We have previously shown that diarrheagenic Escherichia coli (DEC) and non-DEC are prevalent in food sources and irrigation water in South Africa. Recent data suggest that an increased relative abundance of faecal Enterobacteriaceae is associated with poorer health outcomes among children in developing countries. Thus, exposure to non-DEC from environmental sources may incur adverse effects, although the mechanisms underlying these effects remain obscure. To further elucidate this phenomenon, we assayed non-DEC strains from environmental sources in South Africa for phenotypes that may be associated with intestinal dysfunction (ID). DEC strains were also used. The strains had previously been isolated from Producer Distributor Bulk Milk (PDBM), irrigated lettuce, street vendor coleslaw and irrigation water.In-vitro assays identified; biofilm formation (n = 38), extracellular polymeric substance (EPS) formation (n = 38), cytotoxic activity (n = 10), disruption of tight junctions and induction of Interleukin 8 (IL-8) on polarized T-84 cells (n = 20). The number of strains tested for each assay differed, depending on prior molecular and phenotypic characterization that signalled potential pathogenicity in-vitro. Subsequently, all strains having data points for all analyses were used to compute Principal Component Analysis (PCA) plot curves to infer potential associations amongst test strains and a standard DEC pathogenic strain (042).Biofilm formation on glass coverslips after strains were grown in nutrient-rich media (LB and DMEM-F12 + 0.5% D-Mannose) at 37 °C varied based on pathotype (DEC and non-DEC) and source of isolation (food, irrigation water, clinical) suggesting that pathotype and source isolation influence persistence within a defined environmental niche. Additionally, DEC isolated from irrigated lettuce had a significantly higher (p ≤ 0.05) propensity for biofilm formation in both media compared to all strains including DEC standard controls. This suggested the propensity for irrigated lettuce as a potential source of persistent pathogenic strains. Furthermore, all strains were able to form EPS suggesting the ability to form mature biofilms under conditions relevant for food processing (20–25 °C). Of the (60%, 6 out of 10) strains that showed cytotoxic activity, most (83%, 5 out of 6 strains) were non-DEC isolated from food sources many of which are consumed with minimal processing.Mean percentage reduction in initial TEER (a measure of intestinal disruption), did not significantly differ (p = 0.05) in all test strains from that observed in the standard DEC. Additionally, IL-8 induction from strains isolated from PDBM (139 pg/mL), irrigation water (231.93 pg/mL) and irrigated lettuce (152.98 pg/mL) was significantly higher (p ≤ 0.05) than in the commensal strain aafa. PCA categorized strains based on sources of isolation showed potential for use in source tracking especially when comparing many strains from various environmental sources. We show that non-DEC strains along the food chain possess characteristics that may lead to ID. Further investigations using a larger collection of strains may provide a clearer link to these reported observations that could be associated with the high diarrheal disease burden within the country, especially among infants.

Questions related to the safety of alternative water sources, such as recycled water or reclaimed water (including grey water, produced water, return flows, and recycled wastewater), for produce production have been largely un-explored at the detail warranted for protection of public health. Additionally, recent outbreaks of Escherichia coli (E. coli) in fresh produce, in which agricultural water was suspected as the source, coupled with heightened media coverage, have elevated fruit and vegetable safety into the forefront of public attention. Exacerbating these concerns, new Federal regulations released by the U.S. Food and Drug Administration (FDA) as part of implementation of the FDA Food Safety Modernization Act (FSMA), require testing of agricultural water quality for generic E. coli. Here, we present a review of water quality criteria – including surface water, groundwater recreational water, and water reuse – in an attempt to better understand implications of new FDA regulations on irrigated produce. In addition, a Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate risks from pathogen contamination of food crops eaten fresh under the context of FDA regulations to provide perspective on current water reuse regulations across the country. Results indicate that irrigation water containing 126 CFU/100 mL of E. coli correspond to a risk of GI illness (diarrhea) of 9 cases in 100,000,000 persons (a 0.000009% risk) for subsurface irrigation, 1.1 cases in 100,000 persons (a 0.0011% risk) for furrow irrigation, and 1.1 cases in 1000 persons (a 0.11% risk) for sprinkler irrigation of lettuce. In comparison to metrics in states that currently regulate the use of recycled water for irrigation of food crops eaten fresh, the FDA FSMA water quality metrics are less stringent and therefore the use of recycled water presents a reduced risk to consumers than the FDA regulations. These findings, while limited to a one-time exposure event of lettuce irrigated with water meeting FSMA water quality regulations, highlight the need for additional assessments to determine if the scientific-basis of the regulation is protective of public health.

With a rapidly growing urban population in Kumasi, Ghana, the consumption of street food is increasing. Raw salads, which often accompany street food dishes, are typically composed of perishable vegetables that are grown in close proximity to the city using poor quality water for irrigation. This study assessed the risk of gastroenteritis illness (caused by rotavirus, norovirus and Ascaris lumbricoides) associated with the consumption of street food salads using Quantitative Microbial Risk Assessment (QMRA). Three different risk assessment models were constructed, based on availability of microbial concentrations: 1) Water — starting from irrigation water quality, 2) Produce — starting from the quality of produce at market, and 3) Street — using microbial quality of street food salad. In the absence of viral concentrations, published ratios between faecal coliforms and viruses were used to estimate the quality of water, produce and salad, and annual disease burdens were determined. Rotavirus dominated the estimates of annual disease burden (~10−3Disability Adjusted Life Years per person per year (DALYs pppy)), although norovirus also exceeded the 10−4DALY threshold for both Produce and Street models. The Water model ignored other on-farm and post-harvest sources of contamination and consistently produced lower estimates of risk; it likely underestimates disease burden and therefore is not recommended. Required log reductions of up to 5.3 (95th percentile) for rotavirus were estimated for the Street model, demonstrating that significant interventions are required to protect the health and safety of street food consumers in Kumasi. Estimates of virus concentrations were a significant source of model uncertainty and more data on pathogen concentrations is needed to refine QMRA estimates of disease burden.

Saeng-sikis a powdered ready-to-eat food with very low moisture that contains dried raw materials such as grains, fruits,mushrooms, and seaweeds. This product is consumed as a convenient and nutritious meal replacement. The objective of this study was to develop a mathematical model for predicting the survival of Clostridium perfringens vegetative cells and spores in saeng-sikas a function of temperature and to validate the model using saeng-siksamples with different microbial communities analyzed by matrix-assisted laser desorptionionization time-of-flight mass spectrometry. Kinetic data for C. perfringens survival in saeng-sikfit well to the Weibull model with high goodness off it (R(2) = 0.92 to 0.98). The obtained δ values (required time for first decimal reduction) for each temperature were 19.62 to 864.86 h, and concave curves (p < 1) were observed under all experimental conditions (5 to 40 degree C). Kinetic parameters were further described in a secondary model as a function of temperature using a Davey model (R(2) =0.99). The developed model was validated by the bias factor, accuracy factor, and root mean square error, and the values were within acceptable ranges for predictive models, even for saeng-sik samples with different microbial communities. When saeng-sikwas rehydrated according to the manufacturer’s recommendations, germination and outgrowth of C. perfringens was observed when the sample was subjected to unusual temperatures during storage, such as at 30 degree C for 15 h. C. perfringens spores survived in saeng-sik with very low water activity. Because C. perfringens could germinate and grow under such conditions, care must be taken to avoid initial contamination of C. perfringens during the manufacturing process. Our model developed with samples with different microbial communities provides useful information for next-generation microbiological risk assessment taking into consideration the ecology of the food-associated microbial community.

A predictive model was made for the logarithm of the thermal decimal reduction time (logD) of Salmonella enterica (D = time to 90% reduction by inactivation). The model was fitted with multiple linear regression from 521 logD-values reported in literature for laboratory media and foods highly varying in water activity and pH. The single regression model with temperature as the only variable had a high residual standard error (RSE) of 0.883 logD and no predictive value (fraction of variance explained (R²) < 0.001). Adding water activity, sugar content and pH as predictors resulted in a model with a lower RSE of 0.458 logD and an adjusted R² of 0.73. The model was validated by comparing 985 predicted with observed logD for S. enterica from other publications. The model was subsequently validated with 1498 published logD-values for inactivation of vegetative cells of nine other pathogenic bacteria genera (mainly Listeria monocytogenes, Escherichia coli, Clostridium perfringens, Cronobacter spp., Staphylococcus aureus, Yersinia enterocolitica) in or on a variety of laboratory media, meat, fish, dairy, nuts, fruits and vegetables. Regression analyses for validation with the 985 logD of S. enterica and 2483 logD of all genera show deviations from the expected slope of 1 (both 0.81) and the expected intercept of 0 (0.04 and 0.19 logD respectively). However, only 0.7% and 2% respectively of the new logD (expected: 0.5%) were observed above the 99% prediction interval of the original S. enterica model based on 521 logD. The findings suggest that i) the variability of thermal resistance of strains within species is larger than between genera and species; ii) one generic predictive model, also accounting for variability, suffices for designing the thermal inactivation of a variety of vegetative pathogenic bacteria in many food types.