title: Antimicrobial resistance: molecular approaches to track antimicrobial resistance gene spread from decentralised septic tank wastewater
Creators: name: Okonkwo, Valentine Obinna
Publisher: name: University of Glasgow
Year: 2023

description: Antimicrobial resistance (AMR) is a major global public health and wastewater treatment (WWT), including septic tanks, are now recognised as hotspots and potential sources of AMR genes to the environment. However, compared to centralised WWT (e.g., municipal WWT), an in-depth understanding of the contributions of septic tanks in the dissemination of AMR and mobile genetic elements (MGEs) remains scarce. Nonetheless, effective AMR gene monitoring from polluted settings such as WWT to the environment remains challenging primarily due to multiple AMR genes found in WWT. The class 1 integronintegrase (intI1) gene was proposed as a proxy for inferring potential AMR pollution elevates challenges associated with multiple monitoring. Yet, the suitability of this gene as an adequate and reliable proxy for inferring AMR pollution remains unclear. To this end, this thesis focused on using state-of-the-art molecular tools to:- 1) Evaluate and validate the suitability of the intI1 gene as a proxy for inferring overall AMR abundance using wastewater samples from septic tanks from Thailand treating household and healthcare wastewater; and 2) Evaluate the contributions of conventional septic tanks (CST) associated with household and healthcare usage, and the newly developed solar septic tank (SST) associated with household usage in the dissemination of AMR genes and MGE to the environment. The results from this study proposed one primer set (F3-R3) for intI1 quantification of genes and transcripts. However, it found that none of the current intI1 primers can distinguish between intI1 (highly conserved intI1 variant; &gt;98% protein similarity to pVS1 intI1 reference protein) and intI1-like (lesser conserved intI1 variant; &lt;98% protein similarity to pVS1), therefore, potentially contributes to an overestimation of quantified intI1 gene abundance. Furthermore, the relative abundance (relative to the 16S rRNA gene) of a fewer number of AMR genes correlated positively and significantly to the abundance of intI1 compared to intI3. Therefore, taken together, indicates that intI1 cannot serve as a proxy for overall AMR gene abundance. The septic tanks were found significant source of AMR gene subtypes and abundance. However, depending on the molecular method used the tank posing the highest risk of AMR or integrase, dissemination to the environment differed. HT-QPCR, after careful validation of the array, identified the CST-household tank, among the three reactors, as potentially the higher contributor of AMR and integrases (intI1 and intI3) gene abundance to the environment via its sludge and effluent. In contrast, shotgun metagenomics identified the CST-healthcare septic tank, among the three reactors, as potentially the highest contributor of ARG abundance to the environment through its effluent and sludge (if applied directly to the environment). Therefore, emphasises the trade-off that must be considered when selecting a molecular tool for effective AMR monitoring. This study has provided valuable insights into contributions from septic tanks in disseminating AMR and integrases (intI1, intI2, intI3) genes to the environment.