Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7.

The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections.IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

Increased Iron Availability Aggravates the Infection of Escherichia coli O157:H7 in Mice.

Iron plays an important role both in bacterial pathogenicity and in host defense mechanisms, which has frequently been underestimated. The primary purpose of this study was to investigate the influence of iron supplementation on the progression of bacterial infection. We used mice as an experimental model to supplement iron after Escherichia coli (E. coli) O157:H7 infection and found that iron supplementation exacerbated clinical symptoms of bacterial infection by increasing mortality and reducing body weight. Iron supplementation promoted the colonization of bacteria and enhanced inflammatory responses by increasing C-reaction protein level and the phagocytic capacity of PBMCs, as well as upregulating the expression of TNF-α and IL-1ß in E. coli O157:H7-challenged mice. In vitro cell experiment confirmed that an excess of iron would enhance the growth of E. coli O157:H7 and worsen the outcome of bacterial infection. Therefore, it is certainly plausible that iron supplementation in bacterial infection may worsen rather than improve host outcome.

Efficacy of activated persulfate in inactivating Escherichia coli O157:H7 and Listeria monocytogenes.

Concerns have been on the rise regarding the use of chlorine-based sanitizers for fresh produce sanitation due to the production of toxic disinfection by-products (DBPs). This study was undertaken to evaluate the efficacy of activated persulfate in inactivating Escherichia coli O157:H7 and Listeria monocytogenes in pure culture. The objectives were to study the effect of persulfate to activator ratios and determine the major contributing radical in pathogen inactivation. A five-strain cocktail of each pathogen was treated with sodium persulfate activated by ferrous sulfate or sodium hydroxide for 60 s or 120 s. Non-selective agars supplemented with sodium pyruvate were used for pathogen enumeration. The steady-state concentrations of free radicals were quantified using HPLC-DAD. Radical scavengers (tert-butanol, isopropanol, and benzoquinone) were used to determine the major contributing radical in pathogen inactivation. The results showed more than 7 log CFU/mL reductions can be achieved in 120 s for both pathogens at appropriate activation conditions. For ferrous activation, the persulfate to ferrous ratio played an important role in the overall inactivation efficacy. The maximum pathogen reduction (7.77 log CFU/mL for E. coli O157:H7 and 7.25 log CFU/mL for L. monocytogenes) was achieved at persulfate to ferrous molar ratio of 1:0.33 when the initial persulfate concentration was set at 40 mmol/L. Further increase or decrease of ferrous ratio always leads to lower pathogen reductions. For alkaline activation, the inactivation efficacy increased with more initial sodium hydroxide. The maximum reduction was achieved at 40 mmol/L persulfate with 30 mmol/L sodium hydroxide for E. coli O157:H7 (6.21 log CFU/mL reduction) and at 500 mmol/L persulfate with 350 mmol/L sodium hydroxide for L. monocytogenes (8.64 log CFU/mL reduction). Also, persulfate activated by sodium hydroxide always achieved significantly (P < 0.05) higher microbial reductions than sodium hydroxide or persulfate alone. L. monocytogenes was generally more resistant against the activated persulfate treatment compared with E. coli O157:H7, which might be due to the different cell envelop structures between Gram-positive and Gram-negative bacteria. Hydroxyl radical was demonstrated to be the major radical to inactivate both pathogens in ferrous activation while superoxide radical was demonstrated to be the major radical to inactivate both pathogens in alkaline activation.

Effect of nanoliposomes containing Zataria multiflora Boiss. essential oil on gene expression of Shiga toxin 2 in Escherichia coli O157:H7.

AIMS: Enterohaemorrhagic Escherichia coli serotype O157:H7 as a major human pathogen is responsible for food borne outbreaks, bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome and even death. In this study, the antibacterial activity of the Zataria multiflora essential oil (ZMEO) and nanoliposome-encapsulated ZMEO was evaluated on the pathogenicity of E. coli O157:H7. METHODS AND RESULTS: The minimum inhibitory concentrations (MIC) of essential oil (EO) were determined against the bacterium before and after encapsulation into nanoliposome. Then, the effect of subinhibitory concentrations was evaluated on Shiga toxin 2 (Stx2) production. The effect of free and nanoliposomal EO was also studied on the gene expression of Stx2 by real-time PCR. It was found that inhibitory activity of EO was improved after incorporation into nanoliposomes (P < 0·05). The MIC of free EO against E. coli O157:H7 was 0·03% (v/v), while this value decreased to 0·015%, after encapsulation of EO into nanoliposomes. Furthermore, subinhibitory concentrations of liposomal EO (50 and 75% MIC) had significantly higher inhibitory effect on Stx2 titre than its free form (P < 0·05). Sub-MICs of nanoencapsulated EO also showed a better activity in reduction of Stx2A gene expression than free EO. Using 75% MIC of nanoliposomal EO, the relative transcriptional level of Stx2A gene was decreased from 0·721 to 0·646. CONCLUSIONS: The findings of present study suggest that application of nanoliposomes can improve the antibacterial effect of EOs like ZMEO. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the enhancement of antimicrobial activity, nanoencapsulation of plant EOs and extracts may increase their commercial application not only in food area but also in the pharmaceutics, cosmetics and health products.

Internalization of Escherichia coli O157:H7 gfp+ in rocket and Swiss chard baby leaves as affected by abiotic and biotic damage.

Internalization of human pathogens in edible parts of vegetables eaten raw is a major concern, since once internalized they are protected from sanitizing treatments. In this study, we examined the invasion of gfp-labelled Escherichia coli O157:H7 into intact and biotically (infection with Xanthomonas campestris/Pseudomonas syringae) and abiotically (grating with silicon carbide) damaged leaves of wild rocket (Diplotaxis tenuifolia) and Swiss chard (Beta vulgaris subsp. cicla) using laser scanning confocal microscopy. Bacterial cells were found in internal locations of the tissue, irrespective of tissue health status. Contaminated leaf sections of biotically and abiotically damaged wild rocket leaves showed higher susceptibility to microbial invasion, while the pathogen was internalized in greater numbers into intact Swiss chard leaf sections when abiotically, but not biotically, damaged. The greatest differences were observed between the plant species; after surface sanitization, E. coli O157:H7 was still detected in wild rocket leaves, but not in Swiss chard leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of leafy vegetables with Escherichia coli O157:H7 is a growing problem, as reported outbreaks are increasing. However, establishment of this human pathogen in the phyllosphere is not completely understood. Using laser scanning confocal microscopy, we demonstrated that E. coli O157:H7gfp+ can invade plant tissue of Swiss chard and wild rocket leaves and that the bacterium is more sensitive to surface sanitization of Swiss chard leaves. Damage to leaf tissue promoted leaf invasion, but the nature of the damage (abiotic or biotic) and plant species had an impact.

Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

This study evaluated the inhibitory effect of cinnamon oil against Escherichia coli O157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil against E. coli O157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and the stx2 mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where the stx2 gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited the qseBC and luxS expression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genes oxyR, soxR, and rpoS was increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction in E. coli O157:H7 in multiple ways. IMPORTANCE: This study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production, stx2 mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction in E. coli O157:H7, which may be due to the suppression of RNA polyadenylation enzyme PNPase at 0.25× to 1.0× MIC and the downregulation of bacterial SOS response key regulator RecA and RNA polyadenylation enzyme PAP I at 0.75× or 1.0× MIC. Cinnamon oil at higher levels (0.75× and 1.0× MIC) eliminated quorum sensing and oxidative stress. Therefore, cinnamon oil has potential applications as a therapeutic to control E. coli O157:H7 infection through inhibition of bacterial growth and virulence factors.

Selenium reduces enterohemorrhagic Escherichia coli O157:H7 verotoxin production and globotriaosylceramide receptor expression on host cells.

AIM: This study investigated the efficacy of selenium (Se) in reducing Escherichia coli O157:H7 verotoxin production and toxin gene expression. Additionally, the effect of Se on globotriaosylceramide (Gb3) receptor in human lymphoma cells was determined. MATERIALS & METHODS: The effect of Se on verotoxin synthesis was determined by standard ELISA, whereas its effect on Gb3 receptor was determined by flow cytometry and real-time quantitative PCR. RESULTS & CONCLUSIONS: Se reduced extracellular and intracellular verotoxin concentration by 40-60% and 80-90%, respectively (p < 0.05), and downregulated verotoxin genes (p < 0.05). Se reduced Gb3 receptor synthesis in lymphoma cells, and real-time quantitative PCR data revealed a significant downregulation of LacCer synthase gene (GalT2) involved in Gb3 synthesis. Further studies are warranted to validate these results in an appropriate animal model.

Antibacterial Activity of Carum copticum Essential Oil Against Escherichia Coli O157:H7 in Meat: Stx Genes Expression.

This work were aimed to (a) determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Carum copticum essential oil (EO) against Escherichia. coli O157:H7 in vitro Trypticase Soy Broth, (TSB) and in ground beef; (b) evaluation of the effect of sub-inhibitory concentrations (sub-MICs) of EO on the growth of bacterium in TSB over 72 h (at 35 °C) and ground beef over 9 days (at 4 °C); and (c) investigation of gene expression involved in Shiga toxins production using relative quantitative real-time PCR method. The MIC in broth and ground beef medium were determined as 0.05 (v/v) and 1.75 % (v/w), respectively. In comparison with control cultures, the EO concentration of 0.03 % in broth caused reduction of colony counting as 1.93, 1.79, and 2.62 log10 CFU ml(-1) after 24, 48, and 72 h at 35 °C, and similarly EO (0.75 %) in ground beef resulted to reduction of colony counting as 1.03, 0.92, 1.48, and 2.12 log10 CFU g (-1) after 2, 5, 7, and 9 days at 4 °C, respectively. An increase and decrease in gene expression were observed as result of EO addition (0.03 %) to broth and (0.5 %) to ground beef was noticed, respectively.

Immune-stimulating properties of 80% methanolic extract of Ludwigia octovalvis against Shiga toxin-producing E. coli O157:H7 in Balb/c mice following experimental infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Ludwigia octovalvis is an aquatic plant widely distributed throughout the tropical and sub-tropical regions. It is commonly consumed as a health drink and traditionally used for treating various ailments such as dysentery, diarrhea, diabetes, nephritisn and headache. No information is available on its in vivo antibacterial activity against an important foodborne pathogen, Shiga toxin producing Escherichia coli O157:H7. MATERIALS AND METHODS: Male Balb/c mice were orally administered with the extract at doses of 200 or 400mg/kg body weight for one week before the infection with E. coli O157:H7 and continued for 14 consecutive days after infection. Serum antibody (IgA, IgG and IgM) levels were quantified at days 7 and 14 post-challenge by an ADVIA(®) 2400 Clinical Chemistry Auto Analyzer. Nitroblue tetrazolium (NBT) and Ceruloplasmin, as nonspecific immune parameters, were determined enzymatically. RESULTS: A significant increase (p<0.05) in IgA serum level was indicated on the 7th day post-challenge with the pathogen in the experimental group received 400mg/kg of the extract in comparison with other groups. Total IgA serum levels on day 7 post-challenge in groups of PBS negative control, E. coli O157:H7 positive control, E. coli O157:H7+200mg/kg extract group and E. coli O157:H7+400mg/kg extract group were 709.4 ± 149.6, 1655.8 ± 139.7, 1728.6 ± 64.3 and 1971.4 ± 135.6 µg/ml, respectively. Serum IgG and IgM did not significantly change among different groups. The extract administered orally to infected Balb/c mice did not affect the NBT as well as ceruloplasmin levels. CONCLUSIONS: The extract of L. octovalvis contains biologically active principles which increased systemic immune response to E. coli O157:H7 via potentiating the synthesis of IgA antibodies.

Inhibition of cytotoxicity of Shiga toxin of Escherichia coli O157:H7 on vero cells by Prosopis alba Griseb (Fabaceae) and Ziziphus mistol Griseb (Rhamnaceae) extracts.

The capacity of Prosopis alba Griseb. and Ziziphus mistol Griseb. fruit extracts to inhibit the toxic action of Shiga toxin (Stx) was investigated. Purification of Stx from Escherichia coli O157:H7 was performed by saline precipitation and affinity chromatography using a column with globotriaosylceramide, while the fruits were subjected to ethanolic or aqueous extractions. The protective action of both fruits was determined by pre-, co-, and postincubation of one 50% cytotoxic dose per ml of Stx with different concentrations of ethanolic and aqueous extracts in confluent monolayers of Vero cells for 72 h at 37°C (5% CO2). The inhibition of the cytotoxic effect of Stx by fruit extracts was determined by the neutral red vital staining technique. The extraction of the polyphenols and flavonoids was effective, and more polyphenols per milligram of dissolved solids were obtained from P. alba than from Z. mistol. However, there were more flavonoids in Z. mistol than in P. alba. Components of both fruits increased the viability of cells treated with Stx when the extracts were preincubated with Stx for 1 h before being applied to the cell cultures, with the ethanolic extract of P. alba showing 95% cell viability at a concentration of 2.45 mg/ml. The extracts were less effective in protecting cells when Stx, extracts, and cells were coincubated together without a previous incubation of Stx; only the concentrations of 19.46 mg/ml for the P. alba aqueous extract and 3.75 mg/ml for the Z. mistol ethanolic extract resulted in the inhibition of cytotoxicity, with 52 and 56% cell viability occurring, respectively. Investigation into this difference in the protection of cells indicated that the protein molecule of Stx suffered degradation to advanced oxidative protein products during preincubation with extracts, principally with P. alba, which exhibited a greater amount of nonflavonoid polyphenols than Z. mistol. The prooxidant action on Stx favored the cells and enhanced the protective action of both fruits.

AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria.

(-)-Epigallocatechin-3-gallate (EGCG), a main constituent of tea catechins, affects Gram-positive and Gram-negative bacteria differently; however, the underlying mechanisms are not clearly understood. Atomic force microscopy (AFM) was used to compare morphological alterations in Gram-positive and Gram-negative bacteria induced by EGCG and by H(2)O(2) at sub-minimum inhibitory concentrations (MICs). EGCG initially induced aggregates in the cell envelopes of Staphylococcus aureus and eventually caused cell lysis, which was not observed in cells treated with H(2)O(2). It initially induced nanoscale perforations or microscale grooves in the cell envelopes of Escherichia coli O157:H7 which eventually disappeared, similar to E. coli cells treated with H(2)O(2). An E. coli O157:H7 tpx mutant, with a defect in thioredoxin-dependent thiol peroxidase (Tpx), was more severely damaged by EGCG when compared with its wild type. Similar differing effects were observed in other Gram-positive and Gram-negative bacteria when exposed to EGCG; it caused aggregated in Streptococcus mutans, while it caused grooves in Pseudomonas aeruginosa. AFM results suggest that the major morphological changes of Gram-negative bacterial cell walls induced by EGCG depend on H(2)O(2) release. This is not the case for Gram-positive bacteria. Oxidative stress in Gram-negative bacteria induced by EGCG was confirmed by flow cytometry.

YkgM and ZinT proteins are required for maintaining intracellular zinc concentration and producing curli in enterohemorrhagic Escherichia coli (EHEC) O157:H7 under zinc deficient conditions.

Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.

Increased advanced oxidation of protein products and enhanced total antioxidant capacity in plasma by action of toxins of Escherichia coli STEC.

Shiga toxin (Stx) and hemolysin (Hly) of Escherichia coli O157:H7 produced an increase of reactive oxygen species (ROS) in normal human blood. In vitro assays showed that stimuli of ROS with these toxins oxidized proteins to carbonyls in plasma and raised the degradation of oxidized macromolecules, with the AOPP/carbonyl relationship also increasing. The oxidative stress generated by toxins during the Hemolytic Uremic Syndrome (HUS) produced oxidation of blood proteins with a rise in advanced oxidation protein products (AOPP) in children with HUS. There was a response from the antioxidant system in these patients, evaluated through the determination of the total antioxidant capacity of plasma by the Ferric Reducing Antioxidant Power (FRAP), which reduced the stimuli of ROS during in vitro incubation with Stx or Hly. The application of natural antioxidants was sufficient to reduce in vitro the oxidative stress provoked by both toxins in blood.

Effect of heat-shock treatment on the survival of Escherichia coli O157:H7 and Salmonellaenterica Typhimurium in dairy manure co-composted with vegetable wastes under field conditions.

This study investigated the survival of heat-shocked (HS) and non-heat-shocked (NHS) Escherichia coli O157:H7 and Salmonellaenterica Typhimurium when co-composting dairy manure and vegetable wastes in a field setting. In the summer, HS E. coli O157:H7 and Salmonella survived for 7 and 2 days longer at the surface and bottom locations of the compost heaps, respectively, than NHS cultures. Both HS and NHS E. coli O157:H7 and Salmonella were detectable in all compost samples for more than 60 days in the winter. The results indicate that composting dairy manure with vegetable wastes under sub-optimal conditions may allow extended survival of pathogens in the heap at low ambient temperature. Analysis of covariance revealed that the heat-shock treatment may have induced cross-resistance to desiccation, allowing extended survival of HS E. coli O157:H7 and Salmonella at the surface of the compost heaps during the summer.

Screening and analysis of spices with ability to suppress verocytotoxin production by Escherichia coli O157.

To reduce the amounts of verocytotoxin (VT) produced by Escherichia coli O157:H7, various spices were screened for their ability to suppress VT production. Extracts of these spices were prepared with 70% ethyl alcohol. When E. coli O157:H7 cells were grown to the stationary phase at 37 degrees C in Luria-Bertani medium supplemented with 0.02% allspice extract, the production of both VT1 and VT2 was significantly reduced. Neither growth inhibition nor a delay in the lag phase was observed when the cells were cultured in the presence of 0.02% allspice extract. An active component of the allspice extract was purified by HPLC and was identified as eugenol. When we examined the suppressive effect of eugenol on VT production by E. coli O157:H7, the amounts of both intracellular and extracellular VTs were found to decrease with an increase in eugenol concentration. Our results suggest that eugenol is useful for reducing the virulence of E. coli O157:H7.

Gnotobiotic piglet infection model for evaluating the safe use of antibiotics against Escherichia coli O157:H7 infection.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli (STEC), especially O157:H7, cause bloody diarrhea, and in 3%-15% of individuals the infection leads to hemolytic uremic syndrome (HUS) or other complications. Use of antibiotics to treat STEC infections is controversial. Here, we describe the use of piglets to evaluate the efficacy and mechanism of action of antibiotics in these infections. METHODS: The effects of 2 antibiotics on STEC toxin production and their mechanisms of action were first determined by enzyme-linked immunosorbent assay and subsequently evaluated clinically in the gnotobiotic piglet infection model. RESULTS: In vitro treatment of clinical and isogenic strains with ciprofloxacin increased the production of Stx2 via phage induction but not the production of Stx1. Azithromycin caused no significant increase in toxin production. After treatment with ciprofloxacin, infected piglets had diarrhea and the severe fatal neurological symptoms associated with Stx2 intoxication. Characteristic petechial hemorrhages in the cerebellum were more severe in ciprofloxacin-treated animals than in control animals. In contrast, azithromycin-treated piglets survived the infection and had little or no brain hemorrhaging. CONCLUSIONS: The increased in vitro toxin production caused by ciprofloxacin was strongly correlated with death and an increased rate of cerebellar hemorrhage, in contrast to the effect of azithromycin. The piglet is a suitable model for determining the effectiveness and safety of antibiotics available to treat patients.

Bovine colostrum contains immunoglobulin G antibodies against intimin, EspA, and EspB and inhibits hemolytic activity mediated by the type three secretion system of attaching and effacing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of gamma-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing E. coli. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing E. coli strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity.

Influence of dietary catechols on the growth of enteropathogenic bacteria.

The dietary constituents that may act, in the broadest sense, as co-factors to enable bacterial enteropathogens to replicate in gastrointestinal environments are still largely unknown. Recent work has demonstrated that certain non-nutritional components of food, such as the catecholamines, can contribute to the ability of Gram-negative pathogens to replicate in iron-restrictive media that may be reflective of gastrointestinal environments. The present report examines whether other, non-catecholamine, dietary catechols, which occur widely in plant foods, can also influence enteropathogen growth in an iron-restrictive environment such as might be found in the gastrointestinal tract. In the present study, we have examined the ability of a range of catechol-rich foodstuffs, ranging from beverages (tea and coffee) to fruit and vegetable extracts, as well as purified preparations of commonly consumed dietary catechols (catechins, chlorogenic acid, caffeic acid and tannic acid), to modulate the growth of the Gram-negative enteric pathogens Escherichia coli O157:H7 and Salmonella enterica SV Enteriditis. Time-dependent growth in response to dietary catechols (0.05-5.0% v/v of beverage or fruit/vegetable extracts; 10-200 microM of purified catechols) was examined in an iron-replete, rich medium as well as in an iron-limited, basal medium designed to reflect the iron-restricted environment that is more characteristic of human and animal tissues. Results obtained in iron-replete, rich medium demonstrated dose-dependent bacteriostatic effects for certain catechols, consistent with previous studies. However, in iron-restricted medium, all of the dietary catechols produced marked growth stimulation of up to 4 logs greater than non-supplemented controls. Mechanistic studies measuring the uptake of radiolabelled (55)Fe from (55)Fe-labelled lactoferrin and transferrin in bacteria grown in the presence or absence of dietary catechols demonstrated that the ability of catechols to stimulate bacterial growth was dependent on the provision of iron from iron-sequestering glycoproteins. Urea gel analysis of transferrin incubated in the presence of the dietary catechols confirmed that these compounds were directly chelating and removing transferrin-complexed iron. Analysis using E. coli O157:H7 entA and tonB mutants further showed that a functional siderophore synthesis and uptake system was required for the growth-stimulatory response. In contrast to previous studies, which have reported the anti-microbial activity of dietary catechols, the present study demonstrates that these non-nutritional components of foods can, under iron-restrictive conditions, provide iron and enable the growth of enteric bacterial pathogens.

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use.

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru).

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.