Recently, a novel coagulase-positive staphylococcal species, Staphylococcus argenteus, was distinguished from on-pigmented Staphylococcus aureus (Holt et al., 2011; Tong et al., 2015). It was first identified as S. aureus according to its phenotype; however, according to whole-genome sequence analysis, the two species exhibit identical 16S rRNA genes but are distinct in terms of average nucleotide identity (ANI) (87%) and digital DNA–DNA hybridization results (34%) (Ng et al., 2009; Ruimy et al., 2009; Tong et al., 2015). Staphylococcus argenteus was first found in northern Australia in 2002 (McDonald et al., 2006). Since then, several studies have reported sporadic cases of S. argenteus infections, mainly in Sweden, Denmark, France, Belgium, China, Japan, New Zealand, Fiji, Cambodia, French Guiana, and Trinidad and Tobago (Ruimy et al., 2009, 2010; Jenney et al., 2014; Monecke et al., 2014; Ritchie et al., 2014; Dupieux et al., 2015; Thaipadungpanit et al., 2015; Chantratita et al., 2016; Hansen et al., 2017; Schuster et al., 2017; Jiang et al., 2018; Olatimehin et al., 2018; Tång Hallbäck et al., 2018; Kitagawa et al., 2020), which confirmed that this species is globally distributed. Based on multilocus sequence typing (MLST), S. argenteus is easy to identified. Theoretically, several arcC alleles (36, 151, 207, and 272) and pta alleles (39, 107, 145, 175, 198, 256, 268, and 287) clustered in a single branch containing known S. argenteus isolates and were not shared with S. aureus (Thaipadungpanit et al., 2015). Clonal complex 75 (CC75), CC2250, CC1223, CC2485, and CC2198 were the common sequence types (STs) found in this species, most of which have been previously reported as causes of community infections (Chantratita et al., 2016).

Initially, S. argenteus was thought to be less virulent than S. aureus due to its lack of staphyloxanthin, but subsequent deaths have shown its clinical importance (Thaipadungpanit et al., 2015; Chantratita et al., 2016; Chen et al., 2018). Moreover, genes encoding toxins such as staphylococcal enterotoxin, hemolysin, and Panton-Valentine leukocidin have been associated with S. argenteus (Zhang et al., 2017). A recent study indicated the ability of S. argenteus to cause staphylococcal food poisoning, revealing that this bacterium is highly toxigenic and is also a potential foodborne pathogen (Wakabayashi et al., 2018). Thus, it is important to understand the genetic factors that allow some strains to spread aggressively, whereas others exist asymptomatically.

The epidemiology of S. argenteus and the phenotypic diversity present among different strains are reflected in their genotypes. Unfortunately, classical genotyping methods, such as pulsed-field gel electrophoresis (PFGE) or MLST, rely on the evaluation of highly conserved housekeeping genes that are representative of the vertical gene pool and thus do not provide a sufficient resolution for the prediction of disease phenotypes (Bosi et al., 2016). Therefore, it may be possible to analyse multiple S. argenteus genomes to discover factors that could be predictors of disease phenotypes and virulence capabilities. This approach to comparative genomics analysis might be more effective for characterizing the breadth of the gene repertoire accessible to individual microbes and understanding the amount of additional genomic data required for the proper characterization of this repertoire (Tettelin et al., 2008). The definition of the pangenome of a bacterium sheds light on its biology and lifestyle and has implications for the definition of the species itself. Currently, more than one hundred S. argenteus genome sequences are available in the National Center for Biotechnology Information (NCBI) Genome database. In addition, we obtained multiple S. argenteus strains in our previous work (Wu et al., 2020). In the present study, we intended to construct a comparative genomic analysis of the S. argenteus species and provide insight into the evolutionary divergence of this bacterium that represents the breadth of its genetic, phenotypic, and geographical characteristics.

As a recently named novel species, only a few genomic studies of S. argenteus have offered comprehensive information regarding its genetic diversity, epidemiological characteristics, AMR genes, virulence genes and other profiles (Zhang et al., 2017). Therefore, in the present study, we performed a large-scale genomic comparison of this species previously described as a divergent lineage of S. aureus. Accordingly, we found that S. argenteus consists of seven genetically distinct clades, the population structures of which are roughly similar to those described by Moradigaravand et al. (2017). We also revealed the existence of three new clades, among which C5 (ST5057) and C7 (ST1850) were most closely related to the previously described branch C1 (ST2250), while C6 (mainly ST2793) was most closely related to branch C2 (ST1223) (Figure 1B). However, the numbers of strains in these clades were rather limited, and we therefore obtained very little new information on the general features of these clades. Further acquisition of more S. argenteus strains or the extraction of potential S. argenteus genome sequences from an increasing number of environmental and clinical metagenomics datasets may help promote a higher-genetic resolution understanding of the evolutionary divergence of this bacterium.

Previously, S. argenteus was considered less virulent than S. aureus. On the basis of a study in Australia, it was reported that this species was associated mainly with skin and soft tissue infections but rarely with bacteremia (3/220 cases) (Tong et al., 2013). Additionally, a comparative study of 311 cases of sepsis caused by S. argenteus and S. aureus in Thailand showed a similar outcome after 28 days (Chantratita et al., 2016). Moreover, S. argenteus lacks staphyloxanthin, which is a carotenoid pigment that confers resistance against oxidative stress and neutrophil killing (Liu et al., 2005; Clauditz et al., 2006). In this study, we determined the distribution of 75 VFs in S. argenteus genomes, including multiple genes related to cell adhesion properties, hemolysin genes, type VII secretion system genes, PC synthesis-related genes, iron-regulated protein-coding genes and a subset of enterotoxin genes. These VFs play pivotal roles in host immune evasion and the infection of animals and humans (McAdow et al., 2012; Zecconi and Scali, 2013). Importantly, some specific virulence genes, such as PVL and sak, have also been detected in S. argenteus genome. As we know, PVL is a bacteriophage-encoded bicomponent leukotoxin that is in some strains of S. aureus and plays a key role in leukocytolysis and tissue necrosis (Shallcross et al., 2013). Many studies have reported an association between PVL genes and invasive disease, indicating that PVL is an epidemiological marker of a syndrome of severe infection (Gillet et al., 2002; Ellis et al., 2004; Lee et al., 2005; Pannaraj et al., 2006). In fact, the risk of death associated with PVL-positive S. aureus has been reported to be higher than that associated with non-PVL-producing S. aureus (Gillet et al., 2002). In addition, staphylokinase, which is encoding by sak gene, is a serine protease-like molecule produced by S. aureus, to interact with and neutralize α-defensins, important effector molecules of the host innate and adaptive immune system (Jin et al., 2004). As a result, staphylokinase expression may act as an important factor for bacterial persistence in susceptible groups of immunocompromised and elderly patients (Bokarewa et al., 2006). Therefore, the emergence of these virulence genes in S. argenteus is not a good phenomenon.

Moreover, prophages, which are important vectors carrying VFs in S. aureus (Feng et al., 2008), show diverse distributions in S. argenteus. As we know, S. aureus bacteriophages as important contributors to pathogenesis and the evolution of staphylococcal genomes (Novick, 2003; van Wamel et al., 2006). In this study, some of these elements were prevalent in both S. aureus and S. argenteus, such as S. aureus phage phiNM2, which revealed essential contributions to the pathogenesis of staphylococcal infection (Bae et al., 2006). By Bae et al. (2006)’s report, S. aureus phage phiNM2 were first identified in the genome of Staphylococcus aureus Newman, a human clinical isolate. This prophage occurs in culture and during animal infection, displayed organ specific virulence defects in a murine model of abscess formation. Thus, our findings indicate that S. argenteus may show a similar level of pathogenicity to that of S. aureus.

Pangenome analysis was performed to investigate the evolutionary differences in the overall gene contents within different S. argenteus clusters. In this study, the pangenome of S. argenteus was in accordance with a previous report in S. aureus, which also possesses an open pangenome (Bosi et al., 2016). The pangenome curve of S. argenteus was not saturated, suggesting the existence of an open pangenome in S. argenteus (Supplementary Figure 5). This inference was further supported by the high prevalence of “cloud genes” (genes found in less than 15% of the strains), which corresponded to approximately 59% of the pangenome. Although the genomic homology and differentiation of S. argenteus with respect to S. aureus deserve long-term attention, the genetic diversity within S. argenteus itself is also a point of interest. As two of the most prevalent clusters within S. argenteus, C1 and C2 showed different distributions within isolated sources and might represent two different evolutionary directions of this species. One of the differences between these two clusters is in the number of accessory genes. The greater number of accessory genes in C2 than in C1 indicates that C1 may be specialized for a specific environment, while C2 may have a better capacity to adapt to different environments (Merhej et al., 2013; Weinert et al., 2015; Pang et al., 2019). This is also in accord with the source distributions of the two clusters: C1 strains are mainly found in human infections, while C2 strains are attributed to multiple sources. As a result, the VFs preferentially found in C1 (e.g., PVL) may promote the infective potential of corresponding strains (Otto, 2013). In contrast, the greater number of sucrose synthases found in C2 may be a strategy for the better utilization of carbon sources in diverse environments. However, the possibility of transmission of this bacterium between humans and the environment is worthy of attention.

Interestingly, our results showed that the geographical structure of S. argenteus is geographical distribution. The global distribution of this bacterium has been previously shown (McDonald et al., 2006; Thaipadungpanit et al., 2015; Zhang et al., 2017), and this study further suggested that S. argenteus strains stabilized in certain regions might adapt to special niches by horizontally acquiring particular elements. One type of region-specific element is the SCCmec element. SCCmec elements can transport determinants of resistance to antimicrobial agents, virulence determinants and other genes important for bacterial survival under stress conditions, and it was shown in this study that the strains isolated from Europe were more likely to carry this type of element than those isolated from Asia. This finding is in accord with previous studies, in which MRSA-related S. argenteus has never been found in Asia but has occasionally been reported in Europe (McDonald et al., 2006; Yeap et al., 2017; Tång Hallbäck et al., 2018; Wu et al., 2020). In addition, some plasmids were preferentially located in particular regions (Supplementary Figure 6). All of these findings reveal potential gene exchange between S. argenteus strains and local S. aureus strains.

In fact, significant geographical specialization of S. argenteus is observed in strains isolated in the East Asia region. Bacterial strains in this region include multiple environmental isolates (Supplementary Table 1) and harbor a number of specific genes associated with antibiotic resistance, quorum sensing and biofilm formation. In fact, most S. argenteus strains isolated in China have been reported to show a strong or moderate ability to produce biofilms (Wu et al., 2020). This remarkable capacity to form biofilms may also account for the broad distribution of biofilms among different environmental sources and their transmission between the environment and humans (Zhang et al., 2017; Wu et al., 2020). Considering the presence of antibiotic resistance-conferring plasmids and their spread in the environment, the potential risk of plasmid-mediated resistance from environmental S. argenteus sources deserves more attention.

In summary, we used a multiscale comparative genomic approach to provide insights into the diversity of S. argenteus species. Our results revealed that S. argenteus may show a high level of pathogenicity similar to that of S. aureus, and one significant driving force behind the evolutionary divergence of S. argenteus may be the geographical distribution because of that the panmobilome of this species is closely related to the environmental bacteria they contacting in a specific region. New high-throughput DNA sequencing technologies and analysis techniques will continue to improve our ability to track and distinguish this pathogen. Furthermore, with the increase in the S. argenteus genome, a more extensive analysis combining disciplines such as evolutionary biology, climate science, and phylogeography will provide new insights into the complex interactions between these populations and the variable ecological conditions of their surrounding environments during the process of diversification, which are aspects that are critical for understanding the basis of the mechanisms driving the evolution of this novel species and the initiation of geographic expansion, specialization and epidemic radiation.

The data presented in the study are deposited in the NCBI database under BioProject PRJNA769826.

QWu, JZ, SW, and RP conceived and designed the experiments. RP, JH, and FZ performed the experiments. SW, RP, and LX analyzed the data. QG, MC, ZC, QWa, YD, and JW contributed the reagents, materials, and analysis tools. SW, RP, and QWu contributed to wrote the manuscript. All authors contributed to the article and approved the submitted version.

ZC and QWa are employed by Guangdong Huankai Microbial Science and Technology Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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