Highly sensitive detection of antimicrobial resistance genes in hospital wastewater using the multiplex hybrid capture target enrichment

Abstract

Wastewater can be useful in monitoring the spread of antimicrobial resistance (AMR) within a hospital. The abundance of antibiotic resistance genes (ARGs) in hospital effluent was assessed using metagenomic sequencing (mDNA-seq) and hybrid capture (xHYB). mDNA-seq analysis and subsequent xHYB targeted enrichment were conducted on two effluent samples per month from November 2018 to May 2021. Reads per kilobase per million (RPKM) values were calculated for all 1,272 ARGs in the constructed database. The monthly numbers of patients with presumed extended-spectrum β-lactamase (ESBL)-producing and metallo-β-lactamase (MBL)-producing bacteria, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) were compared with the monthly RPKM values of bla CTX-M , bla IMP , mecA , vanA , and vanB by xHYB. The average RPKM value for all ARGs detected by xHYB was significantly higher than that of mDNA-seq (665, 225, and 328, respectively, and P < 0.05). The average number of patients with ESBL producers and RPKM values of bla CTX-M-1 genes in 2020 were significantly higher than that in 2019 (17 and 13 patients per month and 921 vs 232 per month, respectively, both P < 0.05). The average numbers of patients with MBL-producers, MRSA, and VRE were 1, 28, and 0 per month, respectively, while the average RPKM values of bla IMP , mecA , vanA , and vanB were 6,163, 6, 0, and 126 per month, respectively. Monitoring ARGs in hospital effluent using xHYB was found to be more useful than conventional mDNA-seq in detecting ARGs including bla CTX-M , bla IMP, and vanB , which are important for infection control. IMPORTANCE Environmental ARGs play a crucial role in the emergence and spread of AMR that constitutes a significant global health threat. One major source of ARGs is effluent from healthcare facilities, where patients are frequently administered antimicrobials. Culture-independent methods, including metagenomics, can detect environmental ARGs carried by non-culturable bacteria and extracellular ARGs. mDNA-seq is one of the most comprehensive methods for environmental ARG surveillance; however, its sensitivity is insufficient for wastewater surveillance. This study demonstrates that xHYB appropriately monitors ARGs in hospital effluent for sensitive identification of nosocomial AMR dissemination. Correlations were observed between the numbers of inpatients with antibiotic-resistant bacteria and the ARG RPKM values in hospital effluent over time. ARG surveillance in hospital effluent using the highly sensitive and specific xHYB method could improve our understanding of the emergence and spread of AMR within a hospital.

Comparison of three sequencing methods for identifying and quantifying antibiotic resistance genes (ARGs) in sewage

“…Similarly, clinically relevant carbapenems ARGs blaNDM-1, blaVIM, blaKPC and blaIMP were only detected by QIAseq xHYB AMR panel, albeit in low abundance (< 0.00001). Similar results were also observed in a study in Japan [22].…”

“…Based on the proportions of ARGs detected, tetracycline ( abundance (< 0.00001). Similar results were also observed in a study in Japan [22].…”

“…The presence of ARGs in sulfonamide antibiotic class such as sul1 in sewage implicate strong anthropogenic contamination and increased rates of horizontal gene transfer (HGT) [22]. QIAseq xHYB AMR panel and shotgun sequencing were the only methods that identified sul1, sul2 and sul4 as dominant ARGs.…”

“…The presence of ARGs in sulfonamide antibiotic class such as sul1 in sewage implicate strong anthropogenic contamination and increased rates of horizontal gene transfer (HGT) [22].…”

A proof of concept for a targeted enrichment approach to the simultaneous detection and characterization of rickettsial pathogens from clinical specimens

“…It has previously been demonstrated that a variety of in-solution hybridization-based enrichment ( Gnirke et al, 2009 ) designs can increase the sensitivity of HTS for the detection of viruses or bacteria in complex sample matrices ( Christiansen et al, 2014 ; Matranga et al, 2014 ; Bonsall et al, 2015 ; Briese et al, 2015 ; Mate et al, 2015 ; Wylie et al, 2015 ; Brown et al, 2016 ; O'Flaherty et al, 2018 ; Lim et al, 2019 ; Paskey et al, 2019 ; Furtwängler et al, 2020 ; Li et al, 2020 ; Elliott et al, 2021 ; Wylezich et al, 2021 ; Kuchinski et al, 2022 ) although there can also be challenges in terms of false positive identification of pathogens and the bioinformatic methods involved ( Kapel et al, 2023 ). Hybridization enrichment sequencing has also been demonstrated to enrich the sensitivity of detecting antimicrobial resistance genes in wastewater samples ( Baba et al, 2023 ). Here, we extend the application of this strategy to investigate the possibility that a whole genome, targeted enrichment approach could widen the diagnostic window for R. prowazekii and O. tsutsugamushi and, unlike previous studies, including an O. tsutsugamushi study ( Elliott et al, 2021 ) we apply this approach not just to whole genome sequence-based detection and characterization, but also to qPCR-based detection.…”