A molecular study investigating the effect of sulfamethoxazole on the bacterial community of two biological wastewater reactors

With the efficacy of antibiotics diminishing, owing to global increase in bacterial resistance, health authorities are becoming increasingly concerned. Strategies for identifying and remediating potential hot spot sites for bacterial antibiotic resistance gene (ARG) transfer have thus come to the fore. One such site is the wastewater treatment plant (WWTP) found in municipalities worldwide. There are typically two types of biological systems; the conventional activated sludge (CAS) reactor and the membrane bioreactor (MBR). In WWTPs, particularly those used in industry, the MBR is slowly replacing the conventional system due to its smaller footprint and cleaner effluent. Both systems have high microbial density, high nutrient content often with low levels of excreted or disposed of antibiotics, ideal conditions for promoting ARG transfer within the reactors bacterial community. The MBR process has a longer bacterial retention time which may be exacerbating the multi-resistance crisis. The published reports of effective removal of bacterial antibiotic resistance by the various treatment plants are conflicting. Some describe effective removal of bacterial antibiotic resistance by WWTPs prior to environmental release, others show otherwise. Currently, the release of effluent and solid waste from the WWTP is not monitored for multi-resistant bacteria. The concern is that ARGs are entering the environment either via treated effluent into the waterways or waste sludge spread onto fields. The aim of this study was to ascertain whether the MBR facilitates higher levels of antibiotic resistance within the bacterial community compared to the CAS reactor, and secondly to determine if lateral gene transfer could proceed in the presence of low (1ngL-1) and background (200ngL-1) antibiotic levels. Samples from two pilot-scale MBR and CAS bioreactors fed synthetic wastewater were subjected to various molecular studies (DGGE, LH-PCR, qPCR) designed to determine the impact of sulfamethoxazole (smx) on bacterial community dynamics. The potential for lateral gene transfer of the sulfonamide resistance genes (sul1, sul2) was determined using PCR, hybridisations and conjugation mating experiments. The study revealed that low smx levels did not lead to smx resistance gene increases in either reactor; however, high levels of ARGs were maintained in the MBR at typical WWTP background smx concentrations as demonstrated by quantitative PCR (qPCR). Potential for lateral gene transfer was verified with the sul resistant genes localised within integrons and plasmids. One particular plasmid, harbouring a sul1 gene within a Class 1 integron, was observed to be highly promiscuous transferring across species. According to denaturing gradient gel electrophoresis (DGGE) analysis, 690 cultured isolates comprised 16 different species types, all of which are reportedly associated with lateral transference of genes. All 16 representative bacterium harbored more than one antibiotic resistance trait, some to at least seven. High levels of cultiviable smx resistant isolates were found in both the CAS and MBR rectors (60 and 70%, respectively). The high levels were most likely due to clonality rather than acquired resistance based on the low diversity shown by SDI diversity plots. This implies that the total number of ARGs within the bioreactor community must be enormous. Based on the results of this study, recommendations have been made for WWTP bioreactor waste sludge to be subjected to a treatment that effectively destroys its microbial DNA content prior to environmental discharge, thus eliminating the release of highly antibiotic resistant gene pools into the environment