum of chickens intended to be slaughtered. Antibiotic susceptibility was tested against 21 antimicrobial agents and ESBL phenotype was assessed by double-disk synergy test. The presence of STEC virulence genes stx1, stx2,eaeA and ehxA was detected by PCR. The identification of STEC serogroups was realised by PCR amplification. Additive virulence genes, phylogenetic groups and integrons were examined among the STEC isolates. 3. Out of 222 E.coli isolates, 72 (32%) were identified as STEC strains and the most predominant serogroups were O103, O145 and O157. Shiga toxin gene 1 (stx1) was found in 84.7% (61/72) of the STEC strains, and eae and stx2 were detected in 38.8% and 13.8%, respectively. The ESBL phenotype was documented in 48.6% (35/72) of isolates. Most of the isolates (90.3%) carried class 1 integron with the gene cassette encoding resistance to trimethoprim (dfrA) and streptomycin (aadA) in 31.9% of the isolates. Class 2 integron was identified in 36.1% of isolates. 4. Broilers can be considered as a reservoir of STEC strains which have high virulence factors and integrons that might be transmitted to other chickens, environments and humans. It is important to undertake surveillance and efficient control measures in slaughterhouses and farms to control measures of STEC bacteria.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

Evaluation of UVC Radiation and a UVC-Ozone Combination as Fresh Beef Interventions against Shiga Toxin–Producing Escherichia coli, Salmonella, and Listeria monocytogenes and Their Effects on Beef Quality

This research study was conducted to evaluate treatments with UVC light and a combination of UVC and ozone that have recently received attention from the beef processing industry as antimicrobial interventions that leave no chemical residues on products. The effectiveness of UVC and UVC plus gaseous ozone treatments was evaluated for inactivation of pathogenic bacteria on fresh beef and for any impact on fresh beef quality. Fresh beef tissues were inoculated with cocktails of Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O26, O45, O103, O111, O121, O145, and O157:H7), Salmonella, and Listeria monocytogenes. Inoculated fresh beef tissues were subjected to UVC or UVC-ozone treatments at 106 to 590 mJ/cm2. UVC treatment alone or in combination with ozone reduced populations of STEC, Salmonella, L. monocytogenes, and aerobic bacteria from 0.86 to 1.49, 0.76 to 1.33, 0.5 to 1.14, and 0.64 to 1.23 log CFU, respectively. Gaseous ozone alone reduced populations of E. coli O157:H7, Salmonella, and L. monocytogenes by 0.65, 0.70, and 0.33 log CFU, respectively. Decimal reduction times (D-values) for STEC serotypes, Salmonella, and L. monocytogenes on surfaces of fresh beef indicated that the UVC-ozone treatment was more effective (P ≤ 0.05) than UVC light alone for reducing pathogens on the surface of fresh beef. Exposure to UVC or UVC plus gaseous ozone did not have a deleterious effect on fresh meat color and did not accelerate the formation of oxidative rancidity. These findings suggest that UVC and UVC in combination with gaseous ozone can be useful for enhancing the microbial safety of fresh beef without impairing fresh beef quality.

Immunomagnetic Capture of Big Six Shiga Toxin–Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix

Having reliable methods for detecting Shiga toxin-producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed "The Big Six." We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non-Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

Evolution of STEC virulence: Insights from the antipredator activities of Shiga toxin producing E. coli

Shiga toxin-producing Escherichia coli (STEC) are a diverse group of strains that are implicated in over 270,000 cases of human illness annually in the United States alone. Shiga toxin (Stx), encoded by a resident temperate lambdoid bacteriophage, is the main STEC virulence factor. Although the population structure of E. coli O157:H7, the most common disease-causing STEC strain, is highly homogenous, the range of clinical illness caused by this strain varies by dramatically outbreak, suggesting that human virulence is evolving. However, the factors governing this variation in disease severity are poorly understood. STEC evolved from an O55:H7-like progenitor into a human pathogen. In addition to causing human disease, Stx released from STEC kill bacterivorous protist predators and enhance bacterial survival in the face of protist predation. Cattle are the primary reservoir for STEC and protists and bacteria occur together within the ruminant intestinal tract. Cattle associated STEC are not highly pathogenic to humans. These observations suggest that disease causing STEC strains evolved from cattle-associated “antipredator” STEC strains. To test this idea and to gain insight into the features that govern the evolution of STEC from a commensal strain of ruminants strain to virulent human pathogen, we compared the predation resistance of STEC strains isolated from asymptomatic infected cows and human patients. We find that STEC O157:H7 progenitor lineages and clades are more effective than human associated ones at killing the types of protist predators. In addition, our results indicate that the presence of Stx2c-containing bacteriophage is associated with more efficient amoeba killing. Also, these phage apparently also encode Q21-like version of the Q antitermination protein, the protein that controls expression of Stx.

Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli (STEC) on New Zealand Dairy Farms: Application of a Culture-Independent Assay and Whole-Genome Sequencing.

ABSTRACTNew Zealand has a relatively high incidence of human cases of Shiga toxin-producing Escherichia coli (STEC), with 8.9 STEC cases per 100,000 people reported in 2016. Previous research showed living near cattle and contact with cattle feces as significant risk factors for STEC infections in humans in New Zealand, but infection was not linked to food-associated factors. During the 2014 spring calving season, a random, stratified, cross-sectional study of dairy farms (n = 102) in six regions across New Zealand assessed the prevalence of the “Top 7” STEC bacteria (serogroups O157, O26, O45, O103, O111, O121, and O145) in young calves (n = 1,508), using a culture-independent diagnostic test (PCR/MALDI-TOF). Twenty percent (306/1,508) of calves on 75% (76/102) of dairy farms were positive for at least one of the “Top 7” STEC bacteria. STEC carriage by calves was associated with environmental factors, increased calf age, region, and increased number of calves in a shared calf pen. The intraclass correlation coefficient (ρ) indicated strong clustering of “Top 7” STEC-positive calves for O157, O26, and O45 serogroups within the same pens and farms, indicating that if one calf was positive, others in the same environment were likely to be positive as well. This finding was further evaluated with whole-genome sequencing, which indicated that a single E. coli O26 clonal strain could be found in calves in the same pen or farm, but different strains existed on different farms. This study provides evidence that would be useful for designing on-farm interventions to reduce direct and indirect human exposure to STEC bacteria.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) bacteria that can cause bloody diarrhea and kidney failure in humans if ingested. New Zealand has relatively high numbers of STEC cases, and contact with cattle feces and living near cattle are risk factors for human infection. This study assessed the national prevalence of STEC in young dairy cattle by randomly selecting 102 farms throughout New Zealand. The study used a molecular laboratory method that has relatively high sensitivity and specificity compared to traditional methods. “Top 7” STEC was found in 20% of calves on 75% of the farms studied, indicating widespread prevalence across the country. By examining the risk factors associated with calf carriage, potential interventions that could decrease the prevalence of “Top 7” STEC bacteria at the farm level were identified, which could benefit both public health and food safety.

Virulence Genes and Phenotypic Evaluation of the Antibiotic Resistance of Vero Toxin Producing Escherichia coli Recovered From Milk, Meat, and Vegetables

Background: Resistant and virulent strains of Shiga toxin producing Escherichia coli (STEC) are one of the main prevalence sources of food infection due to consumption of meat, milk, and vegetables. Objectives: This study was conducted to assess the frequency of virulence factors and phenotypes of antibiotic- resistant STEC bacteria recovered from meat, milk, and vegetable samples. Methods: In this study, 280 samples were assessed. Escherichia coli isolates were assessed by PCR and disk diffusion. Results: Out of 280 samples, 59 (21.07%) were contaminated with E. coli. Vegetables had the highest prevalence (31.25%), while raw meat (14%) had the lowest. Stx1, eae, and ehly genes were found in all enterohemorrhagic Escherichia coli (EHEC) bacteria. Moreover, 21 out of 59 E. coli strains were STEC (35.59%). Shiga toxin producing E. coli strains showed the most resistance against ampicillin (100%), gentamycin (90.47%), tetracycline (85.71%), and ciprofloxacin (71.42%). Shiga toxin producing E. coli strains harbored resistance to at least 2 antibiotics (100%). Conclusions: Judicious prescription of imipenem and cotrimoxazole based on the results of disk diffusion and also proper washing of the vegetables, cooking of the meat, and boiling of the milk before consumption can control and reduce the risk of incidence of food poisoning due to the virulent and resistant STEC strains.

Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California.

Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area—around or within city—with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology between free STEC-specific phages and STEC bacteria for further pre-harvest food safety management in produce environment.

Heat inactivation of Shiga toxin-producing Escherichia coli in a selection of low moisture foods

Survival during temporary storage and heat inactivation of Shiga toxin-producing Escherichia coli (STEC) were investigated in a selection of four different low moisture foods with water activities between 0.26 and 0.63. A cocktail of nine strains of O157 and non-O157 STEC was selected for this study, giving preference to those strains of public health concern associated with food consumption. The data from previous studies on STEC survival and heat resistance in wheat flour were taken into account for the selection of the relevant target serotypes and strains. No significant difference was observed on STEC viable counts among the four products stored at 16 °C for 7 days. The viable counts decreased by maximum 1 log CFU/g by day 28, as simulation of temporary storage of bulk raw materials before processing. The level of STEC inactivation by dry heat treatments varied with the food formulation. The STEC bacteria showed a lower heat resistance in savory seasoning and pet food formulations compared to confectionery formulation and chicken meat powder. We also identified the minimum heating temperature and time combinations for each food to achieve a 5 log reduction of STEC using linear and Weibull models. Our results show the capability of obtaining a 5 log reduction of STEC in pet food, savory seasoning and confectionery formulations, and chicken meat powder by heating to product temperatures such as 90.9, 92.7, 99.1, and 106.0 °C, respectively. High product temperatures can be achieved by heating food samples in thermal cells hermetically sealed and submerged in a thermo-fluid bath at temperatures of 110 °C or above. The use of thermal cells allowed to simulate in the laboratory the high processing temperatures such as those applied in food extrusion or continuous treatment without moisture loss during heating.

An in vitro combined antibiotic-antibody treatment eliminates toxicity from Shiga toxin-producing Escherichia coli.

Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the "big six" serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

Virulence characteristics of Shiga toxin-producing Escherichia coli from raw meats and clinical samples.

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae-negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non-O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.

Detection of Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) Serogroups with Hyperspectral Microscope Imaging Technology

Abstract. Non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups such as O26, O45, O103, O111, O121, and O145 are recognized as serious risks to human health due to their toxicity. The conventional microbiological detection method of cell counting on agar plates is laborious and time-consuming. Because optical methods are promising for real-time, in situ foodborne pathogen detection, a hyperspectral microscope imaging (HMI) method based on acousto-optic tunable filters (AOTF) was developed for detecting pathogenic bacteria with a capability to differentiate the spectral characteristics of each bacterial cell from microcolony samples. Using the AOTF-based HMI method, a total of 89 contiguous spectral images were acquired within approximately 45 s with 250 ms exposure time. In this study, we developed a protocol for successfully immobilizing live cells on glass slides to acquire quality spectral images of STEC bacterial cells using a modified drying method. Among the contiguous spectral imagery between 450 and 800 nm, the intensities at 458, 498, 522, 546, 570, 586, 670, and 690 nm were distinct for STEC bacteria under dark-field illumination. With two different classification algorithms, i.e., support vector machine (SVM) and sparse kernel-based ensemble learning (SKEL), STEC serogroup O45 could be classified with 92% detection accuracy. However, the mean accuracies in identifying the six STEC serogroups with SVM and SKEL were not high enough for use in classification models.

English

Domestic ruminants, especially sheep and cattle, are the main reservoirs of Shiga toxin-producing Escherichia coli (STEC) and may transmit this pathogen to human. Probiotics are "live organisms that, when administered in adequate amounts, confer a health benefit on the host", decreasing the infection and its dissemination. However, these benefits are conferred only after the colonization of probiotic strains in the gut of the animal, which may be impaired by normal microbiota. The aims of this study were to determine whether the inoculation of sheep with probiotic strains decreases the shedding of STEC and to determine whether the age of sheep interferes with this protective effect. Sheep that received oral inoculums at a concentration of 2 × 109 cells per mL of viable STEC bacteria, which are carriers of stx1, stx2 and eae genes, were compared with other sheep that did not receive inoculums. When probiotic bacteria were inoculated together with the STEC, the number of pathogenic bacteria in the population was similar to the control. The protective effect of probiotic strains was largest in groups with younger animals than with older animals. These findings suggest that the use of probiotic strains in sheep may decrease the intestinal shedding by STEC as well as the fact that the age of the sheep may interfere in the protective effect of probiotics against colonization by STEC.   Key words: Probiotic, protective effect, Escherichia coli, sheep.

Escherichia coli O104:H4 outbreak--have we learnt a lesson from it?

Shiga toxin-producing Escherichia coli (STEC) strains belong to the group of pathogens that cause bloody diarrhea and hemorrhagic colitis with often severe complications. The main problem with human pathogenic E. coli strains, including STEC, is a wide spectrum of phenotypes and clinical manifestations. It is related to a variety of exchangeable genetic elements, like plasmids, bacteriophages, transposons and pathogenicity islands, that take part in horizontal gene transfer which influences creation of new dangerous bacterial strains. A good example of this phenomenon is a novel Shiga toxin-producing E. coli O104:H4 serotype that was associated with a widespread and severe foodborne disease outbreak in Germany in 2011. The O104:H4 strain was created by a number of horizontal gene transfer events between two distinct pathogens, resulting in the emergence of the new, atypical strain. That outbreak proved that also rare and unusual serotypes of STEC may be a significant risk factor and that the procedures recommended for STEC detection were not suitable to deal with this kind of pathogens. With respect to new combinations of chromosomal and extrachromosomal elements in susceptible bacterial hosts, epidemics and frequent human infections caused by STEC strains, we suggest that more attention should be paid to the development and improvement of diagnostic methods. It is difficult to determine STEC bacteria by general microbiological, biochemical and immunological assays, because strains can vary dramatically in their phenotypic and serotypic properties. It is postulated that standardized genetic tests, based on detection of features most frequently presented by STEC, particularly those located on easily exchangeable elements (such as Shiga toxin-encoding phages), can be more adequate for STEC detection.

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods. Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx 1 gene, 44 (67.7%) carried the stx 2 gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.

Diversity of virulence patterns among shiga toxin-producing Escherichia coli from human clinical cases — need for more detailed diagnostics

Intestinal infections due to shiga toxin-producing Escherichia coli bacteria (STEC) reveal a broad range of clinical symptoms and a large scale of virulence properties of the respective pathogens. The question whether all STEC variants or only a particular group of them need to be considered for clinical and epidemiological purposes was answered throughout this study. Using the PCR technique for the identification of 25 different virulence-associated genes, 266 E. coli strains belonging to 81 different E. coli serotypes from various clinical origins were investigated. A great genetic diversity of the virulence properties and a broad range of virulence marker combinations have been identified. However, distinct virulence marker combinations (e.g. Stx2/LEE/pO157 as well as Stx2dac/pO113) were found to be associated with the same notified clinical symptoms (e.g. HUS). Such an association speaks either for the “shiga toxin-only concept” or for several redundant, but clinically or epidemiologically important virulence properties.

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR.

The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.

Development of a PCR-based method for specific identification of genotypic markers of shiga toxin-producing Escherichia coli strains.

A simple, rapid and specific PCR-based method for identification of shiga toxin-producing Escherichia coli (STEC) was developed. The procedure involves amplification of the E. coli-specific universal stress protein A (uspA) gene (uspa-PCR), with the primer pair described by other authors, which allows differentiation of E. coli (STEC and non-STEC) from other gram-negative bacteria followed by identification of the main genetic virulence traits of the uspA-positive isolates. For this purpose, two multiplex PCR assays, based on previously published primer sequences, were established. Assay 1 (mPCR-1) uses three primer pairs and detects the genes encoding O157 (rfb), enterohemolysin (ebly) and shiga toxin (stx), generating amplification products of 420, 534 and 230 bp, respectively. Assay 2 (mPCR-2) uses four primer pairs specific for rfb (E. coli O157), eaeA (intimin), stx1 and stx2 (shiga toxin 1 and 2, respectively), generating PCR amplicons of 420, 840, 348 and 584 bp, respectively. These two assays were validated by testing several E. coli reference strains and 202 previously characterized E. coli isolates originating from calves and from children, and 100% agreement with previous results was obtained. The method developed can be used for specific identification of STEC bacteria including those of the O157 serogroup.

Survival of Shiga toxin-producing Escherichia coli O157 in marine water and frequent detection of the Shiga toxin gene in marine water samples from an estuary port

Shiga toxin-producing Escherichia coli (STEC) O157 was investigated with respect to its halotolerance and whether it can survive in marine water. STEC O157 could multiply in a medium containing 5% NaCl and in sterilized marine water, and could survive in unsterilized marine water for at least 15 days. On the basis of these results, we postulated that STEC O157 may survive in natural marine water, and attempted to isolate the bacterium and Shiga toxin gene (stx) from marine water in Japan. The stx, comprising stx1 and stx2, was detected from marine water samples by PCR. STEC and other stx-positive bacteria, however, could not be isolated from these samples in this study. These results indicate that stx-positive bacteria may survive in marine water and suggest the necessity of a survey.

Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli O157:H7 infection in infant rabbits.

We examined colonization patterns of Shiga toxin-producing Escherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (approximately 10(3) CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration of Lactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. casei administration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration of L. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration of L. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.