Measuring Microbial Community-Wide Antibiotic Resistance Propagation via Natural Transformation in the Human Gut Microbiome

Abstract

1) 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/625464v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d9dfc0org.highwire.dtl.DTLVardef@f0df34org.highwire.dtl.DTLVardef@1ce04bforg.highwire.dtl.DTLVardef@99a6e2_HPS_FORMAT_FIGEXP M_FIG C_FIG 2) ImportanceInfections from antibiotic resistant bacteria in the human gut microbiome are a growing public health concern. Antibiotic resistance may develop in gut microbiota from exposure to environmentally prevalent extracellular antibiotic resistance genes (eARGs). This work explores the impact of eARG exposure on a complex human gut microbial community. It quantifies significant accumulation and expression of eARG-borne genes by endogenous gut microorganisms, thereby demonstrating that natural transformation may play a role in resistance propagation in the human gut. It also demonstrates the highly variable changes in gut taxonomy in response to eARG exposure, implying that eARGs may impact gut composition and therefore downstream human health effects. These data may be useful in characterizing and mitigating resistance propagation in the human gut, and in general, the suite of genotypic and phenotypic measurements used constitute a quantitative framework to characterize the effects of perturbations on complex microbiomes.