Potential use of bacteriophages for enteropathogen reduction in treated wastewater and sludge from Ruai and Kariobangi treatment plants

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Wastewater treatment plants (WWTPs) are large repositories of enteropathogenic bacteria as they serve as the foundation for environmental enteropathogenic pollution and disease transmission in the environment. This threatens public health, thus undermining wastewater effluent and sludge reuse. Convetional biological methods used in Kenya WWTPs have limitations in controlling multidrug resistant (MDR) pathogens.Therefore, better biocontrol strategies to reduce the enteropathogenic load and control MDR pathogens in sewage effluent and sludge for reuse are required. Bacteriophages are viruses that infect and lyse bacteria. They have emerged as promising biocontrol agents in reducing bacterial loads including MDR strains following ONE HEALTH strategies including wastewater treatment. However their application in wastewater biocontrol remains under explored in Kenya.This study aimed in isolating and characterizing enteropathogens and bacteriophages in Ruai and Kariobangi WWTPs. Wastewater and sludge samples were aseptically collected, transported under cold chain conditions, and processed as soon as possible. Bacterial isolates were obtained through standard culture methods and characterized using morphological, biochemical following API20E system, and molecular techniques, including 16S rRNA sequencing. The 18 isolates molecularly confirmed were tested against 8 antibiotics are carriers of two resistant genes following PCR techniques. Bacteriophages were isolated following direct plate method and tested for pH, temparute stability, host range activity and growth curve determined. Data from this work show that the CFU counts of treated wastewater (5962028 per 100 mL) and treated sludge (6019053.667 per 100 mL) were above the World Health Organization (WHO) and National Environmental Management (NEMA), which stipulate that a CFU count of 1000.0/100 mL and below is allowed for discharge. The dominant genera included Escherichia, Enterobacter, Klebsiella, Citrobacter, and Serratia. Antibiotic susceptibility testing of 18 molecularly confirmed isolates revealed that14 were multidrug-resistant, with high resistance rates to ampicillin, co-trimoxazole, and sulfamethoxazole. PCR analysis identified 12 isolates carrying the sul2 gene and 11 carrying the Amp gene, with seven harboring both. The five lytic bacteriophages Psvu, Encl, Klpn, Sepr, and Enfa targeted P. vulneris, E. cloacae, K. pneumoniae, S. proteamaculans, and E. faecalis, respectively.The Phage stability studies showed that lytic activity peaked at pH 7.5 and 37 °C, with substantial variation in burst sizes and plaque morphologies among the isolates.The phages were used to prepare a cocktail for a laboratory-based CFU reduction experiment. The CFU count in the effluent was reduced by 20.24-fold and in sludge by 22.25-fold. These findings highlight the prevalence of multidrug resistance and associated resistance genes in WWTP enteropathogen isolates and demonstrate the potential of locally isolated bacteriophages as effective biocontrol agents for reducing enteropathogenic loads. This study provides evidence of the application of bacteriophage therapy in environmental biotechnology, paving the way for scalable, sustainable interventions in enteropathogen management in treated wastewater.

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