Hosts and hotspots of environmental antibiotic resistance: impact of extracellular DNA, matrix, and the pandemic
Creators: name: Deshpande, Aishwarya Santosh
Publisher: name: No Publisher Supplied
Year: 2023

description: Antibiotic resistance is a global public health crisis. A human health risk assessment for antibiotic resistance has yet to be performed and requires an understanding of the hosts and genetic context of antibiotic resistance genes (ARGs) in addition to studying their abundance. This work fills critical data gaps to improve our understanding of the hazard posed by environmental ARGs in riverine water, sediment, and biofilms as well as wastewater treatment plant water and bioaerosols. In this study, the relative abundance, diversity, hosts, and genetic context of environmental ARGs were studied in 1) rivers to understand the impact of extracellular/ putative “non-viable” cell DNA on metagenomics analyses, 2) wastewater influent to characterize any changes in antibiotic resistome during the pandemic compared to pre-pandemic and 3) air to study the antibiotic resistance with paired wastewater samples. First, sediment, water, and biofilm samples were collected along a land use gradient in the Raritan River, New Jersey. DNA was extracted to separate extracellular DNA (eDNA) and intracellular DNA (iDNA), and qPCR was performed for select ARGs and the 16S rRNA gene. Shotgun metagenomic sequencing was performed on different DNA fractions of sediment samples and two different bioinformatics pipelines for host assignment were compared to evaluate their performance in terms of number and diversity of linkages. The overall microbial community structure was more similar for iDNA and total DNA compared to eDNA and generally clustered by sampling site for sediment samples. ARGs associated with mobile genetic elements increased in iDNA for the downstream sites. Both the bioinformatics pipelines assigned ARGs to putative pathogenic hosts and other comparisons between the pipeline with respect to efficiency and accuracy were made. To study putative “viable-cell” DNA, biofilm and water samples were treated with propidium monoazide (i.e., PMA-DNA). Metagenomic sequencing was performed on PMA-DNA and total DNA extracts revealing significant differences between the two for bacterial community structure and ARG profiles. Removing PMA-bound DNA improved N50 and assembly mapping compared to total DNA extracts. A different measure of the potential hazards associated with ARGs was observed for PMA-DNA, which was indicated by the ARG profiles, microbial community, and host assignments. Next, to study the impact of the pandemic and the speculated impact of increased use of disinfectants and changes in antibiotic prescription rates on the wastewater resistome, DNA extracts of wastewater samples collected from a separate and a combined sanitary sewer system from June 2020- March/May 2021 (early- and later- pandemic), and archived samples from the combined sanitary sewer collected in 2015-16 (pre-pandemic) were analyzed for select ARGs using qPCR. Samples from the combined sanitary sewer along with pre-pandemic samples were sequenced using the long-read Nanopore sequencing platform. Significant differences in qPCR-measured ARGs (qacE, sul1, and tet(G)) were observed between the time phases. Metagenomics analysis revealed significant differences in the abundance of macrolide and sulfonamide ARG classes between the time phases. A positive correlation was observed between the disinfectants/antiseptics class of ARGs and COVID-19 cases while the sulfonamide and aminoglycoside resistance classes showed strong negative correlations with COVID-19 cases. The microbiome analysis revealed biomarkers for the early- and later-pandemic samples, shedding light on the potential co-infections during the pandemic. Finally, given that air is an understudied matrix with respect to antibiotic resistance and the bioaerosol resistome’s relationship to the water resistome at WWTP is poorly understood, paired air and wastewater influent/ effluent samples were collected from two locations in a WWTP: grit chamber and UV disinfection room. Samples were analyzed through culture-dependent and culture-independent techniques. Cultivation of ‘total’ bacteria was performed on Trypticase soy agar (TSA) to study the colony forming units (CFUs) and of methicillin resistant Staphylococcus aureus (MRSA) on CHROMagar MRSA to detect the presence of this opportunistic pathogen. Culture- independent study involved qPCR analysis for select ARGs and shotgun metagenomic sequencing through the Illumina short-read platform. TSA-cultivatable organisms were observed and similar at both sampling locations while MRSA was detected in most air samples from the grit chamber (N=4/5) and some from the UV disinfection room (N=2/5). The ARG sul1 was detected in 3/10 air samples from both sites despite detection of 16S rRNA gene copies in all air samples. The results of this study provide new insights into the influence of eDNA/ putative “non-viable” cell DNA on the resistome, microbiome, mobilome, and hosts in riverine matrices (i.e., water, bed sediments, and biofilms). Additionally, it provides evidence of changes in the wastewater resistome during the COVID-19 pandemic on antibiotic resistance and observations of ARGs and MRSA in bioaerosols at a wastewater treatment plant. Overall, these results provide new insights into environmental resistomes including guidance on environmental sample preparation (i.e., removing eDNA/non-viable cell DNA), the impact of bioinformatics pipelines, matrices of interest, and potential changes during the pandemic with the ultimate aim of helping to inform human health risk assessment.