Comprehensive genomic landscape of antibiotic resistance in Staphylococcus epidermidis

ABSTRACT Staphylococcus epidermidis, a common commensal bacterium found on human skin, can cause infections in clinical settings, and the presence of antibiotic resistance genes (ARGs) impedes the treatment of S. epidermidis infections. However, studies characterizing the ARGs in S. epidermidis with regard to genomic and ecological diversities are limited. Thus, we performed a comprehensive and comparative analysis of 405 high-quality S. epidermidis genomes, including those of 35 environmental isolates from the Han River, to investigate the genomic diversity of antibiotic resistance in this pathogen. Comparative genomic analysis revealed the prevalence of ARGs in S. epidermidis genomes associated with multi-locus sequence types. The genes encoding dihydrofolate reductase (dfrC) and multidrug efflux pump (norA) were genome-wide core ARGs. β-Lactam class ARGs were also highly prevalent in the S. epidermidis genomes, which was consistent with the resistance phenotype observed in river isolates. Furthermore, we identified chloramphenicol acetyltransferase genes (cat) in the plasmid-like sequences of the six river isolates, which have not been reported previously in S. epidermidis genomes. These genes were identical to those harbored by the Enterococcus faecium plasmids and associated with the insertion sequence 6 family transposases, homologous to those found in Staphylococcus aureus plasmids, suggesting the possibility of horizontal gene transfer between these Gram-positive pathogens. Comparison of the ARG and virulence factor profiles between S. epidermidis and S. aureus genomes revealed that these two species were clearly distinguished, suggesting genomic demarcation despite ecological overlap. Our findings provide a comprehensive understanding of the genomic diversity of antibiotic resistance in S. epidermidis. IMPORTANCE A comprehensive understanding of the antibiotic resistance gene (ARG) profiles of the skin commensal bacterium and opportunistic pathogen Staphylococcus epidermidis needs to be documented from a genomic point of view. Our study encompasses a comparative analysis of entire S. epidermidis genomes from various habitats, including those of 35 environmental isolates from the Han River sequenced in this study. Our results shed light on the distribution and diversity of ARGs within different S. epidermidis multi-locus sequence types, providing valuable insights into the ecological and genetic factors associated with antibiotic resistance. A comparison between S. epidermidis and Staphylococcus aureus revealed marked differences in ARG and virulence factor profiles, despite their overlapping ecological niches.

harmless on the skin of approximately 30% of healthy individuals and survives well on inanimate surfaces, facilitating further infection (2).
Staphylococcus epidermidis, S. haemolyticus, and S. saprophyticus are well-known coagulase-negative staphylococci (CoNS).With the increase in the incidence of CoNS infection, there is growing awareness that they should be considered pathogens (3).CoNS members are more frequently isolated from patients in the intensive care unit than S. aureus during bloodstream infections (4).Historically, they have been consid ered culture contaminants, as they are widely distributed throughout the human body, including in the mouth and throat.These characteristics render the ability to distinguish between the infecting strains and normal bacterial flora difficult and recognition of their infection challenging.Most symptoms of CoNS infection differ markedly from those of S. aureus and are usually subacute or chronic with no apparent symptoms (5).CoNS infections rarely develop into life-threatening cases if treated promptly and appropri ately, but in immunocompromised patients, fatal outcomes can occur (6).
S. epidermidis is a common human skin commensal bacterium isolated from the entire body surface of healthy individuals (7).Previously, S. epidermidis was considered a beneficial skin commensal bacterium involved in promoting homeostasis, defense against pathogens, and skin development (8).Currently, S. epidermidis is considered an opportunistic pathogen under certain conditions in which the skin barrier is disrupted by either genetic mutations or physical means (8,9).The infections usually do not develop into life-threatening diseases due to their low toxicity compared to S. aureus; however, they are considered important in the clinical setting because of the high infection frequency and complexity of treatment required (9).Approximately 250,000 intravascular catheter-associated bloodstream infections are reported annually in the USA with S. epidermidis being the major cause of sepsis (10).
One of the challenges in treating infections caused by S. epidermidis is the presence of antibiotic resistance genes (ARGs).Clinical studies have revealed that antibiotic-resistant S. epidermidis strains associated with sepsis and orthopedic infections possess specific genotypes, and they are ubiquitous in clinical settings (11,12).Notably, S. epidermidis strains isolated from patients with orthopedic infections harbor a higher proportion of ARGs and mobile genetic elements (MGEs) than strains isolated from healthy individ uals or normal skin that are not the sites of infection (13).Furthermore, multi-locus sequence typing (MLST) analysis revealed the presence of 1,133 sequence types (STs) in S. epidermidis, indicating substantial strain-level diversity (14).However, previous studies on S. epidermidis have predominantly focused on clinical isolates, such as ST2 and ST23 strains of S. epidermidis associated with the nosocomial infection with limited represen tation of its genetic and ecological diversity.Considering the increasing prevalence of S. epidermidis infections in clinical settings, a genome-wide comparative analysis of S. epidermidis strains isolated from various sources should be performed to improve our understanding regarding the transmission dynamics of ARGs with the One-Health perspective (15,16).
Here, we aimed to perform a comparative analysis of ARGs in 405 high-quality S. epidermidis genome sequences, including those from 35 isolates from the Han River, South Korea.Our study included both clinical and environmental isolates, enabling a more comprehensive evolutionary assessment of the genomic diversity of antibiotic resistance in S. epidermidis.We also compared the ARGs and virulence factors (VFs) present in S. epidermidis genomes with those in S. aureus, the results of which suggested genomic demarcation despite their ecological overlap.

Whole-genome sequences of S. epidermidis strains isolated from the Han River
In total, 35 strains of S. epidermidis were isolated from the Han River, South Korea.The 16S rRNA gene sequences of the isolates showed the highest similarities to S. epidermidis NCTC 11047 T (99.1%-100%).The whole-genome sequences were determined using the Oxford Nanopore Technologies sequencing platform.The assembled genomes were generally of high quality with a median of four contigs.The average nucleotide identity (ANI) values of the genomes with S. epidermidis NCTC 11047 T ranged from 99.5% to 96.8%, indicating that all strains were taxonomically identified to be S. epidermidis (17).The genomic features of S. epidermidis isolates are summarized in Table S1.The 35 draft genomes were approximately 2.5 ± 0.18 Mbp in size with an average GC content of 32.1%.Each genome contained 2,554 ± 314 genes, consisting of 2,474 ± 314 protein-cod ing genes, 19 rRNA genes, and 60 ± 1 tRNA genes (Table S1).

Phylogenomic analyses of S. epidermidis
A phylogenomic tree was constructed using the concatenated sequences of 640 core genes shared among the 1,002 S. epidermidis genomes (Fig. 1).The results showed that genome phylogeny was closely associated with the sequence type.Most S. epidermidis strains were isolated from humans, and ST2 was the most abundant lineage, which is known to spread globally in recent years (14).In contrast, hospital-acquired ST23 lineage, which has recently emerged as a significant global threat to patient health, accounted for a very low percentage (0.7%) (12).The phylogeny of the Han River isolates showed wide distribution, belonging to ST57 (n = 14), ST35 (n = 5), ST558 (n = 5), ST2 (n = 1), ST59 (n = 1), ST329 (n = 1), ST1094 (n = 1), and ST1099 (n = 1), while six strains could not be classified using the MLST scheme.
The ARG profiles of the Han River isolates were similar to those of the entire S. epidermidis genomes obtained from the public database with a notable prevalence of β-lactam and MLSB class ARGs in most of the genomes (Fig. 4).The presence of ARGs against β-lactams, phenicol, tetracycline, and MLSB was highly consistent with the resistance phenotypes of these isolates, whereas that of ARGs against aminoglycosides, fluoroquinolones, and trimethoprim was not (Fig. 4).Notably, the plasmid-like sequences of the Han River isolates belonging to ST558 (n = 5) and ST1099 (n = 1) harbored chloramphenicol acetyltransferase genes (cat) identical to those found in the plasmids of Enterococcus faecium (Fig. 5).These plasmid-like sequences were associated with insertion sequence 6 family transposases, homologous to those found in the plasmids of S. aureus (Fig. 5), which do not contain cat gene.

Comparison of the ARG and VF profiles between S. epidermidis and S. aureus genomes
Phylogenetically adjacent S. epidermidis and S. aureus are both common commensals on human skin and are major causes of nosocomial infections.The overlapping ecological niches of these two species suggest that various inter-species interactions may occur.Therefore, we performed a comparative genomic analysis focusing on ARGs and VFs.Our results revealed that each species has a distinct ARG profile with certain genes found exclusively in each genome (Fig. 7A and B). S. epidermidis genomes displayed relatively more diverse ARG profiles than S. aureus genomes (Fig. 7C).S. aureus possessed a larger number of ARGs per genome (9.4 ARG copies) than S. epidermidis (6.2 ARG copies) (Fig. 7D).The genome-wide core ARGs in S. epidermidis were dfrC and norA, while lmrS, S. aureus norA, mepA, and tet (38) were the core ARGs in the S. aureus genomes; core ARGs were not shared between the two species (Fig. 7A).The S. epidermidis and S. aureus genomes encoded the β-lactam class ARGs, blaZ (76% and 75% of each genome, respectively), mecA (39% and 60%), mecR1 (35% and 53%), and mecI (10% and 23%), and the MLSB class ARGs, msrA (33% and 16%) and mphC (25% and 15%) (Fig. 7A).
The VF profiles of S. epidermidis and S. aureus were more clearly distinguishable than their ARG profiles (Fig. S4A and B).The genome-wide core VFs of S. epidermidis included tufa (100% of S. epidermidis genomes), clpP (99%), acpXL (99%), and aur (97%).In contrast, S. aureus consisted of 53 core VFs, including the four core VFs of S. epidermidis (Fig. S4A), indicating that the VF composition of S. aureus genomes was considerably more diverse (Fig. S4C) and abundant than that of S. epidermidis genomes.Notably, esaG, which is associated with the type VII secretion system, was the most abundant VF across all genomes (99% of S. aureus genomes) (Fig. S4A).S. aureus possessed an enormously larger number of VFs per genome (80.8 VF copies) than S. epidermidis (4.8 VF copies) (Fig. S4D).

DISCUSSION
The increasing prevalence of multidrug-resistant S. epidermidis has become a serious problem constraining treatment options in clinical settings.It has a relatively small genome of approximately 2.5 Mb, and 20% of its genome is composed of variable structures (19), which suggests that this opportunistic pathogen has an open pangenome and high potential to acquire new genetic traits via MGEs (19).Furthermore, evidence of horizontal gene transfer between S. epidermidis genomes from different skin sites involving antibiotic resistance and virulence genes has been reported (20).The flexible genome structures of S. epidermidis and horizontal gene transfer contribute significantly to the unpredictability of the S. epidermidis genome (7).
In this study, we highlighted the ARGs present in S. epidermidis genomes by analyzing comprehensive and high-quality genomes obtained from diverse habitats.Previous genomic studies have focused only on the clinical isolates, which may lead to biased views and hinder our understanding of the genetic structures of antibiotic resistance across different habitats.Therefore, a comparison of the entire S. epidermidis genomes from the One-Health perspective is critical.Although S. epidermidis is primarily found on human skin, 33% of the genomes analyzed in our study was obtained from various habitats other than human skin, including the environment (30%) and animals (3%).
In the present study, we isolated 35 environmental strains of S. epidermidis from the Han River and those genomes were analyzed.The ARG profiles of these isolates were similar to those of the entire S. epidermidis genomes obtained from the public database.The genome-predicted ARG profiles were not perfectly consistent with experimentally determined resistance phenotype (Fig. 4).This inconsistency may be due to several reasons: ARGs may not be expressed under certain conditions.ARGs may be mutated and become non-functional.The presence of multidrug efflux pumps can also make resistance phenotype to certain antibiotics unpredictable.
ARGs not yet discovered may confer resistance phenotype.Interestingly, genomes of the six environmental isolates from the Han River sequenced in this study contained plasmid-like sequences harboring cat genes identical to those of E. faecium.These ARGs have never been identified in S. epidermidis genomes.Furthermore, these sequences were associated with IS6 family transposases, which are homologous to those of S. aureus.These results suggest that ARGs structured in the transposon of plasmid may have been transmitted between these Gram-positive pathogens (21,22).
Previous studies have suggested that the genotype of S. epidermidis is associated with infection and host disease (12,23), and its ARG profile may also be closely related to its sequence type (12,24).Our results revealed that S. epidermidis ARG profiles based on their genomes could be distinguished according to different sequence types.Hospitalassociated ST2 was found to have relatively conserved ARG profiles, whereas sequence types isolated from various habitats, such as ST73, ST59, ST57, and ST5, possessed more diverse ARG profiles, suggesting that ARG diversity within a specific sequence type may be influenced by ecological niches.Our study revealed that the ARG and VF profiles of S. epidermidis and S. aureus could be clearly distinguished at the genome level.Despite overlapping ecological niches, gene transfer between staphylococcal species is believed to be rare owing to the presence of restriction modification systems and clustered regularly interspaced short palindromic repeats (CRISPR) loci that degrade foreign DNAs (25,26).However, several studies have indicated that the proportion of CRISPR loci within staphylococci is lower than that reported previously (27).In addition, multiple lines of evidence regarding the inter-species transfer of genes related to antibiotic resistance, pathogenicity, and metal resistance between S. epidermidis and S. aureus have been documented (22,28,29).The transfer of genes may herald the emergence of S. aureus lineages with increased antibiotic resistance and virulence, as the genomic flexibility of S. epidermidis renders it likely to act as a genetic reservoir for pathogens such as S. aureus (20,30).
In conclusion, our results underscore that the genome-wide ARG profiles of S. epidermidis are well characterized among their sequence types.Our study contributes to a more comprehensive understanding of S. epidermidis antibiotic resistance at the pangenome level and provides a genomic basis for variations in resistance and virulence between staphylococcal pathogens S. epidermidis and S. aureus.Addressing how and why these two species from a similar ecological niche have evolved differently to possess distinct antibiotic resistance and virulence genes are the next steps for elucidating genomic demarcation.

Whole-genome sequencing and assembly
A total of 35 environmental isolates from the Han River were subjected to whole-genome sequencing.Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) and prepared using the ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies, Oxford, UK), following the manufacturer's protocols.Wholegenome sequencing was performed using Oxford Nanopore MinION flow cells (R9.4.1 FLO-MIN106; Oxford Nanopore Technologies) at DNA Link (Seoul, Korea).Basecalling on the raw signal data was performed using the Guppy v6.0.7.The draft genomes were de novo assembled using Flye, Miniasm, and Raven implemented in Trycycler v0.5.3 (32).

FIG 1
FIG 1 Whole-genome phylogeny of Staphylococcus epidermidis associated with their sequence types and isolation sources.A genome-based phylogenetic tree was reconstructed by the maximum-likelihood method using the concatenated alignments of 640 core genes among S. epidermidis genomes.S. aureus DSM 20231 was used as an outgroup.sequence types with n ≥ 10 among the S. epidermidis genomes are color-shaded on strain names.Colors in the outer circle indicate the isolation source of each strain.Etc indicates isolation sources other than humans, animals, and the environment.Han River isolates are marked with black circles.

FIG 2
FIG 2 ARG profile of Staphylococcus epidermidis pangenome.The ARG profile-based clustering was performed using the Manhattan clustering method.Black color in the upper bar indicates the Han River isolates.Colors in the middle bar indicate the isolation sources of each strain.Etc indicates isolation sources other than humans, animals, and the environment.Colors in the lower bar indicate sequence types.CAT 1 , CAT 2 , and CAT 3 indicate chloramphenicol acetyltransferase (CAT) from Enterococcus faecalis, E. faecium, and S. intermedius, respectively.The heatmap shows the copy number of ARGs.

FIG 4
FIG 4 Genome-wide ARG profiles and resistance phenotypes of Staphylococcus epidermidis from the Han River isolates.(A) ARG profiles of S. epidermidis genomes from Han River isolates.The ARG profile-based clustering was performed using the Manhattan clustering method.Colors on the right side of the tree nodes indicate sequence types.The heatmap shows the copy number of the ARGs.(B) Antimicrobial susceptibility test results for the Han River isolates.Black, gray, and yellow colors indicate susceptible, intermediate, and resistant, respectively.

FIG 6 8 FIG 7
FIG 6 MLST distribution and ARG profiles of Staphylococcus epidermidis genomes among isolation sources.(A) MLST distribution among different isolation sources was analyzed using Fisher's exact test and Cramer's V. (B) Plot coordinates were determined by non-metric multidimensional scaling analysis of Bray-Curtis dissimilarity matrix, based on the ARG profiles.