High genomic diversity of multi-drug resistant wastewater Escherichia coli

Abstract

Wastewater treatment plants play an important role in the emergence of antibiotic resistance. They provide a hot spot for exchange of resistance within and between species. Here, we analyse and quantify the genomic diversity of the indicator Escherichia coli in a German wastewater treatment plant and we relate it to isolates' antibiotic resistance. Our results show a surprisingly large pan-genome, which mirrors how rich an environment a treatment plant is. We link the genomic analysis to a phenotypic resistance screen and pinpoint genomic hot spots, which correlate with a resistance phenotype. Besides well-known resistance genes, this forward genomics approach generates many novel genes, which correlated with resistance and which are partly completely unknown. A surprising overall finding of our analyses is that we do not see any difference in resistance and pan genome size between isolates taken from the inflow of the treatment plant and from the outflow. This means that while treatment plants reduce the amount of bacteria released into the environment, they do not reduce the potential for antibiotic resistance of these bacteria.

Figure 1

Wastewater plays an important role in antibiotic resistance development. Wastewater Escherichia coli isolates were tested for antibiotic resistance and sequenced. Many isolates are multi-drug resistant and have markers often found in pathogenic isolates. Their large pan-genome is a source of potentially novel resistance genes.

Figure 2

The pan-genome at the outflow has the same size as at the inflow, suggesting that highly flexible Escherichia coli emerge from a treatment plant. The wastewater pan-genome is larger than a clinical pan-genome and of similar size to (see Table 1) highly diverse samples comprising pathogenic, commensal, and lab Escherichia coli, as well as Shigella.

Figure 3

1178 Wastewater Escherichia coli isolates were tested for antibiotic resistance to 20 antibiotics covering 4 main classes as well as the Miscellenous class (chloramphenicol and fosfomycin). Nearly all isolates are multi-drug resistant. Isolates were highly susceptible to carbapenems (meropenem and imipenem) which are beta-lactams. Isolates were also more susceptible to fluoroquinolones than to tetracyclins and aminoglycosides. The outflow isolates (n = 322) show similar resistance as inflow (n = 856) (p-value 0.0001), suggesting that wastewater treatment is not reducing resistance development.

Figure 4

Top 10 correlating genes for 18 antibiotics from correlation of genomes to resistance phenotype. Antibiotics were color-coded based on antibiotic class following the scheme in Fig. 3. The highlighted yellow boxes represent genes involved in resistance to the respective antibiotics based on available literature.

Figure 5

Phylogeny of wastewater Escherichia coli from the inflow (n = 50) and the outflow (n = 42) of a wastewater treatment plant. Phylogenetic tree, multi-locus sequence types (shown as numbers in black), and phylogroups of 92 sequenced wastewater Escherichia coli isolates reveal 16 potential ExPEC isolates (marked with a black star) in phylogroups B2 (yellow) and D (green), which are associated with pathogenicity. Half of these 16 isolates stem from the outflow of the treatment plant.