Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting

Aims:  Zero‐valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. Methods:  A field‐scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c. 8·5 log CFU 100 ml^?1 of Escherichia coli O157:H12 was introduced to all three column treatments in 20‐l doses. Filtered waters were subsequently overhead irrigated to ‘Tyee’ spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. Results:  ZVI filters inactivated c. 6 log CFU 100 ml^?1E. coli O157:H12 during filtration on day 0, significantly (P < 0·05) more than S filter (0·49 CFU 100 ml^?1) when compared to control on day 0 (8·3 log CFU 100 ml^?1). On day 0, spinach plants irrigated with ZVI‐filtered water had significantly lower E. coli O157 counts (0·13 log CFU g^?1) than spinach irrigated with either S‐filtered (4·37 log CFU g^?1) or control (5·23 log CFU g^?1) water. Soils irrigated with ZVI‐filtered water contained E. coli O157:H12 populations below the detection limit (2 log CFU g^?1), while those irrigated with S‐filtered water (3·56 log CFU g^?1) were significantly lower than those irrigated with control (4·64 log CFU g^?1). Conclusions:  ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. Significance and Impact of the Study:  Zero‐valent ion treatment may be a cost‐effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.     

Survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity

Aims: To determine survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity (r.h.). Methods and Results: Spinach leaves were inoculated with suspensions of E. coli O157:H7 in distilled water (DW) and 0·1% peptone water (PW) and incubated at 4, 12 and 25°C and 43, 85 and 100% r.h. The number of E. coli O157:H7 on leaves (5·6 or 1·9 log CFU per leaf) inoculated using DW as a carrier medium increased significantly at 25°C and 100% r.h. within 120 h but remained constant or decreased significantly under other test conditions. E. coli O157:H7 on leaves (5·4 log CFU per leaf) inoculated using PW as a carrier increased significantly within 72 and 24 h, respectively, at 12 or 25°C and 100% r.h.; counts using a low inoculum (2·2 log CFU per leaf) increased significantly within 24 h at 25°C. Conclusions: Escherichia coli O157:H7 can colonize on spinach leaves at 12 or 25°C in a 100% r.h. environment. Organic matter in the inoculum carrier may provide protection and nutrients which enhance survival and colonization. Significance and Impact of the Study: Colonization of E. coli O157:H7 on spinach leaves as affected by organic matter in the inoculum, temperature and r.h. was determined. These observations will be useful when developing strategies to prevent growth of E. coli O157:H7 on pre‐ and postharvest spinach.     

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Antimicrobial efficacy of lactoferrin, its amidated and pepsin-digested derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens

Aims: To evaluate the in vitro bactericidal efficacy of lactoferrin (LF), its amidated (AMILF) and pepsin‐digested (PDLF) derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens. Methods and Results: PDLF exhibited the most potent bactericidal efficacy on E. coli O157:H7 (>2·5 log₁₀ CFU ml^?1 reduction at concentrations ≥1 mg ml^?1), and AMILF on Ser. liquefaciens (1 log₁₀ CFU ml^?1 reduction at 0·25–0·50 mg ml^?1). Some combinations of LF with PDLF or AMILF showed a slight synergy on E. coli O157:H7 and Ser. liquefaciens. However, all combinations of AMILF with PDLF were less active than the sum of the individual effects of the two antimicrobials. Production of capsular polysaccharide by bacteria might be involved in antimicrobial resistance. Conclusions: Escherichia coli O157:H7 and Ser. liquefaciens showed marked differences in the sensitivity to LF and its derivatives. E. coli O157:H7 was strongly inhibited by PDLF, whereas the effect of LF and its derivatives on Ser. liquefaciens was weak to negligible. Significance and Impact of the Study: PDLF was the most promising of the tested antimicrobials on E. coli O157:H7. However, the resistance of Ser. liquefaciens to LF and its derivatives hinders their use in the food industry.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Proposed mechanism of inactivating Escherichia coli O157:H7 by ultra‐high pressure in combination with tert‐butylhydroquinone

Aims: Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert‐butylhydroquinone (TBHQ) during ultra‐high pressure (UHP) treatment. Methods and Results: Escherichia coli O157:H7 EDL‐933, and 14 E. coli K12 strains with mutations in selected genes, were treated with dimethyl sulfoxide solution of TBHQ (15–30 ppm), and processed with UHP (400 MPa, 23 ± 2°C for 5 min). Treatment of wild‐type E. coli strains with UHP alone inactivated 2·4–3·7 log CFU ml^?1, whereas presence of TBHQ increased UHP lethality by 1·1–6·2 log CFU ml^?1; TBHQ without pressure was minimally lethal (0–0·6 log reduction). Response of E. coli K12 mutants to these treatments suggests that iron–sulfur cluster‐containing proteins ([Fe–S]‐proteins), particularly those related to the sulfur mobilization (SUF system), nitrate metabolism, and intracellular redox potential, are critical to the UHP–TBHQ synergy against E. coli. Mutations in genes maintaining redox homeostasis and anaerobic metabolism were associated with UHP–TBHQ resistance. Conclusions: The redox cycling activity of cellular [Fe–S]‐proteins may oxidize TBHQ, potentially leading to the generation of bactericidal reactive oxygen species. Significance and Impact of the Study: A mechanism is proposed for the enhanced lethality of UHP by TBHQ against E. coli O157:H7. The results may benefit food processors using UHP–based preservation, and biologists interested in piezophilic micro‐organisms.     

The growth potential of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in dairy manure‐based compost in a greenhouse setting under different seasons

Aim: The pathogen growth in dairy compost was studied in a greenhouse setting under different seasons. Methods and Results: The five‐strain mixtures of each Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes were inoculated separately into dry compost to yield c. 1 log CFU g^?1. After acclimation at room temperature, the inoculated compost was initially adjusted to moisture levels of 10–50% and then kept in a greenhouse under different seasons. The populations of all three pathogens increased by 2·1–3·9 log CFU g^?1 within 3 days in autoclaved compost with initial moisture content of at least 40%. Listeria monocytogenes multiplied up to 2·4 log CFU g^?1 in compost with initial moisture content of 30% and was detected up to 28 days for all seasons, whereas populations of both E. coli O157:H7 and Salmonella increased by c. 1 log in compost with initial moisture content of 30% during winter months only. No pathogen growth in nonautoclaved compost was detected. Conclusion: Bacterial species, temperature, light intensity and moisture content affected the growth potential and survival of pathogens in compost when the population of background microflora was low. Significance and Impact of the Study: Keeping compost as dry as possible and maintaining certain levels of background microflora may be critical to prevent the growth of pathogens.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

European starlings (Sturnus vulgaris) challenged with Escherichia coli O157 can carry and transmit the human pathogen to cattle

Aims: European starlings (Sturnus vulgaris) are an invasive species in the United States and are considered a nuisance pest to agriculture. The goal of this study was to determine the potential for these birds to be reservoirs and/or vectors for the human pathogen Escherichia coli O157:H7. Materials and Results: Under biosecurity confinement, starlings were challenged with various doses of E. coli O157:H7 to determine a minimum infectious dose, the magnitude and duration of pathogen shedding, and the potential of pathogen transmission among starlings and between starlings and cattle. Birds transiently excreted E. coli O157:H7 following low‐dose inoculation; however, exposure to greater than 10^5.5 colony‐forming units (CFUs) resulted in shedding for more than 3 days in 50% of the birds. Colonized birds typically excreted greater than 10³ CFU g^?1 of faeces, and the pathogen was detected for as long as 14 days postinoculation. Cohabitating E. coli O157:H7‐positive starlings with culture‐negative birds or 12‐week‐old calves resulted in intra‐ and interspecies pathogen transmission within 24 h. Likewise, E. coli O157:H7 was recovered from previously culture‐negative starlings following 24‐h cohabitation with calves shedding E. coli O157:H7. Conclusions: European starlings may be a suitable reservoir and vector of E. coli O157:H7. Significance and Impact of the Study: Given the duration and magnitude of E. coli O157:H7 shedding by European starlings, European starlings should be considered a public health hazard. Measures aimed at controlling environmental contamination with starling excrement, on the farm and in public venues, may decrease food‐producing animal and human exposure to this pathogen.     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

Effect of continuous ohmic heating to inactivate Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice

Aims: The purpose of this study was to investigate the efficacy of continuous ohmic heating for reducing Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice. Methods and Results: Orange juice and tomato juice were treated with electric field strengths in the range of 25–40 V cm^?1 for different treatment times. The temperature of the samples increased with increasing treatment time and electric field strength. The rate of temperature change for tomato juice was higher than for orange juice at all voltage gradients applied. Higher electric field strength or longer treatment time resulted in a greater reduction of pathogens. Escherichia coli O157:H7 was reduced by more than 5 log after 60‐, 90‐ and 180‐s treatments in orange juice with 40, 35 and 30 V cm^?1 electric field strength, respectively. In tomato juice, treatment with 25 V cm^?1 for 30 s was sufficient to achieve a 5‐log reduction in E. coli O157:H7. Similar results were observed in Salm. Typhimurium and L. monocytogenes. The concentration of vitamin C in continuous ohmic heated juice was significantly higher than in conventionally heated juice (P < 0·05). Conclusions: Continuous ohmic heating can be effective in killing foodborne pathogens on orange juice and tomato juice with lower degradation of quality than conventional heating. Significance and Impact of the Study: These results suggest that continuous ohmic heating might be effectively used to pasteurize fruit and vegetable juices in a short operating time and that the effect of inactivation depends on applied electric field strengths, treatment time and electric conductivity.     

Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli

Aims: The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes. Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml^?1 reductions at 37°C. Multiplex‐PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes. Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes. Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa. Methods and Results: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g^?1 in manure and manure‐amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro‐Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high‐density inocula and enhanced the decline of low‐density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure‐amended soil than in manure. The 7 log CFU g^?1E. coli O157:H7 and Salm. Typhimurium survived for 49–84 and 63–98 days, while at 4 log CFU g^?1, persistence was 21–28 and 35–42 days, respectively. Conclusions: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. Significance and Impact of the Study: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.     

Prevalence of Escherichia coli O157:H7 on hides and faeces of ruminants at slaughter in two major abattoirs in Nigeria

Aim: To determine the occurrence of Escherichia coli O157: H7 in hides and faeces of slaughtered ruminants in Nigeria. Methods and Results: A total number of 320 animals were sampled from January to December covering the wet and harmattan seasons. Samples were obtained from the hides and faeces of animals at slaughter. The ISO (ISO 16654:2001, Microbiology of food and animal feedingstuffs – horizontal method for the detection of Escherichia coli O157) method for enrichment and isolation of E. coli O157 incorporating selective enrichment using modified tryptone soya broth with novobiocin (mTSBn),immunomagnetic separation and plating on sorbitol‐MacConkey agar with cefixime tellurite (CT‐SMAC) was used. Overall cattle had a prevalence rate of 49·4% followed by sheep and goats with rates of 6·3% and 2·5%, respectively. There was a significant difference in carriage of E. coli O157 among two different cattle breeds. Conclusions: The prevalence of E. coli O157: H7 is substantial from two abattoirs in the country. The carriage and shedding of E. coli O157: H7 did not differ with season but differed among groups of ruminants and among breeds of cattle in a tropical country. Significance and Impact of the Study: This is the first study on E. coli O157: H7 from abattoir operations in Nigeria. The study emphasizes the risk of E. coli O157: H7 along the meat chain and the need for concerted effort to limit it through best hygiene practices.     

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy

Aims: To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol. Methods and Results: Fourier transform‐infrared (FT‐IR) spectroscopy was used to analyse E. coli O157:H7 cells, after filtration or immunomagnetic collection. Partial least squares analysis of the spectra quantified live E. coli O157:H7 in the presence of dead cells with an R² > 0·996. Canonical variate analysis (CVA) not only differentiated between spectra of 100% dead and 100% live cells but also between 1% live : 99% dead and 100% dead. CVA using principal components also differentiated between the spectra of the differentially treated cells at a 95% confidence level, and Cooman plots showed clear separation between clusters of spectra of bacteria exposed to the different inactivation treatments. Mahalanobis distances (MD) corroborated the results of CVA. Conclusions: These results demonstrated the effectiveness of rapid cell collection and FT‐IR spectroscopy techniques to differentiate between live and dead E. coli O157:H7 cells. Significance and Impact of the Study: This technique has potential applications for use with foods subjected to various inactivation treatments.     

Recovery of E. coli O157 strains after exposure to acidification at pH 2

Aims: Rapid detection and selective isolation of E. coli O157:H7 strains have been difficult owing to the potential interference from background microflora present in high background food matrices. To help selectively isolate E. coli O157H7 strains, a useful plating technique that involved acidifying the cultures to pH 2 was evaluated with a large number of E. coli O157:H7 strains to ensure response to treatment was consistent across strains. Methods and Results: Escherichia coli O157, 46 strains including ATCC 35150, were acidified to pH 2 following enrichment and plated onto Tryptic Soy Agar + 0·6% Yeast Extract (TSA‐YE) and Sorbitol MacConkey Agar + cefixime and tellurite (CT‐SMAC). Samples were enumerated and modest decreases in plate counts were observed on TSA‐YE media, with a greater reduction observed on CT‐SMAC. Conclusions: The acid‐resistant character of E. coli O157:H7 is a consistent trait and may be used for improved isolation of the organism from mixed cultures. Significance and Impact of the Study: There was little difference observed between the commonly used laboratory strain E. coli O157:H7 35150 and 45 other strains of E. coli O157 when subjected to acidifying conditions prior to plating, demonstrating that an acid rinse procedure was equally effective across a wide variety of E. coli O157 strains and broadly applicable for isolating unknown strains from food samples.     

Leaching of bioluminescent Escherichia coli O157:H7 from sheep and cattle faeces during simulated rainstorm events

Aims: Development of a novel inoculation technique to improve the current methods of determining the leaching of Escherichia coli O157:H7 from faeces. Methods and Results: Ruminant faeces were inoculated with a high [c. 10⁷ colony forming units (CFU) g⁻¹] or low (c. 10⁴ CFU g⁻¹) load of a lux-marked strain of E. coli O157:H7 via injection, and subjected to four simulated heavy rainfall events. The population density and metabolic activity of E. coli O157:H7 recovered within the leachate was determined following each simulated rain event and compared with the indigenous E. coli population. The concentration of E. coli O157:H7 in the leachates followed a similar trend to that of nonpathogenic E. coli. Significantly greater densities of generic and pathogenic E. coli were recovered in the leachates generated from sheep faeces compared with cattle faeces. Pathogen metabolic activity was also significantly greater in sheep faeces. Conclusions: Our findings show that E. coli O157:H7 may readily leach from ruminant faeces during rain events. The bacterium leaches more freely from sheep faeces than from cattle faeces and displays greater metabolic activity within sheep leachate. Significance and Impact of the Study: A novel inoculation technique was developed that allowed the determination of both population density and cellular activity of E. coli O157:H7 in leachate derived from faeces.