Genetic Characteristics of Methicillin-Resistant Staphylococcus argenteus Isolates Collected in the Dutch National MRSA Surveillance from 2008 to 2021

Abstract

Staphylococcus argenteus is a recently described member of the Staphylococcus aureus complex (SAC) and is associated with human disease. The frequency and intensity of infections caused by S. argenteus are similar to those of Staphylococcus aureus. S. argenteus can harbor antibiotic resistance genes and a variety of virulence factors analogous to methicillin-resistant S. aureus (MRSA). The aim of our study was to analyze a collection of isolates in the Dutch national MRSA surveillance from January 2008 until March 2021 that were nontypeable by multilocus variable-number tandem-repeat analysis (MLVA). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) was used for identifying the S. argenteus isolates, and whole-genome sequencing and SeqSphere were used to generate an in-house whole-genome multilocus sequence typing (wgMLST) scheme for typing the isolates. Furthermore, the presence of antibiotic resistance genes, replicons, and virulence genes was determined. Of 52,467 isolates submitted as MRSA from January 2008 until March 2021, 64 isolates (0.12%) were nontypeable with MLVA, and 54 of them were identified with mass spectrometry (MALDI-ToF MS) as S. argenteus. It appeared in retrospect that the first methicillin-resistant S. argenteus (MRSArg) was already submitted in 2008. An in-house-developed S. argenteus wgMLST scheme revealed that S. argenteus isolates clustered in 5 genomic groups which were characterized by distinct MLST types, resistomes, plasmid replicon families, and virulence factors. All but one isolate carried the staphylococcal chromosomal cassette mec (SCCmec) type IV harboring the methicillin resistance gene mecA and represent MRSArg. Most of the isolates with SCCmec subtype IVc(2B) had a trimethoprim resistance gene, dfrG, and harbored a blaZ-carrying plasmid, and most MRSArg isolates have the immune-modulating genes scn and sak. Nine of the 47 isolates carried enterotoxin-encoding genes seg, sei, sem, seo, and seu, which might be able to cause food poisoning. In some persons there was long-term persistence of MRSArg, and there were several genetically related MRSArg isolates in people living in close proximity, suggesting direct human-human transmission. IMPORTANCE We show that MRSArg has been circulating in the Netherlands since at least 2008. Although MRSArg is distinct from MRSA, it has a comparable population structure and carries similar resistance and virulence genes. The Dutch national MRSA surveillance has been expanded to include other methicillin-resistant members of the S. aureus complex, such as S. argenteus and Staphylococcus schweitzeri.

Keywords: MRSArg; SCCmec subtype IVc(2B); SCCmec type IV; Staphylococcus argenteus; enterotoxins; resistance genes; virulence factors.

FIG 1

The wgMLST-based population structure of Dutch S. argenteus isolates. (A) MLST sequence types and wgMLST genogroups are indicated in the minimum spanning tree (MST). Each circle represents the first unique isolate per person, and the circle size indicates the number of isolates. The colors of the circles represent genetic clusters (right bottom). Genogroups (GG) are indicated with gray halos, while specific MLST STs are highlighted with colored halos. A genetic cluster of S. argenteus isolates is defined as ≥2 isolates that differ by ≤20 alleles. (B) Overview of the allelic distances between the genogroups from the wgMLST MST.

FIG 2

International comparison of S. argenteus isolates by wgMLST. (A) In-house wgMLST MST of S. argenteus obtained in the Netherlands and from other countries, visualized in a minimum spanning tree (MST) with only the first unique isolate per person. GG indicates genogroups, which are encircled with gray halos, except for GG2250, which is light yellow. The Dutch MRSArg isolates are colored dark blue, and the Dutch MSSArg isolates are colored light blue. The MRSArg isolates from other countries are green, while the MSSArg isolates from other countries are white. An international genetic cluster with an S. argenteus isolate from the Netherlands and two isolates from the United States is depicted in red. (Inset) Closeup of GG2250 to provide greater detail of the international S. argenteus cluster. (B) Overview of the genogroups and isolates from different countries in the genogroups. The ISO 3166 code is used for country abbreviation. *1, these isolates had an incomplete MLST profile. The sequence type nearest the incomplete profile is used as the genogroup name. (C) Overview of the allelic distances between the genogroups.

FIG 3

International comparison of SCCmec type, resistance genes, and virulence determinants of S. argenteus. (A) Resistance genes, virulence factors, and SCCmec cassette types as identified through ResFinder, VirulenceFinder, and SCCmecFinder found in the Dutch MRSArg isolates compared with the findings in MRSArg isolates from other countries. (B) Resistance genes and virulence factors as identified through ResFinder and VirulenceFinder found in all the MRSArg isolates compared with the findings in all MSSArg isolates.