Pneumocystis sp. is a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly

Pneumocystis sp. is a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pneumocystis sp. is a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly Fabien Magne, Susana Ruiz-Ruiz, Vicente Pérez-Brocal, Carolina A. Ponce, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4766014/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Background. Early life gut microbiota is being increasingly recognized as a major contributor to short and/or long-term human health and diseases. However, little is known about these early-life events in the human microbiome of the lower respiratory tract. Objective. To explore fungal and bacterial colonization in the lower airways over the first year of life using lung tissue from autopsied infants. Method. The fungal and bacterial communities of lung tissue samples from 54 autopsied infants were characterized by NGS of ITS and 16S rRNA genes respectively. Results. Our study highlights a high degree of inter-individual variability in both fungal and bacterial communities inhabiting the infant lung, providing insights into the diverse nature of the lower respiratory tract microbiome. Mainly transient microorganisms composed the lower respiratory tract microbiota, but some genera were identified as inhabiting the lung, indicating their potential role in lung physiology or disease. At 3-4 months of age, important dynamic changes to the microbial community were observed, which might correspond to a transitional time period in the maturation of the lung microbiome. This timeframe represents a susceptibility period for the colonization of pathogens such as Pneumocystis. The asymptomatic colonization of Pneumocystis was associated with changes in the fungal and bacterial communities. Conclusions. These findings suggest that the period of 2-4 months of age is a “critical window” early in life. Pneumocystis sp. could be a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly, and to the future health of children. Biological sciences/Microbiology/Microbial communities/Microbiome Health sciences/Medical research/Paediatric research Early life window of opportunity Lower respiratory tract Lung microbiome Pneumocystis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.xlsx Table S1: Relative abundance of fungal and bacterial genera identified in negative controls during the DNA extraction process. TableS2.xlsx Table S2: Average proportions of the dominant fungal and bacterial genera present in the lung tissue samples (n=53). Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4766014","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":337199374,"identity":"bb5a1ac8-2539-4280-b0dd-4f714325f714","order_by":0,"name":"Fabien Magne","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBAC9gYwZcPAIMHYeADIkiGohecAmEoDaWkAsXmI1XIYqIWBgUgtYocfvy6oOJ/YP7u54cAHBjsitEinmVnPOHM7ccadgw0HZzAkE9ZiL51gZszbdjtxg0Riw2EehgPE2JL+zZj33zmIlj/EackxfszbcACihYFILWXMPMeSjWfcSGw42GNAhF+ADtv8mafGTrZ/RvrDBz8q7OQIagECNgkE24AYDQwMzB+IUzcKRsEoGAUjFgAAfT49X9nieL4AAAAASUVORK5CYII=","orcid":"","institution":"University of Chile School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Fabien","middleName":"","lastName":"Magne","suffix":""},{"id":337199375,"identity":"4aa06ffa-29be-415b-842b-6333f82b3cde","order_by":1,"name":"Susana Ruiz-Ruiz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Ruiz-Ruiz","suffix":""},{"id":337199376,"identity":"521c9929-1619-4d20-a38e-2e08233c6f4c","order_by":2,"name":"Vicente Pérez-Brocal","email":"","orcid":"https://orcid.org/0000-0002-1927-9676","institution":"FISABIO","correspondingAuthor":false,"prefix":"","firstName":"Vicente","middleName":"","lastName":"Pérez-Brocal","suffix":""},{"id":337199377,"identity":"cdc37e1a-f069-4458-962d-421aa97d6548","order_by":3,"name":"Carolina A. 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Vargas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"L.","lastName":"Vargas","suffix":""},{"id":337199383,"identity":"1b762c61-7def-41e5-a056-f431545e09d3","order_by":9,"name":"Andres Moya","email":"","orcid":"","institution":"University of Valencia","correspondingAuthor":false,"prefix":"","firstName":"Andres","middleName":"","lastName":"Moya","suffix":""}],"badges":[],"createdAt":"2024-07-19 03:55:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4766014/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4766014/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64602269,"identity":"febc30ad-d503-4208-9747-2673b7f18959","added_by":"auto","created_at":"2024-09-16 12:20:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24788,"visible":true,"origin":"","legend":"\u003cp\u003eBlank controls cluster separately from the lung tissue samples. CCA plots of bacterial (A) and \u0026nbsp;fungal (B) microbiomes according to sample type. Red characters represent lung tissue samples. Blue \u0026nbsp;characters are blank controls.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/ca09183d0d6e31587fedb53d.png"},{"id":64602271,"identity":"6ed5b6c3-2495-4314-aa29-57c83c9844d8","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214876,"visible":true,"origin":"","legend":"\u003cp\u003eEarly-life lung fungal communities. (A) Relative abundance of the most abundant fungal \u0026nbsp;genera identified in lung tissue samples (present in almost 37% of samples). Sequencing of the ITS \u0026nbsp;region was carried out on 53 lung tissue samples using the Illumina MiSeq platform. A complete list of \u0026nbsp;taxa is provided in Table S1. (B) Plot of fungal genus prevalence versus relative abundance across \u0026nbsp;samples. Each point corresponds to a different or unique taxon. Red dotted line represents the 30% of \u0026nbsp;prevalence.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/f423a0ea7a02931fa3fac0a7.png"},{"id":64602277,"identity":"634d5720-3f5c-471d-ba5d-9c4aeedcb7fd","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270226,"visible":true,"origin":"","legend":"\u003cp\u003eEarly-life lung bacterial communities. (A) Relative abundance of the most abundant bacterial \u0026nbsp;genera identified in lung tissue samples (present in almost 50% of samples). Sequencing of the 16S rRNA \u0026nbsp;gene region was carried out on 53 lung tissue samples using the Illumina MiSeq platform. A complete list \u0026nbsp;of taxa is provided in Table S3. (B) Plot of bacterial genus prevalence versus relative abundance across \u0026nbsp;samples. Each point corresponds to a different or unique taxon. Red dotted line represents the 50% of \u0026nbsp;prevalence.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/f3e44bcdd1330d462c80595c.png"},{"id":64603144,"identity":"5f177441-36a3-4d82-829f-157af582ba90","added_by":"auto","created_at":"2024-09-16 12:28:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":529151,"visible":true,"origin":"","legend":"\u003cp\u003ePneumocystis load determines the fungal community assembly at 3-4 months of age. \u0026nbsp;Dynamic changes of the most abundant fungal (\u0026gt; 37% of prevalence) (A) and bacterial (\u0026gt; 50% of \u0026nbsp;prevalence) (B) genera during the six months of life. Samples are clustered by age and samples over 6 \u0026nbsp;months of age were ranged in the same age class. The changes of the most prevalent taxa were \u0026nbsp;modeled using the edgeR package in R.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/7c29ea97f51af09bc8ecc6f8.png"},{"id":64602273,"identity":"ca4a2b01-026a-4582-85b3-fb9c2a036038","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":988630,"visible":true,"origin":"","legend":"\u003cp\u003eEarly-life fungal community clusters into four distinct microbiota profiles. (A) Composition of \u0026nbsp;fungal profiles (MPs) identified by PAM clustering in the total cohort (n = 53), based on the most \u0026nbsp;abundant fungal genera (present in almost 37% of samples). (B) Cumulative distribution of samples over \u0026nbsp;the first year of life, stratified by MPs. (C) Prevalence of Pneumocystis at [0-2], [2-4] and [\u0026gt;4] months life. \u0026nbsp;(D) Relative abundance of Pneumocystis at [0-2], [2-4] and [\u0026gt;4] months life. Pneumocystis detection was \u0026nbsp;confirmed by qPCR.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/95f4508726386f3732677aba.png"},{"id":64602278,"identity":"17a17780-daa3-4f3a-ae55-ab5969263f85","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82399,"visible":true,"origin":"","legend":"\u003cp\u003eEarly-life bacterial community clusters into two distinct microbiota profiles. (A) Composition \u0026nbsp;of bacterial profiles (MPs) identified by PAM clustering in the total cohort (n = 53), based on the most \u0026nbsp;abundant bacterial genera (present in almost 50% of samples). (B) Cumulative distribution of samples \u0026nbsp;over the first year of life, stratified by MPs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/1b9ea32bee693e1ab8678ad2.png"},{"id":64602272,"identity":"f5d320fd-a260-40e1-8eae-1ab7078b46fd","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":40845,"visible":true,"origin":"","legend":"\u003cp\u003eLevel of Pneumocystis colonization affects the early-life fungal community. A) Canonical \u0026nbsp;correspondence analysis (CCA) plots of lung mycobiome according to the load of Pneumocystis. B) \u0026nbsp;Canonical correspondence analysis (CCA) plots of lung mycobiome in the three groups after removing \u0026nbsp;the Pneumocystis sequencing reads. C) LEfSe analysis of lung mycobiome composition (after release of \u0026nbsp;Pneumocystis sequencing reads). Histogram of the LDA scores reveals the most differentially abundant \u0026nbsp;taxa among different level of Pneumocystis colonization.\u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/646cb1f5f1cca55ff1df29d2.png"},{"id":64603143,"identity":"94b418e9-726e-49f7-9496-4e682b865150","added_by":"auto","created_at":"2024-09-16 12:28:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":32310,"visible":true,"origin":"","legend":"\u003cp\u003eLevel of Pneumocystis colonization moderately affects the early-life bacterial 633 community. A) \u0026nbsp;Canonical correspondence analysis (CCA) plots of lung microbiome. B) LEfSe analysis of lung microbiome \u0026nbsp;composition. Histogram of the LDA scores reveals the most differentially abundant taxa among different \u0026nbsp;level of Pneumocystis colonization.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/92b2b9f439162e1293399cec.png"},{"id":64603601,"identity":"f6e555b2-f66c-449a-b57e-353c779d6008","added_by":"auto","created_at":"2024-09-16 12:36:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040206,"visible":true,"origin":"","legend":"","description":"","filename":"Microbiomepneumo20240526CommunicationsBiology.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1_covered_c0bce73b-a1aa-461a-a920-7063d7abcd69.pdf"},{"id":64602270,"identity":"4c644138-a504-4eff-842c-6751e91d581d","added_by":"auto","created_at":"2024-09-16 12:20:27","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18792,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1: Relative abundance of fungal and bacterial genera identified in negative controls during the\u003c/p\u003e\n\u003cp\u003eDNA extraction process.\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/3f09176868a5252ca5dc6d2f.xlsx"},{"id":64602275,"identity":"f889f82a-0355-4eaa-906f-4ffa1beeba8e","added_by":"auto","created_at":"2024-09-16 12:20:28","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":72376,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2: Average proportions of the dominant fungal and bacterial genera present in the lung tissue\u003c/p\u003e\n\u003cp\u003esamples (n=53).\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4766014/v1/e515bcf479b2f6bfa6e1b941.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Pneumocystis sp. is a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Early life, window of opportunity, Lower respiratory tract, Lung, microbiome, Pneumocystis","lastPublishedDoi":"10.21203/rs.3.rs-4766014/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4766014/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground. \u003c/strong\u003eEarly life gut microbiota is being increasingly recognized as a major contributor to short \u0026nbsp;and/or long-term human health and diseases. However, little is known about these early-life events in \u0026nbsp;the human microbiome of the lower respiratory tract. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective.\u003c/strong\u003e To explore fungal and bacterial colonization in the lower airways over the first year of life \u0026nbsp;using lung tissue from autopsied infants. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod.\u003c/strong\u003e The fungal and bacterial communities of lung tissue samples from 54 autopsied infants were \u0026nbsp;characterized by NGS of ITS and 16S rRNA genes respectively. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e Our study highlights a high degree of inter-individual variability in both fungal and bacterial \u0026nbsp;communities inhabiting the infant lung, providing insights into the diverse nature of the lower \u0026nbsp;respiratory tract microbiome. Mainly transient microorganisms composed the lower respiratory tract \u0026nbsp;microbiota, but some genera were identified as inhabiting the lung, indicating their potential role in lung \u0026nbsp;physiology or disease. At 3-4 months of age, important dynamic changes to the microbial community \u0026nbsp;were observed, which might correspond to a transitional time period in the maturation of the lung \u0026nbsp;microbiome. This timeframe represents a susceptibility period for the colonization of pathogens such as \u0026nbsp;Pneumocystis. The asymptomatic colonization of Pneumocystis was associated with changes in the \u0026nbsp;fungal and bacterial communities. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e These findings suggest that the period of 2-4 months of age is a “critical window” early in \u0026nbsp;life. Pneumocystis sp. could be a pivotal ecological driver contributing to shifts in microbial equilibrium \u0026nbsp;during the early-life lower airway microbiome assembly, and to the future health of children.\u003c/p\u003e","manuscriptTitle":"Pneumocystis sp. is a pivotal ecological driver contributing to shifts in microbial equilibrium during the early-life lower airway microbiome assembly","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 12:20:23","doi":"10.21203/rs.3.rs-4766014/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aeab2b92-a750-4a7a-93c0-c245bddc342a","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":35716559,"name":"Biological sciences/Microbiology/Microbial communities/Microbiome"},{"id":35716560,"name":"Health sciences/Medical research/Paediatric research"}],"tags":[],"updatedAt":"2025-02-25T12:17:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 12:20:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4766014","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4766014","identity":"rs-4766014","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}