Control of a hospital outbreak with methicillin-resistant Staphylococcus argenteus in The Netherlands, June–September 2023

Abstract

Background

Staphylococcus argenteus belongs to the Staphylococcus aureus complex and causes similar disease. Methicillin-resistant S. argenteus (MRSArg) is rare in the Netherlands. We describe the first hospital outbreak and its management.

Methods

After a healthcare worker linked to a patient with MRSArg bacteremia tested positive, cohort screening of healthcare workers, patients, and family members was performed. Carriers received eradication therapy. Whole-genome multilocus sequence typing (wgMLST) and antimicrobial susceptibility testing were used to characterize isolates and mupirocin resistance.

Results

Fifteen (2.6%) MRSArg carriers were detected: 9/317 (2.8%) healthcare workers, 4/238 (1.7%) patients, 2/16 (12.5%) family members; age range 22–88 years, median 55 years. Nine (60%) were complicated and six (40%) uncomplicated carriers. After transfer of one initially negative patient to a nursing home, subsequent positivity led to two carriers among nursing home staff. wgMLST confirmed a single outbreak cluster of 15 isolates, all MLST ST2250. The isolates carried mecA, dfrG, and blaZ, but not PVL. Eradication therapy was started in 13/15 (86.7%) persons, with 84.6% (11/13) successful eradication. Six isolates carried a V588F-mutated ileS gene and had mupirocin MICs of 2–32 mg/L; nine were sensitive.

Conclusions

We report the first successfully controlled MRSArg hospital outbreak in the Netherlands. The outbreak strain was clonally related and showed low-level mupirocin resistance linked to a mutated ileS gene. This work supports the importance of infection prevention and control measures that are applied for MRSA are also effective for MRSArg.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13756-026-01756-1.

Background

Staphylococcus argenteus is part of the Staphylococcus aureus-complex and was first described in 2006. It was shown to belong to multilocus sequence typing (MLST) clonal complex 75 (CC75) of S. aureus, but these CC75 isolates were distinct from S. aureus and were designated S. argenteus as novel species in 2015 [1, 2]. In the Netherlands, the first methicillin-resistant S. argenteus (MRSArg) isolate was submitted for typing in 2008 to the National Institute for Public Health and the Environment (RIVM) for national MRSA surveillance via Type-Ned. A retrospective analysis of 64 isolates which were non-typeable with multilocus variable-number tandem-repeat analysis MLVA designed for methicillin-resistant S. aureus (MRSA), revealed 54 MRSArg of 52,467 (0.1%) MRSA isolates submitted as MRSA from January 2008 until March 2021 [3]. Most of the MRSArg isolates in the Netherlands carried the SCCmec subtype IVc(2B), had a trimethoprim resistance gene, dfrG, and harbored a blaZ-carrying plasmid. These Dutch S. argenteus isolates belonged to ST2250, which represents a globally spread lineage of S. argenteus. Other MRSArg sequence types found in the Netherlands are ST1223, ST1850, and ST2793 (3). Overall, the prevalence of MRSArg in diagnostic S. argenteus isolates the Netherlands is still very low, around 1%, and comparable to the prevalence of MRSA, based on data from the Dutch national surveillance system of antimicrobial resistance (ISIS-AR) [4].

Various reports and studies showed that S. argenteus causes similar disease as S. aureus [5, 6]. However, data on carriage, person-to-person transmission and outbreaks in the European Union/European Economic Area (EU/EEA) are scarce. Two studies report carriage rates of 0.9% (5/563) in nares of healthy food handlers in Myanmar and 4.1% (6/146) of S. argenteus in nares of healthy individuals in a dental clinic in the United Arab Emirates [7, 8]. Another study reported a cluster of S. argenteus strains in a small group (4/40) of Royal Marine recruits with potential transmission events within the troop or presence of spread from a common source [9]. Transmission of methicillin-resistant S. argenteus (MRSArg) in a family has been described in Stockholm, Sweden [10].

Mupirocin is the cornerstone of nasal carriage eradication therapy of MRSA and alternatives for local treatment are less effective [11, 12]. Thus far, no evidence has been found for the presence of mupirocin resistance determinants in the MRSArg population in the Netherlands. In contrast, two types of mupirocin resistance have been described in MRSA, with high-level resistance being associated with the plasmid-mediated mupA gene and low-level resistance being associated with chromosomal point mutations in the isoleucyl-tRNA synthetase (ileS) gene, respectively [13–15]. In Europe, 8% of the MRSA isolates carried high-level resistance for mupirocin [15]. As of January 2024, mupirocin resistance has not been detected in methicillin-susceptible or -resistant Staphylococcus argenteus (MSSArg/MRSArg) isolates in the Netherlands, according to data from the ISIS-AR surveillance system [16]. Members of the ESCMID Study Group for Staphylococci and Staphylococcal Diseases (ESGS) suggest that the same infection prevention and control measures must be applied for MRSArg as for MRSA [6], but data to support the effectiveness of the same measures are unknown. Here, we describe the successful control of the first large hospital outbreak of MRSArg in the Netherlands and the EU/EEA in patients and health care workers with a panton valentine leucocidin (PVL)-negative strain of MRSArg ST2250, carrying a mutated (V588F) ileS gene in a number of isolates.

Methods

Clinical setting

The Spaarne Gasthuis is a secondary teaching hospital in the Netherlands with 560 beds and 54,000 annual visits to the emergency department divided over two separate locations, and admitting 55,000 patients per year. In June 2023, a dialysis patient was admitted on the internal ward for 2 months due to a central venous line infection with a methicillin-susceptible S. aureus. Blood cultures taken during the patient’s stay later became positive for MRSArg. In the same week, a health care worker (HCW), who was screened for MRSA in contact tracing around another patient, revealed nasal and throat carriage of MRSArg. Since there was an epidemiological link between the HCW and the patient with MRSArg, an outbreak investigation was initiated. All infection control strategies and investigations were performed in accordance with the national recommendations for MRSA outbreak investigation guidelines of the Netherlands [17]. Swabs for culture of nose, throat and rectum from HCW patients and family members were collected in COPAN eSwab^®.

Infection control measures

The ‘Search and Destroy’ policy in the Spaarne Gasthuis, normally used for MRSA, focuses on infection control through active screening, isolation, and decolonization of high-risk groups [17]. These include MRSA-positive patients, individuals with recent unprotected contact with MRSA carriers, patients recently admitted to Dutch healthcare institutions with ongoing MRSA outbreaks, or those recently admitted to foreign healthcare facilities. Additional high-risk groups include individuals with recent contact with commercially kept pigs, veal calves, or broiler chickens, foreign dialysis patients treated in the Netherlands, children adopted from abroad at first healthcare contact, and individuals recently residing in refugee reception centers. High-risk patients are pre-emptively isolated until screening results are available. Low-risk groups are screened but not isolated unless positive; these include patients with intensive unprotected healthcare contact with an MRSA-positive healthcare worker, recent hemodialysis abroad, previous admission to a foreign healthcare facility (2–12 months prior) with additional risk factors, patients in follow-up after previous MRSA carriage with repeated negative tests, and patients with persistent exposure but a recent negative MRSA test. Screening involved swabs from the throat, nose, and rectum for inpatients, outpatients and dialysis patients. For HCWs, throat and nose swabs were obtained, with an additional rectal swab performed if the initial screening was positive.

Personal protective equipment (PPE), including gloves, gowns, and surgical masks (type IIR), is mandatory for contact with isolated patients. Positive patients receive decolonization treatment. MRSA decolonization therapy typically consists of either topical treatment alone (nasal mupirocin ointment and antiseptic skin wash therapy) for uncomplicated carriers defined by limited, primarily nasal colonization without active infection or risk factors or a combination of topical and systemic oral anti-staphylococcal agents for complicated carriers, characterized by extranasal colonization, resistance, or relevant clinical risk factors [18].

Additional cleaning protocols are implemented daily and post-discharge for isolation rooms, with extra measures for intensive care settings. Contact tracing and screening are conducted for unexpected MRSA cases, with work suspension and decolonization treatment for MRSA-positive HCWs.

MRSArg culture and molecular analysis

Staphylococcus argenteus was distinguished from Staphylococcus aureus using MALDI-TOF MS (Bruker, BioTyper Smart, database V12.0 [10833–11897] used 2022) containing reference spectra for S. argenteus. Detection of MRSArg was performed using Muller Hinton Broth + 6,5% NaCl (Thermo Fisher Scientific, Waltham, US) and selective chromogenic agar plates (Brilliance™ MRSA 2 Agar) (Thermo Fisher Scientific, Waltham, US), incubated 24 h at 35 °C. Antimicrobial susceptibility testing was performed using the VITEX-2 system (Vitex 2XL, BioMérieux, Marcy-l’Étoile, France) and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards for clinical breakpoints (version 13.1). Breakpoints for mupirocin used for nasal decolonization (S ≤ 1 mg/L; R > 256 mg/L) were applied according to the EUCAST topical breakpoint criteria (version 13.1). Isolates were screened by cefoxitin disk diffusion and molecular screening was performed by Xpert^® MRSA NxG for detection of the mecA/mecC gene. All isolates were sent to the RIVM for confirmation and whole-genome sequencing.

Whole-genome sequencing (WGS) and genomic analysis

S. argenteus isolates were subjected to WGS using both Illumina short-read and Oxford Nanopore Technologies (ONT) long-read sequencing. DNA of S. argenteus was isolated using The Maxwell^® (Promega) and the Maxwell RSC Cultured Cells DNA kit (AS1620) kit was used as described previously [19]. WGS data was used for interrogation of ResFinder, PlasmidFinder, VirulenceFinder and SCCmec Finder databases from the Center for Genomic Epidemiology (CGE) and the Comprehensive Antibiotic Resistance Database (CARD) for the identification of resistance genes, replicons, virulence determinants and the SCCmec type, respectively. A threshold of 95% was used for identity and 60% for the minimum length for ResFinder and PlasmidFinder. To determine the genetic location of ileS and ileS-V588F, outbreak MRSArg isolates were sequenced with Nanopore long-read sequencing and WGS data used in hybrid assembly with short-read WGS data to analyse the genomic elements.

wgMLST analysis of Staphylococcus argenteus

S. argenteus-specific wgMLST scheme was used as described previously [3]. The wgMLST scheme comprised 2,168 core genes and 387 accessory genes. WGS data of all S. argenteus isolates from the Netherlands in the MRSA surveillance database (Type-Ned MRSA, n = 59 from 2008 to 2023) and publicly available S. argenteus (n = 273 from 2022 to 2025) were used to place the MRSArg outbreak (n = 15) in context. Off the 273 internationally retrieved S. argenteus sequences 40 were MRSArg and 233 MSSArg. A genetic cluster e.g. outbreak is defined as ≥ 2 S. argenteus isolates that differ ≤ 20 wgMLST alleles. The allelic wgMLST profiles were imported in BioNumerics (v8.1, Applied Maths, Sint-Martens-Latem, Belgium) and used to assess genetic relationships between isolates, which were visualized in a minimum spanning tree (MST). Missing alleles were ignored and not counted as allelic differences.

Results

Outbreak description

The identification of an epidemiological link between the MRSArg-positive healthcare worker (HCW) and the patient led to the implementation of cohort screening and the formation of an outbreak management team (OMT) (Fig. 1). The cohort screening identified a total of 15 persons with MRSArg carriership among 571 screened individuals (2.6%). Of these, 9 out of 317 screened HCWs (2.8%), 4 out of 238 screened patients (1.7%) and 2 out of 16 family members (12.5%) were positive. Incidental MRSA isolates were also detected: MRSA was found in 2/238 (0.8%) patients, 2/317 (0.6%) healthcare workers (HCWs), and 0/16 (0%) family members; all isolates were unrelated. The MRSArg-positive cohort (n = 15; range: 22–88 years, median: 55 years) included 60% (n = 9) complicated and 40% (n = 6) uncomplicated carriers (Table 1). Antimicrobial susceptibility profiles of all MRSarg isolates are reported in Supplementary Table 1. Decolonization therapy was initiated in 86.7% (n = 13/15) of the positive individuals, for treatment details see Table 1. One patient did not receive eradication treatment due to death from an unrelated cause (aged 68 years), one patient had transient colonization and lost MRSArg carriage. Eradication therapy was successful in 85% (n = 11/13) of patients treated.

Fig. 1.

Timeline of newly detected persons with positive cultures of MRSArg from patients, health care workers and family members, related to the outbreak, The Netherlands, June-September 2023 (n = 15)

Table 1.

Characteristics of patients, health care workers and family members with MRSArg, the Netherlands, June-September 2023 (n = 15)

Number	Group	Age	Type of carriage	MIC mupirocin (mg/L)	V588F mutation ileS gene	Eradication therapy	Eradication result
1	Patient	65	Complicated	32	Yes	Rifampicin/clindamycin/mupirocin	Success
2	Patient	76	Complicated	2	Yes	Doxycyline/rifampicin/mupirocin	No success
3	Patient	88	Complicated	≤ 1	No	Doxycyline/rifampicin/mupirocin	Success
4	Patient	68	Uncomplicated	≤ 1	No	No therapy (deceased)	–
5	HCW	55	Complicated	2	Yes	Doxycycline/rifampicin/mupirocin	Success
6	HCW	23	Complicated	≤ 1	No	Doxycycline/rifampicin/mupirocin	Success
7	HCW	22	Complicated	≤ 1	No	Doxycycline/rifampicin/mupirocin	Success
8	HCW	63	Uncomplicated	2	Yes	Mupirocin	Success
9	HCW	46	Uncomplicated	≤ 1	No	Mupirocin	Success
10	HCW	36	Uncomplicated	32	Yes	No therapy	–
11	HCW	50	Complicated	≤ 1	No	Doxycycline/rifampicin/mupirocin	No success
12	HCW	27	Complicated	≤ 1	No	Doxycycline/rifampicin/mupirocin	Success
13	HCW	24	Uncomplicated	32	Yes	Fusidic acid	Success
14	Family	67	Complicated	≤ 1	No	Doxycycline/rifampicin/mupirocin	Success
15	Family	26	Uncomplicated	≤ 1	No	Mupirocin	Success

MIC: minimum inhibitory concentration

A patient with complicated MRSArg carriage who underwent decolonization therapy was transferred to a nursing home. After two consecutive negative follow-up cultures, infection control measures were discontinued too early according to the national guidelines. However, the third follow-up culture from the nose and throat was positive again for MRSArg. Following this, additional screening of HCWs at the nursing home identified two new cases of nasal MRSArg carriage among HCWs (Fig. 1). The analysis of the MRSArg outbreak isolates in context of MRSArg obtained in the national surveillance in 2008–2025, revealed geographic distribution in the province of North-Holland in The Netherlands (Fig. 2a).

Fig. 2.

Geographic location and minimum spanning tree with hospital outbreak-associated methicillin-resistant Stapylococcus argenteus isolates (n = 15) as well as isolates collected in The Netherlands in 2023 (n = 9) and other countries (n = 273), January 2008-May 2025 (n = 297)

Genetic relatedness of MRSArg

Whole-genome multilocus sequence typing (wgMLST) was performed on 15 MRSArg isolates from screening samples (Table 1; Fig. 2) to determine the genetic relatedness in comparison with MRSArg from the Netherlands (n = 59) and internationally retrieved S. argenteus whole-genome sequences (n = 273, 2022–2025). These sequences comprised n = 25 from United States, n = 17 from Canada, n = 76 from Thailand, n = 14 from China, n = 38 from Japan, n = 25 from Denmark, and a variety of unknown or other countries (n = 78) between 2005 and 2022. The MRSArg outbreak-associated isolates from the Netherlands clustered together and varied between 1 and 2 wgMLST alleles in the minimum spanning tree (MST) (Fig. 2b). The closest neighbor of outbreak-associated isolates in the MST was 65 allelic distances from a Dutch isolate and grouped with isolates from other countries in genomic group (GG)2250, a globally epidemic lineage.

Susceptibility of outbreak MRSArg ST2250 to mupirocin

Of the 15 MRSArg isolates, 6 isolates (6/15; 40%) exhibited low-level resistance to mupirocin. The highest measured minimum inhibitory concentration (MIC) was 32 mg/L (Table 1). These 6 isolates demonstrated a mutation in the isoleucyl-tRNA synthetase (ileS) gene as assessed by short-read sequencing leading to a Valine-588 to Fenylalanine (V588F) amino acid change, which is associated with low-level mupirocin resistance in S. aureus. In the hybrid assembled short-read and long-read data analysis, nine of 15 MRSArg yielded complete hybrid assemblies. The MRSArg outbreak isolates had a 2.8–2.9 Mb chromosome carrying either ileS (100% identity) or mutated ileS-V588F (97.27% identity), and the resistance genes dfrG, tet(38), fosB, and mecA (Fig. 3). The incomplete assemblies were omitted in this study. The genes encoding for panton valentine leucocidin, lukS-PV/lukF-PV were not detected in the outbreak strain. The outbreak strain carried one 20.6 kb plasmid with blaZ and a rep16 replicon, while a smaller plasmid of 3.8 kb did not carry known resistance genes or plasmid replicon annotations and was not detected in all isolates (Fig. 3).

Fig. 3.

Distribution of antimicrobial resistance genes on chromosomes versus plasmids of methicillin-resistant S. argenteus strains, June 2023–September 2023 (n = 9 isolates). C, chromosome; p, plasmid. The presence of AMR genes found by CARD is indicated as squares. Colors of the squares indicate % identity to the reference gene in CARD. The chromosome or plasmid size in bp is indicated.

Discussion

This study reports the first large hospital outbreak of MRSArg in the Netherlands and in the EU/EEA in 2023. The prevalence of MRSArg among diagnostic isolates in the Netherlands is low, at approximately 1% [4]. So far, no healthcare-related outbreaks were reported and only a few studies showed the transmission and carriage rates of S. argenteus and MRSArg. In the reported outbreak, fifteen MRSArg carriers were identified, of whom nine (60%) had complicated carriership according to the definitions from the national guidelines. This study demonstrates that existing infection prevention measures and treatment protocols for MRSA, which were applied in this MRSArg outbreak, proved to be sufficient to contain the hospital outbreak. Phenotypic testing showed that MRSArg in this outbreak was low-level resistant to mupirocin in 6 of the 15 (40%) MRSArg isolates. In depth WGS data analysis revealed that these isolates harboured the V588F mutation in the chromosomal gene (ileS) encoding isoleucyl-tRNA synthetase, which encodes low-level resistance to mupirocin in S. aureus, highlighting a potential emerging challenge in decolonization efforts.

However, mupirocin was used alone for decolonization in one case. In the remaining cases, it was either used in combination with other agents or replaced by alternative agents.

Various studies report nasal eradication rates of 43% to 83% for uncomplicated carriage and 74% in complicated carriage of mupirocin susceptible S. aureus or MRSA, which is comparable to our findings, despite the presence of 6 persons with low-level mupirocin resistance [20–23]. However, direct comparisons between studies remain challenging due to methodological differences and substantial variations in follow-up periods and patient populations. This cohort is too small to draw firm conclusions for the use of mupirocin for the eradication in low-level resistant isolates.

A notable feature of this outbreak was the high transmission rate towards healthcare workers. A possible explanation for this rapid transmission is the skin condition of the index patient, who had very dry skin with extensive flaking. This may have facilitated the dispersal of MRSArg through skin particles, increasing the risk of transmission via direct contact or through environmental contamination. There is evidence that patients with psoriasis have a higher risk of staphylococcal colonization than healthy controls, which may contribute to an increased risk of S. aureus transmission from individuals with flaky skin conditions due to enhanced carriage and skin shedding [24].

Although no outbreaks of MRSArg have been described in literature so far, the most relevant comparison can be made with MRSA in the Dutch healthcare setting. One study analysed all contact investigations of 774 MRSA index patients, where MRSA transmission was observed in 0.15% (41/27,377) of contacts, comprising 19/21,488 (0.09%) healthcare workers and 22/4,816 (0.46%) patients [25]. Comparison with our findings - where 9 out of 317 screened healthcare workers (2.9%), 4 out of 238 screened patients (1.7%), and 2 out of 16 family members (12.5%) tested positive - demonstrates substantially higher transmission rates in our outbreak. This marked difference may be largely attributable to the characteristics of the index patient.

The appearance of the V588F mutation in this outbreak may be linked to the widespread use of mupirocin in dialysis patients, where this antimicrobial is commonly applied to prevent infections related to intravenous catheter use. Over time, this selective pressure could promote the emergence of low-level mupirocin resistance, as seen with the V588F mutation, as also described for MRSA in literature [26]. The V588F mutation spread does not result in phenotypic low-level mupirocin resistance in all patients. It is possible that a mixed population of wild-type MRSArg exists within the patient. This study benefits from a well-characterized outbreak, including the use of short-read and long-read sequencing, providing high-resolution insights into the genetic makeup and resistance mechanisms of MRSArg. A key limitation is that the findings are based on a single outbreak, making it challenging to assess whether the transmission potential of MRSArg is comparable to that of MRSA in a wider variety of settings. The findings of this study have several important implications. First, the current MRSA treatment guidelines appear to be effective against MRSArg, indicating that no immediate modifications are required to address this rare highly-resistant microorganism. Second, mupirocin remains an appropriate first-line therapy for managing carriage of MRSArg associated with low-level mupirocin resistance, as observed in cases involving the ileS-V588F mutation. IleS is located on the chromosome and not on S. argenteus plasmids, suggesting low risk of spread of this resistance mechanism. Finally, MRSArg exhibits similar clinical and microbiological behaviour to traditional MRSA, suggesting that existing diagnostic and therapeutic approaches for MRSA are also applicable to this variant.

Conclusion

We report the first hospital outbreak by MRSArg in the Netherlands, which was successfully controlled. Heightened vigilance in clinical practice is crucial for the timely identification of MRSArg cases, and attention in national surveillance systems is important to monitor changes in prevalence. Given its potential for transmission and the emergence of low-level mupirocin resistance, incorporating MRSArg into routine diagnostic and surveillance frameworks will enable early detection of outbreaks and provide valuable insights into its epidemiological trends. This work supports the importance of infection prevention and control measures that are applied for MRSA are also effective for MRSArg.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualisation: SS, DS, BLH and APAH; data curation: DS, SW, SS, EdV and APAH; processing of NGS data: SW and AdH; formal analysis: SS, DS, SW and APAH; visualisation: SS, DS, SW and APAH; supervision: SS, APAH, BLH, DWN and AFS; original draft: SS, DS, EdV and APAH; review and editing: SS, DS and APAH; review and approval of final manuscript: SS, DS, EdV, BLH, AdH, SW, DWN, AFS, APAH.

Funding

This study was carried out as part of the Dutch National Methicillin-resistant Stapylococcus aureus complex surveillance, as part of the regular activities of the RIVM, financed by the Dutch Ministry of Health, Welfare and Sport.

Data availability

All data generated or analysed during this study are included in this published article. The sequence data generated and analysed in this study are available in the Sequence Read Archive (SRA) under projects PRJNA1105730 (https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA1105730/) and PRJNA1105858 (https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA1105858/).

Declarations

Ethics approval and consent to participate

Ethical approval was not needed for the study, since it is solely based on surveillance data. Samples from which the bacterial isolates were cultured were all collected as part of routine clinical practice. No identifiable personal data were collected and data were analysed and processed anonymously; written or verbal patient consent was not required. According to the Dutch Medical Research Involving Human Subjects Act (WMO), this study was exempt from review by an Institutional Review Board.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.