Genomic Analysis of Carbapenem-Resistant Comamonas in Water Matrices: Implications for Public Health and Wastewater Treatments

Abstract

Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired bla_GES-5, bla_OXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All bla_GES-5- and bla_OXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to "last-resort drugs," such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and inorganic substrates is well documented, some species are also opportunistic pathogens. However, data regarding AMR in Comamonas spp. are limited. Here, through the genomic analyses of 39 carbapenem-resistant Comamonas isolates, we make several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encoding carbapenemase bla_GES-5 or extended-spectrum β-lactamase bla_OXA alleles. Given the importance of Comamonas species in potential wastewater bioaugmentation and bioremediation strategies, as well as their status as emerging pathogens, the acquisition of critically important antibiotic resistance genes on genomic islands warrants future monitoring.

Keywords: Comamonas; Comamonas denitrificans; antimicrobial resistance (AMR); blaGES-5; carbapenemase; genomic islands; incP-1; wastewater; whole-genome sequencing (WGS).

FIG 1

Comamonas phylogeny. Midpoint-rooted maximum-likelihood phylogeny and associated metadata of 88 Comamonas species using Phylosift. Isolates from this collection are colored in purple. Bootstrap values greater than 0.9 are shown as pink dots. The blue strip designates isolates carrying antimicrobial-resistant genes (ARGs). AMR, antimicrobial resistance.

FIG 2

C. dentrificans phylogeny. Phylogenetic relationships of Australian C. denitrificans. The midpoint-rooted maximum-likelihood tree was generated from a single-nucleotide polymorphism (SNP)-based alignment via Parsnp, with C. denitrificans JAFNM01 used as a reference. Clades are colored in descending order: light green, maroon, gray, yellow, green, purple, blue, and pink.

FIG 3

C. denitrificans pangenome. Pangenome analyses of 33 C. denitrificans from Australian aquatic environments and C. denitrificans JAFNEM01 sourced from GenBank. Color groupings match the clades designated in Fig. 2. Phylogenetic clustering is by accessory genomes.

FIG 4

Antimicrobial resistance genes (ARGs) in Comamonas spp. Heat map of ARGs identified in all available Comamonas spp. genomes adjacent to a midpoint-rooted maximum-likelihood phylogenetic tree generated using Phylosift. Specific ARGs, detergent resistance genes, and integrases are shown in green. Resistance conferred by gene carriage is shown in purple. NSBL, narrow spectrum β-lactamase.

FIG 5

Genomic islands (GIs) carrying AMR determinants. (A) Comparison of three GIs carrying bla_GES-5 at the chromosomal insertion site. (B) Comparison of two GIs, one carrying bla_OXA-10, the other bla_OXA-5-like. GI in the same genetic context as panel A. (C) Comparison of GI carrying blaGES-5. The genetic context differs to panels A and B and is associated with integrase intIPac. ORF, open reading frame.

FIG 6

BRIG comparisons of IncP-1 plasmids. (Left) Plasmid map of putative IncP-1 plasmid from ERQ0628 and BLAST alignments to other IncP-1 plasmids. Contig breaks are noted in the tra operon. (Right) Plasmid map of D. acidovorans pUO1 and BLAST alignments to other IncP-1 plasmids.