Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated from the intensive care unit environment in Amhara region, Ethiopia

Mizan Kindu; Feleke Moges; Degu Ashagrie; Zemene Tigabu; Baye Gelaw

doi:10.1371/journal.pone.0295286

Peer Review History

Original SubmissionJuly 4, 2023
1 Aug 2023 Decision Letter - Arghya Das, Editor PONE-D-23-20426Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated from the intensive care unit environment in Amhara Region, EthiopiaPLOS ONE Dear Dr. Kindu, 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 Sep 15 2023 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. 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Additional Editor Comments: Line 22: ‘amied’ Comment: typo Lines 23-24: “……..gram negative bacteria (K. pneumoniae, E. coli, P. aeruginosa and Acinetobacter species) and their antibiotic resistance on ICU environmental surfaces” Comment: Slightly modify the sentence “……..Gram negative bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter species) on ICU environmental surfaces and their antibiotic resistance……….” Lines 46-47: “Moreover, the majority of carbapenemase-encoding genes are carried by mobile elements, such as plasmids, which will ease the vertical transmission of resistance across other GNB in the same setting” Comment: In context of mobile genetic elements, probably the authors wanted to mean horizontal transmission of resistance determinants but wrote ‘vertical transmission’. Line 73: “High-touch surfaces are those with frequent contact with the hands, which poses the greatest risk of transmission of microorganisms” Comment: It will be better to mention that the authors targeted high-touch surfaces for environmental sampling before defining high-touch surfaces. Line 74: ‘bed masters’ Comment: Probable typo Line 82: “Single distinct colonies were isolated and purified by subculturing in fresh BAP medium to obtain pure culture isolates” Comment: Authors should explain how they have chosen single distinct colonies for sub-culture. Environmental samples are likely to yield multiple organisms. Have authors considered culturing all types of isolates? Lines 83-84: “Isolates were identified using different biochemical tests such as triple sugar iron agar, indole, motility, urease production, hydrogen sulfide production, citrate utilization, lysine decarboxylase tests, and oxidase strip tests” Comment: Mentioned tests may not be enough to correctly identify Acinetobacter spp. Please explain. Line 122: “A total of 384 environmental samples from neonates (246/384=64.1%) and other intensive care units (150/384=35.9%) were analyzed”. Comment: Please check the statistics mentioned in the line. Line 123: “Of 126(32.8%) culture positive samples, 162 critical GNB isolates……” Comment: Write “From 126(32.8%) culture positive samples, 162 critical GNB isolates….” Line 166: “Moreover, the magnitudes of MDR critical GNB were 75/95(78.9%) and 53/67(80.5%) at FHSCH, respectively” Comment: Please check the above sentence. It seems incomplete. Lines 166-168: “The neonatal intensive care unit of UoGCSH was more contaminated by MDR and CP-critical GNB than other ICUs. In contrast, at the FHCSH, the ICUs for pediatric, adult, and surgical critical care patients were more contaminated than the neonatal units”. Comment: Just based on the proportions, it will not be prudent to mention on the level of contamination of different areas without doing relevant statistical analysis. Lines 191-192: CP K. pneumoniae isolates were 25-87.5% resistant to amikacin, ciprofloxacin, chloramphenicol, piperacillin-tazobactam, gentamicin, sulfamethoxazole-trimethoprim and tetracycline, respectively. Comment: It is difficult to follow the actual magnitude of resistance to different antibiotics from the above sentences. Authors are advised to either write to refer to Table 5 or mention resistance rates of all antibiotics individually. Lines 283: “LMIC’ Comment: Please expand ‘LMIC’ where it is first used. Table 2: ‘R’ refers to resistant to classes of antibiotics as per the footnote. But in reality, authors have considered types of antibiotics for calculation of ‘R1 to R15’ not antibiotic classes. Few statistics are little bit misleading because not all drugs were tested for all organisms. Therefore, the higher values of R like ‘R15’ should be denoted as ‘-‘ not as ‘zero’. Table 3: Please delete the number of samples collected from the second row. Table 4: The column totals of few columns are not matching with what mentioned in the table. Please check Overall comment: Please mention the strengths and limitation of the present study toward the end of the Discussion. Please also mention the measures which are planned to control environmental contamination with critical Gram-negative bacteria at the study centers. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I would like to congratulate the authors for doing this valuable study. There are few clarifications needed which must be addressed in the manuscript. These are: 1. Please define MDR in the manuscript (resistance to how many antibiotics) 2. The samples have grown only gram negative organisms. No gram positive (S. aureus/ CONS) have been reported in any of the samples collected. This is in contrast to that seen in most other studies such as Darge, A., Kahsay, A.G., Hailekiros, H. et al. Bacterial contamination and antimicrobial susceptibility patterns of intensive care units medical equipment and inanimate surfaces at Ayder Comprehensive Specialized Hospital, Mekelle, Northern Ethiopia. BMC Res Notes 12, 621 (2019). 3. It would be more useful if in the discussion the authors could give a table of the observations of the other similar studies. This would help identify the local epidemiology of the pathogens based on the regions 4. It would be useful if the authors could add if any of the patients in the ICU developed infection with these organisms. A detailed profile of the infective pathogens and the infections caused in the ICU patients during this timeframe would help understand the relevance of these isolates. Reviewer #2: The manuscript reports important results on the burden of Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated in hospital setups, particularly in ICU’s. Overall, the manuscript is well written and the data flow is clear. But some minor points need to be considered: 1. The terminology 'healthcare-associated infection' instead of 'hospital acquired infection' would be better suited here. Please make similar changes wherever applicable throughout the manuscript. 2. Sampling technique: Did the number of environmental sample collection took place at random, or any criteria (like sample size calculation) for sample collection was used? Please clarify in the main manuscript. 3. The acronym "BAP" appears in line no. 82, I imagine that the authors refer to: "blood agar plates." elaborate the acronym "BAP” since I did not find the acronym in another part of the text. 4. In antibiotic susceptibility result by disk diffusion method the authors have not mentioned anything about the intermediate results in the isolates. Were they counted as resistant or susceptible? 5. sCIM method for testing carbapenemase production in A. baumannii is not recommended by standard guidelines. Hence, few isolates should also be validated for the same by some other methods. 6. There are spacing errors in the text throughout the manuscript that should be corrected. ****** 6. 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: Dr Priyam Batra Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. 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Revision 1
13 Sep 2023 Author Response Response to reviewers Response to the academic editor 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: all the formatting for headings, tables, reference brackets in the body, etc… are corrected according to the journal style and all highlighted in the manuscript 2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: ● The name of the colleague or the details of the professional service that edited your manuscript ● A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a supporting information file) ● A clean copy of the edited manuscript (uploaded as the new manuscript file) Response: Edited and attached as suggested The manuscript is edited by Solomon Taye Sima, PhD; Postdoctoral Research Scientist; Center for Immunity and Inflammation; New Jersey Medical School; Rutgers University. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: All relevant data are within the manuscript. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. 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Response: corrected as suggested and highlighted in lines 118-121 Additional Editor Comments: Response to additional editor comments Line 22: ‘amied’ Comment: typo Response: corrected and highlighted in line 22 Lines 23-24: “……..gram negative bacteria (K. pneumoniae, E. coli, P. aeruginosa and Acinetobacter species) and their antibiotic resistance on ICU environmental surfaces” Comment: Slightly modify the sentence “……..Gram negative bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter species) on ICU environmental surfaces and their antibiotic resistance……….” Response: corrected as suggested and indicated in lines 23-24 Lines 46-47: “Moreover, the majority of carbapenemase-encoding genes are carried by mobile elements, such as plasmids, which will ease the vertical transmission of resistance across other GNB in the same setting” Comment: In context of mobile genetic elements, probably the authors wanted to mean horizontal transmission of resistance determinants but wrote ‘vertical transmission’. Response: corrected and highlighted in line 47 Line 73: “High-touch surfaces are those with frequent contact with the hands, which poses the greatest risk of transmission of microorganisms” Comment: It will be better to mention that the authors targeted high-touch surfaces for environmental sampling before defining high-touch surfaces. Response: corrected as suggested Line 74: ‘bed masters’ Comment: Probable typo Response: corrected and highlighted in line 73 Line 82: “Single distinct colonies were isolated and purified by subculturing in fresh BAP medium to obtain pure culture isolates” Comment: Authors should explain how they have chosen single distinct colonies for sub-culture. Environmental samples are likely to yield multiple organisms. Have authors considered culturing all types of isolates? Response: Thank you for the comment. Single distinct colonies means these colonies preliminary differentiated based on morphology on media. Morphologically deferent means that difference in size, shape, edge, lactose fermentation, mucoid or dry etc… Then, biochemical test was done for all morphologically deferent isolates. Lines 83-84: “Isolates were identified using different biochemical tests such as triple sugar iron agar, indole, motility, urease production, hydrogen sulfide production, citrate utilization, lysine decarboxylase tests, and oxidase strip tests” Comment: Mentioned tests may not be enough to correctly identify Acinetobacter spp. Please explain. Response: I really appreciate the comment. But, through the editing process, an additional test, the catalase test, was deleted from the list of mentioned tests. In this study, most of the Acinetobacter colonies on MacConkey agar were non-lactose fermenters, with raised and smooth-edged colonies, but very few were sticky and flat. In addition to colony morphology characteristics, the above-mentioned biochemical tests, catalase and oxidase strip tests, were used to differentiate Acinetobacter spp. Line 122: “A total of 384 environmental samples from neonates (246/384=64.1%) and other intensive care units (150/384=35.9%) were analyzed”. Comment: Please check the statistics mentioned in the line. Response: I accept the commented and highlighted in line 134 Line 123: “Of 126(32.8%) culture positive samples, 162 critical GNB isolates……” Comment: Write “From 126(32.8%) culture positive samples, 162 critical GNB isolates….” Response: corrected as suggested and highlighted in lines 135-136 Line 166: “Moreover, the magnitudes of MDR critical GNB were 75/95(78.9%) and 53/67(80.5%) at FHSCH, respectively” Comment: Please check the above sentence. It seems incomplete. Response: corrected and highlighted in line 165 Lines 166-168: “The neonatal intensive care unit of UoGCSH was more contaminated by MDR and CP-critical GNB than other ICUs. In contrast, at the FHCSH, the ICUs for pediatric, adult, and surgical critical care patients were more contaminated than the neonatal units”. Comment: Just based on the proportions, it will not be prudent to mention on the level of contamination of different areas without doing relevant statistical analysis. Response: I really accept the comment. But, when we did the analysis, the contamination rate among units was not statistically significant. Therefore, I corrected the statement and indicated in lines 165-168 Lines 191-192: CP K. pneumoniae isolates were 25-87.5% resistant to amikacin, ciprofloxacin, chloramphenicol, piperacillin-tazobactam, gentamicin, sulfamethoxazole-trimethoprim and tetracycline, respectively. Comment: It is difficult to follow the actual magnitude of resistance to different antibiotics from the above sentences. Authors are advised to either write to refer to Table 5 or mention resistance rates of all antibiotics individually. Response: I accept the comment and the corrected sentence highlighted in lines 186-187 and 189-190 Lines 283: “LMIC’ Comment: Please expand ‘LMIC’ where it is first used. Response: corrected and highlighted in line 257 Table 2: ‘R’ refers to resistant to classes of antibiotics as per the footnote. But in reality, authors have considered types of antibiotics for calculation of ‘R1 to R15’ not antibiotic classes. Few statistics are little bit misleading because not all drugs were tested for all organisms. Therefore, the higher values of R like ‘R15’ should be denoted as ‘-‘ not as ‘zero’. Response: Thank you for valuable comment. The table is amended as it was mentioned in the footnote and highlighted in the table Table 3: Please delete the number of samples collected from the second row. Response: corrected as suggested Table 4: The column totals of few columns are not matching with what mentioned in the table. Please check Response: corrected and highlighted in the table Overall comment: Please mention the strengths and limitation of the present study toward the end of the Discussion. Response: corrected and highlighted in lines 262-266 Please also mention the measures which are planned to control environmental contamination with critical Gram-negative bacteria at the study centers. Response: Thank you for the comment. As researcher, we already informed concerned bodies (head of hospitals) of two sites to take actions. Review Comments to the Author Reviewer #1: 1. Please define MDR in the manuscript (resistance to how many antibiotics) Response: I accept the comment. It is added in manuscript and highlighted in line 93 2. The samples have grown only gram negative organisms. No gram positive (S. aureus/ CONS) have been reported in any of the samples collected. This is in contrast to that seen in most other studies such as Darge, A., Kahsay, A.G., Hailekiros, H. et al. Bacterial contamination and antimicrobial susceptibility patterns of intensive care units medical equipment and inanimate surfaces at Ayder Comprehensive Specialized Hospital, Mekelle, Northern Ethiopia. BMC Res Notes 12, 621 (2019). Response: I really appreciate the comment given by the reviewer; however, my objective was only on the critical GNB as mentioned in the manuscript. 3. It would be more useful if in the discussion the authors could give a table of the observations of the other similar studies. This would help identify the local epidemiology of the pathogens based on the regions Response: Thank you for the comment. But, as per my search similar studies have been already discussed. 4. It would be useful if the authors could add if any of the patients in the ICU developed infection with these organisms. A detailed profile of the infective pathogens and the infections caused in the ICU patients during this timeframe would help understand the relevance of these isolates. Response: Thank you for valuable comment. However, this paper is one part of my phd works. The final objective of this work was to see the transmission link or to detect the sources of infection for carbapenemase producing critical GNB in ICUs. Therefore, I collected environmental, clinical and rectal swab samples from ICUs. However, due to resource limitation for gene detection and whole genome sequencing other works were not yet finished. Reviewer #2: 1. The terminology 'healthcare-associated infection' instead of 'hospital acquired infection' would be better suited here. Please make similar changes wherever applicable throughout the manuscript. Response: corrected as suggested throughout the manuscript 2. Sampling technique: Did the number of environmental sample collection took place at random, or any criteria (like sample size calculation) for sample collection was used? Please clarify in the main manuscript. Response: I accept the comment. It is clarified and highlighted in line 71 3. The acronym "BAP" appears in line no. 82, I imagine that the authors refer to: "blood agar plates." elaborate the acronym "BAP” since I did not find the acronym in another part of the text. Response: I accept the comment. It is elaborated in line 80 4. In antibiotic susceptibility result by disk diffusion method the authors have not mentioned anything about the intermediate results in the isolates. Were they counted as resistant or susceptible? Response: I accept the comment. Critical GNB isolates with intermediate results detected were very few in numbers, however, for analysis counted as resistant. 5. sCIM method for testing carbapenemase production in A. baumannii is not recommended by standard guidelines. Hence, few isolates should also be validated for the same by some other methods. Response: I really appreciate comment given by the reviewer. However, due to lack of resource confirmatory molecular test was not done and this is indicated as limitation of this study 6. There are spacing errors in the text throughout the manuscript that should be corrected. Response: corrected as suggested Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0295286.r002
27 Oct 2023 Decision Letter - Ali Amanati, Editor PONE-D-23-20426R1Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated from the intensive care unit environment in Amhara Region, EthiopiaPLOS ONE Dear Dr. Kindu, 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 11 2023 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, Ali Amanati Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Editorial comments Dear authors The manuscript's overall presentation improved after amendments and is now ‎more readable‎. Although ‎your replies to the reviewers' concerns are satisfactory, ‎the manuscript ‎still needs to be improved.‎ 1. Address reviewers' comment one-by-one. Editor comments: #Authors do not explicitly mention the specific study design used in the research. #The following reviewers' comment has not been addressed correctly. Line 73: “High-touch surfaces are those with frequent contact with the hands, which poses the greatest risk of transmission of microorganisms” Comment: It will be better to mention that the authors targeted high-touch surfaces for environmental sampling before defining high-touch surfaces. #Line 120: The "Registration number" should be accessible by a valid link. #Although the information presented in Table 2 is interesting, such a classification is not familiar in the field of clinical microbiology, so I recommend that you present it based on standard interpretations instead of such an explanation: pan-sensitive, MDRو XDR, and PDR [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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: N/A Reviewer #2: No ****** 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 ****** 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 appreciate the work done by the team and recommend acceptance of the manuscript. I recommend that the authors should publish the clinical data as well Reviewer #2: Most of the issues raised were successfully addressed by the authors. A few still need to be further clarified: Line 18. Insert (HAI) after healthcare-associated infections and then the abbreviation can be used throughout the abstract. Line 66. Owing to innovation or renovation? Line 71. Better to use, samples from the hospital environment instead of environmental samples. Please do similar corrections wherever applicable Line 92. I think second generation cephalosporins are also comes under extended-spectrum cephalosporins if so then, either write first & second generation cephalosporins or remove ‘non-extended cephalosporins’ or cefuroxime might be added to the extended-spectrum cephalosporins category in the above line 89-90. Line 136. ‘162 critical GNB isolates were identified and 36 samples showed mixed growth. Add the percentage for these two results also Line 152-153. ‘The most common MDR isolate was K. pneumoniae’. Remove this line, as the next statement is enough to explain that the K. pneumoniae was the most common MDR organism. Line 177-178. I think it would be better to write, two different CP critical GNB rather than two types of CP critical GNB as its confusing. Figure is of low resolution. Please endeavour to get a high quality figure which again can be changed in size without losing quality/resolution. -Some minor grammatical as well as typographical errors are still present in the manuscript that needs to be corrected. I’m pointing a few as follows- Line 18. Insert ‘are’ between ‘infections’ and ‘common’ Line 31. Change to ‘were detected on most of the surfaces from the hospital environment’ Line 32. Insert ‘from the’ between ‘especially’ and ‘baby bed sets and incubators’. Line 34. Correct carpapenemase Line 47. Replace ‘will’ with ‘might’ ease the horizontal transmission of Line 80. use ‘into’ instead of ‘in’ before subculturing Line 99 & 104. Correct 0.5McFarl to 0.5 McFarland Line 121. Insert ‘of the’ between ‘environmental surface’ ‘ICUs’. Line 151-152. Remove ‘of’ after 128(79 %) ****** 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: Dr Priyam Batra Reviewer #2: Yes: Dr. Swati Sharma ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. 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Revision 2
13 Nov 2023 Author Response Response to reviewers Journal requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Author response: It is complete and corrected in the document Author response to academic editor Editor comments: #Authors do not explicitly mention the specific study design used in the research. Author response: Thank you for the comment. It is corrected and included in line 72. Editor comment:-# The following reviewers' comment has not been addressed correctly. Line 73: “High-touch surfaces are those with frequent contact with the hands, which poses the greatest risk of transmission of microorganisms”. Comment: It will be better to mention that the authors targeted high-touch surfaces for environmental sampling before defining high-touch surfaces. Author response: Thank you for the comment. I mention the targeted high-touch surfaces in the study before the definition. Line 72-75 of the document. Editor comment:#Line 120:The "Registration number" should be accessible by a valid link. Author response: In our institution there is no online access for registration number to the ethical letter. The institution simply gives the hardcopy letter for the researchers. This document is already attached to PLOS ONE during submission. Editor comment: #Although the information presented in Table 2 is interesting, such a classification is not familiar in the field of clinical microbiology, so I recommend that you present it based on standard interpretations instead of such an explanation: pan-sensitive, MDRو XDR, and PDR Authors Response:Thank you for the valuable comment. The focus of the current study is on MDR and carbapenemase producing isolates (already included in Table 2). On the other hand, according to the international experts to say XDR (nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories i.e., bacterial isolates remain susceptible to only one or two categories) and PDR (nonsusceptibility to all agents in all antimicrobial categories) bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories. (https://www.sciencedirect.com/science/article/pii/S1198743X14616323) The current study was not able to say PDR since the isolates were not tested to all of the antimicrobialagents within the antimicrobial categories listed by international experts such as aztreonam, fosfomycin and colistin. Moreover, the table 2 was prepared by looking recent published papers in PLOS ONE journal (https://doi.org/10.1371/journal.pone.0264818 and https://doi.org/10.1371/journal.pone.0256556.t002), used R0, R1…. Finally, if I have to remove the columns for R0-R11, I am willing to do so. Author response to the reviewers Review Comments to the Author Reviewer comment#1: I appreciate the work done by the team and recommend acceptance of the manuscript. I recommend that the authors should publish the clinical data as well Author response: Thank you for all the valuable comments. I will send clinical data manuscript for publication the as soon as possible. Reviewer comment #2: Most of the issues raised were successfully addressed by the authors. A few still need to be further clarified: Line 18. Insert (HAI) after healthcare-associated infections and then the abbreviation can be used throughout the abstract. Author response: Thank you for the comment. It is corrected as suggested and indicated in line 18,19 and 20. Reviewer comment: Line 66. Owing to innovation or renovation? Author response: It is renovation and corrected in line 66. Reviewer comment: Line 71. Better to use, samples from the hospital environment instead of environmental samples. Please do similar corrections wherever applicable Author response: It is corrected as suggested throughout the document. Reviewer comment Line 92. I think second generation cephalosporins are also comes under extended-spectrum cephalosporins if so then, either write first & second generation cephalosporins or remove ‘non-extended cephalosporins’ or cefuroxime might be added to the extended-spectrum cephalosporins category in the above line 89-90 Author response: It is corrected by removing non-extended and extended cephalosporin replacing with first and second, and third and fourth generation cephalosporins, respectively. It is highlighted in line 90, 91, 93 and in table-1 in the main document. Reviewer comment: Line 136. ‘162 critical GNB isolates were identified and 36 samples showed mixed growth. Add the percentage for these two results also Authors response: corrected as suggested and included in line138 Reviewer comment: Line 152-153. ‘The most common MDR isolate was K. pneumoniae’. Remove this line, as the next statement is enough to explain that the K. pneumoniae was the most common MDR organism. Author response: corrected as suggested Reviewer comment: Line 177-178. I think it would be better to write, two different CP critical GNB rather than two types of CP critical GNB as its confusing Authors response: corrected as suggested (line 178) Reviewer comment:-Figure is of low resolution. Please endeavour to get a high quality figure which again can be changed in size without losing quality/resolution. Author response: corrected as suggested Reviewer comment:-Some minor grammatical as well as typographical errors are still present in the manuscript that needs to be corrected. I’m pointing a few as follows- Line 18. Insert ‘are’ between ‘infections’ and ‘common’ Authors response: corrected as suggested in line 18 Reviewer comment:-Line 31. Change to ‘were detected on most of the surfaces from the hospital environment’ Authors response: corrected as suggested in line 31 and 32 Reviewer comment:-Line 32. Insert ‘from the’ between ‘especially’ and ‘baby bed sets and incubators’. Authors response: corrected as suggested in line 32 Reviewer comment:-Line 34. Correct carpapenemase Author response: corrected as suggested in line 34 Reviewer comment Line 47. Replace ‘will’ with ‘might’ ease the horizontal transmission of Author response: corrected as suggested in line 47 Reviewer comment: Line 80. use ‘into’ instead of ‘in’ before subculturing Author response: corrected as suggested in line 81 Reviewer comment:-Line 99 & 104. Correct 0.5McFarl to 0.5 McFarland Authors response: corrected as suggested in line 101 and 106 Reviewer comment:-Line 121. Insert ‘of the’ between ‘environmental surface’ ‘ICUs’. Authors response: corrected as suggested in line 123 Reviewer comment: Line 151-152. Remove ‘of’ after 128(79 %) Author response: corrected as suggested Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0295286.r004
20 Nov 2023 Decision Letter - Ali Amanati, Editor Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated from the intensive care unit environment in Amhara Region, Ethiopia PONE-D-23-20426R2 Dear Dr. Mizan Kindu, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Ali Amanati Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have satisfactorily addressed my concerns.‎ I thank the authors for ‎their very detailed ‎‎replies to my comments.‎ https://doi.org/10.1371/journal.pone.0295286.r005
Formally Accepted
23 Nov 2023 Acceptance Letter - Ali Amanati, Editor PONE-D-23-20426R2 Multidrug-resistant and carbapenemase-producing critical gram-negative bacteria isolated from the intensive care unit environment in Amhara Region, Ethiopia Dear Dr. Kindu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Ali Amanati Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0295286.r006

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