Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella enterica serovar Typhimurium selected following exposure to disinfectants

Abstract

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-TolC for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

FIG. 1.

Colony morphology of the wild type (WT), OXCR1, QACFGR2, and TOPR2 grown on brilliant green agar at 20°C for 2 weeks.

FIG. 2.

Growth monitored by determining the optical densities at 600 nm (OD600) of wild-type Salmonella serovar Typhimurium strain SL1344 (open circle), OXCR1 (filled triangle), QACFGR2 (grey diamond), and TOPR2 (grey circle) in LB broth at 37°C with shaking. Experiments were performed in triplicate, and the error bars indicate standard deviations.

FIG. 3.

Reductions in the numbers of viable wild-type Salmonella serovar Typhimurium strain SL1344 (open circle), OXCR1 (filled triangle), QACFGR2 (grey diamond), and TOPR2 (grey circle) cells after desiccation at 20°C for 13 days. Cells were grown until stationary phase before treatment in LB broth at 37°C. Experiments were performed in duplicate, and the error bars indicate standard deviations. An asterisk indicates a statistically significant difference compared to the wild type (P < 0.05).

FIG. 4.

Invasiveness of wild-type Salmonella serovar Typhimurium strain SL1344 (WT), OXCR1, QACFGR2, and TOPR2 in Caco-2 human colon adenocarcinoma cells at 37°C. Experiments were performed in triplicate, and the error bars indicate standard deviations. An asterisk indicates a statistically significant difference compared to the wild type (P < 0.05).

FIG. 5.

LPS profiles of Salmonella serovar Typhimurium strains. The graphs show the band intensities expressed as the inverse of the relative light intensity for the LPS profiles of TOPR2 (A), QACFGR2 (B), and OXCR1 (C) compared with the profile of the wild type (WT). Gel electrophoresis and analyses were repeated three times to confirm the reproducibility, and representative results are shown.