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Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to dissemination of antibiotic resistance genes (ARGs) in the gut microbiota. The gut microbiota often suffers from various disturbances. It is not clear whether and how disturbed microbiota may affect ARG mobility under antibiotic treatments. For proof of concept, in the presence or absence of streptomycin pre-treatment, mice were inoculated orally with a β-lactam-susceptible Salmonella enterica serovar Heidelberg clinical isolate (recipient) and a β-lactam resistant Escherichia coli O80:H26 isolate (donor) carrying a blaCMY-2 gene on an IncI2 plasmid. Immediately following inoculation, mice were treated with or without ampicillin in drinking water for 7 days. Faeces were sampled, donor, recipient and transconjugant were enumerated, blaCMY-2 abundance was determined by quantitative PCR, faecal microbial community composition was determined by 16S rRNA amplicon sequencing and cecal samples were observed histologically for evidence of inflammation. In faeces of mice that received streptomycin pre-treatment, the donor abundance remained high, and the abundance of S. Heidelberg transconjugant and the relative abundance of Enterobacteriaceae increased significantly during the ampicillin treatment. Co-blooming of the donor, transconjugant and commensal Enterobacteriaceae in the inflamed intestine promoted significantly (PblaCMY-2 gene in the gut microbiota of mice that received the combination of streptomycin pre-treatment and ampicillin treatment (Str–Amp) compared to the other mice. Following cessation of the ampicillin treatment, faecal shedding of S. Heidelberg transconjugant persisted much longer from mice in the Str–Amp group compared to the other mice. In addition, only mice in the Str–Amp group shed a commensal E. coli O2:H6 transconjugant, which carries three copies of the blaCMY-2 gene, one on the IncI2 plasmid and two on the chromosome. The findings highlight the significance of pre-existing gut microbiota for ARG dissemination and persistence during and following antibiotic treatments of infectious diseases.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to the dissemination of antibiotic-resistance genes in the gut microbiota and the development of antibiotic-resistant bacterial infection, a significant threat to animal and public health. Food or water may be contaminated with multiple resistant bacteria, but animal models on gene transfer were mainly based on single-strain infections. In this study, we investigated the mobility of β-lactam resistance following infection with single- versus multi-strain of resistant bacteria under ampicillin treatment. We characterized three bacterial strains isolated from food-animal production systems, Escherichia coli O80:H26 and Salmonella enterica serovars Bredeney and Heidelberg. Each strain carries at least one conjugative plasmid that encodes a β-lactamase. We orally infected mice with each or all three bacterial strain(s) in the presence or absence of ampicillin treatment. We assessed plasmid transfer from the three donor bacteria to an introduced E. coli CV601gfp recipient in the mouse gut, and evaluated the impacts of the bacterial infection on gut microbiota and gut health. In the absence of ampicillin treatment, none of the donor or recipient bacteria established in the normal gut microbiota and plasmid transfer was not detected. In contrast, the ampicillin treatment disrupted the gut microbiota and enabled S. Bredeney and Heidelberg to colonize and transfer their plasmids to the E. coli CV601gfp recipient. E. coli O80:H26 on its own failed to colonize the mouse gut. However, during co-infection with the two Salmonella strains, E. coli O80:H26 colonized and transferred its plasmid to the E. coli CV601gfp recipient and a residential E. coli O2:H6 strain. The co-infection significantly increased plasmid transfer frequency, enhanced Proteobacteria expansion and resulted in inflammation in the mouse gut. Our findings suggest that single-strain infection models for evaluating in vivo gene transfer may underrepresent the consequences of multi-strain infections following the consumption of heavily contaminated food or water.