Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Sample Type and Processing Plant Differences in the Proportion of Enterohemorrhagic Escherichia coli O157 and Non-O157 Serogroups in Feces and on Hides of Cull Dairy Cattle at Slaughter.

The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific (rfbE), Shiga toxin (stx1 and/or stx2), and intimin (eae) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx1 and/or stx2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Regulation Mechanisms of Virulence Genes in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common E. coli pathotypes reported to cause several outbreaks of foodborne illnesses. EHEC is a zoonotic pathogen, and ruminants, especially cattle, are considered important reservoirs for the most common EHEC serotype, E. coli O157:H7. Humans are infected indirectly through the consumption of food (milk, meat, leafy vegetables, and fruits) and water contaminated by animal feces or direct contact with carrier animals or humans. E. coli O157:H7 is one of the most frequently reported causes of foodborne illnesses in developed countries. It employs two essential virulence mechanisms to trigger damage to the host. These are the development of attaching and effacing (AE) phenotypes on the intestinal mucosa of the host and the production of Shiga toxin (Stx) that causes hemorrhagic colitis and hemolytic uremic syndrome. The AE phenotype is controlled by the pathogenicity island, the locus of enterocyte effacement (LEE). The induction of both AE and Stx is under strict and highly complex regulatory mechanisms. Thus, a good understanding of these mechanisms, major proteins expressed, and environmental cues involved in the regulation of the expression of the virulence genes is vital to finding a method to control the colonization of reservoir hosts, especially cattle, and disease development in humans. This review is a concise account of the current state of knowledge of virulence gene regulation in the LEE-positive EHEC.

Bile Salts Differentially Enhance Resistance of Enterohemorrhagic Escherichia coli O157:H7 to Host Defense Peptides.

During passage through the human gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) is exposed to membrane-damaging bile in the small intestine. We previously reported that EHEC treatment with a physiological bile salt mixture upregulates basRS, encoding a two-component system, and arnBCADTEF, encoding the aminoarabinose lipid A modification pathway (J. V. Kus, A. Gebremedhin, V. Dang, S. L. Tran, A. Serbanescu, and D. Barnett Foster, J Bacteriol 193: 4509-4515, 2011, https://doi.org/10.1128/JB.00200-11). The present study examined the effect of bile salt mix (BSM) treatment on EHEC resistance to three human gastrointestinal defense peptides-HD-5, HNP-1, and LL-37-as well as the role of basRS and arnT in the respective responses. After BSM treatment, EHEC resistance to HD-5 and HNP-1 was significantly increased in a BSM-, defensin dose-dependent manner. The resistance phenotype was dependent on both basRS and arnT However, the BSM treatment did not alter EHEC resistance to LL-37, even when the ompT gene, encoding an LL-37 cleavage protease, was disrupted. Interestingly, enteropathogenic E. coli, a related pathogen that infects the small intestine, showed a similar BSM-induced resistance phenotype. Using a model of EHEC infection in Galleria mellonella, we found significantly lower survival rates in wax moth larvae infected with BSM-treated wild-type EHEC than in those infected with a BSM-treated basS mutant, suggesting that treatment with a physiological BSM enhances virulence through a basS-mediated pathway. The results of this investigation provide persuasive evidence that bile salts typically encountered during transit through the small intestine can serve as an environmental cue for EHEC, enhancing resistance to several key host defense peptides.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.

Effects of antimicrobials on Shiga toxin production in high-virulent Shiga toxin-producing Escherichia coli.

PURPOSE: Antimicrobial treatment of Shiga toxin-producing Escherichia coli (STEC) infections is controversial because antimicrobials may stimulate Shiga toxin (Stx) production, and thereby increase the risk of developing haemolytic uremic syndrome (HUS). Previous in vitro studies have shown this mainly in infections caused by STEC serotype O157:H7. The aim of this study was to investigate induction of Stx transcription and production in different serotypes of STEC isolated from severely ill patients, following their exposure in vitro to six different classes of antimicrobials. METHODS: We investigated Stx transcription and production in 12 high-virulent STEC strains, all carrying the stx2a gene, of six different serotypes following their exposure to six classes of antimicrobials. Liquid cultures of the STEC strains were incubated with sub-inhibitory concentrations of the antimicrobials. We used reverse-transcription quantitative PCR to measure the relative expression of Stx2a mRNA and an enzyme-linked immunosorbent assay to quantify Stx production. RESULTS: In general the antibiotics tested showed only minor effects on transcriptional levels of Stx2a. Ciprofloxacin caused an increase of Stx production in all but two strains, while gentamicin, meropenem and azithromycin did not induce Stx production in any of the STEC strains examined. STEC O104:H4 was the serotype that in greatest extent responded to antimicrobial exposure with an increase of stx2a transcription and Stx production. CONCLUSION: Gentamicin, meropenem and azithromycin exposure did not result in elevated Stx production. We recommend that this finding is investigated further in the search for candidates for future antimicrobial treatment of STEC.

Redox, amino acid, and fatty acid metabolism intersect with bacterial virulence in the gut.

The gut metabolic landscape is complex and is influenced by the microbiota, host physiology, and enteric pathogens. Pathogens have to exquisitely monitor the biogeography of the gastrointestinal tract to find a suitable niche for colonization. To dissect the important metabolic pathways that influence virulence of enterohemorrhagic Escherichia coli (EHEC), we conducted a high-throughput screen. We generated a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. This unraveled that the cysteine-responsive regulator, CutR, converges with the YhaO serine import pump and the fatty acid metabolism regulator FadR to optimally control virulence expression in EHEC. CutR activates expression of YhaO to increase activity of the YhaJ transcription factor that has been previously shown to directly activate the EHEC virulence genes. CutR enhances FadL, which is a pump for fatty acids that represses inhibition of virulence expression by FadR, unmasking a feedback mechanism responsive to metabolite fluctuations. Moreover, CutR and FadR also augment murine infection by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. This high-throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire toward successful infection of the host.

Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing.

Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials.IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest.IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

Seasonal Prevalence of Shiga Toxin-Producing Escherichia coli on Pork Carcasses for Three Steps of the Harvest Process at Two Commercial Processing Plants in the United States.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that has a significant impact on public health, with strains possessing the attachment factor intimin referred to as enterohemorrhagic E. coli (EHEC) and associated with life-threatening illnesses. Cattle and beef are considered typical sources of STEC, but their presence in pork products is a growing concern. Therefore, carcasses (n = 1,536) at two U.S. pork processors were sampled once per season at three stages of harvest (poststunning skins, postscald carcasses, and chilled carcasses) and then examined using PCR for Shiga toxin genes (stx), intimin genes (eae), aerobic plate count (APC), and Enterobacteriaceae counts (EBC). The prevalence of stx on skins, postscald, and chilled carcasses was 85.3, 17.5, and 5.4%, respectively, with 82.3, 7.8, and 1.7% of swabs, respectively, having stx and eae present. All stx-positive samples were subjected to culture isolation that resulted in 368 STEC and 46 EHEC isolates. The most frequently identified STEC were serogroups O121, O8, and O91 (63, 6.7, and 6.0% of total STEC, respectively). The most frequently isolated EHEC was serotype O157:H7 (63% of total EHEC). Results showed that scalding significantly reduced (P < 0.05) carcass APC and EBC by 3.00- and 2.50-log10 CFU/100 cm2, respectively. A seasonal effect was observed, with STEC prevalence lower (P < 0.05) in winter. The data from this study show significant (P < 0.05) reduction in the incidence of STEC (stx) from 85.3% to 5.4% and of EHEC (stx plus eae) from 82.3% to 1.7% within the slaughter-to-chilling continuum, respectively, and that potential EHEC can be confirmed present throughout using culture isolation.IMPORTANCE Seven serogroups of STEC are responsible for most (>75%) cases of severe illnesses caused by STEC and are considered adulterants of beef. However, some STEC outbreaks have been attributed to pork products, although the same E. coli are not considered adulterants in pork because little is known of their prevalence along the pork chain. The significance of the work presented here is that it identifies disease-causing STEC, EHEC, demonstrating that these same organisms are a food safety hazard in pork as well as beef. The results show that most STEC isolated from pork are not likely to cause severe disease in humans and that processes used in pork harvest, such as scalding, offer a significant control point to reduce contamination. The results will assist the pork processing industry and regulatory agencies to optimize interventions to improve the safety of pork products.

Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the "neck" and "beak" regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

After the Fact(or): Posttranscriptional Gene Regulation in Enterohemorrhagic Escherichia coli O157:H7.

To adapt to ever-changing environments, pathogens quickly alter gene expression. This can occur through transcriptional, posttranscriptional, or posttranslational regulation. Historically, transcriptional regulation has been thoroughly studied to understand pathogen niche adaptation, whereas posttranscriptional and posttranslational gene regulation has only relatively recently been appreciated to play a central role in bacterial pathogenesis. Posttranscriptional regulation may involve chaperones, nucleases, and/or noncoding small RNAs (sRNAs) and typically controls gene expression by altering the stability and/or translation of the target mRNA. In this review, we highlight the global importance of posttranscriptional regulation to enterohemorrhagic Escherichia coli (EHEC) gene expression and discuss specific mechanisms of how EHEC regulates expression of virulence factors critical to host colonization and disease progression. The low infectious dose of this intestinal pathogen suggests that EHEC is particularly well adapted to respond to the host environment.

Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

Long-Term Survival and Thermal Death Kinetics of Enterohemorrhagic Escherichia coli Serogroups O26, O103, O111, and O157 in Wheat Flour.

Wheat flour has been associated with outbreaks of enterohemorrhagic Escherichia coli (EHEC), but little is known on EHEC's survival during storage and thermal processing. The objective of this study was to determine long-term viability and thermal inactivation kinetics of EHEC serogroups O26, O103, O111, and O157. Wheat flour samples were inoculated with a cocktail of five strains of a single serogroup and stored at 23 and 35°C. Inoculated samples were heated at 55, 60, 65, and 70°C. Viability was determined by plate counting. Decimal reduction time (D) and first decimal reduction time (δ) values were calculated with log-linear and Weibull models, respectively. At 23°C, EHEC counts declined gradually for 84 days and samples tested positive from 84 to 280 days. The thermal resistance (D and δ) values ranged from 7.5 to 8.2 and 3.1 to 5.3 days, respectively, but there were no significant differences among serogroups (P ≤ 0.05). At 35°C, no EHEC was quantifiable by day 7 and no positive samples were detected after 49 days. Heating at 55 and 65°C resulted in δ-value ranges of 15.6 to 39.7 min and 3.0 to 3.9 min, respectively, with no significant difference among serogroups either. Z values were 12.6, 6.7, 10.2, and 13.4°C for O26, O103, O111, and O157, respectively. Thermal death kinetics of EHEC in flour were better described using the Weibull model. Survival and inactivation rates of four serogroups were remarkably similar. These findings indicated that all EHEC serovars tested remained viable for at least 9 months at room temperature and survived for up to 60 min at 70°C in wheat flour.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella have recently caused several gastroenteritis outbreaks and recalls of wheat flour. Because EHEC can cause illness with very low doses and there is very scarce information regarding their ability to survive storage and heating in flour, the present study was undertaken to assess the long-term survival of EHEC serogroups O26, O103, O111, and O157 in flour. These findings are relevant, as we report that EHEC can survive for more than 9 months in wheat flour during storage. In addition, results obtained suggest that thermal inactivation at 65°C for 30 min or 2 months of storage at 35°C may be feasible strategies to mitigate the risk of most EHEC serovars in wheat flour.

Adherence factors of enterohemorrhagic Escherichia coli O157:H7 strain Sakai influence its uptake into the roots of Valerianella locusta grown in soil.

Increasing numbers of outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) are associated with the consumption of contaminated fresh produce. The contamination of the plants may occur directly on the field via irrigation water, surface water, manure or fecal contamination. Suggesting a low infectious dose of 10 to 102 cells, internalization of EHEC into plant tissue presents a serious public health threat. Therefore, the ability of EHEC O157:H7 strain Sakai to adhere to and internalize into root tissues of the lamb's lettuce Valerianella locusta was investigated under the environmental conditions of a greenhouse. Moreover, the influence of the two adherence and colonization associated genes hcpA and iha was surveyed regarding their role for attachment and invasion. Upon soil contamination, the number of root-internalized cells of EHEC O157:H7 strain Sakai exceeded 102 cfu/g roots. Deletion of one or both of the adherence factor genes did not alter the overall attachment of EHEC O157:H7 strain Sakai to the roots, but significantly reduced the numbers of internalized bacteria by a factor of between 10 and 30, indicating their importance for invasion of EHEC O157:H7 strain Sakai into plant roots. This study identified intrinsic bacterial factors that play a crucial role during the internalization of EHEC O157:H7 strain Sakai into the roots of Valerianella locusta grown under the growth conditions in a greenhouse.

Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle.

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥104 colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 102 to 3.0 × 105 CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx1 but positive for stx2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

Genetic and Mechanistic Analyses of the Periplasmic Domain of the Enterohemorrhagic Escherichia coli QseC Histidine Sensor Kinase.

The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates.IMPORTANCE In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.

Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7.

Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli Our data support the existing recommendations to avoid antibiotics for treatment of these infections.