Impact of plasmid incompatibility groups on conjugation dynamics under biocide exposure

Purpose: Evidence suggests that biocides at sub-inhibitory concentrations in aquatic environments may stimulate the dissemination of antimicrobial resistance via conjugation. Currently, we lack an understanding of the types of chemicals that exert that effect, the influence of the incompatibility group of plasmids, and donor/recipient combinations that display it.

Methods: To achieve this goal, we conducted conjugation assays using conjugative plasmids, RP4, R27, and R1, belonging to IncP-1ɑ, IncHI1, and IncFII, respectively. The plasmids were harbored by E. coli MG1655/P. putida KT2440 (only RP4) while plasmid-free E. coli MG1655 Nal^R Rif^R/P. putida KT2442 Rif^R was used as the recipient strain. Cell densities at approximately 10⁸ CFU/ml of the donors and recipients were used in a 1:1 ratio and mating was carried out in PBS to avoid growth bias. Two different sub-inhibitory concentrations of biocides were used to expose the cells during mating. Non-exposure controls (at the beginning of the experiment t₀ and end-of-experiment time point t₈) were used as the baseline for the detection of conjugal stimulation. The transconjugants, recipients, and donors were enumerated using a Most-Probable-Number method in a 96-well microplate format.

Results: For the three plasmids harbored in E. coli and for RP4 in P. putida, we did a complete assessment for six biocides at two different exposure concentrations below the minimal inhibitory concentration. In addition, E. coli harboring RP4 was further screened against six other biocides. Exposure to these biocides resulted in statistically significant stimulation of horizontal gene transmission when exposed to copper sulphate and silver nitrate. By increasing the number of replicates, we also found that both triclosan and chlorhexidine were capable of stimulating conjugation. Furthermore, we observed inhibition of conjugation for the R27 and R1 suggesting plasmid-dependent interactions with the biocides.

Conclusions: The findings give valuable insight into the risk of AMR plasmid dissemination associated with exposure to environmental concentrations of biocides and lay the foundations for doing an environmental risk assessment.