Drane, Kezia, Huerlimann, Roger, Jones, Rhondda, Whelan, Anna, Sheehan, Madoc, Ariel, Ellen, and Kinobe, Robert (2025) Concordance in molecular methods for detection of antimicrobial resistance: A cross sectional study of the influent to a wastewater plant. Journal of Microbiological Methods, 228. 107069.

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Abstract

Methods that are used to characterise microbiomes and antimicrobial resistance genes (ARGs) in wastewater are not standardised. We used shotgun metagenomic sequencing (SM-Seq), RNA sequencing (RNA-seq) and targeted qPCR to compare microbial and ARG diversity in the influent to a municipal wastewater treatment plant in Australia. ARGs were annotated with CARD-RGI and MEGARes databases, and bacterial diversity was characterised by 16S rRNA gene sequencing and SM-Seq, with species annotation in SILVA/GreenGenes databases or Kraken2 and the NCBI nucleotide database respectively. CARD and MEGARes identified evenly distributed ARG profiles but MEGARes detected a richer array of ARGs (richness = 475 vs 320). Qualitatively, ARGs encoding for aminoglycoside, macrolide-lincosamide-streptogramin and multidrug resistance were the most abundant in all examined databases. RNA-seq detected only 32 % of ARGs identified by SM-Seq, but there was concordance in the qualitative identification of aminoglycoside, macrolide-lincosamide, phenicol, sulfonamide and multidrug resistance by SM-Seq and RNA-seq. qPCR confirmed the detection of some ARGs, including OXA, VEB and EREB, that were identified by SM-Seq and RNA-seq in the influent. For bacteria, SM-Seq or 16S rRNA gene sequencing were equally effective in population profiling at phyla or class level. However, SM-Seq identified a significantly higher species richness (richness = 15,000 vs 3750). These results demonstrate that SM-Seq with gene annotation in CARD and MEGARes are equally sufficient for surveillance of antimicrobial resistance in wastewater. For more precise ARG identification and quantification however, MEGARes presented a better resolution. The functionality of detected ARGs was not confirmed, but general agreement on the putative phenotypic resistance profile by antimicrobial class was observed between RNA-Seq and SM-Seq.

Item ID:	84196
Item Type:	Article (Research - C1)
ISSN:	1872-8359
Copyright Information:	© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Date Deposited:	25 Nov 2024 22:25
FoR Codes:	31 BIOLOGICAL SCIENCES > 3103 Ecology > 310305 Marine and estuarine ecology (incl. marine ichthyology) @ 25% 32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320799 Medical microbiology not elsewhere classified @ 25% 41 ENVIRONMENTAL SCIENCES > 4105 Pollution and contamination > 410599 Pollution and contamination not elsewhere classified @ 50%
SEO Codes:	18 ENVIRONMENTAL MANAGEMENT > 1802 Coastal and estuarine systems and management > 180201 Assessment and management of coastal and estuarine ecosystems @ 50% 28 EXPANDING KNOWLEDGE > 2801 Expanding knowledge > 280102 Expanding knowledge in the biological sciences @ 50%
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