Pathogen-Phage Geomapping to Overcome Resistance

Reviewer #1 (Public review):

Summary:

In the manuscript "Pathogen-Phage Geomapping to Overcome Resistance," Do et al. present an impressive demonstration of using geographical sampling and metagenomics to guide sample choice for enrichment in human-associated microbes and the pathogen of interest to increase the chances of success for isolating phages active against highly resistant bacterial strains. The authors document many notable successes (17!) with highly resistant bacterial isolates and share a thoughtfully structured phage discovery effort, potentially opening the door to similar geomapping efforts across the field. While the work is methodologically strong and valuable for the community, there are a few areas where additional clarification and analysis could better align the claims with the data presented.

Strengths:

(1) The manuscript describes a well-executed and transparent example of overcoming a major obstacle in therapeutic virus identification, providing a practical success story that will resonate with researchers in microbiology and medicine.

(2) Many phage researchers have anecdotally experienced a similar phenomenon, that a particular wastewater treatment plant always seems to have the pathogens you need. Quantifying this with metagenomics modernizes and adds evidence to this phenomenon in a way that could help researchers reproduce this success in a methodical way.

(3) The methodology of combining environmental sampling, viral screening, and host-range analysis is clearly articulated and reproducible, offering a valuable blueprint for others in the field.

(4) The data are presented with appropriate analytical rigor, and the results include robust sequencing and metagenomic profiling that deepen understanding of local viral communities.

(5) The 17 successes yielding 35 phages have a lot of phylogenetic novelty beyond what the Tailor labs have typically found with previous methods.

(6) The work highlights a practical and innovative solution to an increasingly important clinical problem, supporting the development of personalized antiviral strategies.

Weaknesses:

(1) The central concept of geomapping as a broadly applicable strategy is wonderfully supported by the 17 successes documented in the paper. While this is actually, of course, a strength, the study does not include a comparative analysis across multiple sites with varying sampling outcomes for different bacterial types, which would be necessary to validate this claim more generally.

(2) Some elements, such as beta diversity comparisons and the metagenomics analysis of viral dark matter, would benefit from additional statistical analysis and clearer context.

(3) Claims about therapeutic cocktails would be better framed as speculative and/or moved to the discussion section.

(4) The manuscript could be strengthened by elaborating on the scope and composition of the phage and bacterial isolate collections, which are important for interpreting the broader significance of the findings.

Reviewer #2 (Public review):

Summary:

The manuscript by Do and colleagues aims to develop a workflow for isolating and identifying bacteriophages with potential applications in phage therapy against antibiotic-resistant pathogens. The workflow integrates geΦmapping as a strategy to identify potential phage sources, ΦHD as a device for phage concentration, and RΦ as a phage library constructed from the initial sampling, resulting in the discovery of 36 new phages. The paper is overall interesting, and the proposed method appears robust and effective.

Strengths:

The methods proposed combined state-of-the-art strategies to solve an ever-increasing problem of antibiotic resistance. The methods are robust, and the controls are appropriate. The integration of environmental sampling, concentration strategies, and downstream genomic characterization is a clear strength and provides a potentially scalable framework for identifying candidate therapeutic phages. The manuscript is clearly written overall, and the results support the main conclusions.

Weaknesses:

While the authors acknowledge several limitations, some aspects require clearer framing or additional clarification. The proposed workflow focuses exclusively on aquatic environments as sources of phages, which may limit the diversity of hosts and phage types recoverable using this approach. Some interpretations, particularly regarding taxonomic classification and sampling saturation, would benefit from more cautious wording given current limitations in viral taxonomy and the observed data.