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 ... -  《-》

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 《-》

Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein.

Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.

Bohez L ，Ducatelle R ，Pasmans F ，Botteldoorn N ，Haesebrouck F ，Van Immerseel F ... -  《VETERINARY MICROBIOLOGY》

Comparison of genetic and physiological properties of Salmonella enterica isolates from chickens reveals one major difference between serovar Kentucky and other serovars: response to acid.

For unknown reasons, Salmonella enterica Kentucky has become the serovar most frequently isolated from chickens and chicken carcasses in the United States. In an attempt to identify traits that may underlie this phenomenon, genetic and physiological features of 30 serovar Kentucky chicken isolates were compared with those of chicken isolates belonging to a range of other S. enterica serovars. Most of the well-known Salmonella virulence genes were detected in the serovar Kentucky isolates by PCR, but the cdtB, spvB, spvC, and pefA genes were not found. The serovar Kentucky isolates were as invasive as the non-Kentucky isolates in in vitro assays involving chicken embryo hepatocytes, but were less invasive than the Enteritidis, Mbandaki, and Typhimurium isolates when incubated with human HCT-8 cells. Statistical comparison of growth, biofilm formation, and stress survival data from the serovar Kentucky and those from the serovar Enteritidis, Hadar, Mbandaka, Senftenberg, Typhimurium, and Worthington isolates demonstrated either no differences or differences with only a few of the serovars; however, three data sets were different. These data sets included the OD(600) values of cultures grown in tryptic soy broth (TSB) adjusted to pH 5.5 with acetic acid and survival counts of cells grown in either TSB pH 7 or TSB adjusted to pH 5.5 with acetic acid and then transferred into TSB adjusted to pH 2.5 with HCl. Most notable was the log(10) reduction for acetic acid pre-exposed Kentucky isolates of 3.1 versus <1 log(10) for the other isolates upon transfer to pH 2.5. The connection, if any, between this acid response phenotype and the prevalence of the serovar Kentucky in poultry remains to be elucidated, but it is possible that slightly better growth in the presence of acetic acid in conjunction with not mounting a strong, energy-consuming acetic acid-induced adaptive acid response provides a small competitive advantage to this serovar in low acid environments such as the cecum where the pH is around 5.5.

Joerger RD ，Sartori CA ，Kniel KE 《-》

Expression of the hilA Salmonella typhimurium gene in a poultry Salm. enteritidis isolate in response to lactate and nutrients.

Pathogens express virulence genes in response to the combination of environmental conditions present in the host environment. The crop is the first gastrointestinal environment encountered in birds. However, feed withdrawal alters the crop environment resulting in an increased pH, and decreased concentrations of lactate, glucose and amino acids compared with unmoulted birds. Salmonella enteritidis infections increase significantly in hens that have been force moulted by feed withdrawal. The present study examined the effects of pH, carbohydrate sources, amino acids and lactate on expression of Salm. enteritidis virulence by measuring expression of hilA. The hilA gene encodes a transcriptional activator that regulates expression of Salmonella virulence genes in response to environmental stimuli. HilA expression was determined using a poultry isolate of Salm. enteritidis carrying a hilA-lacZY transcriptional fusion from Salm. typhimurium. The media used were Luria Bertani (LB) broth and LB broth diluted 1:5 (DLB). The expression of hilA was 2.9-fold higher in DLB broth compared with LB broth which suggested that there is a nutritional component to the regulation of hilA. Addition of 0.2% glucose, fructose or mannose to LB and DLB reduced hilA expression 1.5 to twofold. Addition of 0.2% Casaminoacids, arabinose, fucose, or lactose had little effect on hilA expression. Lactate (25 and 50 mmmol 1-1) reduced hilA expression at pH 6, 5 and 4, with the lowest expression occurring at pH 4. Based on these results it appears that the composition of the crop lumen could potentially influence Salm. enteritidis virulence expression.

Durant JA ，Corrier DE ，Stanker LH ，Ricke SC ... -  《JOURNAL OF APPLIED MICROBIOLOGY》