Staphylococcus lugdunensis was considered a commensal coagulase-negative staphylococcal species (CoNS) until the emergence of nosocomial infections, after which it became an important pathogen (Yeh et al., 2015). Unlike other CoNS, S. lugdunensis showed similar pathogenicity to Staphylococcus aureus (Argemi et al., 2015; Douiri et al., 2016; Heilbronner and Foster, 2021) and caused various infections, such as skin and soft tissue infections, bone and joint infections, bacteraemia, and infective endocarditis (Wu et al., 2011; Yeh et al., 2015, 2016; Douiri et al., 2016). The mortality rate of endocarditis caused by S. lugdunensis infection is nearly 40% (Liu et al., 2010), while that caused by other CoNS is only 14.3% (Fernandez-Rufete et al., 2012; Molina et al., 2013), suggesting that S. lugdunensis is more virulent than other CoNS. Previous epidemiological surveillance showed that most isolates remained susceptible to oxacillin (Lin et al., 2015; Yeh et al., 2015, 2016); however, a significantly high proportion of oxacillin-resistant S. lugdunensis (ORSL) strains were found in nosocomial infections (Lin et al., 2015), indicating that transfer of SCCmec (staphylococcal cassette chromosome mec) should be monitored in hospitals. In fact, our previous investigation identified an endemic spreading in northern Taiwan caused by a group of SCCmec II, ST6 strains (Cheng et al., 2015). Further characterization of their SCCmec cassette structure revealed similarities with S. aureus, which suggested that ORSL may act as an interspecies for SCCmec transfer to S. aureus in hospitals (Chang et al., 2019).

It is interesting to note that the endemic spread was caused by SCCmec II, ST6 strains, since most ORSL isolates belong to SCCmec V strains (Yeh et al., 2015, 2016). According to our previous studies regarding ORSL antimicrobial susceptibilities, SCCmec II strains showed more multidrug-resistant phenotypes than SCCmec V strains (Yeh et al., 2015), suggesting that drug resistance may enhance SCCmec II endemic spread. In addition to the drug resistance, virulence factors may also enhance the endemic spread. Although few studies have revealed virulence factors associated with their pathogenicity (Giormezis et al., 2015; Argemi et al., 2017b; Heilbronner and Foster, 2021), the above evidence originated from OSSL, while studies involving ORSL infections are still limited.

Whole-genome sequencing analysis can provide more comprehensive information to investigate virulence factors, drug resistance, pathogenesis, and other factors, which have been widely used in S. aureus studies (Kuroda et al., 2001; Laabei et al., 2014). A prospective study of these reports found that most virulence factors were encoded by mobile genetic elements (MGEs), such as plasmids, prophages, or pathogenicity islands (Malachowa and DeLeo, 2010). Staphylococcus lugdunensis N920143 was one of the earlier strains that provided complete genome sequence and information about virulence factors, although few MGEs were found in this strain compared to S. aureus (Heilbronner et al., 2011). Recent comparative genomic analyses have identified several novel MGEs in S. lugdunensis, and few of the pathogenicity islands have been reported thus far (Argemi et al., 2017a, 2018; Lebeurre et al., 2019).

Recently, the first complete genome sequence of S. lugdunensis ORSL JICS135 revealed the presence of several virulence features compared to other OSSLs (Shibuya et al., 2020). The SCCmec element of JICS135 contained two genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM)-like proteins, which are considered responsible for strain pathogenesis (Patti et al., 1994). However, structural comparisons indicated that partial regions of SCCmec_JICS135 were similar to those of two previously reported ORSL SCCmec V strains: CMUH 22 and CMUH25 (Chang et al., 2017). To elucidate the undiscovered virulence factors in SCCmec II, ST6 ORSL, whole-genome sequence analysis was adopted to decipher its genome structure. The present study demonstrated that SCCmec II and ST6 strains contained unique MGEs encoding a putative virulence factor and antimicrobial resistance genes, which may be responsible for its endemic spread.

Epidemiological surveillance showed that most clinical isolates of S. lugdunensis belonged to OSSL, and only a few isolates belonged to ORSL (McHardy et al., 2017). Among the ORSL clinical isolates, SCCmec V strains were the most frequently isolated strains, yet our previous studies showed that some isolates belonging to SCCmec II were the strains possibly involved in the endemic transmission, which makes them important in the hospital environment (Cheng et al., 2015; Yeh et al., 2015). To further understand the factors contributing to transmission of SCCmec II strains, one of these isolates, CGMH-SL118, was selected for whole-genome sequencing and compared with other OSSLs and SCCmec V ORSL deposited in the NCBI database. The complete genome sequence of S. lugdunensis CGMH-SL118 showed a different genome composition compared to that of OSSL or SCCmec V ORSL. Comparative genome analysis showed that CGMH-SL118 contained two prophages, which may cause an increase in genome size (Table 2). In addition to the prophage, only CGMH-SL118 contained one plasmid, and the above information indicated that MGEs played key roles in differences observed in SCCmec II ORSL compared to other ORSLs and OSSLs.

Mobile genetic elements are known for their horizontal gene transfer abilities and are involved in the dissemination of drug-resistant genes and virulence factors among various strains. Our results showed that multiple drug-resistant genes were present in the CGMH-SL118 genome, and most were located inside the MGEs. The SCCmec II element of CGMH-SL118 contained not only the mecA gene, but also two additional drug-resistant genes, ermA and ant (9)-Ia. Since these two genes confer erythromycin and clindamycin resistance, SCCmec II strains are probably more difficult to treat than SCCmec V strains. According to epidemiological surveillance, few resistant isolates of the above three antibiotics have been reported in previous studies (Taha et al., 2019). Therefore, these two resistant genes were not commonly found in S. lugdunensis. Among ST6 clinical isolates, almost all ORSLs showed resistance to erythromycin and clindamycin, but only a few OSSLs showed similar phenotypes (Table 4), indicating that erythromycin and clindamycin resistances were highly correlated with the SCCmec cassette.

Most of these antibiotic-resistant genes were located on MGEs, which suggested that antibiotic resistance genes horizontally transferred between bacterial strain and indeed species. This possibility is supported by our previous studies showing that the SCCmec cassette of CGMH-SL118 was structurally similar to that of S. aureus N315, and both belonged to SCCmec II (Chang et al., 2019), but structurally different from SCCmec V carried by JICS135 (SCCmec_MRSL-JICS135) (Shibuya et al., 2020). In this study, a transposon Tn554 containing ermA and adjacent ant(9)-Ia was identified in the SCCmec II element of CGMH-SL118 (Chang et al., 2019), and three copies of Tn554 were found in the CGMH-SL118 genome. This phenomenon was also found in the S. aureus N315 genome, suggesting that multiple copies of Tn554 were commonly observed in SCCmec II containing staphylococci.

In addition to the SCCmec cassette, our genome sequencing data revealed the presence of two prophage regions, which was the major difference between SCCmec II and SCCmec V ORSL. The φSPbeta region contained three antibiotic resistance genes and a putative virulence factor sasX gene, all of which might able to enhance the competitiveness of this strain in the healthcare environment. Ant (6′)-Ia, aph (3′)-III, and aac (6′)-aph (2′) all belong to the aminoglycoside-resistant genes, which also contributed to the gentamicin-resistant phenotype. The varying prevalence of these three genes among ST6 ORSL and OSSL indicated that their distribution was highly correlated with the MGEs, since all three genes were located in the φSPbeta region (Table 3). This result suggesting that the dissemination of drug-resistant determinants may carried out by the prophage φSPbeta through a horizontal transfer process.

In addition to aminoglycoside resistance genes, a putative virulence factor SasX reportedly plays critical roles in strain pathogenesis (Li et al., 2012). The SasX found in S. aureus is a homolog of SesI (S. epidermidis surface protein) and contains the LPXTG motif, which can be recognized by sortase A (SrtA) and anchored on the cell wall (Soderquist et al., 2009). Virulence studies of S. epidermidis showed that the sesI gene was only present in clinical isolates but not in the flora of healthy individuals, which indicates a virulence factor associated with strain pathogenesis (Soderquist et al., 2009). Further epidemiological surveillance of S. aureus showed that sasX was transferred by prophage φSPbeta from S. epidermidis and broadly existed in ST239 S. aureus, which was continuously transferred into ST59 S. aureus and considered to be a critical factor for increased clonal spreading (Li et al., 2012). In coagulase-negative staphylococci, sasX has been observed mainly in S. haemolyticus and S. epidermidis isolates, but not in S. lugdunensis isolates (Tekeli et al., 2020). To the best of our knowledge, this is the first report of sasX found in S. lugdunensis. Moreover, further epidemiological surveillance of ST6 clinical isolates showed that both sasX and prophage φSPbeta were present in most ORSLs, but only in one OSSL clinical isolate, suggesting that this putative virulence factor may enhance the ST6 ORSL during the endemic infection.

Among the various drug resistances identified in S. lugdunensis, fusidic acid resistance is one of the phenotypes that should be noted. Surveillance of CoNS found that less than 10% of CoNS showed fusidic acid-resistant phenotypes (Farrell et al., 2016). Similar results have been reported in previous studies of S. lugdunensis and indicated that fusidic acid resistance was low in S. lugdunensis (Hellbacher et al., 2006; Zaaroura et al., 2018). The whole-genome sequence revealed that CGMH-SL118 contained the fusB gene, which is responsible for the fusidic acid-resistant phenotype. Further investigation showed that both OSSL and ORSL ST6 isolates were more than 20% resistant (Table 4), suggesting that fusidic acid resistance is becoming a threatening issue in S. lugdunensis. Previous studies of fusidic acid resistance in S. epidermidis showed that fusB-mediated fusidic acid resistance was carried by various types of phage-related resistant islands (Chen et al., 2011, 2013), which were responsible for the high fusidic acid resistance phenotype in S. epidermidis (Chen et al., 2011). It has been suggested that fusB RI may be easily transferred and spread among different species (Chen et al., 2011). The structure of SlRI_fusB-118 was almost identical to that of SeRI_fusB-5907 and contained partial portions of SaRI, suggesting that the fusB-resistant island may be transferred between two CoNS species and further transferred to S. aureus. Although this phenomenon was rarely mentioned in S. lugdunensis (Bocher et al., 2009; Taha et al., 2019), it appears consistent with our previous studies that CGMH-SL118 may act as a reservoir for interspecies transfer of MGEs, such as SCCmec elements, to S. aureus in hospitals. This drug resistance gene was only found in SCCmec II ORSL in our analysis, suggesting that the antibiotic resistance properties of SCCmec II strains may differ from those of other SCCmec type ORSLs. In addition, our ST6 clinical isolates showed an increased fusidic acid-resistant phenotype, which may be considered as an alternative factor causing ST6 endemic transmission in hospital environments.

This study investigated the whole-genome sequence of SCCmec II, ST6 ORSL, and identified several MGEs containing multidrug-resistant genes and virulence factor, which could account for the differences observed when compared to SCCmec V ORSL and OSSL. The unique genome composition of CGMH-SL118 suggests that it may take advantage of these multidrug-resistant genes and virulence factor to compete with other OSSLs and ORSLs in hospital environments. After further surveillance of clinical isolates, we found that drug-resistant phenotypes similar to that of CGMH-SL118 were commonly found among ST6 ORSLs. Taken together, our results demonstrate that MGEs containing multidrug-resistant genes and virulence factor may play critical roles in ST6-SCCmec II ORSL infections and may be one of the reasons for endemic transmission in hospitals.

Raw data of the CGMH-SL118 complete genome sequence were submitted to the NCBI BioProject and approved with accession number CP048008.

This study was approved by the Ethics Committee of Linkou Chang Gung Memorial Hospital. Because this study only experimented on bacteria and did not affect the patients adversely, the Review Board agreed the usage of the requested bacterial materials from Linkou Chang Gung Memorial Hospital bacterial storage.

S-CC, L-CL, and J-JL conceived the study, analyzed the data, prepared the tables and figures, and contributed to the writing of the manuscript. All authors read and agreed to the published version of the manuscript.

This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 110-2320-B-182A-006-MY3, 108-2320-B-182A-013, and 110-2811-B-182A-505).

The 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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