ABSTRACT
Mupirocin, a topical antimicrobial agent, is an important component in the eradication of methicillin-resistant Staphylococcus aureus (MRSA) colonization. The molecular characteristics of 46 mupirocin-resistant MRSA (MR-MRSA) clinical isolates were analyzed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec element (SCCmec) typing, spa typing, and analysis of virulence genes. All 26 MRSA isolates with low-level mupirocin resistance possessed a V588F mutation in ileS. Among 20 MRSA isolates with high-level resistance to mupirocin, all carried mupA; 2 isolates also possessed the V588F mutation in ileS, and 1 possessed the V631F mutation in ileS (isoleucyl-tRNA synthetase). The majority of MR-MRSA isolates were resistant to erythromycin, clindamycin, tetracycline, ciprofloxacin, and gentamicin, but the rates of resistance to rifampin and fusidic acid were 8.7% and 6.5%, respectively. Eight sequence types (STs) were found among the 46 MR-MRSA isolates, of which ST764 was the most prevalent (76.1%). The most frequent spa type identified was t1084 (52.2%). The SCCmec type most frequently found was type II (80.4%). The most common clone among low-level MR-MRSA isolates was ST764-MRSA-SCCmec type II-t1084 (23 isolates), while ST764-MRSA-SCCmec type II-t002 (9 isolates) was the most common clone among high-level MR-MRSA isolates. Additionally, all toxin genes except the seb gene were not identified among ST764 isolates. Among clonal complex 5 (CC5) isolates, immune evasion cluster (IEC)-associated genes (chp, sak, and scn) and seb were present in ST764 but absent in ST5, while sec, sel1, tsst-1, and hlb genes were identified in ST5 but absent in ST764. In conclusion, the spread of CC5 clones, especially a novel ST764-MRSA-SCCmec type II-t1084 clone with high-level resistance to mupirocin, was responsible for the increase in mupirocin resistance. These findings indicated that the emergence of the ST764 MR-MRSA clone involves a therapeutic challenge for treating serious MRSA infections.
IMPORTANCE Mupirocin, a topical antibiotic that is commonly used for the nasal decolonization of MRSA and methicillin-sensitive Staphylococcus aureus in hospital settings and nursing homes, was introduced as a highly effective antibiotic against MRSA. Mupirocin acts by competitively binding isoleucyl-tRNA synthetase, thereby disrupting protein synthesis. This drug shows bacteriostatic and bactericidal activity at low and high concentrations, respectively. However, with the increase in mupirocin use, low-level and high-level resistance during nasal mupirocin treatment has been reported. In a previous study, the proportion of MRSA strains with high-level mupirocin resistance in a Canadian hospital increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% (2000 to 2004).
KEYWORDS: Staphylococcus aureus, mupirocin resistance, molecular characteristics, ST764
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA), which is a common human pathogen in nosocomial infections, has emerged as a global pathogen in hospital and community settings; it imposes a large burden on health care resources and significantly contributes to morbidity and deaths (1). A major risk factor for nosocomial infections is the extensive use of central venous catheters, heart valves, artificial lenses, and prosthetic joints (2). Moreover, colonization with MRSA has been linked to some invasive MRSA infections and greatly increases the risk of MRSA infection.
Mupirocin (pseudomonic acid A), which is a topical antibiotic that is commonly used for the nasal decolonization of MRSA and methicillin-sensitive S. aureus (MSSA) in hospital settings and nursing homes, was introduced as a highly effective antibiotic against MRSA (3). Mupirocin acts by competitively binding isoleucyl-tRNA synthetase, thereby disrupting protein synthesis (3). This drug shows bacteriostatic and bactericidal activities at low and high concentrations, respectively. However, with the increase in mupirocin use, low- and high-level resistance during nasal mupirocin treatment has been reported (3). Low-level resistance to the antibiotic (MICs of 8 to 256 μg/mL) arises by mutation of the mupirocin target, isoleucyl-tRNA synthetase (ileS), whereas expression of a new isoleucyl-tRNA synthesis by a plasmid mupA gene causes high-level mupirocin resistance (MICs of ≥512 μg/mL) (4). The mupA gene is typically located on mobile genetic elements and is plasmid mediated. In addition to the mupA gene, another mechanism of high-level mupirocin resistance, mediated by a novel locus (mupB), has been reported (5). The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% with ileS. In a previous study, the proportion of MRSA strains in a Canadian hospital with high-level mupirocin resistance increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% (2000 to 2004) (4, 6). There seems to be no mandated testing for MRSA colonization in China.
Two major clones, i.e., sequence type 239 (ST239)-MRSA-staphylococcal cassette chromosome mec element (SCCmec) type III and ST5-MRSA-SCCmec type II, were shown to be prevalent in China in the past few decades (7). The distribution of MRSA clones is geographically dynamic. In a previous study, phenotypic high-level mupirocin resistance and mupA were predominantly found in ST8 and ST36, while phenotypic low-level mupirocin resistance and V588F were predominantly found in ST239/ST241, as well as ST8 and ST36 (8). Rates of high-level mupirocin resistance and low-level mupirocin resistance were low (<4%) in the currently dominant UK MRSA clone ST22 and community/sporadic MRSA isolates (8).
At present, the prevalence of mupirocin-resistant MRSA (MR-MRSA) in China is limited. We determined the prevalence of mupirocin resistance among MRSA isolates from six provinces in China. We compared the MR-MRSA clinical isolates by SCCmec typing, spa typing, multilocus sequence typing (MLST), virulence genes, and antibiotic resistance profiling.
RESULTS
Prevalence of mupirocin resistance among MRSA clinical isolates.
Of 457 MRSA isolates tested, 46 (10.07%) were confirmed to be resistant to mupirocin (MICs of >8 μg/mL). Twenty MRSA isolates showed high-level mupirocin resistance, representing 43.48% of the MR-MRSA isolates (mupirocin MICs of ≥256 μg/mL).
Among the MR-MRSA isolates screened, 25 isolates (54.3%) were identified in Guangzhou, 13 (28.3%) in Shanghai, 4 (8.7%) in Chengdu, 2 (4.3%) in Wuhan, 1 (2.2%) in Nanchang, and 1 (2.2%) in Wenzhou. The sources of the 46 isolates included sputum (24/46 isolates [52.2%]), blood (13/46 isolates [28.3%]), and pus (9/46 isolates [19.6%]). Among the blood samples, 76.9% of the isolates were resistant to high levels of mupirocin; among the sputum and pus samples, the proportions were 29.17% and 33.33%, respectively.
The resistance of MR-MRSA isolates to 16 antibiotics is shown in Table 1. All 46 MR-MRSA clinical isolates were susceptible to vancomycin, linezolid, daptomycin, sulfamethoxazole-trimethoprim, dalbavancin, teicoplanin, and quinupristin-dalfopristin. The majority of MR-MRSA isolates were resistant to erythromycin (93.5%), clindamycin (93.5%), tetracycline (89.1%), ciprofloxacin (93.5%), and gentamicin (78.3%), but the rates of resistance to rifampin and fusidic acid were 8.7% and 6.5%, respectively.
TABLE 1.
Antimicrobial resistance profiles of 46 MR-MRSA isolates, including high-level MR-MRSA and low-level MR-MRSA isolates and ST764 and non-ST764 MRSA isolates
| Druga | No. (%) of MR-MRSA isolates with resistance (n = 46) | % with resistanceb |
|||||
|---|---|---|---|---|---|---|---|
| HLMR (n = 26) | LLMR (n = 20) | P for HLMR vs LLMR | ST764 (n = 35) | Non-ST764 (n = 11) | P for ST764 vs non- ST764 | ||
| Erythromycin | 43 (93.5) | 100.0 | 89.5 | 0.085 | 100.0 | 72.7 | 0.0014 |
| Clindamycin | 43 (93.5) | 96.3 | 84.2 | 0.152 | 100.0 | 72.7 | 0.0014 |
| Tetracycline | 41 (89.1) | 92.6 | 84.2 | 0.368 | 94.3 | 72.7 | 0.045 |
| Ciprofloxacin | 43 (93.5) | 96.3 | 89.5 | 0.356 | 97.1 | 81.8 | 0.073 |
| Gentamicin | 36 (78.3) | 81.5 | 82.4 | 0.528 | 88.6 | 54.5 | 0.0025 |
| Fusidic acid | 4 (8.7) | 15.8 | 3.7 | 0.152 | 5.7 | 1.8 | 0.200 |
| Rifampin | 3 (6.5) | 7.4 | 5.9 | 0.772 | 0 | 27.3 | 0.000 |
| SXT | 0 (0) | 0 | 0 | 0 | 0 | ||
| Dalbavancin | 0 (0) | 0 | 0 | 0 | 0 | ||
| Vancomycin | 0 (0) | 0 | 0 | 0 | 0 | ||
| Q/D | 0 (0) | 0 | 0 | 0 | 0 | ||
| Linezolid | 0 (0) | 0 | 0 | 0 | 0 | ||
| Daptomycin | 0 (0) | 0 | 0 | 0 | 0 | ||
| Teicoplanin | 0 (0) | 0 | 0 | 0 | 0 | ||
SXT, trimethoprim-sulfamethoxazole; Q/D, quinupristin-dalfopristin.
HLMR, high-level MR-MRSA; LLMR, low-level MR-MRSA.
Prevalence and geographical differences of mupirocin resistance determinants.
As shown in Table 2, all 26 isolates with low-level mupirocin resistance possessed a V588F mutation in ileS. We found that 20 isolates (100%) with high-level resistance to mupirocin (MICs of ≥256 μg/mL) were positive for mupA. Additionally, among 20 isolates with high-level resistance to mupirocin, 2 isolates also possessed the V588F mutation in ileS and 1 isolate also possessed the V631F mutation.
TABLE 2.
Distribution of mupirocin MIC values according to resistance determinants among MR-MRSA isolates
| Resistance determinant | No. of isolates | No. of isolates with mupirocin MIC of: |
||||||
|---|---|---|---|---|---|---|---|---|
| 8 μg/mL | 16 μg/mL | 32 μg/mL | 256 μg/mL | 512 μg/mL | 1,024 μg/mL | >1,024 μg/mL | ||
| ileS mutation | 29 | 2 | 23 | 1 | 1 | 2 | ||
| V588F | 28 | 2 | 23 | 1 | 1 | 1 | ||
| V631F | 1 | 1 | ||||||
| mupA | 20 | 1 | 2 | 12 | 5 | |||
Among the 25 MR-MRSA isolates isolated in Guangzhou, only 4 isolates carried the mupA gene and 22 isolates carried the V588F mutation; 21 isolates had low-level resistance to mupirocin. A total of 13 MR-MRSA isolates were detected in Shanghai, including 1 isolate (with low-level resistance to mupirocin) with the V588F mutation and the other 12 isolates (with high-level resistance to mupirocin) all with mupA mutations. The V631F mutation was observed only from Wuhan.
Molecular characteristics of MR-MRSA clinical isolates.
Five different clonal complex (CC) types (CC5, CC45, CC8, CC59, and CC1) were observed among MR-MRSA isolates. CC5 (82.6% [38/46 isolates]) was the predominant type, followed by CC45 (6.5% [3/46 isolates]) and CC8 (6.5% [3/46 isolates]). Eight STs were found among 46 isolates, of which ST764 was the most prevalent, accounting for 76.1% (35/46 isolates). Eleven spa types (t1084, t002, t030, t1081, t992, t015, t062, t127, t264, t437, and t2460), as reported previously, were found in 45 isolates, and 1 ST764 isolate had an unknown type. The most frequent spa types identified were t1084 (n = 24), t002 (n = 9), and t030 (n = 3). The SCCmec type found most frequently was type II (n = 37), followed by type IV (n = 4) and type III (n = 3) (Table 3).
TABLE 3.
Molecular characteristics and resistance determinants among MR-MRSA isolates
| Characteristic (no. of isolates) |
No. of isolates with: |
MIC (μg/mL) (no. of isolates) | Sample type (no. of isolates) | Region (no. of isolates) | ||||
|---|---|---|---|---|---|---|---|---|
| CC | ST | Spa type | SCCmec type | mupA | ileS mutation | |||
| CC5 (38) | ST764 (35) | t1084 (24) | II (24) | 1 | 24 | 8 (2), 16 (21), 512 (1) | Blood (2), sputum (15), pus (7) | Guangzhou (22), Nanchang (1), Wuhan (1) |
| t002 (9) | II (9) | 9 | 0 | 256 (1), 512 (1), 1,024 (5), >1,024 (2) | Blood (4), sputum (5) | Shanghai (9) | ||
| t992 (1) | II (1) | 1 | 0 | 1,024 (1) | Blood (1) | Shanghai (1) | ||
| Unknown (1) | II (1) | 1 | 0 | 1,024 (1) | Sputum (1) | Shanghai (1) | ||
| ST5 (2) | t2460 (1) | II (1) | 1 | 1 | 1,024 (1) | Blood (1) | Wuhan (1) | |
| t264 (1) | II (1) | 0 | 1 | 16 (1) | Blood (1) | Shanghai (1) | ||
| ST965 (1) | t062 (1) | IV (1) | 1 | 0 | 1,024 (1) | Blood (1) | Wenzhou (1) | |
| CC45 (3) | ST45 (2) | t1081 (2) | V (2) | 2 | 0 | 1,024 (1), >1,024 (1) | Blood (2) | Guangzhou (2) |
| ST508 (1) | t15 (1) | IV (1) | 1 | 0 | >1,024 (1) | Blood (1) | Guangzhou (1) | |
| CC8 (3) | ST239 (3) | t030 (3) | III (3) | 1 | 3 | 16 (1), 32 (1), 1,024 (1) | Sputum (3) | Chengdu (3) |
| CC59 (1) | ST59 (1) | t437 (1) | IV (1) | 1 | 0 | 1,024 (1) | Pus (1) | Chengdu (1) |
| CC1 (1) | ST1 (1) | t127 (1) | IV (1) | 1 | 0 | >1,024 (1) | Pus (1) | Shanghai (1) |
The ST764 clone spread in Guangzhou (22/35 isolates [62.9%]), Shanghai (11/35 isolates [31.4%]), Nanchang (1/35 isolates [2.9%]), and Wuhan (1/35 isolates [2.9%]), with Shanghai having the highest mupirocin resistance rate. Except for rifampin, the antimicrobial resistance rates of ST764 isolates were higher than those of non-ST764 MRSA isolates. The erythromycin, clindamycin, tetracycline, and gentamicin resistance rates of ST764 isolates were significantly (P < 0.05) higher than those of non-ST764 isolates (Table 1).
ST764-MRSA-SCCmec type II-t1084 was the most prevalent clone (24/46 isolates [52.2%]), with the ileS V588F mutation and low-level resistance. ST764-MRSA-SCCmec type II-t002 was the second most prevalent clone (9/46 isolates [19.6%]), with the mupA mutation and high-level resistance.
High-level MR-MRSA and low-level MR MRSA isolates.
An aminoglycoside resistance phenotype was more frequently observed among high-level MR-MRSA than low-level MR-MRSA isolates, but no significant differences were found between them (Table 1). It is noteworthy that the rate of resistance of high-level MR-MRSA isolates to fusidic acid was 15.7%.
As shown in Table 4, among the high-level MR-MRSA isolates, 60.0% (12/20 isolates) belonged to ST764 and 10.0% (2/20 isolates) belonged to ST45, 45.0% (9/20 isolates) belonged to spa type t002, and 65.0% (13/20 isolates) belonged to SCCmec type II. Similarly, among the low-level MR-MRSA strains, 88.5% (23/26 isolates) belonged to ST764, and 92.3% (24/26 isolates) belonged to SCCmec type II. However, 88.5% of low-level MR-MRSA isolates (23/26 isolates) belonged to spa type t1084.
TABLE 4.
Molecular characteristics of high-level MR-MRSA and low-level MR-MRSA isolates
| Characteristic | Findings (no. of isolates) fora: |
|
|---|---|---|
| HLMR (n = 20) | LLMR (n = 26) | |
| CC | CC5 (14), CC45 (3), CC1 (1), CC8 (1), CC59 (1) | CC5 (24), CC8 (2) |
| ST | ST764 (12), ST45 (2), ST1 (1), ST5 (1), ST59 (1), ST239 (1), ST508 (1), ST965 (1) | ST764 (23), ST239 (2), ST5 (1) |
| Spa type | t002 (9), t1081 (2), t015 (1), t030 (1), t062 (1), t1084 (1), t127 (1), t2460 (1), t437 (1), t992 (1) | t1084 (23), t030 (2), t264 (1) |
| SCCmec type | II (13), III (1), IV (4), V (2) | II (24), III (2) |
HLMR, high-level MR-MRSA; LLMR, low-level MR-MRSA.
Virulence genes.
In this study, 81 virulence factors were analyzed for 46 MR-MRSA isolates. As shown in Fig. 1, adhesion genes, including cap8H, cap8I, cap8J, and cap8K, were not present in CC5 MR-MRSA isolates. The adhesion associated with fnbA, clfB, aur, and sdrD was more frequently carried by CC5 than by CC59 and CC8. All toxin genes except for the seb gene were not identified among ST764 isolates. Among CC5 isolates, immune evasion cluster (IEC)-associated genes (chp, sak, scn) and seb were present in ST764 isolates but absent in ST5 isolates, while sec, sel1, tsst-1, and hlb genes were identified in ST5 isolates but absent in ST764 isolates.
FIG 1.
Virulence genotypes of different STs (CCs).
DISCUSSION
With the increasing prevalence of MRSA strains worldwide, many states in the United States have mandated testing for MRSA colonization on admission to the intensive care unit (ICU), but there seems to be no mandatory requirement in China (9). According to literature data, MRSA colonization may even proceed faster and more easily than colonization with MSSA isolates, and the risk of symptomatic infection with MRSA is higher than that for MSSA colonization. In instances in which decolonization is to be attempted, it is important to maintain awareness of the susceptibilities of MRSA strains to antimicrobial agents that could be used for decolonization. Mupirocin is often used for this purpose. Mupirocin resistance is very important for infection control personnel who are engaged in MRSA control efforts. In this study, 10.07% of patients had MRSA isolates that were phenotypically either low-level MR-MRSA or high-level MRSA, with the prevalence of low-level MR-MRSA (5.7%) being higher than that of high-level MR-MRSA (4.4%). This is in accordance with the findings of Kim et al. (10), Chaturvedi et al. (11), and Ohadian Moghadam et al. (12), who reported that the incidence rates of mupirocin-resistant isolates were 9.5%, 18.3%, and 10.26%, respectively. Liu et al. reported that 53/803 MRSA isolates (6.6%) were confirmed to be highly resistant to mupirocin, which was significantly lower than the rate in the present study (13). However, a study in a tertiary care facility in the United States over 18 months reported mupirocin resistance among MRSA-positive patients at hospital admission in 20/591 cases (3.4%); high-level mupirocin resistance occurred in 0.62% and low-level mupirocin resistance in 2.9% (14). Also, Chen et al. stated that 26 (1.95%) of 1,333 Staphylococcus aureus clinical isolates from a Chinese hospital in Wenzhou were found to be resistant to mupirocin, including 18 (1.35%) with high-level mupirocin resistance and 8 (0.6%) with low-level mupirocin resistance (15). These differences may be attributed to the different regional policies regarding the use of antibiotics and accurate adherence to the mupirocin course for eradication.
The resistance profiles of the clinical isolates suggested that most of the MRSA isolates were resistant to most of the antibiotics. Interestingly, most high-level MR-MRSA isolates and low-level MR-MRSA isolates were resistant to antibiotics except for rifampin and fusidic acid. Vancomycin, trimethoprim-sulfamethoxazole, rifampin, dalbavancin, quinupristin-dalfopristin, linezolid, daptomycin, and teicoplanin maintained high activity against essentially all MR-MRSA isolates. No association between multidrug resistance and high-level MR-MRSA was found, as described by Perez-Roth et al. (16).
Previous studies generally showed a high level of concordance between the carriage of mupA and high-level mupirocin resistance (4). Point mutations in the ileS gene are the main mechanisms determining low-level MR. V588F and V631F are well-identified frequent mutations in IleS responsible for low-level mupirocin resistance (17). Similarly, all MRSA isolates with high-level mupirocin resistance had the mupA gene and all MRSA isolates with low-level mupirocin resistance possessed a V588F mutation in ileS in the present work. Among 26 high-level MR-MRSA isolates, 3 isolates carried both ileS and mupA genes. The result corresponded with those of Walker et al., who observed that there are populations of MR-MRSA strains involving both mupA and ileS genes (18). In addition, because genes for coresistance to macrolides, gentamicin, and tetracycline may be located alongside mupA on the same plasmid, mupirocin use could select for increased drug resistance in MRSA (19).
Additionally, virulence genes of ST764 were different from those of other clones. Previous studies reported that some virulence genes are found in virtually all S. aureus strains, while others are linked to specific molecular types. Compared to other STs, ST764 harbors few toxin genes except seb, which may help ST764 be transmitted among populations in China. S. aureus enterotoxin type B (SEB) is a superantigen. Since SEB is also considered to play a role in immune evasion upon staphylococcal infection, SEB may contribute to community infection (20). It was reported that tsst-1-positive CC5 isolates were associated with higher mortality rates. However, ST764 strains that belonged to CC5 did not harbor tsst-1.
Regarding clonal dissemination in this study, the vast majority of the molecularly analyzed MRSA clinical isolates belonged to a single clone, ST764, which was first reported in Japan (21) and was a single-locus variant of a ST5 nosocomial MRSA clone with or without the arginine catabolic mobile element (ACME) (a feature of community-acquired MRSA strains). In recent years, three studies reported ST764 S. aureus clones in China (15, 22, 23). Among them, Chen et al. found that 31.6% of mupirocin-resistant isolates were ST764 (23). Chen et at. found that ST965 was the predominant clone, accounting for 23.08% of MR-MRSA isolates, and only 2 isolates were ST764 (15). In the present study, ST764 was the most prevalent, accounting for 76.1% of isolates, and was isolated from four regions, including Shanghai, Guangzhou, Wuhan, and Nanchang, indicating clonal transmission. However, previous studies reported that, among high-level MR-MRSA isolates, 97.5% belonged to ST125 and 2.5% to ST5, being widely distributed in hospital and community settings in Spain (24). In the case of high-level MR-MRSA, clones ST22 (CC22) and ST36 (CC30) existed as the dominant UK clones between 1999 and 2009 (8). Interestingly, ST764, ST125, and ST5 are included in CC5. Dissemination of the ST764 MRSA clone, especially with multidrug resistance, should be a concern in China.
SCCmec type III is the predominant type in Asian countries. Similar to observations from Iran, the most prevalent SCCmec subtypes in China in 2010 were type III (13). Most high-level MR-MRSA isolates carried SCCmec type IV in Spain and the United States (25, 26). In contrast, the data in our study revealed that the most prevalent SCCmec subtypes were type II (79.8%).
In Spain, spa type t067 was the predominant type (82%), although this spa type was also frequently observed in the mupirocin-susceptible group (52%) (25). In China, the most frequent spa type identified was t030 (n = 23) (13). In the present study, t1084 (n = 24) was the predominant spa type, and almost all spa type t1084 strains (n = 23) were low-level MR-MRSA isolates.
The most common clone among low-level MR-MRSA isolates was ST764-MRSA-SCCmec type II-t1084 (23 isolates), while ST764-MRSA-SCCmec type II-t002 (9 isolates) was the most common clone among high-level MR-MRSA isolates. In conclusion, most of the high-level MR-MRSA isolates belonged to ST764-MRSA-SCCmec type II-t1084. This specific lineage is predominant in our area and is associated with resistance to aminoglycosides and macrolides. Based on our findings, we recommended future inclusion of MRSA testing in hospital laboratories. Monitoring for mupirocin resistance in MRSA, especially in ST764-MRSA-SCCmec type II-t1084, is necessary to monitor the usefulness of this antimicrobial drug for the treatment of MRSA infections and for infection control.
MATERIALS AND METHODS
Bacterial isolates.
The 46 MR-MRSA clinical isolates were isolated from six cities (Shanghai, Guangzhou, Chengdu, Nanchang, Wenzhou, and Inner Mongolia) in China between 2004 and 2020. Identification of MRSA from clinical samples was performed by using matrix-assisted laser desorption ionization–time of flight mass spectrometry (Vitek). Escherichia coli ATCC 8739 was used as a control strain for the identification. All isolates were stored at −80°C for later use. Information on all isolates is presented in Table S1 in the supplemental material.
Antibiotic susceptibility testing.
Antibiotic susceptibility was assessed based on MIC values determined using the microdilution method. Results were interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (27). The antimicrobial agents tested included ciprofloxacin, clindamycin, tetracycline, erythromycin, quinupristin-dalfopristin, ceftaroline, rifampin, sulfamethoxazole-trimethoprim, gentamicin, daptomycin, mupirocin, teicoplanin, linezolid, fusidic acid, vancomycin, dalbavancin, and cefoxitin. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 29213 were used as quality control strains.
Molecular typing methods.
All S. aureus isolates were sequenced using the NovaSeq sequencing platform (Illumina Inc., San Diego, CA), with 150-bp paired-end reads. The whole-genome sequencing (WGS) data were used for genotypic characterization and analysis of mupirocin resistance determinants. MLST was performed by submitting sequences to the MLST database (https://cge.food.dtu.dk/services/MLST). SCCmec typing was performed by using the SCCmecFinder database (https://cge.food.dtu.dk/services/SCCmecFinder). The spa typing was performed by using the spaTyper spa database (https://cge.food.dtu.dk/services/spatyper). Resistance-associated mutations in ile and mupA were assessed by using the ResFinder database (https://cge.food.dtu.dk/services/ResFinder). Furthermore, virulence genes were identified by using VirulenceFinder software, with a minimum query coverage of 80% and a similarity threshold value of 90%.
Ethics statement.
This study was approved by the Ethics Committee of Shanghai Pulmonary Hospital. Because this retrospective study experimented only on bacteria and did not affect the patients adversely, the review board exempted the study from requesting informed consent.
Data availability.
The Illumina sequences of the 46 S. aureus isolates in this study are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA881641.
ACKNOWLEDGMENTS
This study was financially supported by the National Natural Science Foundation of China (grant 81902122).
Y.G. and L.X. conceived and designed the experiments. B.W., L.R., and Y.X. performed the experiments. X.W. analyzed the data. H.Z., J.Y., and Y.Z. contributed reagents/materials/analysis tools. Y.G. wrote the manuscript. Y.G. and F.Y. edited the manuscript. All authors contributed to the article and approved the submitted version.
Footnotes
Supplemental material is available online only.
Contributor Information
Fangyou Yu, Email: wzjxyfy@163.com.
Kunyan Zhang, University of Calgary.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Download spectrum.03794-22-s0001.xlsx, XLSX file, 0.04 MB (42.8KB, xlsx)
Data Availability Statement
The Illumina sequences of the 46 S. aureus isolates in this study are available in the Sequence Read Archive (SRA) under BioProject accession number PRJNA881641.