Transcriptomic Approach for Understanding the Adaptation of Salmonella enterica to Contaminated Produce

Abstract

Salmonellosis is a form of gastroenteritis caused by Salmonella infection. The main transmission route of salmonellosis has been identified as poorly cooked meat and poultry products contaminated with Salmonella. However, in recent years, the number of outbreaks attributed to contaminated raw produce has increased dramatically. To understand how Salmonella adapts to produce, transcriptomic analysis was conducted on Salmonella enterica serovar Virchow exposed to fresh-cut radish greens. Considering the different Salmonella lifestyles in contact with fresh produce, such as motile and sessile lifestyles, total RNA was extracted from planktonic and epiphytic cells separately. Transcriptomic analysis of S. Virchow cells revealed different transcription profiles between lifestyles. During bacterial adaptation to fresh-cut radish greens, planktonic cells were likely to shift toward anaerobic metabolism, exploiting nitrate as an electron acceptor of anaerobic respiration, and utilizing cobalamin as a cofactor for coupled metabolic pathways. Meanwhile, Salmonella cells adhering to plant surfaces showed coordinated upregulation in genes associated with translation and ribosomal biogenesis, indicating dramatic cellular reprogramming in response to environmental changes. In accordance with the extensive translational response, epiphytic cells showed an increase in the transcription of genes that are important for bacterial motility, nucleotide transporter/metabolism, cell envelope biogenesis, and defense mechanisms. Intriguingly, Salmonella pathogenicity island (SPI)-1 and SPI-2 displayed up- and downregulation, respectively, regardless of lifestyles in contact with the radish greens, suggesting altered Salmonella virulence during adaptation to plant environments. This study provides molecular insights into Salmonella adaptation to plants as an alternative environmental reservoir.

Keywords: Salmonella enterica; epiphytic; planktonic; produce; transcriptomics.

Fig. 1. Growth of Salmonella enterica serovar Virchow FORC_038 in contact with radish greens.

(A) Bacterial growth curve in the presence of radish greens. Salmonella cells were added to phosphate-buffered saline (PBS) containing fresh-cut radish greens at 2 × 10⁷ colony-forming units (CFU)/ml and the optical density at 600 nm (OD₆₀₀) was measured. PBS containing only radish greens was used a control to examine the contamination with indigenous bacteria. The data from three independent tests were averaged. (B) Bacterial enumeration in the presence of radish greens. The numbers of planktonic cells (CFU/ml) and epiphytic cells (CFU/g) adhering to plant tissues were counted using XLD agar plating. Bacterial viability in PBS was measured as a control. The average values of three biological replicates were plotted.

Fig. 2. Characterization of differentially regulated genes (DEGs).

(A) Numbers of DEGs that were up- or downregulated in planktonic or epiphytic cells in contact with radish greens. Genes with altered transcription of three-fold or more in the presence of plants were counted, and the numbers are shown in Venn diagrams. (B) Functional categorization of DEGs. Genes that were up- or downregulated three-fold or more in the presence of plants were grouped based on Clusters of Orthologous Groups (COG) analysis, and their percentages in each COG group are illustrated using bars of different colors depending on lifestyles.

Fig. 3. DEGs observed in both planktonic and epiphytic cells at 24 h post-contact with plants.

(A) Heat maps of genes that were up- or downregulated in both lifestyles were represented using fold-changes (log₂[TMM(plant)/TMM(PBS)]) and depicted using a colorimetric gradient: downregulation in blue and upregulation in red. P 1, planktonic 1 h; P 24, planktonic 24 h; E 24, epiphytic 24 h; TMM, Trimmed Mean of M-value. (B) Validation of DEGs using quantitative reverse transcription polymerase chain reaction (qRT-PCR). For each gene, the ΔCt values of PBS-treated cells were subtracted from the ΔCt values of planktonic or epiphytic cells, and the mean values of three independent tests were plotted with their standard deviations (SDs).

Fig. 4. Expression of genes associated with anaerobic respiration in contact with plants.

(A) Heat maps of genes associated with nitrate/nitrite reductase activity and cobalamin biosynthesis were represented using fold-changes (log₂[TMM(plant)/TMM(PBS)]) and depicted using a colorimetric gradient: downregulation in blue and upregulation in red. P 1, planktonic 1 h; P 24, planktonic 24 h; E 24, epiphytic 24 h; TMM, Trimmed Mean of M-value. (B) Validation of DEGs using qRT-PCR. For each gene, the ΔCt values of PBS-treated cells were subtracted from the ΔCt values of planktonic or epiphytic cells, and the mean values of three independent tests were plotted with their SDs.

Fig. 5. Expression of genes associated with ribosomal biogenesis in contact with plants.

(A) Heat maps of genes associated with the bacterial translation process were represented using fold-changes (log₂[TMM(plant)/TMM(PBS)]) and depicted using a colorimetric gradient: downregulation in blue and upregulation in red. P 1, planktonic 1 h; P 24, planktonic 24 h; E 24, epiphytic 24 h; TMM, Trimmed Mean of M-value. (B) Validation of DEGs using qRT-PCR. For each gene, the ΔCt values of PBS-treated cells were subtracted from the ΔCt values of planktonic or epiphytic cells, and the mean values of three independent tests were plotted with their SDs.

Fig. 6. Expression of genes associated with Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs) in contact with plants.

(A) Heat maps of genes associated with SPI-1/SPI-2 T3SSs were represented using fold-changes (log₂[TMM(plant)/TMM(PBS)]) and depicted using a colorimetric gradient: downregulation in blue and upregulation in red. P 1, planktonic 1 h; P 24, planktonic 24 h; E 24, epiphytic 24 h; TMM, Trimmed Mean of Mvalue. (B) Validation of DEGs using qRT-PCR. For each gene, the ΔCt values of PBS-treated cells were subtracted from the ΔCt values of planktonic or epiphytic cells, and the mean values of three independent tests were plotted with their SDs.

Fig. 7. Effect of phytic acid on Salmonella growth in contact with plants.

PBS solutions containing radish greens were supplemented with different concentrations of phytic acid from 0 mM to 7.5 mM and inoculated with S. Virchow FORC_038. Bacterial growth was measured every hour, and the OD₆₀₀ values were averaged from three independent tests. PBS containing only radish greens without Salmonella was tested in parallel as a control to examine the contamination with indigenous bacteria.