Evalvuation of efficacy of saclac globule formulation of indigenous bacteriophages against multidrug resistant bacterial pathogens in water microcosm

Water is an important fundamental requirement of the mankind. Water covers nearly 75 % of the earth surface. Water resources are becoming limited due to the contamination problems. Unfortunately, life- threatening bacterial pathogens contaminate waters that we drink thus can be responsible for significant health risks. Waterborne bacterial pathogens are now a days developing resistance to chlorine that is used for the disinfection purpose. They are also developing resistance to antibiotics. To overcome such problems, associated with the use of chemical disinfectant like chlorine, an alternative strategy could be use of Lytic bacteriophages as biological disinfectant. Bacteriophages are the viruses of prokaryotes that can either instantly kill a bacterial cell or integrate their genome into the host chromosome. In this study, potent lytic phages were isolated from the natural resources. They were characterized and their infectivity abilities against specific pathogens were checked. Once the phages are produced in large scale, there is a need of specific carriers to maintain their stability. Therefore, there is a need of acceptable, eco-friendly formulations that will increase the longevity of phage particles. In order to achieve better efficacy of phage formulations in the bioremediation of water bodies’, selection of the carrier to make good formulation is an important and mandatory step. Saclac globule (it’s a globule made up of goat milk and sugar) formulations using efficient phages were prepared and their shelf-life was checked at 4ºC and 30 ºC. As theses globules contain goat milk sugar, this component supports the pathogen to remain in the metabolically active state. Bacteria in this state will be killed rapidly by phages, secondly, Saclac globules are water soluble that get dissolved rapidly as compared to other carriers used in the formulation. In this study, capability of the bacteriophages, viz., φSPB,BVPaP-3 and KPP was evaluated in the bioremediation of water microcosms prepared using the Pavana river water and swimming pool water spiked independently with the bacterial pathogens, viz., Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneuminiae. In this study, water microcosms were loaded with an individual pathogen, and then treated with the monovalent saclac globule phage formulation of., φSPB,BVPaP-3 and KPP independently and also in a cocktail form (φSPB,BVPaP-3 and KPP) at different conditions. Efficacy of saclac globule monovalent formulations of indigenous φ SPB (Salmonella phage) and BVPaP-3 (Pseudomonas phage) were evaluated against Salmonella enterica serovar Paratyphi B and Pseudomonas aeruginosa respectively in the water microcosms prepared using river water and swimming pool water spiked independently with the mentioned respective pathogens at different environmental parameters,viz.,growth phase of the cell (log and stationary), incubation conditions (shaking and static) and varied MOI (multiplicity of infection) values (MOIË‚1, MOI=1, MOI=50 and MOI=100). In case of application of monovalent phage formulation, at different MOI values, there was a great difference in the inactivation rate of Salmonella enterica serovar Paratyphi B and Pseudomonas aeruginosa, with the maximum inactivation rate at MOI = 100. There was a significant difference (P Ë‚ 0.05) in pathogen inactivation rate at shaking and static condition.The rate of inactivation of the log phase cells was higher than stationary phase cells both at shaking and static conditions at MOI 50 and 100.In case of Klebsiella pneuminiae no inactivation of the cells by KPP was observed. There was no reduction in the number of pathogens in the filter-sterilized water micrtocosms (containing natural phages) spiked with the individual pathogens (Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneuminiae) treated with respective phage ( φSPB,BVPaP-3 and KPP). Results also indicated that the application of phage cocktail did not affect pathogen population. Saclac globules contain goat milk sugar that keeps the bacteria in a log phase of growth for long period of time. At different MOI values, there was a great difference in the inactivation rate of Salmonella enterica serovar paratyphI B and Pseudomonas aeruginosa with maximum inactivation rate at MOI=100.There was a significant difference (PË‚ 0.05) in pathogen inactivation rate at shaking and static condition. The rate of inactivation of log phase cells were higher than the stationary phase cells at shaking as well as static conditions at MOI=50 and MOI=100. Result presented in this study strongly support that saclac globule formulation of bacteriophages in the monovalent form can be used as an effective biological disinfectant for killing many kinds of water borne bacterial pathogens.

Author(s): Sangeeta Ahiwale

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