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Thank you for thoroughly addressing the reviewers' comments.
# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #
In particular, please address the concerns of Reviewer #2 regarding statistics and taxonomic classification of the Vibrio.
This paper describes the resistance of Vibrios to triclosan. The paper is extremely well written and clear. Appropriate literature is cited and sufficient background and context is provided. The article has nice figures and tables and provides the raw data.
The supplementary .txt file would not open for me. When I opened it, it was gibberish. The authors should confirm it has the correct information in it.
The research questions are well designed and thoroughly tested using sound statistical methods. Appropriate controls are used. Methods are provided in sufficient detail.
The findings are robust and clear. The results are novel and useful.
This paper was very well written and the research was done well. I only have minor comments:
L102. Is there any reason to believe these concentrations are stable? Or do you think they may vary? I think its important to state whether these were the concentrations at the time of sampling or if they were at a different time and to note that they are likely to change given various periodic and aperiodic sources fate and transport mechanisms in the coastal system.
L156. What if the duplicates did not agree? Or did they always agree?
This manuscript meets basic reporting requirements. The current study measures levels of triclosan resistance in environmental Vibrionaceae isolates from three sampling locations in South Carolina and Florida. Triclosan is a widely-used contaminant of emerging concern that is poorly removed in wastewater treatment plants and thus can be detected at measurable concentrations in coastal waters around the US. A better understanding of how this antimicrobial compound impacts the composition and antibiotic resistance of coastal microbial communities is needed. This study provides some valuable information to close the knowledge gap, but some revisions are needed to ensure the findings are presented in a valid and supported manner (see below).
The use of Kruskal-Wallis one-way ANOVA assumes homoscedacity among the groups. Was this assumption verified? I am fairly confident that the data violates this assumption and could lead to inflated false positives particularly with non-normal data and the small sample sizes used in many of the comparisons.
The methods section on Identification of Environmental Vibrionaceae Isolates doesn't actually describe how isolates were assigned to species and clades based on the hsp60 sequences. Fully document how the assignment process was conducted (OTU clustering? Closest BLAST hit?). I also request that the authors document the level of confidence in those assignments and discuss how the varying confidence among isolates could impact the differences observed among groups. The statistical results are highly dependent on these categorizations and the groupings are likely to vary depending on which assignment method is applied. Overall, the references cited are appropriate, but for taxonomic assignment, I would recommend using Dikow, R. B. (2011) Systematic relationships within the Vibrionaceae (Bacteria: Gammaproteobacteria): steps toward a phylogenetic taxonomy. Cladistics.
Why was a BLASTN search of the V. cholerae fabV allele considered an appropriate in silico experiment? If the gene of interest has any degree of vertical gene transmission, searching for nucleotide similarity will certainly return more hits from closely related taxa compared to other bacteria. For finding homologous genes in less closely related taxa, a search at the protein level may be more appropriate, but NCBI has better options available for searching proteins that have already been annotated as fabV. For example, the first result in a search of protein clusters for fabV includes a cluster of 766 proteins from 450 organisms conserved across the domain bacteria. Only 185 of these proteins are from 60 Vibrionaceae taxa indicating much broader representation outside the Vibrionaceae than suggested by the BLASTN search. This in silico anaylsis is thus flawed and should be redone or removed.
The last paragraph of the introduction is vague and does not fully present the state of current knowledge versus the new information provided by the current study. Provide a few references that summarize the data previously collected on triclosan resistance in Vibrionaceae.
The interpretations in the third paragraph of the discussion (Lines 237-242) are not supported by referenced sources. DeLorenzo et al. 2016 found increased Vibrio relative abundance at 0.33 ppm and Lydon et al. 2017 only found a relative enrichment at >4 ppm, not at ~5 ppb triclosan levels. Specifically, the assertion that Vibrio abundance increases in the parts per billion range is not justified. Given that measured environmental concentrations were 10,000 times lower than the dosage that experimentally resulted in Vibrio enrichment, the interpretations are overstated regarding potential enrichment in coastal waters under current concentrations. Moreover, the enrichment effect could be temporary, so more work is needed to understand how persistent exposure to low concentrations of triclosan could affect coastal microbial communities.
The authors hypothesize that triclosan resistance is an evolutionary inherited trait rather than due to environmental exposure (Lines 256, 266), but the results of this study are not directly targeted at that hypothesis. To more directly address this hypothesis, a different analysis such as using a Mantel test relating a distance matrix of triclosan MICs versus a second matrix based on phylogenetic distance (used to construct the tree in Figure 5). Another advantage of this approach is that it avoids the need to classify the hsp60 sequences into clades and/or species, which is inherently difficult to do well for bacteria with high gene transfer rates.
I think it bears mentioning in the discussion that the lower MICs for the Cholerae clade could be complicated by the fact that five of the six organisms tested for this clade were previously characterized isolates rather than those collected from your sampling sites. If the authors only consider the isolates characterized in this study, is a significant difference among clades still observed? While Figure 4 shows no difference among isolates from the three sampling locations in the current study, previously characterized isolates are omitted from the comparison since they all differ is sampling location.
(I added line numbers to the manuscript and will reference them in my comments)
Line 27 (3rd line of abstract): The Vibrionaceae family is singular, but "among" refers to multiple subjects, so "across" may be a better choice.
Lines 33, 35: Since the authors are using non-parametric statistics, it makes sense to summarize group MICs using the median value, but in a few instances the authors switch to mean MICs, which is confusing since the data are not normally distributed.
Lines 40-42: The last sentence of the abstract is partially redundant and could be better used to highlight the potential implications.
Lines 81-82: the phrase "…given the rising health burden of this group." is vague and should be made more specific.
Line 105: year for Preheim et al. citation should be 2011 instead of 2005.
Line 173: "resistance triclosan" should be "triclosan resistance"
Line 175: > 100 ug/ml should be ≥ 100 ug/ml
Lines 223-224: what evidence supports that collected environmental isolates have "known variability in prior environmental triclosan exposure"? The fact that environmental isolates were collected from sites containing different triclosan concentrations at time of isolation doesn't imply variability in past exposure.
Line 247: I believe the authors mean "plastic" (flexible content) rather than "elastic" (expanding and contracting genome size) genomes.
Lines 274-275: Should funding source(s) be included in the acknowledgements?
Figure 3: Should V. coralliilyticus be included?
Figure 4: For consistency, format this figure in the same way as Figures 2 and 3.
Figure 5: Branch support numbers are unreadable and should have larger text size. It would also be helpful to overlay taxonomic assignments (clades, species) onto this tree.
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