Identification of Salmonella enterica serovar Typhimurium genes important for survival in the swine gastric environment

Abstract

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.

FIG. 1.

Survival of the rpoS, phoP, and fur mutants in the SSC assay. SX 2 (wild type), SX 21 (rpoS), SX 46 (phoP), and SB 270 (fur) strains were grown in minimal EG medium to a mid-logarithmic phase of an OD₆₀₀ of 0.4 (∼2 × 10⁸ CFU/ml). The cultures were adapted at pH 4.4 for 1 h. Acid-adapted cells were washed and resuspended in an equal volume of filter-sterilized SSC. Viable counts were determined at 0, 10, and 20 min for determination of percent survival.

FIG. 2.

Effect of pH on the survival of serovar Typhimurium in the SSC assay. Wild-type serovar Typhimurium UK1 (SX 2) was grown to a mid-log phase of an OD₆₀₀ of 0.4. Following acid adaptation at pH 4.4 for 1 h, the cells were pelleted and resuspended in an equal volume of SSC at pHs 3.87, 4.4, and 7.0 (adjusted with NaOH) as well as E buffer at pH 3.87. Viable counts were determined at 0, 10, 30, 60, and 120 min for determination of percent survival.

FIG. 3.

Effects of SSC filtration on survival of signature-tagged mutants. The SSC was filtered consecutively through centricon centrifugal filter units with molecular mass cutoffs of 30, 10, and 3 kDa. The filtrate from each was employed to challenge mid-log-phase, pH 4.4-adapted STM pool A for 0 or 10 min. The Southern hybridization patterns of STM pool A were determined following challenge in the filtered and unfiltered SSC. STM pool A input represents the 16 mutants present prior to challenge in the SSC assay, whereas the output pools indicate the absence or presence of signature-tagged mutants following challenge. The signature-tagged mutants represented in STM pool A are as follows: A = SB 29; B = SB 271; C = SB 110; D = SB 117; E = SB 104; F = SB 116; G = SB 272; H = SB 273; I = SB 156; J = SB 126; K = SB 114; L = SB 134; M = SB 113; N = SB 135; O = SB 274; P = SB 275. The controls are indicated in the key by circling (negative) or underlining (positive) the strain designation.

FIG. 4.

Survival of serovar Typhimurium in synthetic gastric fluid containing lactic acid. Wild-type serovar Typhimurium UK1 (SX 2) was grown to mid-log phase, adapted at pH 4.4 for 1 h, pelleted, and resuspended in an equal volume of synthetic gastric fluid at pH 3.87 containing lactic acid at 0, 5, 10, or 20 mM. Viable counts were determined at 0, 15, 30, 60 and 90 min for determination of percent survival.

FIG. 5.

Survival of serovar Typhimurium mutants in synthetic gastric fluid containing 10 mM lactic acid. SX 2 (wild type), SX 21 (rpoS), SX 46 (phoP), SB 270 (fur)m and SB 130 (pnp) were grown in minimal EG medium to an OD₆₀₀ of 0.4. Following acid adaptation at pH 4.4 for 1 h, the cells were washed and resuspended in an equal volume of synthetic gastric fluid containing 10 mM lactic acid. Viable counts were determined at 0, 30, and 60 min for determination of percent survival.