PONE-D-24-41634First Time Detail Virulence and antibiogram profiling of Pseudomonas aeruginosa isolated from apparently healthy Japanese quail (Coturnix japonica) in BangladeshPLOS ONE

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“CONFLICT OF INTEREST

19th September 2024

Title: First Time Detail Virulence and antibiogram profiling of Pseudomonas aeruginosa isolated from apparently healthy Japanese quail (Coturnix japonica) in Bangladesh

DECLARATION AND STATEMENT

We know of no conflicts of interest associated with the publication. We declare that this manuscript is original and has not been submitted to anywhere else for publication. The manuscript nicely falls under the scope of your journal.

Sincerely yours,

Mahbubul Pratik Siddique, PhD

Corresponding author

and

Professor

Department of Microbiology and Hygiene

Bangladesh Agricultural University

Mymensingh-2202

Cell Phone: +880-1751-945123

Email: mpsiddique@bau.edu.bd

     https://www.researchgate.net/profile/Mahbubul-Siddique

     https://scholar.google.com/citations?user=YmxVEzEAAAAJ&hl=en”

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Reviewers' comments:

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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

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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: Comments to authors:

-The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

-The manuscript should be revised for English editing and grammar mistakes.

-Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and the references section.

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

- Add the full expression before the abbreviations.

-Introduction: (it needs to be more informative):

-Give a hint about the virulence factors and the mechanism of disease occurrence , and infecions caused by P. aeruginosa.

-Illustrate the mechanism of action different virulence factors of P. aeruginosa.

-Rephrase the aim of the work to be clear and better sound.

Material and methods:

-Support all methods with updated specific references.

•Add the company, city, and country of the used chemicals and reagents.

-Isolation and identification:

Discuss in detail the methods of isolation and identification of P. aeruginosa. Besides, specific references should be added.

•Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

-PCR based detection of other antimicrobial resistance genes should be performed. Afterwards, the correlation between phenotypic and genotypic multidrug resistance should be performed.

-MLST should be carried out to illustrate the genetic relatedness among the retrieved isolates (If available).

-Results:

-Please add a starting paragraph to the results section to briefly introduce the topic, your goals and hypothesis and a short summary of what you did in this work.

-Antimicrobial susceptibility testing:

• Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates as the following (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).The antibiotic-resistance genes

-Tables 6 and 7: Address the multidrug resistance papperns in the tables. I couldn't find it (MDR, XDR, and PDR)

-MLST should be carried out to illustrate the genetic relatedness among the retrieved isolates.

-Support your findings with more illustrating figures. Besides, increase the resolution of all figures (must be 600 dpi).

-Discussion:

-The authors are advised to illustrate the real impact of their findings without repetition of results.

-Please illustrate the role of virulence genes and virulence determinants of P. aeruginosa in the pathogenesis of the disease.

-Please illustrate different mechanisms of antibacterial resistance P. aeruginosa.

-Illustrate the essential role of biofilm in the occurence of infection.

-Conclusion

-Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Reviewer #2: Dear Editor:-

I think this article is very good regarding the originality of the idea at which the field of study is so interesting and medically important.

The topic of manuscript is timely and will be of interest to the readers of the journal. Also, the manuscript is very well written and the ideas flow logically.

Dear authors you are doing well and that’s look great work. A few issues, however, need to be addressed;

In line 13 page 2 Abstract: Abstract was too long, must be 250 words.

In line 13 page 2 Give a brief explanation of the significance of Pseudomonas aeruginosa for bird health and its effects on the poultry trade. This might facilitate the setting of your research.

In line 17 page 2 Provide details on how the quail were selected for sampling. Also, mention whether the birds were healthy or showing signs of illness.

What effects do the virulence genes (exoA, rhlR, etc.) that have been found have on P. aeruginosa's pathogenicity in quail? Could you explain more about the relevance of other genes' varied distribution?

Is there a biological or environmental reason why female quail are more likely than male quail to host Pseudomonas spp.?

In line 28 page2 What biological or environmental factors could explain the increased odds of female quail harboring Pseudomonas spp. compared to males?

In line 35 page2 Given that exoU, lasI, and lasA were not detected in any isolates, how might this influence the virulence potential of these strains?

In line 84 page 5 Regarding P. aeruginosa from quails, how does the rise in multidrug resistance (MDR) correspond to developments in resistance in human medicine?

In line 88 page 5 To enhance introduction section add the following reference: Rawaa A. Hussein, Shaymaa H. AL-Kubaisy, Mushtak T.S. Al-Ouqaili. The influence of efflux pump, outer membrane permeability and β-lactamase production on the resistance profile of multi, extensively and pandrug resistant Klebsiella pneumoniae. Journal of Infection and Public Health, Volume 17, Issue 11, 2024. https://doi.org/10.1016/j.jiph.2024.102544.

In line 90 page 5 Why are quail farms mentioned in this particular setting? Could you explain on how common AMR is, particularly in quail farms as opposed to other livestock?

In line 100 page 6 To enhance introduction section add the following reference: Alduhaidhawi AHM, AlHuchaimi SN, Al-Mayah TA, Al-Ouqaili MTS, Alkafaas SS, Muthupandian S, Saki M. Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater. Infect Drug Resist. 2022 Mar 19;15:1143-1154. doi: 10.2147/IDR.S358248. PMID: 35340673; PMCID: PMC8942119.

In line 118 page 7 What guidelines were applied when choosing the particular areas for the sample—Jamalpur, Mymensingh, Gazipur, and Narsingdi? Do these regions reflect the larger quail farming sector in Bangladesh?

In line 119 page 7 Why were oral and cloacal swabs the only ones used to get samples? Would other sample types—like environmental or fecal samples—offer more thorough insights?

In line 122 page 7 Why are samples from male and female quails being collected in equal numbers, and why specifically? Regarding illness susceptibility or AMR prevalence, were gender variations taken into account?

In line 149 page 8 On one hand authors worked with boiling and thawing for DNA extraction technique, but why Ethidium bromide was used. Ethidium bromide- a carcinogen!

In line 153 page 8 What is the clinical significance of detecting the specified virulence-associated genes in your study? How do the presence of these genes correlate with pathogenicity or virulence in Pseudomonas?

In line 232 page 14 In what ways was turbidity assessed or quantified in your cultures? When evaluating the turbidity levels in LB broth, nutritional broth, and BHI broth, did you employ any particular methods or tools?

In line 267 page 16 Results by sample type and gender are shown in Table 2. Why are the prevalence statistics for male and female birds presented differently, in your opinion? What knowledge does this analysis of gender differences offer?

In line 273 page 16 What underlying variables could be responsible for the higher probability of Pseudomonas spp. being present in quail from Narsingdi as opposed to Mymensingh Sadar?

In line 290 page 17 The absence of genes such as exoU, lasI, and lasA is noteworthy. What implications does this have for the virulence potential of these isolates? Could their absence indicate a particular environmental adaptation?

In line 293 page 17 Has any genetic diversity study been done on the 25 isolates of P. aeruginosa? In what ways could genetic variation affect the way virulence genes are distributed?

In line 308 page 19 It is remarkable that 72% of specimens have intermediate streptomycin resistance. In the setting of treatment, how should this be understood, and what kind of follow-up testing is recommended?

In line 326page 20 Nine of the 18 antibiotics evaluated in the study demonstrated susceptibility. How did these antibiotics come to be chosen? Were they chosen because they were commonly used antibiotics in clinical settings, or because they were relevant to veterinary medicine?

In line 326page 23 Could you provide more details about how your research on quail birds in particular advances our knowledge of P. aeruginosa's potential harm to public health? In what way does this study contribute to the overall understanding of P. aeruginosa epidemiology?

In line 326page 24 To enhance discussion section add the following reference: Murshid RM, Al-Ouqaili MTS, Kanaan BAJ. Microbial Vaginosis and its Relation to Single or Multi-Species Biofilm in Iraqi Women: Clinical and Microbiological Study. Pak J Biol Sci. 2024 Jul;27(8):404-412. doi: 10.3923/pjbs.2024.404.412. PMID: 39300677.

In line 388 page 24 According to result of this research, the likelihood of contracting P. aeruginosa is six times higher in young birds (less than 8 weeks old). In younger birds, what particular biochemical or immunological pathways could explain this heightened vulnerability? Has the literature put forth or examined any plausible theories?

In line 401page 24 To enhance discussion section add the following reference: •Saleh RO, Al-Ouqaili MTS, Ali E, Alhajlah S, Kareem AH, Shakir MN, Alasheqi MQ, Mustafa YF, Alawadi A, Alsaalamy A. lncRNA-microRNA axis in cancer drug resistance: particular focus on signaling pathways. Med Oncol. 2024 Jan 9;41(2):52. http://doi.org/ 10.1007/s12032-023-02263-8.

In line 450 page 27 Conclusion should be objective with further perspective or should add at least a few sentences about future study/future perspective of it

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: Reviewer comments-PONE-D-24-41634.pdf

First Report of MDR Virulent Pseudomonas aeruginosa in Apparently Healthy Japanese Quail (Coturnix japonica) in Bangladesh

PONE-D-24-41634R1

Dear Dr. Mahbubul Pratik Siddique,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Morteza Saki

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

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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: The authors have carried out significant changes to the manuscript. They have addressed most of the suggested corrections and comments. Really, it's an interesting study that has a significant impact. Now, the manuscript could be accepted.

Congratulations.

Reviewer #2: Dear author

You are doing well. The manuscript now is so arranged and more acceptable for publication

Regard

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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: No

Reviewer #2: Yes: Mushtak T.S. Al-Ouqaili

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