Physicochemical factors that favor conjugation of an antibiotic resistant plasmid in non-growing bacterial cultures in the absence and presence of antibiotics

Authors: Headd, Brendan; Bradford, Scott

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2018
Publication Date: 9/11/2018
Citation: Headd, B.J., Bradford, S.A. 2018. Physicochemical factors that favor conjugation of an antibiotic resistant plasmid in non-growing bacterial cultures in the absence and presence of antibiotics. Frontiers in Microbiology. 9:2122. https://doi.org/10.3389/fmicb.2018.02122.
DOI: https://doi.org/10.3389/fmicb.2018.02122

Interpretive Summary: The spread of antibiotic resistant bacteria has become a global health threat. Much of the spread has been attributed to horizontal gene transfer, whereby bacteria are able to spread genes encoding resistance to neighboring bacteria via transformation, transduction, or conjugation. Most studies have only examined conjugation in growing cells, despite the fact that most bacteria in the environment are not growing. We examined conjugation of a plasmid that encodes resistance to multiple antibiotics and heavy metals in growing and non-growing E. coli. We found that shaking bacterial cultures generally resulted in increased conjugation frequencies regardless of if the culture was growing or non-growing. The optimal salinity for conjugation in non-growing E. coli was 9.1mM NaCl, lower and higher salinity solutions had much less conjugation. This salinity effect was attributed to changes in how the bacteria interact with one another in low and high salinity solutions. In the presence of antibiotics we found that non-growing bacterial cultures had up to nine times more conjugation than growing bacteria cultures in the presence of the antibiotic Cefotaxime. The proposed mechanism for this increased conjugation was the absence of a protein that Cefotaxime normally binds to in growing cells, but is not present in non-growing cells. In its absence, Cefotaxime was free to interact with the plasmid carrying the antibiotic resistance genes and induce conjugation. Our results suggest that more attention may need to be paid to horizontal gene transfer in non-growing bacteria.

Technical Abstract: Horizontal gene transfer of antibiotic resistance genes has received increased scrutiny from the scientific community in recent years owing to the public health threat associated with antibiotic resistant bacteria. Most studies have examined horizontal gene transfer in growing cultures. We examined conjugation in growing and non-growing cultures of E. coli using a conjugative multi antibiotic and metal resistant plasmid to determine physiochemical parameters that favor horizontal gene transfer. The conjugation frequency in growing and non-growing cultures was generally greater under shaken than non-shaken conditions, presumably due to increased frequency of cell collisions. Non-growing cultures in 9.1mM NaCl had a similar conjugation frequency to that of growing cultures in Luria-Bertaini broth, whereas those in 1 mM or 90.1 mM NaCl were much lower. This salinity effect on conjugation has not been previously reported, and was attributed to differences in cell-cell interactions and conformational changes in cell surface macromolecules. In the presence of antibiotics, the conjugation frequencies of growing cultures did not increase, but in non-growing cultures of 9.1mM NaCl supplemented with Cefotaxime the conjugation frequency was as much as nine times greater than that of growing cultures. The mechanism responsible for the increased conjugation in non-growing bacteria was attributed to the likely lack of penicillin-binding protein 3 (the target of Cefotaxime), in non-growing cells that enabled Cefotaxime to interact with the plasmid and induce conjugation. Our results suggests that more attention may be owed to horizontal gene transfer in non-growing bacteria as most bacteria in the environment are likely not growing and the proposed mechanism for increased conjugation may not to be unique to the bacteria/plasmid system we studied.