Intestinal flora metabolites indole-3-butyric acid and disodium succinate promote IncI2 mcr-1- carrying plasmid transfer

Abstract

Introduction: Plasmid-driven horizontal transfer of resistance genes in bacterial communities is a major factor in the spread of resistance worldwide. The gut microbiome, teeming with billions of microorganisms, serves as a reservoir for resistance genes. The metabolites of gut microorganisms strongly influence the physiology of their microbial community, but the role of the metabolites in the transfer of resistance genes remains unclear.

Methods: A dual-fluorescence conjugation model was established. We assessed the effects of different concentrations of indole-3-butyric acid (IBA) and disodium succinate (DS) on plasmid transfer using conjugation assays. The growth of bacteria (donors, recipients, and transconjugants), the reactive oxygen species (ROS) levels and membrane permeability were measured under IBA and DS exposure. The plasmid copy number, and transcriptional levels of conjugation-related genes (including the related genes of the regulation of ROS production, the SOS response, cell membrane permeability, pilus generation, ATP synthesis, and the type IV secretion system (T4SS) ) were evaluated by qPCR.

Results: In this study, we demonstrated that IBA and DS at low concentrations, which can also be ingested through diet, enhance the interspecies transfer ratio of IncI2 mcr-1-carrying plasmid in Escherichia coli. At 20 mg/L, the transfer ratios in the presence of IBA or DS increased by 2.5- and 2.7-fold compared to that of the control, respectively. Exposure to this concentration of IBA or DS increased the production of reactive oxygen species (ROS), the SOS response, cell membrane permeability, and plasmid copy number. The transcription of genes of the related pathways and of pilus, ATP, and the T4SS was upregulated.

Discussion: Our findings revealed that low-dose gut microbiota metabolites-particularly those with dietary origins-promote plasmid-mediated resistance gene dissemination through multifaceted mechanisms involving oxidative stress, SOS activation, and conjugation machinery enhancement. This highlights potential public health risks associated with microbiota metabolites, especially those utilized in food production.

Keywords: DS; IBA; IncI2; conjugation; intestinal flora metabolite; mcr-1-carrying plasmid.

Figure 1

The effects of IBA and DS on the transfer ratios of IncI2 pMCR-1 in E. coli conjugation pairs. Fold changes in plasmid transfer ratios after 18 h of IBA (A) and DS (B) treatment were shown. The results represent the mean ± SD of six biological samples. Significant differences between the IBA or DS treatment groups at the different concentrations and the control group were tested by t-test and indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

The effects of IBA and DS on the growth of donor, recipient, transconjugant. The growth situation of the recipient, donor and transconjugant with IBA (A, B, E) and DS (C, D, F) were shown. The results represent the mean ± SD of three biological samples. Significant differences between the IBA or DS treatment groups at the different concentrations and the control group were tested by t-test and indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Changes related to the ROS production and the SOS respone after exposure to IBA and DS. Fold changes in ROS production by E. coli MG1655 and E. coli CNE6 under IBA (A) and DS (B) exposure for 2 h were shown. The ratios of conjugative transfer under IBA (C) and DS (D) in the presence and absence of the ROS scavenger were shown. The results represent the mean ± SD of six biological samples. The * on the numbers indicated inter-group differences, while the * in the graph represented intra-group differences. Fold changes in the transcription levels of ROS (E) and SOS (F) related genes in the conjugation systems were shown. The results represent the mean ± SD of three biological samples. Significant differences between the IBA or DS treatment groups at the different concentrations and the control group were tested with t-test and indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Changes related to the cell membrane permeability after exposure to IBA and DS. Fold changes in the cell membrane permeability by E. coli MG1655 and E. coli CNE6 after 2 h treatment of IBA (A) and DS (B) were shown. The results represent the mean ± SD of six biological samples. Fold changes in the transcription levels of cell membrane-related genes (C) in the conjugation systems were shown. The results represent the mean ± SD of three biological samples. Significant differences between the IBA or DS treatment groups at the different concentrations and the control group were tested with t-test and indicated by *p < 0.05, **p<0.01, and ***p< 0.001.

Figure 5

Changes related to the plasmid copies after exposure to IBA and DS. Fold changes in the plasmid copy numbers in the conjugation systems after 18 h treatment with IBA (A) and DS (B) were shown. The results represent the mean ± SD of three biological samples. Significant differences between the IBA or DS treatment groups at the different concentrations and the control group were tested with t-test and indicated by *p < 0.05.

Figure 6

Changes related to the transcription levels of genes associated with pilus generation, ATP synthesis, and T4SS after exposure to IBA and DS. Fold changes in the transcription levels of genes related to pilus generation and ATP synthesis in the conjugation systems, as well as the T4SS of donor after 2 h treatment with IBA (A) and DS (B) were shown. The results represent the mean ± SD of three biological samples. Significant differences between the IBA or DS treatment groups and the control group were tested with t-test and indicated by *p < 0.05, **p<0.01, and ***p< 0.001.

Figure 7

The metabolic pathway changes associated with the accelerated conjugative transfer of ARGs mediated by the IncI2 pMCR-1 under the influence of IBA and DS. The schematic diagram was created with BioRender.com.