Emergence of a novel carbapenem-resistant Pseudomonas aeruginosa sequence type (ST) 5380 causing a hospital outbreak in Northern Italy, May to September 2024

Emergence of a novel carbapenem-resistant Pseudomonas aeruginosa sequence type (ST) 5380 causing a hospital outbreak in Northern Italy, May to September 2024 | 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 Short Report Emergence of a novel carbapenem-resistant Pseudomonas aeruginosa sequence type (ST) 5380 causing a hospital outbreak in Northern Italy, May to September 2024 Batignani Virginia, Francesca Saluzzo, Di Marco Federico, Moghaddasi Kiarash, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9345832/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Between May and September 2024, a carbapenem-resistant Pseudomonas aeruginosa outbreak occurred in a Northern Italian tertiary hospital's intensive care unit. Whole-genome sequencing identified nine patients infected with a novel sequence type, then named ST5380. Genomic analysis revealed homogeneous resistome and virulome, while epidemiological data supported patient-to-patient transmission. This study highlights the emergence of a previously unreported clone and emphasizes the critical role of genomic surveillance in detecting and controlling hospital outbreaks. Carbapenem-resistant Pseudomonas aeruginosa Whole-genome sequencing (WGS) Hospital acquired infections Sequence type ST5380 Genomic surveillance Figures Figure 1 Figure 2 Figure 3 Figure 4 Event description At IRCCS San Raffaele hospital (Milan, Italy), a large tertiary 1350 beds tertiary-care hospital[ 1 ], molecular surveillance of multidrug-resistant organisms has been routinely performed since 2019, including carbapenem-resistant Pseudomonas aeruginosa (CR-PA), using whole genome sequencing (WGS) on the Illumina platform[ 2 ]. Between 2021 and 2024, a progressive increase in CR-PA incidence was observed, reaching 118 isolates in 2024 (Fig. 1 ). Between May and September 2024, the largest CR-PA outbreak recorded at the hospital was identified in the intensive care unit (ICU), involving nine patients. Eight isolates were obtained from bronchoalveolar lavage (BAL) samples and one from an anal swab. Patients had a mean age of 74 years (range: 41–81), and seven were male. All patients were admitted to the ICU and had central venous and urinary catheters; eight of nine patients were mechanically ventilated. Epidemiological investigation identified probable transmission events involving five patients, supported by the shared room occupancy for at least two days. Specifically, patient PA36-24 likely transmitted the strain to patient PA39-24 who subsequently transmitted it to PA40-24. A separate transmission event was observed between patients PA26-24 and PA54-24 during their stay in another ward (Fig. 2 ). For the remaining four patients no clear epidemiological link was identified, but environmental transmission could not be excluded. All patient clinical data including length of stay and reason of admission are reported in Supplementary Table 1 . Multilocus sequence typing (MLST) revealed an unknown allelic profile. Submission to the BIGSdb database ( https://bigsdb.pasteur.fr ) confirmed a novel sequence type, designed ST5380 (Fig. 3 ). Genomic characterization Antimicrobial susceptibility testing showed that all isolates were resistant to carbapenems (imipenem and meropenem). Although no carbapenemase genes were detected, resistome analysis identified the presence of the β-lactamase genes bla OXA-486 and the chromosomal AmpC bla PDC-3, which are associated with carbapenem resistance in P. aeruginosa . In addition, all isolated carried resistance genes to aminoglycoside, chloramphenicol and fosfomycin ( aph(3')-IIb , catB7 and fosA ) ( Supplementary Table 2 ). Virulome analysis identified a mean of 221 ± 3.9 virulence-associated gene per isolate. These included type VI secretion system (T6SS) components and effectors involved in interbacterial competition and host cell apoptosis (e.g. Hcp1, Tse1, Tse2, Tse3), type IV pili genes mediation host cell adhesion, and elastases LasA and LasB secreted via type II secretion system (T2SS) pathway. All isolates also carried the type III secretion system (T3SS) effectors exoS , exoT and exoY , which are associated with host cell invasion and tissue damage. The newly identified ST5380 was genetically distant from all other CR-PA strains collected at our hospital (with minimum spanning tree nearest distance found of 2901 alleles). Similarly, phylogenetic analysis including our isolates and representative sequences from all types reported in PubMLST showed that ST5380 did not cluster to any other reported STs (Fig. 4 ; Supplementary Fig. 1 ). The nearest related lineages corresponded to rare or poorly characterized STs in global P. aeruginosa MLST data. Discussion Pseudomonas aeruginosa (PA) is a Gram-negative opportunistic pathogen identified as a cause of hospital outbreaks in several regions’ worldwide patients[ 3 – 5 ]. PA genome has been extensively studied as model for bacterial gene expression, antibiotic resistance, virulence mechanisms, quorum sensing and biofilm formation. It is characterized by a large genome size ranging from 5.5 to 7.76 Mbp with a high genetic variability, largely due to the presence of regions with highly genomic plasticity, encoding accessory genes involved in catabolic pathway and antibiotic resistance[ 6 ]. Italy is among the European countries with a high burden of PA infections in ICUs, where outbreaks are commonly driven by the clonal expansion of a limited number of high-risk STs, most notably ST111 and ST235, which are strongly associated with carbapenem resistance and environmental persistence [ 7 – 9 ]. In contrast, the outbreak described here was caused by a previously unreported ST, ST5380, highlighting that novel clones can rapidly emerge and sustain transmission in critical care settings. All patients involved in the outbreak were eventually discharged or transferred to other healthcare facilities with microbiological clearance, except for one patient who died from causes unrelated to PA infection. No further cases associated with the outbreak clone were detected following the implementation of enhanced infection prevention and control measures, suggesting effective containment. WGS demonstrated a high degree of genomic homogeneity among ST5380 isolates, supporting recent transmission events and corroborating epidemiological links between patients. Despite the absence of acquired carbapenemase genes, all isolates exhibited phenotypic resistance to carbapenems, likely mediated by the combined effect of chromosomal β-lactamases ( bla PDC-3 and bla OXA-486) and additional resistance determinants ( aph(3')-IIb , catB7 and fosA ). The conserved virulome, including type III and type VI secretion system components, indicates that ST5380 harbors traits associated with host interaction and bacterial competition that may support its circulation in ICU settings[ 10 ]. Phylogenetic analyses confirmed that ST5380 is genetically distant from both previously circulating hospital strains and known global P. aeruginosa lineages, indicating an independent emergence rather than local evolution of established high-risk clones. These findings underscore the importance of continuous genomic surveillance for the early detection of emerging lineages that may not yet be recognized as high risk but are capable of causing clinically significant outbreaks. In conclusion, this study documents the emergence of a novel carbapenem-resistant P. aeruginosa sequence type responsible for an ICU outbreak in Italy and highlights the critical role of integrated genomic and epidemiological surveillance in identifying, characterizing, and controlling hospital outbreaks caused by both established and emerging clones. Abbreviations CR-PA carbapenem-resistant Pseudomonas aeruginosa WGS Whole Genome Sequencing ICU Intensive Care Unit BAL bronchoalveolar lavage MLST Multilocus sequence typing ST sequence type Declarations Ethics approval and consent to participate Ethical approval was not necessary as data were collected during routine surveillance activities Consent for publication Not applicable Availability of data and materials The sequences of the isolates can be accessed on NCBI repository with bioproject number PRJNA1422792 Competing interests The authors declare that they have no competing interests Funding Not applicable Authors' contributions Conceptualization: V.B., F.S., D.M.C. Methodology: V.B, F.S., J.V Formal analysis: V.B., K.M., F.DM Investigation: V.B., F.S., M.M., A.B., P.N. Resources: V.B., J.V. Data curation: V.B., F.DM, A.B., P.N., M.M. Writing - Original Draft: V.B. Writing - Review & Editing: V.B., F.S., F.DM., M.K., P.N., A.B., J.V., G.M., M.M., D.M.C. Supervision: F.S., D.M.C. Project Administration: F.S., D.M.C. Acknowledgements Not applicable References San Raffaele Hospital Website ( https://www.hsr.it/ ). Badalucco Ciotta F, Saluzzo F, Pescò A, Di Marco F, Carletti S, Ripa M, et al. Genetic characterization of Klebsiella pneumoniae carbapenemase -producing Klebsiella pneumoniae isolates with different susceptibility to ceftazidime/avibactam in patients with blood-stream infections. Clinical Microbiology and Infection. Volume 29. Elsevier B.V.; 2023. pp. 1610–2. Gravningen K, Kacelnik O, Lingaas E, Pedersen T, Iversen BG. Pseudomonas aeruginosa countrywide outbreak in hospitals linked to pre-moistened non-sterile washcloths, Norway, October 2021 to April 2022. Eurosurveillance. 2022;27(18). Iversen BG, Eriksen HM, Boø G, Hagestad K, Jacobsen T, Engeset E et al. Pseudomonas aeruginosa contamination of mouth swabs during production causing a major outbreak. Ann Clin Microbiol Antimicrob. 2007;6. Yetiş Ö, Ali S, Coen P, Wilson P. Clinical risk factors associated with nosocomial Pseudomonas aeruginosa bacteraemia in patients within a tertiary care healthcare setting – a case control study. Antimicrob Resist Infect Control. 2025;14(1). Ambreetha S, Zincke D, Balachandar D, Mathee K. Genomic and metabolic versatility of Pseudomonas aeruginosa contributes to its inter-kingdom transmission and survival. Volume 73. Journal of Medical Microbiology. Microbiology Society; 2024. Ecdc. Healthcare-associated infections acquired in intensive care units - Annual epidemiological report 2021. Rath A, Kieninger B, Fritsch J, Caplunik-Pratsch A, Blaas S, Ochmann M, et al. Whole-genome sequencing reveals two prolonged simultaneous outbreaks involving Pseudomonas aeruginosa high-risk strains ST111 and ST235 with resistance to quaternary ammonium compounds. J Hosp Infect. 2024;145:155–64. Debast SB, van den Bos-Kromhout MI, de Vries-van Rossum SV, Abma-Blatter SEM, Notermans DW, Kluytmans JAJW, et al. Aquatic reservoir-associated outbreaks of multi-drug-resistant bacteria: a hospital outbreak report of Pseudomonas aeruginosa in perspective from the Dutch national surveillance databases. J Hosp Infect. 2025;162:310–8. Liao C, Huang X, Wang Q, Yao D, Lu W. Virulence Factors of Pseudomonas Aeruginosa and Antivirulence Strategies to Combat Its Drug Resistance. Frontiers in Cellular and Infection Microbiology. Volume 12. Frontiers Media S.A.; 2022. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTablesandFigure.docx Cite Share Download PDF Status: Posted 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-9345832","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":623441212,"identity":"22b709ca-ee44-42b9-8af2-33605dfe31d1","order_by":0,"name":"Batignani Virginia","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Batignani","middleName":"","lastName":"Virginia","suffix":""},{"id":623441213,"identity":"75a8ccc9-938e-4929-96af-b50db5b0bcaf","order_by":1,"name":"Francesca Saluzzo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFAC5gYwxd4AE2DvAZESeLQwQtTyHIAJ8JwBa8GjB0OLRA4DXmvk2w+2Sf74Y8PAI3342YMfFXXy/DPfHv5cwWBRh0uLwZnENgkJnjQGHr40c8OeM4cNZ9zOS5M8g8dhBgxALQYShxnseRjMpBnbDiQw3M4xAzoWtxb5/odtEgkGhxl4eNi/STP+q0uQv3nG+CM+LQw3gLYcSABp4QHa0sCcYHCDx0ASnxaDGw+bLRsOpAF18JRJ9hw7bLjxTI6ZZIOBBFAbLoclH7wJDDE5oMO2SfyoqZOXOw5yWEUdP06HQQEPuu2ENIyCUTAKRsEowAcAxbdL2zzwjh0AAAAASUVORK5CYII=","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":true,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Saluzzo","suffix":""},{"id":623441214,"identity":"ef32971d-63c7-49d6-b161-fe205e4b53d3","order_by":2,"name":"Di Marco Federico","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"Marco","lastName":"Federico","suffix":""},{"id":623441218,"identity":"382b8916-b300-440a-a30b-779d8b6d9fd7","order_by":3,"name":"Moghaddasi Kiarash","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Moghaddasi","middleName":"","lastName":"Kiarash","suffix":""},{"id":623441220,"identity":"e1d47d46-1922-451f-a336-8c48d8a1cf3e","order_by":4,"name":"Nizzero Paola","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Nizzero","middleName":"","lastName":"Paola","suffix":""},{"id":623441221,"identity":"d9939c5e-6da4-46db-929d-ef52694d527d","order_by":5,"name":"Biancardi Anna","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Biancardi","middleName":"","lastName":"Anna","suffix":""},{"id":623441222,"identity":"cd8eaced-d9f4-4c9e-9ad3-268641d5bffc","order_by":6,"name":"Vitali Jessica","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Vitali","middleName":"","lastName":"Jessica","suffix":""},{"id":623441223,"identity":"444f77f9-e363-458c-8534-e7d30875ab83","order_by":7,"name":"Monti Giacomo","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Monti","middleName":"","lastName":"Giacomo","suffix":""},{"id":623441224,"identity":"06a080ac-1d1f-49a7-bc57-16f598a8c757","order_by":8,"name":"Moro Matteo","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Moro","middleName":"","lastName":"Matteo","suffix":""},{"id":623441225,"identity":"434452aa-dc7b-4b26-971f-04366cfe10ff","order_by":9,"name":"Cirillo Daniela Maria","email":"","orcid":"","institution":"IRCCS Ospedale San Raffaele","correspondingAuthor":false,"prefix":"","firstName":"Cirillo","middleName":"Daniela","lastName":"Maria","suffix":""}],"badges":[],"createdAt":"2026-04-07 13:27:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9345832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9345832/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107484949,"identity":"c24d2b42-7dd3-466e-9cd0-9526822b1c27","added_by":"auto","created_at":"2026-04-22 02:33:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30738,"visible":true,"origin":"","legend":"\u003cp\u003eAnnual number of carbapenem-resistant P. aeruginosa isolates, IRCCS San Raffaele Hospital, 2021–2024.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/01e9b15d80300faec7bc6926.png"},{"id":107257744,"identity":"7b35fd10-a2ac-4096-9b24-8d6bf3728a54","added_by":"auto","created_at":"2026-04-19 12:33:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83725,"visible":true,"origin":"","legend":"\u003cp\u003eTracking of patients in hospital wards during the outbreak period. The lower portion of the figure illustrates the daily hospital stay for each patient in the different wards (W) and intensive care unit (ICU), whereas the upper portion indicates the timing of isolate collection.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/719e9d4b61ca54c260eac4eb.png"},{"id":107484719,"identity":"c63b6330-b681-45ac-aaf1-821fd94e3af1","added_by":"auto","created_at":"2026-04-22 02:32:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37429,"visible":true,"origin":"","legend":"\u003cp\u003eCore genome phylogenetic tree of ST5380 isolates generated using Ridom SeqSphere+ version 10.0.5, annotated MLST allelic profile.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/9606f201397c4aa1e47cce9e.png"},{"id":107483298,"identity":"66cdca4f-236f-4c96-a2c5-469357f6770d","added_by":"auto","created_at":"2026-04-22 02:27:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":232102,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree comparing outbreak isolates (pink) with global sequences belonging to all reported STs in PubMLST database with known ST indicated in the inner circle, followed by the country of isolation and year of collection.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/0d3d911ad4ac6fb9a7c30a4e.png"},{"id":109067450,"identity":"f1fce520-0d57-4b71-8da0-9ea0a4c00af9","added_by":"auto","created_at":"2026-05-12 09:52:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":413275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/7d744b2c-282f-4170-b28e-eca2460bc3fd.pdf"},{"id":107257742,"identity":"6f3859fe-fe4d-4633-9ddb-34807c5fc97f","added_by":"auto","created_at":"2026-04-19 12:33:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":278052,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesandFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-9345832/v1/659db60f77fca671ce5ccf76.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emergence of a novel carbapenem-resistant Pseudomonas aeruginosa sequence type (ST) 5380 causing a hospital outbreak in Northern Italy, May to September 2024","fulltext":[{"header":"Event description","content":"\u003cp\u003eAt IRCCS San Raffaele hospital (Milan, Italy), a large tertiary 1350 beds tertiary-care hospital[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], molecular surveillance of multidrug-resistant organisms has been routinely performed since 2019, including carbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (CR-PA), using whole genome sequencing (WGS) on the Illumina platform[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBetween 2021 and 2024, a progressive increase in CR-PA incidence was observed, reaching 118 isolates in 2024 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBetween May and September 2024, the largest CR-PA outbreak recorded at the hospital was identified in the intensive care unit (ICU), involving nine patients. Eight isolates were obtained from bronchoalveolar lavage (BAL) samples and one from an anal swab. Patients had a mean age of 74 years (range: 41\u0026ndash;81), and seven were male. All patients were admitted to the ICU and had central venous and urinary catheters; eight of nine patients were mechanically ventilated. Epidemiological investigation identified probable transmission events involving five patients, supported by the shared room occupancy for at least two days. Specifically, patient PA36-24 likely transmitted the strain to patient PA39-24 who subsequently transmitted it to PA40-24. A separate transmission event was observed between patients PA26-24 and PA54-24 during their stay in another ward (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For the remaining four patients no clear epidemiological link was identified, but environmental transmission could not be excluded. All patient clinical data including length of stay and reason of admission are reported in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMultilocus sequence typing (MLST) revealed an unknown allelic profile. Submission to the BIGSdb database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigsdb.pasteur.fr\u003c/span\u003e\u003cspan address=\"https://bigsdb.pasteur.fr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) confirmed a novel sequence type, designed ST5380 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e "},{"header":"Genomic characterization","content":"\u003cp\u003eAntimicrobial susceptibility testing showed that all isolates were resistant to carbapenems (imipenem and meropenem). Although no carbapenemase genes were detected, resistome analysis identified the presence of the β-lactamase genes \u003csub\u003ebla\u003c/sub\u003eOXA-486 and the chromosomal AmpC \u003csub\u003ebla\u003c/sub\u003ePDC-3, which are associated with carbapenem resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e. In addition, all isolated carried resistance genes to aminoglycoside, chloramphenicol and fosfomycin (\u003cem\u003eaph(3')-IIb\u003c/em\u003e, catB7 and \u003cem\u003efosA\u003c/em\u003e) (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e). Virulome analysis identified a mean of 221\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 virulence-associated gene per isolate. These included type VI secretion system (T6SS) components and effectors involved in interbacterial competition and host cell apoptosis (e.g. Hcp1, Tse1, Tse2, Tse3), type IV pili genes mediation host cell adhesion, and elastases \u003cem\u003eLasA\u003c/em\u003e and \u003cem\u003eLasB\u003c/em\u003e secreted via type II secretion system (T2SS) pathway. All isolates also carried the type III secretion system (T3SS) effectors \u003cem\u003eexoS\u003c/em\u003e, \u003cem\u003eexoT\u003c/em\u003e and \u003cem\u003eexoY\u003c/em\u003e, which are associated with host cell invasion and tissue damage.\u003c/p\u003e \u003cp\u003eThe newly identified ST5380 was genetically distant from all other CR-PA strains collected at our hospital (with minimum spanning tree nearest distance found of 2901 alleles). Similarly, phylogenetic analysis including our isolates and representative sequences from all types reported in PubMLST showed that ST5380 did not cluster to any other reported STs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). The nearest related lineages corresponded to rare or poorly characterized STs in global \u003cem\u003eP. aeruginosa\u003c/em\u003e MLST data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PA) is a Gram-negative opportunistic pathogen identified as a cause of hospital outbreaks in several regions\u0026rsquo; worldwide patients[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PA genome has been extensively studied as model for bacterial gene expression, antibiotic resistance, virulence mechanisms, quorum sensing and biofilm formation. It is characterized by a large genome size ranging from 5.5 to 7.76 Mbp with a high genetic variability, largely due to the presence of regions with highly genomic plasticity, encoding accessory genes involved in catabolic pathway and antibiotic resistance[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eItaly is among the European countries with a high burden of PA infections in ICUs, where outbreaks are commonly driven by the clonal expansion of a limited number of high-risk STs, most notably ST111 and ST235, which are strongly associated with carbapenem resistance and environmental persistence [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, the outbreak described here was caused by a previously unreported ST, ST5380, highlighting that novel clones can rapidly emerge and sustain transmission in critical care settings.\u003c/p\u003e \u003cp\u003eAll patients involved in the outbreak were eventually discharged or transferred to other healthcare facilities with microbiological clearance, except for one patient who died from causes unrelated to PA infection. No further cases associated with the outbreak clone were detected following the implementation of enhanced infection prevention and control measures, suggesting effective containment.\u003c/p\u003e \u003cp\u003eWGS demonstrated a high degree of genomic homogeneity among ST5380 isolates, supporting recent transmission events and corroborating epidemiological links between patients. Despite the absence of acquired carbapenemase genes, all isolates exhibited phenotypic resistance to carbapenems, likely mediated by the combined effect of chromosomal β-lactamases (\u003csub\u003ebla\u003c/sub\u003ePDC-3 and \u003csub\u003ebla\u003c/sub\u003eOXA-486) and additional resistance determinants (\u003cem\u003eaph(3')-IIb\u003c/em\u003e, catB7 and \u003cem\u003efosA\u003c/em\u003e). The conserved virulome, including type III and type VI secretion system components, indicates that ST5380 harbors traits associated with host interaction and bacterial competition that may support its circulation in ICU settings[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhylogenetic analyses confirmed that ST5380 is genetically distant from both previously circulating hospital strains and known global \u003cem\u003eP. aeruginosa\u003c/em\u003e lineages, indicating an independent emergence rather than local evolution of established high-risk clones. These findings underscore the importance of continuous genomic surveillance for the early detection of emerging lineages that may not yet be recognized as high risk but are capable of causing clinically significant outbreaks.\u003c/p\u003e \u003cp\u003eIn conclusion, this study documents the emergence of a novel carbapenem-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e sequence type responsible for an ICU outbreak in Italy and highlights the critical role of integrated genomic and epidemiological surveillance in identifying, characterizing, and controlling hospital outbreaks caused by both established and emerging clones.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR-PA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole Genome Sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntensive Care Unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebronchoalveolar lavage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultilocus sequence typing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esequence type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was not necessary as data were collected during routine surveillance activities\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences of the isolates can be accessed on NCBI repository with bioproject number PRJNA1422792\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: V.B., F.S., D.M.C.\u003c/p\u003e\n\u003cp\u003eMethodology: V.B, F.S., J.V\u003c/p\u003e\n\u003cp\u003eFormal analysis: V.B., K.M., F.DM\u003c/p\u003e\n\u003cp\u003eInvestigation: V.B., F.S., M.M., A.B., P.N.\u003c/p\u003e\n\u003cp\u003eResources: V.B., J.V.\u003c/p\u003e\n\u003cp\u003eData curation: V.B., F.DM, A.B., P.N., M.M.\u003c/p\u003e\n\u003cp\u003eWriting - Original Draft: V.B.\u003c/p\u003e\n\u003cp\u003eWriting - Review \u0026amp; Editing: V.B., F.S., F.DM., M.K., P.N., A.B., J.V., G.M., M.M., D.M.C.\u003c/p\u003e\n\u003cp\u003eSupervision: F.S., D.M.C.\u003c/p\u003e\n\u003cp\u003eProject Administration: F.S., D.M.C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSan Raffaele Hospital Website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.hsr.it/\u003c/span\u003e\u003cspan address=\"https://www.hsr.it/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadalucco Ciotta F, Saluzzo F, Pesc\u0026ograve; A, Di Marco F, Carletti S, Ripa M, et al. Genetic characterization of Klebsiella pneumoniae carbapenemase -producing Klebsiella pneumoniae isolates with different susceptibility to ceftazidime/avibactam in patients with blood-stream infections. Clinical Microbiology and Infection. Volume 29. Elsevier B.V.; 2023. pp. 1610\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGravningen K, Kacelnik O, Lingaas E, Pedersen T, Iversen BG. Pseudomonas aeruginosa countrywide outbreak in hospitals linked to pre-moistened non-sterile washcloths, Norway, October 2021 to April 2022. Eurosurveillance. 2022;27(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIversen BG, Eriksen HM, Bo\u0026oslash; G, Hagestad K, Jacobsen T, Engeset E et al. Pseudomonas aeruginosa contamination of mouth swabs during production causing a major outbreak. Ann Clin Microbiol Antimicrob. 2007;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYetiş \u0026Ouml;, Ali S, Coen P, Wilson P. Clinical risk factors associated with nosocomial Pseudomonas aeruginosa bacteraemia in patients within a tertiary care healthcare setting \u0026ndash; a case control study. Antimicrob Resist Infect Control. 2025;14(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbreetha S, Zincke D, Balachandar D, Mathee K. Genomic and metabolic versatility of Pseudomonas aeruginosa contributes to its inter-kingdom transmission and survival. Volume 73. Journal of Medical Microbiology. Microbiology Society; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEcdc. Healthcare-associated infections acquired in intensive care units - Annual epidemiological report 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRath A, Kieninger B, Fritsch J, Caplunik-Pratsch A, Blaas S, Ochmann M, et al. Whole-genome sequencing reveals two prolonged simultaneous outbreaks involving Pseudomonas aeruginosa high-risk strains ST111 and ST235 with resistance to quaternary ammonium compounds. J Hosp Infect. 2024;145:155\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebast SB, van den Bos-Kromhout MI, de Vries-van Rossum SV, Abma-Blatter SEM, Notermans DW, Kluytmans JAJW, et al. Aquatic reservoir-associated outbreaks of multi-drug-resistant bacteria: a hospital outbreak report of Pseudomonas aeruginosa in perspective from the Dutch national surveillance databases. J Hosp Infect. 2025;162:310\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao C, Huang X, Wang Q, Yao D, Lu W. Virulence Factors of Pseudomonas Aeruginosa and Antivirulence Strategies to Combat Its Drug Resistance. Frontiers in Cellular and Infection Microbiology. Volume 12. Frontiers Media S.A.; 2022.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbapenem-resistant Pseudomonas aeruginosa, Whole-genome sequencing (WGS), Hospital acquired infections, Sequence type ST5380, Genomic surveillance","lastPublishedDoi":"10.21203/rs.3.rs-9345832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9345832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBetween May and September 2024, a carbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e outbreak occurred in a Northern Italian tertiary hospital's intensive care unit. Whole-genome sequencing identified nine patients infected with a novel sequence type, then named ST5380. Genomic analysis revealed homogeneous resistome and virulome, while epidemiological data supported patient-to-patient transmission. This study highlights the emergence of a previously unreported clone and emphasizes the critical role of genomic surveillance in detecting and controlling hospital outbreaks.\u003c/p\u003e","manuscriptTitle":"Emergence of a novel carbapenem-resistant Pseudomonas aeruginosa sequence type (ST) 5380 causing a hospital outbreak in Northern Italy, May to September 2024","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-19 12:33:22","doi":"10.21203/rs.3.rs-9345832/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c3b574b-49a1-470b-bdd3-dd2100308db8","owner":[],"postedDate":"April 19th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-04-30T16:41:51+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T16:55:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-19 12:33:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9345832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9345832","identity":"rs-9345832","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}