Prolonged cold stress response of Escherichia coli O157 and the role of rpoS

Phenotypic analyses were performed using an enterohemorrhagic Escherichia coli O157 (EHEC) strain (B-1) and a commensal E. coli K-12 strain, exposed to prolonged cold stress. The EHEC E. coli O157 showed significantly (P < 0.05) higher resistance to cold stress compared to non-pathogenic E. coli K-12 DH5α. Further, it was found that RpoS sigma factor plays a significant (P < 0.05) role in the cold stress physiology of the enterohemorrhagic E. coli strain. Using comparative proteomic analysis of hypo-thermally adapted E. coli O157 wild-type and rpoS mutant strains, we identified 21 proteins that were differentially expressed upon cold temperature shifts or rpoS mutation. All identified proteins of cold post-acclimation stimulons fell into two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. This prolonged cold stress response included proteins involved in mRNA turnover, cell replication efficiency, conditional and post-synthetic modification of membrane lipid bilayers, biosynthetic processes, and the uptake of different sugars. The RpoS sigma factor had no control over the key stress proteins, polynucleotide phosphorylase and elongation factor G, in prolonged stress stimulon. However, RpoS was shown to regulate the expression of proteins involved in homeoviscous adaptation during cold shock, as well as various proteins involved in central metabolic pathways of this food-borne pathogen.     

Acid stress, starvation, and cold stress affect poststress behavior of Escherichia coli O157:H7 and nonpathogenic Escherichia coli.

The effects of acid shock, acid adaptation, starvation, and cold stress of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant (FRIK 816-3), and nonpathogenic E. coli (ATCC 25922) on poststress heat resistance and freeze-thaw resistance were investigated. Following stress, heat tolerance at 56 degrees C and freeze-thaw resistance at -20 to 21 degrees C were determined. Heat and freeze-thaw resistance of E. coli O157:H7 and nonpathogenic E. coli was enhanced after acid adaptation and starvation. Following cold stress, heat resistance of E. coli O157:H7 and nonpathogenic E. coli was decreased, while freeze-thaw resistance was increased. Heat and freeze-thaw resistance of the rpoS mutant was enhanced only after acid adaptation. Increased or decreased tolerance of acid-adapted, starved, or cold-stressed E. coli O157:H7 cells to heat or freeze-thaw processes should be considered when processing minimally processed or extended shelf-life foods.     

Forage feeding to reduce preharvest Escherichia coli populations in cattle,a review

Although Escherichia coli are commensal organisms that reside within the host gut, some pathogenic strains of E. coli can cause hemorrhagic colitis in humans. The most notable enterohemorrhagic E. coli (EHEC) strain is O157:H7. Cattle are asymptomatic natural reservoirs of E. coli O157:H7, and it has been reported that as many as 30% of all cattle are carriers of this pathogen, and in some circumstances this can be as high as 80%. Feedlot and high-producing dairy cattle are fed large grain rations in order to increase feed efficiency. When cattle are fed large grain rations, some starch escapes ruminal microbial degradation and passes to the hind-gut where it is fermented. EHEC are capable of fermenting sugars released from starch breakdown in the colon, and populations of E. coli have been shown to be higher in grain fed cattle, and this has been correlated with E. coli O157:H7 shedding in barley fed cattle. When cattle were abruptly switched from a high grain (corn) diet to a forage diet, generic E. coli populations declined 1000-fold within 5 d, and the ability of the fecal generic E. coli population to survive an acid shock similar to the human gastric stomach decreased. Other researchers have shown that a switch from grain to hay caused a smaller decrease in E. coli populations, but did not observe the same effect on gastric shock survivability. In a study that used cattle naturally infected with E. coli O157:H7, fewer cattle shed E. coli O157:H7 when switched from a feedlot ration to a forage-based diet compared with cattle continuously fed a feedlot ration. Results indicate that switching cattle from grain to forage could potentially reduce EHEC populations in cattle prior to slaughter; however the economic impact of this needs to be examined.     

Colicin concentrations inhibit growth of Escherichia coli O157:H7 in vitro

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present Study, the efficacy of three pore-forming colicins (El, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations <0.1 microg/ml. These potent antimicrobial proteins may potentially provide an effective and environmentally sound preharvest strategy to reduce E. coli O157:H7 in food animals.     

Protective effect of exopolysaccharide colanic acid of Escherichia coli O157:H7 to osmotic and oxidative stress

Many strains of Escherichia coli O157:H7 produce, under stress, an exopolysaccharide (EPS) comprised of colanic acid (CA) and form mucoid colonies on minimal glucose agar (MGA) at ambient temperature. Previous research conducted in our laboratory involving a CA-proficient (W6-13) and a CA-deficient (M4020; wcaD::Ekan(r)) strain of E. coli O157:H7 revealed that CA conferred acid and heat tolerance to E. coli O157:H7. Cells covered with CA were more persistent during acid (pH 4.5, 5.5, and 6.5) and heat (55 and 60 degrees C) treatment. The goal of this research was to study the effect of CA on the fate of E. coli O157:H7 under osmotic and oxidative stress. Cells of W6-13 and M4020 were exposed to various concentrations of NaCl (0.5, 1.5, and 2.5 M) and H2O2 (0, 10, and 20 mM) in minimal glucose broth (MGB) at 22 degrees C. Viable counts of E. coli O157:H7 were determined within 48 h of the osmotic stress and 3 h of the oxidative stress. The results suggest that cells of E. coli O157:H7 deficient in CA production are more susceptible than its wild-type parent to NaCl ( P< 0.05) and H2O2 (P< or = 0.05). This indicates that CA plays a role in protecting E. coli O157:H7 from osmotic and oxidative stress.     

Effects of environmental stresses on the activities of the uspA, grpE and rpoS promoters of Escherichia coli O157:H7

Heat shock proteins and RNA polymerase sigma factor play an important role in protecting cells against environmental stresses, including starvation, osmotic and oxidative stresses, and cold shock. In this study, the effect of environmental stresses on activity of the auto-fluorescent Escherichia coli O157:H7 generated by the fusion of gfp(uv) to E. coli uspA, grpE and rpoS promoters were examined. Osmotic shock caused about a 4-fold increase in green fluorescence of E. coli O157:H7 harboring uspA::gfp(uv) or rpoS::gfp(uv) at 37 degrees C and room temperature whereas osmotic shock at 5 degrees C did not induce green fluorescence. When starved, E. coli O157:H7 possessing grpE::gfp(uv) was more sensitive for evaluating stress at low temperature while uspA::gfp(uv) was better suited for detecting the stress response at higher temperature. The uspA, grpE and rpoS promoters were up-regulated to varying degrees by stresses commonly encountered during food processing.     

Distribution of Escherichia coli O157:H7 in beef processed in a table-top bowl cutter

Beef-processing equipment can be contaminated with pathogens such as Escherichia coli O157:H7 and Salmonella spp. The bowl cutter has wide application in particle-size reduction and blending of meat products. This study was undertaken to determine (i) the distribution patterns of E. coli O157:H7 in equipment components and ground beef produced with a table-top bowl cutter under different operational conditions and (ii) the likelihood that pathogen contamination can be transferred to subsequent batches after a batch of beef contaminated with E. coli O157:H7 has been processed in the same bowl cutter. A beef trim (44.6 +/- 29.5 g) inoculated with 2 log CFU of an E. coli O157:H7 mutant strain resistant to rifampicin (E. coli O157:H7rif) was fed by hand into an uncontaminated beef-trim batch under two different batch sizes (2 and 4 kg), three processing times (60, 120, and 240 s), and two feeding modes (running and stoppage fed). There were no significant differences (P > or = 0.05) among all the treatments for the averages of the counts of E. coli O157:H7rif distributed in the ground beef. Regardless of the processing time and the method used to feed the beef trims into the bowl cutter, the whole batch and the following subsequent batch became contaminated when previously contaminated beef was processed. Areas of the bowl cutter most likely to be contaminated with E. coli O157:H7 were (i) the material left on the top of the comb/knife guard and (ii) the knife. Material that overflowed the bowl cutter, when processing the batch with E. coli O157:H7rif, contaminated the equipment surroundings. A Pearson V probability distribution function was determined to describe the distribution of pathogenic organisms in the ground beef, a distribution that can also be applied when conducting process risk analyses on mixing-particle reduction operations for beef trims.     

ESCHERICHIA COLI O157 IN IRISH FEEDLOT CATTLE: A LONGITUDINAL STUDY INVOLVING PREHARVEST AND HARVEST PHASES OF THE FOOD CHAIN

The aim of this study was to investigate fecal shedding and transmission of E. coli O157 in cohorts of cattle within a feedlot, to assess subsequent contamination of carcasses with this pathogen and to identify risk factors associated with fecal shedding of E. coli O157. A cohort of 133 heifers housed infour adjacent pens was examined over a five month period, from entering the feedlot to slaughter. Individual rectal fecal samples and pen environmental samples were taken at monthly intervals. The entire outer and inner surfaces of a carcass side of each animal were swabbed immediately following slaughter.
E. coli O157 was isolated from 136 (23%) of the 600 rectal fecal samples; 96% of which contained virulent markers. One hundred and sixty environmental samples were examined and E. coli O157 was isolated from 46 (29%), all of which contained virulent markers. E. coli O157 was not isolated from any of the dressed carcasses. The prevalence of E. coli O157 fecal shedding may be related to the pen and E. coli O157 contamination of the pen floor feces, water trough and feed.
E. coli O157 should be considered as a pathogen shed in the feces of a substantial proportion of feedlot cattle. However, with good hygienic practice at harvest, a very low level of this pathogen can be achieved on dressed carcasses.     

A review of quantitative microbial risk assessment in the management of Escherichia coli O157:H7 on beef

Since Escherichia coli O157:H7 first emerged as a food borne pathogen in the mid 1980s, it has been linked to many cases of food poisoning across the world. While multiple sources and routes of transmission for this pathogen are now recognised, beef and beef products remain an important vehicle of the pathogen and continue to be linked to outbreaks across the developed world. Much research has been directed at E. coli O157:H7 transmission, survival and control in the beef chain and this paper presents an overview of current knowledge on this pathogen in the beef chain from primary production through slaughter, processing, distribution, final preparation and cooking. In order to strategically manage E. coli O157:H7 and to devise approaches to reduce the public health risk posed, many national and international groups have applied quantitative risk assessment techniques to model the risk posed by E. coli O157:H7 in beef, particularly in ground/minced beef which is most often linked with infection. This paper reviews these quantitative risk assessments and their application in managing the risk posed by E. coli O157:H7 in beef.     

A comparison of the survival in feces and water of Escherichia coli O157:H7 grown under laboratory conditions or obtained from cattle feces

Escherichia coli O157:H7 is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic uremic syndrome. Cattle feces and fecally contaminated water are important in the transmission of this organism on the farm. In this study, the survival of E. coli O157:H7 in feces and water was compared following passage through the animal digestive tract or preparation in the laboratory. Feces were collected from steers before and after oral inoculation with a marked strain of E. coli O157:H7. Fecal samples collected before cattle inoculation were subsequently inoculated with the marked strain of E. coli O157:H7 prepared in the laboratory. Subsamples were taken from both animal and laboratory-inoculated feces to inoculate 5-liter volumes of water. E. coli O157:H7 in feces survived up to 97 days, and survival was not affected by the method used to prepare the inoculating strain. E. coli O157:H7 survived up to 109 days in water, and the bacteria collected from inoculated cattle were detected up to 10 weeks longer than the laboratory-prepared culture. This study suggests that pathogen survival in low-nutrient conditions may be enhanced by passage through the gastrointestinal tract.     

uspA of Shigella sonnei

One of the strategies that bacteria utilize to combat environmental stress is to synthesize stress-responding proteins. In Escherichia coli, adverse environmental factors, such as starvation, heat, and the presence of acid, oxidants, heavy metals, and antibiotics, trigger the expression of the universal stress protein (USP). The gene of the USP, uspA, in E. coli K-12 and E. coli O157:H7 has been identified and sequenced. In this study, the nucleotide sequence of uspA in a strain of Shigella sonnei implicated in the 1998 parsley-related outbreak of shigellosis was determined. Within an 800-bp region sequenced, there were 17 bp mismatches between the uspA of S. sonnei and that of E. coli K-12. Among the 17 mismatched nucleotides, 8 were within the structure gene of uspA. A total of 12 bp variations were identified between the uspA of S. sonnei and that of E. coli O157:H7, of which 5 bp were internal to the coding region of uspA. However, unlike the mismatches between the uspA of E. coli K-12 and the same gene of E. coli O157:H7 and S. sonnei that resulted in a single amino acid substitution and changed an alanine to an arginine at position 140, the mismatches between S. sonnei and E. coli O157:H7 were silent and did not result in any amino acid substitution.     

Horizontal transmission of Escherichia coli O157:H7 during cattle housing

Ruminant livestock, particularly cattle, is considered the primary reservoir of Escherichia coli O157:H7. This study examines the transmission of E. coli O157:H7 within groups of cattle during winter housing. Holstein Friesian steers were grouped in six pens of five animals. An animal inoculated with and proven to be shedding a marked strain of E. coli O157: H7 was introduced into each pen. Fecal (rectal swabs) and hide samples (900 cm2 from the right rump) were taken from the 36 animals throughout the study. Water, feed, and gate or partition samples from each pen were also examined. Within 24 h of introducing the inoculated animals into the pens, samples collected from the drinking water, pen barriers, and animal hides were positive for the pathogen. Within 48 h, the hides of 20 (66%) of 30 cohort animals from the six pens were contaminated with E. coli O157:H7. The first positive fecal samples from the noninoculated cohort animals were detected 3 days after the introduction of the inoculated steers. During the 23 days of the study, 15 of 30 cohort animals shed the marked E. coli O157:H7 strain in their feces on at least one occasion. Animal behavior in the pens was monitored during a 12-h period using closed circuit television cameras. The camera footage showed an average of 13 instances of animal grooming in each pen per hour. The study suggests that transmission of E. coli O157:H7 between animals may occur following ingestion of the pathogen at low levels and that animal hide may be an important source of transmission.     

Thermal resistance parameters for Shiga toxin-producing Escherichia coli in apple juice

The purpose of the present study was to determine the heat resistance of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison to E. coli O157:H7 in single-strength apple juice without pulp. The thermal parameters for stationary-phase and acid-adapted cells of E. coli strains from serogroups O26, O45, O103, O111, O121, O145, and O157:H7 were determined by using an immersed coil apparatus. The most heat-sensitive serotype in the present study was O26. Stationary-phase cells for serotypes O145, O121, and O45 had the highest D(56°C)-value among the six non-O157 serotypes studied, although all were significantly lower (P < 0.05) than that of E. coli O157:H7. At 60°C E. coli O157:H7 and O103 demonstrated the highest D-values (1.37 ± 0.23 and 1.07 ± 0.03 min, respectively). The D(62°C) for the most heat-resistant strain belonging to the serotype O145 was similar (P > 0.05) to that for the most resistant O157:H7 strain (0.61 ± 0.17 and 0.60 ± 0.09 min, respectively). The heat resistance for stationary-phase cells was generally equal to or higher than that of acid-adapted counterparts. Although E. coli O157:H7 revealed D-values similar to or higher than the individual six non-O157 STEC serotypes in apple juice, the z-values for most non-O157 STEC tested strains were greater than those of E. coli O157:H7. When data were used to calculate heat resistance parameters at a temperature recommended in U.S. Food and Drug Administration guidance to industry, the D(71.1°C) for E. coli O157:H7 and non-O157 STEC serotypes were not significantly different (P > 0.05).     

Sensitivity of two techniques to detect Escherichia coli O157 in naturally infected bovine fecal samples

The aim of this study was to investigate the sensitivity and reliability of two techniques commonly used for the isolation of Escherichia coli O157: (i) buffered peptone water (BPW) containing vancomycin, cefsulodin and cefixime followed by immunomagnetic separation (IMS-VCC) and (ii) modified E. coli (EC) broth supplemented with novobiocin (m ECn), both followed by culturing on cefixime tellurite sorbitol McConkey (ctSMAC) agar plates. Over a 2-year period, 24 feedlots located over a large geographical area (approximately 600 x 450 km) were screened for the presence of E. coli O157. A total of 194 E. coli O157 isolates were identified; 151 (77.4%) using IMS-VCC and 108 (55.4%) using m ECn. The recovery rates of IMS-VCC varied from 100% to 47%, whereas for m ECn ranged from 100% to 16%. All isolates were grouped, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), into 3 major clusters that comprised 39 different subtypes, providing evidence of considerable genetic heterogeneity. The results from this study revealed false negatives in IMS-VCC technique, most probably due to the high genetic diversity of environmental E. coli O157 isolates and antibiotic sensitivity. Using only IMS-VCC as a method for detection may result in significant underestimation of the pathogen. Performing two different enrichment steps in parallel can lead to markedly improved recovery rates of E. coli O157 isolates from naturally infected samples.     

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2 degrees C, and -20 degrees C

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.     

Use of deodorized yellow mustard powder to control Escherichia coli O157:H7 in dry cured Westphalian ham

Dry cured (uncooked) hams with low water activity and pH ≥5.6 seem a likely food vehicle for Escherichia coli O157:H7. In previous work, isothiocyanates produced from mustard glucosinolates by bacterial myrosinase-like activity converted deodorized mustard into a potent antimicrobial in dry sausage. In this study its value in controlling E. coli O157:H7 survival in Westphalian ham was investigated. Hams were inoculated with a 7.5 log cfu g(-1) cocktail of E. coli O157:H7, 4% or 6% (w/w) deodorized yellow mustard powder was surface applied and monitored 80 d for pathogen survival. In one trial to accelerate formation of isothiocyanate, a Staphylococcus (S.) carnosus meat starter culture was added to hams at 45 d (after salt equilibration). At 21 d, E. coli O157:H7 was reduced by 3 log cfu g(-1) on hams treated with mustard powder compared to only a 1 log cfu g(-1) reduction in the control. By 45 d, mustard powder caused a reduction of >5 log cfu g(-1)E. coli O157:H7, whereas it took 80 d for numbers in control hams to be similarly reduced. Although the commercial process used caused a 5 log cfu g(-1) reduction of E. coli O157:H7 in 80 d, 4% or 6% deodorized mustard accelerated this reduction and the S. carnosus starter culture may have contributed to the maintenance of this effect.     

Influence of transportation stress and animal temperament on fecal shedding of Escherichia coli O157:H7 in feedlot cattle

To test the influence of transportation stress and temperament on shedding of Escherichia coli O157:H7, cattle (n=150) were classified at various stages of production as Excitable, Intermediate or Calm based on a variety of disposition scores. Presence of E. coli O157:H7 was determined by rectal swabs from live animals and from colons collected postmortem. Percentage of cattle shedding E. coli O157:H7 at arrival at the feedlot was approximately equal among temperament groups. Before shipment to the processing facility, a higher (P=0.03) proportion of cattle from the Calm group shed E. coli O157:H7 compared to the other temperament groups. When pooled across all sampling periods, cattle from the Calm group had a greater percentage test positive for E. coli O157:H7. Neither the acute stressor of transportation nor a more excitable temperament led to increased shedding of E. coli O157:H7 in cattle.     

Survival of antibiotic resistant and antibiotic sensitive strains of E. coli O157 and E. coli O26 in food matrices

Escherichia coli O157:H7 or E. coli O26, which were AS (antibiotic sensitive), AR (laboratory created antibiotic resistant mutants), or naturally MAR (multi-antibiotic resistant), were inoculated into laboratory media, yoghurt or orange juice and their growth/survival monitored during enrichment at 37 degrees C or storage at 4 degrees C. The strains were also inoculated into minced beef and their thermal inactivation (D-values) examined at 55 degrees C, with and without a prior heat shock at 48 degrees C. The growth kinetics (lag phases, growth rates) of the VTEC (verocytotoxigenic E. coli), incubated over 24 h at 37 degrees C in laboratory media, were similar regardless of the presence or absence of antibiotic resistance. In yoghurt and orange juice, E. coli O157:H7 MAR died off significantly faster (P<0.05) than any of other VTEC strains examined. E. coli O157:H7 MAR was also found to be significantly more heat sensitive (P<0.05) than the other VTEC strains tested. The reasons for the observed differences in survival of the different VTEC strains and the link between antibiotic resistance and survival in VTEC organisms are discussed.     

Survival of Escherichia coli O157:H7 in set yogurt as influenced by the production of an exopolysaccharide, colanic acid

Previous studies conducted in our laboratory revealed that Escherichia coli O157:H7 cells capable of producing colanic acid (CA), the acidic polysaccharide of mucoid slime, had increased tolerance to sublethal heat and the extreme pH of microbiological culture media. This study was undertaken to determine the effect of CA on the fate of E. coli O157:H7 during the processing and storage of an acid food: yogurt. Pasteurized and homogenized whole milk was inoculated with a wild-type E. coli O157:H7, its CA-deficient mutant, or a mixture (1:1) of the two strains. Set yogurt was processed from the contaminated milk and stored at 4 degrees and 15 degrees C for 3 weeks. Samples of milk and yogurt were withdrawn during processing and storage and analyzed for total plate counts and populations of E. coli O157:H7 and starter cultures. The results showed that E. coli O157:H7 survived longer in yogurt stored at 15 degrees C than at 4 degrees C. Cells of E. coli O157:H7 deficient in CA production died off more rapidly than those of the parent strain. This suggests that CA plays a role in protecting cells of E. coli O157:H7 from stress during the processing and storage of set yogurt.