Genotyping of Acinetobacter baumannii isolates from a tertiary care hospital in Cochin, South India

  1. Vineeth Rajan
  2. Ardhra Vijayan
  3. Ambili Puthiyottu Methal
  4. Justus Lancy
  5. Gopalan Krishnan Sivaraman

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Abstract

Acinetobacter baumannii poses a significant challenge in healthcare settings across the globe, with isolates exhibiting carbapenem resistance at unprecedented rates. Here, we characterized a collection of A. baumannii isolates ( n =64) recovered during the period September 2020 – November 2021 at a teaching hospital in Cochin, South India. The species identity of the isolates was confirmed with bla OXA-51-like PCR. The major carbapenemase determinants identified were bla OXA-23-like (45, 70.3 %) and bla NDM-1 (31, 48.4 %); co-occurrence of these genes was also observed in 27 (42.2 %) isolates. Other resistance genes identified included bla PER (34, 53.1 %), aac(6')-Ib-cr (42, 65.6 %), qnrS (25, 39.1 %), sul1 (32, 50 %), sul2 (33, 51.6 %), strA/strB (36, 56.3 %), aphA1-Iab (35, 54.7 %) and tetB (32, 50 %). Mapping PCR revealed the insertion element, IS AbaI upstream of bla OXA-23-like in all isolates possessing this gene. Concerning disinfectant resistance, all isolates carried the quaternary ammonium compound (QAC) resistance gene, qacEΔ1 . Minimal inhibitory concentration (MIC) of benzalkonium chloride was high among the isolates and ranged from 8 to 128 µg ml −1 . However, low MICs were observed for chlorhexidine and triclosan, with the majority (54, 80.6 %) of isolates showing an MIC of 2 µg ml −1 for chlorhexidine and all isolates exhibiting MICs of ≤0.125 µg ml −1 for triclosan. Further, all isolates were strong biofilm-producers, as assessed by the crystal violet-based microtitre plate assay. The Apa I-pulsed-field gel electrophoresis (PFGE) revealed the multi-clonal nature of the isolates, with 16 clusters and 16 unique pulsotypes identified at a cut-off of 80 %. In short, this study provides useful data on the molecular features of A. baumannii from this region, which could be helpful to assess the local epidemiology of this pathogen and also to devise infection control strategies.

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  1. Version published to 10.1099/acmi.0.000662.v4 on Access Microbiology
  2. Access Microbiology

    I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. The work presented is clear and the arguments well formed. The manuscript is well written and contributes to the literature. Thank you for addressing all reviewers comments satisfactorily and in a timely manner.

  3. Version published to 10.1099/acmi.0.000662.v3 on Access Microbiology
  4. Access Microbiology

    Thank you for addressing all reviewers comments satisfactorily and in a timely manner. The revised manuscript was sent for review and further amendments are required before this manuscript can be accepted for publication. I agree with the reviewer regarding the biofilm formation section and think a plot showing the results for the whole isolate cohort should be included. I will be pleased to consider a revised manuscript along with your response to the reviewer.

  5. Access Microbiology

    Comments to Author

    I would like to commend the authors for their efforts in implementing my suggestions in the manuscript and respond to my comments. I really think this is a valuable contribution to the field. However, I still have concerns with the respect to the biofilm formation section. I agree that it reads much clearer now, but mentioning a range of values does not really show the results for the whole isolate cohort. I would like to insist in showing this represented in any form of plot (either a bar chart or a heat map would be appropriate).

    Please rate the manuscript for methodological rigour

    Very good

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

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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.0.000662.v2 on Access Microbiology
  7. Access Microbiology

    Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.

  8. Access Microbiology

    Comments to Author

    In Rajan, et al., the authors describe a basic genotypic and phenotypic characterisation of an A. baumannii isolate collection from a hospital in South India. Formally, the text is very well written and the descriptions, as well as the figures, give a clear picture of the findings. Please find below some points that need clarification, suggestions and minor amendments. Major comments L100-104: the authors used an automated system to determine the antibiotic susceptibility profile of the isolate collection. In all honesty, I am not familiar with this method, as might be the case of many potential readers. Could you please clarify in this paragraph the culture media used, if it follows the EUCAST or CLSI guidelines and the criteria for determining an isolate sensitive or resistant to a certain antibiotic? L139-140: different genes are targeted to find disinfectant resistance determinants using previously established protocols in E. coli. Can the authors clarify if the PCR primers used will still work efficiently to detect those genes in A. baumannii? L146-147: In my experience working with A. baumannii, it forms stronger biofilms when grown in a shaking incubator. This might just be a lab-to-lab variability of the assay, but I strongly suggest to remove the "…to favour biofilm formation" statement from this sentence, as it is not really necessary and may add some controversy. L152-158: the typical biofilm formation value from a crystal violet assay is given as the raw OD value of the well minus the value of the uninoculated well, but here the authors give a more complex normalisation criterion comparing the normalised OD value (after subtracting that of the uninoculated well) to the OD value of the uninoculated well. Can the authors provide any reference for this or justify the criteria? Also, as all the isolates are considered strong biofilm formers (L234), I see unnecessary to give a definition for non-biofilm producers, weak producers… The biofilm section needs to be simplified and a bar chart with the results described in L232-234 needs to be shown. L145, L161: For different experiment, A. baumannii is grown using different media formulations (brain heart infusion, tryptic soy, plus that used for AST (see comment above). The use of Mueller-Hinton (L134) would be justified as per the CLSI guidelines, but I struggle to understand the changes in media formulation, rather than running all experiments with one standard culture media. Could the authors justify this? Also, it is mentioned in L145 that brain heart infusion broth is supplemented with glucose. However, the majority of A. baumannii strains cannot grow on hexoses as glucose (PMID: 32989034). What would be the reason for this supplementation? Table 2: I think this table would be made more comprehensive by adding the SI numbers from Table 1 as appropriate and including the resistance levels to the different disinfectants. Please take this as a suggestion for improvement to be made at the authors' discretion, the information is correctly given as it is. L216-220: Please reword this paragraph or try representing it with a Venn diagram. It is a bit hard to picture the matching between the ISAbaI element and the blaOXA genes. L228: is the qacΔ1 gene specific to any disinfectant or is it a broad-spectrum resistance gene? L309-311: "This mainly included genes conferring resistance…". As these genes were specifically targeted, the author could only find those. Please remove "mainly". L363-367: The systems mentioned here are indeed involved in biofilm formation in A. baumannii, but except for the quorum sensing system (please include the original reference for this, not a review), the rest of the systems mentioned are not regulators. Please correct. Minor ammendments L52: Please correct the acronym for the Acinetobacter calcoaceticus-baumannii complex and use only one. L74: change "and also" for "including". L75-76, L194, L206: in these lines there is a mention to the blaOXA-51 gene as an A. baumannii marker, but no bibliographic reference is given. Please include an appropriate reference. L76-78: Please include a reference L83: change "challenge" to "challenges" L86-87: "Here, in this study…" sounds a bit redundant. Please choose "here" or "in this study". For referring to β-lactamases, please use the Greek symbol instead of the spelling "beta" throughout the manuscript. I hope the authors find these comments helpful for the improvement of the manuscript.

    Please rate the manuscript for methodological rigour

    Very good

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

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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

    This is an interesting paper investigating epidemiology of MDR A. baumannii clinical isolates. Some issues are raised to authors for addressing. -What is the study type? -What are study guidelines/checklist used in the study to ensure validity? -Authors should mention valid statement considering ethics in human research -Why authors used 64 isolates with blaOXA-51? -What is the Reference guidelines for susceptibility testing? How can authors interpret the antibiogram to individual antibiotics? -What are definitions for MDR and XDR if any? -Why authors do not confirm CR via conventional methods? -What is the ref. for MIC of disinfectants? Why authors do not use quality control in such experiment? -Discussion is too long. -Where are limitations of this study?

    Please rate the manuscript for methodological rigour

    Very good

    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?

    No: Authors should mention valid statement considering ethics in human research

  10. Version published to 10.1099/acmi.0.000662.v1 on Access Microbiology