The emergence and global spread of antibiotic resistance among life-threatening pathogens are serious public health threats. Conjugative plasmids are considered the leading cause of spreading antibiotic-resistant genes among pathogens. The human gut microbiota is considered an important reservoir of antibiotic resistance genes. However, little is known about the frequencies and mechanistic drivers of the plasmids-mediated spread of antimicrobial resistance in the human gut. This study aims to determine the frequency and transferability of conjugative RP4 plasmid among enteric bacteria in both in-vitro settings and in-vivo mouse models transplanted with human gut microbiota.We performed in-vitro experiments to determine the primary and secondary frequencies of a broad host range plasmid RP4 to multiple naïve host bacteria. We demonstrated that the RP4 plasmid transferred from human gut commensal donor E. coli LM715-1 to Citrobacter rodentium, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas putida, Vibrio cholerae, and three different strains of E. coli. However, the plasmid transfer frequency (TF) differed greatly between specific donor-recipient pairings, ranging from 10-2 to 10-8. We also observed that recipients of RP4 further transferred that plasmid to commensal E. coli. Furthermore, we examined the effect of the antibiotic ampicillin on RP4 plasmid transfer frequency from human gut commensal E. coli LM715-1 to Citrobacter rodentium, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas putida, Vibrio cholerae, and E. coli. A serial passage plasmid persistence assay showed that the RP4 plasmid imposed a fitness cost on its host, E. coli LM715-1, resulting in the loss of the plasmid over time. However, plasmid-bearing cells persisted at a low proportion of the population for at least ten transfers. Next, we performed in vivo experiments to develop a tractable mouse model transplanted with the adult human gut microbiota to study the RP4 plasmid-mediated spread of ARGs from a human gut commensal donor E. coli LM715-1 to resident bacteria of the human gut microbiota. We found that commensal donor strain E. coli LM715-1 colonized the mouse gut and persisted throughout the ten-day experiment, while the laboratory donor strain E. coli MG1655 was not recovered after 48 hours. Next, we tracked donor and recipient bacteria in a complex microbial community using flow cytometry to sort the transconjugant bacteria. The flow cytometry of the treatment group fecal samples showed an increased spread of detectable cells with the tagged plasmid when compared to those before the gavage. Donor and transconjugant bacteria were recovered from fecal samples and sorted by FACS. A 16S sequencing analysis of sorted cells showed Lachnospiraceae, Clostridiaceae, Pseudomonadaceae, Rhodanobacteraceae, Erysipelotrichaceae, Oscillospiraceae, and Butyricicoccaceae are the primary target bacterial families of RP4 plasmid acquisition. In summary, the findings from these studies have paved a path for addressing the spread and persistence of antibiotic-resistant bacteria through horizontal gene transfer in complex environments like the human gut. The transplanted mouse model would likely to serve an important tool to study epidemiologic, evolutionary, and ecological aspects of antibiotic resistance in the human gut.
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