Zhiming He

Mating assays are common laboratory experiments for measuring the conjugation frequency, i.e. efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, conjugation frequencies may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating duration. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 harbored in Escherichia coli at a fixed cell density and mating conditions, inoculated a serial dilution of the mating mixture on transconjugant-selective plates or in transconjugant-selective broth, and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (liquid vs solid), the enumeration of transconjugants becomes significantly biased if the plated cell density exceeds 28 Colony Forming Unit (CFU)/mm² (or 1.68•10⁵ CFU/standard 9 cm Petri dish). This threshold is determined with a 95% confidence interval of ± 4 CFU/mm² (± 2.46•10⁴ CFU/standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the conjugation frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate at this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay. As an alternative, a liquid enumeration method can be utilized to increase the signal-to-noise ratio and allow for more accurate enumeration of transconjugants.

A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance.