Staphylococcus aureus, a major Gram-positive coagulase-positive pathogen, is a spherical bacterium approximately 1 μm in diameter that forms grape-like clusters (Lakhundi and Zhang, 2018). This bacterium possesses a range of virulence factors and has the capability to develop resistance to most antibiotics and antiseptics, further compounded by the continual emergence of new clones. The clinical use of methicillin has led to the rise of methicillin-resistant S. aureus (MRSA). MRSA can adapt its genetic content and expression to generate new strains with enhanced virulence and colonization abilities. As a highly virulent and challenging pathogen, MRSA is a prevalent cause of both community-acquired and hospital-associated infections (Lakhundi and Zhang, 2018). Colonization with MRSA is a critical step in the pathogenesis of active infection and a key factor in the epidemiology of MRSA infections. Colonizing strains can act as endogenous reservoirs for overt clinical infections or spread to other patients, potentially leading to serious bacterial infections (Abad et al., 2013).

The novel Staphylococcus aureus cell wall-anchored protein gene, sasX, plays a critical role in enhancing nasal colonization, immune evasion, and overall virulence (Li et al., 2012). In addition to its role in nasal colonization, sasX is involved in biofilm formation and mechanisms of immune evasion. MRSA strains carrying sasX gene have been identified as potential causes of severe diseases, including pulmonary infections and abscess formation (Alam et al., 2022). The SasX protein, which is carried on the φSPβ-like prophage, was initially identified in the sequence type (ST) 239 TW20 isolated in the UK (Holden et al., 2010; Baines et al., 2015; Harris et al., 2010); However, sasX has also been detected in other STs, suggesting its association with diverse phenotypic expressions (Li et al., 2012; Holden et al., 2010; Wang et al., 2014).

Mupirocin, a topical antibiotic produced by Pseudomonas fluorescens, is used for the decolonization of both methicillin-susceptible S. aureus (MSSA) and MRSA in patients and healthcare personnel. It exhibits high efficacy against staphylococci, streptococci, and certain Gram-negative bacteria, including Haemophilus influenzae and Neisseria gonorrhoeae (Sutherland et al., 1985). Mupirocin is commonly employed to treat local skin and soft tissue infections caused by S. aureus and streptococcal species. Resistance to mupirocin is associated with the presence of the plasmid-encoded mupA gene, which is linked to high-level mupirocin resistance and therapeutic failure (Udo et al., 2001; Simor et al., 2007; Chaves et al., 2004; Cadilla et al., 2011). High-level mupirocin resistance, defined as a minimum inhibitory concentration (MIC) ≥512 mg/L, is mediated by the mupA gene that encodes an alternate isoleucyl-tRNA synthetase (IleRS-2) (Hetem et al., 2016), located on mobile genetic elements (Hetem and Bonten, 2013; Hodgson et al., 1994).

Staphylococcus aureus strains have shown decreased sensitivity to chlorhexidine, an antiseptic solution used globally since the 1950s (Milstone et al., 2008). This resistance is attributed to the presence of the qacA/B genes, which encodes proton-motive force-dependent export pumps (Mayer et al., 2001). The qacA/B genes are typically located on multiresistance plasmids and can co-exist with antimicrobial resistance genes, contributing to the persistence and survival of qacA/B-positive MRSA strains (Sidhu et al., 2002).The presence of the qacA/B genes is associated with elevated minimum bactericidal concentrations of chlorhexidine and failures in MRSA decolonization protocols (Warren et al., 2016).

Multilocus sequence typing (MLST) has been employed to study the genetic relatedness among MRSA isolates from clinical specimens. Among 119 European MRSA isolates, only two harbored sasX, and both were of the ST239 type (De Backer et al., 2019). MRSA ST239 is known for producing exotoxins that cause a range of severe infections (Abimanyu et al., 2012). The recent dissemination of sasX from ST239 to other invasive clones indicates that this ST shift may be a significant factor driving the MRSA epidemic in Asia (Li et al., 2012).

Detecting the sasX, qacA/B, and mupA genes, along with identifying circulating MRSA strains that carry these specific genes, has significant implications for nasal decolonization of S. aureus, reducing the infection rate, and guiding rational drug use in clinical practice. It provides crucial insights for optimizing therapeutic strategies and informs the development of more effective decontamination protocols currently in place.

A total of 77 MRSA isolates were analyzed, revealing the presence of sasX, mupA, and qacA/B genes across various STs, as detailed in Table 2 . PCR results indicated that 18 isolates (23.4%) were positive for the sasX gene, 26 isolates (33.8%) for the mupA gene, and 61 isolates (79.2%) for the qacA/B genes. The predominant STs with sasX were ST59 (5 isolates, 6.5%), ST239 (3 isolates, 3.9%), and ST764 (3 isolates, 3.9%). For mupA, the most common STs were ST5 (6 isolates, 7.8%), ST59 (3 isolates, 3.9%), and ST764 (3 isolates, 3.9%). The most frequent STs for qacA/B were ST59 (15 isolates, 19.5%), ST5 (6 isolates, 7.8%), ST398 (6 isolates, 7.8%), and ST764 (4 isolates, 5.2%). Co-existence of sasX and qacA/B genes was observed exclusively in 6.5% (n = 5) of MRSA ST59, and in 3.9% (n = 3) of MRSA ST239 and ST764. The simultaneous presence of sasX, qacA/B, and mupA genes occurred in 1.3% (n = 1) of MRSA ST6290 and 1.3% (n = 1) of MRSA ST88 (see Table 3 ).

The proportional distribution of CA-MRSA and HA-MRSA strains across various specimens is shown in Figure 1 . No significant differences were observed in the detection rates of individual genes between hospital-acquired and community-acquired MRSA. The detection frequencies of the three genes in adults and children are presented in Figure 2 , where the detection rate of mupA in adults was higher than that in children (P < 0.001).

The icaA and clfA genes were the most frequently detected adhesion genes, with detection rates of 100% and 96.1%, respectively. However, the detection rate of sdrD was only 64.9% ( Supplementary Table S1 ). The sdrC, sdrD, sdrE, icaA, and clfA genes were detected in all ST1-MRSA isolates and most ST5-MRSA isolates. Relatively low detection rates of sdrD were observed among ST59-MRSA and ST398-MRSA isolates. Only two MRSA isolates from ST398 harbored the sdrE gene, as shown in Supplementary Table S1 .

The antimicrobial resistance profiles of 77 S. aureus isolates are displayed in Supplementary Figure S1 . All strains were resistant to penicillin, oxacillin, and cefoxitin, but fully susceptible to several tested antibiotics, including quinupristin/dalfopristin, linezolid, vancomycin, and tigecycline. Notably, forty-seven (61%) S. aureus BSI strains were multidrug-resistant, with the majority being resistant to penicillin, oxacillin, erythromycin, and clindamycin.

Overall, 26 STs were identified among the MRSA strains. Phylogenetic analysis revealed significant genetic variability among the MRSA isolates, as shown in Figure 3 . Three predominant clones—ST59, ST5, and ST764—were identified. The phylogenetic tree demonstrated that MRSA isolates obtained from clinical blood culture samples were clonal. The phylogram, reflecting the evolutionary relationships among clinical epidemic MRSA isolates from southern China (n = 77), is based on MLST homology ( Figure 3 ).

MRSA remains one of the most successful contemporary pathogens, demonstrating remarkable adaptability as both a commensal organism and a leading cause of severe infections in healthcare and community settings. It is responsible for a broad spectrum of clinical manifestations, including bacteremia, endocarditis, skin and soft tissue infections, bone and joint infections, and hospital-acquired infections (Turner et al., 2019). MRSA colonization significantly increases the risk of developing ventilator-associated pneumonia following intubation, particularly in intensive care units, and is associated with elevated mortality rates among hospitalized patients (Percival et al., 2015). Patients who have MRSA nasal colonization are at high risk of developing subsequent infections. The epidemiology of MRSA is characterized by the serial emergence of epidemic strains, posing a persistent clinical threat with consistently high morbidity and mortality rates (Turner et al., 2019). Analyzing the distribution of sasX, qacA/B, and mupA genes in MRSA clones is crucial for understanding the pathogen’s evolution and dissemination across different regions.

The sasX gene, although rarely detected in clinical strains, has been identified at varying frequencies across different studies. Nakaminami et al. reported a sasX prevalence of only 0.3% among isolated strains (Nakaminami et al., 2017), while Kong et al. detected it in 5.8% of their isolates (Kong et al., 2018). Surprisingly, sasX was relatively frequently identified in the study by Zieliński W et al., with 69% of sasX-positive isolates (75 out of 108) being S. aureus. A study conducted in a Malaysian hospital detected the sasX gene in 14.9% of cases (Alam et al., 2022), with one case involving ST4649 and the remainder (n = 13) exclusively from ST239 (Alam et al., 2022). This finding is consistent with a previous study (Zarizal et al., 2018). Another study conducted in a Chinese hospital found sasX in 36.7% of cases (Song et al., 2013). Despite the common association of the sasX gene with ST239, Nair et al. reported in 2013 that the sasX gene was not detected in any MRSA isolates, including those of ST239, which may reflect its low prevalence in the Mongolian hospital studied (Nair et al., 2013). Moreover, recent evidence suggests that sasX has spread from ST239 to other invasive clones across different sequence types (Li et al., 2012). In our study, 23.4% (n = 18) of MRSA isolates carried the sasX gene. Among these, five were from ST59 (5/17, 29.4%), three from ST239 (3/3, 100%), and three from ST764 (3/8, 37.5%) strains. Notably, all three ST239 strains in this study contained the sasX gene, making ST239 the dominant lineage among sasX-positive isolates, with a 100% detection rate within this sequence type. The sasX gene, a colonization-virulence factor, likely contributes to the success of MRSA ST239 in Asia (De Backer et al., 2019). Our data underscore the dissemination of sasX to non-ST239 sequence types, highlighting its broader impact on MRSA epidemiology.

Additionally, antiseptic resistance genes qacA/B were detected in 79.2% (n = 61) of MRSA isolates in our study. This finding aligns with the report by Shamsudin MN et al., who in 2012 observed a high prevalence of qacA/B-positive MRSA at 83.3% (50 out of 60) in Malaysian isolates (Shamsudin et al., 2012). However, a more recent study from Malaysia reported a much lower prevalence, with qacA/B genes detected in only 7.4% (n = 7) of MRSA isolates. These findings, including those from our study and others, suggest significant regional differences in the prevalence of qacA/B genes among MRSA isolates. Our results also indicate a notable association between qacA/B genes and specific sequence types. For example, qacA/B genes were frequently present in ST59 (15/17, 88.2%), ST5 (6/9, 66.7%), ST398 (6/7, 85.7%), ST764 (4/8, 50.0%), and ST239 (3/3, 100%) isolates. This observation is consistent with a report by Lu et al., who found that 88.0% (22/25) of ST239 MRSA isolates were qacA/B-positive (Lu et al., 2015). Similarly, Ho et al. reported a high frequency of qacA/B genes (88.9%) in ST239 isolates (Ho and Branley, 2012). Conversely, Kong H et al. observed that the highest incidence of qacA/B genes was in ST5 clones (34.1%), which were the predominant clone in their MRSA isolates from the region (Kong et al., 2018). In Taiwan, several studies screening MRSA isolates from various hospitals over different periods consistently found that the genotypic resistance rate to chlorhexidine, attributed to the presence of qacA/B genes, ranged from 35.4% to 55.4% (Sheng et al., 2009; Wang et al., 2008; Lee et al., 2022). Among different sequence types, ST239 MRSA isolates exhibited the highest resistance to both chlorhexidine and other antimicrobial agents (Sheng et al., 2009; Lee et al., 2022), likely due to the widespread use of chlorhexidine in hospital settings (Wang et al., 2008).

Previous studies have also reported an increased incidence of mupirocin resistance in MRSA, leading to the failure of decolonization treatments (Lu et al., 2015). Several MRSA decolonization studies have shown that 6.9%–10.9% of MRSA isolates were mupirocin-resistant, and all carried the mupA gene (Warren et al., 2016; Hayden et al., 2016; Muñoz-Gallego et al., 2016). Notably, no mupirocin-resistant MRSA isolates were reported in Taiwan before 2010 (Chen et al., 2014); however, by later years, 91.5% of MRSA isolates harbored the mupA gene, despite mupirocin not being routinely used for MRSA decolonization in the country (Lee et al., 2022).

In our study, the mupA gene was detected in 26 (33.8%) of the MRSA isolates. This gene was predominantly found in ST5 (6/9, 66.7%), ST59 (3/17, 17.6%), ST764 (3/8, 37.5%), and ST15 (2/2, 100%) isolates. Notably, all ST15 isolates exhibited a 100% detection rate for the mupA gene, whereas none of the ST239 isolates harbored the mupA gene. This contrasts with an earlier Malaysian study, which reported that 70% (11 out of 16) of ST239 isolates were positive for the mupA gene (Ghasemzadeh-Moghaddam et al., 2014). In contrast, studies by Alam NNNB et al. and Nejabat et al. found no mupA gene presence in any of their MRSA isolates (Alam et al., 2022). Our study underscores the importance of MLST data analysis in assessing the genetic variability of MRSA isolates, investigating the association between the sasX, qacA/B, and mupA genes with different STs, and examining the genetic relatedness among MRSA strains from different regions. Notably, this study revealed the co-existence of the sasX, qacA/B, and mupA genes in MRSA isolates, with this gene co-existence occurring exclusively in ST6290 and ST88 isolates. The gene distribution pattern from all of the isolates showed that sasX+ qacA/B+ mupA- was closely associated with epidemic clones ST59, ST239, and ST764. Both sasX and qacA/B genes were detected in ST239, consistent with previous studies (Alam et al., 2022; Song et al., 2013).

Obtaining relevant information about the sasX, qacA/B, and mupA genes is greatly helpful for nasal decolonization of S. aureus, reducing the infection rate, and guiding rational drug use in clinical practice. The involvement of adhesion genes is also important in contributing to the severity of infections and the pathogen’s endemicity. Adhesion genes were detected in the majority of MRSA isolates. The positivity rates for the icaA and clfA genes were high in our study, and all ST1-MRSA isolates carried the five genes with a 100% detection rate, which were consistent with previous findings (Wang et al., 2018; Foster et al., 2020). However, some findings differ from previous reports. For example, the icaA gene was detected in all isolates except for ST188 (Liu et al., 2023). Only 16.7% (8/48) of isolates harbored the icaA gene (Zamani et al., 2022). The carrying rates for the sdrC, sdrD, and sdrE genes were 76.7%, 20.0%, and 91.7%, respectively (Yang et al., 2017).Phylogenetic analysis revealed genetic diversity among MRSA isolates. The most common MRSA clones in southern China were ST59, ST5, ST764, and ST398, as identified from blood samples. Notably, 47 (61%) MRSA BSI strains were multidrug-resistant, with the highest resistance observed against penicillin, erythromycin, and clindamycin. These findings are consistent with those reported in previous studies (Wang et al., 2018).

In clinical practice, one of the critical hygienic measures to prevent the spread of S. aureus is the decontamination of potentially contaminated rooms, utensils, and colonized patients. Therefore, the isolation of S. aureus strains carrying antiseptic resistance genes from clinical samples is concerning (Kong et al., 2018). Our findings could be instrumental in developing more effective control and prevention strategies for nosocomial MRSA infections. However, this study has limitations. We did not perform a correlation validation between laboratory results and clinical outcomes, which would be a valuable direction for future research. Additionally, differences in study approaches and sample sizes limit the comparability of our data with that of other studies.

In conclusion, we analyzed a total of 77 MRSA isolates from a healthcare setting, revealing the presence of the sasX, mupA, and qacA/B genes, which are associated with increased mortality rates in hospitalized patients. The detection rates of these genes across all isolates were 23.4%, 33.8%, and 79.2%, respectively. Our findings demonstrated that circulating MRSA genotypes exhibit varying virulence and resistance determinants, with significant differences in gene prevalence among STs. Notably, ST59 (5/17), ST239 (3/3), and ST764 (3/8) isolates had the highest prevalence of sasX, while ST59, ST398, and ST239 showed the highest rates of qacA/B. Additionally, ST5 and ST15 isolates exhibited higher incidences of the mupA gene compared to other STs. Despite these findings, our understanding of the distribution and associations of sasX, qacA/B, and mupA genes in MRSA isolates remains limited, highlighting the need for ongoing, intensive research. Continued studies are crucial to further elucidate the genetic diversity, evolution, and epidemiology of MRSA.