intI1 gene abundance from septic tanks in Thailand using validated intI1 primers

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 (intI1) gene abundance as a proxy for AMR. A systematic review of the literature found 65 intI1 primers. We evaluated the coverage and specificity of each, including a new MGB TaqMan primer-probe, against clinical and environmental intI1, intI1-like, and non-intI1 databases. The three best primers were selected, laboratory validated for DNA and mRNA quantification, and used to quantify septic tank intI1 gene abundance. No primer set could distinguish between intI1 and intI1-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 intI1 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 intI1 they remain a source to the surrounding environment. Toward the goal of achieving standardization across studies, we recommend the F3-R3 primer for intI1 quantification.

Automated summary

Antimicrobial resistance poses a serious global health threat, and septic tanks in Thailand are found to be a source of this resistance. The abundance of the intI1 gene, a proxy for antimicrobial resistance, was monitored in conventional and solar septic tanks in Thailand. It was found that the gene abundance was highest in influent, lowest in the effluent of solar septic tanks, and 31% to 42% of the gene was removed by the conventional septic tank. Different primers used for quantification of the gene could lead to different interpretations of antimicrobial resistance risk. The F3-R3 primer is recommended for standardization in future studies.