Methicillin-Resistant Staphylococcus Aureus Transmission and Hospital-Acquired Bacteremia in a Neonatal Intensive Care Unit in Greece

Methicillin-Resistant Staphylococcus Aureus Transmission and Hospital-Acquired Bacteremia in a Neonatal Intensive Care Unit in Greece | 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 Research Article Methicillin-Resistant Staphylococcus Aureus Transmission and Hospital-Acquired Bacteremia in a Neonatal Intensive Care Unit in Greece ANASTASSIOS GEORGE DOUDOULAKAKIS, Iris Spiliopoulou, Nikolaos Giormezis, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-231274/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Nov, 2021 Read the published version in Journal of Infection and Chemotherapy → Version 1 posted You are reading this latest preprint version Abstract The epidemiology of methicillin-resistant S. aureus (MRSA) colonization and infections in a 30-bed, level III university-affiliated neonatal intensive care unit was retrospectively investigated (2014-2018). Virulence, resistance genes and clonality of 46 isolates were determined by PCRs and MLST. Of 1538 neonates, 77 (5%) had a positive culture for MRSA; four bacteremias occured. One major clone was identified, ST225 (23/40, 58%), imported from the same maternity hospital. Another clone, ST217, was predominant (4/6) among colonized health care workers. Four isolates classified as ST80 were PVL-positive, four tst- positive, and two etb -positive. Strengthening of infection control measures with emphasis on hand hygiene was applied. General Microbiology Infectious Diseases Staphylococcus aureus MRSA ST225 neonates clones Greece Background Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of healthcare associated infections worldwide. Neonates constitute a special group in which MRSA infections can pose a significant burden of morbidity and mortality. Neonatal Intensive Care Units (NICUs) are an important reservoir of introduction and transmission of various MRSA clones among parents, health care workers (HCWs) and neonates [1]. The acquisition rate of initially non-colonized neonates admitted in a NICU has been reported to be around 6% with a median time of acquisition of nine days [2]. Around 20% of colonized neonates will develop an infection with a mortality rate 3-28% [3]. The majority of MRSA infections worldwide are due to a limited number of clonal lineages. The most common MLST types in neonatal wards are ST1, 5, 8 and 22 [3]. In Greece, the ST80-SCC mec IV prevailed in the community over 12 years (61.6% in total and 88.8% in CA-MRSA), and ST239-III in hospitals (22.5% in total and 60.8% in HA-MRSA) [4]. An observational study was performed to assess the prevalence of MRSA carriage and invasive infections in a 30-bed level III university-affiliated NICU at the P. & A. Kyriakou Children’s Hospital between January 1 st , 2014 and December 31 st , 2018. The NICU admits a large proportion of outborn infants, prematures included as well as neonates with malformations or complex conditions requiring surgical care. Surveillance and clinical care cultures of neonates that grew MRSA were retrospectively reviewed for the period 2014-2018, after a case outbreak of MRSA bacteremias during the last months of 2017. Following this outbreak, all HCWs were investigated for MRSA carriage too, with repeated nasal cultures. NICU-acquired MRSA colonization or infection is defined when a positive surveillance/clinical care culture is associated with a minimum stay of three days in the NICU of our hospital [5]. If admission culture or culture within two days of admission grows MRSA, then NICU-imported MRSA is considered. Identification and susceptibility testing of S. aureus was performed with conventional methods according to EUCAST guidelines [6]. All isolates with a cefoxitin inhibition zone of ≤21mm were tested with a latex agglutination test (Slidex MRSA Detection; bioMérieux®) for the presence of penicillin-binding protein 2a, and with PCR for mecA and mec C [7]. Amplification of genes encoding Panton-Valentine Leukocidin (PVL, lukS/lukF-PV ), exfoliative toxins ( eta, etb ), toxic shock syndrome toxin ( tst ), and the resistance gene fusB (fusidic acid) was performed by PCRs among 46 representative strains (40 from patients and six from HCWs), as described [7-9] . The 46 selected S. aureus strains were characterized by MLST (http://mlst.net) [10]. Results were analyzed by the application of eBURST algorithm. Clonal complexes were defined by using the default setting. During the study period 1538 neonates accounting for 26673 patient days where admitted to the NICU with a median length of stay 17 days (IQR: 11 to 56 days). All patients were either transferred from another NICU or from maternity hospitals (MHs), In total, 77 neonates (5%) had a positive culture for MRSA. Fifty-one (66%) were boys. Twenty three of 77 (29.9%) were NICU-acquired and 54/77 (70.1%) were imported cases. The hospitalization period before colonization ranged from 4 to 131 days (median 28, IQR: 11-65days). Four colonized boys (5.2%) developed MRSA bacteremia. Teicoplanin was successfully administered to all for seven days. Most isolates were multi-resistant, with higher resistance percentages observed against kanamycin (71%), macrolides (49%), lincosamides (47%) and ciprofloxacin (39%). All were susceptible to teicoplanin and vancomycin. All 37 HCWs were also tested and six among them were found positive for MRSA. All 46 (40 obtained from neonates and six from HCWs) molecularly analyzed strains were mecA -positive. Ten fusidic acid-resistant isolates were fusB -positive. One major clone was identified, ST225, among 40 tested neonatal strains (23/40, 58%). Of these, 14/23 were imported from the same MH. Another clone, ST217 comprising seven isolates, was predominant among HCWs (4/6) found to be colonized during screening performed on January 2018. ST30 and ST80 with seven and four strains respectively, were also identified. NICU-acquired bacteremia occurred in four neonates on Nov16, Aug17, Oct17 and Jan18 due to ST217 and ST225, three and one cases, respectively. Four isolates classified as ST80 were PVL-positive. Four additional strains carried tst (10%), belonging to ST30 and ST225 (two strains each), and two etb (5%, ST225). Decolonization treatment with nasal mupirocin and chlorexidine baths was initiated and successfully performed in all colonized neonates and HCWs and all were negative for staphylococcal carriage upon subsequent screening. The implicated MH was notified for the problem and strengthening of infection control measures with emphasis on hand hygiene was applied. In this study we describe the epidemiology of MRSA colonization and infection in a Greek referral NICU as well as the molecular characteristics of the implicated strains. To our knowledge, this is the first study concerning molecular characteristics of MRSA strains in a NICU in Greece. Four clones (ST225, ST217, ST30, ST80) were found colonizing the neonates and of these, two (ST225 and ST80) were implicated in bacteremias. The sources of this variable population were located in both within and outside the hospital. In our NICU, colonization rate was 5%. In a study from USA among 3536 neonates from 2007 to 2011, 2% had a culture grow MRSA [3]. A rate of 5,2% MRSA colonization among 536 neonates was recently reported from China for the period 2015-2016 [11]. Similar findings come from USA, where 3,9% of 3700 neonates from a single NICU were colonized [12]. In a recent multicenter epidemiological study conducted with the participation of 16 Greek NICUs for the period 2012-2015, S. aureus accounted for only two late onset septic episodes among 459 in total and the implicated strains were MSSA [13]. In the current study 5,2% of colonized neonates developed MRSA bacteremia. This rate is lower than that reported by Dong et al, where one out of five MRSA colonized neonates may develop bacteremia [2]. Regarding the detected clones, ST225 was the most common healthcare associated (imported) clone introduced by a sole ΜΗ of Athens, followed by ST217 and ST30. Τhe MH was immediately notified and infection control bundles such as cohorting-isolation of colonized newborns, use of contact precautions, reinforcement of hand hygiene to personnel, education and training of new staff and outset of active surveillance shortly after the notified outbreak were imposed. In a report from Greece, among 194 erythromycin-resistant MRSA isolates, phylogenetic analysis showed that ST225, which belongs to CC5, was the most prevalent clone, accounting for 137 MRSA isolates. Sequencing of two isolates revealed a plethora of toxin genes of the enterotoxin family increasing its pathogenicity [15]. ST225-MRSA-II is a single locus variant (SLV) of the ST5-MRSA-II pandemic CC5 strain [16]. Isolates of ST5/ST225-MRSA-II have been recovered in Austria, Croatia, Hong-Kong (China), Hungary, Japan, Portugal, Taiwan, UK and USA [16]. In our study, 20/23 ST225 isolates were erythromycin and clindamycin resistant too, exhibiting a MDR phenotype. erm(A) and the aminoglycoside resistance gene aadD are commonly present in ST5/ST225-MRSA-II isolates [16]. Only two of ST225 isolates were gentamicin-resistant in this study. In three out of four neonates with bacteremia, ST217 was implicated. This clone was NICU-acquired and was isolated from 4/6 HCWs. Contact with HCWs is an important factor for colonization of neonates [14]. Although we had limited data on HCW MRSA colonization prevalence during the study period, we identified HCWs as the main bacterial reservoir for institutional transmission and subsequent bacteremia. Very few ST217 strains are currently found in the MLST database ( http://saureus.mlst.net ), and data on such strains are scant in the literature. In particular, ST217-MRSA-IV was one of the dominant MRSA lineages isolated from patients in a hospital in Switzerland and Italy and was detected in food samples of animal origin in Spain [17]. ST217 is a single-locus variant of EMRSA-15 and might have been evolved from the ST22-MRSA-IV clone. The presence of ST217 was documented in India in 2012 [18]. The ST80 clone was reported for the first time in Greece in 2003, which accounted for 9.3% (11/118) of all MRSA strains isolated [19]. Since then, several studies reporting different percentages of MRSA-ST80 among all MRSA isolates have been published, including a 12 year survey (2001 to 2012) from geographically diverse areas of the country which showed the epidemic proportion of this clone in the community, accounting for 2838 isolates (88.8% CA-MRSA and 11.2% HA-MRSA isolates). This clone is predominantly lukS/lukF-PV positive. In the same study, the ST30 clone accounted for 453 isolates (70.4% CA-MRSA and 29.6% HA-MRSA) [4]. These two clones, widely distributed in the community, are introduced on a regular basis in the NICU from the HCWs and people caring for the neonates. Continuous investigation of MRSA prevalence is useful to uncover reservoirs for on-going MRSA transmission in NICUs and has proved challenging. Well-known nosocomial MRSA clones are being constantly introduced and transmitted through tranfers from MHs, parents and HCWs. Effective infection control requires constant vigilance, since the best strategy to avoid neonatal MRSA infections lies in prevention rather than treatment. Declarations Funding: This study was supported by funds of the participating laboratories and by funding of the University of Patras, Greece, Grant number 39540000 under the scientific responsibility of IS. Conflicts of interest: The authors have no conflicts of interest to declare that are relevant to the content of this article The Ethics Committee of “P. & A. Kyriakou Children’s Hospital” approved this study and waived the need for informed consent, number 9956 Authors contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Anastassios Doudoulakakis, Nikolaos Giormezis, Angeliki Nika, Elisavet Bozavoutoglou, Maria Militsopoulou, Georgios Kalogeras, Evangelia Lebessi. Molecular analysis was performed by Nikolaos Giormezis and Maria Militsopoulou. The first draft of the manuscript was written by Anastassios Doudoulakakis and Garyfallia Syridou and all authors commented on previous versions of the manuscript. Supervision of the study as well review and editing of the study were performed by Iris Spiliopoulou, Maria Tsolia and Evangelia Lebessi. All authors have read and approved the final manuscript. Reference Zervou FN, Zacharioudakis IM, Ziakas PD, Mylonakis E. MRSA colonization and risk of infection in the neonatal and pediatric ICU: a meta-analysis. Pediatrics. 2014 Apr;133(4):e1015-23. doi: 10.1542/peds.2013-3413. Dong Y, Glaser K, Speer CP. New Threats from an Old Foe: Methicillin-Resistant Staphylococcus aureus Infections in Neonates. Neonatology . 2018;114(2):127-134. doi:10.1159/000488582 Popoola VO, Budd A, Wittig SM, et al. Methicillin-resistant Staphylococcus aureus transmission and infections in a neonatal intensive care unit despite active surveillance cultures and decolonization: challenges for infection prevention. Infect Control Hosp Epidemiol . 2014;35(4):412-418. doi:10.1086/675594 Drougka E, Foka A, Liakopoulos A, Doudoulakakis A, Jelastopulu E, Chini V, Spiliopoulou A, Levidiotou S, Panagea T, Vogiatzi A, Lebessi E, Petinaki E, Spiliopoulou I. A 12-year survey of methicillin-resistant Staphylococcus aureus infections in Greece: ST80-IV epidemic? Clin Microbiol Infect. 2014 Nov;20(11):O796-803. doi: 10.1111/1469-0691.12624. NHSN Patient Safety Component. CDC/NHSN Surveillance Definitions for Specific Types of Infections. Jun.2011 The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.0, 2018. http://www.eucast.org. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, Nesme X, Etienne J, Vandenesch F. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun. 2002 Feb;70(2):631-41. doi: 10.1128/iai.70.2.631-641.2002. Gomes AR, Vinga S, Zavolan M, de Lencastre H. Analysis of the genetic variability of virulence-related loci in epidemic clones of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2005 Jan;49(1):366-79. doi: 10.1128/AAC.49.1.366-379.2005. Hung WC, Chen HJ, Lin YT, Tsai JC, Chen CW, Lu HH, Tseng SP, Jheng YY, Leong KH, Teng LJ. Skin Commensal Staphylococci May Act as Reservoir for Fusidic Acid Resistance Genes. PLoS One. 2015 Nov 18;10(11):e0143106. doi: 10.1371/journal.pone.0143106. Thomas JC, Vargas MR, Miragaia M, Peacock SJ, Archer GL, Enright MC. Improved multilocus sequence typing scheme for Staphylococcus epidermidis . J Clin Microbiol. 2007 Feb;45(2):616-9. doi: 10.1128/JCM.01934-06. Geng W, Qi Y, Li W, McConville TH, Hill-Ricciuti A, Grohs EC, Saiman L, Uhlemann AC. Epidemiology of Staphylococcus aureus in neonates on admission to a Chinese neonatal intensive care unit. PLoS One. 2020 Feb 13;15(2):e0211845. doi: 10.1371/journal.pone.0211845. Reich PJ, Boyle MG, Hogan PG, Johnson AJ, Wallace MA, Elward AM, Warner BB, Burnham CA, Fritz SA. Emergence of community-associated methicillin-resistant Staphylococcus aureus strains in the neonatal intensive care unit: an infection prevention and patient safety challenge. Clin Microbiol Infect. 2016 Jul;22(7):645.e1-8. doi: 10.1016/j.cmi.2016.04.013. Gkentzi D, Kortsalioudaki C, Cailes BC, Zaoutis T, Kopsidas J, Tsolia M, Spyridis N, Siahanidou S, Sarafidis K, Heath PT, Dimitriou G; Neonatal Infection Surveillance Network in Greece. Epidemiology of infections and antimicrobial use in Greek Neonatal Units. Arch Dis Child Fetal Neonatal Ed. 2019 May;104(3):F293-F297. doi: 10.1136/archdischild-2018-315024. Giuffrè M, Bonura C, Cipolla D, Mammina C. MRSA infection in the neonatal intensive care unit. Expert Rev Anti Infect Ther. 2013 May;11(5):499-509. doi: 10.1586/eri.13.28. Sarrou S, Malli E, Tsilipounidaki K, Florou Z, Medvecky M, Skoulakis A, Hrabak J, Papagiannitsis CC, Petinaki E. MLS B -Resistant Staphylococcus aureus in Central Greece: Rate of Resistance and Molecular Characterization. Microb Drug Resist. 2019 May;25(4):543-550. doi: 10.1089/mdr.2018.0259. Monecke S, Coombs G, Shore AC, et al. A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus . PLoS ONE 6(4): e17936. https://doi.org/10.1371/journal.pone.0017936 Vignaroli C, Mancini A, Varaldo PE. Composite SCCmec element in single-locus variant (ST217) of epidemic MRSA-15 clone. Emerg Infect Dis. 2014 May;20(5):905-7. doi: 10.3201/eid2005.130934. Bouchiat C, El-Zeenni N, Chakrakodi B, Nagaraj S, Arakere G, Etienne J. Epidemiology of Staphylococcus aureus in Bangalore, India: emergence of the ST217 clone and high rate of resistance to erythromycin and ciprofloxacin in the community. New Microbes New Infect. 2015 May 14;7:15-20. doi: 10.1016/j.nmni.2015.05.003. Aires de Sousa M, Bartzavali C, Spiliopoulou I, Sanches IS, Crisóstomo MI, de Lencastre H. Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras, Greece. J Clin Microbiol. 2003 May;41(5):2027-32. doi: 10.1128/jcm.41.5.2027-2032.2003. Cite Share Download PDF Status: Published Journal Publication published 01 Nov, 2021 Read the published version in Journal of Infection and Chemotherapy → 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-231274","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":11592459,"identity":"d473bb8d-f2aa-4e79-a9b5-2672411a1a7b","order_by":0,"name":"ANASTASSIOS GEORGE DOUDOULAKAKIS","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYPACZjkDMG1gQVgtD1SLsQEDM0iLBPFaEjeAtTAQocWe/fjjDx/+WKdvZ+8/uuFHgQQDf3t3An5beHLMJGe2pefu7DnMdrMH6DCJM2c3EHBYDhszb8Ph3A03ktlu8AC1GEjkEtDC//zxZ54/h9MNgFpu/iFKi0SCgTQP2+EEkJbbxNly4w3YL4Ybzhw2uy1jIMFD0C/s/engEJM3ON747OabPzZy/O29+LVgWkua8lEwCkbBKBgFWAEA6ChD8Gyml94AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5286-5896","institution":"Department of Microbiology, \"P. \u0026amp; A. Aglaia Kyriakou\" Children's Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"ANASTASSIOS","middleName":"GEORGE","lastName":"DOUDOULAKAKIS","suffix":""},{"id":11592460,"identity":"def2b939-2ad6-4f45-a072-d5a3e0e47818","order_by":1,"name":"Iris Spiliopoulou","email":"","orcid":"","institution":"University of Patras Department of Medicine: Panepistemio Patron Tmema Iatrikes","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Iris","middleName":"","lastName":"Spiliopoulou","suffix":""},{"id":11592461,"identity":"3f0118a6-d909-4d04-ae6a-17fe8e61fcf8","order_by":2,"name":"Nikolaos Giormezis","email":"","orcid":"","institution":"University of Patras Department of Medicine: Panepistemio Patron Tmema Iatrikes","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nikolaos","middleName":"","lastName":"Giormezis","suffix":""},{"id":11592462,"identity":"b423e932-a8b4-44eb-ba6c-2360c483d8b8","order_by":3,"name":"Garyfallia Syridou","email":"","orcid":"","institution":"Panepistemiako Geniko Nosokomeio Attikon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Garyfallia","middleName":"","lastName":"Syridou","suffix":""},{"id":11592463,"identity":"4dfc0bdb-e55d-412b-ad61-01897d0aef66","order_by":4,"name":"Angeliki Nika","email":"","orcid":"","institution":"Neonatal Intensive Care Unit, \"P. \u0026amp; A. 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Kyriakou\" Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Evangelia","middleName":"","lastName":"Lebessi","suffix":""}],"badges":[],"createdAt":"2021-02-11 02:57:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-231274/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-231274/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.jiac.2021.07.013","type":"published","date":"2021-11-01T04:49:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":15514743,"identity":"38b8f7d3-b322-4235-86c3-a0aa83cb6958","added_by":"auto","created_at":"2021-11-14 04:49:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":193259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-231274/v1/797428f4-a032-4a25-931e-1178e2834b83.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMethicillin-Resistant Staphylococcus Aureus Transmission and Hospital-Acquired Bacteremia in a Neonatal Intensive Care Unit in Greece\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus \u003c/em\u003e(MRSA) is a leading cause of healthcare associated infections worldwide. Neonates constitute a special group in which MRSA infections can pose a significant burden of morbidity and mortality. Neonatal Intensive Care Units (NICUs) are an important reservoir of introduction and transmission of various MRSA clones among parents, health care workers (HCWs) and neonates [1]. The acquisition rate of initially non-colonized neonates admitted in a NICU has been reported to be around 6% with a median time of acquisition of nine days [2]. Around 20% of colonized neonates will develop an infection with a mortality rate 3-28% [3].\u003c/p\u003e\n\u003cp\u003eThe majority of MRSA infections worldwide are due to a limited number of clonal lineages. The most common MLST types in neonatal wards are ST1, 5, 8 and 22 [3]. In Greece, the ST80-SCC\u003cem\u003emec\u003c/em\u003eIV prevailed in the community over 12 years (61.6% in total and 88.8% in CA-MRSA), and ST239-III in hospitals (22.5% in total and 60.8% in HA-MRSA) [4].\u003c/p\u003e\n\u003cp\u003eAn observational study was performed to assess the prevalence of MRSA carriage and invasive infections in a 30-bed level III university-affiliated NICU at the P. \u0026amp; A. Kyriakou Children\u0026rsquo;s Hospital between January 1\u003csup\u003est\u003c/sup\u003e, 2014 and December 31\u003csup\u003est\u003c/sup\u003e, 2018. The NICU admits a large proportion of outborn infants, prematures included as well as neonates with malformations or complex conditions requiring surgical care.\u003c/p\u003e\n\u003cp\u003eSurveillance and clinical care cultures of neonates that grew MRSA were retrospectively reviewed for the period 2014-2018, after a case outbreak of MRSA bacteremias during the last months of 2017. Following this outbreak, all HCWs were investigated for MRSA carriage too, with repeated nasal cultures.\u003c/p\u003e\n\u003cp\u003eNICU-acquired MRSA colonization or infection is defined when a positive surveillance/clinical care culture is associated with a minimum stay of three days in the NICU of our hospital [5]. If admission culture or culture within two days of admission grows MRSA, then NICU-imported MRSA is considered.\u003c/p\u003e\n\u003cp\u003eIdentification and susceptibility testing of \u003cem\u003eS. aureus\u003c/em\u003e was performed with conventional methods according to EUCAST guidelines [6]. All isolates with a cefoxitin inhibition zone of \u0026le;21mm were tested with a latex agglutination test (Slidex MRSA Detection; bioM\u0026eacute;rieux\u0026reg;) for the presence of penicillin-binding protein 2a, and with PCR for \u003cem\u003emecA\u003c/em\u003e and \u003cem\u003emec\u003c/em\u003eC [7].\u003c/p\u003e\n\u003cp\u003eAmplification of genes encoding Panton-Valentine Leukocidin (PVL, \u003cem\u003elukS/lukF-PV\u003c/em\u003e), exfoliative toxins (\u003cem\u003eeta, etb\u003c/em\u003e), toxic shock syndrome toxin (\u003cem\u003etst\u003c/em\u003e), and the resistance gene \u003cem\u003efusB\u003c/em\u003e (fusidic acid) was performed by PCRs among 46 representative strains (40 from patients and six from HCWs), as described [7-9] .\u003c/p\u003e\n\u003cp\u003eThe 46 selected \u003cem\u003eS. aureus\u003c/em\u003e strains were characterized by MLST (http://mlst.net) [10]. Results were analyzed by the application of eBURST algorithm. Clonal complexes were defined by using the default setting.\u003c/p\u003e\n\u003cp\u003eDuring the study period 1538 neonates accounting for 26673 patient days where admitted to the NICU with a median length of stay 17 days (IQR: 11 to 56 days). All patients were either transferred from another NICU or from maternity hospitals (MHs), In total, 77 neonates (5%) had a positive culture for MRSA. Fifty-one (66%) were boys. Twenty three of 77 (29.9%) were NICU-acquired and 54/77 (70.1%) were imported cases. The hospitalization period before colonization ranged from 4 to 131 days (median 28, IQR: 11-65days). Four colonized boys (5.2%) developed MRSA bacteremia. Teicoplanin was successfully administered to all for seven days. Most isolates were multi-resistant, with higher resistance percentages observed against kanamycin (71%), macrolides (49%), lincosamides (47%) and ciprofloxacin (39%). All were susceptible to teicoplanin and vancomycin. All 37 HCWs were also tested and six among them were found positive for MRSA. All 46 (40 obtained from neonates and six from HCWs) molecularly analyzed strains were \u003cem\u003emecA\u003c/em\u003e-positive. Ten fusidic acid-resistant isolates were \u003cem\u003efusB\u003c/em\u003e-positive. One major clone was identified, ST225, among 40 tested neonatal strains (23/40, 58%). Of these, 14/23 were imported from the same MH. Another clone, ST217 comprising seven isolates, was predominant among HCWs (4/6) found to be colonized during screening performed on January 2018. ST30 and ST80 with seven and four strains respectively, were also identified. NICU-acquired bacteremia occurred in four neonates on Nov16, Aug17, Oct17 and Jan18 due to ST217 and ST225, three and one cases, respectively. Four isolates classified as ST80 were PVL-positive. Four additional strains carried \u003cem\u003etst \u003c/em\u003e(10%), belonging to ST30 and ST225 (two strains each), and two \u003cem\u003eetb \u003c/em\u003e(5%, ST225). Decolonization treatment with nasal mupirocin and chlorexidine baths was initiated and successfully performed in all colonized neonates and HCWs and all were negative for staphylococcal carriage upon subsequent screening. The implicated MH was notified for the problem and strengthening of infection control measures with emphasis on hand hygiene was applied.\u003c/p\u003e\n\u003cp\u003eIn this study we describe the epidemiology of MRSA colonization and infection in a Greek referral NICU as well as the molecular characteristics of the implicated strains. To our knowledge, this is the first study concerning molecular characteristics of MRSA strains in a NICU in Greece. Four clones (ST225, ST217, ST30, ST80) were found colonizing the neonates and of these, two (ST225 and ST80) were implicated in bacteremias. The sources of this variable population were located in both within and outside the hospital.\u003c/p\u003e\n\u003cp\u003eIn our NICU, colonization rate was 5%. In a study from USA among 3536 neonates from 2007 to 2011, 2% had a culture grow MRSA [3]. A rate of 5,2% MRSA colonization among 536 neonates was recently reported from China for the period 2015-2016 [11]. Similar findings come from USA, where 3,9% of 3700 neonates from a single NICU were colonized [12]. In a recent multicenter epidemiological study conducted with the participation of 16 Greek NICUs for the period 2012-2015, \u003cem\u003eS. aureus\u003c/em\u003e accounted for only two late onset septic episodes among 459 in total and the implicated strains were MSSA [13]. In the current study 5,2% of colonized neonates developed MRSA bacteremia. This rate is lower than that reported by Dong et al, where one out of five MRSA colonized neonates may develop bacteremia [2].\u003c/p\u003e\n\u003cp\u003eRegarding the detected clones, ST225 was the most common healthcare associated (imported) clone introduced by a sole \u0026Mu;\u0026Eta; of Athens, followed by ST217 and ST30. \u0026Tau;he MH was immediately notified and infection control bundles such as cohorting-isolation of colonized newborns, use of contact precautions, reinforcement of hand hygiene to personnel, education and training of new staff and outset of active surveillance shortly after the notified outbreak were imposed. In a report from Greece, among 194 erythromycin-resistant MRSA isolates, phylogenetic analysis showed that ST225, which belongs to CC5, was the most prevalent clone, accounting for 137 MRSA isolates. Sequencing of two isolates revealed a plethora of toxin genes of the enterotoxin family increasing its pathogenicity [15]. ST225-MRSA-II is a single locus variant (SLV) of the ST5-MRSA-II pandemic CC5 strain [16]. Isolates of ST5/ST225-MRSA-II have been recovered in Austria, Croatia, Hong-Kong (China), Hungary, Japan, Portugal, Taiwan, UK and USA [16]. In our study, 20/23 ST225 isolates were erythromycin and clindamycin resistant too, exhibiting a MDR phenotype. erm(A) and the aminoglycoside resistance gene aadD are commonly present in ST5/ST225-MRSA-II isolates [16]. Only two of ST225 isolates were gentamicin-resistant in this study.\u003c/p\u003e\n\u003cp\u003eIn three out of four neonates with bacteremia, ST217 was implicated. This clone was NICU-acquired and was isolated from 4/6 HCWs. Contact with HCWs is an important factor for colonization of neonates [14]. Although we had limited data on HCW MRSA colonization prevalence during the study period, we identified HCWs as the main bacterial reservoir for institutional transmission and subsequent bacteremia. Very few ST217 strains are currently found in the MLST database (\u003ca href=\"http://saureus.mlst.net\"\u003ehttp://saureus.mlst.net\u003c/a\u003e), and data on such strains are scant in the literature. In particular, ST217-MRSA-IV was one of the dominant MRSA lineages isolated from patients in a hospital in Switzerland and Italy and was detected in food samples of animal origin in Spain [17]. ST217 is a single-locus variant of EMRSA-15 and might have been evolved from the ST22-MRSA-IV clone. The presence of ST217 was documented in India in 2012 [18].\u003c/p\u003e\n\u003cp\u003eThe ST80 clone was reported for the first time in Greece in 2003, which accounted for 9.3% (11/118) of all MRSA strains isolated [19]. Since then, several studies reporting different percentages of MRSA-ST80 among all MRSA isolates have been published, including a 12 year survey (2001 to 2012) from geographically diverse areas of the country which showed the epidemic proportion of this clone in the community, accounting for 2838 isolates (88.8% CA-MRSA and 11.2% HA-MRSA isolates). This clone is predominantly \u003cem\u003elukS/lukF-PV\u003c/em\u003e positive. In the same study, the ST30 clone accounted for 453 isolates (70.4% CA-MRSA and 29.6% HA-MRSA) [4]. These two clones, widely distributed in the community, are introduced on a regular basis in the NICU from the HCWs and people caring for the neonates.\u003c/p\u003e\n\u003cp\u003eContinuous investigation of MRSA prevalence is useful to uncover reservoirs for on-going MRSA transmission in NICUs and has proved challenging. Well-known nosocomial MRSA clones are being constantly introduced and transmitted through tranfers from MHs, parents and HCWs. Effective infection control requires constant vigilance, since the best strategy to avoid neonatal MRSA infections lies in prevention rather than treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: This study was supported by funds of the participating laboratories and by funding of the University of Patras, Greece, Grant number 39540000 under the scientific responsibility of IS.\u003c/p\u003e\n\u003cp\u003eConflicts of interest: The authors have no conflicts of interest to declare that are relevant to the content of this article\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of \u0026ldquo;P. \u0026amp; A. Kyriakou Children\u0026rsquo;s Hospital\u0026rdquo; approved this study and waived the need for informed consent, number 9956\u003c/p\u003e\n\u003cp\u003eAuthors contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Anastassios Doudoulakakis, Nikolaos Giormezis, Angeliki Nika, Elisavet Bozavoutoglou, Maria Militsopoulou, Georgios Kalogeras, Evangelia Lebessi. Molecular analysis was performed by Nikolaos Giormezis and Maria Militsopoulou. The first draft of the manuscript was written by Anastassios Doudoulakakis and Garyfallia Syridou and all authors commented on previous versions of the manuscript. Supervision of the study as well review and editing of the study were performed by Iris Spiliopoulou, Maria Tsolia and Evangelia Lebessi. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"Reference","content":"\u003col\u003e\n\u003cli\u003eZervou FN, Zacharioudakis IM, Ziakas PD, Mylonakis E. MRSA colonization and risk of infection in the neonatal and pediatric ICU: a meta-analysis. Pediatrics. 2014 Apr;133(4):e1015-23. doi: 10.1542/peds.2013-3413.\u003c/li\u003e\n\u003cli\u003eDong Y, Glaser K, Speer CP. New Threats from an Old Foe: Methicillin-Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Infections in Neonates.\u0026nbsp;\u003cem\u003eNeonatology\u003c/em\u003e. 2018;114(2):127-134. doi:10.1159/000488582\u003c/li\u003e\n\u003cli\u003ePopoola VO, Budd A, Wittig SM, et al. Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e transmission and infections in a neonatal intensive care unit despite active surveillance cultures and decolonization: challenges for infection prevention.\u0026nbsp;\u003cem\u003eInfect Control Hosp Epidemiol\u003c/em\u003e. 2014;35(4):412-418. doi:10.1086/675594\u003c/li\u003e\n\u003cli\u003eDrougka E, Foka A, Liakopoulos A, Doudoulakakis A, Jelastopulu E, Chini V, Spiliopoulou A, Levidiotou S, Panagea T, Vogiatzi A, Lebessi E, Petinaki E, Spiliopoulou I. A 12-year survey of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infections in Greece: ST80-IV epidemic? Clin Microbiol Infect. 2014 Nov;20(11):O796-803. doi: 10.1111/1469-0691.12624.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eNHSN Patient Safety Component. CDC/NHSN Surveillance Definitions for Specific Types of Infections. Jun.2011\u003c/li\u003e\n\u003cli\u003eThe European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.0, 2018. http://www.eucast.org.\u003c/li\u003e\n\u003cli\u003eJarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, Nesme X, Etienne J, Vandenesch F. Relationships between \u003cem\u003eStaphylococcus aureus\u003c/em\u003e genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun. 2002 Feb;70(2):631-41. doi: 10.1128/iai.70.2.631-641.2002.\u003c/li\u003e\n\u003cli\u003eGomes AR, Vinga S, Zavolan M, de Lencastre H. Analysis of the genetic variability of virulence-related loci in epidemic clones of methicillin-resistant \u003cem\u003eStaphylococcus aureus.\u003c/em\u003e Antimicrob Agents Chemother. 2005 Jan;49(1):366-79. doi: 10.1128/AAC.49.1.366-379.2005.\u003c/li\u003e\n\u003cli\u003eHung WC, Chen HJ, Lin YT, Tsai JC, Chen CW, Lu HH, Tseng SP, Jheng YY, Leong KH, Teng LJ. Skin Commensal Staphylococci May Act as Reservoir for Fusidic Acid Resistance Genes. PLoS One. 2015 Nov 18;10(11):e0143106. doi: 10.1371/journal.pone.0143106.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eThomas JC, Vargas MR, Miragaia M, Peacock SJ, Archer GL, Enright MC. Improved multilocus sequence typing scheme for \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e. J Clin Microbiol. 2007 Feb;45(2):616-9. doi: 10.1128/JCM.01934-06.\u003c/li\u003e\n\u003cli\u003eGeng W, Qi Y, Li W, McConville TH, Hill-Ricciuti A, Grohs EC, Saiman L, Uhlemann AC. Epidemiology of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e in neonates on admission to a Chinese neonatal intensive care unit. PLoS One. 2020 Feb 13;15(2):e0211845. doi: 10.1371/journal.pone.0211845.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eReich PJ, Boyle MG, Hogan PG, Johnson AJ, Wallace MA, Elward AM, Warner BB, Burnham CA, Fritz SA. Emergence of community-associated methicillin-resistant \u003cem\u003eStaphylococcus\u0026nbsp;aureus\u003c/em\u003estrains in the neonatal intensive care unit:\u0026nbsp;an\u0026nbsp;infection\u0026nbsp;prevention and patient safety challenge. Clin Microbiol Infect. 2016 Jul;22(7):645.e1-8. doi: 10.1016/j.cmi.2016.04.013.\u003c/li\u003e\n\u003cli\u003eGkentzi D, Kortsalioudaki C, Cailes BC, Zaoutis T, Kopsidas J, Tsolia M, Spyridis N, Siahanidou S, Sarafidis K, Heath PT, Dimitriou G; Neonatal Infection Surveillance Network in Greece. Epidemiology of infections and antimicrobial use in Greek Neonatal Units. Arch Dis Child Fetal Neonatal Ed. 2019 May;104(3):F293-F297. doi: 10.1136/archdischild-2018-315024.\u003c/li\u003e\n\u003cli\u003eGiuffr\u0026egrave; M, Bonura C, Cipolla D, Mammina C. MRSA infection in the neonatal intensive care unit. Expert Rev Anti Infect Ther. 2013 May;11(5):499-509. doi: 10.1586/eri.13.28.\u003c/li\u003e\n\u003cli\u003eSarrou S, Malli E, Tsilipounidaki K, Florou Z, Medvecky M, Skoulakis A, Hrabak J, Papagiannitsis CC, Petinaki E. MLS\u003csub\u003eB\u003c/sub\u003e-Resistant\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u0026nbsp;in Central Greece: Rate of Resistance and Molecular Characterization. Microb Drug Resist. 2019 May;25(4):543-550. doi: 10.1089/mdr.2018.0259.\u003c/li\u003e\n\u003cli\u003eMonecke S, Coombs G, Shore AC, et al. A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. PLoS ONE 6(4): e17936. \u003ca href=\"https://doi.org/10.1371/journal.pone.0017936\"\u003ehttps://doi.org/10.1371/journal.pone.0017936\u003c/a\u003e\u003c/li\u003e\n\u003cli\u003eVignaroli C, Mancini A, Varaldo PE. Composite SCCmec element in single-locus variant (ST217) of epidemic MRSA-15 clone. Emerg Infect Dis. 2014 May;20(5):905-7. doi: 10.3201/eid2005.130934.\u003c/li\u003e\n\u003cli\u003eBouchiat C, El-Zeenni N, Chakrakodi B, Nagaraj S, Arakere G, Etienne J. Epidemiology of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e in Bangalore, India: emergence of the ST217 clone and high rate of resistance to erythromycin and ciprofloxacin in the community. New Microbes New Infect. 2015 May 14;7:15-20. doi: 10.1016/j.nmni.2015.05.003.\u003c/li\u003e\n\u003cli\u003eAires de Sousa M, Bartzavali C, Spiliopoulou I, Sanches IS, Cris\u0026oacute;stomo MI, de Lencastre H. Two international methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e clones endemic in a university hospital in Patras, Greece. J Clin Microbiol. 2003 May;41(5):2027-32. doi: 10.1128/jcm.41.5.2027-2032.2003.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Staphylococcus aureus, MRSA, ST225, neonates, clones, Greece","lastPublishedDoi":"10.21203/rs.3.rs-231274/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-231274/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe epidemiology of methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) colonization and infections in a 30-bed, level III university-affiliated neonatal intensive care unit was retrospectively investigated (2014-2018). Virulence, resistance genes and clonality of 46 isolates were determined by PCRs and MLST. Of 1538 neonates, 77 (5%) had a positive culture for MRSA; four bacteremias occured. One major clone was identified, ST225 (23/40, 58%), imported from the same maternity hospital. Another clone, ST217, was predominant (4/6) among colonized health care workers. Four isolates classified as ST80 were PVL-positive, four \u003cem\u003etst-\u003c/em\u003epositive, and two \u003cem\u003eetb\u003c/em\u003e-positive. Strengthening of infection control measures with emphasis on hand hygiene was applied.\u003c/p\u003e","manuscriptTitle":"Methicillin-Resistant Staphylococcus Aureus Transmission and Hospital-Acquired Bacteremia in a Neonatal Intensive Care Unit in Greece","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-24 22:30:10","doi":"10.21203/rs.3.rs-231274/v1","editorialEvents":[{"type":"communityComments","content":1}],"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":"2cece96c-ab3f-4bfd-abfd-3d8546f4523a","owner":[],"postedDate":"February 24th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2600380,"name":"General Microbiology"},{"id":2600381,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2021-11-14T04:49:25+00:00","versionOfRecord":{"articleIdentity":"rs-231274","link":"https://doi.org/10.1016/j.jiac.2021.07.013","journal":{"identity":"journal-of-infection-and-chemotherapy","isVorOnly":true,"title":"Journal of Infection and Chemotherapy"},"publishedOn":"2021-11-01 04:49:25","publishedOnDateReadable":"November 1st, 2021"},"versionCreatedAt":"2021-02-24 22:30:10","video":"","vorDoi":"10.1016/j.jiac.2021.07.013","vorDoiUrl":"https://doi.org/10.1016/j.jiac.2021.07.013","workflowStages":[]},"version":"v1","identity":"rs-231274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-231274","identity":"rs-231274","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}