Starvation-induced cross protection against osmotic challenge in Escherichia coli.

Stationary-phase Escherichia coli cultures showed enhanced osmotic resistance as compared with cultures in mid-logarithmic growth or preadapted to osmotic stress. The osmotolerance that developed during starvation or osmotic adaptation required de novo protein synthesis. Of the 22 polypeptides induced during osmotic shock, five were also starvation proteins.     

Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2

Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 degrees C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 degrees C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.     

Protection against Escherichia coli O157:H7 challenge by immunization of mice with purified Tir proteins

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections cause serious public health problems worldwide. The translocation intimin receptor (Tir) is responsible for adhesion and attaching and effacing lesions. In the current study, we used a mitomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the recombinant Tir as vaccine. Following immunization, mice were infected with E. coli O157:H7 and faces were monitored for shedding. Mice immunized intrasally with purified Tir proteins produced higher IgG and IgA titers in serum and feces, resulting in significant reductions in fecal shedding of EHEC O157 and higher a survival rate (92.9%), compared with subcutaneous or control immunizations. These results demonstrate the potential for the use of Tir proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7. Therefore, purified Tir protects mice against EHEC challenge after intranasal immunization and is worth further clinical development as a vaccine candidate.     

Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds.

A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E. coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds. Pretreatment with a sublethal dose (60 microM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde. CAR was also observed in WP2uvrA (uvrA-) and ZA12 (umuC-) cells, but not in ZA60 (recA-) and CM561 (lexA- (Ind-] cells. A role of recA and lexA in CAR was further suggested by the lack of beta-galactosidase induction in recA- and lexA- cells by H2O2. CAR and beta-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA- cells, but no induction of beta-galactosidase by H2O2 was observed in cells with the same gene transfer. These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

Superoxide dismutase protects against aerobic heat shock in Escherichia coli.

Exposure of a superoxide dismutase-null (sodA sodB) strain of Escherichia coli to aerobic heat stress (45 to 48 degrees C) caused a profound loss of viability, whereas the same heat stress applied anaerobically had a negligible effect. A superoxide dismutase-competent parental strain was resistant to the lethal effect of the aerobic heating. It follows that aerobic heating imposes an oxidative burden of which O2- must be a major component. This effect is not seen at 53 degrees C, presumably because, at this higher temperature, direct thermolability of vital cell components overrides the effect of superoxide radicals.     

Factors affecting the heat resistance of Escherichia coli O157 : H7

Escherichia coli O157 : H7 has been reported as being not particularly heat resistant. However, several factors which might increase its heat resistance have been investigated in this study using five strains. Increase in growth temperature to 40 °C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 °C, and variable heating rate (1 °C min⁻¹ to 23 °C min⁻¹) had no dramatic effect on heat resistance. Growth phase had a marked impact on heat resistance ; late stationary phase cells were more heat-resistant than were log phase cells. The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth ; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells. The addition of polyphosphates to the heating medium did not increase heat resistance. A reduction in water activity of the heating medium from 0·995 to levels between 0·980 and 0·960 also resulted in a marked increase in heat resistance. This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil. Survivors were detected even after a heat treatment at 60 °C for 1 h or 70 °C for 5 min. It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance.     

Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology

In 2011, Germany experienced the largest outbreak with a Shiga toxin-producing Escherichia coli (STEC) strain ever recorded. A series of environmental and trace-back and trace-forward investigations linked sprout consumption with the disease, but fecal-oral transmission was also documented. The genome sequences of the pathogen revealed a clonal outbreak with enteroaggregative E. coli (EAEC). Some EAEC virulence factors are carried on the virulence plasmid pAA. From an unknown source, the epidemic strains acquired a lambdoid prophage carrying the gene for the Shiga toxin. The resulting strains therefore possess two different mobile elements, a phage and a plasmid, contributing essential virulence genes. Shiga toxin is released by decaying bacteria in the gut, migrates through the intestinal barrier, and is transported via the blood to target organs, like the kidney. In a mouse model, probiotic bifidobacteria interfered with transport of the toxin through the gut mucosa. Researchers explored bacteriophages, bacteriocins, and low-molecular-weight inhibitors against STEC. Randomized controlled clinical trials of enterohemorrhagic E. coli (EHEC)-associated hemolytic uremic syndrome (HUS) patients found none of the interventions superior to supportive therapy alone. Antibodies against one subtype of Shiga toxin protected pigs against fatal neurological infection, while treatment with a toxin receptor decoy showed no effect in a clinical trial. Likewise, a monoclonal antibody directed against a complement protein led to mixed results. Plasma exchange and IgG immunoadsoprtion ameliorated the condition in small uncontrolled trials. The epidemic O104:H4 strains were resistant to all penicillins and cephalosporins but susceptible to carbapenems, which were recommended for treatment.     

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Glutathione in Escherichia coli is dispensable for resistance to H2O2 and gamma radiation.

Escherichia coli devoid of glutathione (because of transposon insertions in the gshA gene) has normal resistance to H2O2, cumene hydroperoxide, heat, or ionizing radiation. Intracellular glutathione thus does not protect E. coli from such lethal oxidative damage. The use of gshA::Tn10 mutants also revealed a glutathione-independent, H2O2-inducible resistance to N-ethylmaleimide.     

Cell envelope protection of alkaline phosphatase against acid denaturation in Escherichia coli.

The effects of a highly acidic environment on the cell-associated alkaline phosphatase activities of a smooth and a rough strain of Escherichia coli O8 have been examined. The observation that cell-associated enzyme is denatured to a lesser degree than purified enzyme suggests that the association of the enzyme with the cell envelope affords it some degree of protection from potentially disruptive agents in the environment. The degree of protection afforded the enzyme from pH denaturation appears to be dependent upon the presence of a complete lipopolysaccharide in the outer membrane of these strains. An abbreviation of the chemical structure of this cell envelope component produces a change in the outer membrane, resulting in increased susceptibility of the cells to a battery of antibiotics and to lysozyme and in a small, but significant, change in the sensitivity of the cell envelope-associated alkaline phosphatase to the denaturing effect of an acidic environment.     

Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157:H7

Aims:  To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7.
Methods and Results:  ZnO NP with sizes of 70 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ and NP-free solutions were used in antimicrobial tests against E. coli O157:H7. ZnO NP showed increasing inhibitory effects on the growth of E. coli O157:H7 as the concentrations of ZnO NP increased. A complete inhibition of microbial growth was achieved at the concentration level of 12 mmol l⁻¹ or higher. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy were used to characterize the changes of morphology and cellular compositions of bacterial cells treated with ZnO NP and study the mode of action of ZnO NP against E. coli O157:H7. The intensity of lipid and protein bands in the Raman spectra of bacterial cells increased after exposure to ZnO NP, while no significant changes in nucleic acid bands were observed.
Conclusions:  ZnO NP were found to have antibacterial activity against E. coli O157:H7. The inhibitory effects increase as the concentration of ZnO NP increased. Results indicate that ZnO NP may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells.
Significance and Impact of the Study:  These results suggest that ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety.     

Experimental Escherichia coli O157:H7 carriage in calves.

Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding.     

rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7.

An rpoS mutant (rpoS::pRR10) of Escherichia coli O157:H7 ATCC 43895 was generated. Stationary-phase acid, heat, and salt tolerance was significantly reduced, and starvation-induced acid tolerance did not develop in the mutant. RpoS was also important for survival of E. coli O157:H7 in dry, fermented sausage.     

UV-inducible proteins and UV-induced cross-protection against acid,ethanol, H2O2 or heat treatments in Lactococcus lactis subsp. lactis

The relationship between UV-irradiation-induced tolerance to different environmental stresses and change in protein synthesis was examined in Lactococcus lactis subsp lactis IL1403. The results showed that preirradiation of cultures of L. lactis subsp. lactis with UV_254nm light led to increased tolerance of usually lethal challenges to acid (pH 4.0), ethanol (20%, v/v), H₂O₂ (15 mM), or heat (52° C). This suggests that there is an overlapping regulation between the UV-induced pathway and the other stress responses. Whole-cell protein extracts from UV-treated (100 J/m²) and untreated cultures were compared using two-dimensional polyacrylamide gel electrophoresis. At least 14 polypeptides were induced in response to damage after UV irradiation, which indicated an SOS-like response in this species. The RecA protein, however, seemed not to be significantly induced in Lactococcus lactis subsp. lactis IL1403. Some of the UV-induced polypeptides overlaped with stress proteins induced by the other treatments.     

Escherichia coli O157:H7 in free-ranging deer in Nebraska.

In order to determine the prevalence and distribution of the human pathogen, Escherichia coli O157:H7, in free-ranging deer, hunters were asked to collect and submit fecal samples from deer harvested during a regular firearm season (14-22 November 1998). Prior to the season, 47% of the hunters with permits in the southeastern Nebraska (USA) study area indicated a willingness to participate in the study. Approximately 25% of successful hunters in the area submitted deer fecal samples. Escherichia coli O157:H7 was cultured from four (0.25%) of 1,608 total samples submitted. All of the fecal samples that were properly identified (1,426) and all that were positive for E. coli O157:H7 were from white-tailed deer (Odocoileus virginianus). We were unable to detect a statistically significant geographic distribution pattern of E. coli O157:H7. The presence of E. coli O157:H7 in the feces of free-ranging deer has implications not only for hunters, consumers of venison, and others in contact with deer or deer feces, but also for the development of strategies aimed at reducing and/or controlling this pathogen in water sources and domestic livestock.     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.     

Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7

An amphiphilic, cationic peptide composed of eight leucines and six lysines was synthesized by solid phase peptide synthesis (SPPS). The synthetic peptide was bactericidal within 10 min at concentrations as low as 3 microg ml - 1 against mid-exponential Escherichia coli O157:H7 suspended in buffer. Concentrations of 25 microg ml - 1 caused up to 7 log10 cfu ml - 1 reductions. When tested against E. coli O157:H7 grown in TSB, the peptide was bactericidal and bacteriostatic at concentrations of 50 and 25 microg ml - 1, respectively. An inhibitory effect was also observed against stationary phase cells. The synthetic peptide caused the release of u.v.-absorbing materials from the E. coli O157:H7 as well as an increase in its O.D.600 nm. Intracellular K+ and ATP depletion were also observed. These results suggest that the peptide increased the cell membrane permeability but it did not lyse the cells.     

Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7

AIMS: To quantify the antibacterial properties of five essential oils (EO) on a non-toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures. METHODS AND RESULTS: Five EOs known to exhibit antibacterial properties were screened by disc diffusion assay and the most active were selected for further study in microdilution colorimetric assays. Oregano (Origanum vulgare) and thyme (Thymus vulgaris; light and red varieties) EO had the strongest bacteriostatic and bactericidal properties, followed by bay (Pimenta racemosa) and clove bud (Eugenia caryophyllata synonym: Syzygium aromaticum) EO. Oregano oil was colicidal at 625 microl l(-1) at 10, 20 and 37 degrees C. The addition of 0.05% (w/v) agar as stabilizer reinforced the antibacterial properties, particularly at 10 degrees C, whereas 0.25% (w/v) lecithin reduced antibacterial activity. Scanning electron micrographs showed extensive morphological changes to treated cells. CONCLUSIONS: Oregano and thyme EO possess significant in vitro colicidal and colistatic properties, which are exhibited in a broad temperature range and substantially improved by the addition of agar as stabilizer. Bay and clove bud EO are less active. Lecithin diminished antibacterial properties. The bactericidal concentration of oregano EO irreversibly damaged E. coli O157:H7 cells within 1 min. SIGNIFICANCE AND IMPACT OF THE STUDY: Oregano and light thyme EO, particularly when enhanced by agar stabilizer, may be effective in reducing the number or preventing the growth of E. coli O157:H7 in foods.