Please use this identifier to cite or link to this item: https://hdl.handle.net/10321/4459
Title: Evaluation of antibiotic-resistant bacteria and genes associated with tuberculosis treatment regimens from wastewater treatment plants in South Africa
Authors: Mtetwa, Hlengiwe Nombuso 
Keywords: Infectious diseases;Tuberculosis (TB);Multidrug-resistant tuberculosis (MDR-TB);Wastewater-based analysis;Wastewater-based epidemiology;TB transmission
Issue Date: 13-May-2022
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.
Description: 
Submitted in fulfillment of the requirements for the degree of Master of Health Sciences in Environmental Health, Durban University of Technology, Durban, South Africa, 2021.
URI: https://hdl.handle.net/10321/4459
DOI: https://doi.org/10.51415/10321/4459
Appears in Collections:Theses and dissertations (Health Sciences)

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