Reaction of Surrogate Escherichia coli Serotype O157:H7 and Non-O157 Strains to Nutrient Starvation: Variation in Phenotype and Transcription of Stress Response Genes and Behavior on Lettuce Plants in the Field.

Preharvest contamination with bacteria borne by irrigation water may result in leafy vegetables serving as vehicles for transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The influence of starvation-associated stress on the behavior of non-toxin-producing strains of E. coli serotype O157:H7 and serotypes O26, O103, O111, and O145 was examined subsequent to their introduction to the phyllosphere of field-grown romaine lettuce as inocula simulating starved (96 h in sterile deionized water) and nutrient-depleted (24 h broth culture) cells. As with E. coli O157:H7, leaf populations of the non-O157 strains declined rapidly during the first 72 h postinoculation, displaying the biphasic decay curve typical of serotype O157:H7 isolates. Preinoculation treatment appeared not to influence decay rates greatly (P > 0.5), but strain-specific differences (persistence period and attachment proficiency) indicated that serotype O103:H2 strain PARC445 was a better survivor. Also assessed was the impact of preinoculation treatment on phenotypes key to leaf colonization and survival and the expression of starvation stress-associated genes. The 96-h starvation period enhanced biofilm formation in one strain but reduced motility and autoinducer 2 formation in all five study strains relative to those characteristics in stationary-phase cells. Transcription of rpoS, dps, uspA, and gapA was reduced significantly (P < 0.05) in starvation-stressed cells relative to that for exponential- and stationary-phase cultures. Strain-specific differences were observed; serotype O103:H2 PARC445 had greater downturns than did serotype O157:H7 and other non-O157 strains. Within this particular cohort, the behavior of the representative serotype O157:H7 strain, PARC443 (ATCC 700728), was not predictive of behavior of non-O157 members of this STEC group.

Aerated Steam Sanitization of Whole Fresh Cantaloupes Reduces and Controls Rind-Associated Listeria but Enhances Fruit Susceptibility to Secondary Colonization.

Recent bacterial illnesses and outbreaks associated with the consumption of fresh and fresh-cut fruit and vegetables emphasize the need to supply produce that is microbiologically safe while retaining its quality and nutrient value. We assessed the capacity of aerated steam to reduce initial levels and control the posttreatment proliferation of a 4-strain mixture of Listeria innocua, a surrogate for L. monocytogenes, and microflora native to the rind of whole cantaloupes. Studies were conducted at the pilot-scale level by passing deliberately contaminated melons through a prototype stainless-steel, continuous-feed heating device. Exposure for 240 s to aerated steam heated to 85 °C achieved a mean reduction in surface-inoculated L. innocua of 3.9 ± 0.6 log10 CFU/cm2 (n = 3) and decreased background microorganisms (yeast, moulds, and coliforms) to undetectable levels. No significant outgrowth of surviving L. innocua or yeast and moulds was observed on heat-treated melons during their storage at 4, 7, and 10 °C for 14 days. Treated fruit continued to respire. Although rind quality was altered, edible fleshy portions remained largely unaffected. Cantaloupe inoculated with L. innocua subsequent to its exposure to aerated steam provided a suitable environment for surrogate growth (mean 3.3 log10 increase in rind density over 10 days at 7 °C), whereas its proliferation was restricted on nonheated cantaloupe (mean 0.7 log10 increase). Steam sanitization provides an effective means for the control of pathogen and spoilage organisms, but the proliferation of surrogate organisms on heated cantaloupes raises concern regarding the impact of postprocessing contamination on consumer health risk. PRACTICAL APPLICATION: Water vapor (steam) at a high temperature can be used to sanitize the surface of fresh, whole cantaloupe melons in a continuous-feed manner. Both Listeria bacteria and spoilage organisms are markedly reduced from initial levels and survivor outgrowth severely restricted during subsequent refrigerated storage. This approach to microorganism control is likely most applicable in situations where rinds and flesh are to be separated immediately via further processing.

Reach specificity in sediment E. coli population turnover and interaction with waterborne populations.

Sediment-borne Escherichia coli can elevate waterborne concentrations through sediment resuspension or hyporheic exchange. This study sought to correlate hydrological, sediment transport, and water quality variables with: (i) the temporal stability of sediment E. coli populations [concentrations, strain richness and similarity (Raup-Crick index)]; and (ii) the contribution of sediment E. coli to the water column as defined through a library-dependent microbial source tracking approach that matched waterborne E. coli isolates to sediment E. coli populations. Three monitoring locations differing in their hydrological characteristics and adjacent upland fecal sources (dairy operation, low-density residential, and tile-drained cultivated field) were investigated. Sediment E. coli population turnover was influenced by sediment transport at upstream, high-energy reaches, but not at the downstream low-energy reach. Sediment contributions to the water column averaged 13% and 18%, and fecal sources averaged 17% and 21% at the upstream sites adjacent to dairy operations and low-density residential areas, respectively. Waterborne E. coli at the downstream site had low matches to E. coli from reach sediments (1%), higher matches to the upstream sediments (27% and 12%), and an average of 14% matches to the tile drained field. The percentage of waterborne E. coli matching sediment-borne E. coli at each stream reach varied in correlations to hydrological and sediment transport variables, suggesting reach-specific differences in the role of sediment resuspension and hyporheic exchange on E. coli transport.

Evaluation of different approaches for modeling Escherichia coli O157:H7 survival on field lettuce.

The ability to predict the behavior of Escherichia coli O157:H7 on contaminated field lettuce is essential for the development of accurate quantitative microbial risk assessments. The survival pattern of the species was assessed from several data sets derived from field-based experiments, which were analyzed by regression analysis fitting one monophasic model (log-linear) and two biphasic (Weibull and Cerf's model) models. Probabilistic models were also simulated with @RISK™, integrating the fitted monophasic and biphasic models in order to analyze their impact on the estimate of the extent of die-off subsequent to a contamination event in the field. Regression analysis indicated that E. coli O157:H7 followed a biphasic decay pattern in most cases, with the Weibull and Cerf's model showing similar good fit to individual and pooled survival data. Furthermore, results from the stochastic analysis demonstrated that using the log-linear model could lead to different risk estimates from those obtained with biphasic models, with a lower prevalence in the former scenario as no tailing is assumed in this model. The models and results derived from this work provide the first suitable mathematical base upon which to build probabilistic models to predict the fate of E. coli O157:H7 on field-grown leafy green vegetable.

Characterizing spatial structure of sediment E. coli populations to inform sampling design.

Escherichia coli can persist in streambed sediments and influence water quality monitoring programs through their resuspension into overlying waters. This study examined the spatial patterns in E. coli concentration and population structure within streambed morphological features during baseflow and following stormflow to inform sampling strategies for representative characterization of E. coli populations within a stream reach. E. coli concentrations in bed sediments were significantly different (p = 0.002) among monitoring sites during baseflow, and significant interactive effects (p = 0.002) occurred among monitoring sites and morphological features following stormflow. Least absolute shrinkage and selection operator (LASSO) regression revealed that water velocity and effective particle size (D 10) explained E. coli concentration during baseflow, whereas sediment organic carbon, water velocity and median particle diameter (D 50) were important explanatory variables following stormflow. Principle Coordinate Analysis illustrated the site-scale differences in sediment E. coli populations between disconnected stream segments. Also, E. coli populations were similar among depositional features within a reach, but differed in relation to high velocity features (e.g., riffles). Canonical correspondence analysis resolved that E. coli population structure was primarily explained by spatial (26.9­31.7 %) over environmental variables (9.2­13.1 %). Spatial autocorrelation existed among monitoring sites and morphological features for both sampling events, and gradients in mean particle diameter and water velocity influenced E. coli population structure for the baseflow and stormflow sampling events, respectively. Representative characterization of streambed E. coli requires sampling of depositional and high velocity environments to accommodate strain selectivity among these features owing to sediment and water velocity heterogeneity.

Effect of hillslope position and manure application rates on the persistence of fecal source tracking indicators in an agricultural soil.

The influence of liquid dairy manure (LDM) application rates (12.5 and 25 kL ha) and soil type on the decay rates of library-independent fecal source tracking markers (host-associated and mitochondrial DNA) and persistent (>58 d) population structure was examined in a field study. The soils compared were an Aquic Haplorthod and a Typic Haplorthod in Nova Scotia, Canada, that differed according to landscape position and soil moisture regime. Soil type and LDM application rate did not influence decay rates (0.045-0.057 d). population structure, in terms of the occurrence of abundance of strain types, varied according to soil type ( = 0.012) but did not vary by LDM application rate ( = 0.121). Decay of ruminant-specific (BacR), bovine-specific (CowM2), and mitochondrial DNA (AcytB) markers was analyzed for 13 d after LDM application. The decay rates of BacR were greater under high-LDM application rates (0.281-0.358 d) versus low-LDM application rates (0.212-0.236 d) but were unaffected by soil type. No decay rates could be calculated for the CowM2 marker because it was undetectable within 6 d after manure application. Decay rates for AcytB were lower for the Aquic Haplorthod (0.088-0.100 d), with higher moisture status compared with the Typic Haplorthod (0.135 d). Further investigation into the decay of fecal source tracking indicators in agricultural field soils is warranted to assess the influence of soil type and agronomic practice on the differential decay of relevant markers and the likelihood of transport in runoff.

Evaluation of statistical models for predicting Escherichia coli particle attachment in fluvial systems.

Modeling surface water Escherichia coli fate and transport requires partitioning E. coli into particle-attached and unattached fractions. Attachment is often assumed to be a constant fraction or is estimated using simple linear models. The objectives of this study were to: (i) develop statistical models for predicting E. coli attachment and virulence marker presence in fluvial systems, and (ii) relate E. coli attachment to a variety of environmental parameters. Stream water samples (n = 60) were collected at four locations in a rural, mixed-use watershed between June and October 2012, with four storm events (>20 mm rainfall) being captured. The percentage of E. coli attached to particles (>5 µm) and the occurrences of virulence markers were modeled using water quality, particle concentration, particle size distribution, hydrology and land use factors as explanatory variables. Three types of statistical models appropriate for highly collinear, multidimensional data were compared: least angle shrinkage and selection operator (LASSO), classification and regression trees using the general, unbiased, interaction detection and estimation (GUIDE) algorithm, and multivariate adaptive regression splines (MARS). All models showed that E. coli particle attachment and the presence of E. coli virulence markers in the attached and unattached states were influenced by a combination of water quality, hydrology, land-use and particle properties. Model performance statistics indicate that MARS models outperform LASSO and GUIDE models for predicting E. coli particle attachment and virulence marker occurrence. Validating the MARS modeling approach in multiple watersheds may allow for the development of a parameterizing model to be included in watershed simulation models.

Comparative examination of Escherichia coli O157:H7 survival on romaine lettuce and in soil at two independent experimental sites.

Little is known about the influence of abiotic factors such as climate and soil chemistry on the survival of Escherichia coli O157:H7 in field lettuce. We applied a nalidixic acid-resistant derivative of strain ATCC 700728 to field-grown romaine lettuce in two regions in Canada characterized by large variances in soil type and climate. Surviving populations in soil and on lettuce leaves were estimated on sorbitol MacConkey agar supplemented with nalidixic acid. Data were fitted with the Weibull decline function to permit comparison of decay rates in the two experimental sites. E. coli O157:H7 populations fell from 105 to <10² CFU/g on leaves, and <10³ CFU/g in soil within 7 days after inoculation. Analysis revealed there was no significant difference between decay rates at the two experimental sites in either environment. The results of this study suggest that the inherent ecological fitness of E. coli O157:H7 ATCC 700728 determines the extent of survival in the production environment.