PONE-D-20-09321

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PLOS ONE

Dear Dr. Stratmann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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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: Partly

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: 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: No

Reviewer #4: No

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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: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: 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: The manuscript titled 'Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer' tries to show the importance of multi-drug resistant organisms on survival and mortality of patients suffering from non-small cell lung cancer. The main limitation of this investigation is the use of a very small sample to draw an important/significant conclusion.

Major comments:

1. The study sample size is small, only 24 out of 295 (8%) patients were studied. The sample size must be significantly larger than this to draw any valid conclusion.

2. In Pg 2 Lines 42-46, the authors mention that 'There was a significantly higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p<0.001) with a trend towards an increased rate of fatal infections in MDROpos patients (p=0.05).

Also, Pg 11, Lines 231-232 states 'When stratified for disease stage (Fig. 2A), median OS in the MDROpos study group showed a significantly inferior median survival time in patients with advanced (IIIB) or metastatic disease (IV). Then again, in Pg 14, Line 302, the authors say ‘Approximately 80% of non-relapse mortality in the MDROpos group was infection-related.’

These are strong statements. Patients in advanced stages of cancer are more likely to suffer from mortality when compared to those in early stages of cancer simply because of their physiological conditions and not just because of bacterial colonization.

Bacterial infections in patients at early stage of cancer can actually augment progression to more critical stages/metastasis according to the existing literature. So, it is the severity of the infection at the early stage which determines or is one of the predisposing factors to progression to a more critical stage.

3. There are many existing literature that the authors can compare their work with such as:

i. Sarihan S, Ercan I, Saran A, Cetintas SK, Akalin H, Engin K. Evaluation of infections in non-small cell lung cancer patients treated with radiotherapy. Cancer Detect Prev. 2005;29(2):181-188. doi:10.1016/j.cdp.2004.11.001

ii. https://www.cancernetwork.com/view/infectious-complications-lung-cancer.

4. Recent literature suggests the presence of the bacterial genus Acidovorax particularly in lung cancer patients. In the manuscript, the authors did not provide details of the bacterial genera found. The author might consider comparing their findings with the paper and comment on why their microbiome could be different:

Greathouse, K.L., White, J.R., Vargas, A.J. et al. Interaction between the microbiome and TP53 in human lung cancer. Genome Biol 19, 123 (2018). https://doi.org/10.1186/s13059-018-1501-6.

5. Since the authors emphasize on the effect of bacteria on mortality, the author must include one paragraph on what has already been published on the possible role of microbial infections on mortality of cancer patients. Recent literature such as the following could have been cited.

Translational Oncology. VOLUME 14, ISSUE 12, P2097-2108, DECEMBER 01, 2019

Gram-Negative Pneumonia Augments Non–Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4. Stephen D. Gowing, Simon C. Chow, Jonathan J. Cools-Lartigue, Simon Rousseau, Salman T. Qureshi, Lorenzo E. Ferri, https://doi.org/10.1016/j.jtho.2019.07.023

Koslow M, Epstein Shochet G, Matveychuk A, Israeli-Shani L, Guber A, Shitrit D. The role of bacterial culture by bronchoscopy in patients with lung cancer: a prospective study. J Thorac Dis. 2017;9(12):5300-5305. doi:10.21037/jtd.2017.10.150

Ye M, Gu X, Han Y, Jin M, Ren T. Gram-negative bacteria facilitate tumor outgrowth and metastasis by promoting lipid synthesis in lung cancer patients. J Thorac Dis. 2016;8(8):1943-1955. doi:10.21037/jtd.2016.06.47

Kovaleva, O.V.; Romashin, D.; Zborovskaya, I.B.; Davydov, M.M.; Shogenov, M.S.; Gratchev, A. Human lung microbiome on the way to cancer. J. Immunol. Res. 2019, 2019, 1394191.

Chow, S. C. et al. Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation. Int J Cancer 136, 1341–1350, https://doi.org/10.1002/ijc.29111 (2015).

Minor corrections:

6. In Pg 3 line 56 cephalosporine spelling correction to Cephalosporin

7. In Pg 4 Line 83 says '(definition see below)' – no definition given

8. In Pg 4 Line 84 ‘…were included into this analysis…’ consider changing the word 'into' to 'to'.

9. In Pg 4 Line 85 ‘….second malignancy - aside of localized non-melanoma skin cancer….’ consider replacing ‘aside of’ by ‘aside from’

10. In Pg 5 Line 100 ‘…….unit as well as all patients all patients admitted to the thoracic surgery…..’, repetition of ‘all

patients’.

11. In Pg 14, Lines 289-290, ‘We encountered a significantly higher co-occurence of diabetes in patients’ spelling of occurrence.

Reviewer #2: I would like to congratulate the authors for conducting the study. It is a well written protocol. There are no previous studies on MDRO and lung cancer. The authors have clearly discussed the limitations.

Reviewer #3: The authors aimed to reveal that the impact of MDRO colonization in patients who have been diagnosed with Non-small cell lung cancer (NSCLC) who are at known high-risk for invasive infections. However, the data is not accurate enough. The data in this manuscript do not support the conclusion.

Reviewer #4: Authors have presented a very important topic on antimicrobial resistance in patients with NSCLC. Antibiotic resistance is a global problem which need be to tackled worldwide. Despite low prevalence of MDRO in their study, however this data from developed world merit to be shared to scientific community at large.

Overall the manuscript is well written and interesting to read. However, results and discussion sections need to revised. In results section some tables are difficult to understand and statistical interpretations are not well understood.

Specific comment

Abstract:

Line 38: Replace 295 with Two hundred and ninety-five.

Line 44: Add crude and adjusted, plus their 95%CI for both univariate and multivariable analysis, respectively.

Line 44 – 46: “There was a significantly higher rate of non-cancer-related mortality in MDROpos compared to MDROneg patients (p<0.001) with a trend MDROpos towards an increased rate of fatal infections in patients (p=0.05)” from table 3 I found very difficult to interpret. The way p-value which has been presented it looks to reject null hypothesis in favor of alternative hypothesis. Considering NRM cases alone in comparison of non-cancer related mortality might obscure the intended outcome, if possible include all participants in this analysis. Table 3 need to be revised for more clarity.

Introduction:

Line 55–56: Third-generation cephalosporin resistant should be replaced with extended spectrum beta lactamase producers.

Line 57: Replace Staphylococcus aureus with methicillin resistance to Methicillin resistance Staphylococcus aureus

Materials and Methods

ESBL, MRSA and VRE were screened by screening media, these media have high sensitivity. It will be interesting to state how this MDRO were confirmed either by phenotypic or genotypic.

Line 146: Ensure consistency either use MDROpos or MDRO+ .

Results

Line 168: Replace 271 with Two hundred and seventy-one.

Line 173: Diabetes mellitus did not show significant association, revisit supplementary file OR included 1.

Table1: Some variable in a column total % do not add to 100%. For example, in co-morbidity variable total number of MDRO is 25 and not 24. Interpretation along the row could be interesting than along the column. Think of revising this table.

Line 197 – 207: Specify name of specific antibiotics tested instead of using classes of antibiotics. For examples macrolide use either erythromycin or azithromycin, aminoglycosides specify gentamicin or others.

In analysis MDRO+ exposure to OS outcome. The disease stage could be one of the important confounders for OS. See Fig1A for example, for interest I would like to know if you controlled for disease stage as confounder, what happen HR. If you did not do please could you explain to me for interest.

Although this has been explained in discussion, however authors need to dig further on MDRO colonization with inferior overall survival outcome. It is well hypothesized MDRO colonization is a risk for severe MDRO infection with the same bacteria. MDRO infection could have direct effect on overall survival in these group of patients.

Analysis on Fig2B and 2C could be combined (IV/IIIB vs IIIA/II/I) rather than categorized in different group. Since disease staging in itself could predict OS.

Line 246 – 251 looks like figure 2 legend, please move below the figure2

Line 256 – 258: Check comment on abstract.

Table 3: Check comment on abstract

Line 267 – 268: Looks like legend for figure 3.

Discussion:

Well written, however in some part authors need to revise like line 302. If table 3 is revised this statement might need to change, there is a need to include all mortality not NRM only.

In conclusion, line 350 authors did not establish the correlation between MDRO colonization and infection. To state this as a reason “due higher rate of fatal infections mostly involving the lung tissue”. This infection was unrelated to colonization, the statement needs to be revised.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Professor Sunjukta Ahsan, Department of Microbiology, University of Dhaka, Bangladesh, sunjukta@du.ac.bd

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PONE-D-20-09321R1

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PLOS ONE

Dear Dr. Stratmann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 18 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Joel Manyahi

Academic Editor

PLOS ONE

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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: (No Response)

**********

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: The authors have considered all suggestions and corrections addressed by the reviewer. Comments have been added and additional references have been cited as suggested. Minor corrections have also been addressed. The most outstanding limitations were acknowledged. This makes the manuscript more acceptable from a readers point of view.

However, the author needs to correct one spelling mistake, that is 'thirdly' in line 326. Otherwise, the manuscript is acceptable.

Reviewer #2: The authors have answered all queries. I have no further comments, The manuscript will be useful for the readers.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Sunjukta Ahsan, Department of Microbiology, University of Dhaka, Bangladesh

Reviewer #2: No

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Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PONE-D-20-09321R2

Dear Dr. Stratmann,

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

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Joel Manyahi

Guest Editor

PLOS ONE

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