Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice.

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.

Biofilm formation by Non-O157 Shiga toxin-producing Escherichia coli in monocultures and co-cultures with meat processing surface bacteria.

This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by non-O157 Shiga toxin-producing Escherichia coli (STEC), and potential links between biofilm formation by STEC and biofilm-related genes in their genomes. Biofilm development by 50 MPB and 6 STEC strains in mono- and co-cultures was assessed by the crystal violet staining method, and their expression of curli and cellulose was determined using the Congo red agar method. Genes (n = 141) associated with biofilm formation in the STEC strains were profiled. Biofilm formation in general correlated with cellulose and curli expression in both mono- and co-cultures. Most MPB strains had antagonistic effects on the biofilm formation of the STEC strains. Of the genes investigated, 81% were common among the STEC strains and there seems to be a gene-redundancy in biofilm formation. The inability of the O26 strain to form biofilms could be due to mutations in the rpoS gene. Truncation in the mlrA gene in the O145 strain seems not affecting its biofilm formation alone or with MPB. The O45 strain, despite having the greatest number of biofilm-related genes, did not form measurable biofilms. Overall, biofilm formation of STEC was affected by curli-cellulose expression and companion strains.

Ruminant-Related Risk Factors are Associated with Shiga Toxin-Producing Escherichia coli Infection in Children in Southern Ghana.

Livestock can provide benefits to low-income households, yet may expose children to zoonotic enteropathogens that cause illness and negative long-term health outcomes. The aim of this cross-sectional study was to determine whether livestock-related risk factors, including animal ownership, exposure to animal feces, and consumption of animal-source foods, were associated with bacterial zoonotic enteropathogen infections in children 6-59 months old in Greater Accra, Ghana. Stool samples from 259 children and 156 household chickens were analyzed for atypical enteropathogenic Escherichia coli (aEPEC), Campylobacter jejuni/coli (C. jejuni/coli), Salmonella, and Shiga toxin-producing Escherichia coli (STEC) using quantitative polymerase chain reaction (qPCR). aEPEC, C. jejuni/coli, STEC, and Salmonella were detected in 45.6%, 11.6%, 4.3%, and 0.8% of children's stool samples, respectively. In adjusted logistic regression models, household ownership of goats or sheep was associated with STEC detection in children (odds ratio [95% confidence interval]: 4.30 [1.32, 14.08]), as were positive detection of STEC in chicken feces (7.85 [2.54, 24.30]) and frequent consumption of fresh cow's milk (3.03 [1.75, 5.24]). No livestock-related risk factors were associated with aEPEC or C. jejuni/coli infection in children. Our findings suggest that ruminant ownership in southern Ghana may expose children to STEC through household fecal contamination and foodborne routes. The lack of association between livestock risk factors and the more commonly detected pathogens, aEPEC and C. jejuni/coli, warrants further research, particularly to help explain how animal-keeping and sanitation practices affect transmission of fecal pathogens that were highly prevalent in chicken feces.

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.

Changes in STEC and bacterial communities during enrichment of manufacturing beef in selective and non-selective media.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from manufacturing beef is challenging and it may be affected by microbial changes during enrichment. This study was designed to understand population changes during enrichment of beef from an integrated (Samples A and B) and a fragmented (Samples C and D) abattoir. The samples were enriched in buffered peptone water (BPW), Assurance GDS MPX top 7 STEC mEHEC®, BAX® E. coli O157:H7 MP and PDX-STEC media then were processed for 16 S rRNA sequencing. Escherichia dominated Sample B enrichment broths regardless of the media used (71.6-97.9%) but only in mEHEC broth (79.6%) of Sample A. Escherichia was dominant in Sample C in mEHEC (95.2%) and PDX-STEC (99.2%) broths but less in BPW (58.5%) and MP (64.9%) broths. In Sample D, Clostridium dominated in mEHEC (65.5%), MP (80.2%) and PDX-STEC (90.6%) broths. O157 STEC was isolated from Sample C only. The study suggested that MP may not be as effective as mEHEC and PDX-STEC and that Clostridium could interfere with enrichment of Escherichia. Understanding the ecological changes during enrichment provides meaningful insight to optimising the enrichment protocol for STEC and subsequently enhance the efficiency of STEC detection.

Modeling the effect of simultaneous use of allyl isothiocyanate and cinnamaldehyde on high hydrostatic pressure inactivation of Uropathogenic and Shiga toxin-producing Escherichia coli in ground chicken.

BACKGROUND: A combination of high-pressure processing (HPP) and antimicrobials is a well-known approach for enhancing the microbiological safety of foods. However, few studies have applied multiple antimicrobials simultaneously with HPP, which could be an additional hurdle for microbial inactivation. The present study applied a full factorial design to investigate the impact of HPP (225-325 MPa; 10-20 min), allyl isothiocyanate (AITC) (0.3-0.9 g kg-1 ) and trans-cinnamaldehyde (tCinn) (1.0-2.0 g kg-1 ) on the inactivation of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and uropathogenic E. coli (UPEC) in ground chicken meat. RESULTS: The regulatory requirement of 5-log reduction was achieved at 305 MPa, 18 min, 0.8 g kg-1 AITC and 1.7 g kg-1 tCinn for STEC O157:H7 and at 293 MPa, 16 min, 0.6 g kg-1 AITC and 1.6 g kg-1 tCinn for UPEC, as specified by response surface analysis and verified via experiments. The surviving population was eliminated by post-treatment storage of 9 days at 10 °C. The developed linear regression models showed r2 > 0.9 for the E. coli inactivation. The developed dimensionless non-linear regression models covered a factorial range slightly wider than the original experimental limit, with probability Pr > F (< 0.0001). CONCLUSION: Simultaneous use of AITC and tCinn reduced not only the necessary concentration of each compound, but also the intensity of high-pressure treatments, at the same time achieving a similar level of microbial inactivation. STEC O157:H7 was found to be more resistant than UPEC to the HPP-AITC-tCinn stress. The developed models may be applied in commercial application to enhance the microbiological safety of ground chicken meat. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Label-Free Immunoassay for Multiplex Detections of Foodborne Bacteria in Chicken Carcass Rinse with Surface Plasmon Resonance Imaging.

The frequent outbreaks of foodborne pathogens have stimulated the demand of biosensors capable of rapid and multiplex detection of contaminated food. In this study, surface plasmon resonance imaging (SPRi) was used in simultaneous label-free detection of multiple foodborne pathogens, mainly Salmonella spp. and Shiga-toxin producing Escherichia coli (STEC), in commercial chicken carcass rinse. The antibodies were immobilized on the same SPRi sensor chip as a label-free immunoassay. Their immobilization concentrations were optimized to be ranging from 0.25 to 1.0 mg/mL, and independent of pH values. This label-free immunoassay achieved 106 colony-forming unit (CFU)/mL limit of detection for Salmonella, which was further improved to 1.0 CFU/mL with overnight bacteria enrichment. The injected samples with different bacteria, Salmonella Enteritidis, STEC, and Listeria monocytogenes, have been identified by the same biochip. Moreover, the SPRi signals revealed complex interference effects among coexisting bacteria species in heterogeneous bacteria solutions. This SPRi-based immunoassay demonstrates the great potential in high-throughput screening of multiple pathogenic bacteria coexisting in chicken carcass rinse. The reliability of antibody immobilization and cross-reactions of different antibodies on the same biochip are the major challenges of practical application of SPRi.

Characterization of Shiga toxin-producing Escherichia coli in raw beef from informal and commercial abattoirs.

Shiga toxin-producing Escherichia coli are foodborne pathogens that are mostly associated with beef products and have been implicated in human illness. E.coli-associated illness range from asymptomatic conditions of mild diarrhoea to haemorrhagic colitis which can progress into life threatening haemolytic uremic syndrome (HUS). Beef from cattle are regarded as the main reservoir of Shiga toxin-producing E. coli (STEC) pathogen. The aim of this study was to assess the level and sources of contamination of raw beef with STEC, and determine the incidences of STEC strains in raw beef from informal and commercial abattoirs in Windhoek, Namibia. A total of 204 raw beef samples, 37 equipment and 29 hand swabs were collected and tested for STEC. The meat samples were first enriched with pre-warmed buffered peptone water, cultured on Tryptone Bile X-Glucuronide and CHROMagar STEC, and then sub-cultured on nutrient agar. The presence of E.coli in the samples was confirmed by using VITEK 2 E.coli identification cards and PCR. The overall prevalence of STEC in the meat samples from both the abattoirs was 41.66% raw beef samples; 5.40% equipment swabs; and none of the hand swabs was STEC positive. From the STEC positive meat samples 29.41% contained one of the major STEC strains. Moreover, 52% of the 25 samples that contained the major STECs were characterised by eae and stx1, 8% characterised by eae and stx2 while 40% were characterised by eae, stx1 and stx2 virulence genes. This study has revealed the necessity for proper training on meat safety (for meat handlers) as well as the development, implementation and maintenance of effective sanitary dressing procedures at abattoirs to eliminate beef contamination by STECs thereby ensuring the production of wholesome meat, and to prevent the occurrences of STEC infections.

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells.

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae+ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae+ STEC.

Bacteriophage biocontrol of Shiga toxigenic Escherichia coli (STEC) O145 biofilms on stainless steel reduces the contamination of beef.

Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P < 0.01) of planktonic cells grown in M9 broth at 24 °C for 24 h, while reductions were 2.0 log10 CFU/mL if these cells were grown for 48 h or 72 h prior to phage treatment. STEC O145 biofilms formed on SS coupons for 24, 48 and 72 h were reduced (P < 0.01) 2.9, 1.9 and 1.9 log10 CFU/coupon by phages. STEC O145 cells in biofilms were readily transferred from the surface of the SS coupon to beef (3.6 log10 CFU/coupon) even with as little as 10 s of contact with the meat surface. However, transfer of STEC O145 cells from biofilms that formed on SS coupons for 48 h to beef was reduced (P < 0.01) by 3.1 log10 CFU by phage (2 × 1010 PFU/mL) at 24 °C. Scanning electron microscopy revealed that bacterial cells within indentations on the surface of SS coupons were reduced by phage. These results suggest that bacteriophage AZO145A could be effective in reducing the viability of biofilm-adherent STEC O145 on stainless steel in food industry environments.

Metabolic Traits of Bovine Shiga Toxin-Producing Escherichia Coli (STEC) Strains with Different Colonization Properties.

Cattle harbor Shiga toxin-producing Escherichia coli (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STECper) or only sporadically (STECspo) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STECspo strains produced significantly more biofilm than STECper when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STECspo, but not or only by some STECper. Genomic sequences of the respective glc and rha operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STECper. These findings suggest that STECspo conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.

Viability of Shiga Toxin-Producing Escherichia coli, Salmonella, and Listeria monocytogenes within Plant versus Beef Burgers during Cold Storage and following Pan Frying.

ABSTRACT: The viability of Shiga toxin-producing Escherichia coli (STEC), Salmonella, and Listeria monocytogenes within plant- and beef-based burgers was monitored during storage and cooking. When inoculated (ca. 3.5 log CFU/g) into 15-g portions of plant- or beef-based burgers, levels of STEC and Salmonella decreased slightly (≤0.5-log decrease) in both types of burgers when stored at 4°C, but increased ca. 2.4 and 0.8 log CFU/g, respectively, in plant-based burgers but not beef-based burgers (≤1.2-log decrease), after 21 days at 10°C. For L. monocytogenes, levels increased by ca. 1.3 and 2.6 log CFU/g in plant burgers after 21 days at 4 and 10°C, respectively, whereas pathogen levels decreased slightly (≤0.9-log decrease) in beef burgers during storage at 4 and 10°C. Regarding cooking, burgers (ca. 114 g each) were inoculated with ca. 7.0 log CFU/g STEC, Salmonella, or L. monocytogenes and cooked in a sauté pan. Cooking plant- or beef-based burgers to 62.8°C (145°F), 68.3°C (155°F), or 73.9°C (165°F) delivered reductions ranging from ca. 4.7 to 6.8 log CFU/g for STEC, ca. 4.4 to 7.0 log CFU/g for L. monocytogenes, and ca. 3.5 to 6.7 log CFU/g for Salmonella. In summary, the observation that levels of all three pathogens increased by ca. 1.0 to ca. 2.5 log CFU/g in plant-based burgers when stored at an abusive temperature (10°C) highlights the importance of proper storage (4°C) to lessen risk. However, because all three pathogens responded similarly to heat in plant-based as in beef-based burgers, well-established cooking parameters required to eliminate STEC, Salmonella, or L. monocytogenes from ground beef should be as effective for controlling cells of these same pathogens in a burger made with plant-sourced protein.

Inactivation of Shiga Toxin-Producing Escherichia coli and Listeria monocytogenes within Plant versus Beef Burgers in Response to High Pressure Processing.

ABSTRACT: We evaluated high pressure processing to lower levels of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes inoculated into samples of plant or beef burgers. Multistrain cocktails of STEC and L. monocytogenes were separately inoculated (∼7.0 log CFU/g) into plant burgers or ground beef. Refrigerated (i.e., 4°C) or frozen (i.e., -20°C) samples (25 g each) were subsequently exposed to 350 MPa for up to 9 or 18 min or 600 MPa for up to 4.5 or 12 min. When refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of STEC were reduced by ca. 0.7 to 1.3 log CFU/g. However, when refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of L. monocytogenes remained relatively unchanged (ca. ≤0.3-log CFU/g decrease) in plant burger samples but were reduced by ca. 0.3 to 2.0 log CFU/g in ground beef. When refrigerated plant or beef burger samples were treated at 600 MPa for up to 4.5 min, levels of STEC and L. monocytogenes were reduced by ca. 0.7 to 4.1 and ca. 0.3 to 5.6 log CFU/g, respectively. Similarly, when frozen plant and beef burger samples were treated at 350 MPa up to 18 min, reductions of ca. 1.7 to 3.6 and ca. 0.6 to 3.6 log CFU/g in STEC and L. monocytogenes numbers, respectively, were observed. Exposure of frozen plant or beef burger samples to 600 MPa for up to 12 min resulted in reductions of ca. 2.4 to 4.4 and ca. 1.8 to 3.4 log CFU/g in levels of STEC and L. monocytogenes, respectively. Via empirical observation, pressurization did not adversely affect the color of plant burger samples, whereas appreciable changes in color were observed in pressurized ground beef. These data confirm that time and pressure levels already validated for control of STEC and L. monocytogenes in ground beef will likely be equally effective toward these same pathogens in plant burgers without causing untoward effects on product color.

Biofilm formation by South African non-O157 Shiga toxigenic Escherichia coli on stainless steel coupons.

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air-liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air-liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.

Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures.

A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P < 0.05) at a lower multiplicity of infection and sampling time (6-10 h), when STEC O157:H7 was also included in the culture. This phage was able to simultaneously reduce numbers of STEC and Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

Application of chlorine dioxide and peroxyacetic acid during spray chilling as a potential antimicrobial intervention for beef carcasses.

Enteric pathogens such as Shiga-toxin producing Escherichia coli (STEC) and Salmonella spp. continue to be a major food safety concern for the beef industry. Currently, no single method is completely effective in controlling these pathogens during carcass processing. Previous research, however, suggested that STEC might become more susceptible to oxidative damage when exposed to carcass chilling (King et al., 2016). We aimed to test that hypothesis by evaluating the antimicrobial effects of an oxidant (chlorine dioxide, ClO2 or peroxyacetic acid, PAA) on beef meat during a simulated spray chilling process (sprayed for 4 s every 15 min for 36 cycles) and/or when applied (sprayed for 144 s) prior to spray chilling with water. In all experiments, the inactivating effects of oxidants were greatest on fat surfaces and much less effective on lean surfaces. ClO2 at 15 ppm, a non-lethal level for E. coli under optimal growth conditions, caused higher log reductions in E. coli numbers (approximately 3-log reduction) when applied during spray chilling than when applied immediately prior to 'normal' spray chilling (approximately 1-log reduction). This confirms the hypothesis that E. coli are more susceptible to oxidative stress during spray chilling. In subsequent studies, both ClO2 and PAA at lethal levels (at ≥20 and ≥ 200 ppm, respectively) applied during spray chilling resulted in pronounced inactivation of both E. coli and Salmonella enterica strains, achieving a ≥4-log reduction at the end of chilling. These results indicate that an oxidant-based application during spray chilling as an antimicrobial intervention could be effective to minimise the problems associated with enteric pathogen contamination on beef meat.

Shiga toxin-producing Escherichia coli survives storage in wheat flour for two years.

Wheat flour has recently been recognised as an exposure vehicle for the foodborne pathogen Shiga toxin-producing Escherichia coli (STEC). Wheat flour milled on two sequential production days in October 2016, and implicated in a Canada wide outbreak of STEC O121:H19, was analysed for the presence of STEC in November 2018. Stored in sealed containers at ambient temperature, the water activity of individual flour samples was below 0.5 at 6 months post-milling and remained static or decreased slightly in individual samples during 18 months of additional storage. STEC O121 was isolated, with the same genotype (stx2a, eae, hlyA) and core genome multilocus sequence type as previous flour and clinical isolates associated with the outbreak. The result of this analysis demonstrates the potential for STEC to persist in wheat flour at levels associated with outbreak infections for periods of up to two years. This has implications for the potential for STEC to survive in other foods with low water activity.

Escherichia coli O157:H7 Stationary-Phase Acid Resistance and Assessment of Survival in a Model Vegetable Fermentation System.

ABSTRACT: Escherichia coli O157:H7 (STEC) acid resistance may aid the pathogen's ability to cross the human gastric barrier, which makes it an organism of concern in acidic foods. Our objective was to determine how STEC acid resistance may correlate with survival during vegetable fermentations. Seven E. coli O157:H7 strains were screened to assess acid resistance in simulated stomach acid at pH 2. The strains were separated into two groups that differed in acid resistance (P < 0.05), with three being acid sensitive and four acid resistant. The growth rates of these strains were measured in a Luria broth at pH values from 4.2 to 6.8. Two strains having similar growth kinetics, B201 (acid sensitive) and B241 (acid resistant), were selected for further analysis. B201 was found to be missing (compared with B241) two glutamic acid decarboxylase regulatory genes required for acid resistance, gadE and gadX. These strains were challenged in lactic acid (100 mM) solutions, including cucumber juice (CJ) media at pH 3.3. As expected, B201 was more acid sensitive than B241, and a filtered fermented CJ was more inhibitory than similarly acidified CJ. In competitive growth studies with Lactobacillus plantarum LA445 in CJ, B201 or B241 grew from approximately 104 to 108 CFU/mL within 24 h, but the STEC strains were below the limit of detection by 48 h. In all fermentations, L. plantarum reached 108 CFU/mL by 48 h. However, in three of four independent fermentation experiments, strain B201 survived longer than B241. This was possibly due to buffering in B241-LA445 fermentation brines that had increased lactic acid for a given pH compared with B201-LA445. These data indicate that stationary-phase acid resistance may not accurately predict STEC survival during vegetable fermentations.

Enhanced formation of shiga toxin-producing Escherichia coli persister variants in environments relevant to leafy greens production.

Bacterial persistence is a form of phenotypic heterogeneity in which a subpopulation, persisters, has high tolerance to antibiotics and other stresses. Persisters of enteric pathogens may represent the subpopulations capable of surviving harsh environments and causing human infections. Here we examined the persister populations of several shiga toxin-producing Escherichia coli (STEC) outbreak strains under conditions relevant to leafy greens production. The persister fraction of STEC in exponential-phase of culture varied greatly among the strains examined, ranging from 0.00003% to 0.0002% for O157:H7 strains to 0.06% and 0.08% for STEC O104:H4 strains. A much larger persister fraction (0.1-11.2%) was observed in STEC stationary cells grown in rich medium, which was comparable to the persister fractions in stationary cells grown in spinach lysates (0.6-3.6%). The highest persister fraction was measured in populations of cells incubated in field water (9.9-23.2%), in which no growth was detected for any of the STEC strains examined. Considering the high tolerance of persister cells to antimicrobial treatments and their ability to revert to normal cells, the presence of STEC persister cells in leafy greens production environments may pose a significant challenge in the development of effective control strategies to ensure the microbial safety of fresh vegetables.

Analysis of individual patient data to describe the incubation period distribution of Shiga-toxin producing Escherichia coli.

Shiga-toxin producing Escherichia coli (STEC) is a pathogen that can cause bloody diarrhoea and severe complications. Cases occur sporadically but outbreaks are also common. Understanding the incubation period distribution and factors influencing it will help in the investigation of exposures and consequent disease control. We extracted individual patient data for STEC cases associated with outbreaks with a known source of exposure in England and Wales. The incubation period was derived and cases were described according to patient and outbreak characteristics. We tested for heterogeneity in reported incubation period between outbreaks and described the pattern of heterogeneity. We employed a multi-level regression model to examine the relationship between patient characteristics such as age, gender and reported symptoms; and outbreak characteristics such as mode of transmission with the incubation period. A total of 205 cases from 41 outbreaks were included in the study, of which 64 cases (31%) were from a single outbreak. The median incubation period was 4 days. Cases reporting bloody diarrhoea reported shorter incubation periods compared with cases without bloody diarrhoea, and likewise, cases aged between 40 and 59 years reported shorter incubation period compared with other age groups. It is recommended that public health officials consider the characteristics of cases involved in an outbreak in order to inform the outbreak investigation and the period of exposure to be investigated.