Staphylococcus argenteus bacteremia in the Republic of Korea

ABSTRACT

In 2015, Staphylococcus argenteus and Staphylococcus schweitzeri were proposed as new species, distinct from Staphylococcus aureus and collectively referred to as the S. aureus complex. However, no clinical reports of these new species exist in Korea. Upon the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for all bloodstream isolates since September 2022, S. argenteus was identified in one patient. Therefore, we aimed to search for new species among the archives of the S. aureus bacteremia cohort and describe their clinical and microbiological characteristics. Among the 691 archived S. aureus isolates between 2012 and 2018, one was identified as S. argenteus via MALDI-TOF MS. Both S. argenteus isolates (one in 2022) were obtained from patients with extensive pneumonia accompanied by bacteremia and both cases had fatal outcomes. They harbored multiple virulence genes (clfA, clfB, fnbpA, sdrC, sdrD, sdrE, bbp, cna, see, seg, sei, blaZ, fnbpB, and map) but did not harbor mecA and pvl. No matched sequence type (ST) was found in either isolate, and both S. argenteus isolates were closely related to ST1594, ST1593, ST1793, and ST1303, which belonged to S. argenteus. S. argenteus accounted for <1% of the S. aureus complex but had clinical characteristics similar to S. aureus. Therefore, clinicians should be aware of these factors to avoid misidentifying these strains as coagulase-negative staphylococci, and appropriate reporting is required to minimize confusion.

IMPORTANCE

Staphylococcus argenteus, a member of Staphylococcus aureus complex, has been reported as an important pathogen that causes clinically invasive infections in humans similar to S. aureus. Clinical isolates of S. argenteus have been reported across the world, showing a large geographical difference in prevalence and genomic profile. However, there have been no clinical reports regarding this new species in Korea. This is the first report to investigate the clinical and genetic characteristics of S. argenteus identified in patients with bacteremia, and the proportion of S. argenteus bacteremia among S. aureus bacteremia cohort in Korea.

OBSERVATION

Staphylococcus aureus is a clinically important pathogen that causes various infections ranging from skin and soft tissue infections to infective endocarditis (1). In 2015, Staphylococcus argenteus and Staphylococcus schweitzeri were identified as new species, distinct from S. aureus (2). These new species are indistinguishable from S. aureus using conventional routine diagnostics, such as microscopy, colony morphology, and coagulase assays and are collectively referred to as the S. aureus complex. However, they can be distinguished via peptidoglycan composition analysis, molecular typing, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), genotyping, including multi-locus sequence typing (MLST), and whole-genome sequencing (3). Since new diagnostic methods based on molecular typing or genotyping have been introduced and are widely used, clinicians may encounter S. argenteus more frequently based on clinical microbiology laboratory results. However, no clinical reports on these new species exist in Korea; thus, they remain largely unrecognized. Therefore, we aimed to investigate the presence of these species in an archived S. aureus bacteremia cohort and describe their clinical and microbiological characteristics.

Since September 2022, MALDI-TOF MS has been applied to all positive blood cultures of patients with bacteremia at the study hospital. S. argenteus was identified in the blood culture of one patient (strain 1) on 4 December 2022. Stored isolates from the S. aureus bacteremia cohort were re-evaluated using MALDI-TOF MS to identify possible misidentifications. This study involved the S. aureus bacteremia cohort treated between May 2012 and December 2018. Four cohorts from which data were prospectively collected over the study period were included, and their detailed information is described in the Acknowledgments section (4–7).

Isolates were evaluated using a MALDI Biotyper Sirius (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) with the MBT compass reference library (version 11.0) containing S. argenteus and S. schweitzeri in the strain list, which was highly sensitive and specific for distinguishing S. argenteus from S. aureus (8). MLST was performed and the sequence type (ST) was analyzed based on the MLST scheme for S. aureus using the S. aureus pubMLST database (https://pubmlst.org/organisms/staphylococcus-aureus) (9). Phylogenetic analysis was performed by aligning concatenated MLST data using CLUSTALW and constructing the phylogenetic tree using the neighbor-joining method in MEGA version 11. S. argenteus isolates used in this study were distinguished from the reference collection based on phylogenetic clustering. Furthermore, 73 STs were downloaded from the MLST database and selected as reference sequences based on the initial analysis of each S. aureus MLST locus using the same database (not shown).

Genomic DNA was extracted using an E.Z.N.A. Stool DNA Kit (Omega Bio-tek, USA) according to the manufacturer’s protocol. Library preparation from isolated DNA and de novo whole-genome sequencing (WGS) were performed by Macrogen (Seoul, Republic of Korea). High-quality DNA was used for constructing the library with the help of the TruSeq Nano DNA kit. The WGS was done on the Illumina sequencing by synthesis platform (Illumina Inc., San Diego, CA, USA).

The antimicrobial susceptibility of S. argenteus isolates was determined using the Sensititre Gram-positive GPALL1F AST Plate (Thermo Fisher Scientific, MA, USA), a commercially available broth microdilution method-based kit. The test was performed manually according to the manufacturer’s instructions, and the results were interpreted based on the Clinical and Laboratory Standards Institute breakpoints for S. aureus (10). The isolates were investigated for the presence of genes encoding mecA, blaZ, Panton-Valentine leukocidin, adhesins, enterotoxins, toxic shock syndrome toxin, and exfoliative toxins using primers as described in previous reports (11–13). The 95% confidence interval of the proportion of S. argenteus among S. aureus complexes was estimated using the binomial exact method.

Among the 691 isolates of S. aureus bacteremia cohort from the study hospital, one was identified as S. argenteus (strain 2). The estimated proportion of S. argenteus among S. aureus complex was 0.14% (95% confidence interval 0%–0.8%). The clinical and microbiological characteristics of both S. argenteus isolates (strains 1 and 2) are presented in Table 1. Both isolates were identified from the blood cultures of patients with extensive pneumonia accompanied by bacteremia. Both patients died from multi-organ failure despite intensive care.

TABLE 1.

Clinical and microbiological characteristics of two cases of Staphylococcus argenteus bacteremia^b

Clinical characteristics	Case 1 (2022)	Case 2 (2017)
Age	70	81
Sex	Male	Male
Relevant comorbidities	Video-assisted pulmonary lobectomy, chemotherapy, neutropenia (ANC 980 cells/mm3)	Chronic obstructive pulmonary disease
Symptom
Onset	5 days before admission	1 day before admission
Fever	+	+
Dyspnea	+	+
Cough	+	−
Infection type	Healthcare-associated pneumonia	Community-acquired pneumonia
Clinical sample	Blood	Blood
Date of sample collection	04.12.2022	20.02.2017
Treatment
Empirical antibiotics	Ceftizoxime, amikacin	Piperacillin/tazobactam
Definitive antibiotics	Cefazolin	Nafcillin
Mechanical ventilation	+	+
Continuous renal replacement therapy	−	+
Outcome	Deceased	Deceased
Microbiological characteristics
Multi-locus sequence typing (allelic profile)
arcC	151	151
aroE	755	47
glpF	20	8
gmk	101	34
Pta	145	175
Tpi	150	180
yqiL	131	169
Sequence type	8342	8343
Antimicrobial susceptibility test (MIC, mg/L)a
Oxacillin	0.5 (S)	≤0.25 (S)
Rifampin	≤0.5 (S)	≤0.5 (S)
Clindamycin	≤0.5 (S)	≤0.5 (S)
Vancomycin	1 (S)	2 (S)
Linezolid	4 (S)	4 (S)
Levofloxacin	>4 (R)	≤0.25 (S)
Trimethoprim-sulfamethoxazole	≤0.5/9.5 (S)	≤0.5/9.5 (S)

^a Antimicrobial susceptibility test was performed using the Sensititre Gram-positive GPALL1F AST Plate (Thermo Fisher Scientific, MA, USA), a commercially available broth microdilution method-based kit, and interpreted according to Clinical and Laboratory Standards Institute breakpoints for Staphylococcus aureus.

^b ANC, absolute neutrophil count and MIC, minimal inhibitory concentration.

No ST was consistent with both isolates using the S. aureus pubMLST database on 17 April 2023. Therefore, the allelic profiles of both isolates were deposited in the MLST database, and the new STs were classified as 8342 and 8343. Phylogenetic analysis revealed that strains 1 and 2 were closely related to ST1594, ST1593, ST1793, and ST1303, which belonged to S. argenteus (Fig. 1). Both isolates were susceptible to oxacillin, rifampin, and vancomycin (Table 1). Strain 1 was resistant to levofloxacin. Both isolates harbored genes associated with virulence (clfA, clfB, fnbpA, sdrC, sdrD, sdrE, bbp, cna, see, seg, and sei). Additionally, blaZ, fnbpB, and map existed only in strain 1. Neither isolate had mecA and pvl.

Fig 1.

Phylogenetic tree of two S. argenteus isolates and 73 STs downloaded from the MLST website (http://www.mlst.net). The tree was constructed based on concatenated sequences of seven MLST loci using the neighbor-joining method.

This is the first report to investigate the clinical and microbiological characteristics of S. argenteus identified in patients with bacteremia and the proportion of S. argenteus bacteremia among S. aureus bacteremia in Korea. S. argenteus has been reported worldwide, particularly high in Australia, Thailand, and Taiwan accounting for 11.9%–25% of S. aureus-associated infections (14–16). However, <1% have been reported in Europe and East Asia, including the Netherlands, Belgium, Sweden, Denmark, China, and Japan, revealing a large geographical difference (17–22). The proportion of S. argenteus among S. aureus complex in this study was consistent with that of reports from neighboring countries, China and Japan (20, 21). The estimated proportion of S. argenteus in this study was low; nonetheless, the incidence of S. argenteus infection may change as global interaction/immigration increases and requires further monitoring.

Both S. argenteus isolates were associated with community- and healthcare-acquired pneumonia resulting in fatal outcomes, consistent with previously reported high virulence (15, 16). Strains 1 and 2 were classified as new ST8342 and ST8343, respectively. The most common ST in S. argenteus was ST2250, followed by ST1123 (23). As ST composition indicates geographical diversity, further studies are needed to determine whether the S. argenteus ST in Korea differs from those of other regions (23). Various virulence genes encoding adhesins, enterotoxins, toxic shock syndrome toxin, and exfoliative toxin were identified in this study, consistent with previous reports that S. argenteus and S. aureus share a significant proportion of genes encoding virulence factors (3, 24). Both isolates were methicillin-susceptible, as S. argenteus is associated with lower antimicrobial resistance than S. aureus (16, 17). However, penicillin-resistant S. argenteus is relatively common and carries blaZ, similar to strain 1 (23).

Although, S. argenteus accounted for <1% of the S. aureus complex in Korea, it was highly virulent and associated with severe infections as S. aureus. However, S. argenteus may be misrecognized as coagulase-negative staphylococci in clinical settings because it is an unfamiliar species, especially in regions with a low incidence, such as Korea. Therefore, we believe reporting S. argenteus as a member of the S. aureus complex is necessary to avoid confusion with coagulase-negative staphylococci, as suggested by the ESCMID Study Group for Staphylococci and Staphylococcal Diseases in 2019 (3).

AFTER EPUB

[This article was published on 10 January 2024 with an error in Acknowledgments. The error was corrected in the current version, posted on 16 January 2024.]

DATA AVAILABILITY

The complete genome sequences of the two S. argenteus strains have been deposited in GenBank under accession numbers SAMN38109731 (SNUBHSAr1) and SAMN38109732 (SNUBHSAr2).

ETHICS APPROVAL

The study protocol (no. B-2302-810-101) was approved by the institutional review board of the Seoul National University Bundang Hospital. The study mainly focused on archived clinical information and isolates of S. aureus bacteremia. The need for informed consent was waived owing to the study’s retrospective nature and minimal risk.