Research gaps and priorities for quantitative microbial risk assessment (QMRA).

The coronavirus disease 2019 pandemic highlighted the need for more rapid and routine application of modeling approaches such as quantitative microbial risk assessment (QMRA) for protecting public health. QMRA is a transdisciplinary science dedicated to understanding, predicting, and mitigating infectious disease risks. To better equip QMRA researchers to inform policy and public health management, an Advances in Research for QMRA workshop was held to synthesize a path forward for QMRA research. We summarize insights from 41 QMRA researchers and experts to clarify the role of QMRA in risk analysis by (1) identifying key research needs, (2) highlighting emerging applications of QMRA; and (3) describing data needs and key scientific efforts to improve the science of QMRA. Key identified research priorities included using molecular tools in QMRA, advancing dose-response methodology, addressing needed exposure assessments, harmonizing environmental monitoring for QMRA, unifying a divide between disease transmission and QMRA models, calibrating and/or validating QMRA models, modeling co-exposures and mixtures, and standardizing practices for incorporating variability and uncertainty throughout the source-to-outcome continuum. Cross-cutting needs identified were to: develop a community of research and practice, integrate QMRA with other scientific approaches, increase QMRA translation and impacts, build communication strategies, and encourage sustainable funding mechanisms. Ultimately, a vision for advancing the science of QMRA is outlined for informing national to global health assessments, controls, and policies.

Prevalence and Clonal Diversity of over 1,200 Listeria monocytogenes Isolates Collected from Public Access Waters near Produce Production Areas on the Central California Coast during 2011 to 2016.

A 5-year survey of public access surface waters in an agricultural region of the Central California Coast was done to assess the prevalence of the foodborne pathogen Listeria monocytogenes. In nature, L. monocytogenes lives as a saprophyte in soil and water, which are reservoirs for contamination of preharvest produce. Moore swabs were deployed biweekly in lakes, ponds, streams, and rivers during 2011 to 2016. L. monocytogenes was recovered in 1,224 of 2,922 samples, resulting in 41.9% prevalence. Multiple subtypes were isolated from 97 samples, resulting in 1,323 L. monocytogenes isolates. Prevalence was higher in winter and spring and after rain events in some waterways. Over 84% of the isolates were serotype 4b. Whole-genome sequencing was done on 1,248 isolates, and in silico multilocus sequence typing revealed 74 different sequence types (STs) and 39 clonal complexes (CCs). The clones most isolated, CC639, CC183, and CC1, made up 27%, 19%, and 13%, respectively, of the sequenced isolates. Other types were CC663, CC6, CC842, CC4, CC2, CC5, and CC217. All sequenced isolates contained intact copies of core L. monocytogenes virulence genes, and pathogenicity islands LIPI-3 and LIPI-4 were identified in 73% and 63%, respectively, of the sequenced isolates. The virulence factor internalin A was predicted to be intact in all but four isolates, while genes important for sanitizer and heavy metal resistance were found in <5% of the isolates. These waters are not used for crop irrigation directly, but they are available to wildlife and can flood fields during heavy rains. IMPORTANCE Listeria monocytogenes serotype 4b and 1/2a strains are implicated in most listeriosis, and hypervirulent listeriosis stems from strains containing pathogenicity islands LIPI-3 and LIPI-4. The waters and sediments in the Central California Coast agricultural region contain widespread and diverse L. monocytogenes populations, and all the isolates contain intact virulence genes. Emerging clones CC183 and CC639 were the most abundant clones, and major clones CC1, CC4, and CC6 were well represented. CC183 was responsible for three produce-related outbreaks in the last 7 years. Most of the isolates in the survey differ from those of lesser virulence that are often isolated from foods and food processing plants because they contain genes encoding an intact virulence factor, internalin A, and most did not contain genes for sanitizer and heavy metal resistance. This isolate collection is important for understanding L. monocytogenes populations in agricultural and natural regions.

Strain, Soil-Type, Irrigation Regimen, and Poultry Litter Influence Salmonella Survival and Die-off in Agricultural Soils.

The use of untreated biological soil amendments of animal origin (BSAAO) have been identified as one potential mechanism for the dissemination and persistence of Salmonella in the produce growing environment. Data on factors influencing Salmonella concentration in amended soils are therefore needed. The objectives here were to (i) compare die-off between 12 Salmonella strains following inoculation in amended soil and (ii) characterize any significant effects associated with soil-type, irrigation regimen, and amendment on Salmonella survival and die-off. Three greenhouse trials were performed using a randomized complete block design. Each strain (~4 log CFU/g) was homogenized with amended or non-amended sandy-loam or clay-loam soil. Salmonella levels were enumerated in 25 g samples 0, 0.167 (4 h), 1, 2, 4, 7, 10, 14, 21, 28, 56, 84, 112, 168, 210, 252, and 336 days post-inoculation (dpi), or until two consecutive samples were enrichment negative. Regression analysis was performed between strain, soil-type, irrigation, and (i) time to last detect (survival) and (ii) concentration at each time-point (die-off rate). Similar effects of strain, irrigation, soil-type, and amendment were identified using the survival and die-off models. Strain explained up to 18% of the variance in survival, and up to 19% of variance in die-off rate. On average Salmonella survived for 129 days in amended soils, however, Salmonella survived, on average, 30 days longer in clay-loam soils than sandy-loam soils [95% Confidence interval (CI) = 45, 15], with survival time ranging from 84 to 210 days for the individual strains during daily irrigation. When strain-specific associations were investigated using regression trees, S. Javiana and S. Saintpaul were found to survive longer in sandy-loam soil, whereas most of the other strains survived longer in clay-loam soil. Salmonella also survived, on average, 128 days longer when irrigated weekly, compared to daily (CI = 101, 154), and 89 days longer in amended soils, than non-amended soils (CI = 61, 116). Overall, this study provides insight into Salmonella survival following contamination of field soils by BSAAO. Specifically, Salmonella survival may be strain-specific as affected by both soil characteristics and management practices. These data can assist in risk assessment and strain selection for use in challenge and validation studies.

A Predictive Model for Survival of Escherichia coli O157:H7 and Generic E. coli in Soil Amended with Untreated Animal Manure.

This study aimed at developing a predictive model that captures the influences of a variety of agricultural and environmental variables and is able to predict the concentrations of enteric bacteria in soil amended with untreated Biological Soil Amendments of Animal Origin (BSAAO) under dynamic conditions. We developed and validated a Random Forest model using data from a longitudinal field study conducted in mid-Atlantic United States investigating the survival of Escherichia coli O157:H7 and generic E. coli in soils amended with untreated dairy manure, horse manure, or poultry litter. Amendment type, days of rain since the previous sampling day, and soil moisture content were identified as the most influential agricultural and environmental variables impacting concentrations of viable E. coli O157:H7 and generic E. coli recovered from amended soils. Our model results also indicated that E. coli O157:H7 and generic E. coli declined at similar rates in amended soils under dynamic field conditions.The Random Forest model accurately predicted changes in viable E. coli concentrations over time under different agricultural and environmental conditions. Our model also accurately characterized the variability of E. coli concentration in amended soil over time by providing upper and lower prediction bound estimates. Cross-validation results indicated that our model can be potentially generalized to other geographic regions and incorporated into a risk assessment for evaluating the risks associated with application of untreated BSAAO. Our model can be validated for other regions and predictive performance also can be enhanced when data sets from additional geographic regions become available.

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production.

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech's Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields.

A Mathematical Model for Pathogen Cross-Contamination Dynamics during the Postharvest Processing of Leafy Greens.

We developed a probabilistic mathematical model for the postharvest processing of leafy greens focusing on Escherichia coli O157:H7 contamination of fresh-cut romaine lettuce as the case study. Our model can (i) support the investigation of cross-contamination scenarios, and (ii) evaluate and compare different risk mitigation options. We used an agent-based modeling framework to predict the pathogen prevalence and levels in bags of fresh-cut lettuce and quantify spread of E. coli O157:H7 from contaminated lettuce to surface areas of processing equipment. Using an unbalanced factorial design, we were able to propagate combinations of random values assigned to model inputs through different processing steps and ranked statistically significant inputs with respect to their impacts on selected model outputs. Results indicated that whether contamination originated on incoming lettuce heads or on the surface areas of processing equipment, pathogen prevalence among bags of fresh-cut lettuce and batches was most significantly impacted by the level of free chlorine in the flume tank and frequency of replacing the wash water inside the tank. Pathogen levels in bags of fresh-cut lettuce were most significantly influenced by the initial levels of contamination on incoming lettuce heads or surface areas of processing equipment. The influence of surface contamination on pathogen prevalence or levels in fresh-cut bags depended on the location of that surface relative to the flume tank. This study demonstrates that developing a flexible yet mathematically rigorous modeling tool, a "virtual laboratory," can provide valuable insights into the effectiveness of individual and combined risk mitigation options.

Escherichia coli transfer from simulated wildlife feces to lettuce during foliar irrigation: A field study in the Northeastern United States.

Wildlife intrusion has been associated with pathogen contamination of produce. However, few studies have examined pathogen transfer from wildlife feces to pre-harvest produce. This study was performed to calculate transfer coefficients for Escherichia coli from simulated wildlife feces to field-grown lettuce during irrigation. Rabbit feces inoculated with a 3-strain cocktail of non-pathogenic E. coli were placed in a lettuce field 2.5-72 h before irrigation. Following irrigation, the E. coli concentration on the lettuce was determined. After exclusion of an outlier with high E. coli levels (Most Probable Number = 5.94*108), the average percent of E. coli in the feces that transferred to intact lettuce heads was 0.0267% (Standard Error [SE] = 0.0172). Log-linear regression showed that significantly more E. coli transferred to outer leaves compared to inner leaves (Effect = 1.3; 95% Confidence Interval = 0.4, 2.1). Additionally, the percent of E. coli that transferred from the feces to the lettuce decreased significantly with time after fecal placement, and as the distance between the lettuce and the feces, and the lettuce and the sprinklers increased. These findings provide key data that may be used in future quantitative risk assessments to identify potential intervention strategies for reducing food safety risks associated with fresh produce.

Survival of Escherichia coli on Lettuce under Field Conditions Encountered in the Northeastern United States.

Although wildlife intrusion and untreated manure have been associated with microbial contamination of produce, relatively few studies have examined the survival of Escherichia coli on produce under field conditions following contamination (e.g., via splash from wildlife feces). This experimental study was performed to estimate the die-off rate of E. coli on preharvest lettuce following contamination with a fecal slurry. During August 2015, field-grown lettuce was inoculated via pipette with a fecal slurry that was spiked with a three-strain cocktail of rifampin-resistant nonpathogenic E. coli. Ten lettuce heads were harvested at each of 13 time points following inoculation (0, 2.5, 5, and 24 h after inoculation and every 24 h thereafter until day 10). The most probable number (MPN) of E. coli on each lettuce head was determined, and die-off rates were estimated. The relationship between sample time and the log MPN of E. coli per head was modeled using a segmented linear model. This model had a breakpoint at 106 h (95% confidence interval = 69, 142 h) after inoculation, with a daily decrease of 0.70 and 0.19 log MPN for 0 to 106 h and 106 to 240 h following inoculation, respectively. These findings are consistent with die-off rates obtained in similar studies that assessed E. coli survival on produce following irrigation. Overall, these findings provide die-off rates for E. coli on lettuce that can be used in future quantitative risk assessments.

Inactivation of Escherichia coli O157:H7 on Romaine Lettuce When Inoculated in a Fecal Slurry Matrix.

A field trial was conducted in July 2011 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, CA. One-half milliliter of rabbit fecal slurry, containing 6.3 × 107 CFU of E. coli O157:H7, was inoculated onto the upper (adaxial) surface of a lower leaf on 240 heads of lettuce within 30 min after a 2.5-h irrigation event. Forty-eight romaine lettuce heads were collected per event at 2.5 h (day 0.1), 19.75 h (day 0.8), 43.25 h (day 1.8), 67.25 h (day 2.8), and 91.75 h (day 3.8) postinoculation and were analyzed for the concentration of E. coli O157:H7 (Ct). E. coli O157:H7 was detected on 100% of collected heads in concentrations ranging from 340 to 3.40 × 1010 most probable number (MPN) per head. Enumeration data indicate substantial growth of E. coli O157:H7 postinoculation (2.5 h), leading to elevated concentrations, 1 to 3 log above the starting inoculum concentration (Co). By the end of the 92-h trial, we observed a net 0.8-log mean reduction of E. coli O157:H7 compared with Co; however, after accounting for the substantial bacterial growth, there was an overall 2.3-log reduction by the final sampling event (92 h). On the basis of two different regression models that used either the raw data for Ct or log-transformed values of Ct/Co during the period 2.5 to 91.75 h postinoculation, there was an estimated 76 to 80% reduction per day in bacterial counts; however, more accurate predictions of MPN per head of lettuce were generated by using non-log-transformed values of Ct. This study provides insight into the survival of E. coli O157:H7 transferred via splash from a contaminated fecal source onto produce during irrigation. Moreover, these findings can help generate inactivation times following a potential contamination incident.

Distribution of Animal Drugs between Skim Milk and Milk Fat Fractions in Spiked Whole Milk: Understanding the Potential Impact on Commercial Milk Products.

Seven animal drugs [penicillin G (PENG), sulfadimethoxine (SDMX), oxytetracycline (OTET), erythromycin (ERY), ketoprofen (KETO), thiabendazole (THIA), and ivermectin (IVR)] were used to evaluate the drug distribution between milk fat and skim milk fractions of cow milk. More than 90% of the radioactivity was distributed into the skim milk fraction for ERY, KETO, OTET, PENG, and SDMX, approximately 80% for THIA, and 13% for IVR. The distribution of drug between milk fat and skim milk fractions was significantly correlated to the drug's lipophilicity (partition coefficient, log P, or distribution coefficient, log D, which includes ionization). Data were fit with linear mixed effects models; the best fit was obtained within this data set with log D versus observed drug distribution ratios. These candidate empirical models serve for assisting to predict the distribution and concentration of these drugs in a variety of milk and milk products.

Transfer of Escherichia coli O157:H7 from simulated wildlife scat onto romaine lettuce during foliar irrigation.

A field trial in Salinas Valley, California, was conducted during July 2011 to quantify the microbial load that transfers from wildlife feces onto nearby lettuce during foliar irrigation. Romaine lettuce was grown using standard commercial practices and irrigated using an impact sprinkler design. Five grams of rabbit feces was spiked with 1.29 × 10(8) CFU of Escherichia coli O157:H7 and placed - 3, - 2, and - 1 days and immediately before a 2-h irrigation event. Immediately after irrigation, 168 heads of lettuce ranging from ca. 23 to 69 cm (from 9 to 27 in.) from the fecal deposits were collected, and the concentration of E. coli O157:H7 was determined. Thirty-eight percent of the collected lettuce heads had detectable E. coli O157:H7, ranging from 1 MPN to 2.30 × 10(5) MPN per head and a mean concentration of 7.37 × 10(3) MPN per head. Based on this weighted arithmetic mean concentration of 7.37 × 10(3) MPN of bacteria per positive head, only 0.00573% of the original 5 g of scat with its mean load of 1.29 × 10(8) CFU was transferred to the positive heads of lettuce. Bacterial contamination was limited to the outer leaves of lettuce. In addition, factors associated with the transfer of E. coli O157:H7 from scat to lettuce were distance between the scat and lettuce, age of scat before irrigation, and mean distance between scat and the irrigation sprinkler heads. This study quantified the transfer coefficient between scat and adjacent heads of lettuce as a function of irrigation. The data can be used to populate a quantitative produce risk assessment model for E. coli O157:H7 in romaine lettuce to inform risk management and food safety policies.

Prevalence of shiga toxin producing Escherichia coli, Salmonella enterica, and Listeria monocytogenes at public access watershed sites in a California Central Coast agricultural region.

Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks.