MUNICIPAL WASTEWATER TREATMENT PLANT AS A RESERVOIR OF ANTIBIOTIC RESISTANCE

Abstract

Easy availability at cheap prices has led to the widespread and indiscriminate use of antibiotics. The presence of antibiotic residues in the environment has caused the bacteria to adapt to develop resistance against the antibiotics. Even at low concentrations (ng/L), long term exposure of antibiotics to the microbe-rich environment could create a selective pressure on bacteria for the development of resistance against antibiotics. While it is obvious that high antibiotic concentrations may naturally select the antibiotic-resistant bacteria (ARB), but even the low antibiotic concentrations play a typical role in selecting resistant mutants. Researchers have proposed that even very low antibiotic concentrations at several orders of magnitude lower than the minimum inhibitory concentration (MIC) can target susceptible bacteria to develop resistance against antibiotics (Gullberg et al. 2011). The results from this study also indicated that low antibiotic concentrations found in the natural environment might enrich and maintain resistance in the bacterial populations. In Municipal wastewater treatment plants (MWTPs) conducive conditions are provided to the bacteria for their abundant growth so that the organic content in the sewage can be degraded at a fast rate within a small footprint. Considering that there is the presence of antibiotics at low concentrations in the influent and high loads of bacteria in the biological treatment unit when kept in close contact, this could expose bacteria to antibiotics that exert selective pressure on bacteria indicating the risk of resistance development. Influent raw wastewater to the MWTPs is known to contain sub-MIC levels of antibiotics as about 60-90% of antibiotics consumed are excreted from the body in the un-metabolized or partially metabolized state and these antibiotics ultimately find their way to MWTPs through wastewater. When the microbes in the biological treatment of MWTPs are continuously exposed to several low concentrations of antibiotics, the selection of ARB may take place. Also, biological treatment with activated sludge process or for sequential batch reactors generally consists of an aeration tank coupled with a separate or in-built sedimentation tank from where the sludge mass, mainly containing active bacteria, is recycled back into the aeration tank so as to maintain high loads of bacteria for the degradation of the organic content in the wastewater. The chances of selective pressure on bacteria are more in these conditions as bacteria are continuously recycled and exposed to the antibiotics. Therefore, the logical hypothesis is that MWTPs can act as the hub for the growth of ARB. The positive selection of resistant bacteria is demonstrated by few studies outside India but there is still a knowledge gap existing in support of this hypothesis(Auerbach, Seyfried, and McMahon 2007)(Marcinek et al. 1998; Da Silva et al. 2006). There are contradicting results that indicated that no selection occurred in the biological treatment of MWTPs (Martins da Costa, Vaz-Pires, and Bernardo 2006). Therefore, the studies so far could not conclusively establish whether the MWTPs act as the hub for the growth and proliferation of the ARB. The reason for such a lack of conclusion may be because of the presence of a low concentration of antibiotics in the wastewater reaching the MWTPs. Also, there were varied results from different studies because numerous other factors might play roles to influence the selection process inside the biological treatment, such as influent wastewater characteristics, type of the biological treatment and size of the plant, hydraulic retention time etc. India has a high infectious disease burden and easy availability of antibiotics over-the-counter, which could be a reason for having high concentrations of antibiotics in the influent raw wastewater to the Indian MWTPs. Therefore, this particular study was designed to provide evidence on whether the biological treatment process in the MWTPs of the Indian subcontinent induces selection pressure in favor of ARB. Quantification of antibiotics, antibiotic-resistant bacteria and resistance genes before and after biological treatment were performed in this study as evidence for the selection process taking place. Also, MWTPs play as a significant point source to release antibiotics and ARB and antibiotic resistance genes into the environment. Consequently, one objective of this study was also to determine the removal efficiencies of ARB in the MWTPs. India stands as the world’s largest consumer of antibiotics in 2010. Till now, no comprehensive study has been taken up for reporting the occurrence of antibiotics and antibiotic-resistant bacterial patterns in the Indian MWTPs. This work showcases the eventual effect of high antibiotic consumption on the occurrence of antimicrobial resistance patterns in MWTPs. This study has focused on the quantification of fluoroquinolone (FQs) and FQ resistant bacteria in MWTPs located in India. The MWTPs selected in this study are situated in Haridwar and Rishikesh, these MWTPs were selected because these cities, being famous for pilgrimage, receive a high number of visitors throughout the year from all corners of India. Hence, the chances of detecting different levels and distribution of antibiotic resistance patterns could be higher at these sites. Although there is no data available on the consumption of antibiotics in the city, it is highly likely that the extent of use of antibiotics may cause high concentrations of these antibiotics in the wastewater received in the treatment plant. FQs have been used as representative antibiotics because according to the recent literature available, the consumption of FQs has been reported to be rising in the country (Laxminarayan and Chaudhury 2016) and also FQs are over the counter antibiotics and majorly used for common bacterial infections The MWTPs selected in this study are: 1) Activated sludge-based (ASP) MWTP that can treat 18 million liters per day (MLD) located at Haridwar, India. 2) Cyclic activated sludge process (Sequential batch reactor-SBR-H) based MWTP that can treat 27 million liters per day (MLD) located at Haridwar, India. 3) Cyclic activated sludge (Sequential batch reactor-SBR-R) based MWTP that can treat 3 million liters per day (MLD) located at Rishikesh, India. Samples were collected from three points, ASP (Bio-inlet, Bio-outlet, Final effluent), SBR-H (SBR-inlet, SBR-outlet, Final effluent), SBR-R (SBR-inlet, SBR-outlet, Final effluent). Microbial resistance and quantification of antibiotics were examined on Norfloxacin (NOR), Ciprofloxacin (CIP) and Ofloxacin (OFL) antibiotics belonging to the class FQs, which are most widely used. We used phenotypic methods and molecular techniques for the detection of resistant bacteria. The results from a one-year-long study on the different processes in the MWTPs suggested that there is an abundance of FQ- resistant bacteria and FQs concentrations in the MWTPs. The influent concentrations for FQs ranged from 4.2 to 16.4 μg/L in three MWTPs. The concentrations detected in this study were much higher than the predicted sub-MIC levels needed to induce the selection of bacteria (Bengtsson-Palme and Larsson 2016). Further, the maximum removal rates were OFL (96.6%), NOR (79.6%), CIP (66.6%). No apparent differences in the removal rates were observed in this study. The ubiquitous presence of FQs in the influent wastewater might be a significant factor in this process. On average, FQ resistant bacteria frequency ranged from 6.1×103–2.5×103 CFU/mL in the influents of three MWTPs. The maximum removal rate was observed for OFL (>95%). Also, the SBR-R plant which is a small plant with longer hydraulic retention which is 14.6 hours showed maximum removal rates among three MWTPs tested. When the samples from Bio or SBR inlet are compared with the outlet samples, the fraction of FQ resistant bacteria over the total bacteria is observed to increased indicating there is a selection pressure within the biological treatment unit of the treatment plant. All the three MWTPs investigated showed the increase in the FQ resistant bacteria. The disinfection process was partially effective but caused the release of a sizeable loads of resistant species in the surface water along with the treated water. The heterotrophic plate counts of final effluent ranged from 4x102 CFU/mL to not detected for the three MWTPs. Also, the data collected from the samples before (E1) and after (E2) effluent discharge site of MWTP showed that besides OFL resistant bacteria (with removal >95%), the densities of NOR and CIP resistant bacteria are significantly high (p<0.02) at E2 site indicating MWTPs acted as significant point sources to release resistant bacteria into the environment.Analysis of the genetic basis of FQ resistant bacteria was performed, so as to determine the occurrence of resistance genes - whether in plasmid DNA or in genomic DNA. The results from this study showed that no transformation of isolated plasmids occurred stating resistance genes are present elsewhere – integrons, transposons, genomic DNA. Forward from this, resistance determination was narrowed down to ASP-based MWTP. The isolates from Bio-inlet and Bio-outlet samples showed two-point mutations in gyrA gene sequence S83L and D87N. Among 80 isolates tested (40 from Bio-inlet and 40 from Bio-outlet), 75% isolates showed signature mutations in QRDR (Quinolone resistance determining region) region. Also, these particular mutations identified in this study are associated with a high prevalence of FQs in a particular environment. Furthermore, 16S rRNA gene sequencing revealed that the antibiotic-resistant isolates belonged to the family Enterobacteriaceae of which Escherichia, Salmonella, and Shigella were dominant species irrespective of the sampling point. Antibiotic susceptibility tests were also performed on most commonly used antibiotics which revealed the presence of multidrug resistance in the samples. The multi-antibiotic resistance (MAR) index also significantly increased (p<0.05), indicating the growth and emergence of multi-drug resistant bacteria within the MWTP increased from 0.62 to 0.65 in the bio-outlet and to 0.66 in the final effluent samples. The results of this study established that the biological treatment process (both ASP and SBR) in MWTPs indeed plays a significant role in the selection and proliferation of ARB leading to even the generation of multi-drug resistant bacteria. The biological treatment process in these findings reiterates that irrespective of the type of biological treatment employed in the treatment of sewage, the conditions maintained in the biological treatment process favor the growth of ARB. The disinfection process was inefficient in removing the ARB causing the discharge of the treated wastewater to contain multi-drug resistant species. Although we did not find the presence of plasmid-mediated horizontal transfer of the antibiotic-resistance genes in the fluoroquinolone-resistant species, however, the same may not be true for resistance to the other antibiotics. Thus, unless remedial measures are taken, MWTPs can be a significant source for the spread of antibiotic-resistant species in the environment.