Evaluation of antibiotic-resistant bacteria and genes associated with tuberculosis treatment regimens from wastewater treatment plants in South Africa

Abstract

Essential components of a strong public health system include an efficient surveillance system which helps in early detection and prevention of infectious diseases. This is particularly important for tuberculosis (TB) and multidrug-resistant tuberculosis (MDR-TB), due to increasing globally infections and the associated economic burdens. TB and MDR-TB infections are high in several countries, with South Africa contributing almost 3% of total infections globally. This advocates for improved surveillance systems to help health authorities respond effectively in developing effective policies for managing and controlling diseases. The reliance on clinical case reports, hospital admissions and clinical surveys, as surveillance methods, has proven to be a challenge in developing countries like South Africa, where there are other competing interests for scarce resources. The development and implementation of alternative surveillance tools for identifying disease severity, the emergence of novel strain and resistance patterns is, therefore, a top priority. One such strategy is the use of sewage or wastewater-based analysis, commonly referred to as wastewater-based epidemiology (WBE), which has received attention lately due to its role in developing early warning and surveillance of SARS-CoV-2 (COVID-19) infections. This study evaluates, method development for utilizing WBE approach for monitoring TB and MDR-TB infections via the detection and quantification of tuberculosis-causing mycobacteria and genes (ARGs) associated with resistance to TB treatment in untreated wastewater. Furthermore, the study contributes towards the understanding potential TB transmission through wastewater. To achieve these, conventional and advanced polymerase chain reaction (droplet digital PCR) assays were optimized for the detection and quantification of total mycobacteria, members of the Mycobacterium tuberculosis complex (MTBC) and ARGs associated with resistance to first and second-line TB drugs. The mycobacteria targeted in this study were total mycobacteria, M. tuberculosis complex, M. tuberculosis, M. africanum, M. bovis and M. caprae. The ARGs (and the antibiotic they encode resistance to, in parenthesis) targeted in this study were; katG (isoniazid), rpoB (rifampicin), embB (ethambutol), pncA (pyrazinamide), rrs (streptomycin), gyrA (ofloxacin), gryB (moxifloxacin), atpE (bedaquiline), ethR (ethionamide), eis (kanamycin/amikacin). Untreated and treated (post-chlorination) wastewater samples from three wastewater treatments plants (WWTPs) in the city of Durban, South Africa were used for this study. All wastewater samples (untreated and treated) analyzed in this study contained total mycobacteria and MTBC at varying percentages per WWTP studied. The human and animal MTBC pathogens such as M. tuberculosis, M. bovis and M. caprae showed a similar prevalence, except for M. africanum, which was less common compared to the others. The highest median concentration detected in untreated wastewater was 4.9(±0.2) Log10 copies/ml for total mycobacteria, 4.0(±0.85) Log10 copies/ml for MTBC, 3.9(±0.54) Log10 copies/ml for M. tuberculosis, 2.7(±0.42) Log10 copies/ml for M. africanum, 4.0(±0.29) Log10 copies/ml for M. bovis and 4.5(±0.52) Log10 copies/ml for M. caprae. A statistically significant difference (p-value ≤ 0.05) in concentrations of each organism was observed between the plants. A significant reduction in copy numbers from untreated to treated samples were observed. However, the log reduction in each WWTP did not show any statistically significant differences when compared between the three WWTPs, irrespective of the organism or group of organisms (p-value ≥0.05). Furthermore, all targeted ARGS were detected in all samples analyzed at varying concentrations. The most abundant ARG in the untreated wastewater was rrs, associated with resistance to the aminoglycosides, specifically streptomycin. In contrast, pncA gene associated with resistance to the TB drug pyrazinamide was the least detected. Furthermore, the resistant gene associated with bedaquiline (aptE) was also detected in all samples, albeit at low concentrations. This antibiotic is a new addition to the TB treatment regimen in South Africa and it is concerning that resistance has already been detected. The occurrence and concentration of these ARGs were lower in the treated wastewater in most instances, ranging from 1 log copy/ml to over 4 log copies/ml except for selected genes at few instances. The study makes novel major contributions, firstly, the detection of M. tuberculosis complex members in the untreated wastewater at high concentrations signifies a potentially high prevalence of TB in the study area. Secondly, the detection of M. africanum in South African wastewater also signifies that some of the TB infections in the communities could be caused by this pathogen. M. africanum is the main causative agent of TB in West Africa but is not frequently reported clinically in South Africa. Finally, the presence of diverse ARGs associated with TB drugs also points towards an association between the drug use and resistance profile in the area. These results further support the potential application of WBE to gather data on MDR-TB within communities with limited or no clinical data. The detection of the aptE gene also shows that resistance to the new drug, bedaquiline, could already be developing in the communities. The study also observed that the wastewater treatment plant configuration did not significantly influence the removal of these mycobacteria. Furthermore, selective conditions in the WWTPs may contribute to increased concentrations of ARGs during the treatment processes as indicated by increased concentrations for certain ARGs detected in the treated wastewater. This warrants further studies to determine whether the genes detected in the effluent are extracellular or carried in viable microorganisms, to assess the viability and infectivity of the microorganisms carrying these genes in the effluent samples and therefore the potential public health risks associated with the exposure to wastewater. In conclusion, this study establishes the potential of molecular surveillance of wastewater for monitoring TB and MDR-TB infections in communities and supports the use of WBE as a public health strategy to combat infectious diseases.