Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters

Abstract

The dissemination of antimicrobial resistance (AMR) is an escalating problem and a threat to public health. Comparative metagenomics was used to investigate the occurrence of antibiotic resistant genes (ARGs) in wastewater and urban surface water environments in Singapore. Hospital and municipal wastewater (n = 6) were found to have higher diversity and average abundance of ARGs (303 ARG subtypes, 197,816 x/Gb) compared to treated wastewater effluent (n = 2, 58 ARG subtypes, 2,692 x/Gb) and surface water (n = 5, 35 subtypes, 7,985 x/Gb). A cluster analysis showed that the taxonomic composition of wastewaters was highly similar and had a bacterial community composition enriched in gut bacteria (Bacteroides, Faecalibacterium, Bifidobacterium, Blautia, Roseburia, Ruminococcus), the Enterobacteriaceae group (Klebsiella, Aeromonas, Enterobacter) and opportunistic pathogens (Prevotella, Comamonas, Neisseria). Wastewater, treated effluents and surface waters had a shared resistome of 21 ARGs encoding multidrug resistant efflux pumps or resistance to aminoglycoside, macrolide-lincosamide-streptogramins (MLS), quinolones, sulfonamide, and tetracycline resistance which suggests that these genes are wide spread across different environments. Wastewater had a distinctively higher average abundance of clinically relevant, class A beta-lactamase resistant genes (i.e., blaKPC, blaCTX-M, blaSHV, blaTEM). The wastewaters from clinical isolation wards, in particular, had a exceedingly high levels of blaKPC-2 genes (142,200 x/Gb), encoding for carbapenem resistance. Assembled scaffolds (16 and 30 kbp) from isolation ward wastewater samples indicated this gene was located on a Tn3-based transposon (Tn4401), a mobilization element found in Klebsiella pneumonia plasmids. In the longer scaffold, transposable elements were flanked by a toxin–antitoxin (TA) system and other metal resistant genes that likely increase the persistence, fitness and propagation of the plasmid in the bacterial host under conditions of stress. A few bacterial species (Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, Pseudomonas aeruginosa) that were cultured from the isolation ward wastewaters on CHROMagar media harbored the blaKPC-2 gene. This suggests that hospital wastewaters derived from clinical specialty wards are hotspots for the spread of AMR. Assembled scaffolds of other mobile genetic elements such as IncQ and IncF plasmids bearing quinolone resistance genes (qnrS1, qnrS2) and the class A beta-lactamase gene (blaTEM-1) were recovered in wastewater samples which may aid the transfer of AMR.

Geospatial distribution of viromes in tropical freshwater ecosystems

“…In total, 38 libraries and one negative control library using Illumina TruSeq Nano DNA Library Kit were constructed. The sequencing libraries with a corresponding insert size and adapters were prepared accordingly as previously described (Ng et al, 2017). The Illumina's PhiX control library was used as standard.…”

PathRacer: racing profile HMM paths on assembly graph

“…Different non-overlapping paths on the Figure 4 represents several different families of bla CTX−M gene: green corresponds to CTX-M-15 family, while blue and red corresponds to CTX-M-9 and CTX-M-14 respectively (differs from each other only by 2 SNPs). Note that Table S5 in [12] mentiones the presence of CTX-M-15 and CTX-M-18 beta-lactamase families in this dataset, however we found that the extracted graph path that corresponds to CTX-M-9 aligned to the Fig. 5.…”

“…In [12] comparative metagenomics was used to investigate the occurrence of antibiotic resistant genes in wastewater and urban surface water environments in Singapore. For this experiment we took H1 (clinical isolation ward, SRA acc.…”

“…Red path corresponds to TEM-1 family reference with 100% identity. Since the amino acid sequence of the CTX-M-18 beta-lactamase differs from that of the CTX-M-9 beta-lactamase by an Ala-to-Val change at position 231 [14], the additional checking of the results of [12] might be necessary (in [12] different gene sequences were searched in reads rather than assemblies and therefore might be prone to sequencing errors).…”

Genomic and Metagenomic Approaches for Predictive Surveillance of Emerging Pathogens and Antibiotic Resistance

“…A metagenomic characterization study found bla NDM-1 in hospital wastewater, indicating wastewaters as a hotspot for ARGs. 43 An important aspect of understanding and combating the rising prevalence of antibiotic resistance is to survey and monitor environmental reservoirs for infectious agents and accumulation of ARGs. The human microbiome is regularly exposed and influenced by environmental bacteria.…”