Chowdhury, N. N.; Forry, S. P.; Servetas, S.; Hunter, M. E.; Dootz, J. N.; Dunkers, J. P.; Jackson, S. A.

This work explored the role of natural transformation - a mechanism by which bacteria uptake and express extracellular genes - in driving antibiotic resistance propagation in the human gut microbiome. The model extracellular antibiotic resistance gene (eARG) - a plasmid containing a kanamycin resistance (kanR) gene and a green fluorescence protein (GFP) gene - was dosed into pooled and homogenized human stool and incubated anaerobically. Cellular uptake of the eARG was assessed via droplet digital PCR, the expression of newly acquired genes was assessed by culturing on selective media and fluorescent microscopy, newly resistant isolates were identified by long-read Nanopore sequencing and the impacts on the taxonomy of the gut microbiome was assessed using shotgun Illumina sequencing. Significant gene uptake of both kanR and GFP was quantified in gut microbes, and extent of gene accumulation correlated with background kanamycin levels. Gut microbes dosed with background kanamycin expressed kanamycin resistance acquired by the eARG (as quantified by CFU on kanamycin-containing media). Newly resistant isolates, identified as Enterococcus faecium by long-read sequencing, also expressed green fluorescence acquired from the eARG. Though compositional changes of the kanamycin-resistant subpopulation were observed in the gut microbiome in response to eARG and antibiotic exposure, these changes were not reproducible among replicates and trends in taxonomy due to transformation could not be identified. This comprehensive analysis therefore establishes the significant propagation of antibiotic resistance within the human gut microbiome due to eARG exposure, while evaluating the utility of various measurements in characterizing transformation in a complex microbial community.