intI 1 gene abundance from septic tanks in Thailand using validated intI 1 primers

Abstract

ABSTRACT Antimicrobial resistance (AMR) poses a serious global health threat, and wastewater treatment (WWT), including septic tanks, is 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 tanks (SST) in Thailand using the class 1 integron-integrase ( intI 1) gene abundance as a proxy for AMR. A systematic review of the literature found 65 intI 1 primers. We evaluated the coverage and specificity of each, including a new MGB TaqMan primer-probe, against clinical and environmental intI 1, intI 1-like, and non- intI 1 databases. The three best primers were selected, laboratory validated for DNA and mRNA quantification, and used to quantify septic tank intI 1 gene abundance. No primer set could distinguish between intI 1 and intI 1-like sequences. While primer choice did not 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 intI 1 gene abundance was greatest in influent > effluent > sludge. intI 1 gene abundance was lowest in the effluent of the SST-household < CST-household < CST-healthcare. 31% to 42% of intI 1 was removed by the CST-household tank, indicating while septic tanks remove some intI 1 they remain a source to the surrounding environment. Toward the goal of achieving standardization across studies, we recommend the F3-R3 primer for intI 1 quantification. IMPORTANCE Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI 1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI 1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI 1 quantification toward the goal of achieving standardization across studies.