Physicochemical Factors That Favor Conjugation of an Antibiotic Resistant Plasmid in Non-growing Bacterial Cultures in the Absence and Presence of Antibiotics

Abstract

Horizontal gene transfer (HGT) 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 HGT 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 9.1 mM 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 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 9.1 mM 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 HGT in non-growing bacteria as most bacteria in the environment are likely not growing and the proposed mechanism for increased conjugation may not be unique to the bacteria/plasmid system we studied.

The Conjugation Window in an Escherichia coli K-12 Strain with an IncFII Plasmid

“…The highest conjugation frequencies were observed in 9.1 mM NaCl supplemented with 25 g/ml of CFX, with the nonantibiotic control having lower conjugation frequencies. These trends are consistent with our earlier findings (25).…”

“…coli K-12 ER1793 is a streptomycin-resistant and restriction enzyme-deficient strain obtained from New England BioLabs (Ipswich, MA) that was modified to produce a strain that contained a chromosomal blue fluorescent protein (mTagBFP2) and pUUH239.2 (herein referred to as ER1793_mTag/pUUH239.2) and a strain that contained a chromosomal yellow fluorescent protein gene (SYFP2) and a chromosomal chloramphenicol resistance gene (herein referred to as ER1793_SYFP2-Cat). Details on the strain modification have been previously described (25).…”

“…Antibiotics. MIC assays of strains used in conjugation experiments were carried out previously (25) to establish selective concentrations of antibiotics for use in conjugation experiments. The following antibiotics were used to maintain strains and/or used in conjugation experiments: ampicillin sodium salt (AMP; Fisher Scientific, Waltham, MA), cefotaxime sodium salt (CFX; ACROS Organics, Thermo Fisher Scientific, Waltham, MA), kanamycin monosulfate (KAN; Fisher Scientific), gentamicin sulfate (GEN; Sigma-Aldrich, St. Louis, MO), tetracycline hydrochloride (TET; RPI, Mount Prospect, IL), and chloramphenicol (CAT; Fisher Scientific, Waltham, MA).…”

“…A limited number of studies have shown that conjugation can still occur in nutrientdepleted natural and artificially created environments (17)(18)(19)(20)(21)(22)(23)(24). Recently, it was demonstrated that the conjugation frequency can even be higher under nongrowing rather than growing conditions (25). The underlying factors that control the dynamics of conjugation under nongrowing conditions and their relationship to growing conditions remain unresolved.…”

Modelling environmental antibiotic-resistance gene abundance: A meta-analysis

“…Given the complexity of this relationship, there are numerous potential factors that can influence gene transfer and abundance, e.g. temperature, metals, pH and salinity (Headd and Bradford, 2018;Liang et al, 2013;Miller et al, 2014;Seiler and Berendonk, 2012). Moreover, environmental co-occurrence of antibiotics and ARGs does not necessarily indicate a causal relationship, since they are generally emitted into the environment simultaneously and follow similar environmental pathways.…”