Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates

  1. Charlotte Michel
  2. Maria De Los Angeles Argudín
  3. Magali Wautier
  4. Fedoua Echahidi
  5. Benoit Prevost
  6. Olivier Vandenberg
  7. Delphine Martiny
  8. Marie Hallin

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Abstract

Introduction. Haemophilus influenzae ( Hi ) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018–2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae , as they presented an unusual multi-resistant profile.

Methods. All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology.

Results. The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin–clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B , mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility.

Conclusion. The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae . To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

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  1. Version published to 10.1099/acmi.0.000649.v3 on Access Microbiology
  3. Access Microbiology

    Comments to Author

    The author has responded all addressed comments and review. I agreed the revision and i have no further comments and inputs.

    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: The research did not involved human or animal subject

  4. Access Microbiology

    Comments to Author

    I thank the authors for their thoughtful consideration of the comments and rewriting of the manuscript. Please change "genomic DNA" to "Genomic DNA" at line 175 in methods section of Whole genome sequencing.

    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?

    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

  5. Version published to 10.1099/acmi.0.000649.v2 on Access Microbiology
  7. Access Microbiology

    Comments to Author

    Review manuscript ACMI-D-23-00111 Title: "Multiple interspecies recombination events documented by whole genome sequencing in multidrug resistant Haemophilus influenzae clinical isolates." The manuscript entitled Multiple interspecies recombination events documented by whole genome sequencing in multidrug resistant Haemophilus influenzae clinical isolates has described the recombination of murF, murE, and Ftsl gene with Haemophilus haemolyticus and haemophilus parainfluenzae according to the sequence of genes. This manuscript will describe the possible horizontal transfer causing intra and inter-species recombination causing increasing resistance of antibiotics. The manuscript has been well-written and well described. However, in majority the authors might add some information about possible transfer mechanism or events causing this recombination instead of describing the role of genes in supporting antimicrobial resistance, and how the recombination can be occurred to the NTHi if it is from the cell capacity or the environment support. The authors might also compare the recombination events with other serotypes of Haemophilus influenzae if it is already reported that will support the cell capacity explanation related to the recombination events. In general, the manuscript data are well presented and well described. Few minor suggestions might be useful for the authors to improve the quality of the manuscript. The table and pictures and how the data are presented must follow the journal requirements that should be re-checked by the authors. After the revision, I recommend this manuscript to be published in the Access Microbiology Journal Abstract: Please describe the method the author used for the study briefly in 3 or 4 sentences, and reduce the results description. Please follow the journal guideline, should the abstract be provided in separated section (introduction, Methods, Results, conclusion) or not ? provide a concise conclusion to accommodate the methods information Belgian National Reference Centre (NRC) for Hi should be written as Belgian National Reference Centre (NRC) for Haemophilus influenzae. Introduction Please provide a clear statement in introduction, can be in last paragraph, few sentences describing why the recombination event should be described and should be known. It is unclearly stated in the introduction. Results Line 250. Figure 1 information should be accordance with the narrative, similarity of murE found in 763 and 1151 should be 90,1% or 90,2% ? Line 263. The reference (Georgiou et al. 1996) should be revised. Line 265. The reference ((Watts et al. 2021) should be revised.

    Please rate the manuscript for methodological rigour

    Good

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

    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?

    No: the manuscript did not involve the animal or human subject

  8. Access Microbiology

    Comments to Author

    Comments on manuscript Manuscript Number: ACMI-D-23-00111 Title: Multiple interspecies recombination 1 events documented by whole genome sequencing in multidrug resistant Haemophilus influenzae clinical isolates. Comments: Authors demonstrated the whole genome sequencing of clinical isolates of Haemophilus influenzae (Hi) bacterium aiming to identify the antibiotic resistance genes that are acquired through horizontal gene transfer. Mutations by comparing the USS of desired AMR genes. Studying the critical genes and mechanism of AMR acquirement is receiving wide attention nowadays, so the topic of this work is sound. Nevertheless, the data presented here is of only 3 clinical isolates although compared with a wide database of isolates. Also, for manuscript some major improvements should be made before considering for publication. 1. Page 3 Line 82- Authors may give more explanation on the significance of USS that are exchanged/acquired by Hi related to the reported AMR genes. 2. The methods section is significantly lacking references and needs to be thoroughly referenced to support their choice of antibiotics, software and methodology used in the current study. 3. Are these clinical isolates being re cultured in the laboratory for DNA extraction for WGS analysis? Explanation on the isolates culture conditions is required. 4. Similar to above comment WGS analysis is being core of the manuscript, a step wise explanation of the process is required or to be supported by the references. An explanation on percentage of error during sequence analysis compared to WT and isolated might be necessary. 5. Including a list of isolates and WT strains used in the study as a table along with the AMR genes tested of them, would be recommended in order to understand the data. 6. The phylogenetic tree in supplementary figure is missing the scale bar. 7. A clear explanation on the lowest similarity of USS of murF- murE- ftsl genes aligned with referenced isolates is necessary in the discussion. A previous work reference needs to be added in results section expanding Fig 1 on the percentage of gene similarity. 8. Dats set showing the ompP2 gene similarity comparison like murF- murE- ftsl is recommended in all figure sets and tables. 9. Did authors compare the sequence alignments with any other resistant bacteria that reside along with Hi in the human system, in order to explain the inter species resistant gene transfer? An explanation is recommended in the discussion section. Minor points: * The overall manuscript needs to be uniformly arranged especially in terms of reference style according to journals requirements. Page 11, line 263,265. * Captions for Figure 2 and supplementary figure need to be added.

    Please rate the manuscript for methodological rigour

    Good

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

    Satisfactory

    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

    Dear Authors, I completely read it. I appreciate the whole work, My suggestions and comments are mentioned below 1. Kindly check the spelling of antibiotics 2. line 128 Materials and methods title with blank citation 3. line 224 add 'for' in between 868 and NRCH180136 4. line 287 check susceptibility spelling 5. concentrate on the language editing and follow the author's guidelines provided by the journal I wish your future success

    Please rate the manuscript for methodological rigour

    Good

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

    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

  10. Version published to 10.1099/acmi.0.000649.v1 on Access Microbiology