Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant pathogens encountered in both hospital and community settings1. MRSA bacteremia is characterized by high incidence and mortality rates and has the potential to cause metastatic or complicated infections, such as infective endocarditis or sepsis2,3. Vancomycin is the first-line treatment for MRSA infections; however, the clinical utility of vancomycin is limited due to the emergence of vancomycin-intermediate and -heteroresistant MRSA isolates4,5,6.

In addition to vancomycin, several viable alternative antimicrobial agents exhibit in vitro activity against MRSA, such as daptomycin and dalbavancin. Although daptomycin is becoming an integral addition to the “gold standard arsenal” for the treatment of invasive MRSA infections, daptomycin resistance has been demonstrated to have a positive correlation with vancomycin resistance7. Dalbavancin, an FDA-approved lipoglycopeptide since 2014, treats acute bacterial skin and skin structure infections from Gram-positive bacteria. This semi-synthetic antibiotic binds to cell wall precursors, disrupting bacterial division and demonstrating potent bactericidal effects8. Its enhanced lipophilicity and dimerization enable sustained activity, surpassing traditional glycopeptides9. Initially, dalbavancin was administered as two doses (1000 mg followed by 500 mg a week later)10. Yet, recent evidence supports a single 1500 mg dose, maintaining efficacy and safety11. This flexible dosing may offer more options for clinicians and patients, potentially prompting to broader clinical use of dalbavancin.

In this study, we describe two MRSA isolates (an initially susceptible isolate and a subsequently non-susceptible isolate) from a patient diagnosed with MRSA bacteremia. The initial susceptible isolate developed non-susceptibility to vancomycin, dalbavancin, and daptomycin after treatment with vancomycin. Through whole-genome sequencing and a series of phenotypic experiments on both the initially susceptible and subsequently non-susceptible MRSA isolates, we analyzed the adaptation strategy of MRSA under vancomycin pressure.

Materials and methods

Bacterial isolates and culture conditions

MRSA isolates, designated XF1 and XF2, were obtained from blood samples of a single patient at the First Affiliated Hospital of Nanchang University (Jiangxi, China). Following standardized isolation protocols, the isolates were stored in glycerol broth at −80 °C for future experiments. For routine culturing, the isolates were grown in tryptic soy broth (TSB) at 37 °C with shaking at 220 rpm.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was conducted using multiple methods to evaluate the resistance profiles of the bacterial isolates. The primary testing was performed using the VITEK 2 automated system (bioMérieux, France) with AST-GP67 cards for a broad range of antibiotics. To specifically assess cross-resistance to lipoglycopeptide antibiotics (daptomycin and dalbavancin), cation-adjusted Mueller-Hinton broth (MHB) was employed. The medium was supplemented with 50 mg/L calcium for daptomycin testing and 0.002% polysorbate 80 (PS80; Sigma-Aldrich) for dalbavancin testing. The minimum inhibitory concentrations (MICs) of vancomycin, oxacillin, cefoxitin, cefotaxime, and piperacillin-tazobactam were determined using the microbroth dilution method, which was conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (document M100-S32)12. The lowest concentration that visibly inhibited bacterial growth was recorded as the MIC. All these antibiotics were purchased from MCE (Shanghai, China).

Population analysis

Population analysis was performed to assess bacterial subpopulation responses to vancomycin, daptomycin, and dalbavancin, following the method previously described by Wootton et al.13, with minor modifications. Bacterial suspensions were prepared by adjusting overnight cultures to approximately 108 CFU/mL, based on a 0.5 McFarland standard suspension. Serial 10-fold dilutions were performed in sterile saline, and 100 µL of each dilution was plated on agar supplemented with increasing concentrations of the respective antibiotics. Vancomycin population analysis was conducted using Brain Heart Infusion (BHI) agar, while daptomycin and dalbavancin population analyses were performed on Mueller-Hinton agar supplemented with 50 mg/L calcium and 0.002% polysorbate 80 (PS80; Sigma-Aldrich), respectively. The agar plates were prepared with increasing concentrations of the respective antibiotics. Plates were incubated at 37 °C for 48 h, the number of colony-forming units (CFU) was counted for each antibiotic concentration. The limit of detection was 10 CFU/mL, based on the lowest countable dilution.

Whole genome sequencing and genomic analysis

Genomic DNA was extracted using the Bacterial Genomic DNA Kit (Tiangen-DP302, China) from overnight cultures. After qualification of the DNA samples, sequencing was performed on the Illumina NovaSeq platform. Raw reads were processed to obtain clean reads using fastp v0.20.114. De novo genome assembly of the WGS data was conducted with Unicycler v0.4.815. Multilocus sequence typing (MLST) was performed using the online MLST 1.8 tool (https://cge.cbs.dtu.dk/services/MLST/). Spa typing was performed using the online SpaServer (https://cge.food.dtu.dk/services/spaTyper). The staphylococcal cassette chromosome mec (SCCmec) typing was carried out with SCCmecFinder16. Virulence and resistance genes were identified using the ABRicate v1.0.0 (https://github.com/tseemann/abricate), based on the VFDB and CARD database17,18. Variant detection in the assembled genomes of the two isolates was executed using Snippy v4.6.0 (https://github.com/tseemann/snippy).

Transmission electron microscopy (TEM)

Overnight cultures of XF1 and XF2 isolates grown in TSB were harvested by centrifugation at 5000 g for 10 min, washed with PBS, and fixed in 2.5% glutaraldehyde at 4 °C overnight. Following the initial fixation, the cells were washed with PBS and post-fixed in 1% osmium tetroxide for 1 h at 4 °C. The fixed cells were then processed and embedded in epoxy resin for sectioning. Ultrathin sections were prepared using an ultramicrotome and subsequently stained with uranyl acetate and lead citrate to enhance contrast. The stained sections were examined using a Hitachi TEM system (Hitachi, Japan) to observe cell morphology. Cell wall thickness was quantified by measuring perpendicular angles along the length of 20 individual cells per strain. Measurements were taken using the imaging software integrated with the Hitachi TEM system.

Triton X-100-induced autolysis

The autolysis assay was performed following previously established protocols19. Overnight S. aureus cultures were diluted 1:200 into 50 mL of TSB containing 1 M NaCl and incubated until the OD600 reached 0.6. Cells were then collected by centrifugation at 4000 × g for 20 min at 4 °C. The pellets were gently washed with pre-chilled deionized water to remove any residual medium. Next, the bacterial cells were resuspended in a lysis buffer containing 0.05% Triton X-100 and 50 mM Tris-HCl (pH 7.2), and the OD600 of the suspension was adjusted to 1.0. The suspensions were incubated at 37 °C with shaking at 220 rpm for 3 h, during which OD600 readings were taken hourly to monitor lysis progress. All experiments were performed in triplicate.

Growth kinetics experiment

S. aureus isolates were grown to the logarithmic phase, after which the cells were resuspended in TSB and the OD600 was adjusted to approximately 0.01. The cultures were then incubated at 37 °C with shaking at 220 rpm. A sterile TSB control was included to rule out contamination during the experiment. Bacterial growth was monitored by measuring the OD600 hourly over a 24-hour period. The experiment was conducted in triplicate to ensure reliability. To further assess bacterial viability, CFU/mL were determined at specific time points (0, 4, 8, 12, and 24 h). At each time point, aliquots were serially diluted in 10-fold increments and plated onto Columbia agar plates supplemented with 5% sheep blood. The plates were incubated overnight at 37 °C, and the resulting colonies were counted to quantify bacterial populations.

Hemolytic activity assay

Hemolytic activity of S. aureus isolates XF1 and XF2 was determined using defibrinated rabbit erythrocytes provided by Nanjing Maojie Microbiology Technology Co., Ltd. After 24 h of culture in TSB, the bacteria were centrifuged to obtain the supernatant. Subsequently, 100 µL of the supernatant was mixed with 900 µL of a 3% erythrocyte suspension in PBS. The positive control contained 1% Triton X-100, while the negative control contained PBS. After incubation at 37 °C for 1 h, the mixture was centrifuged to separate the erythrocytes from the supernatant. The absorbance of the supernatant was measured at OD600 to calculate the hemolysis rate.

Galleria Mellonella larvae model

The G. mellonella experiment was conducted with minor modifications based on previous methods20. Each experimental group consisted of 15 G. mellonella larvae, weighing between 250 and 300 mg (obtained from Tianjin Huiyude Biotech, Tianjin, China). A 10 µL aliquot containing 3.0 × 106 CFU of saline bacterial suspension was injected into the last proleg of the larvae. Control group were injected with 10 µL of sterile PBS. After injection, the treated larvae were carefully placed in a sterile petri dish and observed at 37 °C. Survival rates were recorded every 12 h for five days. This experiment was repeated at least three times.

Biofilm formation assay

S. aureus isolates XF1 and XF2 were cultured in TSB at 37 °C for 24 h, diluted 100-fold with TSBG (Tryptic Soy Broth containing 0.5% glucose), and then aliquoted into sterile 96-well plates. The plates were incubated at 37 °C for 24 h to allow biofilm formation. Non-adherent cells were washed away with PBS. The biofilms were fixed with methanol and stained with 0.1% crystal violet for 15 min. The biofilms were dissolved in 30% acetic acid, and the absorbance at 600 nm was measured. S. epidermidis ATCC 35,984 served as the positive control and S. epidermidis ATCC 12,228 as the negative control. Wells containing only the medium served as blank controls. The experiment was performed in triplicate and repeated three times.

Confocal laser scanning microscope (CLSM)

S. aureus were cultured in TSBG in confocal culture dishes (NEST Biotechnology, China) at 37 °C for 24 h. After incubation, biofilms were gently washed twice with PBS to remove non-adherent cells. The biofilms were stained using SYTO9 (3 µM) and propidium iodide (PI, 20 µM) fluorescent dyes (Thermo Fisher Scientific, USA). SYTO9 stains both live and dead cells, whereas PI penetrates and stains only cells with damaged membranes, identifying dead cells. The stained biofilms were incubated in the dark for 10 min to allow adequate dye penetration. Following staining, the biofilms were visualized using a CLSM (Leica, Wetzlar, Germany).

Real-time quantitative PCR (RT-qPCR)

S. aureus were cultured in TSB at 37 °C for 24 h. Total RNA was extracted using the RNA purification kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. The quality and integrity of total RNA were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA) and agarose gel electrophoresis. Complementary DNA (cDNA) synthesis was performed using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) following the manufacturer’s instructions to eliminate genomic DNA contamination. RT-qPCR was conducted using the TB Green™ Premix Ex Taq™ (Takara, Japan) in a final reaction volume of 20 µL. Primer sequences used for RT-qPCR are listed in (Supplementary Table S1). Amplifications were performed on a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) with three technical replicates per reaction. The relative RNA expression levels of target genes were calculated using the 2−ΔΔCt method21, with gyrB as the internal reference gene.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8. Comparisons between two groups were made using an unpaired, two-tailed Student’s t-test. Data are represented as mean ± standard deviation. The following symbols denote levels of statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results

Patient history

A 52-year-old male patient with a history of chronic renal failure was admitted to the hospital on March 10, 2022, for femoral vein catheter placement to facilitate hemodialysis. During the dialysis session, the patient developed a high fever (39.5 °C), accompanied by coughing and chills. A chest CT scan revealed interstitial pulmonary edema and bilateral lung infections, suggesting a possible hematogenous spread of infection. Laboratory tests showed an elevated white blood cell (WBC) count of 14.12 × 109/L, with 88.54% neutrophils. The femoral vein catheter was promptly removed, and blood cultures were obtained before initiating empirical broad-spectrum antibiotic therapy with piperacillin-tazobactam. However, the patient remained febrile over the following days. On March 17, the blood culture results identified the presence of MRSA, designated as isolate XF1, which was susceptible to vancomycin. Consequently, the antibiotic regimen was adjusted, and the patient was started on intravenous vancomycin (1 g every 12 h) on March 18. Following this adjustment, the patient exhibited gradual clinical improvement, with a progressive decline in fever. Repeat blood cultures were negative, suggesting an initial response to treatment. However, on March 21, the patient experienced a recurrence of symptoms, including shortness of breath and chills, raising concerns about a potential persistent or relapsing infection. A repeat blood culture obtained on March 22 identified a second MRSA isolate, designated as XF2, which demonstrated non-susceptibility to vancomycin but remained susceptible to linezolid. Given the failure of vancomycin therapy, the treatment was promptly switched to intravenous linezolid (0.6 g every 24 h) on March 23. Following the initiation of linezolid, the patient showed progressive clinical improvement, and subsequent blood cultures obtained on March 25, March 28, March 31, and April 3 remained sterile, confirming microbiological clearance of the infection. A follow-up blood routine examination on April 17 revealed a WBC count of 6.23 × 10⁹/L, with a neutrophil percentage of 64.80%, indicating that the infection had resolved, and the patient had achieved hematological recovery. A timeline representation of the patient’s infection progression, antibiotic adjustments, and bacterial isolate collection is depicted in (Fig. 1).

Fig. 1
figure 1

Timeline representation of patient information, including infections, treatments administered, and isolate collection.

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Clinical isolate XF2 show reduced susceptibility to vancomycin, dalbavancin, and daptomycin, but increased sensitivity to β-lactam antibiotics

Table 1 summarizes the drug susceptibility profiles of isolates XF1 and XF2. XF1 was sensitive to vancomycin, daptomycin, and dalbavancin, with MICs of 0.25 mg/L, 0.5 mg/L, and 0.125 mg/L, respectively. In contrast, XF2 exhibited significantly reduced susceptibility, with a 16-fold increase in the MIC for vancomycin (0.25 to 4 mg/L), an 8-fold increase for daptomycin (0.5 to 4 mg/L), and a 16-fold increase for dalbavancin (0.125 to 2 mg/L). Remarkably, XF2 demonstrated a 4- to 8-fold reduction in MICs for β-lactam antibiotics, including oxacillin, cefoxitin, and cefotaxime, as well as a 4-fold reduction in piperacillin-tazobactam MIC (from 32/4 mg/L in XF1 to 8/4 mg/L in XF2), indicating the presence of the β-lactam seesaw effect (Table 1).

Table 1 The antimicrobial susceptibility testing of MRSA XF1 and XF2.
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To confirm the differences in antibiotic susceptibility between the two isolates, we performed a population analysis profile assay. As shown in Fig. 2, XF2 exhibited a substantially higher survival rate at elevated antibiotic concentrations compared to XF1. Specifically, for vancomycin, XF1 showed a sharp decline in viable colony counts at 1–2 µg/mL, whereas XF2 maintained viable up to 5 µg/mL, consistent with its increased MIC (Table 1). A similar trend was observed for daptomycin, where XF1 was completely eradicated at 2 µg/mL, while XF2 remained viable at concentrations up to 6 µg/mL. Likewise, XF2 demonstrated higher survival at dalbavancin concentrations of 2–3 µg/mL, whereas XF1 was eradicated at 1 µg/mL. These results collectively confirm that XF2 has developed reduced susceptibility to vancomycin, daptomycin, and dalbavancin, which may have significant clinical implications for antibiotic treatment efficacy.

Fig. 2
figure 2

Population analysis profiles of MRSA isolates XF1 and XF2 in the presence of vancomycin, daptomycin, and dalbavancin.

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Genomic variants associated with nonsusceptibility to vancomycin, daptomycin, and dalbavancin in isolate XF2

To elucidate the genomic underpinnings of vancomycin, daptomycin, and dalbavancin, non-susceptibility in XF2 isolate, we conducted whole genome sequencing on isolates XF1 and XF2. MLST and spa typing analysis categorized both XF1 and XF2 isolates as ST59 (arcC-aroE-glpF-gmk-pta-tpi-yqiL, allele numbers 19-23-15-2-19-20-15) and t172, respectively. The total genome size for XF1 is 2,797,549 bp (approximately 2.80 Mbp), while XF2 has a genome size of 2,798,610 bp (approximately 2.80 Mbp), confirming their close genetic relationship. Analysis of the virulence genes carried by XF1 and XF2 revealed that both isolates harbor genes encoding hemolysins (hla, hld, hlgA, hlgB, and hlgC), adhesion factors (fnbA, fnbB, map, ebpS, sdrC, and sdrE), and the biofilm formation-related ica operon (icaA, icaB, icaC, icaD, and icaR). Additionally, genes encoding the immune evasion cluster (sbi, sak, chp, and scn) and the ESAT-6 secretion system genes (esxA, essA, essB, esaA, esaB) were present in both isolates. Resistance gene analysis showed that both XF1 and XF2 are mecA-positive isolates, and both belong to SCCmec type IV. A comparative genomic analysis identified multiple variations between XF1 and XF2, as shown in (Table 2). We only observed seven variations across the entire genomes, confirming that the isolates are closely related. Among the detected variants, single nucleotide polymorphisms (SNPs) were discovered in the walK (encodes a sensor protein kinase), vraR (encodes a response regulator protein), ltaS (encodes a lipoteichoic acid synthase), clpX (encodes an ATP-dependent Clp protease ATP-binding subunit), and SAOUHSC_01193 (encodes a hypothetical protein). Additionally, frameshift mutations in mgt (encodes a glycosyltransferase) and SAOUHSC_02147 (encodes a hypothetical protein). These findings suggest a potential genomic basis for the observed vancomycin, daptomycin and dalbavancin non-susceptibility in XF2 isolate, offering valuable insights that could inform the development of novel therapeutic interventions.

Table 2 Chromosomal mutations in MRSA XF2.
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The XF2 isolate possess thicker cell walls and a slower autolysis rate

Previous studies have reported that vancomycin-intermediate S. aureus (VISA) strains often exhibit common characteristics, including thickened cell walls, reduced autolysis, and attenuated virulence22,23. To determine whether the VISA strain XF2 shares these features, we utilized TEM to compare the cell wall thickness of isolates XF1 and XF2. TEM analysis revealed that the cell wall of XF2 was thicker than that of the parent strain XF1 (Fig. 3A). Quantitatively, the mean cell wall thickness of XF2 was 33.09 ± 1.26 nm, markedly greater than the XF1 strain, which measured 23.40 ± 1.71 nm (P < 0.0001) (Fig. 3B). To evaluate autolysis activity, we induced autolysis using 0.1% Triton X-100 and monitored the decrease in OD600 over time. The results showed that the VISA strain XF2 exhibited a slower autolysis rate compared to XF1 (Fig. 3C).

Fig. 3
figure 3

The cell wall thickness and Autolytic activity differences between MRSA isolates XF1 and XF2. (A) Transmission electron microscopy images showing cell wall thickness in XF1 and XF2 isolates. Scale bar = 500 nm. (B) Quantitative comparison of cell wall thickness between XF1 and XF2 isolates. Data are presented as mean ± SD. (C) Autolysis rates of XF1 and XF2 isolates induced by 0.1% Triton X-100. Data are presented as mean ± SD of three independent experiments.

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The XF2 isolate exhibits lower growth rate and attenuated virulence, but higher biofilm formation ability compared to the XF1 isolate

Growth curve analysis revealed that XF2 exhibited a significantly slower growth rate during the early logarithmic phase (3–9 h post-inoculation) compared to XF1, suggesting a compensatory growth pattern in XF2 (Fig. 4A). To further investigate the growth dynamics of XF1 and XF2, we measured viable cell counts under identical inoculation and culture conditions. Consistent with the growth curve results, XF2 demonstrated a significant growth defect at multiple time points (Fig. 4B). To evaluate virulence differences between XF1 and XF2, we performed hemolytic activity assays and G. mellonella larvae infection experiments. The hemolytic activity of XF2 was markedly lower than that of XF1 (P < 0.0001), as shown in (Fig. 4C). Additionally, in the G. mellonella infection model, larvae infected with XF2 exhibited a significantly higher survival rate compared to those infected with XF1 (86.67 vs. 40.00%, P = 0.0065, Fig. 4D). Specifically, the survival rate of XF1-infected larvae dropped to 66.67% at 24 h and further declined to approximately 40% by 48 h. These findings suggest that XF2 has attenuated virulence compared to XF1.

Fig. 4
figure 4

Growth rates and virulence characteristics differences between MRSA isolates XF1 and XF2. (A) Growth curves of MRSA isolates XF1 and XF2. TSB serves as a negative control to rule out contamination during the experiment. (B) Viable cell counts (CFU/mL) of XF1 and XF2 at different time points (2, 4, 8, 12, 24 h) under identical culture conditions. (C) Hemolytic activity of XF1 and XF2 isoaltes. (D) Survival probability of G. mellonella larvae infected with XF1 or XF2.

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To further explore phenotypic changes associated with decreased lipoglycopeptide susceptibility in XF2, we assessed biofilm formation using crystal violet staining and CLSM. Quantitative analysis revealed that XF2 exhibited a significantly higher biofilm formation capacity compared to XF1 (OD600: 2.15 ± 0.05 vs. 0.33 ± 0.06, P < 0.0001) (Fig. 5A). For comparison, we included two reference strains, ATCC 35,984 (a strong biofilm-forming strain) and ATCC 12,228 (a weak biofilm-forming strain), and observed that the biofilm formation capacity of XF2 was comparable to that of ATCC 35,984, whereas XF1 demonstrated minimal biofilm formation similar to ATCC 12,228 (Fig. 5A, B).

Fig. 5
figure 5

Biofilm formation ability differences between MRSA isolates XF1 and XF2. (A) Quantification of biofilm formation using crystal violet staining. (B) Optical density (OD600) measurement of biofilm formation. (C) CLSM images of biofilms formed by XF1 and XF2 isolates.

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To further visualize and quantify the biofilm structure, CLSM was employed to examine the biofilms formed by XF1 and XF2. The biofilm produced by XF2 was markedly thicker and denser than that of XF1, as evidenced by both 2D and 3D CLSM images (Fig. 5C). In the 2D images, XF2 displayed a significantly higher density of adherent cells compared to XF1. The 3D images revealed that XF2 formed a biofilm with a more complex and stratified architecture, exhibiting a greater overall thickness and volume. These findings indicate that XF2 possesses a robust ability to form biofilms, which may contribute to its persistence and survival under antibiotic treatment.

Gene expression changes in XF2 isolate

To investigate the molecular basis underlying the phenotypic differences between XF1 and XF2, we performed RT-qPCR to analyze the expression levels of genes associated with cell wall metabolism, autolysis regulation, virulence, and biofilm formation (Fig. 6). The results revealed significant gene expression changes in XF2 compared to XF1, as detailed below. The expression of walR and vraR, which are regulators of cell wall homeostasis and antibiotic resistance, was significantly downregulated in XF2 compared to XF1 (P < 0.001). walR, a key component of the WalKR regulatory system, showed the greatest repression, with a fold change of 0.23 ± 0.05. Similarly, vraR, part of the VraSR stress response system, exhibited a fold change of 0.35 ± 0.04. These changes may reflect alterations in regulatory networks contributing to the adaptive response of XF2. The expression levels of murD, ddl, femA, and dltA, which are involved in peptidoglycan biosynthesis and cell envelope integrity, were significantly upregulated in XF2 compared to XF1. murD and femA showed the most significant increase in expression, with relative fold changes of 2.56 ± 0.41 (P < 0.001) and 3.26 ± 0.17 (P < 0.0001), respectively (Fig. 5). ddl and dltA were also upregulated, with fold changes of 1.30 ± 0.17 (P < 0.05) and 2.01 ± 0.11 (P < 0.001), respectively. These findings suggest enhanced peptidoglycan synthesis and modifications in XF2, potentially contributing to its thickened cell wall phenotype. The expression of autolysis-related genes, including atlA, sle1, lytN, and lrgA, was significantly downregulated in XF2 compared to XF1. The expression of RNAIII, hla, agrC, and agrA, which are central to the Agr quorum-sensing system and virulence regulation, was significantly reduced in XF2. RNAIII, the main effector of the Agr system, showed a 75% reduction in expression in XF2 (P < 0.001). Similarly, the expression levels of hla (encoding α-hemolysin), agrC, and agrA were all substantially decreased (P < 0.001). These findings align with the attenuated hemolytic activity and virulence observed in XF2.

Fig. 6
figure 6

RT-qPCR analysis of the relative expression levels of genes associated with regulators, cell wall metabolism, autolysis, virulence, and biofilm formation in XF1 and XF2 isolates.

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Discussion

The rise of MRSA isolates with acquired non-susceptibility to last-line antibiotics, including vancomycin, daptomycin, and dalbavancin, poses a significant challenge in managing bacteremia. Our study of a MRSA bacteremia case reveals the critical importance of vigilant surveillance for antimicrobial resistance following treatment with these agents. The identification of XF2, a vancomycin-intermediate S. aureus strain, highlights the adaptive potential of S. aureus under antibiotic pressure and underscores the urgent need to decode the mechanisms driving its resistance. The non-susceptibility developed by MRSA isolate post-vancomycin therapy was marked by a notable increase in MICs for both daptomycin and dalbavancin, suggesting an effective adaptive response. This reduced susceptibility may be ascribed to alterations in peptidoglycan cross-linking and membrane anchoring, mechanisms that are believed to facilitate cross-resistance24. Population analysis profile results further confirmed that XF2 exhibited reduced susceptibility to vancomycin, daptomycin, and dalbavancin. However, due to the absence of an hVISA reference strain as a control, we did not calculate the AUC ratio. In contrast, the significant reduction in MICs for β-lactam antibiotics, including oxacillin and cefotaxime, in XF2 reflects the “β-lactam seesaw effect”25. This paradoxical relationship between glycopeptide resistance and β-lactam susceptibility presents an opportunity to strategically repurpose β-lactam antibiotics, alone or in combination with other agents, to combat resistant MRSA strains26,27.

Whole-genome sequencing confirmed that both XF1 and XF2 belong to the ST59-SCCmecIV lineage, a predominant epidemic clone of MRSA in China, particularly in hospital settings28,29. Multiple studies have reported a high prevalence of ST59-SCCmecIV isolates in clinical infections, underscoring their significance in MRSA-associated diseases30,31. Comparative genomic analysis identified seven key mutations in the XF2 strain, including SNPs in walK, vraR, ltaS, clpX, and SAOUHSC_01193, as well as frameshift mutations in mgt and SAOUHSC_02147. These mutations likely play critical roles in resistance development, particularly through modifications in cell wall biosynthesis and stress response pathways. Among these, the Gly223Ser substitution in WalK and the mutations in VraR are of particular interest. Bacterial resilience, as evidenced by their capacity to modulate virulence and metabolic pathways32,33. Two-component signal transduction systems (TCSs), such as WalKR and VraSR, play a critical role in the physiology of S. aureus, influencing a range of functions from cell wall metabolism to antibiotic resistance34,35. Previous studies have associated multiple mutations in WalKR with resistance to vancomycin and daptomycin36,37,38,39. Nevertheless, the involvement of WalKR in resistance to dalbavancin remains to be fully elucidated, with only a solitary genomic study hinting at a potential link40. Our study provides further evidence by identifying a Gly223Ser substitution in WalK, suggesting its potential role in non-susceptibility to vancomycin, daptomycin, and dalbavancin. This mutation likely alters the regulatory activity of the WalKR system, contributing to cell wall thickening and reduced autolysis in the XF2 isolate. This discovery enriches our understanding of the response of S. aureus to vancomycin treatment, and suggests that its resistance may lead to S. aureus resistance to similar other lipoglycopeptide antibiotics.

The VraSR two-component signal transduction system is closely linked to vancomycin resistance in S. aureus41. When exposed to antibiotics that target the cell wall, VraR can induce the activation of the vra operon along with an additional suite of 46 cell wall stress regulon genes, including the penicillin-binding protein Pbp242,43. Evidence from multiple studies demonstrates that mutations in the VraSR system lead to its activation and subsequent upregulation of cell wall synthesis genes, reinforcing the bacterial cell wall synthesis and enhancing resistance to vancomycin38,44,45. Furthermore, the VraSR system has been implicated in promoting the survival of S. aureus within the host by increasing its adhesion abilities and biofilm formation46. In XF2, the Gly223Ser mutation in VraR could promote the activation of the vra operon and other cell wall stress regulon genes, reinforcing cell wall synthesis and contributing to biofilm formation. These phenotypic adaptations were evident in XF2, which displayed a significantly thicker cell wall, slower autolysis, and enhanced biofilm formation compared to XF1. The increased biofilm-forming ability of XF2 is clinically significant, as biofilms are known to shield bacteria from antibiotics and immune responses, promoting chronic infection and persistence in clinical environments. These findings suggest that mutations in VraSR not only augment resistance but also enhance biofilm-related survival strategies, making XF2 particularly challenging to eradicate.

Despite its enhanced resistance profile, XF2 exhibited notable phenotypic trade-offs, including a reduced growth rate and attenuated virulence. The diminished hemolytic activity and lower mortality rate in the G. mellonella infection model indicate that XF2’s virulence was compromised, likely due to downregulation of the Agr system and other virulence-associated genes. These changes may reflect a fitness cost associated with resistance development, where the bacterium reallocates resources toward survival under antimicrobial pressure at the expense of its virulence factors. This trade-off may delay clinical detection but could also offer therapeutic opportunities if resistance mechanisms are targeted effectively. Our findings highlight the dual challenges of managing resistant infections: the emergence of phenotypic adaptations, such as biofilm formation, that enhance persistence, and the simultaneous attenuation of virulence that may complicate early diagnosis.

Conclusion

In summary, this study has identified several mutations associated with non-susceptibility to vancomycin, dalbavancin, and daptomycin, underscoring the genomic plasticity of S. aureus under antibiotic pressure. These genomic alterations could lead to a compromised cell wall structure, potentially making the bacteria more susceptible to β-lactam antibiotics that target cell wall synthesis. Exploiting the “β-lactam seesaw effect” through combination therapies or antibiotic rotation could provide a promising avenue for addressing resistant MRSA isolates while minimizing the risk of further resistance development.