Profile of the Spatial Distribution Patterns of the Human and Bacteriophage Virome in a Wastewater Treatment Plant Located in the South of Spain

Round 1

Reviewer 1 Report

First I would like to thanks to the authors for their work.

General comments

Taxonomical units must be in italic - correct it in whole manuscript.

row 58 - 103 - ?? should be 10³, superscript must be checked in whole manuscript (row 151, 153)

row 76 - hm3/year - no SI unit correct it

Material and Methods

Table 1 - environmental parameters - redundant information. Please provide information about wastewater quality this is more important for your research. 

How did you calculate SD for flow rate HRT, SRT, TSS ? More common is to show the range of values, please correct it. 

Table 2 - please provide information about the number of repetitions

Please correct form of manufacturer information (device name, city, country). Also there is lack of information for some instruments example:

centrifugation, filtration, NGS, Kits ... etc, please correct it in whole manuscript. 

if you used Nanodrop for DNA quality check, provide info in methods. 

Please provide info about primers used.

Results and Discussion

row 204-206 redundant information, commonly known fact

Please explain why OTU 0521, 0326, 0092 are in corelation with COD,BOD, TN, TP.

Discussion + Conclusion

There is no discussion and conclusion in accordance with Introduction, where did you mention reuse of wastewater in agriculture. Please provide these information.

Author Response

Dear,

First of all, I would like to thank you for your effort to review the manuscript. We think that all your comment and suggestions have improved the quality and replicability of the manuscript. 

Please, finding attached the answer 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors provide an important contribution in regards to the methodology, approach, and characterization of the virome at various stages in a conventional public/municipal wastewater treatment plant system. In addition, the authors also provide some evidence of deposition locations for various viruses, including potential human pathogens, within the wastewater treatment process. The methodologies employed are rigorous, align with best practices, and the reporting of potentially complicated results was done in a manner understandable by a broader audience. The manuscript demonstrates the use of controls for assessing the integrity of the genetic material extracted, as demonstrated in section 3.1 of the manuscript, and the values presented are all acceptable and some demonstrating very high-quality/near-pure.

In summary, as a reviewer, I have two major concerns, one moderate concern, and provide a number of major and minor editing comments. All of my concerns are likely able to be rectified, except possibly the moderate concern.

Major concern #1: Although it may be implied in the manuscript, it is important to more clearly indicate that the dominant groups from the virome observed (as a whole and throughout the processes) may be similar elsewhere or are unique to this particular wastewater system. Every wastewater plant will have different inputs, and generalisations that may be made by some readers could interpret these findings to be universal. Clarifying the potential for these results to be highly specific to the WWTP investigated would be of benefit.

Major concern #2: The manuscript rightfully suggests E. coli O157 typing phage 7 was quite dominant throughout and overall in the WWTP virome based upon their data. Referring to Line 236: There are a tremendous variety of E. coli phages, and we need to be cautious insinuating that E. coli O157 typing phage 7 is representative of all E. coli phages. Phages have remarkable specificity. E. coli O157 is a pathogen whereas most E. coli are normal gut flora. With respect to the Escherichia coli O157 typing phage 7 observed, what role do the dairy contributions potentially play here? The bacterial strain (E. coli O:157) is associated a lot with cattle, where the bacteria may be spread among the animals asymptomatically, however, in humans, this pathogen (E. coli O:157) can cause problems. To find the associated bacteriophage as a dominant strain in the wastewater may indicate that the dairy contributions are driving this result. The authors need be careful confusing readers that this strain specific phage is indicative of normal wastewater phage populations and may be unique to their dairy contributions. The authors seem to be suggesting that since E. coli (as a group) are commonly shed by humans and animals, which would explain why this specific phage is so dominant. There are a variety of other E. coli phages with stronger linkages to human waste, so the question is open, as to why this specific one dominates? Demonstrating that open question more clearly, unless the authors have other evidence, is important. Even my comments are speculative here as the authors rightfully indicate early on that little has been done in this area. These papers may be of interest in the author’s consideration of there comments in this area around Line 236:

Gulino, K., Rahman, J., Badri, M., Morton, J., Bonneau, R. and Ghedin, E., 2020. Initial Mapping of the New York City Wastewater Virome. Msystems, 5(3).

Goh, S., Newman, C., Knowles, M., Bolton, F.J., Hollyoak, V., Richards, S., Daley, P., Counter, D., Smith, H.R. and Keppie, N., 2002. E. coli O157 phage type 21/28 outbreak in North Cumbria associated with pasteurized milk. Epidemiology & Infection, 129(3), pp.451-457.

Moderate Concern #1: This concern may not be able to be addressed, however, it may have helped explain some of the observations. With respect to the 0.45 um filters, were bacterial DNA investigated or are they planned to be investigated? The alignment of the virome results potentially correlates with the dominant bacterial taxa, and are E. coli O:157 commonly shed in great abundance in the sewershed for this WWTP? This may not be able to be addressed, which is okay and in my opinion, should not compromise eventual publication. 

Major Editing Concerns

#1. Line 57: “Also, viral particles are usually spread in the air, mainly situated above tanks, at a concentration of up to 103 most probable number (MPN) per litre [13].”  The value of 103 MPN/L does not align with the findings of the reference and is off by possibly five or six orders of magnitude. The MPN values presented are all in the 10^(-4) range.

#2. Table 2 (Line 108): The TN and TP measures have presumably the wrong units. (mg/L) should be sufficient. The mg O2/L is presumably an artifact of the earlier rows. Even in those earlier rows, mg/L is sufficient for COD and BOD5 vales. It’s understood that BOD is measuring the mg of oxygen. For TN and TP it is understood these are mg/L as in mg of N per L, etc.

#3. Line 120 and Line 124: What material were the 0.45um and 0.22um  filters? (mixed cellulose ester, nylon, polycarbonate?) Were the filters sterile? Were the filters cation coated?  These bits of information would help others attempting to use the authors methodology in their own future studies. 

Minor Editing Concerns:

1.) Abstract Line 21: “only a limited virome…” should be “only a limited number of virome studies”

2.) Abstract, Line 30: add the period (.) to end abstract.

3.) Introduction: Line 35: Should it be “vegetable” for the first term or “Plant” as plant is a more inclusive term of the plant world?

4.)  Line 39: The most abundant “enteric” viruses. I’m not sure the references of the science back up that those viruses listed are the most abundant worldwide, as I would imagine some soil-related viruses being even more numerous. The references refer to these as the most abundant enteric viruses (or maybe most abundant human-associated viruses).

5.) Line 44: Indicates “few” studies, but only lists one study. There are a few, and possibly more: Wyn-Jones AP, Carducci A, Cook N, D'Agostino M, Divizia M, et al. (2011) Surveillance of adenoviruses and noroviruses in European recreational waters. Water Res 45 (3): 1025–1038

Newton RJ, VandeWalle JL, Borchardt MA, Gorelick MH, McLellan SL (2011) Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor. Appl Environ Microbiol 77 (19): 6972–6981

6.) Line 47: There are stronger references in addition beyond reference 9, including possibly these papers (or the references included within):

Soller JA, Bartrand T, Ashbolt NJ, Ravenscroft J, Wade TJ (2010) Estimating the primary etiologic agents in recreational freshwaters impacted by human sources of fecal contamination. Water Res 44 (16): 4736–4747

Marion JW, Lee C, Lee CS, Wang Q, Lemeshow S, Buckley TJ, Saif LJ, Lee J. Integrating bacterial and viral water quality assessment to predict swimming-associated illness at a freshwater beach: a cohort study. PloS one. 2014 Nov 19;9(11):e112029.

7.) Line 49: E. coli should be italicized. In this instance, the actual bacteria is being referenced, not the phage. Taxonomical norms dictate Escherichia coli should be italicized. When the bacteria name is part of a virus name, such as the phages, then the name should not be italicized. Here though, in line 49, the reference is to the bacteria.

8.) Line 49: biosensors in the field of environmental health refer to real-time monitoring technologies, rather than biological sample based measures. The term “indicator” or “bioindicators” may be more appropriate.

9.) Line 52: The reference used (currently #10) is appropriate, but given the abundance of literature on this topic, more references should support this statement, including #16 in your paper, and #17.

10.) Line 58: “which are 10 to 100 times more abundant THAN in natural environments”. Need to add the word ‘than’.

11.) Line 69: “Water supply is a global alarm” should be reworded. An alarm is a warning signal. “Water supply concerns present a serious global threat that have been intensified with growing water demand.”

12.) Line 138: Concentered or “concentrated”

13.) Line 246: Line 273: Does the E. coli O157 typing phage 7 have advantages to persist? An article to consider. Lu Z, Breidt F. Escherichia coli O157: H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity. Frontiers in microbiology. 2015 Feb 17;6:67. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330901/

14.) Line 283: Need to specify acronym C.D. for Figure 2.

15.) Line 314: Need to specify acronym C.D. for Figure 3.

16.) Line 328: The S-W index value of 2.7 is off-centered.

17.) Line 336: The images in Figure 4 are difficult to read in the current format. Creating this figure in a 1 x 4 image arrangement versus a 2 x 2 arrangement may permit easier reading and interpretation by readers. The axis labels are too small to read.

18.) Line 352: There’s a great review article aligning with the author’s remarks here. See Gerba et al. 2018.

Gerba CP, Betancourt WQ, Kitajima M, Rock CM. Reducing uncertainty in estimating virus reduction by advanced water treatment processes. Water research. 2018 Apr 15;133:282-8. https://dl.uswr.ac.ir/bitstream/Hannan/96182/1/2018%20WaterResearch%20Volume%20133%20April%20%2825%29.pdf

19.) Line 355: Need to specify acronym for C.D. in Figure label.

Author Response

Thank you very much for your comments and suggestions. We think that your comment had served enormously to improve the manuscript. In this sense, we attach the answer and the improvements made.

Kind Regards 

Bárbara 

Author Response File: Author Response.docx

Reviewer 3 Report

This work analyses the presence of viruses in four different steps of a wastewater treatment plant by random metagenomics. The work shows interesting results and the methodology seems to be correct. However, some aspect must be reviewed.

Major comments
Authors analyzed samples from 4 different points through the WWTP process for 6months. However, authors only talk about 4 samples that correspond to the sampling sites. Were the samples mixed? At what step were mixed? Explain in material and methods.
Figure 1 and lines 212-213: Can you diferentiate between animal and plant viruses only with TEM images?
Some works of viral metagenomics are missing: Adriaenssens et al., 2018; Fernandez-Cassi et al., 2018.
Conclusions are very limited and some claims are not explained. Please develop the assertions and the main findings.

Minor comments
Add references for R packages used.
Line 43: Reference work talks about drinking water. Change by a reference of viral pathogens in wastewater.
Line 45 and 51: Add references
Lines 117-118: Abbreviations seems to be in Spanish. If so, change by English abbreviations.
Line 128: What was the temperature and time used for the DNAse treatment? What is “DNAse Treatment and Removal”? If it is a kit, rewrite as “……using DNAse Treatment and Removal (Invitrogen, MANUFACTURER LOCATION).
Line 135: Specify sequencing library, Illumina platform and sequence length used.
Line 140-143: Add reference of the staining method.
Line 145: Raw reads were cleaned? If so, specify software and parameters.
Line 346; Add “in”

Table 1: Add the meaning of HRT, SRT and TSS in the table legend.
Table 2: The table can be more visual if authors differentiate influent, effluent and removal values in columns.
Figures 2 and 3: Add the meaning of “C.D.” in the figure caption.

Author Response

Dear, 

First of all, I would like to thank you for your effort in reviewing the manuscript. After your comments, the manuscript increases its quality and replicability.

Kind Regards 

Bárbara 

Author Response File: Author Response.docx

Reviewer 4 Report

Garcia-Fontana et al. have evaluated the virome in a full-scale WWTP. Since there are limited studies regarding this topic, this manuscript can be of great importance.

However, the current manuscript is not suitable to be published and extensive revision is required.

The introduction doesn’t focus properly on the objective. For example, authors mention about viruses in WWTPs and then discuss what viruses are. The order is not correct there. Many similar instances can be found throughout the introduction. There is repetitions also (Ex: Line 38-39 and 41-43). Line 45 talks about waterborne diseases. The main reason for treatment is to reduce the waterborne disease burden. Therefore, it should be mentioned first.    

Title: Remove full stop

Line 49: E. coli needs to be italic. Please check for other microorganisms also like line 260.

Line 57: abundant than in

Line 58: 103 or 1000?

Line 60: air-borne viruses

Line 60: nuclei population??

Line 63: Please use a citation

Line 69: Global alarm is not very suitable

Line 62-65: This sentence doesn’t seem to fit here.

Line 65-68: Why there are details about adenovirus, after discussing bacteriophages? It is suggested to use two paragraphs for viruses and bacteriophages and discuss their removal by different methods.

Line 69-79: This should come before types of viruses because this is the main reason we treat our wastewater.

Line 80-85: There are many studies about viruses and not much about virome.

It is suggested to rewrite the introduction with a suitable flow. For example water scarcity, water reuse, treatment, treatment methods, viruses, metagenomics or any logical order which fits.

Line 99: Facultative anoxic…….treatment?

Table 2 can be shortened. Inf, eff and removal can be in columns with COD, BOD, TN and TP in rows

Line 114: Sample collection

Line 130: Reverse transcription?

Line 151, 154: Correct 10^-5 and 10^-3

Line 204-206: Sentence seems unnecessary.

Line 212: Eukaryotic viruses?

Line 236 and following paragraphs: Make E. coli italic

Line 241-242: Doesn’t match the discussion because you are not talking about seasonal variations.

Line 247: registered can be replaced with observed

Line 253: 5.1X10^6

Line 255-257: The meaning is not clear

Line 256: Ammonia-oxidizing bacteria

Line 260-265: Are there any available citations?

Line 271: What is the difference of inlet water? Only other samples differ from inlet water.

Figure 2, 3 and so on: What is C.D.? The quality of Figures 2, 3 and 4 is not good.

Line 293: Gammaherpesviridae

Line 295-296: What can be the reason for not detecting hepatitis C virus in mixed liquor, effluent and sludge?

Line 302: Abbreviation (HCV) should be mentioned at first, Line 295.

Line 324: minimizing

Line 325-327: The idea is not very clear here

Line 331-333: What can be the reason for similarities?

Figure 4: It is almost impossible to see anything in the figure.

Line 341-347: The paragraph is not very clear.

Line 348-352: How does this paragraph belong here?

Line 359-361: What can be the reason for this observation?

Line 368-382: This section is not very clearly explained.

Line 370-371: What does this mean by high physicochemical performance?

Figure 7: The symbols in the plot are not explained.

Author Response

Dear, 

Thank you very much for your comments and suggestions. Certainly, your questions had significantly improved the quality of the manuscript. A lot of suggestions have been of great help to us.

Kind Regards

Bárbara 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors, I am satisfied with your manuscript corrections.

Author Response

Dear, 

Thank you very much for your effort. 

Kind Regards

Bárbara 

Reviewer 3 Report

Changes has been made and the work is now accepted for publication. Congratulations.

Author Response

Dear,

Thank you very much for your effort, and thank you for your advice.

Kind Regards.

Bárbara 

Reviewer 4 Report

Garcia-Fontana et al. have modified the manuscript as suggested by the reviewer and the quality of the manuscript is reasonably improved. However, the reviewer finds further modifications which needs to be done before accepting the article. They are as follows.

The introduction is still not completely rearranged. It may be better to completely check the manuscript for English language usage.  

Line 49: indicators

Line 56: Reuse of treated wastewater

Line 34-35 should be included around line 56 or can be removed because the meaning is similar to Line 57-58.

Line 65-66: The main purpose of WWTPs is to remove pathogens rather than accumulation

Line 67: Activated sludge treatment is the main process

Line 69-72: It is important information, but seems a little irrelevant here

Line 72: The population within the viral matrix can be simply written as virus population

Line 78: CAS process

Line 78-80: Please check this article 10.1016/j.envint.2016.03.001

Line 83-84: Environmental virome. The total sentence is not very clear.

Line 84-85: viral particles is challenging from both

Line 88: Also it is essential

Line 93: Biological treatment of activated sludge is wrong. It must be full-scale WWTP from the city of Granada

Line 102: followed by biological treatment using CAS

Line 123: January to April is only 4 months, not 6

Line 138: Remove “and removal”

Line 141: (Thermofisher)

Line 149-150: were out through doesn’t make any sense.

Line 152-155: This step is before library preparation if I am correct.

Line 157: Remove “The virus particles previously” and use “Concentrated virus particles were visualized”

Line 210-211: What does it mean by “sensitive viral phylotypes”

Line 224-225: This sentence is redundant

Line 256: Escherichia coli needs to be italic

Line 260-261: What kind of diversity you discuss here?

Line 261: Remove: in O:157, remove “a lot”

Line 265: urban wastewater treatment processes

Line 266: bacteriophage accounts for more than 10%

Line 270: phage vB were also observed in high abundance

Line 272: P.aeruginosa must be italic

Line 276: Remove “thus”

Line 278: What is the meaning of “The small doses of phages”

Line 290: Remove 00 from 1.00%

Line 296: E. coli italic

Line 306: as per SIMPER analysis

Line 326-328: How is this relevant here?

Line 333: removes the viruses from influent wastewater

Line 349: “minimizing public health risks in receiving water” doesn’t make sense. The risk is the exposure to effluent water containing viruses

Line 352: phage which were

Line 359-361: This should be the opposite. CAS process has a strong influence on the WWTP bacterial composition

Line 389-390: “it did not…” What can be the reason for the observation?

Line 401: What does “to high physiochemical performance” means?

Line 408: “possibly caused by the biological treatment” In what mechanism?

Line 425: Remove “Therefore”

Line 427: role in the viral community composition of all collected samples

Line 428: Collected samples

Line 428-430: In conclusion, please be specific

Author Response

Dear, 

Thank you very much for your effort to improve the manuscript. 

Kind regards

Bárbara

Author Response File: Author Response.pdf