Emergence of Teicoplanin and Linezolid Resistance in Clinical Isolates of Methicillin-Resistant and -Susceptible Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), a critical pathogen according to the World Health Organization, requires new treatments and resistance surveillance. This study compared the antibiotic susceptibility of clinical methicillin-susceptible S. aureus (MSSA) and MRSA isolates to last-line agents, including teicoplanin, linezolid, and daptomycin. A total of 134 S. aureus strains isolated from skin and soft tissue infections (SSTIs) were confirmed by detecting the nuc gene via PCR. MRSA and MSSA were identified by cefoxitin disk diffusion and confirmed by mecA gene amplification. Antibiotic susceptibility was initially screened by the Kirby–Bauer disk diffusion method, with minimum inhibitory concentrations (MICs) of last-resort antibiotics (teicoplanin, linezolid, and daptomycin) precisely determined by broth microdilution. Multidrug-resistant (MDR) phenotypes were defined as resistance to ≥2 non-β-lactam antimicrobial classes for MRSA and ≥3 for MSSA. Statistical analyses were conducted with chi-square and Fisher’s exact tests. MRSA isolates exhibited significantly greater resistance than MSSA to cefazolin, gentamicin, amikacin, azithromycin, tetracycline, clindamycin, ciprofloxacin, and teicoplanin (p < 0.05). While daptomycin remained highly effective (96.3% susceptibility), concerning rates of non-susceptibility were observed for linezolid (41%) and teicoplanin (15.7%), primarily among MRSA isolates. Overall, MDR was prevalent in 41.7% of the S. aureus isolates. Daptomycin and cefazolin remain effective against MRSA and MSSA SSTIs, respectively. However, emerging resistance to last-line agents such as teicoplanin and linezolid is alarming and necessitates enhanced surveillance, prudent antibiotic use, and increased antimicrobial research to counter the growing threat of antibiotic resistance.

Keywords

antibiotic resistance MRSA MSSA daptomycin teicoplanin linezolid

Introduction

Staphylococcus aureus is a leading global cause of infections and can quickly develop antimicrobial resistance (AMR) via mutation or gene transfer. It remains a significant source of community-acquired and health care-associated infections, including skin infections, pneumonia, bacteremia, endocarditis, osteomyelitis, prosthetic joint infections, and catheter-related infections.^1–3 S. aureus is a pathogen responsible for over 250,000 deaths linked to AMR.⁴ The World Health Organization (WHO) in 2022 reports ∼100 K annual deaths directly from methicillin-resistant S. aureus (MRSA).⁴ MRSA resists antibiotics via PBP2a and PBP2c proteins, encoded by mecA and mecC genes. The staphylococcal chromosomal cassette mec (SCCmec), a transposable genetic element, carries mecA or mecC, which encode the penicillin-binding protein PBP2a.^1,5

MRSA is a multidrug-resistant (MDR) “superbug” resistant to β-lactam antibiotics, aminoglycosides, quinolones, macrolides, and lincosamides.⁶ The rapid spread of MDR pathogens has diminished common antibiotics’ effectiveness, making diseases caused by MDR pathogens a global public health concern. The latest WHO report reaffirms MRSA’s place on the Bacterial Priority Pathogens List due to its significant burden and negative impact.^4,7 The prevalence of MRSA varies by region and is influenced by research investment, drug development, and infection control measures.

MRSA remains a major global health concern, recognized as a leading cause of infections acquired in both health care and community settings.⁷ The four last-line antibiotics effective against MRSA infections are linezolid, glycopeptides such as vancomycin and teicoplanin (the latter widely used in Europe), and daptomycin.^8,9 Global glycopeptide resistance in MRSA is a significant clinical concern due to the emergence of vancomycin- and teicoplanin-resistant strains.^10–12 Although high-level vancomycin resistance in S. aureus clinical isolates remains rare, the gradual increase in minimum inhibitory concentration (MIC) in MRSA strains is concerning, as it reduces vancomycin’s effectiveness.^3,13,14 Teicoplanin and vancomycin typically bind to d-alanine-d-alanine subunits of the murein monomer, potentially leading to cross-resistance.¹⁵ Daptomycin, a cyclic lipopeptide, effectively kills MRSA; however, S. aureus daptomycin-resistant mutants frequently emerge during prolonged treatment.^3,16–18 Linezolid, the first Food and Drug Administration (FDA)-approved oxazolidinone antibiotic in 2000, is a highly effective antibiotic with proven in vitro and in vivo activity against MRSA.^9,19

This study aimed to compare antibiotic susceptibility between methicillin-susceptible S. aureus (MSSA) and MRSA strains causing skin and soft tissue infections (SSTIs), focusing on the MICs of the last-line antibiotics daptomycin and teicoplanin.

Material and Methods

Collection of clinical isolates and MRSA identification

From 293 skin and soft tissue swab samples collected at Razi Hospital, Tehran, 134 S. aureus isolates were obtained during routine microbiology diagnosis. To prevent duplicates, only the first isolate per patient within the 15-month study was included; repeated samples from the same patient were excluded. Isolation and initial identification employed standard tests, including Coagulase (tube and slide), DNase, and Mannitol Fermentation. Samples without S. aureus were excluded. Phenotypically confirmed isolates were molecularly identified by PCR targeting the nuc gene using species-specific primers. Each strain was stored at −70°C in Trypticase Soy Broth with 20% glycerol for further analysis.

MRSA isolates were identified by cefoxitin (30 μg) disk diffusion susceptibility testing and mecA gene confirmation. Isolates with cefoxitin inhibition zones ≥22 mm were classified as susceptible, and those ≤21 mm as resistant.²⁰ Multiplex PCR was conducted on all S. aureus isolates using two primer sets: MecAF-MecAR to amplify a 310 bp mecA gene fragment and NucF-NucR to amplify a 279 bp fragment of the S. aureus-specific nuc gene.²¹

Determination of antibiotic resistance profile

Antibiotic resistance was assessed by the disk diffusion method using azithromycin (15 µg), clindamycin (2 µg), ciprofloxacin (5 µg), tetracycline (30 µg), cefazolin (30 µg), gentamicin (10 µg), amikacin (30 µg), penicillin (1 µg), ceftaroline (30 µg), and linezolid (30 µg) disks.

Vancomycin susceptibility testing

Vancomycin susceptibility was assessed using the agar dilution method with pure vancomycin (Sigma-Aldrich, Germany). The study used 1 mg (1,000 μg) of vancomycin to determine the MIC following Clinical and Laboratory Standards Institute (CLSI) protocols, with test concentrations ranging from 0.625 to 1,280 μg/mL.²¹

Daptomycin susceptibility testing

Daptomycin powder (Sigma-Aldrich, Germany) was purchased, and broth microdilution was used to determine the MICs of the isolates. An antibiotic stock solution was prepared based on CLSI guidelines for daptomycin MICs against S. aureus. Following CLSI M100-S31, susceptibility testing employed daptomycin dilutions ranging from 0.06 to 4 μg/mL, with ≤1 μg/mL considered susceptible.²⁰ The manufacturer-provided daptomycin potency was 900 μg/mg, and a stock solution was prepared by dissolving 0.1 g of antibiotic in 11.250 mL sterile distilled water.

Broth microdilution was performed in 96-well microplates according to CLSI M07-A10.²² Daptomycin was twofold serially diluted in 50 mg/L Ca2⁺-supplemented, cation-adjusted Mueller– Hinton Broth by adding 50 μL of an 8 μg/mL solution to the first well containing 45 μL of broth in each row.

An 18–24-hour bacterial culture was used to prepare a suspension in physiological saline, adjusted to 0.5 McFarland standard, containing approximately 1–2 × 10⁸ colony forming unit per milliliter (CFU/mL). This suspension was then diluted 1:20 to achieve a final concentration of 2–8 × 10⁵ CFU/mL.

Microbial suspension (5 μL) was added to each well, except the negative control, resulting in a 1:10 dilution. Microplates were incubated at 37°C for 20 hours, and turbidity was assessed visually and with an ELISA reader. A positive control (bacteria only) monitored bacterial growth, while a negative control (antibiotic solution only) confirmed sterility. MIC result accuracy was confirmed by comparing S. aureus ATCC 29213 to the isolates, accepting results only if the ATCC strain’s MIC fell within the CLSI M100, 2025 range (0.12–1 μg/mL).²⁰

Teicoplanin susceptibility testing

Teicoplanin powder was obtained from Sigma-Aldrich, Germany, and the MICs of the isolates were determined by broth microdilution. The antibiotic stock solution was prepared using a range of teicoplanin MICs against S. aureus based on CLSI guidelines. According to CLSI M100-S31,²⁰ MIC ≤8 μg/mL indicated susceptibility. For testing, teicoplanin dilutions from 0.06 to 128 μg/mL, spanning above and below 8 μg/mL, were used. The teicoplanin powder’s potency was 900 μg/mg. To prepare the stock solution, 1.991 g of antibiotic was dissolved in 7 mL sterile distilled water, according to the required calculations.

Broth microdilution was conducted following CLSI M07-A10²² in 96-well microplates. Doubling dilutions of teicoplanin were prepared in cation-adjusted Mueller–Hinton Broth by adding 45 μL of broth to each well, then 50 μL of a 256 μg/mL teicoplanin solution to the first well of each row, followed by twofold serial dilutions.

Bacterial colonies from an 18–24-hour culture were suspended in physiological saline to a 0.5 McFarland standard, containing approximately 1–2 × 10⁸ CFU/mL. This suspension was then diluted 1:20 to achieve a final concentration of 2–8 × 10⁵ CFU/mL, corresponding to 10⁶ CFU/mL after dilution.

To all prepared wells except the negative control, 5 μL of microbial suspension was added, resulting in a 1:10 bacterial dilution. The microplates were incubated at 37°C for 20 hours, then examined for turbidity both visually and with an ELISA reader. A well inoculated with only bacteria served as the positive control, while a well containing only antibiotic solution served as the negative control to verify sterility. MIC quality was confirmed by comparing results for S. aureus ATCC 29213 with the isolates, considered valid if the MIC for the ATCC strain fell within the CLSI M100, 2025 range (0.25–1 μg/mL).²⁰

Determination of multidrug resistance

MDR in S. aureus strains isolated from SSTIs was determined according to CLSI M100, 35th edition²⁰ and EUCAST v15.0²³ guidelines. MDR was defined as resistance to at least three different antibiotic classes. For MRSA isolates, resistance to methicillin, and thus to β-lactams (penicillins, cephalosporins, and carbapenems except ceftaroline), meant MDR was defined as resistance to at least two additional antibiotic classes. For MSSA isolates, MDR was defined as resistance to three antibiotic classes other than β-lactams.²⁴

Results

Isolation and identification of MRSA strains

A specialist physician diagnosed the SSTIs and skin diseases. S. aureus-induced SSTIs were most prevalent in patients with pemphigus (55.2%) and eczema (14.9%), with other conditions accounting for less than 10% (Fig. 1). PCR test confirmed the presence of the nuc gene in all the isolates. Of the 134 S. aureus strains isolated from patients with SSTI, 78 (58.2%) were MRSA (mecA-positive), while 41.8% were MSSA.

FIG. 1.

Frequency of Staphylococcus aureus isolates obtained from various types of skin and soft tissue infections (SSTIs).

Antibiotic resistance patterns

The S. aureus isolates showed the highest antibiotic resistance to penicillin (91.8%) and cefoxitin (58.2%). Vancomycin (98.5% susceptible), ceftaroline (96.3% susceptible, 3.7% dose-dependent susceptible [SDD]), daptomycin (96.3% susceptible, 3.7% non-susceptible [NS]), teicoplanin (84.3% susceptible, 10.4% intermediate, and 5.2% resistant), and cefazolin (77.6% susceptible) exhibited the highest susceptibility. Linezolid showed the highest percentage of intermediate resistance (31.3%), with 59% of isolates being susceptible and 9.7% resistant (Fig. 2).

FIG. 2.

Antibiotic resistance and susceptibility patterns of Staphylococcus aureus strains isolated from skin and soft tissue infections (SSTIs), showing (A) resistance, susceptibility, and intermediate resistance; (B) resistance patterns; and (C) susceptibility patterns. AN, amikacin; AZM, azithromycin; CIP, ciprofloxacin; CN, clindamycin; CPT, ceftaroline; CZ, cefazolin; DAP, daptomycin; FOX, cefoxitin; GEN, gentamicin; I, intermediate; LZD, linezolid; NS, non-susceptible; P, penicillin; R, resistant; S, susceptible; SDD, ceftaroline dose-dependent susceptibility; TE, tetracycline; TEC, teicoplanin; Van, vancomycin.

The MRSA isolates exhibited significantly higher antibiotic resistance than MSSA isolates (Table 1). Specifically, mecA-positive MRSA demonstrated increased resistance to cefazolin, gentamicin, amikacin, azithromycin, tetracycline, clindamycin, ciprofloxacin, and teicoplanin (p < 0.05). No significant differences in resistance to penicillin, linezolid, teicoplanin, ceftaroline, vancomycin, and daptomycin were observed between MRSA and MSSA isolates.

Table 1.

Comparison of Antibiotic Resistance and Susceptibility Patterns in MRSA and MSSA Strains Isolated from Skin and Soft Tissue Infections

Antibiotics	Staphylococcus aureus isolates						p value
	mecA gene carrier (n = 78)			No mecA gene (n = 78)
	Susceptible	Intermediate	Resistant	Susceptible	Intermediate	Resistant
Cefazolin	(54) 69.2%	0	(24) 30.8%	(50) 89.3%	0	(6) 10.7%	0.006
Gentamicin	(53) 67.9%	(2) 6.2%	(23) 29.5%	(48) 85.7%	(5) 8.9%	(3) 5.4%	0.000
Amikacin	(51) 65.4%	(5) 6.4%	(22) 28.2%	(49) 87.5%	(2) 3.6%	(5) 9.8%	0.009
Azithromycin	(38) 48.7%	(1) 1.3%	(39) 50%	(43) 76.8%	(2) 3.6%	(11) 19.6%	0.001
Tetracycline	(20) 25.6%	(15) 19.2%	(43) 55.1%	(28) 50%	(6) 10.7%	(22) 39.3%	0.01
Clindamycin	(39) 50%	(9) 11.5%	(30) 38.5%	(43) 76.8%	(4) 7.1%	(9) 16.1%	0.006
Ciprofloxacin	(36) 46.2%	(2) 2.6%	(40) 51.3%	(44) 78.6%	(1) 1.8%	(11) 19.6%	0.000
Teicoplanin	(61) 78.2%	(10) 12.8%	(7) 8.9%	(52) 92.8%	(4) 7.1%	0	0.02

MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

In this study, 56 out of 134 isolates (41.7%) exhibited MDR, which was significantly higher in MRSA isolates (p < 0.05). As shown in Table 2, MDR was 59% in mecA-positive S. aureus isolates, compared with 17.9% in the mecA-negative isolates.

Table 2.

Prevalence of Multidrug-Resistant Strains in MSSA Versus MRSA Isolates

Staphylococcus aureus isolates	MDRn (%)	No MDRn (%)	p value
mecA gene carrier (MRSA; n = 78)	46 (59%)	32 (41%)	<0.001
No mecA gene (MSSA; n = 56)	10 (17.9%)	46 (82.1%)	<0.001

MDR, multidrug-resistant; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

Daptomycin susceptibility

The MIC results (Table 3) showed that 129 of 134 S. aureus isolates (96.3%) were susceptible to daptomycin. Among these, 65 isolates (48.5%) had an MIC of 1 μg/mL, 57 (42.6%) had 0.5 μg/mL, 6 (4.5%) had 0.25 μg/mL, and 1 had 0.0625 μg/mL. Non-susceptible isolates had an MIC of 2 μg/mL. From 78 MRSA isolates, 76 (97.4%) were susceptible (MIC ≤1 μg/mL) and 2 (2.6%) were non-susceptible. Among 56 MSSA isolates, 53 (94.6%) were susceptible and 3 (5.4%) were non-susceptible (MIC 2 μg/mL). No isolate had an MIC ≥4 μg/mL. The control strain S. aureus ATCC 29213 had a daptomycin MIC of 1 μg/mL.

Table 3.

Minimum Inhibitory Concentration of Daptomycin for S. aureus Strains Isolated from Skin and Soft Tissue Infections

MIC (µg/mL)	MRSA (n = 78)	MSSA (n = 56)	Total (n = 134)
0.0625	1	0	1 (0.7)
0.125	0	0	0
0.25	4	2	6 (4.5)
0.5	33	24	57 (42.6)
1	38	27	65 (48.5)
2	2	3	5 (3.7)
4	0	0	0

MIC, minimum inhibitory concentration; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

Teicoplanin susceptibility

Table 4 shows that 84.3% (113/134) of S. aureus isolates were susceptible to teicoplanin, while 5.2% (7/134) were resistant. Among 78 MRSA isolates, 17 (21.7%) had MIC ≤16 μg/mL and 7 (9%) were resistant with MIC 32–64 μg/mL. All MSSA isolates (56/56) were susceptible, with no MIC ≥128 μg/mL. The control strain S. aureus ATCC 29213 had a teicoplanin MIC of 0.125 μg/mL.

Table 4.

Minimum Inhibitory Concentration of Teicoplanin for S. aureus Strains Isolated from Skin and Soft Tissue Infections

MIC (µg/mL)	MRSA (n = 78)	MSSA (n = 56)	Total (n = 134) (%)
0.0625	2	3	5 (4)
0.125	14	18	32 (24)
0.25	10	4	14 (10.4)
0.5	8	11	19 (14)
1	9	2	11 (8.2)
2	6	3	9 (6.7)
4	3	7	10 (7.4)
8	9	4	13 (9.7)
16	10	4	14 (10.4)
32	5	0	5 (3.7)
64	2	0	2 (1.5)
128	0	0	0

MIC, minimum inhibitory concentration; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus.

Discussion

The ongoing evolution of AMR in S. aureus, particularly the emergence of resistance to last-resort therapeutics, represents a critical threat to global public health. Our findings illuminate a disquieting trend within clinical isolates from SSTIs, revealing not only the expected MDR phenotype of MRSA but also its concerning propensity for developing resistance to glycopeptide and oxazolidinone antibiotics. The divergence in resistance profiles between MRSA and MSSA isolates underscores a fundamental disparity in their evolutionary trajectories under antimicrobial pressure. Specifically, the emergence of non-susceptibility to teicoplanin (a phenomenon exclusively observed in the MRSA cohort) coupled with a disconcerting prevalence of reduced linezolid susceptibility suggests the potential mobilization of novel genetic determinants that may compromise the efficacy of these cornerstone therapies. This study delineates local antimicrobial susceptibility patterns and suggests potential implications for the management of severe staphylococcal infections within the study setting. The findings underscore the importance of enhanced genomic surveillance and reinforced antimicrobial stewardship to preserve the effectiveness of available therapeutic agents.

Iranian MRSA isolates in a study by Ghorda et al. exhibited resistance rates exceeding 70% to erythromycin, clindamycin, ciprofloxacin, and levofloxacin. This contrasts with MSSA isolates in the same study, where resistance to all tested antibiotics was below 50% (24). Similarly, our study found MRSA isolates demonstrating double or more the antibiotic resistance of MSSA isolates, with peak resistance rates of 50% to azithromycin, 55% to tetracycline, 51.3% to ciprofloxacin, and 93.6% to penicillin.

Given the high rate of penicillin resistance in S. aureus isolates from SSTIs, we investigated cefazolin as an alternative. Cefazolin, a first-generation cephalosporin selected in consultation with an infectious disease specialist, exhibits in vitro activity against MSSA, offers favorable pharmacokinetics allowing for lower dosages than penicillinase-resistant β-lactams such as oxacillin,²⁵ and is suitable for patients with penicillin allergies.²⁶ MSSA isolates showed higher cefazolin susceptibility (89.3%) than MRSA isolates (69.2%). A meta-analysis suggested cefazolin as a viable alternative to penicillinase-resistant antibiotics for MSSA infections.²⁵ Future research should focus on patient-specific factors to refine antibiotic selection for MSSA infections.²⁷

Tetracycline resistance was observed in 55.1% of isolates, second only to penicillin. As tetracycline is recommended for routine testing per CLSI M100 guidelines (35th edition),²⁰ the isolates susceptible to tetracycline can be considered susceptible to doxycycline and minocycline as well. However, isolates with tetracycline resistance or intermediate susceptibility should be tested against doxycycline or minocycline if treatment is necessary, a recommendation supported by the present study. Despite over 60 years of clinical use,²⁸ tetracyclines remain important for treating serious Gram-positive and Gram-negative infections, including MRSA.^29,30 Resistance to older tetracyclines such as doxycycline and tetracycline is increasing globally in clinical settings.^28,30 Newer tetracyclines (eravacycline, omadacycline, and tigecycline) offer a treatment option for bacterial infections due to their broad-spectrum activity.²⁹

In this study, ciprofloxacin resistance was high among MRSA isolates. While fluoroquinolones such as ciprofloxacin, along with clindamycin, minocycline, and trimethoprim-sulfamethoxazole, serve as vancomycin alternatives, ciprofloxacin’s utility is limited by the rapid development of resistance.²⁶ Ciprofloxacin is a fourth-priority antimicrobial agent according to CLSI guidelines, requiring testing and reporting only when other antimicrobial options are suboptimal. In this study, MDR isolates were selected based on physician request, and resistance prevalence was assessed.

MDR was observed in 41.8% of the isolates, with a significantly higher rate in MRSA compared with MSSA. This prevalence is similar to a 2022 study in Tehran by Ghodrati et al., which reported an MDR rate of 48.5% across various infectious samples and hospital departments. Consistent with our findings, the Ghodrati et al. study also found significantly different MDR rates in MRSA (65.5%) and MSSA (24.7%) isolates.²⁴

The two MRSA isolates (1.4%) exhibited intermediate vancomycin resistance (MIC 4–8 μg/mL), which may suggest a risk of developing vancomycin-resistant S. aureus (VRSA). A study in India reported 6.08% VRSA and 46.08% vancomycin-intermediate S. aureus (VISA) isolates among S. aureus, underscoring the clinical challenges of vancomycin resistance.³¹ Clinicians treating VISA/VRSA infections may need to adjust vancomycin dosage or consider alternative therapies for effective treatment.³¹

Besides vancomycin, daptomycin, linezolid, and ceftaroline, teicoplanin is used to treat MRSA infections. In one study, 84.3% of isolates were susceptible to teicoplanin, 5.2% (MRSA isolates with MICs of 32–64 μg/mL) were resistant, and 10.5% were intermediate. Teicoplanin, a glycopeptide antibiotic similar to vancomycin, is commonly used against β-lactam-resistant Gram-positive pathogens, including MRSA, and demonstrates excellent therapeutic efficacy. Several Chinese studies have compared its clinical efficacy and safety with vancomycin.³²

Hosseini et al. (Iran, 2023) reported moderate antibacterial activity of vancomycin and teicoplanin against MDR S. aureus from skin infections, with resistance rates of 26.47% and 34.31%, respectively.³³ In contrast, we observed no vancomycin resistance and a much lower teicoplanin resistance rate of 2.5%. This discrepancy may be due to methodological differences; we used agar dilution for vancomycin, while Hosseini et al. used the broth microdilution method. Although both methods are CLSI-approved, agar dilution is more accurate and less susceptible to reading errors caused by low turbidity or antibiotic precipitation, requiring stringent culture medium quality control.³⁴ The study also lacked replication of results using an alternative method and molecular confirmation of vancomycin-resistant isolates, including confirmation by the Centers for Disease Control and Prevention.

In MRSA isolates, teicoplanin resistance was observed while vancomycin resistance was not, which is notable because both are glycopeptides. Observational studies and case reports suggest teicoplanin resistance can emerge before vancomycin resistance in S. aureus.^35–37 Vancomycin-susceptible revertants of VISA/VRSA isolates can maintain some teicoplanin resistance, and acquiring teicoplanin resistance often slightly increases vancomycin resistance. In vitro, S. aureus PBP2 overexpression increased vancomycin MIC from 1 to 2 μg/mL and teicoplanin MIC from 2 to 8 μg/mL.³⁸ Generalizing vancomycin MICs to teicoplanin treatment can lead to failure, as demonstrated by successful vancomycin treatment after teicoplanin failure.³⁷ A 2019 Indian study by Bakthavatchalam et al. identified multiple mutations in candidate genes, suggesting a complex evolution in TR-MRSA and hVISA strains. Strains with high teicoplanin MICs (16 or 32 μg/mL) showed significantly increased pbp2 expression. Mutations in tcaRAB, vraSR, graSR, and rpoB genes may affect cell wall biosynthesis gene transcription.¹⁵ The observed discordance between teicoplanin and vancomycin susceptibility among certain isolates, together with discrepancies relative to previously published studies, underscores the complexity and context-dependent nature of glycopeptide resistance mechanisms. These findings emphasize the importance of local antimicrobial susceptibility data for guiding clinical decision-making.

In this study, the majority of MRSA isolates were susceptible to daptomycin, with only two exceptions. Daptomycin, a cyclic lipopeptide antibiotic derived from Streptomyces roseosporus,³⁹ disrupts bacterial plasma membrane potential in the presence of calcium ions, independent of lipoteichoic acid.⁴⁰ Its unique mechanism of action allows it to circumvent cross-resistance with other antibiotics, making it effective against MRSA-related skin and bloodstream infections. However, it is ineffective against MRSA pneumonia due to inactivation by alveolar surfactant.⁴¹ Daptomycin and ceftaroline combination therapy has shown synergistic effects against MRSA bloodstream infections.⁴²

Ceftaroline is an effective adjunctive treatment for skin and skin structure infections and community-acquired pneumonia, especially those caused by MRSA.⁴³ In this study, 96.2% of isolates were susceptible to ceftaroline (30 μg disk) with a zone of inhibition ≥25 mm, as determined by CLSI guidelines.²¹

The present study found a 9.7% linezolid resistance rate among isolates via disk diffusion. Linezolid-resistant S. aureus (LRSA) was first isolated in 2001.⁴⁴ The linezolid resistance rate in MRSA isolates was 12.8%. The first linezolid-resistant MRSA strain was reported in a Spanish hospital during an outbreak.⁴⁵ A study in South India reported 2% LRSA in MRSA isolates.⁴⁶ Norma et al. reported a similar prevalence of 2.2% in Mexico.⁴⁷ In Rajasthan, Singh et al. found an LRSA prevalence of 20.3%.⁴⁸ A study of clinical specimens from Imam Reza Hospital in Mashhad (2011–2012) showed that 46.5% of MRSA cases were linezolid-resistant.⁴⁹ Conversely, all Staphylococcus isolates from nasal specimens of health care providers at Mofid Children’s Hospital were susceptible to linezolid and vancomycin, with 21 of 27 isolates being MRSA.^50,51 Tajbakhsh et al. recently reported a 1.7% LRSA prevalence among MRSA isolates from hospitalized children.⁵² The linezolid non-susceptibility rate observed in this study exceeds that reported in many global datasets. While this finding may suggest the emergence of local resistance, it should be interpreted with caution. Potential contributing factors include local antimicrobial prescribing patterns, clonal dissemination of resistant strains, and the inherent limitations of a single-center study. Collectively, these observations highlight the importance of strengthened local surveillance and antimicrobial stewardship, while limiting the extrapolation of these findings to other settings.

Linezolid resistance in Gram-positive bacteria typically arises from mutations in the 23S ribosomal RNA or via plasmid transfer. Resistance can emerge even without prior linezolid exposure and requires careful monitoring. Although linezolid-resistant isolates of enterococci, staphylococci, and streptococci have been reported globally, their prevalence remains low (2–20%) and stable,^53,54 and most Gram-positive bacteria remain susceptible.⁵⁵ The Global Antimicrobial Resistance and Utilization Surveillance System has not reported linezolid resistance.⁵⁴ The 2025 CLSI guideline recommends that S. aureus susceptible to linezolid be considered susceptible to tedizolid, but isolates resistant to linezolid should be tested for tedizolid if treatment is indicated.²⁰

A recent meta-analysis by Gehang et al. indicates that linezolid is more effective than vancomycin and tigecycline for MRSA-related pulmonary and skin/soft tissue infections, though its thrombocytopenia risk requires caution. Daptomycin remains beneficial for bloodstream infections, while vancomycin shows a safer hepatic and renal profile.⁵⁶ These results highlight the need to balance efficacy with safety in selecting optimal therapy.

Chen et al. suggested that linezolid might be superior to teicoplanin for SSTIs and nosocomial pneumonia.⁵⁷ The potential clinical advantage of linezolid in these infections must be weighed against significant public health concerns. The escalating challenge of linezolid-resistant MRSA and the indispensable role of linezolid in WHO guidelines for MDR tuberculosis⁵⁴ underscore the necessity for antimicrobial stewardship. Our findings, together with existing evidence, support the importance of judicious and selective use of linezolid to preserve its long-term effectiveness in comparable epidemiological settings.

This study has several limitations. The single-center design and the inclusion of a specific SSTI patient population may limit the generalizability of the findings to other regions or clinical settings. In addition, reliance on conventional phenotypic antimicrobial susceptibility testing precluded comprehensive characterization of underlying genetic resistance mechanisms. The observed non-susceptibility rates, including those for linezolid, warrant confirmation through molecular analyses and larger multicenter investigations. Accordingly, the present findings are primarily applicable at the local and regional levels and underscore the need for further research and targeted antimicrobial stewardship efforts.

Conclusions

This study demonstrates substantial AMR among S. aureus isolates causing SSTIs, with MRSA exhibiting higher resistance across multiple antimicrobial classes compared with MSSA. The detection of intermediate susceptibility to vancomycin, along with reduced susceptibility to teicoplanin and linezolid, underscores emerging challenges in the therapeutic management of these infections. Collectively, these findings indicate a narrowing spectrum of effective treatment options and highlight the importance of robust local surveillance, accurate antimicrobial susceptibility testing, and sustained antimicrobial stewardship. Strengthened infection control measures remain essential to limit the dissemination of MDR S. aureus and to preserve the effectiveness of currently available therapies.

Authors’ Contributions

Z.F.A.: Methodology, investigation, formal analysis, writing—original draft. M.H.A.: Conceptualization, project administration, supervision, methodology, investigation, formal analysis, data curation, writing—review and editing. H.S.: Methodology, investigation, validation, writing—review and editing.

Footnotes

Acknowledgments

The authors would like to acknowledge the participants for taking part in this study.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

The study design was approved by the Ethics Committee of Shahed University (Approval ID: IR.SHAHED.REC.1401.148), and followed the statements of the Declaration of Helsinki. All participants provided written informed consent.

Disclosure Statement

The authors declare no competing interests.

Funding Information

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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