Ultrahigh-throughput screening of heterogeneous vancomycin-intermediate Staphylococcus aureus based on fluorescence-activated droplet sorting

Background Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is associated with suboptimal glycopeptide treatment outcomes. However, conventional antimicrobial susceptibility testing fails to distinguish hVISA from vancomycin-susceptible Staphylococcus aureus (VSSA), potentially delaying effective therapy. The gold standard population analysis profiling with area under the curve (PAP-AUC) method is labor-intensive and time-consuming, necessitating rapid detection alternatives. Methods This study introduces a microfluidic platform based on fluorescence-activated droplet sorting (FADS) for the rapid detection and isolation of hVISA. Through systematic optimization of key parameters such as droplet generation conditions, bacterial suspension concentration, fluorescence probe selection, fluorescence duration, and vancomycin screening concentration, we established a rapid hVISA screening system. The efficacy of this system was evaluated using the hVISA standard strain Mu3, followed by validation with 15 clinical hVISA isolates. Genomic analysis further elucidated the genetic basis of drug resistance in hVISA strains. Results We developed a high-throughput detection platform for hVISA by integrating a microfluidic system with FADS. Based on a Poisson distribution theoretical model, the bacterial suspension concentration was optimized to 10 6  CFU/mL, achieving a single-bacterium droplet encapsulation rate of approximately 30%. The selected fluorescent probe exhibited detectable fluorescence signals as early as 6 h post-cultivation, with stability maintained for at least 1 week. Drug concentration validation demonstrated that 4 μg/mL vancomycin effectively inhibited susceptible bacteria without interfering with fluorescence detection. This platform successfully isolated resistant subpopulations from the hVISA reference strain Mu3 within 12 h, and its efficacy was further validated using 15 clinical hVISA isolates. Conclusions This study innovatively integrated a microfluidic detection platform and FADS to achieve rapid detection of hVISA. This technology reduced the detection time to just 12 h and significantly improved the enrichment efficiency of drug-resistant bacterial populations.