Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157:H7

The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.     

Survival and virulence properties of multiple antibiotic‐resistant Salmonella Typhimurium under simulated gastrointestinal conditions

The transit tolerance and relative gene expression (acrA, acrB, tolC, stn, hilA and ompD) were observed in antibiotic‐sensitive Salmonella Typhimurium (ASST) and antibiotic‐resistant S. Typhimurium (ARST) exposed to simulated gastric juices (pH 2.0, pH 3.0 and pH 5.0) for 1 or 4 h, followed by small intestinal juices (0.1% and 0.5% bile salts) for 2 h. The relative expression of acrA, acrB, tolC, stn, hilA and ompD in the ARST was upregulated after exposure to simulated gastrointestinal juices. The expression of AcrAB‐TolC efflux system in ARST was highly correlated with the expression of stn and hilA encoding virulence factor and regulator. The cross‐protective effect was observed in ARST against the gastric acid and bile salts. The AcrAB‐TolC involved in enhanced survival and virulence potential, resulting in increased in pathogenicity of ARST. This study provides useful information for assessing the gastrointestinal transit tolerance and virulence potential of multiple antibiotic‐resistant pathogens.     

Review of recent advances in improved lateral flow immunoassay for the detection of pathogenic Escherichia coli O157:H7 in foods

The incidence of foodborne diseases has continuingly increased over the years and resulted in public health problem globally. Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7) is a human pathogen that causes diarrhea and hemorrhagic colitis. E. coli O157:H7 can be found in various foods. It is important to detect this foodborne pathogen to provide safe food supply. A lot of methods, for example, culture and PCR‐based test, used to detect foodborne pathogens are laborious and time consuming. Hence, a variety of methods have been developed for rapid, simple and reliable detection of E. coli O157:H7 as it is required in many food analyses. Lateral flow immunoassay (LFIA) are advantageous over conventional detection methods in terms of their rapidity and simplicity for end user, especially the LFIA can be developed as the strip test for on‐site point‐of‐care test (POCT) products. Gold nanoparticles (AuNPs; colloid gold) are the most commonly used labels in the LFIA for the visual analysis, however, there are still several limitations that restrict their applications of traditional LFIA. Therefore, recent reports on improved LFIA for E. coli O157:H7 detection in foods are continuously reported. This review intends to provide these recent advances in improved LFIA methods for the detection of E. coli O157:H7 in foods.     

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.     

In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices

This study investigated Propionibacterium freudenreichii tolerance to stresses encountered during food technology or in the digestive tract. The impact of food matrices on stress survival was evaluated. The ability of P. freudenreichii strains SI 41 and CNRZ 81 to survive both acid and bile salts stresses was studied in vitro. Stress tolerance was examined after inclusion of bacteria in different food matrices (alginate beads, xanthan–gellan beads, fermented milk) and compared to the stress tolerance of the same strains in pure cultures (either in exponential growth phase, in stationary phase, or after freeze-drying). Fermented milk was determined as the best probiotic vector to protect propionibacteria from this trial. This in vitro investigation gave promising results. Indeed, some food matrices can significantly improve protection of bacterial cells from stress injury. These data, which should be confirmed in an in vivo study, will be taken into consideration for the improvement of technological processes and for the choice of an adequate probiotic vector.     

Increased thermal tolerance in Cronobacter sakazakii strains in reconstituted milk powder due to cross protection by physiological stresses

Cronobacter sakazakii (C. sakazakii) is an opportunistic, neonatal, and food borne pathogen primarily associated with the contamination of powdered infant formula (PIF). The pathogen is reported to overcome the food safety barriers such as increased acidity, heat treatment, and so on. This study evaluates the thermal tolerance of C. sakazakii strains independently at 52, 55, and 58°C in reconstituted PIF after exposure to physiological stresses: refrigeration (4°C for 24 hr), starvation (37°C for 48 hr), and desiccation (25°C for 4 days). The Log₁₀ CFU/ml and D-values indicated that survival rate of all the strains decreased significantly (p < .05) after desiccation as compared to those of the control condition (without stress exposure). However, cold stress increased the thermal tolerance of all strains at all temperatures (52, 55, and 58°C) as indicated by increased D-values. Among the tested strains, C. sakazakii strain N15 was found to be the most resistant to thermal treatment after each stress exposure as depicted by principal component analysis (PCA). No apparent correlation between thermal tolerance and starvation stress was observed. The findings indicate that prior exposure to stress conditions may induce cross protection to thermal treatment in C. sakazakii.     

Interactions of Foodborne Pathogens with Free‐living Protozoa: Potential Consequences for Food Safety

Free‐living protozoa (FLP) are ubiquitous in natural ecosystems where they play an important role in the reduction of bacterial biomass and the regeneration of nutrients. However, it has been shown that some species such as Acanthamoeba castellanii, Acanthamoeba polyphaga, and Tetrahymena pyriformis can act as hosts of pathogenic bacteria. There is a growing concern that FLP might contribute to the maintenance of bacterial pathogens in the environment. In addition to survival and/or replication of bacterial pathogens in FLP, resistance to antimicrobial agents and increased virulence of bacteria after passage through protozoa have been reported. This review presents an overview of FLP in food‐associated environments and on foods, and discusses bacterial interactions with FLP, with focus on the foodborne pathogens Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. The consequences of these microbial interactions to food safety are evaluated.     

AUTOINDUCER-2-MEDIATED QUORUM SENSING IS NOT INVOLVED IN LISTERIA MONOCYTOGENES' ADAPTIVE RESPONSES TO THE FOOD PRESERVATIVES LACTIC ACID AND NISIN

Recent reports indicate the presence of autoinducer‐2 (AI‐2)‐like activity in foods such as milk and tofu; it is unclear what effect this molecule may have on virulence and survivability of foodborne pathogens. Quorum sensing has been implicated in stress responses of certain bacteria, and in this study we investigated AI‐2‐mediated quorum sensing as one of the mechanisms through which the general stress response in Listeria monocytogenes could be triggered. In particular, the ability of L. monocytogenes to acquire resistance to the food preservatives nisin and lactic acid was examined. The cells were pre‐exposed to extracellular AI‐2 and then challenged with the specific stresses so the level of adaptation caused by the pre‐exposure could be assessed. Our data suggested that the resistance to the studied antimicrobials was not mediated through AI‐2‐dependent quorum sensing pathways. No evidence was found suggesting that quorum sensing in general was involved in L. monocytogenes response to stress.     

A rapid detection of Escherichia coli O157:H7 by competition visual antigen macroarray

Pathogenic bacterial contamination is a serious problem for the food industry and in public health. Rapid, accurate and affordable testing for pathogenic bacterial strains is desirable. In this study, a competition visual antigen macroarray (CVAM) for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) has been developed. This array was able to utilize an HRP‐labeled anti‐E. coli O157:H7 MAb at a concentration of 1:20000 while having a similar sensitivity of 10 ⁵ CFU/ml for E. coli O157:H7 detection as double antibody sandwich array and conventional ELISA. The detection time of CVAM was short as 2 h and can be examined directly by the naked eye. Moreover, there was no cross‐reactivity in the detection of tested in this study. Application of CVAM for the detection of E. coli O157:H7 artificially contaminated milk samples was feasible that showed the antigen array could be applied in the detection of food pathogen infections.     

High prevalence of antimicrobial‐resistant Escherichia coli from animals at slaughter: a food safety risk

BACKGROUND: There has been concern about the increase of antimicrobial resistant bacteria and protection of animal and public health, along with food safety. In the present study, we evaluate the incidence of antimicrobial resistance among 192 strains of Escherichia coli isolated from faecal samples of healthy food‐producing animals at slaughter in Portugal. RESULTS: Ninety‐seven % of the pig isolates, 74% from sheep and 55% from cattle were resistant to one or more antimicrobial agents, with the resistances to ampicillin, streptomycin, tetracycline and trimethoprim–sulfamethoxazole the most common phenotype detected. Genes encoding resistance to antimicrobial agents were detected in most of the resistant isolates. Ninety‐three % of the resistant isolates were included in the A or B1 phylogenetic groups, and the virulence gene fimA (alone or in association with papC or aer genes) was detected in 137 of the resistant isolates. Five isolates from pigs belonging to phylogroup B2 and D were resistant to five different antimicrobial agents. CONCLUSION: Our data shows a high percentage of antibiotic resistance in E. coli isolates from food animals, and raises important questions in the potential impact of antibiotic use in animals and the possible transmission of resistant bacteria to humans through the food chain. © 2012 Society of Chemical Industry     

EXPOSURE OF LISTERIA MONOCYTOGENES TO FOOD AND TEMPERATURE ABUSE USING A DIALYSIS TUBING CULTURE METHOD

Foodborne illnesses are often linked to foods that have been contaminated postprocessing and exposed to temperature abuse conditions prior to consumption. Limitations in methods to recover sufficient numbers of a target bacterium from an (inoculated) intentionally contaminated food can hinder genomic/proteomic analysis, and in conducting in vitro assays using the recovered cells. In this study, a dialysis tubing culture technique was developed to facilitate the recovery of high numbers of Listeria monocytogenes exposed to temperature abuse while in association with chocolate milk and frankfurter slurry. The impact of exposure to foods, followed by temperature abuse, on the virulence of L. monocytogenes was investigated using the Caco‐2 cell infection assay. The expression of groEL, associated with stress, was also determined. L. monocytogenes inoculated into brain–heart infusion (BHI) broth (control), chocolate milk or frankfurter slurry was held at 4C for 24 h to simulate short‐term exposure to each food. The L. monocytogenes‐contaminated food was then exposed to a series of temperature shifts to simulate temperature abuse. The final temperature of the abused BHI and chocolate milk was 30C, and the frankfurter slurry was 22C. The stress response gene, groEL, was only induced in the control cells (suspended in BHI) after they had been exposed to temperature abuse conditions. The expression of groEL was also evident in cells exposed to foods and temperature abuse conditions, suggesting that the foods used in this study were a stressful environment for the cells. This study showed that the exposure of L. monocytogenes to chocolate milk or frankfurter slurry, or temperature abuse has no impact on the virulence of L. monocytogenes as demonstrated using the Caco‐2 cell assay. However, the expression of groEL suggests that the foods evaluated can be stressful environments for L. monocytogenes despite its ability to grow or survive in those foods. The dialysis tubing culture technique developed is a simple and highly cost‐effective method for exposure of bacteria to food and the recovery of a large number of cells suitable for additional analysis.     

Bacterial stress response in Listeria monocytogenes: jumping the hurdles imposed by minimal processing

Minimal processing relies on the use of multiple sub-lethal stresses (or processes) to achieve a similar level of microbial control as that traditionally achieved using a single lethal stress. The benefit to the consumer is products which are less obviously processed than a frozen or canned, acidified or heavily salted food item. However, our increasing understanding of how bacteria can adapt to sub-lethal stresses in a manner which can render them less susceptible to additional insults, should be borne in mind when designing safety or extended shelf-life into a minimally processed product. Listeria monocytogenes is a target organism for many minimally processed food manufacturers because of its ability to tolerate adverse conditions such as low Aw and low temperature. In this communication we use L. monocytogenes as a model system to describe some of the consequences of stress adaptation in terms of improved survival in minimally processed foods and, importantly, the consequences in terms of the virulence of the target organism.     

Mechanisms of enterohemorrhagic Escherichia coli spread along the food-chain and precautionary measures

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens implicated in large outbreaks and sporadic cases of bloody diarrhea and the hemolytic uremic syndrome. The main reservoir of EHEC is the intestinal tract of ruminants, in particular cattle. Feces containing these bacteria may act as a source of contamination for the environment and particularly for a variety of foods. E. coli O157:H7 as well as other EHEC-serotypes have been isolated from domestic ruminants and non-ruminant farm animals as well as products produced from them, but also from drinking water, vegetables and dairy products. The main transmission pathway of the pathogens is the ingestion of raw or undercooked contaminated food but human infection can also occur by person-to-person transmission. This article will focus on the prevalence and spread of EHEC by vegetable foods, especially the less common EHEC transmission sources sprouts and leafy greens. It will discuss precautionary measures against the spread of EHEC in food at all stages of the food chain: the primary production, the industrial processing, the retailing as well as the consumer.     

The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroup-specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx ₁ and stx ₂ genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx ₁ and stx ₂ genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Escherichia coli biotransformation of daidzein fermentation products from soy‐based foods—relevance to food oestrogenicity‐based functionality

Oestrogenic isoflavones (e.g. daidzein) present in soy‐based foods are likely to be important in food functionality. Biotransformation of daidzein during fermentation‐based soy‐containing food manufacture forms three trihydroxy metabolites, 6‐ortho‐hydroxydaidzein (6‐OHD), 8‐ortho‐hydroxydaidzein (8‐OHD) and 3′‐ortho‐hydroxydaidzein (3′‐OHD) which alters the oestrogenicity of the final food product. We report that Escherichia coli (a key component of the gut microbiome) metabolism converts 8‐OHD to 6‐OHD with a likely concomitant increase in oestrogenicity. This means that the functionality of 8‐OHD‐containing soy‐based foods is altered by gut microbiome metabolism. This change in oestrogenicity‐based food functionality might have benefits for postmenopausal women, while being a feminising health risk for males and could add to the risk of oestrogen‐mediated precocious puberty in girls.     

Shiga‐Toxin Genes and Genetic Diversity of Escherichia coli Isolated from Pasteurized Cow Milk in Brazil

This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty‐seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paraná state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC‐PCR) and repetitive extragenic palindromic sequence PCR (REP‐PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC‐PCR and REP‐PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin‐producing E. coli in milk produced in the region studied.     

Primers for Amplifying an Alanine Racemase Gene Fragment to Detect E. coli Strains in Foods

Specific oligonucleotide primers for detecting Escherichia coli in various foods were designed based upon the conserved sequences of the E. coli air gene from positions 322 to 345 and from 664 to 687. Bacteria and food samples were treated at 100°C for 10 min in 1% Tween 20 containing 5% NaCl and 1 mM EDTA, then used as templates for polymerase chain reaction (PCR). The oligonucleotide primers were specific to E. coli, except for Shigella species, when tested with 67 strains of E. coli, including such serotypes as O157:H7 and O111, and 32 strains of non-E. coli species. The oligonucleotide primers could prove useful for detecting E. coli in beef, chicken, pork, tomato, soybean, potato, cow's milk, and egg.     

Rapid detection of Escherichia coli O157:H7 in milk,bread, and jelly by lac dye coloration‐based bidirectional lateral flow immunoassay strip

Lac dye, a kind of food additive and textile dye, is extracted from lac insect secretions. The ability of lac dye to stain bacteria was firstly found. Based on the discovery, a new bidirectional lateral flow immunoassay strip (BLFIS) for detecting Escherichia coli O157:H7 (E. coli O157:H7) was successfully developed. The BLFIS has two sheets of nitrocellulose (NC) membranes separated by a sample pad. The anti‐E. coli O157:H7 monoclonal antibodies (McAb) were oppositely dispensed on the NC membranes as the control line and test line, respectively. On the side of the control line, a conjugation pad immobilized with pre‐stained E. coli O157:H7 was assembled. The detection limit of BLFIS was 10⁶ CFU/ml. Furthermore, after pre‐incubation in food sample, the detection sensitivity was significantly increased to 100 colonies of the initial bacterial count. This method may be an improvement over traditional colloidal gold lateral flow immunoassay strips, which does not require the pairing of two antibodies to complete the detection, nor the chromogenic nanomaterials to report the detection results, such as gold nanoparticles. And the BLFIS might provide technical support for detecting E. coli O157:H7 in food and ensuring food safety.     

Characterization and Survival of Environmental Escherichia coli O26 Isolates in Ground Beef and Environmental Samples

In addition to Escherichia coli O157:H7, shiga toxin‐producing E. coli (STEC) O26 was added to the zero‐tolerance adulterant list together with other 5 non‐O157 STEC serogroups in 2012. Four farm O26 isolates were used in this study; they were obtained from a on‐farm survey study conducted in Alabama. The presence of 3 major pathogenic genes (stx1, stx2, and eaeA) was determined through multiplex polymerase chain reaction (PCR). Two major pathogenic gene profiles were observed: 3 of the farm isolates contain only the eaeA gene whereas 1 farm isolate has both the eaeA and the stx1 genes. No significant difference was seen among the 4 farm isolates in the antibiotic resistance tests. To test their survival in ground beef and environmental samples, 2 inoculums were prepared and inoculated at various concentrations into samples of ground beef, bovine feces, bedding materials, and trough water. One inoculum was made of 3 farm isolates containing only the eaeA gene and another inoculum contained the isolate with both the eaeA and stx1 genes. Inoculated beef samples were stored at 4 °C for 10 d and the inoculated environmental samples were stored at ambient temperature for 30 d. Results showed that virulence gene profiles do not have an impact on O26's ability to survive in ground beef and in environment (P > 0.05). The inoculation levels, sample types as well as the storage times are the major factors that impact O26 survival (P < 0.05).