Background: Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes. Objectives: This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates. Materials and Methods: A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR. Results: Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002). Conclusions: There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks. Keywords: Coagulase; Urease; Vancomycin; Methicillin-Resistant Staphylococcus aureus

Introduction: Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is currently problematic. Although the population analysis profile with area under the curve (PAP-AUC) is the gold standard for detecting hVISA strains, this method is time consuming. This study aimed to induce vancomycin non-susceptible Staphylococcus aureus isolates in methicillin-resistant S. aureus (MRSA) and to determine the performance of the vancomycin and teicoplanin disk diffusion test for screening of induced and natural vancomycin non-susceptible isolates. Methodology: Vancomycin resistance was induced in vitro in methicillin-resistant S. aureus by serial passage in media with increasing vancomycin concentrations. All test isolates were confirmed for their susceptibility to vancomycin by using a PAP-AUC method. The performance of the vancomycin and teicoplanin disk diffusion test for detecting both induced and natural hVISA/VISA isolates was analyzed using the MedCal program version 10.2.0. Results: The induction test revealed that 42 of 78 MRSA isolates (53.8%) became hVISA and/or VISA. Using 10, 15, 20, 30 µg vancomycin disks and a 30 µg teicoplanin disk, the highest performance (88.9%) for hVISA/VISA detection (71.1%, sensitivity, 100% specificity, 100% positive predictive value, and 75% negative predictive value) was obtained when a 20 µg vancomycin disk was used at 1.0 McFarland inoculum for a 24-hour incubation. Conclusions: The results indicated that using a 20 µg vancomycin disk and bacterial inoculum of 1.0 McFarland is simple to perform and provides a primary result for hVISA/VISA screening within 24 hours.

We investigated the synergistic effect between vancomycin and ß-lactams against vancomycin-susceptible (VSSA) and non-susceptible MRSA isolates (heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA). A total of 29 MRSA including 6 VISA, 14 hVISA and 9 VSSA isolates were subjected to a microbroth dilution-minimum inhibitory concentration (MIC) checkerboard using vancomycin combined with cefotaxime, imipenem or meropenem. To confirm synergistic activity, the representative strains of VISA, hVISA and VSSA were then selected for the time-kill curve method. The combination of vancomycin with imipenem, meropenem and cefotaxime exhibited synergistic effects against 17 (2 VISA, 9 hVISA and 6 VSSA), 14 (3 VISA, 9 hVISA and 2 VSSA), and 5 (3 VISA and 2 hVISA) isolates respectively. Additive and indifferent effects were found in the remaining isolates but no antagonistic effect was observed. Using time-kill assay, the vancomycin combined with either imipenem or cefotaxime demonstrated synergism against both VISA and hVISA isolates, while the synergistic effect with meropenem was obtained in the VISA isolates only. This study demonstrated in vitro enhanced antibacterial activity of vancomycin plus ß-lactams against clinical hVISA or VISA isolates. These combinations may be an alternative treatment for MRSA infections in clinical practice.

Background/Objectives: Thalassemia is an inherited blood disorder caused by defects in hemoglobin production, where an imbalance or reduction in globin-chain synthesis impairs normal red cell development and results in anemia of varying severity. The disease is classified into α-thalassemia and β-thalassemia according to the affected globin genes. In recent years, infrared (IR) Microspectroscopy has gained increasing attention for blood analysis because it is rapid, label-free, and capable of detecting subtle biochemical alterations. Method: In this study, we analyzed hemoglobin lysate collected from clinically characterized normal, carriers, and thalassemia patients (n = 333) using IR Microspectroscopy combined with multivariate statistical methods, including Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). This approach enabled us to investigate how spectral features correspond to disease status across a range of genotypes commonly encountered in clinical practice. Results: Clear intergroup spectral differences were observed, and the classification models demonstrated diagnostic performance with sensitivity and specificity of approximately 80–90%. Because the technique is non-destructive, requires no chemical reagents, and allows direct biochemical profiling of hemoglobin, it offers practical advantages over conventional hematologic or molecular assays. Conclusions: These findings support the potential of IR-based spectral analysis as a complementary tool for thalassemia screening. Looking ahead, incorporating advanced machine learning algorithms with IR Microspectroscopy may further enhance early detection, improve risk stratification, and strengthen prevention and management strategies in routine clinical workflows.