Simulated Gastric Acid Promotes the Horizontal Transfer of Multidrug Resistance Genes across Bacteria in the Gastrointestinal Tract at Elevated pH Levels

Abstract

The assessment of factors that can promote the transmission of antibiotic resistance genes (ARGs) across bacteria in the gastrointestinal tract is in great demand to understand the occurrence of infections related to antibiotic-resistant bacteria (ARB) in humans. However, whether acid-resistant enteric bacteria can promote ARG transmission in gastric fluid under high-pH conditions remains unknown. This study assessed the effects of simulated gastric fluid (SGF) at different pH levels on the RP4 plasmid-mediated conjugative transfer of ARGs. Moreover, transcriptomic analysis, measurement of reactive oxygen species (ROS) levels, assessment of cell membrane permeability, and real-time quantitative assessment of the expression of key genes were performed to identify the underlying mechanisms. The frequency of conjugative transfer was the highest in SGF at pH 4.5. Antidepressant consumption and certain dietary factors further negatively impacted this situation, with 5.66-fold and 4.26-fold increases in the conjugative transfer frequency being noted upon the addition of sertraline and 10% glucose, respectively, compared with that in the control group without any additives. The induction of ROS generation, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to the increased transfer frequency. These findings indicate that conjugative transfer could be enhanced under certain circumstances in SGF at elevated pH levels, thereby facilitating ARG transmission in the gastrointestinal tract. IMPORTANCE The low pH of gastric acid kills unwanted microorganisms, in turn affecting their inhabitation in the intestine. Hence, studies on the factors that influence antibiotic resistance gene (ARG) propagation in the gastrointestinal tract and on the underlying mechanisms are limited. In this study, we constructed a conjugative transfer model in the presence of simulated gastric fluid (SGF) and found that SGF could promote the dissemination of ARGs under high-pH conditions. Furthermore, antidepressant consumption and certain dietary factors could negatively impact this situation. Transcriptomic analysis and a reactive oxygen species assay revealed the overproduction of reactive oxygen species as a potential mechanism by which SGF could promote conjugative transfer. This finding can help provide a comprehensive understanding of the bloom of antibiotic-resistant bacteria in the body and create awareness regarding the risk of ARG transmission due to certain diseases or an improper diet and the subsequent decrease in gastric acid levels.

Keywords: antibiotic-resistant bacteria; genome-wide RNA sequencing; horizontal gene transfer; pH; simulated gastric fluid.

FIG 1

Effects of SGF pH and exposure time on the frequency of conjugative transfer (left) and fold changes in the frequency of conjugative transfer (right) of the RP4 plasmid from donor E. coli HB101 cells to recipient E. coli K-12 cells (A) and Salmonella Aberdeen cells (B). The bars denote means ± standard deviations (SDs) from three separate experiments. Significant differences are shown with * (P < 0.05) and ** (P < 0.01).

FIG 2

Effects of drug consumption on the frequency of conjugative transfer between E. coli HB101 and Salmonella Aberdeen after exposure to SGF at pH 4.5 for 4 h. The bars denote means ± SDs from three separate experiments. Significant differences are shown with * (P < 0.05 versus the control [0 mg/L]) and ** (P < 0.01 versus the control [0 mg/L]).

FIG 3

Effects of dietary factors on the frequency of conjugative transfer between E. coli HB101 and Salmonella Aberdeen after exposure to SGF at pH 4.5 for 4 h. The bars denote means ± SDs from three separate experiments. Significant differences are shown with * (P < 0.05) and ** (P < 0.01).

FIG 4

Transcriptomic analysis of DEGs in E. coli HB101 (left) and Salmonella Aberdeen (right) between the SGF group exposed at pH 4.5 and the control group exposed at pH 7.0 for 4 h. (A) Volcano map of DEGs. Padjust, adjusted P value. (B) Heat map of DEGs related to cellular antioxidant systems, cell membrane permeability, pilus formation, and ATP synthesis.

FIG 5

Effects of ROS production and cell membrane permeability on conjugative transfer. (A) Comparison of ROS levels (left) and fold changes in ROS production (right) between the SGF group exposed at pH 3.5 and 4.5 and the control group exposed at pH 7.0 for 4 h. (B) Comparison of conjugative transfer frequencies (left) and fold changes in the conjugative transfer frequencies (right) when an ROS scavenger was used versus when it was not used. (C) Comparison of cell membrane permeability (left) and fold changes in cell membrane permeability (right) between the SGF group exposed at pH 3.5 and 4.5 and the control group exposed at pH 7.0 for 4 h. The bars denote means ± SDs from three separate experiments. Significant differences are shown with * (P < 0.05) and ** (P < 0.01).

FIG 6

Differential expression of genes related to pilus formation and conjugative transfer upon exposure to SGF at pH 4.5. Relative concentrations of interesting genes were assessed by RT-qPCR. Fold differences in the expression levels of interesting genes between the SGF group exposed at pH 4.5 and the control group exposed at pH 7.0 were calculated. The primer sequences used are listed in Table S5 in the supplemental material. The bars denote means ± SDs from three separate experiments.