PONE-D-23-43641Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, PakistanPLOS ONE

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PLOS ONE

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Comments to the Author

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

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Reviewer #1: Partly

Reviewer #2: No

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

Reviewer #1: N/A

Reviewer #2: N/A

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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: No

Reviewer #2: No

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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: No

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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: Line 33 Carried the virulence genes bla CTXM and bla TEM - These are antibiotic resistance genes

Line 36 - 37 Multi Locus Sequence Typing (MLST) was performed on twelve (12) - why only 12? why not all 40?

Line 55, 58, 61, 64, 70 - , as documented by (delete)

Line 63 - it's important to note

Line 78 - prevalence of STEC in Pakistan has been reported to vary significantly in various food products...

Line 79 - Antimicrobial resistance is seriously challenging the global health (rephrase this sentence)

Line 82 - Enterobacteriaceae (remove italics) rephrase sentence. It's not making sense.

Line 90 - as documented (FAO 2007) Use more recent refs.

Line 91 -Therefore forty isolated of these bacteria from raw milk from... (rephrase this sentence)

Line 109 - United States of America Materials and Methods

Line 119 - Indole

Line 120 - Voges Proskauer (VP)

Line 130 - Genomic DNA was extracted using the boiling method (17)

Line 146 - to perform an in silico molecular characterization of the sequenced strains. Please indicate which strains were sequenced and on which platform. Also, be clear on the bioinformatics tools that where used for analysis.

Line 163-164 - Unnecessary statement. Delete.

Line 167 - Kirby Bauer CLSI 2013. Why would you use guidelines from over 10 years ago? Please use more recent guidelines.

Line 172 - Antibiogram of the ESBL positive E. coli isolates was performed using antimicrobials (rephrase sentence)

Line 173, 178/179 - Put antibiotic disc concentrations

Line 184 - In Fig. 4

Line 184 - The study identified an E. coli prevalence rate of 40.5% among these samples are described in Table 1, and Fig:1 (rephrase this sentence)

Fig 2 - Very poor fig which is poorly annotated.

Fig 3 - poorly annotated figure. Say more about the presented isolates

Fig 4 - Poor figure. Put a key.

Table 2 - rfb gene mentioned for the first time in this table. The table needs fixing. PCR is a molecular technique, reagents are mixed at microlitre quantities not milliliters.

Table 3 - frequency of positive genes not adding up to 40.

Table 4 - Some antibiotic spellings are wrong e.g Sulphamathoxole, Oxytetracyclin, Ceftriexone. Put antibiotic concentrations. Why use Amoxy & AMC?

Line 257 - Define multi-drug resistance.

Line 267 - However, despite this constraint, the discovery of both bla TM and bla CTXM genes parallel is remarkable (rephrase the sentence). Why is the presence of these genes remarkable?

Reviewer #2: I regret to disappoint authors, but due to numerous methodological errors and ambiguities in the experiment described in the manuscript, I do not recommend this manuscript for publication.

- the authors' goal was to isolate and identify E. coli strains producing shiga toxin, i.e. STEC, from raw milk, because these are strains that may be responsible for various serious infections in humans.

I suggest changing the style in which the introduction was written a bit and removing sentence endings such as: as observed by, as highlighted, as discussed by, according to. They are unnecessary and make understanding difficult. Moreover, authors in such cases should refer to the names of the cited authors, not reference numbers

Lines: 99-102: goal description should be simplified; the sentence is too long and incomprehensible

Line 112: the authors performed selective pre-isolation in the presence of 0.1 mg/ml cefotaxime. According to the CLSI standard, the screening confirmatory test for ESBLs in E. coli uses a 100x lower concentration of cefotaxime. Therefore, with such selective multiplication, most E. coli strains will not grow - a serious methodological error that excludes further results obtained by the authors

Line 146: authors write about molecular characterization based on sequencing. No description of the sequencing procedure (methodology, which strains?)

Line 114: how to check turbidity in a cloudy, even diluted, milk sample?

Lines 157-163: no description of MLST procedure (only genes listed)

Antimicrobial susceptibility profiles:

The procedure for assessing drug susceptibility using the disc diffusion method is a standardized procedure, similar to the screening and confirmatory test for ESBL (the authors described a completely different methodology).

- antimicrobials used only in veterinary medicine were used, for which there are no criteria in the M100S standard. For some antibiotics used in human medicine there are no criteria in this standard either. So what criteria were used to assess drug susceptibility?

Lines 211-212: whether classical serotyping or molecular methods were used to determine serotypes and serogroups? The authors mix the two versions in “materials and methods” vs “results”

Lines217-221: please provide the specific STs obtained.

Due to the presented inconsistencies, the discussion and conclusion do not seem credible.

Minor comments:

Please use susceptible instead of sensitive and animicrobials instead of antibiotics.

Lines 93-95: I suggest deleting these sentences, the authors did not perform WGS

Lines 152-155: I suggest deleting it

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Reviewer #1: Yes: Joshua Mbanga

Reviewer #2: Yes: Aneta Nowakiewicz

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PONE-D-23-43641R1Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, PakistanPLOS ONE

Dear Dr. ullah,

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.

==============================

ACADEMIC EDITOR:Please make an effort to address all comments by the reviewers as they are meant to improve the quality of your work. />==============================

Please submit your revised manuscript by Jul 29 2024 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Sherin Reda Rouby, PhD

Academic Editor

PLOS ONE

Additional Editor Comments :

Please make an effort to address all comments by the reviewer as they are meant to improve the quality of your work.

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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: (No Response)

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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: Partly

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

Reviewer #1: N/A

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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: No

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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: No

**********

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: Check paper for gramma especially abstract, but the whole paper needs to be checked and corrected.

In the initial manuscript there was mention of the ESBL genes bla CTX-M and bla-TEM but they’ve been removed in this revised version. Why? Was the detection of antibiotic resistance genes not done at all?

There was also mention of extracting DNA using the boiling method which has also been removed. Does this mean extraction was only done using the DNA kit mentioned in the revised version of the manuscript?

Always make an effort to address all comments by the reviewers as they are meant to improve the quality of your work. Wrong spellings e.g antibiotics, poorly annotated diagrams and weak grammar make the work less appealing. There’s need to improve on the technical aspects of the manuscript. The presentation of results needs improvement.

Line 28 Rephrase sentence. A total of 800 bovine raw milk samples from milk shops (500), ...

Line 30 write CT-SMA in full when writing for the first time.

Line 30 the positibve isolates were subjected to

Line 31 in silico

Line 32 rephrase sentence. 158 isolates were checked why are the percentages out of 800?

Line 34 say something about the listed serogroups

Line 37 ceftriaxone (correct spelling). Put percentages in brackets. Put a full stop after penicillin (44.5%).

Line 41 list the identified sequence types

Line 43 ...had the potential ability to transfer antibiotic resistance and virulence genes. This assertion is based on what exactly?

Line 132 the 0.5 MacFarland’s standard not 0.05

Line 138 indole, Voges Proskauer (VP)

Line 140 justify why you used the concentration of antibiotic you used. 0.1 gm of cefotaxime (CT-SMAC). Was it based on previous studies or was developed in this study.

Lines 144, 145 A subset of 40 samples matching to the same sampling years as the dairy bovine raw milk isolates was selected and subjected to sequencing. What do you mean by same sampling years?? Wasn’t selection based on the isolates that were positive in the STEC mPCR?

Lines 156 – 158 17µl Nuclease free water + 3µl DNA + 2.5µl PCR buffer + 1µl primers + 2µl dNTPs + 0.5µl Taq = 26µl in total. Please cross check and correct. Which enzyme (manufacturer, city) was used in the PCR?

Line 168 Put antibiotic disc concentrations

Line 171 and the zones... complete the sentence.

172 The CLSI document is an official document that can be cited independently. This must be done here.

Line 174 What was the selection criterion used to select the 12 isolates. This should be made clear.

Line 178 You need to put the web addresses of the tools you used for analysis e.g PATRIC (http://www.patricbrc.org). However PATRIC is now housed under BV-BRC (https://www.bv-brc.org/). Specify the specific tool/s you used for assembly, did you use SPAdes? Please provide all more detail on the bioinformatics analysis so that others may follow what you did.

Line 199 stx1, stx2, eae, ehxA. genes in table 2 and figure 2. Please correct this sentence

Line 200 and street vendors were and... Please correct this sentence

Line 202 100% (40/800) – this is not 100%

Line 204 All raw milk samples showed the presence of ... All means 100%. You can’t say all and then put a % which lower than 100%. Please correct this.

Lines 210 & 211 are the percentages susceptibility or resistance?

Line 212 ceftriaxone (16%) and were highly resistant to amoxycillin (100%)

Line 214 You’ve already mentioned the high resistance to amoxicillin or you meant to write amoxicillin and clavulanic acid (AMC)

Figure 3 – Poorly annotated. Lane 3 has pcr amplicons yet its said to be the negative control. Lanes 9 and 10 have the DNA ladder and can not be test samples. The figure casts doubts over the mPCR results presented in text.

Figure 4. Say something about the isolates in the legend. Multi locus sequence types typically are denoted by ST not SF.

Table 1 - rfb gene mentioned for the first time in this table.

Table 5 - Some antibiotic spellings are wrong e.g Sulphamathoxole, Oxytetracyclin, Ceftriexone.

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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: Yes: Joshua Mbanga

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Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, Pakistan

PONE-D-23-43641R2

Dear Dr. ullah,

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sherin Reda Rouby, PhD

Academic Editor

PLOS ONE

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