Assessing phenotypic virulence of Salmonella enterica across serovars and sources.

Introduction: Whole genome sequencing (WGS) is increasingly used for characterizing foodborne pathogens and it has become a standard typing technique for surveillance and research purposes. WGS data can help assessing microbial risks and defining risk mitigating strategies for foodborne pathogens, including Salmonella enterica. Methods: To test the hypothesis that (combinations of) different genes can predict the probability of infection [P(inf)] given exposure to a certain pathogen strain, we determined P(inf) based on invasion potential of 87 S. enterica strains belonging to 15 serovars isolated from animals, foodstuffs and human patients, in an in vitro gastrointestinal tract (GIT) model system. These genomes were sequenced with WGS and screened for genes potentially involved in virulence. A random forest (RF) model was applied to assess whether P(inf) of a strain could be predicted based on the presence/absence of those genes. Moreover, the association between P(inf) and biofilm formation in different experimental conditions was assessed. Results and Discussion: P(inf) values ranged from 6.7E-05 to 5.2E-01, showing variability both among and within serovars. P(inf) values also varied between isolation sources, but no unambiguous pattern was observed in the tested serovars. Interestingly, serovars causing the highest number of human infections did not show better ability to invade cells in the GIT model system, with strains belonging to other serovars displaying even higher infectivity. The RF model did not identify any virulence factor as significant P(inf) predictors. Significant associations of P(inf) with biofilm formation were found in all the different conditions for a limited number of serovars, indicating that the two phenotypes are governed by different mechanisms and that the ability to form biofilm does not correlate with the ability to invade epithelial cells. Other omics techniques therefore seem more promising as alternatives to identify genes associated with P(inf), and different hypotheses, such as gene expression rather than presence/absence, could be tested to explain phenotypic virulence [P(inf)].

Phenotypic Prediction: Linking in vitro Virulence to the Genomics of 59 Salmonella enterica Strains.

The increased availability of whole-genome-sequencing techniques generates a wealth of DNA data on numerous organisms, including foodborne pathogens such as Salmonella. However, how these data can be used to improve microbial risk assessment and understanding of Salmonella epidemiology remains a challenge. The aim of this study was to assess variability in in vitro virulence and genetic characteristics between and within different serovars. The phenotypic behavior of 59 strains of 32 different Salmonella enterica serovars from animal, human and food origin was assessed in an in vitro gastro-intestinal tract (GIT) system and they were analyzed for the presence of 233 putative virulence genes as markers for phenotypic prediction. The probability of in vitro infection, P(inf), defined as the fraction of infectious cells passing from inoculation to host cell invasion at the last stage of the GIT system, was interpreted as the in vitro virulence. Results showed that the (average) P(inf) of Salmonella serovars ranged from 5.3E-05 (S. Kedougou) to 5.2E-01 (S. Typhimurium). In general, a higher P(inf) on serovar level corresponded to higher reported human incidence from epidemiological reporting data. Of the 233 virulence genes investigated, only 101 showed variability in presence/absence among the strains. In vitro P(inf) was found to be positively associated with the presence of specific plasmid related virulence genes (mig-5, pef, rck, and spv). However, not all serovars with a relatively high P(inf), > 1E-02, could be linked with these specific genes. Moreover, some outbreak related strains (S. Heidelberg and S. Thompson) did not reveal this association with P(inf). No clear association with in vitro virulence P(inf) was identified when grouping serovars with the same virulence gene profile (virulence plasmid, Typhoid toxin, peg operon and stk operon). This study shows that the in vitro P(inf) variation among individual strains from the same serovar is larger than that found between serovars. Therefore, ranking P(inf) of S. enterica on serovar level alone, or in combination with a serovar specific virulence gene profile, cannot be recommended. The attribution of single biological phenomena to individual strains or serovars is not sufficient to improve the hazard characterization for S. enterica. Future microbial risk assessments, including virulence gene profiles, require a systematic approach linked to epidemiological studies rather than revealing differences in characteristics on serovar level alone.

Quantification of Salmonella Survival and Infection in an In vitro Model of the Human Intestinal Tract as Proxy for Foodborne Pathogens.

Different techniques are available for assessing differences in virulence of bacterial foodborne pathogens. The use of animal models or human volunteers is not expedient for various reasons; the use of epidemiological data is often hampered by lack of crucial data. In this paper, we describe a static, sequential gastrointestinal tract (GIT) model system in which foodborne pathogens are exposed to simulated gastric and intestinal contents of the human digestive tract, including the interaction of pathogens with the intestinal epithelium. The system can be employed with any foodborne bacterial pathogens. Five strains of Salmonella Heidelberg and one strain of Salmonella Typhimurium were used to assess the robustness of the system. Four S. Heidelberg strains originated from an outbreak, the fifth S. Heidelberg strain and the S. Typhimurium strain originated from routine meat inspections. Data from plate counts, collected for determining the numbers of surviving bacteria in each stage, were used to quantify both the experimental uncertainty and biological variability of pathogen survival throughout the system. For this, a hierarchical Bayesian framework using Markov chain Monte Carlo (MCMC) was employed. The model system is able to distinguish serovars/strains for in vitro infectivity when accounting for within strain biological variability and experimental uncertainty.

Prevalence and concentration of bacterial pathogens in raw produce and minimally processed packaged salads produced in and for the Netherlands.

Recent outbreaks with vegetable or fruits as vehicles have raised interest in the characterization of the public health risk due to microbial contamination of these commodities. Because qualitative and quantitative data regarding prevalence and concentration of various microbes are lacking, we conducted a survey to estimate the prevalence and contamination level of raw produce and the resulting minimally processed packaged salads as sold in The Netherlands. A dedicated sampling plan accounted for the amount of processed produce in relation to the amount of products, laboratory capacity, and seasonal influences. Over 1,800 samples of produce and over 1,900 samples of ready-to-eat mixed salads were investigated for Salmonella enterica serovars, Campylobacter spp., Escherichia coli O157, and Listeria monocytogenes. The overall prevalence in raw produce varied between 0.11% for E. coli O157 and L. monocytogenes and 0.38% for Salmonella. Prevalence point estimates for specific produce/pathogen combinations ranged for Salmonella from 0.53% in iceberg lettuce to 5.1% in cucumber. For Campylobacter, this ranged from 0.83% in endive to 2.7% in oak tree lettuce. These data will be used to determine the public health risk posed by the consumption of ready-to-eat mixed salads in The Netherlands.

Influence of Reconstitution on Isolation and Enumeration of Listeria monocytogenes from Milk Powder Used for Reference Samples.

To test the performance of the Listeria isolation methods, reference samples consisting of gelatin capsules filled with spray-dried milk powder containing Listeria have been developed. During the spray-drying process the Listeria cells are exposed to heat stress and are susceptible to osmotic stress during the reconstitution procedure. To limit the effect of osmotic shock, the milk powder has to be encapsulated in gelatin in order to guarantee slow dissolution. Furthermore, the capsules have to be preen-riched in a nonselective medium. The practical consequences of these findings are discussed.