Variable agronomic practices, cultivar, strain source and initial contamination dose differentially affect survival of Escherichia coli on spinach

Aims: Greenhouse and field trials were conducted under different agronomic practices and inoculum doses of environmental Escherichia coli and attenuated E. coli O157:H7, to comparatively determine whether these factors influence their survival on leaves and within the rhizosphere. Methods and Results: Hydroponic conditions: E. coli spray‐inoculated at log 4 CFU ml^?1 was recovered from leaf surfaces at a mean population of 1·6 log CFU g^?1 at 15 days. E. coli O157:H7 sprayed at log 2 or 4 CFU ml^?1 levelled off on spinach leaf surfaces at a mean average population of 1·4 log CFU g^?1 after 14 days, regardless of initial dose. Quantitative recovery was inconsistent across leaf developmental age. Field conditions: Average populations of E. coli O157:H7 spray‐inoculated at log 1·45 or 3·4 CFU m^?2 levelled off at log 1·2 CFU g^?1 over a 14‐day period. Pathogen recovery from leaves was inconsistent when compared to regularly positive detection on basal shoot tissue. Pathogen recovery from soil was inconsistent among sampling locations. Moisture content varied up to 40% DW and was associated with 50% (P < 0·05) decrease in positive locations for E. coli O157:H7 but not for E. coli. Conclusions: Overall, similar populations of environmental E. coli and E. coli O157:H7 were recovered from plants despite differences in inoculum dose and agronomic conditions. Strain source had a significant impact on the quantitative level and duration of survival on leaves and in soil. Water availability appeared to be the determinant factor in survival of E. coli and E. coli O157:H7; however, E. coli showed greater environmental fitness. Significance and Impact of the Study: Persistence of surrogate, indicator E. coli and E. coli O157:H7, irrespective of variable growing conditions in spinach is predominantly limited by water availability, strain source and localization within the plant. These findings are anticipated to ultimately be adopted into routine and investigative pathogen testing protocols and mechanical harvest practices of 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.     

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.     

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.     

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.     

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.     

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

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.     

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.     

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.     

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.     

Differences in internalization and growth of Escherichia coli O157:H7 within the apoplast of edible plants,spinach and lettuce,compared with the model species Nicotiana benthamiana

Internalization of food‐borne bacteria into edible parts of fresh produce plants represents a serious health risk. Therefore, internalization of verocytotoxigenic E. coli O157:H7 isolate Sakai was assessed in two species associated with outbreaks, spinach (Spinacia oleracea) and lettuce (Lactuca sativa) and compared to the model species Nicotiana benthamiana. Internalization occurred in the leaves and roots of spinach and lettuce throughout a 10 day time‐course. The plant species, tissue type and inoculum dose all impacted the outcome. A combination of low inoculum dose (~10² CFU) together with light microscopy imaging highlighted marked differences in the fate of endophytic E. coli O157:H7 Sakai. In the fresh produce species, bacterial growth was restricted but viable cells persisted over 20 days, whereas there was > 400‐fold (~2.5 Log₁₀) increase in growth in N. benthamiana. Colony formation occurred adjacent to epidermal cells and mesophyll cells or close to vascular bundles of N. benthamiana and contained components of a biofilm matrix, including curli expression and elicitation, extracellular DNA and a limited presence of cellulose. Together the data show that internalization is a relevant issue in crop production and that crop species and tissue need to be considered as food safety risk parameters.     

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.     

Evaluating the Effect of Environmental Factors on Pathogen Regrowth in Compost Extract

Pathogenic microorganisms may survive the composting process in low numbers and subsequently regrow to high levels under favorable conditions. The objective of this study was to investigate the regrowth potential of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in dairy-based composts under different environmental conditions. Water extract of commercially available dairy compost was used as a model system. Cocktails of five rifampin-resistant strains of each pathogen previously grown in reduced nutrient media (1/2 or 1/10 strength of tryptic soy broth, TSB) were inoculated into water extract of compost of different ratios (1:2,1:5, and 1:10, w/v), and then stored at 35°C or 22°C for 7 days. The strains exhibiting greatest survival or regrowth were identified by pulsed-field gel electrophoresis (PFGE). At 22°C, both E. coli O157:H7 and L. monocytogenes multiplied in all compost extracts, whereas Salmonella spp. regrew in both 1:2 and 1:5 compost extracts but not in 1:10. For all three pathogens, incubation at 22°C provides better conditions for regrowth than at 35°C. Both Salmonella and E. coli O157:H7 previously adapted to nutrient-limited broth (1/10 strength of TSB) regrew in compost extracts to higher populations than the control cultures grown previously in full strength of TSB. In the absence of indigenous microorganisms, all three pathogens regrew even in the most diluted sterile compost extract (1:10) with growth potentials ranging from 2.30 to 3.59 log CFU/ml. In nonsterile compost extract with ca. 5 log CFU/ml of background microorganisms, all three pathogens regrew only in the most concentrated compost extract (1:2) with much less population increases ranging from 0.70 to 1.43 log CFU/ml. Compost extract samples of all ages supported the regrowth of both Salmonella and E. coli O157:H7 with population increases ranging from 0.95 to 2.32 log CFU/ml. The PFGE patterns for E. coli O157:H7 isolates from sterile compost extracts matched with either the spinach outbreak strain or an avirulent B6914 strain. These results demonstrated that compost extract of dairy-based compost contained sufficient nutrients for pathogen regrowth. Cultures previously adapted to low nutrient media regrew to higher populations than control cultures; however, indigenous microflora suppressed the pathogen regrowth in compost extract, especially at 35°C.     

Assessments of Total and Viable Escherichia coli O157:H7 on Field and Laboratory Grown Lettuce

Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 10⁶ CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 10⁴ greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable.