Volatile organic compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivity

Kim F. H. Hintzen; Lionel Blanchet; Agnieszka Smolinska; Marie-Louise Boumans; Ellen E. Stobberingh; Jan W. Dallinga; Tim Lubbers; Frederik-Jan van Schooten; Agnes W. Boots

doi:10.1371/journal.pone.0297086

Peer Review History

Original SubmissionJune 14, 2023
21 Jul 2023 Decision Letter - Awatif Abid Al-Judaibi, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-23-12035Volatile Organic Compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivityPLOS ONE Dear Dr. Hintzen, 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 04 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. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 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: The manuscripts addresses and important health issue and it is very relevant to human health. The authors have addressed the topic well and explained it in a way well understood to everyone. The tables and figures are well explained and clear. Reviewer #2: I have reviewed the manuscript titled “Volatile Organic Compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivity.”. I recommend some revisions before acceptance and publication. Some of my comments are listed below: Title: Article 'the' should be before headspace. Abstract: The author should avoid the use of i.e. Keywords: Volatile Organic Compounds and VOCs shouldn't be used as keywords at the same time. Introduction: Good, but the author should use more recent references (within the last five years). Materials and Methods: � The source of the bacterial isolates was not disclosed by the author � The author should be specific about the antibiotics used for the phenotyping test � Phenotypic and genotypic studies of bacterial isolates were not referenced � For clarity, the author should divide the number of samples in Table. 1 into different columns Results: � The result of the minimum inhibitory concentration (MIC) of the specific antibiotics were not presented � The results of the genotypic analysis were not presented � The exact definitions of "K Pneu," "P Aero," and "S Aur" should be included in a key below Table 2. 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Revision 1
29 Nov 2023 Author Response Reviewer #1 We thank the reviewer for the kind words and compliments. Reviewer #2 Thank you for the extensive reviewing and the valuable comments made to the manuscript. We have carefully considered the comments and made the adjustments as described below. 1. Title: Article ‘the’ should be before headspace Adjusted and replaced “the” 2. Abstract: The author should avoid the use of i.e. Deleted “i.e.” 3. Keywords: Volatile Organic Compounds and VOCs shouldn’t be used as keywords at the same time. Replaced the individual keywords “volatile organic compounds” and “VOCs” by “volatile organic compounds (VOCs)” 4. Introduction: Good, but the author should use more recent references (within the last five years). We understand the importance of using recent references in order to provide up-to-date information. However, findings of studies older than 5 years also make a significant contribution to the description of results already achieved and the realization of this study. Within this relatively small field of research it is difficult to match the proposed ratio of references (more than half within the past 5 years). However, we have again searched the literature and found 9 published papers within the last five years and complemented the introduction. PAGE 3, LINE 10 The most common causes of pneumonia are respiratory microorganisms including the bacteria Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumonia. REMOVED: Parkins MD, Floto RA. Emerging bacterial pathogens and changing concepts of bacterial pathogenesis in cystic fibrosis. J Cyst Fibros. 2015;14(3):293-304. NEW REFERENCE: Niederman MS, Torres A. Severe community-acquired pneumonia. Eur Respir Rev. 2022;31(166). PAGE 3, LINE 13 New reference: Torres A, Niederman MS, Chastre J, Ewig S, Fernandez-Vandellos P, Hanberger H, et al. International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: Guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the European Respiratory Society (ERS), European Society of Intensive Care Medicine (ESICM), European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and Asociación Latinoamericana del Tórax (ALAT). Eur Respir J. 2017;50(3). PAGE 3, LINE 21 Added text: Few in vitro studies already show the potential of VOCs analysis in headspace of bacterial cultures to differentiate between bacteria involved in pulmonary tract infections. New references: Filipiak W, Żuchowska K, Marszałek M, Depka D, Bogiel T, Warmuzińska N, et al. GC-MS profiling of volatile metabolites produced by Klebsiella pneumoniae. Front Mol Biosci. 2022;9:1019290. Kunze-Szikszay N, Euler M, Kuhns M, Thieß M, Groß U, Quintel M, et al. Headspace analyses using multi-capillary column-ion mobility spectrometry allow rapid pathogen differentiation in hospital-acquired pneumonia relevant bacteria. BMC Microbiol. 2021;21(1):69. Steppert I, Schönfelder J, Schultz C, Kuhlmeier D. Rapid in vitro differentiation of bacteria by ion mobility spectrometry. Appl Microbiol Biotechnol. 2021;105(10):4297-307. PAGE 3, LINE 29 Removed: Robroeks CM, van Berkel JJ, Jöbsis Q, van Schooten FJ, Dallinga JW, Wouters EF, et al. Exhaled volatile organic compounds predict exacerbations of childhood asthma in a 1-year prospective study. Eur Respir J. 2013;42(1):98-106. New references: Ibrahim W, Natarajan S, Wilde M, Cordell R, Monks PS, Greening N, et al. A systematic review of the diagnostic accuracy of volatile organic compounds in airway diseases and their relation to markers of type-2 inflammation. ERJ Open Res. 2021;7(3). Ratiu IA, Ligor T, Bocos-Bintintan V, Mayhew CA, Buszewski B. Volatile Organic Compounds in Exhaled Breath as Fingerprints of Lung Cancer, Asthma and COPD. J Clin Med. 2020;10(1). van Vliet D, Smolinska A, Jöbsis Q, Rosias P, Muris J, Dallinga J, et al. Can exhaled volatile organic compounds predict asthma exacerbations in children? Journal of Breath Research. 2017;11(1):016016. PAGE 3, LINE 33 Removed: Fowler SJ, Basanta-Sanchez M, Xu Y, Goodacre R, Dark PM. Surveillance for lower airway pathogens in mechanically ventilated patients by metabolomic analysis of exhaled breath: a case-control study. Thorax. 2015;70(4):320-5. New reference: Felton TW, Ahmed W, White IR, van Oort P, Rattray NJW, Docherty C, et al. Analysis of exhaled breath to identify critically ill patients with ventilator-associated pneumonia. Anaesthesia. 2023;78(6):712-21. 5. Materials and methods: - The source of the bacterial isolates was not disclosed by the author. - The author should be specific about the antibiotics used for phenotypic test. The source of the isolates is described in the methods on page 9 from line 8 onwards. These are the same ATCC reference strains and clinical isolates that were also used in our previous study and have added the reference on line 10. - Phenotypic and genotypic studies of bacterial isolates were not referenced. Indeed no references were provided for the phenotypic and genotypic studies. We have therefore added the following references in the methods on page 4: Line 18 Rijnders MI, Deurenberg RH, Boumans ML, Hoogkamp-Korstanje JA, Beisser PS, Stobberingh EE. Population structure of Staphylococcus aureus strains isolated from intensive care unit patients in the netherlands over an 11-year period (1996 to 2006). J Clin Microbiol. 2009;47(12):4090-5. Line 21 Stobberingh EE, Arends J, Hoogkamp-Korstanje JA, Goessens WH, Visser MR, Buiting AG, et al. Occurrence of extended-spectrum betalactamases (ESBL) in Dutch hospitals. Infection. 1999;27(6):348-54. Line 22 Brink AA, von Wintersdorff CJ, van der Donk CF, Peeters AM, Beisser PS, Stobberingh EE, et al. Development and validation of a single-tube multiple-locus variable number tandem repeat analysis for Klebsiella pneumoniae. PLoS One. 2014;9(3):e91209. - For clarity, the author should divide the number of samples in Table 1 into different columns. The table already distinguishes between the different bacteria and whether or not antibiotic resistance is present and provides the number of samples per separate group. In our opinion, adding additional columns will not contribute to a further clarification. We therefore propose to maintain the table in its current form. 6 . Results: - The result of the minimum inhibitory concentration (MIC) of the specific antibiotics were not presented This comment corresponds with comment number 5 requesting the source of the isolates and the antibiotics used. However, in order to further clarify this we have made the following adjustments in the methods: Removed: Antibiotic resistance was tested phenotypically by determining the minimum inhibitory concentration (MIC) of the specific antibiotics, e.g. methicillin and oxacillin for MRSA and MSSA and ESBL for the resistant and sensitive strains of both K. pneumonia and P. aeruginosa. New text: Antibiotic resistance was tested phenotypically by determining the minimum inhibitory concentration (MIC) of the specific antibiotics. The MRSA and MSSA strains were provided from a previous study. For ESBL cefotaxim and ceftazidim were used with a cut-off value of > 1mg/L followed by a double disk diffusion with clavulanic acid to confirm ESBL production. - The results of the genotypic analysis were not presented As discussed at comment 5, we have added references in the methods to further clarify the genotypic analysis. We therefore believe no further adjustments in the results are needed. - The exact definitions of “K Pneu,” “P Aero,” and “S Aur” should be included in a key below Table 2. We agree that for further clarification the exact definitions should be explained and have included an abbreviations section below Table 2. 7. Discussion: Tables and figures should be cited by the author when discussing. Indeed, it enhances the clarification and discussion of results when referred to the respective tables and figures. We have adjusted this in the discussion. 8. Conclusion: No conclusion was made by the author. “VOC analysis in the headspace of pathogens is a promising as rapid diagnostic tool for differentiating isolated micro-organisms independent of the applied growth media. Additionally, antibiotic-sensitive and -resistant strains of Pseudomonas aeruginosa, could be discriminated. However, validation of our results in appropriate in vivo models is critical to improve translation of breath analysis to clinical applications.” 9. References: More than half of the references should be recent (within the last five years). As already discussed at “4. Introduction”, we understand the importance of using recent references in order to provide up-to-date information. A lot of research in the field of VOCs of pathogens has been published more than 5 years ago and we believe these studies are as important to mention as well as the research that is more recent. However, we have searched the literature and already adjusted the introduction as described above. Additionally we have made the following adjustments in the discussion: PAGE 14, LINE 17 Although the different types of growth media inevitably introduced variation within the excreted VOC profiles of the bacteria, the discriminatory value of the excreted VOCs in this study is still sufficient to enable discrimination between the respective strains. Moreover, the fact that strain-specific VOCs were identified despite the use of different growth media implicates that the discriminative volatiles are directly related to the micro-organism itself. Added text: These findings are consistent with other studies in which multiple types of growth media were evaluated for the discrimination of bacteria based on VOCs in the headspace of S. aureus and P. aeruginosa. New references: Fitzgerald S, Holland L, Morrin A. An Investigation of Stability and Species and Strain-Level Specificity in Bacterial Volatilomes. Front Microbiol. 2021;12:693075. Jenkins CL, Bean HD. Influence of media on the differentiation of Staphylococcus spp. by volatile compounds. Journal of Breath Research. 2019;14(1):016007. PAGE 16, LINE 3 Although in vitro models cannot be extrapolated to in vivo situations, distinguishing various strains independent of their growth media is a step forward towards the clinical applicability of VOC analysis for quick identification of pathogens. Added text: An alternative step in improving the translation of the results from in vitro to in vivo could be the headspace analysis of bacteria in whole blood cultures as a more representative growth method to in vivo situations. One study showed that distinct VOCs could already be identified after 6 hours of incubating E coli, S aureus or P aeruginosa in whole blood samples. Another study detected bacteria specific VOCs within 8 hours of culturing whole blood samples from animals with an E coli induced sepsis. However, this only applies to more widespread infections involving bacteremia and may therefore be less suitable for early detection of infections. New references: Drees C, Vautz W, Liedtke S, Rosin C, Althoff K, Lippmann M, et al. GC-IMS headspace analyses allow early recognition of bacterial growth and rapid pathogen differentiation in standard blood cultures. Appl Microbiol Biotechnol. 2019;103(21-22):9091-101. Euler M, Perl T, Eickel I, Dudakova A, Maguilla Rosado E, Drees C, et al. Blood Culture Headspace Gas Analysis Enables Early Detection of Escherichia coli Bacteremia in an Animal Model of Sepsis. Antibiotics (Basel). 2022;11(8). Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0297086.r002
27 Dec 2023 Decision Letter - Awatif Abid Al-Judaibi, Editor Volatile Organic Compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivity PONE-D-23-12035R1 Dear Dr. Kim F.H. Hintzen, 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, Awatif Abid Al-Judaibi, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0297086.r003
Formally Accepted
15 Jan 2024 Acceptance Letter - Awatif Abid Al-Judaibi, Editor PONE-D-23-12035R1 PLOS ONE Dear Dr. Hintzen, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 Awatif Abid Al-Judaibi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0297086.r004

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