Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5 alpha, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10(-2) to 10(-8)). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10(-2) to 10(-7). A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Automated summary

Many antibiotic-resistant bacteria can transfer resistance genes on plasmids to other bacteria. In this study, we found that the broad-host-range plasmid RP4 can transfer between different enteric bacteria, including clinically relevant species. The transfer frequencies varied among different donor-recipient pairings, and the plasmid also showed the ability to retransfer back to its original host. However, the persistence of the plasmid decreased over time without the presence of antibiotics. These findings highlight the potential for plasmid-mediated spread of antibiotic resistance among bacterial populations.