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Municipal wastewater treatment: assessment of ecotoxicity and dispersion of bacterial antibiotic resistance

Sperelaki Eleftheria

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Summary

Antimicrobial resistance is perhaps the greatest long-term threat to public health. Particularly today, in the era of multi-drug resistance, humanity faces the risk of being unable to treat infections caused by common bacteria. Global health agencies are calling for a restriction on the indiscriminate use of antibiotic drugs, while the scientific community is exploring methods to limit the transmission of bacterial resistance. Municipal wastewater treatment plants, as recipients of human metabolic waste, are ideal environments for the development and spread of antibiotic drug resistance. Furthermore, the presence of antibiotics as chemical pollutants in wastewater poses serious risks to the aquatic ecosystem,s into which they are released after treatment. In order to investigate the above risks, this thesis examines the microbiological quality of urban wastewater (influent and effluent) derived from the WWTP located in Gerani (municipality of Platanias, Chania). The sampling was carried out from October 2021 to February 2022 and May 2022 and the samples were taken from three stages: a) influent, prior to any treatment b) effluent from secondary treatment and before chlorination c) effluent. The aim was to test the efficiency of the treatment methods, regarding the removal and inactivation of important bacterial species, namely, Klebsiella pneumoniae and Staphylococcus aureus and then to test the resistance of the remaining bacterial strains after treatment to specific antibiotics. The antibiotics tested were Amoxicillin, Ciprofloxacin and Sulfamethoxazole and the method referred to the evaluation of the minimum inhibitory concentration, MIC60 , which represents the concentration of the antibiotic that inhibits the growth of 60% of the bacterial population. Most of the bacterial strains isolated from samples before and after wastewater treatment appeared to be resistant to Amoxicillin, while the least resistant strains were related to Ciprofloxacin. In the case of Sulfamethoxazole, the results showed that the strains after treatment were more susceptible to this antibiotic.Further tests included the detection and quantification of the genes sul II and 16S rRNA in wastewater samples (influent and effluent). The sul II is an antibiotic resistance gene associated with the antibiotic Sulfamethoxazole and the 16s rRNA gene corresponds to the whole bacterial genetic material. The results showed a reduction of both genes in the effluents, namely, 2,2 Logs for sul II and 3-6 Logs for 16s rRNA. In contrast, the ratio of sul II to 16s rRNA showed an increase of 4 Logs in the effluents, confirming that wastewater treatment plants have an active role in the transmission of bacterial resistance. The ecotoxicity of the samples was tested using the Artemia nauplii biomarker. The mortality of the bioindicator ranged from 90-100% when influent samples were tested at concentrations of 37-80% v/v. Effluent samples showed lower toxicity levels, as the mortality of Artemia reached 20% of its initial population and the LC50 was recorded in samples containing 41-60% v/v of treated wastewater. In conclusion, municipal wastewater treatment plants are efficient in reducing bacterial load and in removing organic and other pollutants. However, they do not appear to be as efficient in removing resistance genes, increasing the concerns of the scientific community about the spread of bacterial resistance during wastewater treatment. 

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