A metagenomic snapshot of selected wastewater treatment plants in Vhembe Region, Limpopo, South Africa: Investigating the resistome

Abstract

Background: Water is crucial for human life. Rural communities often rely on natural water sources which may become polluted by wastewater due to various activities such as domestic use and agriculture. Antibiotic resistance genes (ARGs) may be transferred from wastewater to the environment and pose a global challenge they affect both human and animal-related sectors. Studying antibiotic resistance in wastewater treatment plants within Vhembe offers a representation of antibiotic resistance genes from entire communities. Knowledge of antibiotic resistance circulating in Vhembe has been sparsely studied. Metagenomics approaches allow for a broad overview of the resistome and the bacterial communities within environmental samples. Aim: To perform wastewater surveillance of antibiotic resistance genes and associated bacteria within Vhembe, Limpopo, South Africa, using a metagenomics approach. Method: A total of 32 sample duplicates were collected from the influents (n=18) and the effluents (n = 14) from nine wastewater treatment plants (WWTPs) around the Vhembe region, Limpopo, South Africa. One hundred milliliter was filtered using sterile cotton gauze and Wattman filter paper to remove debris and membrane filtered through 0.22um membrane filters to capture the bacteria within each sample. DNA was extracted directly from the 0.22 μm filters using a DNA miniprep kit. DNA was quantified using a spectrophotometer. Shotgun 18 metagenomic sequencing was performed on DNA extracts. Open-source bioinformatics pipelines were used to process and analyze raw sequence data, uncovering information of the bacterial community composition and associated ARGs in wastewater. Results: Site observations reveal animal and human activities within and near the sites. ARG analysis revealed an overall number 0f 220 ARGs detected across the WWTPs. Thirty-six genes were common to influent samples and 16 in within effluent samples, encoding predominantly against macrolides, sulfonamides and tetracyclines, beta-lactamases, and aminoglycosides. Some unique ARGS were detected at sites near South African borders. Bacterial Diversity showed the predominance of some genera, such as Arcobacter, Aeromonas, Pseudomonas and Acinetobacter. Pathogens were predominantly enteric and pulmonary, with some being linked to animals in past studies. A notable increase in some members of Mycobactericeae, among other bacteria, was noted in effluents.