Lytic bacteriophages isolated from limestone caves for biocontrol of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen that causes life-threatening infections to humans especially patients who are immunocompromised. Treatment and control of P. aeruginosa infections pose a serious challenge due to extreme resistance towards multiple antimicrobial agents. In this paper, the possibility of screening and isolating lytic bacteriophages from cave samples were studied. The efficiency of these phages to inactivate P. aeruginosa PAO1 cells was performed in in-vitro co-culture assays at 37 °C using five multiplicity of infection (MOI) ratios. The best performing phages were selected and their biocontrol potentials assessed on P. aeruginosa PAO1 immobilized sand samples. A total of 33 phages with distinctive plaque morphologies and titers (10 6 -10 15 PFU mL −1 ) were isolated using an enrichment culture method. At 6 h post-phage treatment, phage cocktail (MOI 10 4 ) showed the highest bacterial log 10 CFU mL −1 reduction of 11.82, while φWCSS4PA (MOI 10 4 ) and φCocktail (MOI 10 2 ) showed the highest bacterial log 10 CFU mL −1 reductions of 10.5 and 10.86, respectively at 24 h post-treatment. Sand decontamination results showed that bacteriophages φFCPA3, φWCSS4PA and φCocktail were capable to significantly reduce 99% of the bacterial load at 6 h post-treatment. However, there was no noticeable reduction of the bacterial cells at 24 h and 48 h post-phage treatment. Optimizing the treatment concentrations and conditions for successful bacterial inactivation at longer incubation period is required. The significance of this study indicates that lytic phages isolated from extreme environments such as limestone cave region can serve as potential biocontrol agents against pathogenic bacteria. • Enrichment culture method was used for phage isolation. • Thirty-three phages with distinctive plaque morphologies were isolated. • Host range analysis and in-vitro bacteriolytic activity of the phages were performed. • Efficiency to control Pseudomonas aeruginosa PAO1 contaminated sands was tested. • Phages φFCPA3, φWCSS4PA and φCocktail can serve as potential biocontrol agents.