PONE-D-23-21745Rapid detection and molecular epidemiology of β-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.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

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: Yes

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: Yes

Reviewer #4: 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: Yes

Reviewer #3: Yes

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: Good work done, well written and described manuscript. This study will definitely contribute to knowledge in often low resources environment. Such method as describe here can definitely be used for surveillance with lower turnaround time.

Reviewer #2: This article presents a modified a multiplex real-time (RT)-PCR that can concurrently detect β-lactamase genes (blaCTX-M, blaTEM, and blaSHV) and Enterobacteriaceae 16S ribosomal RNA and provides useful data on the molecular epidemiology of AMR gene acquisition among Enterobacteriaceae

This study has prospect to be useful to the international AMR community, however there are some major deficiencies that needs to be addressed.

- The use of the acronym “RT-PCR” can be misleading as this is commonly used to represent reverse transcription PCR. Use of “qPCR” will be more appropriate.

- The study conclusion and recommendation seemed to be detached from the main study as the ESKAPE pathogens was not the main focus of this study, but Enterobacteriaceae of which only represent 3 out of the 6 ESKAPE pathogens. Moreover, the recommendation that ESKAPE pathogens be included in surveillance is strange as they are already identified as priority pathogens for AMR surveillance (at least in humans). I suggest conclusion be based on study findings and implication

- The interpretation of sensitivity and specificity should be done with caution considering the limited number of control strains and test isolate tested in this study. Especially the limited number of negative controls used in the control strain (4) and the test strains were selected based on their being able to grow in cefotaxime supplemented plates reducing the including of non ESBL producing strains

- The authors need to provide justification for focusing on the 3 ESBL genes with references to back it up.

- Figure 1 shows 22 control strains as opposed to 21 control strain which was indicated in the method.

- Table 1 adds little or no value to this article since all the reported parameters are 100%. It can be replaced with a sentence indicating 100% sensitivity, specificity, accuracy and precision.

- There is need to include well-articulated study limitations for example limited sample size, limited number of Klebsiella and Enterobacter tested etc

- The discussion in the current state seems to be quite repetitive of study findings. The authors should focus on discussing the key findings and their implication.

Other minor revisions include.

Pg 4 Line 85- “However, methods such as whole genome sequencing…….” This sentence is unwinding and needs to be rephrased.

Page 4 Line 74- I would use “affordable” rather than “cheap.”

Pg 5 Line 98: “evaluate the ability of this assay to rapidly detect these AMR genes in Enterobacteriaceae” I am a bit reserved about the use of “rapid”. PCR based test can hardly be regarded as rapid. Maybe when compared to traditional culture method but still constrained for point-of-care.

Pg 5 Line 106-107: Kindly provide more information on the control strains used in this section for example, how many positive and negative for ESBL of choice. More information about the source of control strains APHA should be provided in this section e.g. location

Pg 7 Line 151: “R gene”??? Do you mean resistant genes. Please provide initial definition

Pg 7 Line 159: Please write WGS in full since referring to “whole genome sequence” and not “whole genome sequencing.”

Pg 8 Line 168: “forward for inclusion any variable…”. Missing the word “of”

Pg 12 Line 227: The use of “as expected” is not necessary and should be expunged. Also quite repetitive across the manuscript

Pg 13 Table 3: Any reason why state level distribution was excluded from this table?

Pg 19 Line 336-346: This whole paragraph is a revision of literature and should be taken to the introduction section. The discussion section should focus on the implication of the study findings.

Pg 21 Line 383-386: “Although in this study, the state from which samples……” Take to study limitation.

Pg 22 Line 420: The sentence seems to be truncated.

Reviewer #3: REVIEWER’S COMMENTS FOR MANUSCRIPT PONE-D-23-21745

The authors purportedly developed a “rapid” RT-PCR assay for the detection of beta-lactamases in Enterobacteriaceae isolated from animal sources and in-contact humans in southeastern part of Nigeria. This is an important area of research given the global menace of antimicrobial resistance and the challenges this has posed to human health. The authors set out with four objectives in mind viz: 1. To develop and validate a multiplex RT-PCR for detection of ESBLs and Enterobacterial 16S rRNA gene

2. Evaluate the ability of the assay to “rapidly” detect the respective genes

3. Investigate factors associated with the presence of beta-lactamase genes

4. Investigate the molecular epidemiology of AMR gene acquisition among Enterobacteriaceae isolated from the same host species and sample origin.

However, while the present manuscript addressed objectives 1, 3 and 4, I could not say the same for objective 3. I could not see anywhere in the current manuscript where the authors made an attempt to evaluate the ability of the assay they developed in the study to “rapidly” detect the target genes. Interestingly, this is a keyword in the title of the manuscript. So, an important research question remains unanswered. How rapidly did this method detected the target genes compared with other methods that have been in existence?

Other minor comments are as below

Line 32: change to cefotaxime-supplemented agar plates

Lines 67-68: Authors should please follow the accepted scientific notation for writing spp. in scientific names. This should be done throughout the manuscript

Line 130: Author should please be specific about the function of AriaMX Agilent technology as a qPCR system

Line 219: Authors should please provide a reference to back up the assertion that blaSHV is intrinsic to K. pneumoniae

Lines 336-338 is more or less a repetition of lines 338-339

Lines 419-420: Incomplete sentence

Reviewer #4: Title: Rapid detection and molecular epidemiology of β-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.

Authors: Olorunleke et al.

General Comments

The article is well written and seems to be the first to conduct in-depth studies on Enterobacteriaceae in three states in the South East of Nigeria. The report of pan resistance in some of these states is quite alarming also. However, there are a few grey areas that need to be explained.

Specific comments

Is it possible to add the cost of multiplex RT-PCR as against the cost of culture in our African setting where qPCR although on the increase in terms of usage due to covid, might not be popular for AMR?

In the supplementary Tables 2 & 6, genus and species names were not italicized.

I was looking out for the common genes that are shared in the states and which ones are found in the three states. In addition, I was looking forward to see at a glance the human isolates according to states and sites and the genes that are similar in each state since human isolates are 34 in number. This table should show any possible similarity with the animal species. I mean the AMR Table not plasmid table. I know there are comprehensive tables as supplementary files, but I would like to see at a glance a table with genes that are common and cut across the states.

Page 22 line 420, has an incomplete sentence, Lines 421 in page 22 to 423 in page 23, needs to be re-casted.

Lines 387 – 390, authors attributed highest prevalence of blaTEM 388 and blaSHV in Abia to be due to its commercial hub, while this may be partly true, it can also be seen from their results that Abia state had the lowest number of samples and so this could also be another attributable factor.

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Reviewer #1: Yes: Babafela Awosile

Reviewer #2: Yes: Emelda Chukwu

Reviewer #3: No

Reviewer #4: No

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Submitted filename: P-ONE-review-08-10-23.docx

Rapid detection and molecular epidemiology of β-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.

PONE-D-23-21745R1

Dear Dr. Olorunleke,

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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Kind regards,

Kwame Kumi Asare, Ph.D

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

Reviewer #4: 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

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: 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

Reviewer #4: 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

Reviewer #4: 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: (No Response)

Reviewer #2: (No Response)

Reviewer #4: The Authors have addressed all my queries. The genus and species names have all been italicized in Tables 2 and 6 and the incomplete sentence has been completed and clearer now. Authors have added a section on limitations of the study in response to my query while the cost between qPCR and culture has been addressed

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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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Reviewer #1: Yes: Babafela Awosile

Reviewer #2: Yes: Chukwu Emelda Eberechukwu

Reviewer #4: Yes: Prof. Stella I. Smith

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