Effect of Sand and Sawdust Bedding Materials on the Fecal Prevalence of Escherichia coli O157:H7 in Dairy Cows

Farm management practices that reduce the prevalence of food-borne pathogens in live animals are predicted to enhance food safety. To ascertain the potential role of livestock bedding in the ecology and epidemiology of Escherichia coli O157:H7 on farms, the survival of this pathogen in used-sand and used-sawdust dairy cow bedding was determined. Additionally, a longitudinal study of mature dairy cattle housed on 20 commercial dairy farms was conducted to compare the prevalence of E. coli O157:H7 in cattle bedded on sand to that in cattle bedded on sawdust. E. coli O157:H7 persisted at higher concentrations in used-sawdust bedding than in used-sand bedding. The overall average herd level prevalence (3.1 versus 1.4%) and the number of sample days yielding any tests of feces positive for E. coli O157:H7 (22 of 60 days versus 13 of 60 days) were higher in sawdust-bedded herds. The choice of bedding material used to house mature dairy cows may impact the prevalence of E. coli O157:H7 on dairy farms.     

Evaluation of Animal Genetic and Physiological Factors That Affect the Prevalence of Escherichia coli O157 in Cattle

Controlling the prevalence of Escherichia coli O157 in cattle at the pre-harvest level is critical to reduce outbreaks of this pathogen in humans. Multilayers of factors including the environmental and bacterial factors modulate the colonization and persistence of E. coli O157 in cattle that serve as a reservoir of this pathogen. Here, we report animal factors contributing to the prevalence of E. coli O157 in cattle. We observe the lowest number of E. coli O157 in Brahman breed when compared with other crosses in an Angus-Brahman multibreed herd, and bulls excrete more E. coli O157 than steers in the pens where cattle were housed together. The presence of super-shedders, cattle excreting >10⁵ CFU/rectal anal swab, increases the concentration of E. coli O157 in the pens; thereby super-shedders enhance transmission of this pathogen among cattle. Molecular subtyping analysis reveal only one subtype of E. coli O157 in the multibreed herd, indicating the variance in the levels of E. coli O157 in cattle is influenced by animal factors. Furthermore, strain tracking after relocation of the cattle to a commercial feedlot reveals farm-to-farm transmission of E. coli O157, likely via super-shedders. Our results reveal high risk factors in the prevalence of E. coli O157 in cattle whereby animal genetic and physiological factors influence whether this pathogen can persist in cattle at high concentration, providing insights to intervene this pathogen at the pre-harvest level.     

Quantification of Survival of Escherichia coli O157:H7 on Plants Affected by Contaminated Irrigation Water

Enterohemorrhagic E. coli O157: H7 (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, with further complications such as hemolytic‐uremic syndrome (HUS). The aim of this study was to use the real‐time PCR method to quantify the survival of Escherichia coli O157:H7/pGFP in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non‐rhizosphere soils (sand and clay) irrigated with contaminated water and compare the results obtained between real‐time PCR method and conventional plate counts. The real‐time PCR probe was designed to hybridize with the (eae) gene of E. coli O157:H7. The probe was incorporated into real‐time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non‐rhizosphere soils irrigated with water artificially contaminated with E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real‐time PCR was 2.3 × 10³ in the rhizosphere and phyllosphere samples. E. coli O157:H7 survived longer in rhizosphere soil than the non‐rhizosphere soil. The concentration of E. coli O157:H7/pGFP in rhizosphere soils was ≥ 10⁴ CFU/g in both soils at day 12 based on both plate count and real time PCR, with the clay soil significantly (P = 0.05) higher than the sandy soil. This data showed that E. coli O157H:7 can persist in the environment for more than 50 d, and this may pose some risk for both animal and human infection and provides a very significant pathway for pathogen recontamination in the environment.     

Examination of indigenous microbiota and survival of Escherichia coli 0157 and Salmonella in a paper milling environment

Aims: A public beach was frequently cited for health advisories because of high Escherichia coli levels, the source suspected to be a paper mill located upstream. This investigation sought to confirm whether or not the paper mill was the pollution source, and to characterize the risk to recreational bathers imposed by the source. Methods and Results: Quantification of E. coli in river water collected at incremental distances showed that paper mill effluent caused elevated E. coli levels in beach samples. Samples collected throughout the mill were variably positive for heterotrophic bacteria, total coliforms and E. coli, but negative for pathogenic E. coli O157 and Salmonella. Escherichia coli O157 or Salmonella spiked into mill samples (4·2 log₁₀ or 5·6 log₁₀ CFU per 100 ml, respectively) fell below detection levels within 14–24 h in raw (unaltered) samples, while in heat‐sterilized replicates, the counts remained at initial levels or increased over 36 h. Conclusions: Pathogenic E. coli O157 and Salmonella were not isolated from paper mill samples. The absence of native bacteria allowed the survival of pathogens, while their presence accelerated pathogen decline. Significance and Impact of the Study: The co‐existence of paper mill and swimming beach may be reasonable for now in spite of the limitations of an E. coli‐based assay for beach water.     

Occurrence of verocytotoxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> in the faeces of free-ranging wild lagomorphs in southwest Spain

Verocytotoxin-producing Escherichia coli (VTEC) is an important group of emerging food-borne pathogens and represents a major public health concern worldwide. The aim of this work was to analyse faecal samples from hunted wild lagomorphs for the presence of E. coli O157:H7 and non-O157 VTEC. During two hunting seasons, faecal samples from 241 animals were collected, including wild rabbit (Oryctolagus cuniculus) and hare (Lepus granatensis) and were examined for VTEC. Overall, VTEC were detected and isolated in four (1.66%) of the 241 animals sampled. E. coli O157:H7 was isolated only from one of 124 (0.81%) wild rabbit faecal samples while non-O157 VTEC were isolated from two of 124 (1.61%) wild rabbit faecal samples and one of 117 (0.85%) hare faecal samples. VTEC isolates obtained in the present study (four in total) belonged to four different O:H serotypes, including two serotypes (O84:H− and O157:H7) previously associated with human infection and in particular with causing the life-threatening haemolytic–uraemic syndrome. Although these results indicate a low prevalence of VTEC infection in free-ranging wild lagomorphs, they may play an important role as a source of exposure to human beings and livestock and as a vehicle for dispersing these pathogens. These findings have implications for the zoonotic risk to hunters, people consuming meat from wild animals and others in contact with wild animal faeces, and also in the development of programmes for controlling VTEC at the farm level.     

Cattle Water Troughs as Reservoirs of Escherichia coli O157

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.     

Dose-Response Envelope for Escherichia coli O157:H7

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

Direct Detection by PCR of Escherichia coli O157 and Enteropathogens in Patients with Bloody Diarrhea

Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 10¹ CFUs/0.5 g of stool sample. PCR was completed within 6 hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy-to-read screening method for the detection of Shiga-like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.     

Soil Conditions That Can Alter Natural Suppression of Escherichia coli O157:H7 in Ohio Specialty Crop Soils

Food-borne pathogen persistence in soil fundamentally affects the production of safe vegetables and small fruits. Interventions that reduce pathogen survival in soil would have positive impacts on food safety by minimizing preharvest contamination entering the food chain. Laboratory-controlled studies determined the effects of soil pH, moisture content, and soil organic matter (SOM) on the survivability of this pathogen through the creation of single-parameter gradients. Longitudinal field-based studies were conducted in Ohio to quantify the extent to which field soils suppressed Escherichia coli O157:H7 survival. In all experiments, heat-sensitive microorganisms were responsible for the suppression of E. coli O157 in soil regardless of the chemical composition of the soil. In laboratory-based studies, soil pH and moisture content were primary drivers of E. coli O157 survival, with increases in pH after 48 h (P = 0.02) and decreases in moisture content after 48 h (P = 0.007) significantly increasing the log reduction of E. coli O157 numbers. In field-based experiments, E. coli O157 counts from both heated and unheated samples were sensitive to both season (P = 0.004 for heated samples and P = 0.001 for unheated samples) and region (P = 0.002 for heated samples and P = 0.001 for unheated samples). SOM was observed to be a more significant driver of pathogen suppression than the other two factors after 48 h at both planting and harvest (P = 0.002 at planting and P = 0.058 at harvest). This research reinforces the need for both laboratory-controlled experiments and longitudinal field-based experiments to unravel the complex relationships controlling the survival of introduced organisms in soil.     

The effect of the native bacterial community structure on the predictability of E. coli O157:H7 survival in manure‐amended soil

Aims: The survival capability of pathogens like Escherichia coli O157:H7 in manure‐amended soil is considered to be an important factor for the likelihood of crop contamination. The aim of this study was to reveal the effects of the diversity and composition of soil bacterial community structure on the survival time (ttd) and stability (irregularity, defined as the intensity of irregular dynamic changes in a population over time) of an introduced E. coli O157:H7 gfp‐strain were investigated for 36 different soils by means of bacterial PCR‐DGGE fingerprints. Methods and Results: Bacterial PCR‐DGGE fingerprints made with DNA extracts from the different soils using bacterial 16S‐rRNA‐gene‐based primers were grouped by cluster analysis into two clusters consisting of six and 29 soils and one single soil at a cross‐correlation level of 16% among samples per cluster. Average irregularity values for E. coli O157:H7 survival in the same soils differed significantly between clusters (P = 0·05), whereas no significant difference was found for the corresponding average ttd values (P = 0·20). The irregularity was higher for cluster 1, which consisted primarily of soils that had received liquid manure and artificial fertilizer and had a significant higher bacterial diversity and evenness values (P < 0·001). Conclusions: Bacterial PCR‐DGGE fingerprints of 36 manure‐amended soils revealed two clusters which differed significantly in the stability (irregularity) of E. coli O157 decline. The cluster with the higher irregularity was characterized by higher bacterial diversity and evenness. Significance and Impact of the Study: The consequence of a high temporal irregularity is a lower accuracy of predictions of population behaviour, which results in higher levels of uncertainty associated with the estimates of model parameters when modelling the behaviour of E. coli O157:H7 in the framework of risk assessments. Soil community structure parameters like species diversity and evenness can be indicative for the reliability of predictive models describing the fate of pathogens in (agricultural) soil ecosystems.     

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.     

The potential for the control of Escherichia coli O157 in farm animals

The presence of Escherichia coli O157 in the faeces of farm animals appears to provide a primary route for human infection, either through physical contact or by contamination of the food chain. Controlling the survival and proliferation of this pathogen in the ruminant gut could offer a measure of protection in the short term, and ultimately complement alternative biotechnological based solutions. Normally, E. coli is greatly outnumbered in the ruminant gut by anaerobic bacteria, producers of weak acids inhibitory to the growth of this species. Withdrawal of feed prior to animal slaughter reduces the concentration of these acids in the gut and may be accompanied by the proliferation of E. coli. There are conflicting reports concerning the effects of changes in the ruminant diet upon faecal shedding of E. coli O157. It is contended that it is important to identify animal husbandry methods or feed additives that may be accompanied by an increased risk of proliferation of this pathogen. Greater understanding of the mechanisms involved in bacterial survival in the presence of weak acids, in the interactions between E. coli and other gut bacteria, and of the effects of some antibacterial plant secondary plant compounds on E. coli, could lead to the development of novel control methods.     

Capacity of the bovine intestinal mucus and its components to support growth of Escherichia coli O157:H7

Colonization of the gastrointestinal tract of cattle by Shiga toxin-producing Escherichia coli increases the risk of contamination of food products at slaughter. Our study aimed to shed more light on the mechanisms used by E. coli O157:H7 to thrive and compete with other bacteria in the gastrointestinal tract of cattle. We evaluated, in vitro, bovine intestinal mucus and its constituents in terms of their capacity to support growth of E. coli O157:H7 in presence or absence of fecal inoculum, with and without various enzymes. Growth of E. coli O157:H7 and total anaerobic bacteria were proportionate to the amount of mucus added as substrate. Growth of E. coli O157:H7 was similar for small and large intestinal mucus as substrate, and was partially inhibited with addition of fecal inoculum to cultures, presumably due to competition from other organisms. Whole mucus stimulated growth to the greatest degree compared with other compounds evaluated, but the pathogen was capable of utilizing all substrates to some extent. Addition of enzymes to cultures failed to impact growth of E. coli O157:H7 except for neuraminidase, which resulted in greater growth of E. coli O157 when combined with sialic acid as substrate. In conclusion, E. coli O157 has capacity to utilize small or large intestinal mucus, and growth is greatest with whole mucus compared with individual mucus components. There are two possible explanations for these findings (i) multiple substrates are needed to optimize growth, or alternatively, (ii) a component of mucus not evaluated in this experiment is a key ingredient for optimal growth of E. coli O157:H7.     

Diversity of Escherichia coli O157 in a longitudinal farm study using multiple-locus variable-number tandem-repeat analysis

Aims: To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). Methods and Results: Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. Conclusions: The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. Significance and Impact of the Study: There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method.     

Longitudinal Study of Fecal Shedding of Escherichia coli O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.     

The history and evolution of Escherichia coli O157 and other Shiga toxin-producing E. coli

Shiga toxin-producing Escherichia coli (STEC) O157 is a formidable human pathogen with the capacity to cause large outbreaks of gastrointestinal illness. The known virulence factors of this organism are encoded on phage, plasmid and chromosomal genes. There are also likely to be novel, as yet unknown virulence factors in this organism. Many of these virulence factors have been acquired by E. coli O157 by transfer from other organisms, both E. coli and non-E. coli species. By examination of biochemical and genetic characteristics of various E. coli O157 strains and the relationships with other organisms, an evolutionary pathway for development of E. coli O157 as a pathogen has been proposed. E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin. During the evolutionary process, Shiga toxins, the pO157 plasmid and other characteristics which enhanced virulence were acquired and other functions such as motility, sorbitol fermentation and β-glucuronidase activity were lost by some strains. It is likely that E. coli O157 is constantly evolving, and changes can be detected in genetic patterns during the course of infection. A variety of mechanisms may be responsible for the development of the virulent phenotype that we see today. Such changes include uptake of as yet uncharacterised virulence factors, possibly enhanced by a mutator phenotype, recombination within virulence genes to produce variant genes with different properties, loss of large segments of DNA (black holes) to enhance virulence and possible adaptation to different hosts. Although little is known about the evolution of non-O157 STEC it is likely that the most virulent clones evolved in a similar manner to E. coli O157. This revised version was published online in November 2006 with corrections to the Cover Date.     

Design of Localized Surface Plasmon Resonance (LSPR) Biosensor for Immunodiagnostic of E. coli O157:H7 Using Gold Nanoparticles Conjugated to the Chicken Antibody

E. coli O157:H7 is one of the most important pathogens in food-borne diseases and is the main cause of the pseudo pandemic development of hemorrhagic colitis and hemolytic uremic syndrome. Also E. coli O157:H7 is the most common serotype of Shiga-toxin-producing E. coli. Traditional methods for detecting E. coli O157:H7 are expensive, time-consuming, and less sensitive. A method with high sensitivity and high-resolution optical detection is utilizes the LSPR property of spherical gold nanoparticles (GNP). In this work, we constructed a novel nano-bio probe to detect E. coli O157:H7 by synthesizing citrate gold nanoparticle conjugated (non-covalent bond) with specific chicken anti-E. coli O157:H7 antibody (IgY) by changing the pH of the nanoparticles’ environment. UV-visible and DLS methods were used to confirm the bonding between the antibody and nanoparticles and the LSPR sensitivity of the nano-bio probe was evaluated by ELISA method. We could optically detect this bacterium in less than 2 h by measuring the LSPR band λ max shifts of GNPs. The sensitivity of this novel biosensor was determined by about 10 CFU/ml, using the LSPR property of spherical gold nanoparticles. So that, the LSPR λ max red shifted from 530 to 543 nm in presence of 10 CFU bacterium. In conclusion, this nano biosensor can be used to detect this important pathogen among the clinical specimens.

     

Viability of zoonotic pathogens Escherichia coli and Salmonella in swine manure slurries with and without a urease inhibitor and thymol

Aims: To determine the effects of urine, a urease inhibitor and/or an odour‐reducing antimicrobial compound, on zoonotic pathogens in swine manure slurries. Methods and Results: Swine faeces were collected and blended with different amounts of urine. Marker strains of Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157:H7 were inoculated into the manure slurries with or without the urease inhibitor, N‐(n‐butyl) thiophosphoric triamide, or the antimicrobial compound thymol. In slurries containing approximately 1 : 1 or 2 : 1 of urine and faeces, the decreases in colony forming units (CFU) of Salm. Typhimurium and E. coli were similar and few counts were observed after 14 days. When the urine content of the slurry was increased to 5 : 1, both strains died off rapidly. When NBPT was added to the slurries, pathogen CFU was not affected in the 1 : 1 slurry. The 2 : 1 and 5 : 1 slurries had higher urea content and NBPT increased CFU of both pathogens. Thymol addition rapidly killed the pathogens and few CFU of Salmonella or E. coli were observed after day 1. Conclusions: These experiments demonstrate that urea hydrolysis in swine manure affects pathogens. Inhibition of urea hydrolysis may promote pathogen viability. Significance and Impact of the Study: Manure amendments that prevent urea hydrolysis may promote pathogen persistence. Additional treatments with antimicrobials may be required to kill pathogens.     

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.