Salmonella enterica serovar Typhimurium hilA-lacZY fusion gene response to iron chelation or supplementation in rich and minimal media.

Virulence expression of Salmonella enterica serovar Typhimurium under iron limited condition was measured by beta-galactosidase (beta-gal) assay using a hilA-lacZY fusion strain and calculated as Miller units. hilA-lacZY beta-galactosidase assays were performed in brain heart infusion (BHI) and minimal media (M9), after iron chelation with 2, 2-dipridyl and iron-supplementation respectively. Before performing virulence assays, concentrations of iron in the media were estimated using ferrozine. Iron content was found to be more in BHI (42.6 microg dL(-1)) as compared to M9 (10.03 microg dL(-1)). beta-gal activity of Salmonella Typhimurium in BHI was generally less than that observed in M9. After exposure to various combinations of iron chelator in BHI, hilA-lacZY activity only increased at the highest concentration of chelator (2001 microM) but decreased in M9 media for all iron concentrations when compared to controls with no iron amendment. These results indicate that iron availability may influence S. Typhimurium hilA expression.

Rishi P ，Woodward CL ，Kim WK ，Ricke SC ... -  《JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART B-PESTICIDES FOOD CONTAMINANTS AND AGRICULTURAL WASTES》

HilA gene expression in SCFAs adapted and inorganic acid challenged Salmonella enterica serovar typhimurium.

Salmonella enterica serovar typhimurium (S. typhimurium) encounters short chain fatty acids (inorganic acids containing propionate, butyrate and acetate) in the intestine as well as in food preservatives. Short chain fatty acids (SCFAs) exposed organisms have been reported to offer resistance to organic acid resulting into enhanced virulence. However, the role of hilA (hyper invasive loci) gene expression has not been assessed in this context. In the present study, S. typhimurium was grown under SCFAs stress condition simulating the in vivo environment and hilA gene expression was evaluated. The gene expression was measured by beta-galactosidase (beta-gal) assay using a hilA-lacZY fusion strain and calculated as Miller units. hilA gene expression was found to be significantly higher in the SCFAs exposed cells than the unexposed ones, after 2 hrs and 4 hrs of exposure. However, no significant difference was observed between the activities at 2 hrs and 4 hrs. It indicates that hilA gene gets expressed by 2 hrs and persists till 4 hrs at least. The beta-gal activity was also measured in the unadapted / SCFAs adapted organisms followed by acid shock for 1 hr. The gene expression was also found to be higher in the SCFAs adapted--acid (pH 3) challenged as compared to the unadapated acid challenged organisms suggesting that SCFAs adaptation may induce organic acid tolerance by modulating the hilA response. This observation indicates that hilA may be the additional gene contributing to acid resistance and thereby increasing virulence of the organism after SCFAs adaptation.

Rishi P ，Ricke S 《-》

Use of a Salmonella typhimurium hilA fusion strain to assess effects of environmental fresh water sources on virulence gene expression.

Many fruits and vegetables are irrigated with water from rivers, lakes and even wastewater systems. Irrigation may be a route for the introduction of Salmonella. Our objectives in this study were to determine survivability and virulence expression in a strain of Salmonella typhimurium when exposed to environmental water sources. Virulence expression was measured using a beta-galactosidase assay on a hilA:lacZY fusion strain of S. typhimurium. Water samples for environmental impact studies were taken from a local pond and specific sites along the Rio Grande River, which serves as a source of irrigation water in southern Texas. There was a significant difference (p<0.05) of virulence expression among the water sites. Certain regions along the Rio Grande River yielded greater amounts of beta-galactosidase activity than others. All sites yielded at least a two-fold greater virulence response than S. typhimurium grown in brain heart infusion. Salmonella survivors were enumerated as colony forming units (CFU)/ml as plated on a selective medium for the duration of 1 week and beta-galactosidase assays were performed to determine a possible correlation between culturable cells and virulence gene expression. Bacterial cells remained viable but decreased after 7 days incubation. In conclusion, water sampled at specific locations and at different times water samples exhibited differences in virulence expression in S. typhimurium.

Nutt JD ，Pillai SD ，Woodward CL ，Sternes KL ，Zabala-Díaz IB ，Kwon YM ，Ricke SC ... -  《WATER RESEARCH》

Effects of Maillard reaction products on hilA expression in Salmonella typhimurium.

Salmonella is a foodborne pathogen causing severe gastroenteritis. Three types of Maillard reaction products (MRP) generated by heat sterilization of D-glucose and L-lysine, L-histidine, and L-arginine were studied at 2 different levels of supplementation (0.5% and 1.0%) for their influence on growth and virulence of Salmonella. Two methods, namely, real-time polymerase chain reaction (RT-PCR) and a beta-galactosidase gene fusion assay, were used to determine the expression of hilA, a regulatory gene for Salmonella pathogenicity. Neither the type of MRP nor their quantities up to 1.0% affected the growth rates of S. Typhimurium EE658 (P > 0.05). When determined by beta-galactosidase assay, lysine MRP in both levels of supplementation were not found to have any effect on the hilA expression compared to the control. The addition of histidine and arginine MRP to M9 media (0.5%) increased by 2-fold hilA induction and up to 6-fold at the higher level (1%) supplementation of these compounds. Although somewhat inconsistent, RT-PCR analyses of hilA expression confirmed the greater induction effect of arginine MRP on hilA compared to lysine MRP. In contrast to beta-galactosidase assay results, however, lysine MRP were found to increase hilA expression compared to the control in both supplementation levels in all trials. The potential of MRP serving as a bacterial virulence modulator may be a factor to be considered in food thermal processing when assessing Salmonella risk for causing foodborne disease.

Kundinger MM ，Zabala-Díaz IB ，Chalova VI ，Ricke SC ... -  《-》

Expression of hilA in response to mild acid stress in Salmonella enterica is serovar and strain dependent.

Salmonella enterica is the leading cause of foodborne illness with poultry and poultry products being primary sources of infection. The 2 most common S. enterica serovars associated with human infection are Typhimurium and Enteritidis. However, Kentucky and Heidelburg and the 2 most prevalent serovars isolated from poultry environments. Given the prevalence of other serovars in poultry products and environments, research is needed to understand virulence modulation in response to stress in serovars other than Typhimurium and Enteritidis. Thus, the objective of this research was to compare hilA gene expression (a master regulator of the virulence pathogenicity island) in response to acid stress among different strains and serovars of Salmonella. A total of 11 serovars consisting of 15 strains of S. enterica were utilized for these experiments. Cultures were suspended in tryptic soy broth (TSB) adjusted to pH 7.2, 6.2, or 5.5 with HCl or acetic acid. Total RNA was extracted from cultures at specific time points (0, 2, 4, and 24 h). Gene expression of hilA was measured with quantitative reverse transcriptase real time PCR (qRT-PCR). Growth and pH were measured throughout the 24 h time frame. Regulation of hilA in response to acid stress varied by serovar and strain and type of acid. The results of these experiments indicate that hilA regulation may have some impact on virulence and colonization of S. enterica. However, these results warrant further research to more fully understand the significance of hilA regulation in response to mild acid stress in S. enterica.

González-Gil F ，Le Bolloch A ，Pendleton S ，Zhang N ，Wallis A ，Hanning I ... -  《-》