Biofilm formation by LEE-negative Shiga Toxin-Producing Escherichia coli strains.

Shiga toxin-producing Escherichia coli (STEC) include several serotypes isolated from cases of hemorrhagic colitis and, hemolytic uremic syndrome. Although O157:H7 is the most predominant STEC serotype, more than 100 non-O157 serogroups cause diseases in humans. Some STEC carry a Locus of Enterocyte Effacement (LEE-positive); however, STEC that do not carry LEE (LEE-negative) have also been associated with illness, mainly those harbouring the Locus of Adhesion and Autoaggregation (LAA). LAA carry some genes such as hes, iha, tpsA, and agn43, related with pathogenicity. One of them is the ability to form biofilms on different environments, which can contaminate food and generate infections while protecting themselves against adverse conditions. Considering that LAA could be responsible for some adherence mechanisms, the aims of this study were to compare different serogroup of LEE-negative STEC strains in their ability to form biofilms and to evaluate the participation of some genes encoding in LAA. A total of 348 LEE-negative STEC strains was analyzed. The presence of hes, iha, tpsA and agn43 were determined by monoplex PCR. From them, 48 STEC strains belonging to serogroups O113, O130, O171, O174 and, O178 were assayed for their ability to form biofilm. The most prevalent genes detected were agn43 (72.1%) and tpsA (69.5%). The iha and hes genes were present in 63.7% and 54% of the strains, respectively. Although all STEC strains were able to form biofilm, it was found a high variability between them. The relation between the biofilm formation and the presence of each gene was not statistically significant, suggesting that biofilm formation is independent of the presence of those genes. Highlighting that there is no treatment for HUS, it is once again notable that prevention measures and control strategies to prevent biofilm formation are important factors in reducing STEC transmission.

Variation in the Distribution of Putative Virulence and Colonization Factors in Shiga Toxin-Producing Escherichia coli Isolated from Different Categories of Cattle.

Shiga toxin-producing Escherichia coli (STEC) are pathogens of significant public health concern. Several studies have confirmed that cattle are the main reservoir of STEC in Argentina and other countries. Although Shiga toxins represent the primary virulence factors of STEC, the adherence and colonization of the gut are also important in the pathogenesis of the bacteria. The aim of this study was to analyze and to compare the presence of putative virulence factors codified in plasmid -katP, espP, subA, stcE- and adhesins involved in colonization of cattle -efa1, iha- in 255 native STEC strains isolated from different categories of cattle from different production systems. The most prevalent gene in all strains was espP, and the less prevalent was stcE. katP was highly detected in strains isolated from young and rearing calves (33.3%), while subA was predominant in those isolated from adults (71.21%). Strains from young calves showed the highest percentage of efa1 (72.46%), while iha showed a high distribution in strains from rearing calves and adults (87.04 and 98.48% respectively). It was observed that espP and iha were widely distributed throughout all strains, whereas katP, stcE, and efa1 were more associated with the presence of eae and subA with the eae-negative strains. A great proportion of eae-negative strains were isolated from adults -dairy and grazing farms- and from rearing calves -dairy and feedlot-, while mostly of the eae-positive strains were isolated from dairy young calves. Data exposed indicate a correlation between the category of the animal and the production systems with the presence or absence of several genes implicated in adherence and virulence of STEC.

Characterization of virulence factors and phylogenetic group determination of Escherichia coli isolated from diarrheic and non-diarrheic calves from Brazil.

This study aimed to detect virulence factors, pathovars, and phylogenetic groups of Escherichia coli strains obtained from feces of calves with and without diarrhea up to 70 days old and to determine the association between occurrence of diarrhea, phylogenetic groups, and pathovars. Phylo-typing analysis of the 336 E. coli strains isolated from calves with Clermont method showed that 21 (6.25 %) belong to phylogroup A, 228 (67.85 %) to phylogroup B1, 2 (0.6 %) to phylogroup B2, 5 (1.49 %) to phylogroup C, 57 (16.96 %) to phylogroup E, and 3 (0.9 %) to phylogroup F. Phylogroup D was not identified and 20 strains (5.95 %) were assigned as "unknown." The distribution of phylogenetic groups among pathovars showed that NTEC belong to phylogroups B1 (17) and C (4); EPEC to phylogroups B1 (6) and E (8); STEC to phylogroups A (5), B1 (56), B2 (2), C (1), and E (15); EHEC to phylogroups B1 (95) and E (5); and ETEC to phylogroups A (3), B1 (7), and E (10). The EAST-1 strains were phylogroups A (13), B1 (47), E (19), and F (3); E. coli strains of "unknown" phylogroups belonged to pathovars EPEC (1), EHEC (2), STEC (7), and EAST-1 strains (6). ETEC was associated with diarrhea (P = 0.002). Our study did not find association between the phylogenetic background and occurrence of diarrhea (P = 0.164) but did find some relationship in phylogenetic group and pathovar. The study showed that EHEC and STEC are classified as phylogroup B1, EAST-1 phylogroup A, ETEC, and EPEC phylogroup E.

Mexican unpasteurised fresh cheeses are contaminated with Salmonella spp., non-O157 Shiga toxin producing Escherichia coli and potential uropathogenic E. coli strains: A public health risk.

Fresh cheeses are a main garnish of Mexican food. Consumption of artisanal fresh cheeses is very common and most of them are made from unpasteurised cow milk. A total of 52 fresh unpasteurised cheeses of five different types were purchased from a variety of suppliers from Tabasco, Mexico. Using the most probable number method, 67% and 63% of samples were positive for faecal coliforms and E. coli, respectively; revealing their low microbiological quality. General hygienic conditions and practices of traditional cheese manufacturers were poor; most establishments had unclean cement floors, all lacked windows and doors screens, and none of the food-handlers wore aprons, surgical masks or bouffant caps. After analysing all E. coli isolates (121 strains) for the presence of 26 virulence genes, results showed that 9 (17%) samples were contaminated with diarrheagenic E. coli strains, 8 harboured non-O157 Shiga toxin producing E. coli (STEC), and one sample contained both STEC and diffusely adherent E. coli strains. All STEC strains carried the stx1 gene. Potential uropathogenic E. coli (UPEC) strains were isolated from 15 (29%) samples; the most frequent gene combination was fimA-agn43. Two samples were contaminated with Salmonella. The results demonstrated that unpasteurised fresh cheeses produced in Tabasco are of poor microbiological quality and may frequently harbour foodborne pathogens. Food safety authorities in Mexico need to conduct more rigorous surveillance of fresh cheeses. Furthermore, simple and inexpensive measures as establishing programs emphasizing good hand milking practices and hygienic manufacturing procedures may have a major effect on improving the microbiological quality of these food items.

Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander.

Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

Presence of non-O157 Shiga toxin-producing Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli and Salmonella in fresh beetroot (Beta vulgaris L.) juice from public markets in Mexico.

BACKGROUND: Unpasteurized juice has been associated with foodborne illness outbreaks for many years. Beetroot is a vegetable grown all over the world in temperate areas. In Mexico beetroot is consumed cooked in salads or raw as fresh unpasteurized juices. No data about the microbiological quality or safety of unpasteurized beetroot juices are available. Indicator bacteria, diarrheagenic Escherichia coli pathotypes (DEP) and Salmonella frequencies were determined for fresh unpasteurized beetroot juice from restaurants. RESULTS: One hundred unpasteurized beetroot juice samples were collected from public markets in Pachuca, Mexico. Frequencies in these samples were 100%, 75%, 53%, 9% and 4% of positive samples, for coliform bacteria, fecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and non-O157 Shiga toxin-producing E. coli (STEC). Identified Salmonella serotypes included Typhimurium and Enteritidis. CONCLUSION: This is the first report of microbiological quality and atypical EPEC, ETEC, non-O157 STEC and Salmonella isolation from fresh raw beetroot juice in Mexico. Fresh raw beetroot juice from markets is very probably an important factor contributing to the endemicity of atypical EPEC, ETEC, non-O157 STEC and Salmonella-related gastroenteritis in Mexico.

Behavior of shiga toxin-producing Escherichia coli, enteroinvasive E. coli, enteropathogenic E. coli and enterotoxigenic E. coli strains on whole and sliced jalapeño and serrano peppers.

The behavior of enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and non-O157 shiga toxin-producing E. coli (non-O157-STEC) on whole and slices of jalapeño and serrano peppers as well as in blended sauce at 25 ± 2 °C and 3 ± 2 °C was investigated. Chili peppers were collected from markets of Pachuca city, Hidalgo, Mexico. On whole serrano and jalapeño stored at 25 ± 2 °C or 3 ± 2 °C, no growth was observed for EPEC, ETEC, EIEC and non-O157-STEC rifampicin resistant strains. After twelve days at 25 ± 2 °C, on serrano peppers all diarrheagenic E. coli pathotypes (DEP) strains had decreased by a total of approximately 3.7 log, whereas on jalapeño peppers the strains had decreased by approximately 2.8 log, and at 3 ± 2 °C they decreased to approximately 2.5 and 2.2 log respectively, on serrano and jalapeño. All E. coli pathotypes grew onto sliced chili peppers and in blended sauce: after 24 h at 25 ± 2 °C, all pathotypes had grown to approximately 3 and 4 log CFU on pepper slices and sauce, respectively. At 3 ± 2 °C the bacterial growth was inhibited.

Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products.

Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 10(10) plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 10(10) PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10(-7)-1.8 × 10(-6)) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in meat products.

Phage biocontrol of enteropathogenic and Shiga toxin-producing Escherichia coli during milk fermentation.

UNLABELLED: Two bacteriophages, isolated from faeces, were assayed as biocontrol agents of pathogenic Escherichia coli during milk fermentation. Phage DT1 was tested on the strain E. coli DH5α, one enteropathogenic E. coli (EPEC) strain and one Shiga toxigenic E. coli O157:H7 (STEC) strain. Phage DT6 was tested on two STEC strains (O157:H7 and non-O157). One additional assay was performed by using a cocktail of both phages against the O157:H7 STEC strain. Streptococcus thermophilus 10-C, the strain used as lactic starter, reached 10(9)  CFU ml(-1) after 4 h, while pH values fell to 4·5 after 8 h, regardless of the presence of E. coli strains and/or phages. In absence of phages, E. coli strains reached 4-6 log CFU ml(-1) at 5-6 h. Escherichia coli DH5α and O157:H7 STEC strains were rapidly and completely inactivated by phage DT1 and phage cocktail, respectively, while O157:H7 STEC was completely inactivated either by DT1 or by DT6, after 8 h. The EPEC strain was not detected at 1 h (<10 CFU ml(-1) ) but grew afterwards, though at lower rates than without phage. For non-O157:H7 STEC, reductions lower than 1 log CFU ml(-1) were observed for all sampling times. Phages DT1 and DT6, either individually or as a cocktail, effectively reduce O157:H7 STEC counts during milk fermentation, without compromising the starter culture performance. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages DT1 and DT6, isolated from faeces and selected on the basis of their host range, showed to be valuable tools for the control of pathogenic Escherichia coli during milk fermentation, without compromising the starter culture performance. Both phages, either individually or as a cocktail, may function as an extra safety barrier beyond traditional pasteurization, effectively reducing O157:H7 Shiga toxin-producing Escherichia coli (STEC) counts during early growth, thus avoiding Shiga toxin production and accumulation.

Differences in Shiga toxin and phage production among stx(2g)-positive STEC strains.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx(2g) was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx(2g)-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx(2g)-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx(2)-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx(2g)-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx(2g)-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx(2)-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx(2g)-positive strains Stx production is phage-regulated.

Determination of adhesin gene sequences in, and biofilm formation by, O157 and non-O157 Shiga toxin-producing Escherichia coli strains isolated from different sources.

Biofilm formation by Shiga toxin-producing Escherichia coli (STEC) has been associated with the expression of different adhesins (type 1 fimbria, curli, Ag43, Cah, and EhaA). In this study, biofilm formation and the presence of adhesin-related gene sequences were determined by PCR in 18 O157 strains and 33 non-O157 strains isolated from different sources (human, animal, food, and water). The expression of different adhesins was also assessed by reverse transcription-PCR (RT-PCR), Congo red agar plates, and mannose-sensitive hemagglutination (MSHA) assay. Biofilm formation occurred in 5/18 (28%) O157 STEC strains and 17/33 (51%) non-O157 STEC strains from different serotypes and sources, when the assays were performed at 28°C for 48 h. Among the non-O157 biofilm-producing isolates, 12/17 (71%) expressed type 1 fimbriae and 11/17 (65%) expressed curli and produced cellulose, while 8/17 (47%) were considered to be Ag43(+) by RT-PCR. Among O157 strains, a close correlation was observed between biofilm formation and expression of curli and cellulose. In non-O157 strains, it seems that, in addition to the presence of curli, the ability to form biofilm is associated with the presence of other factors such as type 1 fimbriae and autotransporter proteins, which may contribute to the persistence of these organisms in the environment.

Interventions to reduce verocytotoxigenic Escherichia coli in ground beef in Argentina: a simulation study.

A stochastic simulation model was used to assess the effects of measures implemented in the agri-food-chain to reduce the contamination of ground beef with verotoxigenic Escherichia coli (VTEC). A published risk assessment model developed in Argentina was used as baseline scenario. Control measures assessed were based on either a reduction in herd prevalence of infection due to vaccination, reduction in opportunity for cross-contamination in the slaughterhouses by the introduction of an on-line hide-wash cabinet, and control of storage temperature in slaughterhouses, retail and home. Additionally, the increase of feedlot production was modelled. Simulations suggested that the greatest potential impact was associated with hide-wash cabinet and vaccination, measures aimed to reduce the VTEC prevalence and concentration in the cattle hides at the beginning of the food-chain. Control of storage temperature was not effective if the carcasses cross-contamination with the pathogen was not prevented or reduced. An increase production (fattening) of cattle in feedlots may raise the risk of VTEC infection and its sequelae. This information can be used as a basis for measures of risk management.

In vitro reactivity and growth inhibition of EPEC serotype O111 and STEC serotypes O111 and O157 by homologous and heterologous chicken egg yolk antibody.

IgY is a chicken egg yolk antibody which has been used for treatment and prophylaxis of gastrointestinal infections. Our aim was to verify if IgY obtained from chickens immunized with EPEC O111, STEC O111 and STEC O157 is able to show in vitro reactivity and biological activity towards the three bacteria. IgY was obtained from eggs laid before and after immunization with each bacterium. The preparations of IgY anti-EPEC O111 and anti-STEC O111 shared high reactivity detected by ELISA and growth inhibition ability towards both bacteria EPEC O111 and STEC O111. Nevertheless, the preparation of IgY anti-STEC O157 showed high reactivity and growth inhibitory effect only towards the homologous strain. Our results showing in vitro biological activity of IgY reinforce its use as an alternative for the treatment or prophylaxis of E. coli infections and encourage the development of in vivo studies for a possible future human therapeutic use.