Enterohemorrhagic Escherichia Coli Cells Research Articles

Bioprotective Lactic Acid Bacteria and Lactic Acid as a Sustainable Strategy to Combat Escherichia coli O157:H7 in Meat.

Human infection by Enterohemorrhagic Escherichia coli (EHEC) constitutes a serious threat to public health and a major concern for the meat industry. Presently, consumers require safer/healthier foods with minimal chemical additives, highlighting the need for sustainable solutions to limit and prevent risks. This work evaluated the ability of two antagonistic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum CRL681 and Enterococcus mundtii CRL35, and their combination in order to inhibit EHEC in beef (ground and vacuum sealed meat discs) at 8 °C during 72 h. The effect of lower lactic acid (LA) concentrations was evaluated. Meat color was studied along with how LAB strains interfere with the adhesion of Escherichia coli to meat. The results indicated a bacteriostatic effect on EHEC cells when mixed LAB strains were inoculated. However, a bactericidal action due to a synergism between 0.6% LA and LAB occurred, producing undetectable pathogenic cells at 72 h. Color parameters (a*, b* and L*) did not vary in bioprotected meat discs, but they were significantly modified in ground meat after 24 h. In addition, LAB strains hindered EHEC adhesion to meat. The use of both LAB strains plus 0.6% LA, represents a novel, effective and ecofriendly strategy to inactivate EHEC in meat.

Possible solution of capturing viable Enterohemorrhagic Escherichia coli (EHEC) in clinical patient stool.

For clinical diagnosis of enterohemorrhagic Escherichia coli (EHEC）, it needs to capture viable EHEC cells from stool sample in the view of medical fee points. However, there is no comprehensive solution for the detection of viable EHEC cells since there are wide variety of serotype and susceptibility against potassium tellurite which is commonly used for selective agent in selective medium for EHEC. In these background, EHEC Clear-HT System (EHEC-CHT）, a novel effective chromogenic medium system for screening comprehensive viable EHEC, was developed. When EHEC-CHT was assessed using 128 microbes including 49 clinical isolated EHEC strains, EHEC-CHT detected all 49 EHEC strains as typical blue-colored colony regardless of both serotype and susceptibility to potassium tellurite. EHEC-CHT was compared with Japanese commercially available tellurite-based EHEC selective media using 107 clinical patient stool samples. EHEC-CHT showed higher detection ratio than conventional tellurite-based selective media compared, and 7% improvement at least in detection ratio in this study.

An Engineered λ Phage Enables Enhanced and Strain-Specific Killing of Enterohemorrhagic Escherichia coli.

ABSTRACTBacteriophages (phages) are ideal alternatives to traditional antimicrobial agents in a world where antimicrobial resistance (AMR) is emerging and spreading at an unprecedented speed. In addition, due to their narrow host ranges, phages are also ideal tools to modulate the gut microbiota in which alterations of specific bacterial strains underlie human diseases, while dysbiosis caused by broad-spectrum antibiotics can be harmful. Here, we engineered a lambda phage (Eλ) to target enterohemorrhagic Escherichia coli (EHEC) that causes a severe, sometimes lethal intestinal infection in humans. We enhanced the killing ability of the Eλ phage by incorporating a CRISPR-Cas3 system into the wild-type λ (wtλ) and the specificity by introducing multiple EHEC-targeting CRISPR spacers while knocking out the lytic gene cro. In vitro experiments showed that the Eλ suppressed the growth of EHEC up to 18 h compared with only 6 h with the wtλ; at the multiplicity of infection (MOI) of 10, the Eλ killed the EHEC cells with ~100% efficiency and did not affect the growth of other laboratory- and human-gut isolated E. coli strains. In addition, the EHEC cells did not develop resistance to the Eλ. Mouse experiments further confirmed the enhanced and strain-specific killing of the Eλ to EHEC, while the overall mouse gut microbiota was not disturbed. Our methods can be used to target other genes that are responsible for antibiotic resistance genes and/or human toxins, engineer other phages, and support in vivo application of the engineered phages.IMPORTANCE Pathogenic strains of Escherichia coli are responsible for 0.8 million deaths per year and together ranked the first among all pathogenic species. Here, we obtained, for the first time, an engineered phage, Eλ, that could specifically and efficiently eliminate EHEC, one of the most common and often lethal pathogens that can spread from person to person. We verified the superior performance of the Eλ over the wild-type phage with in vitro and in vivo experiments and showed that the Eλ could suppress EHEC growth to nondetectable levels, fully rescue the EHEC-infected mice, and rescore disturbed mouse gut microbiota. Our results also indicated that the EHEC did not develop resistance to the Eλ, which has been the biggest challenge in phage therapy. We believe our methods can be used to target other pathogenic strains of E. coli and support in vivo application of the engineered phages.

Development and Improvement of Methods to Disinfect Raw BeefUsing Calcium Hydroxide-Ethanol-Lactate-Based Food Disinfectant for Safe Consumption.

The enterohemorrhagic Escherichia coli (EHEC) group is responsible for outbreaks and sporadic cases around the world annually. EHEC produces a potent protein known as Shiga toxin in the human intestine causing mild to bloody diarrhea. Some cases of EHEC infections may develop life-threatening symptoms, which may lead to human death. It also has other virulent factors that enable the EHEC cells to adhere to a target tissue and invade to some extent to crave more nutrition and escape the external extreme conditions, such as disinfection treatment. For those reasons, beef is not permitted for raw consumption unless guaranteed free of harmful bacteria, including EHEC, or the invading bacterial cells are completely removed or reduced to a safe level. A heat treatment that guarantees a sufficiently high temperature to reach inside the tissue of meat through the surface was established in Japan. This treatment may allow the core part of the meat to be consumed raw. However, it seemed to have some limitations. We aimed at developing a disinfection method with, hypothetically, nutrition-preserving property that is equivalent to the heat treatment or even superior. A combination of calcium hydroxide–ethanol–lactate-based food disinfectant and two proposed physical sterilization methods, assisted with microbial detection methods, exerted sufficient bactericidal activities against EHEC cells adhering to and/or invading the beef. These physical methods showed great usefulness in disinfecting fresh full-size boneless Round-beef of around 12 kg including fat on the outside. The first method applied a commercially available wide-drum washing machine (WM method) while the second method applied a specially designed plastic bag and a commercially available vibration machine (VV method). After trimming out the fat and the denatured surface of the beef (1 cm from the surface), the remaining meat mass showed no signs of denaturation and a significant reduction of viable EHEC cells by a factor of >104 CFU/ml. However, in the WM method, the disinfection process required a large amount of the disinfectant (150 L). The improved method, VV method, implemented a system that consumes a smaller amount of the disinfectant (50 L) while ensuring the targeted disinfection power degree.

Effect of Lactobacillus acidophilus Fermented Broths Enriched with Eruca sativa Seed Extracts on Intestinal Barrier and Inflammation in a Co-Culture System of an Enterohemorrhagic Escherichia coli and Human Intestinal Cells.

Lactic acid bacteria (LAB) “fermentates” confer a beneficial effect on intestinal function. However, the ability of new fermentations to improve LAB broth activity in preventing pathogen-induced intestinal inflammation and barrier dysfunction has not yet been studied. The objective of this study was to determine if broths of LAB fermented with Eruca sativa or Barbarea verna seed extracts prevent gut barrier dysfunction and interleukin-8 (CXCL8) release in vitro in human intestinal Caco-2 cells infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7. LAB broths were assayed for their effects on EHEC growth and on Caco-2 viability; thereafter, their biological properties were analysed in a co-culture system consisting of EHEC and Caco-2 cells. Caco-2 cells infected with EHEC significantly increased CXCL8 release, and decreased Trans-Epithelial Electrical Resistance (TEER), a barrier-integrity marker. Notably, when Caco-2 cells were treated with LAB broth enriched with E. sativa seed extract and thereafter infected, both CXCL8 expression and epithelial dysfunction reduced compared to in untreated cells. These results underline the beneficial effect of broths from LAB fermented with E. sativa seed extracts in gut barrier and inflammation after EHEC infection and reveal that these LAB broths can be used as functional bioactive compounds to regulate intestinal function.

Fate of Salmonella enterica and Enterohemorrhagic Escherichia coli on Vegetable Seeds Contaminated by Direct Contact with Artificially Inoculated Soil during Germination

Contaminated vegetable seeds have been identified as a potential source of foodborne bacterial pathogens. This study was undertaken to observe the behavior of Salmonella and enterohemorrhagic Escherichia coli (EHEC) on vegetable seeds, contaminated by direct contact with artificially inoculated soil, during germination. Sterile sandy soil inoculated with lyophilized cells of four individual strains of Salmonella or EHEC (three O157:H7 strains and one O104:H4 strain) was mixed with sanitized seeds (2 g) of alfalfa, fenugreek, lettuce, and tomato at 20°C for 1 h. The contaminated seeds were germinated on 1% water agar at 25°C for 9 days in the dark. Populations of Salmonella and EHEC on various tissues (seed coat, root, cotyledon, and stem, etc.) of sprouts and seedlings were determined every other day over the germination period. Overall, 70.4 and 72.4% of collected tissue samples (n = 544) tested positive for Salmonella and EHEC, respectively. In general, the mean populations of Salmonella and EHEC on sprout and seedling tissues increased with the prolongation of germination time. Seed coats had the highest bacterial counts (4.00 to 4.06 log CFU/0.01 g), followed by the root (3.36 to 3.38 log CFU/0.01 g), cotyledon (3.13 to 3.38 log CFU/0.01 g), and stem tissues (2.67 to 2.84 log CFU/0.01 g). On average, tissue sections of fenugreek sprouts and lettuce seedlings had significantly higher (P < 0.05) numbers of Salmonella and EHEC cells than that of alfalfa sprouts and tomato seedlings. Data suggest that the growth and dissemination of Salmonella and EHEC cells on alfalfa, fenugreek, lettuce, and tomato sprout and seedling tissues are influenced by the type of vegetable seeds and sprout and seedling tissues involved. The study provides useful information on the fate of two important foodborne bacterial pathogens on selected vegetable seeds, contaminated by direct contact with inoculated soil, during the germination process.

Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

Synbiotic-like effect of linoleic acid overproducing Lactobacillus casei with berry phenolic extracts against pathogenesis of enterohemorrhagic Escherichia coli

BackgroundMajority of enteric infections are foodborne and antimicrobials including antibiotics have been used for their control and treatment. However, probiotics or prebiotics or their combination offer a potential alternative intervention strategy for improving the host health and preventing foodborne pathogen colonization/infections in reservoir. Further, bioengineered probiotics expressing bioactive products to achieve specific function is highly desirable. Recently, we over-expressed mcra (myosin cross-reactive antigen) gene in Lactobacillus casei (Lc) and developed a bioengineered probiotics Lc + CLA which produce higher amounts of metabolites including conjugated linoleic acid (CLA). Furthermore, we also reported that prebiotic like components such as berry pomace (byproduct) phenolic extracts (BPEs) can enhance the growth of probiotics and improved the beneficial effects of probiotics. In this study, we evaluated the antimicrobial effect of modified Lc + CLA in combination of BPEs on growth, survival and pathogenesis of enterohemorrhagic Escherichia coli (EHEC).ResultsIn mixed culture condition, the growth of EHEC was significantly reduced in the presence Lc + CLA and/or BPEs. Cell-free cultural supernatant (CFCS) collected from Lc or Lc + CLA strain also inhibited the growth and survival of EHEC and the inhibitory effects of CFCSs against EHEC were enhanced in the presence of BPEs in concentration dependent manner. Interaction between EHEC and intestinal epithelial INT-407 cells were also altered significantly in the presence of either Lc or Lc + CLA strain or their CFCSs with or without BPEs. The expression of multiple virulence genes and physicochemical properties of EHEC were also altered when the bacterial cells were pretreated with CFCSs and/or BPEs.ConclusionsThese results showed that diet containing bioactive Lc + CLA and natural prebiotic like component such as BPEs might be an effective way to prevent foodborne infections with EHEC.

Effects of fermentation products of the commensal bacterium Clostridium ramosum on motility, intracellular pH, and flagellar synthesis of enterohemorrhagic Escherichia coli.

The flagellum and motility are crucial virulence factors for many pathogenic bacteria. In general, pathogens invade and translocate through motility and adhere to specific tissue via flagella. Therefore, the motility and flagella of pathogens are effectual targets for attenuation. Here, we show that the fermentation products of Clostridium ramosum, a commensal intestinal bacterium, decrease the intracellular pH of enterohemorrhagic Escherichia coli (EHEC) and influence its swimming motility. Quantifications of flagellar rotation in individual EHEC cells showed an increase in reversal frequency and a decrease in rotation rate in the presence of C. ramosum fermentation products. Furthermore, the C. ramosum fermentation products affected synthesis of flagellar filaments. The results were reproduced by a combination of organic acids under acidic conditions. Short-chain fatty acids produced by microbes in the gut flora are beneficial for the host, e.g. they prevent infection. Thus, C. ramosum could affect the physiologies of other enteric microbes and host tissues.

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.

Inhibition and Inactivation of Escherichia coli O157:H7 Biofilms by Selenium

The present study investigated the efficacy of selenium (Se) in reduction of enterohemorrhagic Escherichia coli (EHEC) exopolysaccharide (EPS) synthesis, inhibition of biofilm formation at 25 and 4°C on polystyrene surface, and inactivation of mature EHEC biofilms in combination with hot water. Sterile 96-well polystyrene plates inoculated with EHEC (∼6.0 log CFU per well) were treated with a subinhibitory concentration (SIC) of Se, and biofilms were allowed to mature at 4 and 25°C for 96 h. Biofilm-associated bacterial population was determined by scraping and plating, whereas the extent of EPS production was determined using ruthenium red staining assay. Solid surface assay was used to study the effect of Se on early attachment of EHEC cells to polystyrene. The efficacy of Se in rapid inactivation of preformed, mature EHEC biofilm was investigated by treating biofilms on polystyrene plates with the MBC of Se in combination with hot water at 80°C with a contact time of 0 min, 30 s, 2 min, and 5 min. Furthermore, the effect of Se on EHEC biofilm architecture was visualized using confocal microscopy, whereas the effect of Se on EHEC biofilm genes was determined using real-time quantitative PCR (RT-qPCR). Finally, the potential feasibility of coating stainless steel surfaces with Se nanoparticles to inhibit EHEC biofilm formation was studied. Se reduced early attachment of planktonic cells, biofilm formation, and EPS synthesis in EHEC ( P < 0.05). Se in combination with hot water reduced biofilm-associated bacterial counts by 3 to 4 log CFU/mL at 5 min of exposure compared with the control ( P < 0.05). However, hot water treatment alone decreased biofilm-associated bacterial counts by only 1.0 log CFU/mL. RT-qPCR results revealed that Se down-regulated the transcription of critical genes associated with biofilm synthesis in EHEC ( P < 0.05). The results collectively suggest that Se could potentially be used to control EHEC biofilms in food processing environments, but appropriate applications need to be validated.

Fate of various Salmonella enterica and enterohemorrhagic Escherichia coli cells attached to alfalfa, fenugreek, lettuce, and tomato seeds during germination

Contaminated vegetable seeds have been identified as a potential source of foodborne pathogens. This study was undertaken to observe the physiological behavior of various Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) cells attached to different types of vegetable seeds during germination. Surface-sanitized seeds (2 g) of alfalfa, fenugreek, lettuce, and tomato were exposed to four individual strains of Salmonella (Baildon, Cubana, Montevideo, and Stanley) or EHEC (three O157:H7 and one O104:H4 strain[s]) at 20 °C for 1 h. Vegetable seeds with attached bacterial cells were germinated on 1% water agar at 25 °C in the dark. Populations of Salmonella and EHEC on various tissues of sprouts/seedlings (seed coat, root, cotyledon, stem, etc.) were determined every other day over a 9-day germination period. The mean populations of Salmonella and EHEC on sprout/seedling tissues increased as the prolongation of germination time. Overall, 93.0% and 92.3% of collected tissue samples (n = 544) tested positive for Salmonella and EHEC, respectively. Seed coats had significantly higher (P < 0.05) bacterial counts (7.8 log CFU/g) compared to the root, cotyledon, and stem tissues (7.1–6.2 log CFU/g). On average, lettuce seedlings and alfalfa sprouts had significantly higher (P < 0.05) numbers of Salmonella and EHEC cells, followed by fenugreek sprouts, and tomato seedlings. The E. coli O104:H4 strain established the lowest (P < 0.05) cell population (5.2 log CFU/g) on sprout/seedling tissues among all EHEC strains used in the study. Among the three E. coli O157:H7 strains, F4546 (7.2 log CFU/g) and H1730 (7.0 log CFU/g) had significantly (P < 0.05) higher mean cell population than K4499 (6.8 log CFU/g). The mean population of S. Cubana (7.0 log CFU/g) was significantly higher (P < 0.05) than the populations of the other 3 Salmonella strains (6.5–6.6 log CFU/ g) used in this study. These data suggested that the growth and dissemination of Salmonella and EHEC cells on alfalfa, fenugreek, lettuce, and tomato sprouts/seedlings were influenced by the bacterial strains, type of sprouts/seedlings, and specific sprout/seedling tissues involved.

Effect of RNase E deficiency on translocon protein synthesis in an RNase E-inducible strain of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro.

Attachment and Biofilm Formation by Selected Strains of Salmonella enterica and Entrohemorrhagic Escherichia coli of Fresh Produce Origin.

This study compared the abilities of selected Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) strains of fresh produce origin to form biofilms on polystyrene surface and to attach to alfalfa and bean sprouts. Each of the 7 S. enterica and 4 EHEC inocula (2 mL; 107 CFU/mL) was placed in 6 different broths in 24-well polystyrene tissue culture plates at 28 °C for 1 to 7 d. Developed biofilms were quantified using the crystal violet binding assay. In a separate experiment, alfalfa and mung bean sprouts (5 g) were exposed to 25 mL inocula (107 CFU/mL) of S. enterica or EHEC at 22 °C for 2 h with shaking at 40 rpm. Contaminated sprouts were thoroughly rinsed and homogenized in 0.1% peptone water, and bacteria attached to sprouts were enumerated. Biofilm mass accumulated on polystyrene surface increased with incubation time (P < 0.05). Among the microbiological media used, LB no salt (NaCl) broth better supported biofilm development (P < 0.05). Two EHEC strains formed more biofilms than the Salmonella and other two EHEC strains (P < 0.05). However, more Salmonella cells (5.66 log CFU/g) attached to sprouts than EHEC cells (3.46 log CFU/g). Both Salmonella and EHEC attached in higher numbers to mung bean, than alfalfa, sprouts (P < 0.05).

Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds.

Vegetable seeds have the potential to disseminate and transmit foodborne bacterial pathogens. This study was undertaken to assess the abilities of selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to attach to fungicide-treated versus untreated, and intact versus mechanically damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C. The seeds were then vortexed vigorously for 1 min, and pathogen populations in seed rinse water and soaking buffer were determined using a standard plate count assay. In general, the Salmonella cells had higher attachment ratios than the EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In contrast, individual fenugreek seeds had more attached pathogen cells, followed by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC cells attached to mechanically damaged seeds than to intact seeds (P < 0.05). Although, on average, significantly more Salmonella and EHEC cells were recovered from untreated than fungicide-treated seeds (P < 0.05), fungicide treatment did not significantly affect the attachment of individual bacterial strains to vegetable seeds (P > 0.05), with a few exceptions. This study fills gaps in the current body of literature and helps explain bacterial interactions with vegetable seeds with differing surface characteristics.IMPORTANCE Vegetable seeds, specifically sprout seeds, have the potential to disseminate and transmit foodborne bacterial pathogens. This study investigated the interaction between two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds with differing surface characteristics. This research helps understand whether seed surface structure, integrity, and fungicide treatment affect the interaction between bacterial cells and vegetable seeds.

Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.

Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli.

Virulence of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin-converting phages), present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with H2O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2O2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the “bacterial altruism” and “Trojan Horse” hypotheses, which are connected to the oxidative stress, are discussed.

Survival of pathogenic enterohemorrhagic Escherichia coli (EHEC) and control with calcium oxide in frozen meat products

This study investigated both the level of microbial contamination and the presence of enterohemorrhagic Escherichia coli (EHEC) in frozen meat products, followed by the evaluation of its survival over 180 days under frozen temperature. We also examined the effect of calcium oxide on the populations of EHEC, E. coli O157:H7 and EPEC under both 10 °C and −18 °C storage conditions. Afterward, the morphological changes occurring in EHEC cells in response to freezer storage temperature and calcium oxide (CaO) treatments were examined using transmission electron microscopy. Among the frozen meat products tested, the highest contamination levels of total aerobic counts, coliforms and E. coli were observed in pork cutlets. Examination showed that 20% of the frozen meat products contained virulence genes, including verotoxin (VT) 1 and 2. Over 180 days of frozen storage and after 3 freeze–thaw cycles, the population of EHEC did not change regardless of the type of products or initial inoculated concentration, indicating the strong survival ability of EHEC. Subsequent testing revealed that the growth of three pathogenic E. coli strains was completely inhibited in meat patties prepared with 1% CaO, stored at 10 °C. However, the addition of 2% CaO was necessary to control the survival of EHEC, E. coli O157:H7 and EPEC in meat patties stored at −18 °C. CaO reduced the population of E. coli O157:H7 more effectively than the other EHEC and EPEC strains at both 10 °C and −18 °C. Transmission electron microscopy analysis revealed that exposed EHEC cells were resistant to the freezer storage temperature, although some cells incurred injury and death after several freeze–thaw cycles. Most of the cells exposed to CaO were found to have died or lost their cellular integrity and membranes, indicating that CaO has the potential to be used as a powerful antimicrobial agent for manufacturing frozen meat products.

Effect of heat‐assisted pulsed electric fields and bacteriophage on enterohemorrhagic Escherichia coliO157:H7

Pulsed electric fields (PEF), heat-assisted PEF (H-PEF), and virulent bacteriophage (VP) are non-thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H-PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria-Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature-abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non-thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log10 . For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H-PEF and H-PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non-nutrient RS. Analysis of EHEC recovery on selective and non-selective media indicated no occurrence of sub-lethal damage for VP, PEF/VP, and H-PEF/VP-treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization.

Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were Nal R mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (10 4, 10 5, and 10 6 CFU/mL, respectively). Appropriate controls, BEC8 (approx. 10 6 PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 μg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain.