Growth and virulence properties of biofilm-forming Salmonella enterica serovar typhimurium under different acidic conditions

Abstract

This study was designed to characterize the viability and potential virulence of bofilm-forming Salmonella enterica serovar Typhimurium under different pH levels, ranging from 5 to 7. The plate count method and real-time reverse transcription-PCR (RT-PCR) were used to evaluate the survival of S. Typhimurium grown in Trypticase soy broth (TSB) adjusted to pH 5, 6, and 7 (TSB-5, TSB-6, and TSB-7, respectively) at 37°C for 10 days. In TSB-5 and TSB-6, the numbers of viable cells estimated by using the real-time RT-PCR were greater than the culturable counts enumerated by the plate count method. Reflectance micro-Fourier transform infrared (micro-FTIR) spectroscopy was used to evaluate the biochemical changes in biofilm cells. Considerable changes in chemical components were observed in the biofilm cells grown in TSB-5 and TSB-6 when compared to the cells grown in TSB-7. The enterotoxin production and invasive ability of planktonic and biofilm S. Typhimurium cells were inferred by the relative levels of expression of stn and invA. The levels of expression of stn and invA were significantly increased in biofilm S. Typhimurium cells grown in TSB-5 (1.9-fold and 3.2-fold) and TSB-6 (2.1-fold and 22.3-fold) after 10 days of incubation. These results suggest that the biofilm-forming S. Typhimurium under different pH levels might change the virulence production and stress response mechanisms.

FIG. 1.

Plate and relative counts of planktonic cells of S. Typhimurium in TSB adjusted to pH 5.0 (TSB-5; ▪, □), pH 6.0 (TSB-6; ▴, ▵), and pH 7.0 (TSB-7; •, ○) during 10 days of incubation at 37°C. Viable cells were estimated by the plate count method (▪, ▴, •) and real-time RT-PCR assay (□, ▵, ○). An asterisk indicates counts below the detection limit of 1 log CFU/ml. The viability of S. Typhimurium below the detection limit (1 log₁₀ CFU/ml) was observed by enrichment culture, and negative growth (*) was assigned at 0 for statistical analysis.

FIG. 2.

Plate and relative counts of biofilm cells of S. Typhimurium in TSB adjusted to pH 5.0 (TSB-5; ▪, □), pH 6.0 (TSB-6; ▴, ▵), and pH 7.0 (TSB-7; •, ○) during 10 days of incubation at 37°C. Viable cells were estimated by the plate count method (▪, ▴, •) and real-time RT-PCR assay (□, ▵, ○). The viability of S. Typhimurium below the detection limit (1 log₁₀ CFU/ml) was observed by enrichment culture, and negative growth (*) was assigned at 0 for statistical analysis.

FIG. 3.

Reflectance micro-FTIR spectra of S. Typhimurium biofilm cells on stainless steel coupons immersed in TSB adjusted to pH 5.0 (TSB-5), pH 6.0 (TSB-6), and pH 7.0 (TSB-7) after 10 days at 37°C. a.u., arbitrary units.

FIG. 4.

Relative gene expression of stn in planktonic (A) and biofilm (B) cells of S. Typhimurium in TSB adjusted to pH 5.0 (TSB-5; □), pH 6.0 (TSB-6;,), and pH 7.0 (TSB-7; ▪) during 10 days of incubation at 37°C. Mean values with different letters within the same incubation time (A and B) and the same pH value (a to c) are significantly different at P < 0.05.

FIG. 5.

Relative gene expression of invA in planktonic (A) and biofilm (B) cells of S. Typhimurium in TSB adjusted to pH 5.0 (TSB-5; □), pH 6.0 (TSB-6;), and pH 7.0 (TSB-7; ▪) during 10 days of incubation at 37°C. Mean values with different letters within the same incubation time (A to C) and the same pH value (a to c) are significantly different at P < 0.05.