Identification of Salmonella enterica Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment

Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Effects of physical properties of feed on microbial ecology and survival of Salmonella enterica serovar Typhimurium in the pig gastrointestinal tract

A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of Salmonella enterica serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (P < 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of S. enterica serovar Typhimurium DT12 in content from the stomach (P < 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (P < 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of S. enterica serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (P < 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (P < 0.05) and colon (P < 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of Salmonella during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.     

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Effects of Physical Properties of Feed on Microbial Ecology and Survival of Salmonella enterica Serovar Typhimurium in the Pig Gastrointestinal Tract

A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of Salmonella enterica serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (P < 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of S. enterica serovar Typhimurium DT12 in content from the stomach (P < 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (P < 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of S. enterica serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (P < 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (P < 0.05) and colon (P < 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of Salmonella during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl₂. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 degrees C: optimization and modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20 degrees C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t(s)), the log-linear decline (k(max)), and the magnitude of a critical component (C). The magnitude of k(max) gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar typhimurium

The rpoS gene encodes the alternative sigma factor sigma(S) (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O(6)-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5' flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.     

Acid resistance variability among isolates of Salmonella enterica serovar Typhimurium DT104

AIMS: Acid resistance could be an indicator of virulence since acid resistant strains are able to better survive the human stomach passage and in macrophages. We studied the acid resistance of several Salmonella Typhimurium DT104 strains isolated from food and humans and identified cellular parameters contributing to the enhanced acid resistance of these isolates. METHODS AND RESULTS: Acid resistance was tested in 37 Salmonella enterica Typhimurium serovar DT104 (S. Typhimurium DT104) strains. Acid adaptation at pH 5 followed by exposure for 2 h at pH 2.5 in the 27 human, nine nonhuman, and in two reference strains, revealed strong variation of acid survival. After 2 h at pH 2.5 six strains of S. Typhimurium DT104 were considered high acid resistant as they displayed a level of survival >10%, 14 strains were considered intermediate acid resistant (level of survival was <10% and >0.01%) and 19 strains were considered low acid resistant (level of survival <0.01%). Six strains were selected for further studies and proteomics revealed a relatively high amount of phase 2 flagellin in an acid-sensitive strain and a relatively high amount of the beta component of the H(+)/ATPase in an acid-resistant strain. Two strains were slightly more heat resistant possibly as the result of increased levels of DnaK or GroEL. CONCLUSIONS: A significant difference could be detected between human and food isolates regarding their acid resistance; all high acid-resistant strains were human isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: S. Typhimurium DT104 is known for two decades and has a great impact on human health causing serious food-borne diseases. Our results suggest the existence of a positive correlation between acid resistance and pathogenicity in S. Typhimurium DT104 as all high acid-resistant strains were isolated from humans.     

pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium

The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.