Review Reports - First Isolation of <i>Candida parapsilosis</i> from Water-Related Domestic Environments in Colombia: A Public Health Concern?

Round 1

Reviewer 1 Report

In this manuscript, the authors surveyed 104 households in Villavicencio, Colombia, across two rainfall seasons. Using a filtration workflow designed for enteric bacteria, the authors recovered 22 Candida parapsilosis isolates from 16/104 households (15.4%), predominantly from household water-storage tanks (72.7%). Antifungal susceptibility testing showed one fluconazole-resistant isolate recovered from a storage tank in Commune 3. ERG11 sequencing of this isolate revealed a single nonsynonymous R398I substitution. The authors claim the first report of C. parapsilosis, including a fluconazole-resistant isolate, in domestic water in Colombia, and frame the finding within a One Health context. I provided some comments for the authors to consider as outlined below.

1) “Two water samples were collected from each household visited” with 104 households implies at most 208 samples per period, yet Period 1 reports 209 samples. The total (349) cannot be derived from ≤2 samples per household across 104 households.

2) The Conclusions assert a “significant public health concern” and an “urgent need” for surveillance, based on one borderline-resistant isolate, with no human-exposure or colonization data, no clinical–environmental genetic linkage, and a method that cannot quantify exposure. The Discussion appropriately tempers most of this; the Abstract and Conclusions should be aligned with that measured tone.

3) Previous investigation demonstrated that R398I alone does not reduce fluconazole susceptibility. Further additional or alternative mechanisms, such as ERG11 overexpression, efflux pump-mediated resistance, or lipid-related adaptations, should be evaluated in the present study.

4) The text reports isolate counts per commune (“Commune 3 … n=7”; communes 1, 2, 8 … three positive samples), whereas the figure legend states the numbers represent the proportion of positive households (denominator 13). The map, e.g., shows “6/13” for Commune 3 while the text reports n=7 isolates — readers cannot tell which metric is being displayed.

5) Methods 2.5 cites “M27-A4” for the methodology, but reference [23] is M27M44S (performance standards). Cite the method document and the interpretive document separately and correctly.

Author Response

Dear Reviewers,

We would like to begin by thanking you for your valuable comments and suggestions, and for the time you have dedicated to reviewing our manuscript. We have carefully considered all of your comments and have made the corresponding revisions to the manuscript. Each comment has been addressed individually, and our point-by-point responses are provided below.

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1. “Two water samples were collected from each household visited” with 104 households implies at most 208 samples per period, yet Period 1 reports 209 samples. The total (349) cannot be derived from ≤2 samples per household across 104 households.

Response: We thank the reviewer for this observation. We apologise for the confusion caused by the original wording.

Up to two water samples were collected per household per monitoring period: one from a faucet directly connected to the public or community water supply and one from a faucet connected to a water storage tank. However, not all households were able to provide both samples, particularly during the second monitoring period, because the public water supply in Villavicencio is not continuous due to recurrent operational disruptions.

In the first monitoring period (transitional rainfall), all 104 households provided both samples, yielding 208 samples (156 from storage tanks and 52 from direct connections). In the second monitoring period (ascending rainfall), only 140 samples could be collected (97 from storage tanks and 43 from direct connections), because water from the public network was not always available at the time of the visit. We have revised Section 2.2 to clarify it.

2. The Conclusions assert a “significant public health concern” and an “urgent need” for surveillance, based on one borderline-resistant isolate, with no human-exposure or colonization data, no clinical–environmental genetic linkage, and a method that cannot quantify exposure. The Discussion appropriately tempers most of this; the Abstract and Conclusions should be aligned with that measured tone.

Response: We agree that the original Conclusions overemphasised the public health implications of our findings, particularly given that they rest on a single isolate recovered at the resistance breakpoint, without human exposure data, colonisation data, or evidence of genetic linkage between environmental and clinical strains. We also acknowledge that the culture-based method employed cannot quantify human exposure. We have revised the Conclusions to align with the measured tone of the Discussion.

3. Previous investigation demonstrated that R398I alone does not reduce fluconazole susceptibility. Further additional or alternative mechanisms, such as ERG11 overexpression, efflux pump-mediated resistance, or lipid-related adaptations, should be evaluated in the present study.

Response: We agree that R398I alone has not been shown to independently confer azole resistance, as reported by Thomaz et al. and Štefánek et al. and acknowledged in our Discussion.

We acknowledge that the mechanism underlying the fluconazole resistance phenotype observed in this isolate remains unidentified. The additional assays proposed by the reviewer, ERG11 expression analysis, efflux pump activity, and lipid profiling, were beyond the scope of the present study, which was primarily designed to assess the occurrence of Candida spp. in domestic water. We agree that these analyses are necessary to determine whether the resistance phenotype is mediated by mechanisms other than the R398I substitution, or whether the isolate represents a borderline phenotype at the resistance breakpoint. We have revised the Discussion to state explicitly that the resistance mechanism remains undetermined and to avoid implying that R398I is responsible for the observed phenotype. We are currently seeking funding to conduct a larger study that will include comprehensive molecular and phenotypic characterisation of this and additional isolates, and we consider this a necessary next step to properly assess the clinical and public health relevance of our findings.

4. The text reports isolate counts per commune (“Commune 3 … n=7”; communes 1, 2, 8 … three positive samples), whereas the figure legend states the numbers represent the proportion of positive households (denominator 13). The map, e.g., shows “6/13” for Commune 3 while the text reports n=7 isolates — readers cannot tell which metric is being displayed.

Response: We acknowledge that the original text and figure reported different metrics without clearly distinguishing between them, which caused confusion.

In Commune 3, seven isolates were recovered from six positive households (one household yielded two isolates). Therefore, the isolate count reported in the text (n = 7) differs from the positive-household count shown in the figure (6 of 13 households sampled in that commune). The denominator of 13 corresponds to the number of households sampled per commune, not the number of positive households.

We have revised the figure legend to state explicitly that the numbers represent the proportion of positive households per commune (numerator) relative to the total number of households sampled in that commune (denominator). We have also revised the text to clarify that the percentages reported in Section 3.1 (31.8%, 18.2%, 13.6%, and 9.1%) refer to the proportion of isolates, not households. Overall, 22 isolates were recovered from 16 positive households across the 104 households sampled.

5. Methods 2.5 cites “M27-A4” for the methodology, but reference [23] is M27M44S (performance standards). Cite the method document and the interpretive document separately and correctly.

Response: The reference list has been updated accordingly to include both documents with their correct citations.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors report on the presence of Candida parapsilosis in water used for domestic purposes in Colombia. This work adds to the body of knowledge regarding the possible One Health aspects around pathogenic yeasts. This is an observational study and does not provide any insight into the risk to the population due to the presence of this yeast. No evaluation of the expression of virulence factors or comparison with clinical isolates are made.

Materials and methods

Section 2.1: Please provide clarity regarding the time periods/seasons in which sampling was done. Perhaps include the months of the year.

Results

Section 3.1: The authors state that in Commune 3 the number of positive samples were 7. However, on the figure (Fig 1) it is indicated that there were 6 positive samples. Similarly, the values presented for Commune 6 are also different. The authors should double check all their numbers in the text and figure to make sure they are correct and that the calculations made from them are correct.

Author Response

Dear Reviewers,

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The author has revised the points I focused on.