Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells

AIMS: To develop methods to assess the efficiency of immunomagnetic separation (IMS). METHODS AND RESULTS: The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of 相似文献   

A stable bioluminescent construct of Escherichia coli O157:H7 for hazard assessments of long-term survival in the environment

A chromosomally lux-marked (Tn5 luxCDABE) strain of nontoxigenic Escherichia coli O157:H7 was constructed by transposon mutagenesis and shown to have retained the O157, H7, and intimin phenotypes. The survival characteristics of this strain in the experiments performed (soil at -5, -100, and -1,500 kPa matric potential and artificial groundwater) were indistinguishable from the wild-type strain. Evaluation of potential luminescence was found to be a rapid, cheap, and quantitative measure of viable E. coli O157:H7 Tn5 luxCDABE populations in environmental samples. In the survival studies, bioluminescence of the starved populations of E. coli O157:H7 Tn5 luxCDABE could be reactivated to the original levels of light emission, suggesting that these populations remain viable and potentially infective to humans. The attributes of the construct offer a cheap and low-risk substitute to the use of verocytotoxin-producing E. coli O157:H7 in long-term survival studies.     

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.     

Escherichia coli O157:H7 in environments of culture-positive cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.     

Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach

The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.     

Lettuce cultivar mediates both phyllosphere and rhizosphere activity of Escherichia coli O157:H7

Plant roots and leaves can be colonized by human pathogenic bacteria, and accordingly some of the largest outbreaks of foodborne illness have been associated with salad leaves contaminated by E. coli O157. Integrated disease management strategies often exploit cultivar resistance to provide a level of protection from economically important plant pathogens; however, there is limited evidence of whether the genotype of the plant can also influence the extent of E. coli O157 colonization. To determine cultivar-specific effects on colonization by E. coli O157, we used 12 different cultivars of lettuce inoculated with a chromosomally lux-marked strain of E. coli O157:H7. Lettuce seedlings grown gnotobiotically in vitro did exhibit a differential cultivar-specific response to E. coli O157 colonization, although importantly there was no relationship between metabolic activity (measured as bioluminescence) and cell numbers. Metabolic activity was highest and lowest on the cultivars Vaila-winter gem and Dazzle respectively, and much higher in endophytic and tightly bound cells than in epiphytic and loosely bound cells. The cultivar effect was also evident in the rhizosphere of plants grown in compost, which suggests that cultivar-specific root exudate influences E. coli O157 activity. However, the influence of cultivar in the rhizosphere was the opposite to that in the phyllosphere, and the higher number and activity of E. coli O157 cells in the rhizosphere may be a consequence of them not being able to gain entry to the plant as effectively. If metabolic activity in the phyllosphere corresponds to a more prepared state of infectivity during human consumption, leaf internalization of E. coli O157 may pose more of a public health risk than leaf surface contamination alone.     

Survival of Escherichia coli O157:H7 in ruminal or fecal contents incubated with corn or wheat dried distillers' grains with solubles

Dried distillers' grain with solubles (DDGS) is a by-product of ethanol production, and its use as cattle feed has increased as a result of the expansion of the fuel ethanol industry. However, the inclusion of corn DDGS into feedlot diets may increase the shedding of Escherichia coli O157:H7. This study investigated whether corn or wheat DDGS at 2 concentrations (20% or 40% vs. 100% barley grain) affected the survival of E.?coli O157:H7 in incubations of ruminal digesta and feces. Neither the type nor the level of DDGS had any effect on fermentation or the survival of E. coli O157:H7 in ruminal digesta. However, there was a time by DDGS interaction (p?< 0.05), where the numbers of E.?coli O157:H7 in feces did not differ after 4 or 12?h of incubation but were greater after 24?h in both 40% wheat and 40% corn DDGS as compared with other treatments. Additionally, after 24?h, the numbers of E. coli O157:H7 were greater in fecal incubations with corn DDGS than with wheat DDGS (p?< 0.05). The differences in the numbers of E.?coli O157:H7 were not attributable to changes in pH or in concentrations of volatile fatty acids in the media. These results suggest that the inclusion of high levels of corn or wheat DDGS in feedlot diets of cattle may encourage the survival of E. coli O157:H7 in feces.     

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.     

Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Variable colonization of chickens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs.

Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen.     

Persistence, dissipation, and activity of Escherichia coli O157:H7 within sand and seawater environments

Runoff from agricultural land into watercourses may transport and deposit animal-derived waste contaminated with Escherichia coli O157:H7 onto beaches, which may in turn lead to human infection. To simulate contamination, freshwater mixed with cattle slurry containing E. coli O157:H7 was added to sand from three recreational beaches. The sand was then maintained in a dry state (nontidal) or subjected to a repeated seawater tidal simulation. The pathogen could still be recovered from all sands by day 5. Although survival of the pathogen did not statistically vary between sands of different origin under nontidal conditions, significant differences in numbers occurred between sands when subject to tidal simulation. In the tidal simulations, a considerable proportion of the E. coli O157:H7 rapidly dissipated from sand into the seawater. In a separate experiment, the activity of bioluminescent (lux-marked) E. coli O157:H7 cells was monitored in various mixtures of contaminated runoff water and seawater over 5 days. Pathogen activity declined with increasing seawater concentration; however, cells remained viable in all treatments over the 5-day period. The addition of nutrients to water rapidly increased pathogen activity in all treatments. Our findings highlight the resilience of E. coli O157:H7 in aquatic and marine environments.     

APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7

Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7. The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi. PhiV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E. coli O157:H7 strain R508. PhiV10::luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon. The bacteriophage transduced n-decanal-dependent bioluminescence to E. coli O157:H7 strain R508 that was measurable approximately 1 h post infection.     

Estimating the stability of Escherichia coli O157:H7 survival in manure-amended soils with different management histories

The objective of this study is to describe survival of Escherichia coli O157:H7 populations in manure-amended soils in terms of population stability, i.e. the temporal variation around the decline curve, in relation to soil characteristics indicative of soil health. Cow manure inoculated with E. coli O157:H7 was mixed with 18 pairs of organically and conventionally managed soils (10% of manure, kg kg(-1)). For four of the soil pairs, also three different manure densities (5%, 10% and 20%) were compared. All soil-manure mixtures were incubated for 2 months, and population densities of E. coli O157:H7 were quantified weekly. De-trending of survival data was done by modified logistic regression. The residual values were used to assess variation in the changes of E. coli O157:H7 populations by performing the approximate entropy (ApEn) procedure. The term irregularity is used to describe this variation in ApEn literature. On average, the decline of E. coli O157:H7 was more irregular in conventional and loamy soils than in organic and sandy soils (P < 0.05). Multiple regression analysis of irregularity of E. coli O157:H7 survival on 13 soil characteristics showed a positive relation with the ratio of copiotrophic/oligotrophic bacteria, suggesting greater instability at higher available substrate concentrations. Incremental rates of manure application significantly changed the irregularity for conventional soils only. Estimation of temporal variation of enteropathogen populations by the ApEn procedure can increase the accuracy of predicted survival time and may form an important indication for soil health.     

Effect of Volumetric Water Content and Clover (Trifolium incarnatum) on the Survival of Escherichia coli O157:H7 in a Soil Matrix

Studies aimed at understanding Escherichia coli O157:H7 soil survival dynamics are paramount due to their inevitable introduction into the organic vegetable production systems via animal manure-based fertilizer. Therefore, a greenhouse study was conducted to determine the survival of E. coli O157:H7 in highly controlled soil matrices subjected to two variable environmental stressors: (1) soil volumetric water content (25 or 45 % VWC), and (2) the growth of clover (planted or unplanted). During the 7-week study, molecular-based qPCR analyses revealed that E. coli O157:H7 survival was significantly lower in soils maintained at either near water-holding capacity (45 % VWC) or under clover growth. The significant reduction under clover growth was only observed when E. coli populations were determined relative to all bacteria, indicating the need to further study the competition between E. coli O157:H7 and the total bacterial community in organic soils. Given the significant effect of clover on E. coli O157:H7 survival under different moisture conditions in this greenhouse-based study, this work highlights the antimicrobial potential of clover exudates in arable soils, and future work should concentrate on their specific mechanisms of inhibition; ultimately leading to the development of crop rotations/production systems to improve pre-harvest food safety and security in minimally processed, ready-to-eat and organic production systems.     

Detection of Campylobacter and Escherichia coli O157:H7 from filth flies by polymerase chain reaction

Flies (Diptera: Muscidae) that breed in faeces and other organic refuse (filth flies) have been implicated as vectors of pathogenic bacteria including Escherichia coli O157:H7, which cause haemorrhagic colitis in humans, and Campylobacter, which is the principal causative agent of human enteritis. The potential role of filth flies in the epidemiology of these pathogens in the United States was investigated by examining the prevalence of Campylobacter spp. and E. coli O157:H7 from two Arkansas turkey facilities. Polymerase chain reaction was conducted on DNA extractions of individual Musca domestica Linnaeus, Stomoxys calcitrans (Linnaeus), Hydrotaea aenescens (Wiedemann), Adia cinerella Fallen and turkey faecal samples using primers specific for E. coli H7, O157 and Campylobacter spp. Culturing verified that the flies were carrying viable Campylobacter spp. and E. coli O157:H7. Results from this study indicated that M. domestica, S. calcitrans, H. aenescens and Anthomyids are capable of carrying Campylobacter in North American poultry facilities and that the E. coli O157:H7 is carried by house flies and black dump flies associated with poultry. This PCR method provided a rapid and effective method to identify Campylobacter spp. and E. coli O157:H7 directly from individual filth flies.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Colonization of chicken cecae by Escherichia coli associated with hemorrhagic colitis.

Bacterial enumeration, histologic examination, and immunoperoxidase staining demonstrated the ability of an Escherichia coli strain associated with hemorrhagic colitis (serotype O157:H7) to colonize chicken cecae for up to 90 days postinoculation after a peroral challenge at 1 day of age. The bacteria induced mild, transient, mucous membrane damage confined to the proximal cecae of healthy, normal-appearing chickens, principally at 14 to 28 days postinoculation. Attachment, effacement, and penetration of the cecal surface epithelium by E. coli O157:H7 were observed. With the exception of splenic, hepatic, and cecal tonsil immune-related changes and cecal damage and colonization, no other organ systems or portions of the gastrointestinal tract were affected by the bacteria. Bacterial counts indicated that E. coli O157:H7 was predominantly present in the cecae (often at levels greater than 10(6) CFU/g of tissue and contents) and to a lesser extent in the colon. Our results suggest that E. coli O157:H7 colonizes chicken cecae and is passed through the colon with fecal excrement. The ability of this organism to colonize chicken cecae indicates that chickens may serve as hosts and possibly as reservoirs for E. coli O157:H7.     

Mode of Salmonella and Escherichia coli O157:H7 inactivation by a stabilized oxychloro-based sanitizer

AIM: To determine the mechanisms by which a stabilized oxychloro (SOC)-based sanitizer, applied to decontaminate seeds destined for sprout production, inactivates Escherichia coli O157:H7 ph1 and Salmonella serotype Meleagridis. MATERIALS AND RESULTS: The action of SOC on the metabolism, membrane and DNA integrity of Salmonella and E. coli O157:H7 was studied. In both pathogens, there was an oxidative burst and depletion of intracellular glutathione (GSH) upon initial exposure to 200 ppm SOC. Metabolic activity, measured via bioluminescence, decreased over a 4-h period in E. coli O157:H7 ph1 cells exposed to SOC. Membrane integrity, assessed through viability staining, decreased progressively over 23 h when exposed to SOC. The appearance of auxotrophic mutants suggested that DNA damage had also occurred. Enzymes rich in disulfide bonds (alkaline phosphatase and protease) were sensitive to the chlorite-based sanitizer. Through challenging other microbial types, it was found that Gram positive had higher tolerance to SOC than Gram negatives with the exception of Salmonella. MS2 bacteriophage was highly sensitive; however, Bacillus endospores were not inactivated by SOC. CONCLUSIONS: SOC inactivates E. coli O157:H7 and Salmonella through GSH oxidation and disruption of disulfide bonds. Ultimately, membrane damage resulting from prolonged exposure to SOC leads to the loss of cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide a basis for understanding why extended treatment times are required to inactivate bacteria using SOC.