PREreview of Comparative analysis of culture- and ddPCR-based wastewater surveillance for carbapenem-resistant bacteria

Major Issues

1. The study includes 36 samples from three WWTPs over 12 weeks in one city. This is useful for method comparison, but too limited to support broad claims about community prevalence or wastewater surveillance thresholds. The authors should frame the findings as a short-term, site-specific comparison and avoid overgeneralizing statements such as blaKPC-containing bacteria being “ubiquitous in the population” unless supported by clinical or longer-term surveillance data.

2. The weak correlations are interesting, but they may be expected because ddPCR measures gene copies from viable cells, dead cells, extracellular DNA, and non-target hosts, whereas culture measures viable organisms able to grow under selective conditions. The manuscript would benefit from a clearer conceptual model explaining what degree of correlation should be expected, and under what assumptions culture CFU/mL and ddPCR copies/mL are comparable.

3. The use of mFC agar and ertapenem selects a subset of culturable coliform-like bacteria and may miss non-coliform CRE, non-Enterobacterales, slow-growing organisms, viable-but-nonculturable bacteria, and organisms with different carbapenem-resistance mechanisms. The authors acknowledge this, but the limitations should be emphasized earlier when interpreting culture counts as “carbapenem-resistant bacteria.”

4. The multiplex assay is central to the study. The main text should summarize per-target LOD/LOQ, dynamic range, inhibition assessment, recovery or concentration efficiency, cross-reactivity, target coverage across gene variants, and performance in wastewater matrix. If these are in the supplement, key values should still appear in the main text so readers can interpret non-detects and low-abundance targets.

5. Pearson correlations across 36 samples may be affected by site structure, temporal autocorrelation, non-normality, repeated sampling, and multiple comparisons. The authors checked Durbin-Watson, which is good, but a stronger analysis would include site-stratified correlations, Spearman correlations, log-transformed data handling, mixed-effects or generalized least squares models, and correction for multiple testing.

6. Nanopore sequencing was performed on pooled presumptive CR colonies from nine plates, not the full wastewater community. Therefore, read counts should not be interpreted as quantitative abundance of ARGs in wastewater. The manuscript should make clearer that sequencing describes the enriched culturable fraction and is best used for mechanism exploration, not population-level resistome quantification.

7. The authors suggest that wastewater monitoring could complement clinical surveillance, but there is no comparison with clinical CRE cases, hospital admissions, antibiotic use, or facility-level outbreaks. The conclusion should either soften actionability claims or explicitly propose how future work would link wastewater signals to clinical surveillance metrics.

Minor Issues

1. Define all abbreviations clearly at first use, especially CR, CP, CRE, ARG, ARB, WWTP, and rpoB.

2. Clarify whether triplicate dots in figures represent technical replicates, biological plating replicates, or independent subsamples.

3. Report whether wastewater flow rate, population served, or fecal-strength normalization was considered. Normalization only to rpoB may not fully account for sewershed and sampling differences.

4. The manuscript should state whether ddPCR values were corrected for concentration factor and extraction volume in all plots.

5. Figure 3 is useful but visually dense. A table of key correlations with confidence intervals would improve interpretability.

6. Some claims about site-specific patterns would be stronger with sewershed context, such as hospital contribution, population served, or land-use differences.

7. The Blue-Carba test is important for identifying carbapenemase-producing colonies; please include its expected sensitivity/specificity limitations in mixed wastewater isolates.

8. The manuscript would benefit from separating Results and Discussion sections, or at least making interpretive paragraphs easier to distinguish from primary findings.

9. Data availability includes SRA accession PRJNA1063622; adding analysis code and processed count tables would improve reproducibility.

1. Minor wording issue: “carbapenemase genes are abundant and in US wastewater” should be revised for grammar.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.