Comparative Methodological Evaluation of Three Bacteriophage Isolation Methods From Environmental Water Samples

Bacteriophages are ubiquitous viruses that specifically infect and lyse bacterial hosts and have gained increasing attention for applications in biocontrol, phage therapy, food safety, and diagnostics. Efficient isolation of bacteriophages from environmental sources is essential for both research and applied purposes. This study aimed to comparatively evaluate the efficiency of three bacteriophage isolation methods from environmental water samples.

In this comparative methodological study, 96 environmental water samples were collected from four aquatic sites in Tangail, Bangladesh, over a one-year period (January-December 2025), with sampling conducted twice monthly. Six bacteriophages targeting clinically relevant bacterial hosts were assessed using three isolation methods: direct recovery, polyethylene glycol (PEG)-mediated precipitation, and host-based enrichment. Phage recovery efficiency was compared using Chi-square and Fisher's exact tests, and effect sizes were estimated using odds ratios (ORs) with 95% confidence intervals (CIs).

The average phage recovery rates were 9.38%, 21.35%, and 47.20% for the direct, PEG, and enrichment methods, respectively. A significant difference in recovery proportions was observed among methods (χ² = 224.1, df = 2, p < 0.0001). Pairwise comparisons demonstrated that the enrichment method yielded significantly higher recovery than the PEG method (OR = 3.29, 95% CI: 2.55-4.26, p < 0.0001) and the direct method (OR = 8.65, 95% CI: 6.28-11.97, p < 0.0001).

The modified enrichment method significantly improves bacteriophage recovery compared to the direct and PEG-based approaches, particularly for low-abundance phages. This method represents a robust and sensitive strategy for isolating bacteriophages from environmental water samples and may support future applications in phage-based therapeutics, environmental surveillance, and microbial control.