PONE-D-19-18063

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Metagenomic analysis of antimicrobial resistance (AMR) genes in stool is an area of great interest and utility. Yet, it faces the problem of low detection of organisms present in low abundance (e.g., the Enterobacteriaceae). The authors propose a unique approach involving the selective enrichment of Enterobacteriaceae members before metagenomic analysis to increase detection efficiency. The also compare efficiency of two DNA extraction methods. Although, the results indicate variable success, the results should serve as a stepping stone for future investigations and improvements.

The article does satisfy the criteria listed for publication in PLOS ONE. The study presents the results of primary scientific research that has not been published elsewhere. The experiments are well conceived and well described. The rational for each experiment is adequately discussed. The results are appropriately presented. The conclusions are strongly based on data presented. Statistical analyses are appropriate and satisfactory. The writing style is very good and easy to follow. All ethical considerations are satisfactory. The article adheres to appropriate reporting guidelines and community standards for data availability.

Two minor typos were encountered

Line 172:

Sentence should not begin with a number i.e., “50μl”

Line 544:

“Effect of plasmid extraction of detection” should be “on detection”

Reviewer #2: This is a study focused on testing different methods for enrichment of Enterobacteriaceae for detection of carbapenemase gene and gyrA gene. The goal was to use an in vitro approach of spiking samples with the goal of developing new methodological areas that could be used as a focus for approaches using shotgun sequencing of samples focusing on the resistome (all antimicrobial resistance genes). This is an important area of research given current limitation of sequencing methods for detection of resistance genes in populations that although present in lower abundance, are of great relevance clinically. The methods seem adequate for the goals proposed, but lack enough information that would allow repetition of this approach. Given the strong methodological character of this study, this is of grave importance and need to be addressed. The title of the manuscript is misleading given the focus of the study, and should be re-phrased. The study does not present any statistical approaches, which should be added (at least what tools were used to compared descriptive results). The discussion should include more information about other studies focused on enrichment of samples for resistome analysis, and how outcomes of current method compare (e.g. “Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing . Noyes et al, 2017” - - as a disclosure, the reviewer is not involved with the research group from this manuscript)

Title – the title of this study is misleading given that it focused on specific bacteria (select Enterobacteriaceae) and specific resistance genes. I strongly suggest the title accurately reflect the conditions that were tested in the study.

Introduction

Line 54 – Could you further clarify what you mean with “binary measures of colonization”. It was not very clear to me what specifically you meant in relation to clonal dissemination of resistance.

Line 56-57 – I suggest adding that you mean “resistant” to antimicrobial drugs.

Line 62-64 – You talk about PCR here but make a reference to a microarray approach (reference number 7). I would suggest differentiating PCR (and maybe talking about qPCR instead) and microarray approach for detecting AMR genes.

Line 73 – I would suggest including Salmonella in the list of important enterobacteriaceae.

Line 86-89 – Having a section of the conclusion in the introduction seems atypical. I would suggest including what your hypothesis for the study was instead and presenting the conclusion of the study in the conclusion section.

Method

Line 100 – describe the methods used to label samples as ESBL negative. Culture one or two E coli isolates? Was a phenotypic approach used? Also please provide reference.

Line 126 – How was the 5 MacFarland suspension measured?

Line 128 – What nutrient broth was used?

Line 133- It is not clear what you meant “to allow quantification after sequencing” S. aureus was added to samples.

Line 139- Where was this approach based on? Any references for using this approach (e.g. specific antibiotics used, time of growth (why 8 hours?))? How would you compensate for difference concentration of bacteria affecting higher bacteria growth, given that bacteria have an exponential growth (not a linear growth)?

Line 146- Why were two different DNA extraction kits used, and why different enrichments used (one for each type of DNA extraction kit)?

Line 182- Although you mention different sources you used to annotate the dataset (metagenome, gyrA, plasmids), I did not see in this section what database you used to characterize resistance genes in the samples (e.g. beta-lactam resistance genes).

Line 196 – I understand the goal of the RDE approach, but the formula is not clear to me, and more information on what each variable means may resolve some questions. Also, where are the “4 unreached samples” coming from?

Statistical analysis

- Where is the statistical analysis section of this manuscript?

Results

Line 213-225- Part of this should be in the introduction and methods, not in the results. I suggest you be direct when presenting the results.

Line 230-241 – This should be in the methods. It is very confusing reading the enrichment section in the material in methods in part because this information is not present in that section.

Line 258 – Was this difference between unenriched and enriched significantly different? Where are the statistical methods used for this study?

Line 280 – What do you mean with “relative abundance of the spike organism of >= 90%? Relative abundance could indicate what percent of the bacteria are present in relation to the total population. Here do you mean that, or what % of the total bacteria amount spiked in the sample you are retrieving?

Figure 2 – Enrichment does seems to affect bacteria growth in a matter that is not linear as a greater amount of initial bacteria is spiked in the sample, as expected due to exponential growth of bacteria. How do you account for this effect on the quantification of resistance genes between samples with different initial amount of a bacteria of interest? ( in other words, how to you account for the inflation in the results caused by enrichment towards samples with higher initial AMR bacteria concentration)

Line 294 – By using Staphylococcus after enrichment, you accounted for the impact of all methods starting at and after DNA extraction on output between two or more different samples. However you did not account for the potential for unequal growth of a specific AMR bacteria caused by the enrichment process.

Line 303 – “outnumbered” – correct grammar error

Figure 5 – The legend of figure 5 does not seem to make sense ( I was able to understand what was happening based on the figure description ( the solid and dashed lines)

Discussion

Line 360-372 – None of the findings of the study are discussed in this paragraph and seems a repetition of content from the introduction. I suggest either inserting and linking finding of the study to the content provided here or removing this sentence from the manuscript.

Line 380-382 – Provide more information about what incubation period was selected, and why it was relevant, and how does it compare to other approaches available in literature

Line 392-394 – I think this is relevant information, but it is not clearly outlined here. I suggest better explaining what you meant here. One option is to give an example for a specific bacteria used in the study.

Line 401 - please link this information to the figure where this data is presented.

Conclusion

This study did not look at all AMR genes, but was limited to a small select number of genes. I would suggest adjusting the conclusion of the study to reflect the specific conditions that were tested, which are still very relevant.

Reviewer #3: The authors of this article have developed a very interesting topic of research namely metagenomic sequencing of fecal DNA to characterise an individual’s intestinal resistome. Metagenomics is a method that, instead of sequencing individual genomes, collectively analyzes all DNA isolated from a specific sample, representing all microorganisms. It can provides information about which organisms are present in the sample and what metabolic processes are possible in the community. The Authors of this study aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. They chose culture conditions that would amplify a resistant subpopulation of Enterobacteriaceae of particular clinical importance, specifically those resistant to third generation cephalosporins, with the aim of bringing their resistance genes above the threshold of detection. Performance of the enrichment-sequencing protocol was assessed by spiking fecal samples with known concentrations of CPE. Their finding of such large variation in growth between different strains of Enterobacteriaceae precludes accurate estimation of their starting concentration unless their growth characteristics are known, undermining one of the aims of the assay. Another aspect of this protocol was the use of plasmid DNA extraction, which increased the detection of plasmid-mediated resistance in Enterobacteriaceae in preliminary spiking experiments. It was problematically that, plasmid DNA extraction produced unwanted artefacts because of differential extraction efficiency depending on plasmid size. In the conclusions, the authors stated that, alternative ways of quantifying scarce AMR genes are needed. However, their study demonstrates the need for any method to be validated in a range of well characterized conditions before it can reliably be used to make quantitative comparisons.

In the assessment of this manuscript I state that the concept of the presented research was well planned. In the "Abstract" section, the general assumption of the research undertaken is clearly presented. However, little information was given about the methodology used. Perhaps this is due to the limitations of the text volume in this section. The introduction is short, but it introduces the reader to the research topic in a sufficient way. The "Material and Methods" and "Results" sections present the subsequent stages of the research and the results in a detailed manner. In the "Discussion" section, the authors should compare the results of their own research with the results of other authors.

In summary, I find that the article is written in a way that meets the criteria of PLOS ONE. The applied research methods are well-chosen. In connection with this assessment, I recommend this manuscript for publication in the Journal PLOS ONE.

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Reviewer #1: Yes: Samer Swedan

Reviewer #2: No

Reviewer #3: No

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Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae

PONE-D-19-18063R1

Dear Dr. Peto,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Yung-Fu Chang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I believe that the authors have satisfactorily addressed all points raised by the reviewers. The revised title more appropriately reflects the nature of the study. The abstract and the introduction are satisfactory. The description of the methods is improved and includes important details that are required for replication of experiments by others. The description of the results is improved and easy to follow. The discussion is greatly improved.

I have no additional comments.

Overall, the revised manuscript is suitable for publication in its current state.

Reviewer #2: I commend the authors for carefully considering each comment. All comments have been addressed by authors and I do not have any additional comments or suggestions.

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Reviewer #1: No

Reviewer #2: No