Intl1 gene abundance from septic tanks in Thailand using validated intl1 primers
Antimicrobial resistance (AMR) poses serious global health threat and wastewater treatment (WWT), including septic tanks, are a source of AMR. In Thailand, antibiotics are unregulated, and septic tanks are commonly used. Yet their impact on the spread or mitigation of AMR is unknown. We monitored household and healthcare conventional septic tanks (CST) and household Solar Septic Tank (SST) in Thailand using the class 1 integron-integrase (intl1) gene abundance as a proxy for AMR.  A systematic review of the literature found 65 intl1 primers.  We evaluated the coverage and specificity of each, including a new MGB-TaqMan primer-probe, against clinical and environmental intl1, intl1-like and non-intl1 databases.  Three best primers were selected, laboratory validated for DNA and mRNA quantification and used to quantify septic tank intl1 gene abundance.  No primer set could distinguish between intl1 and intl1-like sequences. While primer choice didn't affect gene abundance of the same sample (p-value >0.05), sometimes when comparing the same samples quantified by different primers, statistical differences were observed for one but not the other primer set. This may lead to different interpretations of AMR risk. Irrespective of primers or reactor type intl1 gene abundance was greatest in influent>effluent>sludge. intI1 gene abundance was lowest in the effluent of the SST-household<CST-household<CST-healthcare. 31 to 42% of intI1 was removed by the CST-household tank, indicating while septic tanks remove some intl1 they remain a source to surrounding environment. Towards the goal of achieving standardisation across studies we recommend the F3-R3 primer for intI1 quantification