Salmonella Genomics and Population Analyses Reveal High Inter- and Intraserovar Diversity in Freshwater.

Freshwater can support the survival of the enteric pathogen Salmonella, though temporal Salmonella diversity in a large watershed has not been assessed. At 28 locations within the Susquehanna River basin, 10-liter samples were assessed in spring and summer over 2 years. Salmonella prevalence was 49%, and increased river discharge was the main driver of Salmonella presence. The amplicon-based sequencing tool, CRISPR-SeroSeq, was used to determine serovar population diversity and detected 25 different Salmonella serovars, including up to 10 serovars from a single water sample. On average, there were three serovars per sample, and 80% of Salmonella-positive samples contained more than one serovar. Serovars Give, Typhimurium, Thompson, and Infantis were identified throughout the watershed and over multiple collections. Seasonal differences were evident: serovar Give was abundant in the spring, whereas serovar Infantis was more frequently identified in the summer. Eight of the ten serovars most commonly associated with human illness were detected in this study. Crucially, six of these serovars often existed in the background, where they were masked by a more abundant serovar(s) in a sample. Serovars Enteritidis and Typhimurium, especially, were masked in 71 and 78% of samples where they were detected, respectively. Whole-genome sequencing-based phylogeny demonstrated that strains within the same serovar collected throughout the watershed were also very diverse. The Susquehanna River basin is the largest system where Salmonella prevalence and serovar diversity have been temporally and spatially investigated, and this study reveals an extraordinary level of inter- and intraserovar diversity.IMPORTANCESalmonella is a leading cause of bacterial foodborne illness in the United States, and outbreaks linked to fresh produce are increasing. Understanding Salmonella ecology in freshwater is of importance, especially where irrigation practices or recreational use occur. As the third largest river in the United States east of the Mississippi, the Susquehanna River is the largest freshwater contributor to the Chesapeake Bay, and it is the largest river system where Salmonella diversity has been studied. Rainfall and subsequent high river discharge rates were the greatest indicators of Salmonella presence in the Susquehanna and its tributaries. Several Salmonella serovars were identified, including eight commonly associated with foodborne illness. Many clinically important serovars were present at a low frequency within individual samples and so could not be detected by conventional culture methods. The technologies employed here reveal an average of three serovars in a 10-liter sample of water and up to 10 serovars in a single sample.

Correlation of Salmonella enterica and Listeria monocytogenes in Irrigation Water to Environmental Factors, Fecal Indicators, and Bacterial Communities.

Outbreaks of foodborne illnesses linked to fresh fruits and vegetables have been key drivers behind a wide breadth of research aiming to fill data gaps in our understanding of the total ecology of agricultural water sources such as ponds and wells and the relationship of this ecology to foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. Both S. enterica and L. monocytogenes can persist in irrigation water and have been linked to produce contamination events. Data describing the abundance of these organisms in specific agricultural water sources are valuable to guide water treatment measures. Here, we profiled the culture independent water microbiota of four farm ponds and wells correlated with microbiological recovery of S. enterica (prevalence: pond, 19.4%; well, 3.3%), L. monocytogenes (pond, 27.1%; well, 4.2%) and fecal indicator testing. Correlation between abiotic factors, including water parameters (temperature, pH, conductivity, dissolved oxygen percentage, oxidation reduction potential, and turbidity) and weather (temperature and rainfall), and foodborne pathogens were also evaluated. Although abiotic factors did not correlate with recovery of S. enterica or L. monocytogenes (p > 0.05), fecal indicators were positively correlated with incidence of S. enterica in well water. Bacterial taxa such as Sphingomonadaceae and Hymenobacter were positively correlated with the prevalence and population of S. enterica, and recovery of L. monocytogenes was positively correlated with the abundance of Rhizobacter and Comamonadaceae (p < 0.03). These data will support evolving mitigation strategies to reduce the risk of produce contamination by foodborne pathogens through irrigation.

Diversity and Dynamics of Salmonella enterica in Water Sources, Poultry Litters, and Field Soils Amended With Poultry Litter in a Major Agricultural Area of Virginia.

The Eastern Shore of Virginia (ESV) is a major agricultural region in Virginia and in the past has been linked to some tomato-associated outbreaks of salmonellosis. In this study, water samples were collected weekly from irrigation ponds and wells in four representative vegetable farms (Farms A-D, each farm paired with one pond and one well) and a creek as well. In addition, water samples from two sites in the Chesapeake Bay on the ESV were collected monthly. Poultry litter was sampled monthly from three commercial broiler farms. Soil samples were collected monthly after fertilization with poultry litter from 10 farms in 2014 and another 14 farms in 2015. A most probable number method was used to detect Salmonella enterica presence and concentration in collected samples. Presumptive Salmonella colonies were confirmed by the cross-streaking method. Molecular serotyping was carried out to determine the Salmonella serovars. The average prevalence of Salmonella in pond, well, creek, and bay water samples was 19.3, 3.3, 24.2, and 29.2%, respectively. There were significant spatial and temporal differences for Salmonella incidence in various water sources. The prevalence of S. enterica in four tested ponds from farms A, B, C, and D were 16, 12, 22, and 27%, respectively. While the prevalence of S. enterica in irrigation wells was significantly lower, some well water samples tested positive during the study. Salmonella Newport was found to be the predominant serovar isolated from water samples. All poultry houses of the three tested broiler farms were Salmonella-positive at certain sampling points during the study with prevalence ranging from 14.3 to 35.4%. Salmonella was found to be able to survive up to 4 months in poultry litter amended soils from the tested farms in 2014, and up to 6 months in 2015. This research examined the dynamics of S. enterica in relationship to water source, poultry litter, and amended soil in a major agricultural area, and provides useful information for food safety risk assessments.

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.

Is the Glasgow Blatchford score useful in the risk assessment of patients presenting with variceal haemorrhage?

BACKGROUND: The Glasgow Blatchford score (GBS) is a pre-endoscopic risk assessment tool for patients presenting with upper gastrointestinal haemorrhage. There are few data regarding use in patients with variceal bleeding, who are generally accepted as being at high risk. AIM: The aim of the study was to assess GBS in correctly identifying patients with subsequently proven variceal bleeding as 'high risk' and to compare GBS, admission and full Rockall scores in predicting clinical endpoints in this group. PATIENTS AND METHODS: Data on consecutive patients with upper gastrointestinal haemorrhage presenting to four UK hospitals were collected. The GBS, admission and full Rockall scores were calculated and compared for the subgroup subsequently shown to have variceal bleeding. Area under the receiver operating curve (AUROC) was used to assess the scores ability to predict clinical endpoints within this variceal bleeding subgroup. RESULTS: A total of 1432 patients presented during the study period. Seventy-one (5%) had a final diagnosis of variceal bleeding. At presentation, none of this group had GBS less than 2, but six had an admission Rockall score of 0. In predicting need for blood transfusion, AUROC scores for GBS, full and admission Rockall scores were 0.68, 0.65 and 0.68, respectively. For endoscopic/surgical intervention the scores were 0.34, 0.51 and 0.55, respectively, and for predicting death the scores were 0.56, 0.72 and 0.70, respectively. None of these AUROC score comparisons were significant. CONCLUSION: At presentation, GBS correctly identifies patients with variceal bleeding as high risk and appears superior to the admission Rockall score. However, GBS and both Rockall scores are poor at predicting clinical outcome within this group.