“Investigating Colistin Resistance in Carbapenem Resistant Acinetobacter baumanii Isolates during a Neonatal Intensive Care Unit outbreak "

  1. Mubashir Raza
  2. Saadullah Jan Khan
  3. Muhammad Faisal Masood.
  4. Laila Jafri
  5. Rehana Rani
  6. Roomana Ali
  7. Sara Sadiq
  8. Nosheen Aakhtar
  9. Bushra Jamil

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Abstract

Purpose: Colistin resistant Acinetobacter baumannii poses a growing challenge in neonatal intensive care units (NICUs) due to the emergence of plasmid-mediated Mobile Colistin Resistance (mcr) genes that can rapidly spread among neonates. This pioneering study conducted a comprehensive analysis of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes in A. baumannii strains isolated from NICU. Notably, this investigation marks the first of its kind in this setting. In addition to genotypic analyses, the study incorporated phenotypic assays to identify the most effective method for detecting colistin resistance in these A. baumannii strains. Methods: In the genotypic investigation, DNA was extracted from strains of colistin-resistant A. baumannii, collected from the NICU of a local hospital of Islamabad, Pakistan. The specific set of primers for each mcr gene was used to detect their presence. Various phenotypic methods such as, disc diffusion, broth macro dilution, Minimum Inhibitory Concentration (MIC), disc elusion and colistin agar methods were performed using predetermined calculations for phenotypic studies. Results:The study confirms the distribution of the mcr-1 gene among the eight strains, which contributes 62% of the total number, whereas mcr-2, 3, 4 and 5 genes were not detected in all strains. Based on phenotypic analysis, A. baumannii had shown resistance against colistin at < 2µg/mL. In contrast, MIC varies from strain to strain. Conclusions: The horizontal transfer of the mcr-1 gene is responsible for developing resistance against colistin among A. baumannii in neonates. All phenotypic methods adopted in this study generated the same results, so the method selection depends upon individual comfort. However, we propose that the colistin agar method offers multiple advantages over other methods as it is more economical, easy to perform, multiple samples can be assessed simultaneously, and fewer calculations are involved for colistin resistance determination and finding out clinical breakpoints.

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  2. Access Microbiology

    Comments to Author

    The study aimed to determine the distribution of Colistin resistance among NICU isolates. Due to the small sample size, the results cannot be generalized. Line by line comments on the methods and results: The two methods: Disk Elution and Colistin Agar tests have not been validated or approved by the CLSI for A.baumanii. Line 168: Add CLSI reference Line 176-177: Authors still mention that they performed Colistin disc susceptibility testing not recommended per CLSI colistin discs (10µg) along with other antibiotic discs (Oxoid, UK) were placed on the inoculated Petri plates. The rational for performing this test is still unclear. Line 188: still not clear whether the positive control was used and which one, E, coli 25922 is not colistin resistant E coli BAA 3170 should rather be used as recommended by CLSI . Growth control and positive control should not be used interchangeably. GC confirms growth/viability of the test isolate so, will thus have only an isolate inoculated while a pos, control is used to test the ability of the method to detect whatever is tested, in this case, detection of colistin resistance. Lines 189-191: Please add how the samples were inoculated for reproducibility Line 243 Fig 5: Please elaborate on the interpretation of this figure. Lines 254-255: The authors are using EUCAST control. While all other testing was performed using CLSI. Can this be clarified? Lines 272-274(Fig 6) and Table 2: These results are expected and therefore do not offer/impart any knowledge. Not sure why this was performed. Would suggest omitting/removing this details on the manuscript Lines 296-297: "All the strains had growth at 16 µg/mL except strain1,7 and 9; while all strains were found sensitive to colistin levels of 32 µg/mL"- This statement is contradictory. Line 315: The sample size it too small to refer to this as prevalence, rather distribution? Line 330-331: Include reference Line 398: Can the authors elaborate why NICU is a good place for the mcr gene to spread from one type of A. baumannii to another?

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  3. Version published to 10.1099/acmi.0.000867.v3 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Overall: The study evaluates methods for determining the prevalence of Colistin Resistance in A.baumanii isolates. However procedures used are ambiguous.The study is limited by the small sample size which limits generalization of the results. The method need more expansion to assist in reproducibility. Some sentences are repeated Line by Line comments Topic: Line 1: The authors should consider writing the abbreviations CRAB and ICU in full on the title. Comment 2 of the second reviewer still not clarified. Design and methodology Aim and objectives of the study are clearly stated Lines 151-152: Specify the biochemical tests used to confirm A.baumanii identification. On what basis were isolates on MacConkey agar were considered suspicious of A.baumanii? Lines 179-180:Rephrase the sentence Line 189;218-220: what is considered a positive control in all the test methods, is actually a growth control. Please correct this in all methods and specify the control strains used. Line 225: After pouring... This statement can be expanded to give more clarity. Results Presentation of results was satisfactory with some minor comments Line 264: Does this confirm only 13 isolates were tested? Lines 270-271: I assume CARB is CRAB? Correct this. Were all Carbapenem resistant isolates considered CRAB? Please clarify the basis for this. Fig 2,6/Table 2: Any rational for testing colistin using a disc given the limitation of the method? Tables 2/3/4 : These can be summarized into 1 table Table 3: 3 isolates (6,7,&9) were considered susceptible although the picture is the same as others. Text mentioned all isolates were resistant. This needs explanation Line 330: Note that 8/13=62% not 66% Line 342: Disc diffusion method does not confirm colistin resistance Discussion Done well with references to other studies Can benefit from being summarized. There is a lot of repetitions

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Version published to 10.1099/acmi.0.000867.v2 on Access Microbiology
  8. Access Microbiology

    Comments to Author

    Dear Authors, Thank you for submitting your manuscript . After a thorough review, we have identified several areas where improvements are necessary to enhance the clarity and quality of your work. Firstly, the overall writing of the article could benefit from more refinement. In particular, the methodology section needs significant attention. Some parts should be summarized to avoid redundancy, while others require more detailed explanations to ensure comprehensibility. Regarding the visual elements in your manuscript, the photos included are not typically used in their current form. If you choose to retain these images, it would be more appropriate to present them as supplementary material. Additionally, creating a flowchart for the methodology would be highly beneficial. This visual aid would provide a clearer and more straightforward depiction of the methods used and the samples included in the study. There are also ambiguities concerning the number of samples included, the infection sites, and the hospital locations where the samples were collected. These details must be explicitly stated to provide a comprehensive understanding of your study. Furthermore, the results section requires a more detailed and descriptive approach. Please include figures and tables that quantitatively demonstrate your findings. This will significantly enhance the presentation and interpretation of your results. We believe that addressing these points will greatly improve the quality and impact of your manuscript.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  9. Access Microbiology

    Comments to Author

    The manuscript entitled 'Investigating mcr Genes in CRAB Isolates during a NICU outbreak and Advancements in Colistin Sensitivity Testing' reports the occurrence of colistin resistant A. baumannii and methods to identify them. The manuscript reports the territorial data which could be use to researchers in that region. Although the manuscript is of interest, there are certain points to be addressed which could improve the overall quality and clarity of the manuscript. Specific comments: 1. Lines 92-94: Repetitive. Seems the sentence has been copy-pasted from other sources. 2. Lines 107 - 109: 'The MDR A. baumannii in Pakistan is alarmingly high, approaching 75.6 %. Data was collected from five different hospitals, and it showed 7.3 % resistance against colistin'. These sentences are contradictory to each other. More clarity could be given. 3. Lines 110 - 111: Already explained in the introduction itself 4. Lines 150 - 151: On what rational these antibiotics have been chosen? 5. Both the broth dilution and broth elution methods are same or identical to each other. In fact the broth dilution method is more accurate in terms of antibiotic concentration than the broth elution as one could not estimate the amount of antibiotic eluted/trapped from/in the discs. What is the purpose behind this? 6. For MIC 'Minimum Inhibitory Concentration' is the correct full form whereas in the manuscript it is mentioned as 'Minimum Inhibition Concentration' at many places. 7. For the MIC and broth dilution assays, instead of taking fixed weight for preparing stock solutions of colistin, the weights corresponding to colistin (say 1 mg) should have been taken and diluted further to achieve 1 mg/ml or 0.1 mg/ml and so on. 8. Assays using colistin extracted from the discs seem inappropriate and inaccurate. 9. Lines 225-234: Repetition of information given in Table - 1. Either of them can be removed. 10. What kind of samples were collected from the hospital and how. How the samples are transported and how the bacteria were isolated. These details are missing. 11. Figure - 4 shows amplification in all the consecutive wells although the samples are not consecutive. Does this mean the samples are re-run excluding the amplification negative samples? 12. As the study claims that the mcr genes are plasmid borne, why have the authors not tried to isolate plasmids and use them for amplification instead of genomic DNA? And no pictorial evidence was given for DNA isolation to show that the method employed extracted/isolated both the genomic DNA and plasmid DNA. DNA isolation gel picture and explanation for this is required. 13. In the results, mcr gene screening has been shown before actual antibiotic screening tests but it is referred to as mcr gene amplifications were done for colistin resistant strains.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    No: Not applicable as the study did not involve any human or animal work.

  10. Version published to 10.1099/acmi.0.000867.v1 on Access Microbiology