Salmonella enterica virulence genes are required for bacterial attachment to plant tissue

Abstract

Numerous Salmonella enterica food-borne illness outbreaks have been associated with contaminated vegetables, in particular sprouted seeds, and the incidence of reported contamination has steadily risen. In order to understand the physiology of S. enterica serovar Newport on plants, a screen was developed to identify transposon mutants that were defective in attachment to alfalfa sprouts. Twenty independent mutants from a pool of 6,000 were selected for reduced adherence to alfalfa sprouts. Sixty-five percentage of these mutants had insertions in uncharacterized genes. Among the characterized genes were strains with insertions in the intergenic region between agfB, the surface-exposed aggregative fimbria (curli) nucleator, and agfD, a transcriptional regulator of the LuxR superfamily, and rpoS, the stationary-phase sigma factor. Both AgfD and RpoS have been reported to regulate curli and cellulose production and RpoS regulates other adhesins such as pili. The intergenic and rpoS mutants were reduced in initial attachment to alfalfa sprouts by 1 log unit compared to the wild type. Mutations of agfA, curli subunit, and agfB in S. enterica serovar Enteritidis differentially affected attachment to plant tissue. The agfA mutation was not reduced in ability to attach to or colonize alfalfa sprouts, whereas the agfB mutation was reduced. Thus, agfB alone can play a role in attachment of S. enterica to plant tissue. These results reveal that S. enterica genes important for virulence in animal systems are also required for colonization of plants, a secondary host that can serve as a vector of S. enterica from animal to animal.

FIG. 1.

Adhesion of S. enterica Newport to alfalfa sprouts. (A) Cells of the S. enterica Newport rpoS mutant (JDB 279) and Tn10:lac:kan insertion in the agfD/agfB intergenic region (JDB 287) were recovered from 3-day-old alfalfa sprouts and compared to wild-type S. enterica Newport after 4-h adhesion assays. The data are averages of three experiments. (B) Cells of JDB 279 recovered from alfalfa sprouts 24 and 48 h postinoculation compared to wild-type S. enterica Newport. (C) Cells of JDB 279 and JDB 423 recovered from alfalfa sprouts 24 h postinoculation compared to wild-type S. enterica Newport. The data for panels B and C are representative experiments with two internal replicates and five sprout samples taken from each. The standard deviations are represented as error bars in all panels.

FIG. 2.

Phenotypic characterization of S. enterica wild-type (section 1), JDB 287 (section 2), JDB 279 (section 3), and JDB 423 (section 4) Newport strains grown on Congo red indicator media (A) and LB no salt containing calcofluor (B) for 48 h at 28°C.

FIG. 3.

Adhesion of S. enterica serovar Enteritidis to alfalfa sprouts. (A) Attachment of cells to 3-day-old alfalfa sprouts in a 4-h attachment assay; (B) attachment and colonization 24 h postinoculation. The data are representative experiments with 10 sprout samples taken for each strain. The standard deviations are represented as error bars in all panels.