Assessing the impact of wastewater irrigation on the dynamics of antibiotic resistance in agricultural setting

Water scarcity is one of the main challenges in sustainable development particularly in

developing countries. The use of wastewater effluent for irrigation of crops is common in most

water-stressed countries as this alleviates pressure on fresh water supply. Wastewater treatment

plants (WWTPs) are regarded as hotspots for antibiotic resistance determinants; antibiotic

resistant bacteria (ARB), antibiotic resistance genes (ARGs) and mobile genetic elements (e.g.

plasmids). These antibiotic resistance determinants are consequently introduced into the soil

and fresh produce through irrigation with effluent wastewater. Microcosm and field

surveillance studies in Palapye and Gaborone respectively, were carried out to evaluate the

impact of wastewater effluent irrigation in soil and vegetables in agricultural settings. Culturebased,

molecular (PCR), 16S rRNA gene metagenomics and shotgun metagenomics methods

were used to determine the occurrence, abundance, diversity and overall dynamics of ARB and

ARGs in effluent irrigated soil and vegetables. Clinically relevant bacteria (Campylobacter,

Listeria, Pseudomonas, E.coli, Enterobacter, Staphylococcus and Shigella species) were

targeted and isolated from wastewater effluent, effluent irrigated soils and selected vegetables.

The results revealed a significant reduction in total viable bacterial quantities in the storage

tank containing effluent used for microcosm irrigation. A shift in bacterial community profile

was observed as notable reduction in proteobacteria and increase in firmicutes phyla from the

microcosm soil following wastewater irrigation. Antibiotic resistance genes; beta-lactamase

resistance gene (blaTEM), tetracycline resistance gene (tetA), aminoglycoside resistance gene

(aadA), sulfonamide resistance gene (sul1), trimethoprim resistant dihydrofolate (dfrA) were

all identified by PCR in Gaborone wastewater treatment plant (GWWTP) effluent but only

blaTEM, aadA and dfrA were detected in the soil from an agricultural field irrigated using

effluent from GWWTP. Shotgun metagenomics revealed diverse ARGs belonging to different

classes of antibiotics; aminoglycoside, beta-lactam, trimethoprim, macrolide, glycopeptide,

tetracycline, sulfonamide, quinolone and oxazolidinone in Palapye wastewater treatment plant

(PWWTP) effluent used in the irrigation of the microcosm experiment. However only blaTEM

and aadA were identified in the microcosm soil, and only beta-lactamase gene blaTEM was

detected on vegetable surfaces following irrigation with PWWTP effluent wastewater. The

results from this study demonstrated the short and long-term impact of wastewater irrigation

which results in persistence and possible dissemination of wastewater-associated ARB and

ARGs into agricultural soils and vegetables. Moreover, this study enhances our understanding

of antibiotic resistance dynamics and highlights the importance of monitoring antibiotic

resistance in agro-systems, which is critical for informing policies aimed at sustainable use of

wastewater effluent in water-stressed countries.