Fate of CMY-2-Encoding Plasmids Introduced into the Human Fecal Microbiota by Exogenous Escherichia coli

Abstract

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical bla_CMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from a human and from poultry meat. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and green fluorescent protein [GFP]), allowing fluorescence-activated cell sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6, and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 10² CFU/ml. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that the plasmids were predominantly acquired by Enterobacteriaceae species, such as E. coli and Hafnia alvei Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Small amounts of the exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment.

Keywords: CoMiniGut model; Escherichia coli; IncI1; cephalosporin.

FIG 1

Pearson correlation coefficient (y axis) between the inoculum concentration and the number of donor cells (D) (black bars), transconjugants (TC) (gray bars) in fecal samples A, E, and O under (a) anoxic (An) and (b) oxic (O) conditions. ns, nonsignificant; *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

FIG 2

Gardner-Altman 2-group mean-difference plot showing the difference between donors (D) transconjugants (TC) and transconjugant/donor (TC/D) ratios at (a) 2 h, (b) 6 h, and (c) 24 h for the poultry meat strain 1061-1-GM (blue) and the human strain C20-GM (orange) in CoMiniGut cultures. The left axis shows the number of donors detected by FACS. On the right axis, the filled curve indicates the complete Δ distribution given the observed data. The human strain C20-GM survives better than the poultry strain 1061-1-GM; however, more transconjugants were detected from the poultry meat strain than the human UTI strain. The low and high bias-corrected and accelerated bootstrap interval values are shown as a density plot on the right side. The confidence interval of the mean differences at 95% is illustrated by the thick black line. Significance was determined by the Mann-Whitney U test.

FIG 3

Pearson correlation coefficient (y axis) indicating the relationship between initial Enterobacteriaceae counts in the fecal samples and (a) numbers of the exogenous strain poultry strain (black bars) and human strain (gray bars) or (b) transconjugants that acquired their plasmids over time. After 24 h, the numbers of the two exogenous strains negatively correlated with the counts of preexisting Enterobacteriaceae in the original fecal sample (a). A significant negative correlation was also seen between counts of preexisting Enterobacteriaceae and the numbers of transconjugants that received the plasmid from poultry strain after 24 h (b). ns, nonsignificant; *, P < 0.05.

FIG 4

Relative abundance at phylum level in 10 fecal samples (A to O) before (a) and 24 h after (b) inoculation of the two exogenous strains of human and poultry origin into the corresponding CoMiniGut culture. The figure shows that the abundance of Proteobacteria increased after inoculation of the exogenous strains, although with marked differences between individual fecal samples.

FIG 5

Relative ASV abundance as a function of fecal donor and strain source; only ASVs detected in the sorted transconjugants from CoMiniGut culture are shown. ASVs with <0.05% relative abundance are grouped in “other.” (a) Fecal sample, (b) CoMiniGut samples, (c) sorted transconjugants. The key shows the lowest taxonomic rank (family/class/genus) that could be confidently attributed to each amplicon sequence variant using Bayesian classification.

FIG 6

Unweighted UniFrac-based principal-coordinate analysis (PCoA) showing the clustering of bacterial communities according to the sample type and strain source. The strain source is human donor assay (circles) or poultry donor assay (triangles). The sample types are CoMiniGut cultures after 24 h (light blue), fecal samples before inoculation (dark blue), and sorted transconjugants from both assays (green). Each dot represents a sample.