Persisting antibiotic resistance gene pollution and its association with human sewage sources in tropical marine beach waters

Abstract

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are pollutants of worldwide concern that threaten human health and ecosystems. Anthropogenic activities and wastewater could be ARB and ARG pollution sources; however, research on ARG abundance and microbial source tracking (MST) of contamination in tropical marine waters is limited. This study examined spatiotemporal variations of six ARGs (bla_NDM, bla_TEM, bla_VIM, mcr-1, sul1, and tetQ) against the widely used antibiotic groups and a class 1 integron-integrase gene (intI1) at two Thai tropical recreational beaches (n = 41). Correlations between ARGs and sewage-specific MST markers (i.e., crAssphage and human polyomaviruses [HPyVs]) and fecal indicator bacteria (i.e., total coliforms, fecal coliforms, and enterococci) were also investigated. Bla_TEM, intI1, sul1, and tetQ were ubiquitous at both beaches (85.4-100% detection rate); intI1 was the most abundant (3-6 orders in log₁₀ copies/100 mL), followed by bla_TEM (2-4 orders), sul1 (2-3 orders), and tetQ (2-4 orders). Bla_NDM was found in 7.3% (up to 4 orders), and no mcr-1 was detected. Interestingly, bla_VIM was prevalent at one beach (2-5 orders; n = 17), but found in only one sample at the other (4 orders). Temporal, but not spatial, differences were noticed; bla_TEM was at higher levels in the wet season. IntI1 correlated with sul1 and tetQ (Spearman's rho = 0.47-0.97), suggesting potential horizontal gene transfer. CrAssphage, but not HPyVs, correlated with intI1, sul1, and tetQ (Spearman's rho = 0.50-0.74). Higher numbers of ARGs tended to co-occur in samples with higher crAssphage concentrations, implying sewage contribution to the marine water, with a persisting ARG background. This study provides insight into the ARG pollution status of tropical coastal waters and suggests crAssphage as a proxy for ARG pollution, which could facilitate effective management policies to minimize ARG dissemination in marine environments.

Keywords: Antibiotic resistance genes; Fecal pollution; Microbial source tracking; Quantitative PCR; Seawater; Southeast asia; crAssphage.