Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus in Belgium, 2013 to 2023

Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus in Belgium, 2013 to 2023 | 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 Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus in Belgium, 2013 to 2023 Nicolas Yin, Charlotte Michel, Nadia Makki, Ariane Deplano, Alisha Milis, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4074618/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2024 Read the published version in Eurosurveillance → Version 1 posted You are reading this latest preprint version Abstract Background Coresistance to both mupirocin and fusidic acid in Staphylococcus aureus affects the treatment of impetigo in Belgium, where they are the only topical treatments available. Aim We investigated resistance to fusidic acid and mupirocin in methicillin-susceptible S. aureus (MSSA) strains involved in community-acquired skin and soft tissue infections (SSTIs). Methods The 10-year variation in fusidic acid and mupirocin resistance in outpatients with SSTI-associated MSSA was studied in two large laboratories. MSSA strains coresistant to fusidic acid and mupirocin and sent to the Belgian Staphylococci Reference Centre were spa -typed and analysed for the presence of the eta and etb virulence genes and the mupA resistance gene. In addition, whole-genome sequencing was performed on isolates collected in the last 2 years. Results Fusidic acid and mupirocin resistance increased over time, and coresistance in children reached 8.9–10.1% in the third quarter 2023. From 2018 to 2023, 64/70 (91.4%) mupirocin-resistant MSSA strains were coresistant to fusidic acid. Whole-genome sequencing revealed that 29/33 (87.9%) of the isolates were sequence type ST121, clonal and more distantly related to the European epidemic fusidic acid-resistant impetigo clone (EEFIC) observed in Belgium in 2020. These strains carried the mupA and fusB genes, which confer resistance to mupirocin and fusidic acid, respectively, and the eta and etb virulence genes. Conclusion We highlight the spread of a mupirocin-resistant EEFIC (M-EEFIC) in children, with a seasonal trend for the third quarter. This is of concern because this variant is resistant to the two main topical antibiotics used to treat impetigo in Belgium. Infectious Diseases Pediatrics Bacteriology Laboratory Diagnostics Molecular Epidemiology Epigenetics & Genomics Staphylococcus aureus Impetigo Child Mupirocin Fusidic Acid Virulence Exfoliatins Belgium Whole Genome Sequencing Figures Figure 1 Figure 2 Figure 3 Introduction The Staphylococcus aureus European epidemic fusidic acid-resistant impetigo clone (EEFIC) was first described in 2007 after an increase in fusidic acid resistance among methicillin-susceptible S. aureus (MSSA) was observed in a number of northern European countries since circa 2000 [ 1 ]. A decade later, EEFIC was described as decreasing in these northern European countries [ 2 , 3 ] and remained rather unnoticed in Belgium at that time. However, a recent study showed not only the existence but also the persistence of EEFIC in Belgium [ 4 ]. This study showed that this clone was particularly involved in childhood impetigo, with a seasonal peak in late summer. The spread of this clone in Belgium is of concern because topical fusidic acid is one of the recommended first-line treatments for localised impetigo in Belgium [ 5 ], as well as in several European countries [ 6 ]. During this surveillance, we observed a small number of strains with coresistance to mupirocin. Such mupirocin-resistant EEFICs had already been described in Greece a few years earlier [ 7 ]. However, in the Greek study, 80.7% of the isolates carried the lukS / lukF genes encoding Panton-Valentine leucocidin toxin (PVL) in addition to the eta and/or etb genes. Topical mupirocin is commonly used to treat impetigo and is recommended for localised forms of impetigo in France [ 8 ] and for those involving methicillin-resistant S. aureus in Belgium [ 5 ]. The Belgian National Reference Centre for Staphylococcus aureus and other species (LHUB-ULB, Brussels, Belgium) routinely analyses strains voluntarily submitted by clinical laboratories as well as those from periodic surveillance requested by national or regional health authorities. Voluntarily submitted strains are usually strains with a particular antimicrobial resistance phenotype or associated with an unusual clinical presentation, such as recurrent or severe infections, or for investigation of clusters. The request form includes information about the time of sampling, the type of sample, whether a cluster was suspected or the patient had recently travelled, and the type of infection. In summer of 2023, the National Reference Centre (NRC) received an unusually high number of MSSA strains coresistant to fusidic acid and mupirocin associated with skin and soft tissue infections (SSTIs). These strains did not seem to be epidemiologically related, as they originated from different clinical laboratories in Flanders and Brussels. Therefore, we performed a 10-year retrospective analysis of the evolution of resistance to fusidic acid and mupirocin among community-associated MSSA strains isolated from skin and soft tissue swabs in two large clinical laboratories. We also reviewed the genetic characteristics of mupirocin- and fusidic acid-associated community-onset MSSA reported to the NRC during the same period. Methods Fusidic acid and mupirocin resistance in MSSA strains isolated from skin and soft tissue swabs Demographic and antimicrobial resistance data of MSSA isolated from skin and soft tissue swabs of outpatients in two large clinical laboratories in Belgium between October 2013 and September 2023 were studied: LHUB-ULB (Brussels), a clinical laboratory serving five university hospitals (with a total capacity of approximately 3,000 beds) as well as a network of general practitioners in the Brussels region covering a service area of 700,000 inhabitants, and AML (Antwerp), a private clinical laboratory belonging to the Sonic Healthcare group covering a large network of general practitioners and sampling sites in the Flanders region. The data collected included the sampling date, age at sampling, resistance to fusidic acid and resistance to mupirocin, as determined by a Vitek® 2 (bioMérieux, Marcy l'Etoile, France) according to EUCAST recommendations. Resistance to fusidic acid and mupirocin was studied over a 1-year period from October of a given year to September of the following year. As impetigo is more common in children and at the end of summer [ 9 ], a separate analysis was performed for children (< 15 years) and during the third quarter of each year (Q3, July to September). Molecular analysis of fusidic acid and mupirocin coresistant SSTI-associated MSSA Belgian clinical laboratories voluntarily send strains to the NRC for investigation of clusters, resistance mechanisms or virulence genes. The strains included in the present study were MSSA strains isolated from skin lesions collected between October 2013 and September 2023 and sent to the NRC for detection of virulence genes. A retrospective analysis was performed on fusidic acid and mupirocin coresistant strains (as determined by disk diffusion according to EUCAST guidelines). As part of the routine analyses carried out by the NRC, resistance to mupirocin was confirmed by end-point PCR detection of the mupA gene [ 10 ], detection of exfoliatins was assessed by end-point PCR detection of the eta and etb genes [ 11 ], and isolates for which at least one exfoliatin gene was detected were further analysed by spa typing (sequencing of the polymorphic X region of the protein A gene) [ 12 ]. Strains received from October 2021 to September 2023 were additionally analysed by whole-genome sequencing. DNA extraction was performed using EZ1&2 Virus Mini Kit v2.0 and the EZ2® Connect MDx instrument (Qiagen, Hilden, Germany). Genomic deoxyribonucleic acid (DNA) was enzymatically fragmented according to the manufacturer's instructions and modified to generate an Illumina-compatible DNA library using NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina. The final libraries were qualified using an AATI Fragment Analyser (Agilent Technologies Inc., Santa Clara, CA, USA) with DNF-474 High Sensitivity NGS Fragment Analysis Kit and quantified using a Qubit 2.0 with Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA). After equimolar pooling, the libraries were sequenced using a NovaSeq 6000 machine (Illumina Inc., San Diego, CA, USA) with NovaSeq 6000 SP Reagent Kit v1.5 (300 cycles) in 2 x 150 bp paired mode. An average coverage of 100× was targeted. De novo assembly was performed using the SPAdes algorithm [ 13 ]. The genome assemblies were deposited at the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA1041362. Resistome, virulome, multilocus sequence typing (MLST), whole-genome MLST (wgMLST) and spa type determination were performed using the BioNumerics 8.1 (bioMérieux, Marcy l'Etoile, France) S. aureus genotyping plugin v1.1 (database S. aureus Virulence KB 2022.12.05 and database S. aureus Resistance KB 2023.10.27), the Spa typing plugin v2.23, the WGS tools plugin v1.08 and MLST for the WGS plugin v1.0. A wgMLST cluster analysis was performed using the categorical distance and UPGMA (unweighted pair group method with arithmetic mean) algorithms with BioNumerics. Thirty genome assemblies of CC121 strains downloaded from the NCBI GenBank database and selected from a previous study by Zhou et al. [ 14 ] to include isolates carrying eta and/or etb and to cover different countries and different spa types and the genome of a mupirocin-susceptible EEFIC from a previous surveillance in Belgium [ 4 ] were included as reference genomes. The list of selected isolates is provided in Supplementary Table S1. Results Fusidic acid and mupirocin rates among MSSA isolated from outpatient SSTI swabs From October 2013 to September 2023, 21,232 MSSA were isolated by culture from SSTI swabs in the LHUB-ULB, including 4,389 from children. In total, 11,271 SSTI-related MSSA strains were isolated from AML patients, including 1941 from children. Overall, resistance to fusidic acid increased from 3.1% (68/2211) to 11.4% (263/2300) in LHUB-ULB and from 17.7% (153/862) to 25.5% (341/1336) in AML from October 2013 to September 2013 (Fig. 1 ). In both cases, a slight decrease was observed during the COVID-19 pandemic, in 2019–2020 for LHUB-ULB and in 2020–2021 for AML. During the same period, resistance to mupirocin increased from 0.5% (10/2211) to 1.7% (38/2300) in LHUB-ULB and from 1.5% (13/862) to 5.6% (75/1336) in AML. Similarly, coresistance to fusidic acid and mupirocin increased from 0.04% (1/2211) to 1.4% (33/2300) in LHUB-ULB and from 0.8% (7/862) to 5.3% (71/1336) in AML. Similar trends, but at higher rates, were observed for children. Fusidic acid resistance increased from 3.6% (19/527) to 18.3% (92/504) in LHUB-ULB and from 32.0% (47/147) to 45.9% (124/270) in AML. Resistance to mupirocin increased from 0.4% (2/527) to 5.2% (26/504) in LHUB-ULB and from 2.7% (4/147) to 9.3% (25/270) in AML. Coresistance to fusidic acid and mupirocin increased from 0% (0/527) to 5.0% (25/504) in LHUB-ULB and from 0% (0/147) to 8.5% (23/270) in AML. For strains isolated from children in Q3 of each year, resistance to fusidic acid increased from 0.7% (1/149) to 23.2% (39/168) in LHUB-ULB and from 45.2% (19/42) to 54.1% (59/109) in AML from 2014 to 2023 (Fig. 2 ). Resistance to mupirocin increased from 0.7% (1/149) to 8.9% (15/168) in LHUB-ULB and from 0% (0/42) to 11.9% (13/109) in AML. Coresistance to fusidic acid and mupirocin increased from 0% (0/149) to 8.9% (15/168) in LHUB-ULB and from 0% (0/42) to 10.1% (11/109) in AML. From October 2018 to September 2023, 91.4% (64/70) of the mupirocin-resistant MSSA strains were coresistant to fusidic acid. The raw data are shown in Supplementary Table S2. Genomic study of fusidic acid and mupirocin coresistant SSTI-associated MSSA From October 2013 to September 2023, the NRC received a total of 58 MSSA strains isolated from SSTIs that were coresistant to mupirocin and fusidic acid from 29 different laboratories across Belgium (Table 1 ). No cluster suspicion or recent travel history was reported on any of the accompanying request forms. No laboratory sent more than 3 strains per period. Thirty-seven MSSA strains originated from Flanders (63.8%), 12 (20.7%) from the Brussels region and 9 (15.5%) from Wallonia. The number of strains received increased from zero in 2013–2014 to 19 in 2022–2023. A total of 40/58 (69.0%) were isolated from children; 51/57 (89.5%) were positive for eta and 47/57 (82.5%) for etb . All strains tested for mupA (39/39) were positive. Of the 54 spa typings performed, 35 (63.6%) were t1994, and 5 had closely related spa types (t162, t2391, t21368), all of which were closely related to t171, which was associated with the original description of EEFIC [ 1 ]. Furthermore, all 40 of these isolates carried eta and etb . Therefore, since October 2017, 40/51 (78.4%) of MSSA strains coresistant to fusidic acid and mupirocin isolated from SSTIs in Belgium are similar to EEFIC, but in addition to their exfoliatins and fusidic acid resistance, they also carry the mupA gene, which confers resistance to mupirocin. Table 1 Fusidic acid- and mupirocin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates from skin and soft tissue infections sent to the Belgian National Reference Centre by different clinical laboratories from October 2014 to September 2023. eta : exfoliatin a gene detection, etb : exfoliatin b gene detection, mupA : mupA gene detection, CC121: S. aureus clonal complex 121 Period N Number of sending laboratories Age < 15 years eta etb mupA CC121-related spa type 10/2014-09/2015 1 1 0/1 0/1 0/1 1/1 0/1 10/2015-09/2016 1 1 0/1 0/1 0/1 1/1 0/1 10/2016-09/2017 1 1 0/1 0/1 0/1 1/1 0/1 10/2017-09/2018 7 3 7/7 7/7 7/7 7/7 6/7 10/2018-09/2019 6 5 3/6 5/6 5/6 5/5 5/6 10/2019-09/2020 4 3 4/4 4/4 3/4 1/1 3/4 10/2020-09/2021 5 5 4/5 5/5 4/5 3/3 4/5 10/2021-09/2022 14 9 8/14 13/14 12/14 5/5 9/13 10/2022-09/2023 19 14 14/19 17/18 16/18 15/15 13/16 Total 58 29 40/58 51/57 47/57 39/39 40/54 Whole-genome sequencing was performed on the 33 MSSA strains received at the NRC between October 2021 and September 2023. These strains were obtained from 19 different laboratories. Twenty strains were from Flanders, 7 from Wallonia and 6 from the Brussels region. Whole-genome sequencing revealed that all the strains carried mupA , with 30/33 (90.9%) being ST121; hence, these strains are CC121 strains. Of these, 28/30 (93.3%) carried eta , and 30/30 carried etb . None of them carried lukS/lukF . Twenty-nine (96.7%) carried the acquired gene fusB , while 1/30 (3.2%) carried the L461K-mutated chromosomal fusA , conferring resistance to fusidic acid. In addition, 26/30 (86.7%) carried the aadD gene, which confers resistance to amikacin, and 6/30 (20.0%) carried the cat(pC194) gene, which confers resistance to chloramphenicol. The remaining three isolates were negative for etb , two are ST45 and negative for eta ( spa type t550), and one is ST15 ( spa type t084) and harboured etb . The wgMLST analysis showed that the 29 ST121 isolates carrying fusB are likely to be clonal. In addition, the location of fusB in these strains was away from groEL , contrary to the original description of EEFIC [ 1 ], but the strains carried the gene encoding epidermal differentiation inhibitor C (EDIN-C) close to etb . These strains clustered with the reference genome of an MSSA isolated in Greece in 2018 (GenBank accession GCA_003605275.1). This MSSA carried mupA , fusB , aadD , eta and etb . These isolates are more closely related to each other than to the ST123 EEFIC observed in Belgium in 2020, while the ST121 MSSA with the fusA mutation is even more distantly related (Fig. 3 ). Additional data are shown in Supplementary Table S3. Discussion Overall, mupirocin resistance seems to have increased since October 2019. It remains at low prevalence (below 2%) in the LHUB-ULB population, whereas it reaches 5.6% in AML. Focusing on the paediatric population, a worrying trend is observed for the last 5 years: the prevalence of mupirocin resistance reached more than 5% for the period October 2022-September 2023 and was almost systematically associated with resistance to fusidic acid (64/70, 91.4%). Focusing on children during Q3, the impetigo season [ 9 ], revealed an even more alarming trend, with mupirocin resistance rates ranging from 8.9–11.9% and an almost systematic association with fusidic acid resistance (26/28, 92.9%). Based on analysis of the isolates received by the NRC since October 2017, 78.4% of the mupirocin and fusidic acid coresistant isolates were confirmed to be related to EEFIC. Whole-genome sequencing analysis of isolates collected during the last 2 years support this trend, with 29/33 clonal isolates (87.9%) sharing the following genetic characteristics: ST121, resistance to fusidic acid conferred by fusB , resistance to mupirocin conferred by mupA , and the presence of the virulence genes eta and etb . In a previous study on subpopulations of S. aureus CC121, Kurt et al. showed that isolates sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, and conjunctivitis) clustered in the same clade, including EEFIC [ 15 ]. Interestingly, wgMLST cluster analysis showed that these strains are closely related to the reference genome of an MSSA isolated in Greece, which shares the same characteristics, while the other reference genomes from SSTI-related isolates are more distantly related. However, in contrast to a previous study in Greece describing the emergence of a similar ST121 clone [ 7 ], none of them carried the lukS / lukF genes. Notably, 1/33 of the strains is ST15 with a t084 spa type and carrying etb . ST15 strains carrying exfoliatin genes have been observed quite frequently in staphylococcal scalded skin syndrome in France [ 16 ]. Treatment of impetigo is often based on the use of topical antibiotics for localised forms, such as fusidic acid cream in Belgium and the Netherlands [ 5 , 6 ] or mupirocin in France [ 8 ]. This option is preferred for localised disease because it has been shown to be at least as effective as oral treatment, with fewer side effects [ 17 , 18 ]. Therefore, the emergence and spread of an MSSA clone coresistant to these two antibiotics is a cause for concern. Other possible topical treatments are retapamulin [ 17 , 19 ] and ozenoxacin [ 20 ]. However, in the European Union, marketing authorisation for retapamulin was withdrawn by the European Commission on 25 February 2017 at the request of the manufacturer. At the time of manuscript writing, ozenoxacin was only available in Italy, Portugal and Spain [ 21 ]. Interestingly, the emerging clone described in the present study frequently carried aadD and cat(pC194) , which confer resistance to amikacin and chloramphenicol, respectively. Although these antibiotics are not used in Belgium, they may be used as topical treatments. Acquisition of these resistance genes is likely a selective advantage for strains with a propensity for superficial skin infections. On the other hand, the efficacy of disinfection as sole treatment for impetigo seems to be inferior to that of topical antibiotics [ 17 ], even though recommendations in the United Kingdom favour the single use of antiseptics [ 22 ]. However, the development of resistance to topical antibiotics may change this consideration, and further studies are needed to determine the best interventions for localised impetigo. Impetigo is known to affect children in summer [ 9 ]. Indeed, in a previous surveillance focused on S. aureus -related SSTIs, we showed that EEFIC was more prevalent in children in late summer [ 4 ]. At that time, resistance to mupirocin was anecdotal. However, one of the limitations of this surveillance was that it was conducted during the summer immediately after onset of the COVID-19 crisis. The social distancing and restrictions that gradually came into force during this period, combined with the workload of the clinical laboratories during the study period, certainly introduced biases. The end of the COVID-19-related restrictions and the increase in mupirocin-resistant strains arriving at the NRC led us to perform the present study using immediately available retrospective laboratory data. As strains are sent to the NRC by clinical laboratories on a voluntary basis, NRC data may not always reflect the true epidemiological situation. To determine the accuracy of the emergence of a new clone, we collected epidemiological data from two large laboratories in Belgium (LHUB-ULB and AML). In a previous study focusing on respiratory viruses, LHUB-ULB was demonstrated to act as a sensor for the whole country due to the large number of analyses and its central location within Belgium [ 23 ]. Inclusion of AML allowed the addition of data from a different type of laboratory (LHUB-ULB is more of a hospital laboratory, whereas AML, located in Flanders, mainly collects samples directly from general practitioners and outpatient sampling centres). However, additional data from a laboratory in the third region of Belgium (Wallonia) could have made the data completely exhaustive. Setting up an actual prospective surveillance would have been more accurate but would have caused a delay in communicating these results. Nevertheless, such a strategy has limitations: there are no data about the representativeness of AML and LHUB-ULB in Belgium for impetigo. Furthermore, the proportion of skin lesions that are sampled before treatment is unknown, and the total number of skin samples sent to laboratories per year is not known, as these data were not collected by health insurance providers. This bias is limited by the overall number of strains included (32,503 MSSA over 10 years) but may lead to some imprecision when focusing on a specific population during a specific time period, as we did in this study for children during the third quarter of the year. The epidemiological differences observed between AML and LHUB-ULB could be due to regional differences and/or differences between the patients sampled (case mix). The impact of the latter is likely mitigated by the fact that we specifically selected samples from outpatients. Nevertheless, AML collects samples from the federal agency for reception of asylum seekers (FEDASIL), and identification of epidemiological clusters at the level of the clinical laboratory is not easy because the context is rarely provided by the prescribers for outpatients. Regional epidemiological differences may exist. For example, in the most recent surveillance study on the EEFIC in Belgium [ 4 ], all EEFICs were from Flanders, and almost half of them (47.4%) were located in the province of Antwerp, where AML is located. From October 2013 to September 2023, the majority of MSSA strains coresistant to mupirocin and fusidic acid analysed by the NRC in this study were from Flanders, followed by Brussels and Wallonia. Beyond an actual regional difference, it is also possible that laboratories and practitioners in Flanders are more aware of the current epidemiological situation due to the EEFIC clusters in 2018 that led to our previous surveillance; in contrast, these clusters were not described in Wallonia. Although very common, impetigo is likely an understudied pathology [ 24 ]. Indeed, there is currently no organised surveillance of impetigo in Belgium. As a consequence, epidemiological data rely on laboratory data, as in this study. At the laboratory level, clinical information on the type of infection is often lacking. We attempted to overcome these problems in our study by including skin samples from outpatients only and by focusing on the more likely target population for impetigo: children sampled in late summer (Q3). Additionally, uncomplicated impetigo may not be sampled. This may lead to bias towards complicated skin infections that should not be treated with topical antibiotics. Nevertheless, this study clearly demonstrated the emergence of a clone combining virulence and resistance. Periodic surveillance is needed to better monitor the true epidemiology of impetigo and adapt treatment recommendations accordingly. In addition, this surveillance may provide useful information for developing intervention trials for impetigo patients. The present study highlights the emergence and spread of a clonal ST121 mupirocin-resistant clone diverging from EEFIC (M-EEFIC), which has acquired the mupA gene in addition to its virulence ( eta and etb ) and resistance ( fusB ) genes. This clone could have a selective advantage in Belgium and similar European countries where there is no other alternative topical treatment. 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Zhou W, Jin Y, Teng G, Chen W, Chen Y, Luo Q, et al. Comparative analysis of genomic characteristics, virulence and fitness of community-associated Staphylococcus aureus ST121 clone causing fatal diseases in China and other CA-MRSA clones. Virulence. 2023 Dec 31;14(1):2242547. Kurt K, Rasigade JP, Laurent F, Goering RV, Žemličková H, Machova I, et al. Subpopulations of Staphylococcus aureus Clonal Complex 121 Are Associated with Distinct Clinical Entities. PLOS ONE. 2013 Mar 7;8(3):e58155. Lamand V, Dauwalder O, Tristan A, Casalegno JS, Meugnier H, Bes M, et al. Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains. Clinical Microbiology and Infection. 2012 Dec;18(12):E514–21. Koning S, Sande R, Verhagen A, Smit L, Morris A, Butler C, et al. Interventions for impetigo. Cochrane database of systematic reviews. 2012;1(1):CD003261-CD003261. Ajay George, Greg Rubin. A systematic review and meta-analysis of treatments for impetigo. Br J Gen Pract. 2003 Jun 1;53(491):480. Yang LPH, Keam SJ. Retapamulin: a review of its use in the management of impetigo and other uncomplicated superficial skin infections. Drugs. 2008;68(6):855–73. Rosen T, Albareda N, Rosenberg N, Alonso FG, Roth S, Zsolt I, et al. Efficacy and Safety of Ozenoxacin Cream for Treatment of Adult and Pediatric Patients With Impetigo: A Randomized Clinical Trial. JAMA Dermatology. 2018 Jul 1;154(7):806. European Medicines Agency. Medicines [Internet]. [cited 2024 Feb 21]. Available from: https://www.ema.europa.eu/en/medicines National Institute for Health and Care Excellence. Impetigo: antimicrobial prescribing [Internet]. National Institute for Health and Care Excellence; 2020 [cited 2023 Feb 17]. Available from: https://www.nice.org.uk/guidance/ng153 Van den Wijngaert S, Bossuyt N, Ferns B, Busson L, Serrano G, Wautier M, et al. Bigger and Better? Representativeness of the Influenza A Surveillance Using One Consolidated Clinical Microbiology Laboratory Data Set as Compared to the Belgian Sentinel Network of Laboratories. Frontiers in Public Health. 2019;7. Gorges H, Hall L, Heal C. Feasibility Study for a Randomised Controlled Trial for the Topical Treatment of Impetigo in Australian General Practice. Tropical Medicine and Infectious Disease. 2021;6(4). Additional Declarations The authors declare no competing interests. Supplementary Files TableS1.docx Information of ST121 isolates analyzed in this study. TableS2.xlsx Number of skin swabs positive for methicillin-susceptible Staphylococcus aureus and their resistance to mupirocin and fusidic acid in 2 Belgian clinical laboratories (AML and LHUB-ULB) from October 2013 to September 2023 TableS3.xlsx Resistome and virulome of 33 methicillin-susceptible Staphylococcus aureus isolates from Belgium from October 2021 to September 2023 as well as 21 reference genomes of ST121 MSSA (Zhou et al.) [13], and 1 ST123 MSSA belonging to the European epidemic fusidic acid-resistant impetigo clone from a Belgium, 2020 (Deplano et al.) [4]. Cite Share Download PDF Status: Published Journal Publication published 09 May, 2024 Read the published version in Eurosurveillance → 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. 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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-4074618","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278290861,"identity":"d1dcad02-0806-45b3-98da-1221cdeabddc","order_by":0,"name":"Nicolas Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3PsQrCMBCA4QuFukTnuKiPEBEUUXyWHIJuIrg4SCkIcfN5uikc1KXg6pi+gS6OaqwIOtQ6CuaHhBvugwTA5frFPHvMY2AmDUHZSQMLC4h6DJ7EjLACAk8C4IuvSKfkpUYtApC7cjzHiCYgUBsW9XNJd+m3pIoJJFVGB0xoZslKsmSUSyRxXyh/ex/aB9SEG3sLpqmAXIKMTO8kFBm5fiaovYzAC9l+IPYvuCZeJd6yy+MZ8HQlMRnmkz2l5ngOapV90jyedG8CpWFsTtEglzzjjbeXqEJgq4ffbLlcLtdfdgO+nlVEqcSH7QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1706-6869","institution":"1.\tNational reference centre for Staphylococcus aureus and other species, department of microbiology, LHUB-ULB, Université libre de Bruxelles","correspondingAuthor":true,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Yin","suffix":""},{"id":278290862,"identity":"1b640947-d5ff-4fe4-8bbe-3d118fc7cadd","order_by":1,"name":"Charlotte Michel","email":"","orcid":"https://orcid.org/0000-0003-0250-2664","institution":"National reference centre for Staphylococcus aureus and other species, department of microbiology, LHUB-ULB, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Michel","suffix":""},{"id":278290863,"identity":"14990c8d-ffdc-49b9-93c6-8363dd9ee57f","order_by":2,"name":"Nadia Makki","email":"","orcid":"","institution":"Department of microbiology, Algemeen Medisch Laboratorium (AML), Antwerp, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Makki","suffix":""},{"id":278290864,"identity":"0c6098fd-4919-4627-9673-2c516a1c3a72","order_by":3,"name":"Ariane Deplano","email":"","orcid":"https://orcid.org/0000-0001-6944-5380","institution":"National reference centre for Staphylococcus aureus and other species, department of microbiology, LHUB-ULB, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Ariane","middleName":"","lastName":"Deplano","suffix":""},{"id":278290865,"identity":"99681171-68c1-415b-9870-10a1b26ee7d6","order_by":4,"name":"Alisha Milis","email":"","orcid":"","institution":"Department of microbiology, Algemeen Medisch Laboratorium (AML), Antwerp, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Alisha","middleName":"","lastName":"Milis","suffix":""},{"id":278290866,"identity":"ef4be2b8-f97b-409f-bb5b-f7b99bd44263","order_by":5,"name":"Benoit Prevost","email":"","orcid":"","institution":"National reference centre for Staphylococcus aureus and other species, department of microbiology, LHUB-ULB, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Benoit","middleName":"","lastName":"Prevost","suffix":""},{"id":278290867,"identity":"c8ca0b52-25f4-47d0-92ac-fe139ab0b19c","order_by":6,"name":"Veronique Yvette Miendje-Deyi","email":"","orcid":"https://orcid.org/0000-0003-3128-3181","institution":"Department of microbiology, LHUB-ULB, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Veronique","middleName":"Yvette","lastName":"Miendje-Deyi","suffix":""},{"id":278290868,"identity":"5712879e-0d4a-49fc-9de9-2f89d76c4fd3","order_by":7,"name":"Marie Hallin","email":"","orcid":"https://orcid.org/0000-0003-4544-0635","institution":"Environmental health research centre (CR4), Public health school, European Plotkin institute for vaccinology (EPIV), Faculty of medecine, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Hallin","suffix":""},{"id":278290869,"identity":"91ec213e-a8c5-40cc-aede-b532be48413e","order_by":8,"name":"Delphine Martiny","email":"","orcid":"https://orcid.org/0000-0001-9307-7418","institution":"National reference centre for Staphylococcus aureus and other species, department of microbiology, LHUB-ULB, Université libre de Bruxelles, Brussels, Belgium","correspondingAuthor":false,"prefix":"","firstName":"Delphine","middleName":"","lastName":"Martiny","suffix":""}],"badges":[],"createdAt":"2024-03-11 13:00:26","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4074618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4074618/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.2807/1560-7917.ES.2024.29.19.2300668","type":"published","date":"2024-05-09T10:17:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52526941,"identity":"83193b32-f9b8-4d35-8dde-1d92790c9138","added_by":"auto","created_at":"2024-03-12 16:06:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107838,"visible":true,"origin":"","legend":"\u003cp\u003eResistance to fusidic acid and mupirocin in methicillin-susceptible Staphylococcus aureus isolated from skin and soft tissue infections from October (Oct) 2013 to September (Sep) 2023 in two clinical laboratories in Belgium (AML and LHUB-ULB), overall (A) and in children (B). The number of resistant isolates per period is indicated on the graphs for the first period, the last period and September 2018 to October 2019.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/c5ac28b2225469640fce262c.png"},{"id":52526943,"identity":"def8d294-5d16-4dc8-bb76-a493c76891d7","added_by":"auto","created_at":"2024-03-12 16:06:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84008,"visible":true,"origin":"","legend":"\u003cp\u003eResistance to fusidic acid and mupirocin in methicillin-susceptible Staphylococcus aureus isolated from skin and soft tissue infections in children during the third quarter (Q3, July to September) of each year from 2014 to 2023 in two clinical laboratories in Belgium (AML and LHUB-ULB). The number of resistant isolates per period is indicated on the graphs for the first period, the last period and September 2018 to October 2019.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/594ac84522a022c40b48e5e3.png"},{"id":52526946,"identity":"3e7bb00e-b348-478c-aebe-ae58d8b735cf","added_by":"auto","created_at":"2024-03-12 16:06:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262698,"visible":true,"origin":"","legend":"\u003cp\u003espa types and UPGMA dendrogram of whole-genome multilocus sequence typing results of 30 methicillin-susceptible ST121 Staphylococcus aureus isolates from Belgium from October 2021 to September 2023 (red squares) and 30 reference genomes of ST121 MSSA (Zhou et al.) [14] (blue squares) and 1 ST123 MSSA belonging to the European epidemic fusidic acid-resistant impetigo clone from Belgium, 2020 (Deplano et al.) [4] (green squares)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/118393b1dbbd1795e731ded0.png"},{"id":56449603,"identity":"b3a476e8-8b7c-4e4c-ace1-11c0b1ecf717","added_by":"auto","created_at":"2024-05-14 10:17:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":747915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/ae6b0e43-ba02-4145-aa29-4dc21e0d3ea5.pdf"},{"id":52526942,"identity":"762fafec-b509-4c55-bf3b-a5927cf3dc92","added_by":"auto","created_at":"2024-03-12 16:06:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInformation of ST121 isolates analyzed in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/3244b739e7e1bb76bba74249.docx"},{"id":52526945,"identity":"fc79f005-79d2-471b-a218-cf1232d5d54c","added_by":"auto","created_at":"2024-03-12 16:06:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16107,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of skin swabs positive for methicillin-susceptible Staphylococcus \u0026nbsp;\u0026nbsp;aureus and their resistance to mupirocin and \u0026nbsp;\u0026nbsp;fusidic acid in 2 Belgian clinical laboratories (AML and LHUB-ULB) from \u0026nbsp;\u0026nbsp;October 2013 to September 2023\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/3b3abe4e18e365e32f5569cd.xlsx"},{"id":52526947,"identity":"4d50ebb1-a064-469a-bd4c-a224c2244507","added_by":"auto","created_at":"2024-03-12 16:06:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15126,"visible":true,"origin":"","legend":"\u003cp\u003eResistome and virulome of 33 methicillin-susceptible Staphylococcus \u0026nbsp;\u0026nbsp;aureus isolates from Belgium from October 2021 to \u0026nbsp;\u0026nbsp;September 2023 as well as 21 reference genomes of ST121 MSSA (Zhou et al.) \u0026nbsp;\u0026nbsp;[13], and 1 ST123 MSSA belonging to the European epidemic fusidic \u0026nbsp;\u0026nbsp;acid-resistant impetigo clone from a Belgium, 2020 (Deplano et al.) [4].\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4074618/v1/b9ffa8d47f438c945f4deede.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eEmergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of \u003cem\u003eStaphylococcus aureus \u003c/em\u003ein Belgium, 2013 to 2023\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe \u003cem\u003eStaphylococcus aureus\u003c/em\u003e European epidemic fusidic acid-resistant impetigo clone (EEFIC) was first described in 2007 after an increase in fusidic acid resistance among methicillin-susceptible \u003cem\u003eS. aureus\u003c/em\u003e (MSSA) was observed in a number of northern European countries since circa 2000 [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. A decade later, EEFIC was described as decreasing in these northern European countries [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e] and remained rather unnoticed in Belgium at that time. However, a recent study showed not only the existence but also the persistence of EEFIC in Belgium [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. This study showed that this clone was particularly involved in childhood impetigo, with a seasonal peak in late summer. The spread of this clone in Belgium is of concern because topical fusidic acid is one of the recommended first-line treatments for localised impetigo in Belgium [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e], as well as in several European countries [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. During this surveillance, we observed a small number of strains with coresistance to mupirocin. Such mupirocin-resistant EEFICs had already been described in Greece a few years earlier [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, in the Greek study, 80.7% of the isolates carried the \u003cem\u003elukS\u003c/em\u003e/\u003cem\u003elukF\u003c/em\u003e genes encoding Panton-Valentine leucocidin toxin (PVL) in addition to the \u003cem\u003eeta\u003c/em\u003e and/or \u003cem\u003eetb\u003c/em\u003e genes. Topical mupirocin is commonly used to treat impetigo and is recommended for localised forms of impetigo in France [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e] and for those involving methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e in Belgium [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe Belgian National Reference Centre for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and other species (LHUB-ULB, Brussels, Belgium) routinely analyses strains voluntarily submitted by clinical laboratories as well as those from periodic surveillance requested by national or regional health authorities. Voluntarily submitted strains are usually strains with a particular antimicrobial resistance phenotype or associated with an unusual clinical presentation, such as recurrent or severe infections, or for investigation of clusters. The request form includes information about the time of sampling, the type of sample, whether a cluster was suspected or the patient had recently travelled, and the type of infection. In summer of 2023, the National Reference Centre (NRC) received an unusually high number of MSSA strains coresistant to fusidic acid and mupirocin associated with skin and soft tissue infections (SSTIs). These strains did not seem to be epidemiologically related, as they originated from different clinical laboratories in Flanders and Brussels. Therefore, we performed a 10-year retrospective analysis of the evolution of resistance to fusidic acid and mupirocin among community-associated MSSA strains isolated from skin and soft tissue swabs in two large clinical laboratories. We also reviewed the genetic characteristics of mupirocin- and fusidic acid-associated community-onset MSSA reported to the NRC during the same period.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eFusidic acid and mupirocin resistance in MSSA strains isolated from skin and soft tissue swabs\u003c/p\u003e\n\u003cp\u003eDemographic and antimicrobial resistance data of MSSA isolated from skin and soft tissue swabs of outpatients in two large clinical laboratories in Belgium between October 2013 and September 2023 were studied: LHUB-ULB (Brussels), a clinical laboratory serving five university hospitals (with a total capacity of approximately 3,000 beds) as well as a network of general practitioners in the Brussels region covering a service area of 700,000 inhabitants, and AML (Antwerp), a private clinical laboratory belonging to the Sonic Healthcare group covering a large network of general practitioners and sampling sites in the Flanders region. The data collected included the sampling date, age at sampling, resistance to fusidic acid and resistance to mupirocin, as determined by a Vitek\u0026reg; 2 (bioM\u0026eacute;rieux, Marcy l'Etoile, France) according to EUCAST recommendations. Resistance to fusidic acid and mupirocin was studied over a 1-year period from October of a given year to September of the following year. As impetigo is more common in children and at the end of summer [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], a separate analysis was performed for children (\u0026lt;\u0026thinsp;15 years) and during the third quarter of each year (Q3, July to September).\u003c/p\u003e\n\u003cp\u003eMolecular analysis of fusidic acid and mupirocin coresistant SSTI-associated MSSA\u003c/p\u003e\n\u003cp\u003eBelgian clinical laboratories voluntarily send strains to the NRC for investigation of clusters, resistance mechanisms or virulence genes. The strains included in the present study were MSSA strains isolated from skin lesions collected between October 2013 and September 2023 and sent to the NRC for detection of virulence genes. A retrospective analysis was performed on fusidic acid and mupirocin coresistant strains (as determined by disk diffusion according to EUCAST guidelines). As part of the routine analyses carried out by the NRC, resistance to mupirocin was confirmed by end-point PCR detection of the \u003cem\u003emupA\u003c/em\u003e gene [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e], detection of exfoliatins was assessed by end-point PCR detection of the \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e genes [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], and isolates for which at least one exfoliatin gene was detected were further analysed by \u003cem\u003espa\u003c/em\u003e typing (sequencing of the polymorphic X region of the protein A gene) [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eStrains received from October 2021 to September 2023 were additionally analysed by whole-genome sequencing. DNA extraction was performed using EZ1\u0026amp;2 Virus Mini Kit v2.0 and the EZ2\u0026reg; Connect MDx instrument (Qiagen, Hilden, Germany). Genomic deoxyribonucleic acid (DNA) was enzymatically fragmented according to the manufacturer's instructions and modified to generate an Illumina-compatible DNA library using NEBNext\u0026reg; Ultra\u0026trade; II FS DNA Library Prep Kit for Illumina. The final libraries were qualified using an AATI Fragment Analyser (Agilent Technologies Inc., Santa Clara, CA, USA) with DNF-474 High Sensitivity NGS Fragment Analysis Kit and quantified using a Qubit 2.0 with Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA). After equimolar pooling, the libraries were sequenced using a NovaSeq 6000 machine (Illumina Inc., San Diego, CA, USA) with NovaSeq 6000 SP Reagent Kit v1.5 (300 cycles) in 2 x 150 bp paired mode. An average coverage of 100\u0026times; was targeted. \u003cem\u003eDe novo\u003c/em\u003e assembly was performed using the SPAdes algorithm [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The genome assemblies were deposited at the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA1041362. Resistome, virulome, multilocus sequence typing (MLST), whole-genome MLST (wgMLST) and \u003cem\u003espa\u003c/em\u003e type determination were performed using the BioNumerics 8.1 (bioM\u0026eacute;rieux, Marcy l'Etoile, France) \u003cem\u003eS. aureus\u003c/em\u003e genotyping plugin v1.1 (database \u003cem\u003eS. aureus\u003c/em\u003e Virulence KB 2022.12.05 and database \u003cem\u003eS. aureus\u003c/em\u003e Resistance KB 2023.10.27), the Spa typing plugin v2.23, the WGS tools plugin v1.08 and MLST for the WGS plugin v1.0. A wgMLST cluster analysis was performed using the categorical distance and UPGMA (unweighted pair group method with arithmetic mean) algorithms with BioNumerics. Thirty genome assemblies of CC121 strains downloaded from the NCBI GenBank database and selected from a previous study by Zhou et al. [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e] to include isolates carrying \u003cem\u003eeta\u003c/em\u003e and/or \u003cem\u003eetb\u003c/em\u003e and to cover different countries and different \u003cem\u003espa\u003c/em\u003e types and the genome of a mupirocin-susceptible EEFIC from a previous surveillance in Belgium [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e] were included as reference genomes. The list of selected isolates is provided in Supplementary Table S1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFusidic acid and mupirocin rates among MSSA isolated from outpatient SSTI swabs\u003c/p\u003e\n\u003cp\u003eFrom October 2013 to September 2023, 21,232 MSSA were isolated by culture from SSTI swabs in the LHUB-ULB, including 4,389 from children. In total, 11,271 SSTI-related MSSA strains were isolated from AML patients, including 1941 from children. Overall, resistance to fusidic acid increased from 3.1% (68/2211) to 11.4% (263/2300) in LHUB-ULB and from 17.7% (153/862) to 25.5% (341/1336) in AML from October 2013 to September 2013 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In both cases, a slight decrease was observed during the COVID-19 pandemic, in 2019\u0026ndash;2020 for LHUB-ULB and in 2020\u0026ndash;2021 for AML. During the same period, resistance to mupirocin increased from 0.5% (10/2211) to 1.7% (38/2300) in LHUB-ULB and from 1.5% (13/862) to 5.6% (75/1336) in AML. Similarly, coresistance to fusidic acid and mupirocin increased from 0.04% (1/2211) to 1.4% (33/2300) in LHUB-ULB and from 0.8% (7/862) to 5.3% (71/1336) in AML.\u003c/p\u003e\n\u003cp\u003eSimilar trends, but at higher rates, were observed for children. Fusidic acid resistance increased from 3.6% (19/527) to 18.3% (92/504) in LHUB-ULB and from 32.0% (47/147) to 45.9% (124/270) in AML. Resistance to mupirocin increased from 0.4% (2/527) to 5.2% (26/504) in LHUB-ULB and from 2.7% (4/147) to 9.3% (25/270) in AML. Coresistance to fusidic acid and mupirocin increased from 0% (0/527) to 5.0% (25/504) in LHUB-ULB and from 0% (0/147) to 8.5% (23/270) in AML.\u003c/p\u003e\n\u003cp\u003eFor strains isolated from children in Q3 of each year, resistance to fusidic acid increased from 0.7% (1/149) to 23.2% (39/168) in LHUB-ULB and from 45.2% (19/42) to 54.1% (59/109) in AML from 2014 to 2023 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Resistance to mupirocin increased from 0.7% (1/149) to 8.9% (15/168) in LHUB-ULB and from 0% (0/42) to 11.9% (13/109) in AML. Coresistance to fusidic acid and mupirocin increased from 0% (0/149) to 8.9% (15/168) in LHUB-ULB and from 0% (0/42) to 10.1% (11/109) in AML. From October 2018 to September 2023, 91.4% (64/70) of the mupirocin-resistant MSSA strains were coresistant to fusidic acid. The raw data are shown in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003eGenomic study of fusidic acid and mupirocin coresistant SSTI-associated MSSA\u003c/p\u003e\n\u003cp\u003eFrom October 2013 to September 2023, the NRC received a total of 58 MSSA strains isolated from SSTIs that were coresistant to mupirocin and fusidic acid from 29 different laboratories across Belgium (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). No cluster suspicion or recent travel history was reported on any of the accompanying request forms. No laboratory sent more than 3 strains per period. Thirty-seven MSSA strains originated from Flanders (63.8%), 12 (20.7%) from the Brussels region and 9 (15.5%) from Wallonia. The number of strains received increased from zero in 2013\u0026ndash;2014 to 19 in 2022\u0026ndash;2023. A total of 40/58 (69.0%) were isolated from children; 51/57 (89.5%) were positive for \u003cem\u003eeta\u003c/em\u003e and 47/57 (82.5%) for \u003cem\u003eetb\u003c/em\u003e. All strains tested for \u003cem\u003emupA\u003c/em\u003e (39/39) were positive. Of the 54 \u003cem\u003espa\u003c/em\u003e typings performed, 35 (63.6%) were t1994, and 5 had closely related \u003cem\u003espa\u003c/em\u003e types (t162, t2391, t21368), all of which were closely related to t171, which was associated with the original description of EEFIC [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Furthermore, all 40 of these isolates carried \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e. Therefore, since October 2017, 40/51 (78.4%) of MSSA strains coresistant to fusidic acid and mupirocin isolated from SSTIs in Belgium are similar to EEFIC, but in addition to their exfoliatins and fusidic acid resistance, they also carry the \u003cem\u003emupA\u003c/em\u003e gene, which confers resistance to mupirocin.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eFusidic acid- and mupirocin-resistant methicillin-susceptible \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MSSA) isolates from skin and soft tissue infections sent to the Belgian National Reference Centre by different clinical laboratories from October 2014 to September 2023. \u003cem\u003eeta\u003c/em\u003e: exfoliatin a gene detection, \u003cem\u003eetb\u003c/em\u003e: exfoliatin b gene detection, \u003cem\u003emupA\u003c/em\u003e: mupA gene detection, CC121: \u003cem\u003eS. aureus\u003c/em\u003e clonal complex 121\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePeriod\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNumber of sending laboratories\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAge\u0026thinsp;\u0026lt;\u0026thinsp;15 years\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eeta\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eetb\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003emupA\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCC121-related \u003cem\u003espa\u003c/em\u003e type\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2014-09/2015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2015-09/2016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2016-09/2017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2017-09/2018\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7/7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7/7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7/7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7/7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6/7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2018-09/2019\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3/6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2019-09/2020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3/4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3/4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2020-09/2021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3/3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2021-09/2022\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8/14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13/14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12/14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5/5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9/13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/2022-09/2023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14/19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17/18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16/18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15/15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13/16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e58\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e40/58\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e51/57\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e47/57\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e39/39\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e40/54\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWhole-genome sequencing was performed on the 33 MSSA strains received at the NRC between October 2021 and September 2023. These strains were obtained from 19 different laboratories. Twenty strains were from Flanders, 7 from Wallonia and 6 from the Brussels region. Whole-genome sequencing revealed that all the strains carried \u003cem\u003emupA\u003c/em\u003e, with 30/33 (90.9%) being ST121; hence, these strains are CC121 strains. Of these, 28/30 (93.3%) carried \u003cem\u003eeta\u003c/em\u003e, and 30/30 carried \u003cem\u003eetb\u003c/em\u003e. None of them carried \u003cem\u003elukS/lukF\u003c/em\u003e. Twenty-nine (96.7%) carried the acquired gene \u003cem\u003efusB\u003c/em\u003e, while 1/30 (3.2%) carried the L461K-mutated chromosomal \u003cem\u003efusA\u003c/em\u003e, conferring resistance to fusidic acid. In addition, 26/30 (86.7%) carried the \u003cem\u003eaadD\u003c/em\u003e gene, which confers resistance to amikacin, and 6/30 (20.0%) carried the \u003cem\u003ecat(pC194)\u003c/em\u003e gene, which confers resistance to chloramphenicol. The remaining three isolates were negative for \u003cem\u003eetb\u003c/em\u003e, two are ST45 and negative for \u003cem\u003eeta\u003c/em\u003e (\u003cem\u003espa\u003c/em\u003e type t550), and one is ST15 (\u003cem\u003espa\u003c/em\u003e type t084) and harboured \u003cem\u003eetb\u003c/em\u003e. The wgMLST analysis showed that the 29 ST121 isolates carrying \u003cem\u003efusB\u003c/em\u003e are likely to be clonal. In addition, the location of \u003cem\u003efusB\u003c/em\u003e in these strains was away from \u003cem\u003egroEL\u003c/em\u003e, contrary to the original description of EEFIC [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e], but the strains carried the gene encoding epidermal differentiation inhibitor C (EDIN-C) close to \u003cem\u003eetb\u003c/em\u003e. These strains clustered with the reference genome of an MSSA isolated in Greece in 2018 (GenBank accession GCA_003605275.1). This MSSA carried \u003cem\u003emupA\u003c/em\u003e, \u003cem\u003efusB\u003c/em\u003e, \u003cem\u003eaadD\u003c/em\u003e, \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e. These isolates are more closely related to each other than to the ST123 EEFIC observed in Belgium in 2020, while the ST121 MSSA with the \u003cem\u003efusA\u003c/em\u003e mutation is even more distantly related (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Additional data are shown in Supplementary Table S3.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOverall, mupirocin resistance seems to have increased since October 2019. It remains at low prevalence (below 2%) in the LHUB-ULB population, whereas it reaches 5.6% in AML. Focusing on the paediatric population, a worrying trend is observed for the last 5 years: the prevalence of mupirocin resistance reached more than 5% for the period October 2022-September 2023 and was almost systematically associated with resistance to fusidic acid (64/70, 91.4%). Focusing on children during Q3, the impetigo season [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], revealed an even more alarming trend, with mupirocin resistance rates ranging from 8.9\u0026ndash;11.9% and an almost systematic association with fusidic acid resistance (26/28, 92.9%). Based on analysis of the isolates received by the NRC since October 2017, 78.4% of the mupirocin and fusidic acid coresistant isolates were confirmed to be related to EEFIC. Whole-genome sequencing analysis of isolates collected during the last 2 years support this trend, with 29/33 clonal isolates (87.9%) sharing the following genetic characteristics: ST121, resistance to fusidic acid conferred by \u003cem\u003efusB\u003c/em\u003e, resistance to mupirocin conferred by \u003cem\u003emupA\u003c/em\u003e, and the presence of the virulence genes \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e. In a previous study on subpopulations of \u003cem\u003eS. aureus\u003c/em\u003e CC121, Kurt et al. showed that isolates sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, and conjunctivitis) clustered in the same clade, including EEFIC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Interestingly, wgMLST cluster analysis showed that these strains are closely related to the reference genome of an MSSA isolated in Greece, which shares the same characteristics, while the other reference genomes from SSTI-related isolates are more distantly related. However, in contrast to a previous study in Greece describing the emergence of a similar ST121 clone [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], none of them carried the \u003cem\u003elukS\u003c/em\u003e/\u003cem\u003elukF\u003c/em\u003e genes. Notably, 1/33 of the strains is ST15 with a t084 \u003cem\u003espa\u003c/em\u003e type and carrying \u003cem\u003eetb\u003c/em\u003e. ST15 strains carrying exfoliatin genes have been observed quite frequently in staphylococcal scalded skin syndrome in France [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTreatment of impetigo is often based on the use of topical antibiotics for localised forms, such as fusidic acid cream in Belgium and the Netherlands [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] or mupirocin in France [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This option is preferred for localised disease because it has been shown to be at least as effective as oral treatment, with fewer side effects [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, the emergence and spread of an MSSA clone coresistant to these two antibiotics is a cause for concern. Other possible topical treatments are retapamulin [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and ozenoxacin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, in the European Union, marketing authorisation for retapamulin was withdrawn by the European Commission on 25 February 2017 at the request of the manufacturer. At the time of manuscript writing, ozenoxacin was only available in Italy, Portugal and Spain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Interestingly, the emerging clone described in the present study frequently carried \u003cem\u003eaadD\u003c/em\u003e and \u003cem\u003ecat(pC194)\u003c/em\u003e, which confer resistance to amikacin and chloramphenicol, respectively. Although these antibiotics are not used in Belgium, they may be used as topical treatments. Acquisition of these resistance genes is likely a selective advantage for strains with a propensity for superficial skin infections. On the other hand, the efficacy of disinfection as sole treatment for impetigo seems to be inferior to that of topical antibiotics [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], even though recommendations in the United Kingdom favour the single use of antiseptics [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the development of resistance to topical antibiotics may change this consideration, and further studies are needed to determine the best interventions for localised impetigo.\u003c/p\u003e \u003cp\u003eImpetigo is known to affect children in summer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, in a previous surveillance focused on \u003cem\u003eS. aureus\u003c/em\u003e-related SSTIs, we showed that EEFIC was more prevalent in children in late summer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. At that time, resistance to mupirocin was anecdotal. However, one of the limitations of this surveillance was that it was conducted during the summer immediately after onset of the COVID-19 crisis. The social distancing and restrictions that gradually came into force during this period, combined with the workload of the clinical laboratories during the study period, certainly introduced biases. The end of the COVID-19-related restrictions and the increase in mupirocin-resistant strains arriving at the NRC led us to perform the present study using immediately available retrospective laboratory data. As strains are sent to the NRC by clinical laboratories on a voluntary basis, NRC data may not always reflect the true epidemiological situation. To determine the accuracy of the emergence of a new clone, we collected epidemiological data from two large laboratories in Belgium (LHUB-ULB and AML). In a previous study focusing on respiratory viruses, LHUB-ULB was demonstrated to act as a sensor for the whole country due to the large number of analyses and its central location within Belgium [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Inclusion of AML allowed the addition of data from a different type of laboratory (LHUB-ULB is more of a hospital laboratory, whereas AML, located in Flanders, mainly collects samples directly from general practitioners and outpatient sampling centres). However, additional data from a laboratory in the third region of Belgium (Wallonia) could have made the data completely exhaustive. Setting up an actual prospective surveillance would have been more accurate but would have caused a delay in communicating these results. Nevertheless, such a strategy has limitations: there are no data about the representativeness of AML and LHUB-ULB in Belgium for impetigo. Furthermore, the proportion of skin lesions that are sampled before treatment is unknown, and the total number of skin samples sent to laboratories per year is not known, as these data were not collected by health insurance providers. This bias is limited by the overall number of strains included (32,503 MSSA over 10 years) but may lead to some imprecision when focusing on a specific population during a specific time period, as we did in this study for children during the third quarter of the year. The epidemiological differences observed between AML and LHUB-ULB could be due to regional differences and/or differences between the patients sampled (case mix). The impact of the latter is likely mitigated by the fact that we specifically selected samples from outpatients. Nevertheless, AML collects samples from the federal agency for reception of asylum seekers (FEDASIL), and identification of epidemiological clusters at the level of the clinical laboratory is not easy because the context is rarely provided by the prescribers for outpatients. Regional epidemiological differences may exist. For example, in the most recent surveillance study on the EEFIC in Belgium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], all EEFICs were from Flanders, and almost half of them (47.4%) were located in the province of Antwerp, where AML is located. From October 2013 to September 2023, the majority of MSSA strains coresistant to mupirocin and fusidic acid analysed by the NRC in this study were from Flanders, followed by Brussels and Wallonia. Beyond an actual regional difference, it is also possible that laboratories and practitioners in Flanders are more aware of the current epidemiological situation due to the EEFIC clusters in 2018 that led to our previous surveillance; in contrast, these clusters were not described in Wallonia.\u003c/p\u003e \u003cp\u003eAlthough very common, impetigo is likely an understudied pathology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Indeed, there is currently no organised surveillance of impetigo in Belgium. As a consequence, epidemiological data rely on laboratory data, as in this study. At the laboratory level, clinical information on the type of infection is often lacking. We attempted to overcome these problems in our study by including skin samples from outpatients only and by focusing on the more likely target population for impetigo: children sampled in late summer (Q3). Additionally, uncomplicated impetigo may not be sampled. This may lead to bias towards complicated skin infections that should not be treated with topical antibiotics. Nevertheless, this study clearly demonstrated the emergence of a clone combining virulence and resistance. Periodic surveillance is needed to better monitor the true epidemiology of impetigo and adapt treatment recommendations accordingly. In addition, this surveillance may provide useful information for developing intervention trials for impetigo patients.\u003c/p\u003e \u003cp\u003eThe present study highlights the emergence and spread of a clonal ST121 mupirocin-resistant clone diverging from EEFIC (M-EEFIC), which has acquired the \u003cem\u003emupA\u003c/em\u003e gene in addition to its virulence (\u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e) and resistance (\u003cem\u003efusB\u003c/em\u003e) genes. This clone could have a selective advantage in Belgium and similar European countries where there is no other alternative topical treatment. The emergence of this new clone, combined with the overall high prevalence of fusidic acid resistance, should lead to reconsideration of the recommendations for first-line treatment of impetigo in Belgium and introduction of active surveillance of impetigo to better assess the situation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical statement\u003c/p\u003e\u003cp\u003eThis was an epidemiological retrospective observational study using aggregated anonymous data; therefore, no ethical approval was needed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eO\u0026rsquo;Neill AJ, Larsen AR, Skov R, Henriksen AS, Chopra I. Characterization of the Epidemic European Fusidic Acid-Resistant Impetigo Clone of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Journal of Clinical Microbiology. 2007;45(5):1505\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eR\u0026oslash;rtveit S, Skutlaberg DH, Langeland N, Rortveit G. The decline of the impetigo epidemic caused by the epidemic European fusidic acid-resistant impetigo clone: an 11.5-year population-based incidence study from a community in Western Norway. Scandinavian Journal of Infectious Diseases. 2014;46(12):832\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eDalager-Pedersen M, S\u0026oslash;gaard M, Sch\u0026oslash;nheyder HC. Staphylococcus aureus skin and soft tissue infections in primary healthcare in Denmark: a 12-year population-based study. European Journal of Clinical Microbiology \u0026amp; Infectious Diseases. 2011 Aug 1;30(8):951\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eDeplano A, Hallin M, Bustos Sierra N, Michel C, Prevost B, Martiny D, et al. Persistence of the Staphylococcus aureus epidemic European fusidic acid-resistant impetigo clone (EEFIC) in Belgium. Journal of Antimicrobial Chemotherapy. 2023 Aug 1;78(8):2061\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eBAPCOC. Guide belge de traitement anti-infectieux en pratique ambulatoire/Belgische gids voor anti-infectieuze behandeling in de ambulante praktijk [Internet]. BAPCOC; 2022 [cited 2024 Mar 1]. Available from: https://organesdeconcertation.sante.belgique.be/fr/documents/guide-belge-de-traitement-anti-infectieux-en-pratique-ambulatoire-2022\u003c/li\u003e\n\u003cli\u003eBons S, Bouma M, Daujer L, Koning S, Mulder L, Warnier M, et al. Bacteri\u0026euml;le huidinfecties (M68) [Internet]. Nederlands Huisartsen Genootschap; 2019 [cited 2023 Feb 17]. Available from: https://richtlijnen.nhg.org/files/pdf/61_Bacteri%C3%ABle%20huidinfecties_mei-2019.pdf\u003c/li\u003e\n\u003cli\u003eDoudoulakakis Anastassios, Spiliopoulou Iris, Spyridis Nikolaos, Giormezis Nikolaos, Kopsidas John, Militsopoulou Maria, et al. Emergence of a Staphylococcus aureus Clone Resistant to Mupirocin and Fusidic Acid Carrying Exotoxin Genes and Causing Mainly Skin Infections. Journal of Clinical Microbiology. 2017 Jul 25;55(8):2529\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eHaute autorit\u0026eacute; de sant\u0026eacute;. Prise en charge des infections cutan\u0026eacute;es bact\u0026eacute;riennes courantes [Internet]. Haute autorit\u0026eacute; de sant\u0026eacute;; 2019 [cited 2023 Feb 17]. Available from: https://www.has-sante.fr/upload/docs/application/pdf/2019-04/prise_en_charge_des_infections_cutanees_bacteriennes_courantes_recommandations.pdf\u003c/li\u003e\n\u003cli\u003eLoffeld A, Davies P, Lewis A, Moss C. Seasonal occurrence of impetigo: a retrospective 8‐year review (1996\u0026ndash;2003). Clinical and Experimental Dermatology. 2005 Sep;30(5):512\u0026ndash;4. \u003c/li\u003e\n\u003cli\u003eRamsey MA, Bradley SF, Kauffman CA, Morton TM. Identification of chromosomal location of mupA gene, encoding low-level mupirocin resistance in staphylococcal isolates. Antimicrobial Agents and Chemotherapy. 1996 Dec;40(12):2820\u0026ndash;3. \u003c/li\u003e\n\u003cli\u003eJarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, et al. Relationships between \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Genetic Background, Virulence Factors, \u003cem\u003eagr\u003c/em\u003e Groups (Alleles), and Human Disease. Infection and Immunity. 2002 Feb;70(2):631\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003eHallin M, Deplano A, Denis O, De Mendon\u0026ccedil;a R, De Ryck R, Struelens MJ. Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections. J Clin Microbiol. 2007 Jan;45(1):127\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eBankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. Journal of Computational Biology. 2012 May 1;19(5):455\u0026ndash;77. \u003c/li\u003e\n\u003cli\u003eZhou W, Jin Y, Teng G, Chen W, Chen Y, Luo Q, et al. Comparative analysis of genomic characteristics, virulence and fitness of community-associated Staphylococcus aureus ST121 clone causing fatal diseases in China and other CA-MRSA clones. Virulence. 2023 Dec 31;14(1):2242547. \u003c/li\u003e\n\u003cli\u003eKurt K, Rasigade JP, Laurent F, Goering RV, Žemličkov\u0026aacute; H, Machova I, et al. Subpopulations of Staphylococcus aureus Clonal Complex 121 Are Associated with Distinct Clinical Entities. PLOS ONE. 2013 Mar 7;8(3):e58155. \u003c/li\u003e\n\u003cli\u003eLamand V, Dauwalder O, Tristan A, Casalegno JS, Meugnier H, Bes M, et al. Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains. Clinical Microbiology and Infection. 2012 Dec;18(12):E514\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eKoning S, Sande R, Verhagen A, Smit L, Morris A, Butler C, et al. Interventions for impetigo. Cochrane database of systematic reviews. 2012;1(1):CD003261-CD003261. \u003c/li\u003e\n\u003cli\u003eAjay George, Greg Rubin. A systematic review and meta-analysis of treatments for impetigo. Br J Gen Pract. 2003 Jun 1;53(491):480. \u003c/li\u003e\n\u003cli\u003eYang LPH, Keam SJ. Retapamulin: a review of its use in the management of impetigo and other uncomplicated superficial skin infections. Drugs. 2008;68(6):855\u0026ndash;73. \u003c/li\u003e\n\u003cli\u003eRosen T, Albareda N, Rosenberg N, Alonso FG, Roth S, Zsolt I, et al. Efficacy and Safety of Ozenoxacin Cream for Treatment of Adult and Pediatric Patients With Impetigo: A Randomized Clinical Trial. JAMA Dermatology. 2018 Jul 1;154(7):806. \u003c/li\u003e\n\u003cli\u003eEuropean Medicines Agency. Medicines [Internet]. [cited 2024 Feb 21]. Available from: https://www.ema.europa.eu/en/medicines\u003c/li\u003e\n\u003cli\u003eNational Institute for Health and Care Excellence. Impetigo: antimicrobial prescribing [Internet]. National Institute for Health and Care Excellence; 2020 [cited 2023 Feb 17]. Available from: https://www.nice.org.uk/guidance/ng153\u003c/li\u003e\n\u003cli\u003eVan den Wijngaert S, Bossuyt N, Ferns B, Busson L, Serrano G, Wautier M, et al. Bigger and Better? Representativeness of the Influenza A Surveillance Using One Consolidated Clinical Microbiology Laboratory Data Set as Compared to the Belgian Sentinel Network of Laboratories. Frontiers in Public Health. 2019;7.\u003c/li\u003e\n\u003cli\u003eGorges H, Hall L, Heal C. Feasibility Study for a Randomised Controlled Trial for the Topical Treatment of Impetigo in Australian General Practice. Tropical Medicine and Infectious Disease. 2021;6(4). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"Université Libre de Bruxelles","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, Impetigo, Child, Mupirocin, Fusidic Acid, Virulence, Exfoliatins, Belgium, Whole Genome Sequencing","lastPublishedDoi":"10.21203/rs.3.rs-4074618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4074618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCoresistance to both mupirocin and fusidic acid in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e affects the treatment of impetigo in Belgium, where they are the only topical treatments available.\u003c/p\u003e\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eWe investigated resistance to fusidic acid and mupirocin in methicillin-susceptible \u003cem\u003eS. aureus\u003c/em\u003e (MSSA) strains involved in community-acquired skin and soft tissue infections (SSTIs).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe 10-year variation in fusidic acid and mupirocin resistance in outpatients with SSTI-associated MSSA was studied in two large laboratories. MSSA strains coresistant to fusidic acid and mupirocin and sent to the Belgian \u003cem\u003eStaphylococci\u003c/em\u003e Reference Centre were \u003cem\u003espa\u003c/em\u003e-typed and analysed for the presence of the \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e virulence genes and the \u003cem\u003emupA\u003c/em\u003e resistance gene. In addition, whole-genome sequencing was performed on isolates collected in the last 2 years.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFusidic acid and mupirocin resistance increased over time, and coresistance in children reached 8.9\u0026ndash;10.1% in the third quarter 2023. From 2018 to 2023, 64/70 (91.4%) mupirocin-resistant MSSA strains were coresistant to fusidic acid. Whole-genome sequencing revealed that 29/33 (87.9%) of the isolates were sequence type ST121, clonal and more distantly related to the European epidemic fusidic acid-resistant impetigo clone (EEFIC) observed in Belgium in 2020. These strains carried the \u003cem\u003emupA\u003c/em\u003e and \u003cem\u003efusB\u003c/em\u003e genes, which confer resistance to mupirocin and fusidic acid, respectively, and the \u003cem\u003eeta\u003c/em\u003e and \u003cem\u003eetb\u003c/em\u003e virulence genes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWe highlight the spread of a mupirocin-resistant EEFIC (M-EEFIC) in children, with a seasonal trend for the third quarter. This is of concern because this variant is resistant to the two main topical antibiotics used to treat impetigo in Belgium.\u003c/p\u003e","manuscriptTitle":"Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus in Belgium, 2013 to 2023","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-12 16:06:30","doi":"10.21203/rs.3.rs-4074618/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":"c1692b32-25e2-4e4e-840f-31d130b9b111","owner":[],"postedDate":"March 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30765599,"name":"Infectious Diseases"},{"id":30765600,"name":"Pediatrics"},{"id":30765601,"name":"Bacteriology"},{"id":30765602,"name":"Laboratory Diagnostics"},{"id":30765603,"name":"Molecular Epidemiology"},{"id":30765604,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2024-05-14T10:17:36+00:00","versionOfRecord":{"articleIdentity":"rs-4074618","link":"https://doi.org/10.2807/1560-7917.ES.2024.29.19.2300668","journal":{"identity":"eurosurveillance","isVorOnly":true,"title":"Eurosurveillance"},"publishedOn":"2024-05-09 10:17:36","publishedOnDateReadable":"May 9th, 2024"},"versionCreatedAt":"2024-03-12 16:06:30","video":"","vorDoi":"10.2807/1560-7917.ES.2024.29.19.2300668","vorDoiUrl":"https://doi.org/10.2807/1560-7917.ES.2024.29.19.2300668","workflowStages":[]},"version":"v1","identity":"rs-4074618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4074618","identity":"rs-4074618","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}