Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.     

Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice

Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice. The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH. On comparing the growth of this serotype with a control strain of E. coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0. The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid. Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid. Control strains of E. coli failed to grow in either types of apple juice. The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.     

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.     

Effect of Cattle Diet on Escherichia coli O157:H7 Acid Resistance

The duration of shedding of Escherichia coli O157 isolates by hay-fed and grain-fed steers experimentally inoculated with E. coli O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed E. coli O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with E. coli O157:H7.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Survival in acidic and alcoholic medium of Shiga toxin-producing Escherichia coli O157:H7 and non-O157:H7 isolated in Argentina

Background

In spite of Argentina having one of the highest frequencies of haemolytic uraemic syndrome (HUS), the incidence of Escherichia coli O157:H7 is low in comparison to rates registered in the US. Isolation of several non-O157 shiga toxin-producing Escherichia coli (STEC) strains from cattle and foods suggests that E. coli O157:H7 is an uncommon serotype in Argentina. The present study was undertaken to compare the survival rates of selected non-O157 STEC strains under acidic and alcoholic stress conditions, using an E. coli O157:H7 strain as reference.

Results

Growth at 37°C of E. coli O26:H11, O88:H21, O91:H21, O111:H^-, O113:H21, O116:H21, O117:H7, O157:H7, O171:H2 and OX3:H21, was found to occur at pH higher than 4.0. When the strains were challenged to acid tolerance at pH as low as 2.5, viability extended beyond 8 h, but none of the bacteria, except E. coli O91:H21, could survive longer than 24 h, the autochthonous E. coli O91:H21 being the more resistant serotype. No survival was found after 24 h in Luria Bertani broth supplemented with 12% ethanol, but all these serotypes were shown to be very resistant to 6% ethanol. E. coli O91:H21 showed the highest resistance among serotypes tested.

Conclusions

This information is relevant in food industry, which strongly relies on the acid or alcoholic conditions to inactivate pathogens. This study revealed that stress resistance of some STEC serotypes isolated in Argentina is higher than that for E. coli O157:H7.     

The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7

The survival characteristics of a non-toxigenic, antibiotic-resistant strain of Escherichia coli O157:H7 in bovine faeces were investigated. Faecal samples were inoculated with 10(8-9) cfu g-1 of the organism and (i) stored in closed plastic containers at 10 degrees C, (ii) stored in closed plastic containers placed outside or (iii) decanted onto the surface of grazing land. Recovery and enumeration on Sorbitol MacConkey Agar (SMAC) and Tryptic Soya Agar (TSA) revealed that the E. coli O157:H7 numbers in both enclosed samples (i and ii) had decreased by 4.5-5.5 log10 cfu g-1 within 99 d. Numbers in samples decanted onto grassland (iii) decreased by 4.0-5.0 log10 cfu g-1 within 50 d but the organism was still detectable in the surrounding soil for up to 99 d. Persistence of E. coli O157:H7 in bovine faeces and contaminated pastures may therefore be an important factor in the initial infection and re-infection of cattle.     

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.     

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.     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production

The influence of heat adaptation (growth at 42 and 45 degrees C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57 degrees C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1omega7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Effects of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log(10) units.     

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.