Interspecies interaction alters the trajectory of antibiotic resistance evolution by amplifying negative fitness epistasis

Interspecies interaction alters the trajectory of antibiotic resistance evolution by amplifying negative fitness epistasis | 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 Interspecies interaction alters the trajectory of antibiotic resistance evolution by amplifying negative fitness epistasis Omar Warsi, Suraya Muzafar, Ramith Nair, Dan Andersson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6047903/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 Interspecies interactions can influence the physiology of competing species, shaping their long-term evolutionary trajectories. While the role of interspecific competition in community dynamics is well-documented, its impact on evolutionary outcomes and their underlying mechanisms is less explored. Here, we investigate how interspecies competition affects antibiotic resistance evolution in the gut pathogen Salmonella enterica within synthetic microbial communities. Specifically, we examine how the presence of an interspecific competitor, Escherichia coli—a key member of the human gut microbiota—modulates resistance evolution at low streptomycin concentrations. Our findings reveal that interspecies competition results in the selection of S. enterica mutants with higher resistance levels and fitness costs. By analyzing resistant mutants (ten each isolated from conditions with and without interspecies competition), we demonstrate that interspecific competition increases the likelihood of accumulating resistance mutations that follow a trajectory of negative fitness epistasis, requiring larger changes in resistance for smaller gains in fitness. We show that this effect is driven by the activation of starvation response pathways in S. enterica, leading to enhanced expression of the cryptic aminoglycoside transferase gene (aadA). Our study links antibiotic resistance evolution to competition-induced physiological changes, emphasizing the interplay between interspecies interaction and genetic adaptation to environmental conditions. Biological sciences/Evolution/Experimental evolution Biological sciences/Ecology/Microbial ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Rawdatacompiled.xls Raw data file SupplementaryTables250211.pdf Supplementary Tables SupplementaryFigures250211.pdf Supplementary Figures 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-6047903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":420096651,"identity":"e9d2898f-8a5e-4749-ab6f-c97d36c7f97f","order_by":0,"name":"Omar Warsi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFADZuYDBxgbJEjSwpZAqhYGHgMGxgYi1PGLHX72gbHNLl++nefjoZs7LKIZJHIMGH9U4NYiOTvNeAZjW7LlhsO8Gw7nnpHIbQBqYeY5g1uLwe0EYwbGbcwGBswgLW1QLYxt+LSkfwZqqTeQb+Z5ANfC+PMfPi05IFsOGzAc5mGAa2HgbcDnl5xihsR/xw0MDrMZgP3SxvOs4DDPMdxa+KXTNzN8OFNtIN9/+PHn3B11uf3syRsf/qjBrQUMEpA5bEB8gICGUTAKRsEoGAUEAACD8UyOWrdeSQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3175-1184","institution":"Uppsala University","correspondingAuthor":true,"prefix":"","firstName":"Omar","middleName":"","lastName":"Warsi","suffix":""},{"id":420096652,"identity":"bbcb2b52-35ce-42b7-a654-3acedc2e018a","order_by":1,"name":"Suraya Muzafar","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Suraya","middleName":"","lastName":"Muzafar","suffix":""},{"id":420096653,"identity":"481e0125-56dd-49d9-9a37-adbecebb8036","order_by":2,"name":"Ramith Nair","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Ramith","middleName":"","lastName":"Nair","suffix":""},{"id":420096654,"identity":"84303afe-bca9-45d5-a899-22c6b9aa12d5","order_by":3,"name":"Dan Andersson","email":"","orcid":"https://orcid.org/0000-0001-6640-2174","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Andersson","suffix":""}],"badges":[],"createdAt":"2025-02-17 12:27:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6047903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6047903/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77185234,"identity":"f15d2530-2ef5-451b-9a05-828821731e79","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85713,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of STR on the long-term dynamics of two-species community of E. \u0026nbsp;coli and S. enterica. A) The natural log-transformed ratio of E. coli and S. enterica in LB and LB with 1 µg/mL STR over 640 generations (equivalent to 80 cycles, 1 \u0026nbsp;cycle ~8 generations). Data are presented for 24 replicates in LB and 22 replicates in \u0026nbsp;LB with STR, and the lines within boxplots represent median values B) The natural \u0026nbsp;log-transformed ratio of E. coli and S. enterica, and C) the Shannon diversity index \u0026nbsp;for lineages after 640 generations. The line in each panel indicates the median \u0026nbsp;value. A Student’s t-test was performed to determine statistically significant differences in data presented in B) and C).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/60f439037d47401863846299.png"},{"id":77185235,"identity":"c72df540-47ef-45ab-8495-27245fba3f7c","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102340,"visible":true,"origin":"","legend":"\u003cp\u003eInterspecific competition results in the upregulation of the stringent-stress pathway and the aadA gene. A) Relative expression levels of genes iraP and aadA in monoculture and co-culture conditions, normalized to the expression of \u0026nbsp;the housekeeping gene hcaT. Expression levels in monoculture is used as the \u0026nbsp;baseline, with its value set as 1. Two biological replicates were used in each case, \u0026nbsp;with the value of each biological replicate obtained by averaging two technical \u0026nbsp;replicates. The error bar represents the standard deviation. A Student’s t-test was \u0026nbsp;used to determine statistically significant differences. B) Outcomes of competition \u0026nbsp;between an ancestral S. enterica and the DaadA S. enterica mutant (plotted as S. \u0026nbsp;enterica/DaadA S. enterica), and C) between these strains and E. coli (plotted as S. \u0026nbsp;enterica/E. coli and DaadA S. enterica/E. coli), in the presence and absence of STR. \u0026nbsp;The Y-axis represents the selection coefficient. Four replicates were used in each case, and error bars represent the standard deviation. A Student’s t-test was \u0026nbsp;performed to determine statistically significant differences.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/48830f6f54abad6f74ebacee.png"},{"id":77186378,"identity":"217edaf1-d6f8-4291-a655-c4dbd544358d","added_by":"auto","created_at":"2025-02-26 03:54:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49800,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of interspecies interaction on resistant levels and growth \u0026nbsp;characteristics of S. enterica mutants selected at sub-MIC of STR. A) \u0026nbsp;Resistance levels of mutants were quantified using Etests. Two biological replicates \u0026nbsp;were used in each case. The Y-axis represents the minimum inhibitory concentration \u0026nbsp;(MIC) for STR, and the X-axis represents the absence (monoculture) or presence \u0026nbsp;(co-culture) of interspecific competition. A Student’s t-test was performed to \u0026nbsp;determine statistically significant differences. B) The fitness costs of resistance \u0026nbsp;mutations on growth rate were determined by measuring the relative exponential \u0026nbsp;growth rate and relative stationary phase density (both normalized to the values for \u0026nbsp;the ancestral S. enterica) for 24 and 22 resistant clones isolated from monoculture \u0026nbsp;and co-culture conditions, respectively. The value of each clone is represented as an \u0026nbsp;average of three biological replicates. The line in the center represents the median \u0026nbsp;value. The Y-axis represents the relative exponential growth rate or relative \u0026nbsp;stationary phase density, and the X-axis represents the absence (monoculture) or \u0026nbsp;presence (co-culture) of interspecific competition. A Student’s t-test was performed to \u0026nbsp;determine statistically significant differences.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/56e27901ff13e4afc608a895.png"},{"id":77186658,"identity":"fead78ac-838e-4f4d-b158-fff94aa4e3cf","added_by":"auto","created_at":"2025-02-26 04:02:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":341281,"visible":true,"origin":"","legend":"\u003cp\u003eFitness-epistasis effects mediated by the resistance mutations and aadA gene. A) Outline of competition experiments performed between resistant mutants \u0026nbsp;and the DaadA S. enterica mutant, DaadA-resistant mutants and the DaadA S. \u0026nbsp;enterica mutant, and S. enterica and DaadA S. enterica mutant. In each case, the \u0026nbsp;selection coefficient is calculated for a given strain with respect to the DaadA S. enterica mutant. B) These competition experiments were used to determine the \u0026nbsp;interactions between the aadA gene and the resistance mutations enriched in co\u0002culture and monoculture conditions. The Y-axis in each case represents selection \u0026nbsp;coefficients and the X-axis represents strain IDs for isolated mutants. In each case, \u0026nbsp;the observed (grey circle) and predicted values (black circle, based on additive \u0026nbsp;effects) are plotted. A Student’s t-test, with Bonferroni’s correction applied for multiple \u0026nbsp;testing, was performed to determine statistically significant differences. C) \u0026nbsp;Prevalence of positive and negative fitness epistasis is depicted for resistant mutants \u0026nbsp;isolated from monoculture and co-culture conditions. The Y-axis represents the \u0026nbsp;difference between observed (Sres+aadA) and predicted (Sres+ SaadA) selection \u0026nbsp;coefficients. Values above 0 represent positive fitness epistasis, and values below 0 \u0026nbsp;represent negative fitness epistasis. Comparison of selection coefficients between D) \u0026nbsp;resistant mutants and E) DaadA-resistant mutants isolated from co-culture and \u0026nbsp;monoculture conditions. A nested ANOVA was performed to determine statistically \u0026nbsp;significant differences for data presented in D and\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/56c183966f297d946701e6a3.png"},{"id":77185238,"identity":"923d73b5-f0c6-4378-bbe3-11b2da302747","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84451,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-genome sequence analysis of resistant mutants selected under \u0026nbsp;sub-MIC of STR in the presence (co-culture) and absence (monoculture) of \u0026nbsp;interspecies competition A) Total number of mutations identified in 18 resistant \u0026nbsp;mutants selected in the absence and presence of interspecies competition. The \u0026nbsp;genes where mutations were observed in two or more clones are shown when B) \u0026nbsp;present in resistant clones isolated from both co-culture and monoculture conditions, \u0026nbsp;C) present only in resistant clones isolated from co-culture conditions, and D) \u0026nbsp;present only in resistant clones isolated from monoculture conditions.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/07765e3ce81cd8ed4fe1c6f2.png"},{"id":77185240,"identity":"c74744e6-d4ca-4c63-8846-87ca159d4a30","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28143,"visible":true,"origin":"","legend":"\u003cp\u003eA fitness epistasis model illustrating how interspecies interaction alters \u0026nbsp;evolutionary outcomes by modifying cellular physiology. Interaction with E. coli shifts the ancestral S. enterica along the fitness-phenotype curve due to increased \u0026nbsp;aadA gene expression (blue circles). This shift positions the ancestral strain at a \u0026nbsp;point where larger phenotypic changes (resistance in this case) are required for \u0026nbsp;small changes in fitness. The starting point can vary depending on the strength of \u0026nbsp;interspecies interaction (represented as different shades of blue). This is in contrast \u0026nbsp;to the starting point of the ancestral S. enterica strain in the absence of interspecific \u0026nbsp;competition (black circle), where small changes in resistance can result in large \u0026nbsp;changes in fitness.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/1e7126d871c6340b8294f230.png"},{"id":83979737,"identity":"4d3b7d8f-3bdc-4fa9-b02b-7ba3e4d5e7b4","added_by":"auto","created_at":"2025-06-05 09:49:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":992429,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Muzafaretal.subMIC250217.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1_covered_c60f5b54-c807-4cc1-b99e-9cdc24d02a8b.pdf"},{"id":77185236,"identity":"a72521cc-c252-4bf6-8d22-fe6c29106fc4","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":145920,"visible":true,"origin":"","legend":"Raw data file","description":"","filename":"Rawdatacompiled.xls","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/9f72af36a0d4487372ad0780.xls"},{"id":77186379,"identity":"98a4c459-ce3d-4b13-8dbe-376de0c342d5","added_by":"auto","created_at":"2025-02-26 03:54:50","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":279554,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupplementaryTables250211.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/50a3e39a798d6ef13a437a8d.pdf"},{"id":77185244,"identity":"9fcbf3ce-8b71-451f-8584-dd383b4e0598","added_by":"auto","created_at":"2025-02-26 03:46:50","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":373341,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFigures250211.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6047903/v1/e8c5eef6a73ed51de57fa145.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Interspecies interaction alters the trajectory of antibiotic resistance evolution\r\nby amplifying negative fitness epistasis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6047903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6047903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Interspecies interactions can influence the physiology of competing species, shaping\r\ntheir long-term evolutionary trajectories. While the role of interspecific competition in\r\ncommunity dynamics is well-documented, its impact on evolutionary outcomes and\r\ntheir underlying mechanisms is less explored. Here, we investigate how interspecies\r\ncompetition affects antibiotic resistance evolution in the gut pathogen Salmonella\r\nenterica within synthetic microbial communities. Specifically, we examine how the\r\npresence of an interspecific competitor, Escherichia coli—a key member of the\r\nhuman gut microbiota—modulates resistance evolution at low streptomycin\r\nconcentrations. Our findings reveal that interspecies competition results in the\r\nselection of S. enterica mutants with higher resistance levels and fitness costs. By\r\nanalyzing resistant mutants (ten each isolated from conditions with and without\r\ninterspecies competition), we demonstrate that interspecific competition increases\r\nthe likelihood of accumulating resistance mutations that follow a trajectory of\r\nnegative fitness epistasis, requiring larger changes in resistance for smaller gains in\r\nfitness. We show that this effect is driven by the activation of starvation response\r\npathways in S. enterica, leading to enhanced expression of the cryptic\r\naminoglycoside transferase gene (aadA). Our study links antibiotic resistance\r\nevolution to competition-induced physiological changes, emphasizing the interplay\r\nbetween interspecies interaction and genetic adaptation to environmental conditions.","manuscriptTitle":"Interspecies interaction alters the trajectory of antibiotic resistance evolution\nby amplifying negative fitness epistasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-26 03:46:46","doi":"10.21203/rs.3.rs-6047903/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"45e2a815-0c5c-4867-b0b5-892d7167fe07","owner":[],"postedDate":"February 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44781729,"name":"Biological sciences/Evolution/Experimental evolution"},{"id":44781730,"name":"Biological sciences/Ecology/Microbial ecology"}],"tags":[],"updatedAt":"2025-06-25T15:40:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-26 03:46:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6047903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6047903","identity":"rs-6047903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}