PROLIFERATION AND ATTENUATION OF ANTIMICROBIAL RESISTANCE IN NATURAL AND ENGINEERED AQUATIC SYSTEMS

Abstract

The rise of antimicrobial resistance (AMR) in environmental matrices has been linked to the rise in the resistant infections in the tertiary care units in the hospitals and it is expected to claim 10 million lives by 2050 each year, making it a global concern. The effluent from sewage treatment plants (STPs), runoff from agricultural catchments, industrial waste, hospital wastewater, etc. make their way into the regional aquatic ecosystems and exert selection pressure on the microbial community. The role of horizontal gene transfer (HGT) becomes critical in the proliferation of the antibiotic resistance genes (ARGs) to the commensal bacteria and other microorganisms. The current study quantifies the regionally problematic and clinically relevant ARGs by utilizing qPCR technique. The study targeted a total of 40 ARGs (different number in different objectives) which included 4 clinically relevant ARGs viz: blaOXA-1, blaOXA-7, blaTEM, mcr5, and tetW; three genetic markers viz: 16S rRNA (marker for bacterial DNA), yccT (marker for fecal coliform bacteria: Escherichia coli), and intI1 (marker for anthropogenic pollution in the environmental matrices). The study also targeted clinical antibiotics: ciprofloxacin, one nonpharmaceutical antimicrobial: triclosan using LC-MS/MS and four heavy metals: copper, chromium, lead and zinc using ICP-MS. The first phase of the current study was dedicated to the quantification of ARGs in the aquatic environment of the metropolitan city: Chennai, with a special focus on the effects of droughts and floods on the Perungudi STP. Twentyone sites were sampled and river water, canal water, groundwater, and STP samples were collected over a period of one year having three seasons viz: dry mild summer, monsoon, mild winter. This study concluded that extreme weather events: flood and drought did not impact the ARGs removal in the targeted Perungudi STP and that the Perungudi STP selected for tetW and blaOXA-1 genes in the maturation pond unit. The median relative abundance of ARGs consistently ranked in the following order: sul1 (log10(x͂) = 0.440671) > ermF (log10(x͂) = -0.0827) > tetW (log10(x͂) = -0.6101) > mcr5 (log10(x͂) = -2.17509) > sul2 (log10(x͂) = -2.38768) > parC (log10(x͂) = -2.39439) > blaOXA-1 (log10(x͂) = -3.13634) throughout the PSTP study period (x͂ = the median relative ARG concentration). The study also revealed that land use patterns had no significant impact on the abundance of the targeted ARGs, seasonally and the areas having less area under cultivation had higher iv relative levels of tetW than the areas having more area under cultivation. The absolute abundance of total bacteria as indicated by the 16S rRNA gene copies was higher in highintensity agricultural area compared to the low-intensity agricultural area. A similar trend was followed in industrial and residential sampling locations. The next phase of the research work quantified the overall impact of mass bathing on the performance of the STPs adjacent to the River Ganga during Kanwar festival. There was no significant reduction in the relative levels of the targeted ARGs in STP-A. On the other hand, in the STP-B, the relative abundance of tetW and sul1 were higher before disinfection than in the influent (tetW: 1.18 orders of magnitude, p = 0.04; sul1: 2.5 orders of magnitude, p = 0.001). Our study showed that there was no change in the performance of the targeted STPs due to shock loading caused by the mass bathing event and none of the targeted STPs were able to significantly reduce the ARGs in the effluent. The last phase of the current research work was focused on the development of the labscale modified sequencing batch reactor (SBR), having equal aeration and anoxic phases, to significantly reduce the abundance of the targeted ARGs in the effluent. The designed SBR was able to significantly reduce the abundance of sul1, acrA, tetA, czcA, and qnrS but was not able to significantly remove the blaTEM gene in the effluent. The relative abundance of blaTEM (log10(x͂) = -3.71) genes was one order of magnitude higher in the outlet (p < 0.01). All the ARGs (except blaTEM) were significantly lower in the outlet when compared with the inlet (log10(x͂) = -4.55). The modified SBR effluent complied with the discharge standard limit set by the central pollution control board (CPCB, New Delhi) and was efficiently removing organic and nutrient loads. The major conclusion from the current study is that the STPs employing conventional technologies are not able to significantly reduce the abundance of the targeted ARGs in the effluent and with modification in the SBR (a conventional treatment technique), it might be possible to reduce the abundance of clinically relevant ARGs; thereby alleviating the overall AMR problem in the environmental matrices due to discharge of poorly treated STP effluent into the surface water bodies.