The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis

Alexandra Tikhomirova; Peter S. Zilm; Claudia Trappetti; James C. Paton; Stephen P. Kidd

doi:10.1371/journal.pone.0271912

Peer Review History

Original SubmissionApril 12, 2022
29 Jun 2022 Decision Letter - Hendrik W. van Veen, Editor PONE-D-22-10862The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis.PLOS ONE Dear Dr. Kidd, Thank you for submitting your manuscript to PLOS ONE and our apologies for the delay in the review process. Your manuscript was reviewed by two expert reviewers whose comments are included at the bottom of this message. Based on their recommendation, we feel that the manuscript has merit but does not yet fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript with minor revisions that address the points raised by the reviewers. Please submit your revised manuscript by Aug 13 2022 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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We will update your Data Availability statement to reflect the information you provide in your cover letter. 5. You should list all authors and all affiliations as per our author instructions and clearly indicate the corresponding author. 6. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: Yes ****** 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. 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 ****** 4. 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: 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: In this manuscript, Tikhomirova and colleagues have studied in vitro competitive interactions among Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, three pathogens that co exist in the human upper respiratory tract and also cause otitis media. They show decreased viability of H. influenzae in co-culture with S. pneumoniae and in triple-species cultures. They demonstrate a key role for arginine in mediating these interactions and further show that arginine stimulates production of ATP by H. influenzae in competitive cultures. Overall the work is straightforward and well done and the data support the conclusions. A limitation of the work is that a limited numbers strains was studied. Individual strains of H. influenzae show substantial variability in growth characteristics and growth requirements between strains. The present work lays a good foundation to assess a broader range of clinical isolates of all three species as different results are likely to be seen. Just the two strains of H. influenzae yielded different results in the present study. The work also establishes observations and methods that will guide work under conditions that better simulate those in the human respiratory tract. Reviewer #2: The authors present an interesting and well-conducted study describing the role of arginine for survival of Haemophilus influenzae in polymicrobial co-existence with S. pneumoniae and M. catarrhalis, relevant co-habitants of the upper respiratory tract and the inner ear, of importance for the development of otitis media. A major comment: In none of the clinical studies I have been involved in focused on bacterial colonization and co-occurrence in the upper respiratory tract I have ever observed a negative correlation between S. pneumoniae and H. influenzae (as opposed to the combination S. pneumoniae and S. aureus), in contrast we have found quite some evidence for synergy. In quite some other clinical studies this observation is also found. Why do the authors focus on the competition and observe reduction in viability when cultures of the different species are combined, while there is hardly or no evidence from human colonization studies for competition rather for inter-dependence? How do the authors explain this important difference between their in vitro results and the in vivo results described by others? Minor comments: -The sentence ‘conditions we previously identified resulted in reduced species competition’ (163-164) is not clear (despite the reference) the authors should elaborate a bit more to explain which conditions they refer to. -It is not entirely clear why two unencapsulated H. influenzae strains were used. It is important to motivate this choice. ****** 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. 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0271912.r001
Revision 1
6 Jul 2022 Author Response Tikhomirova et al Rebuttal PONE-D-22-10862 The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis. PLOS ONE Reviewer #1 In this manuscript, Tikhomirova and colleagues have studied in vitro competitive interactions among Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, three pathogens that co-exist in the human upper respiratory tract and also cause otitis media. They show decreased viability of H. influenzae in co-culture with S. pneumoniae and in triple-species cultures. They demonstrate a key role for arginine in mediating these interactions and further show that arginine stimulates production of ATP by H. influenzae in competitive cultures. Overall the work is straightforward and well done and the data support the conclusions. A limitation of the work is that a limited numbers strains was studied. Individual strains of H. influenzae show substantial variability in growth characteristics and growth requirements between strains. The present work lays a good foundation to assess a broader range of clinical isolates of all three species as different results are likely to be seen. Just the two strains of H. influenzae yielded different results in the present study. The work also establishes observations and methods that will guide work under conditions that better simulate those in the human respiratory tract. Response; The point the Reviewer is making is very valuable. This study was performed as a focussed work aiming to establish an in-depth molecular characterisation of the interactions between S. pneumoniae, H. influenzae and M. catarrhalis, and it was unfortunately not within the scope of this work to include a deeper characterisation of a larger number of strains. However, the interesting results presented provide an alluring basis for the analysis of strain variation on the outcomes of the interactions between these three species, which we are eager to pursue in the future. Reviewer #2: The authors present an interesting and well-conducted study describing the role of arginine for survival of Haemophilus influenzae in polymicrobial co-existence with S. pneumoniae and M. catarrhalis, relevant co-habitants of the upper respiratory tract and the inner ear, of importance for the development of otitis media. A major comment: In none of the clinical studies I have been involved in focused on bacterial colonization and co-occurrence in the upper respiratory tract I have ever observed a negative correlation between S. pneumoniae and H. influenzae (as opposed to the combination S. pneumoniae and S. aureus), in contrast we have found quite some evidence for synergy. In quite some other clinical studies this observation is also found. Why do the authors focus on the competition and observe reduction in viability when cultures of the different species are combined, while there is hardly or no evidence from human colonization studies for competition rather for inter-dependence? How do the authors explain this important difference between their in vitro results and the in vivo results described by others? Response: We thank the reviewer for this interesting and thoughtful comment. There certainly are numerous studies suggesting cooperation between these bacterial otopathogens. However, it is worth noting that there are indeed opposing studies. We discuss this in lines 43-57. This is not only from in vitro and animal studies (ref 14, ref 16) but some indication from clinical studies. This has now been added into the revised introduction – line 51-55. For example, in children with upper respiratory tract infections, an antagonistic relationship between S. pneumoniae and H. influenzae has been suggested (ref 15 - Littorin et al, 2021). Ref 14 -Pettigrew et al, 2008 also observed negative associations between S. pneumoniae and H. influenzae during respiratory tract infection. In addition, clinical studies have shown strain specificity to affect inter-species interactions (ref 16 - Dagan et al, 2013), as well as significant variation in inter-species associations between anatomical sites, where although a high dominance of all three species was observed in the nasopharynx, the ear displayed increased dominance and abundance of H. influenzae compared to S. pneumoniae or M. catarrhalis (Smith-Vaughan et al, 2013 – ref 5). Clearly, in a clinical setting if there is a competitive outcome (such as, as a consequence of hydrogen peroxide being generated by S. pneumoniae inhibiting growth of the other bacterial species, as has been suggested, (ref 19 Ikryannikova et al, 2017)) then there will only be isolation and identification of S. pneumoniae from that sample, seemingly as a mono-culture colonisation or infection. The other bacteria that have not been cultured, exactly because they were outcompeted. There would seem to be opportunities for either synergistic or competitive interactions. This field has been equivocal, the outcomes of the interplay between bacteria seems to be dependent on specific factors. Indeed, our previous, published studies (ref 9 and 10) demonstrated that depending on environmental conditions (pH, nutrient availability and the growth phase) there is synergistic or competitive interactions between S. pneumoniae and H. influenzae. These are the references we mention above and are now added within our manuscript: Littorin, N, et al., Decreased prevalence of Moraxella catarrhalis in addition to Streptococcus pneumoniae in children with upper respiratory tract infection after introduction of conjugated pneumococcal vaccine: a retrospective cohort study. 2021. Clinical Microbiology and Infection, 27(4):630e1-e6. Pettigrew, M, M, et al., Microbial interactions during upper respiratory tract infections. 2008. Emerging Infections diseases, 14(10):1584–1591 Dagan, R, et al., Mixed Pneumococcal–Nontypeable Haemophilus influenzae otitis media is a distinct clinical entity with unique epidemiologic characteristics and pneumococcal serotype distribution. 2013. The Journal of Infectious Diseases, 208(7): 1152–1160. Minor comments: -The sentence ‘conditions we previously identified resulted in reduced species competition’ (163-164) is not clear (despite the reference) the authors should elaborate a bit more to explain which conditions they refer to. Response: Specifically, we reported (ref 9; 10) that there can be either positive interactions or a negative, competitive interactions as a consequence of the distinct growth parameters: pH, nutrient availability or the stage of growth phase. We have added in this text to make this clear (lines 163-167). -It is not entirely clear why two unencapsulated H. influenzae strains were used. It is important to motivate this choice. Response: As noted by the Reviewers, this is an original study that creates a platform for further in-depth research. As such both the model H. influenzae laboratory strain Rd KW20 was used in (as performed in our previously published analyses, ref 9 and 10), as well as the genetically distinct, nontypeable, and clinical OM isolate 86-028NP. Nontypeable H. influenzae (NTHi) do not have a polysaccharide capsule and are a leading cause of OM. Furthermore, NTHi have specifically been associated with polymicrobial OM (Barkai, 2009), hence were highly relevant to the assessment of bacterial interactions in an OM context. Barkai G, et al., Potential contribution by nontypeable Haemophilus influenzae in protracted and recurrent acutre otitis media. 2009. The Pediatric Infectious Disease Journal, 28(6): 466-471. Attachments Attachment Submitted filename: Tikhomirova et al 2022 PONE Letter_Response to Reviewers v2.docx https://doi.org/10.1371/journal.pone.0271912.r002
11 Jul 2022 Decision Letter - Hendrik W. van Veen, Editor The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis. PONE-D-22-10862R1 Dear Prof. Kidd, 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, Hendrik W. van Veen, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271912.r003
Formally Accepted
14 Jul 2022 Acceptance Letter - Hendrik W. van Veen, Editor PONE-D-22-10862R1 The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis. Dear Dr. Kidd: 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 plosone@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 Prof. Hendrik W. van Veen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271912.r004

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