Evidence from an In Vitro Study: Is Oxacillin Plus Vancomycin a Better Choice for Heteroresistant Vancomycin-Intermediate Staphylococcus aureus?

A total of 50 non-duplicate S. aureus isolated from the blood culture between 2016 and 2017 at a 2600-bed tertiary care hospital, Christian Medical College, Vellore, India, were included in this study. Isolates were identified and characterized using standard culture and biochemical methods [24]. Of 50 isolates, 11 were MSSA, 10 were MRSA and 29 were confirmed as hVISA, using population analysis profile-area under curve (PAP-AUC). In this study, S. aureus strains that were resistant to cefoxitin and completely susceptible to vancomycin without any heteroresistant subpopulation were specified as MRSA, while MRSA strains expressing vancomycin heteroresistance were called hVISA. The hVISA strains were subcultured and maintained in brain Heart infusion agar (BHIA) were supplemented with 1 μg/ml of vancomycin and stored at − 70 °C. The study was approved by the institutional review board (IRB. Min. No. 10643 dated April, 2017).

Methicillin resistance was detected by disk diffusion testing using a 30-μg cefoxitin disk as recommended by the CLSI guidelines [25]. The MICs of oxacillin and vancomycin were determined by the CLSI-recommended broth microdilution method [26]. Oxacillin and vancomycin powders were obtained from commercial sources (Sigma, St Louis, MO, USA). Cation-adjusted Mueller–Hinton broth (CA-MHB) containing 2% NaCl was used for oxacillin MICs determination. Oxacillin and vancomycin were tested at the concentration of 0.03–512 μg/ml and 0.03–16 μg/ml, respectively. The MICs of oxacillin and vancomycin were read after incubation at 35 °C for 24 h. S. aureus ATCC 29213 and S. aureus ATCC 43300 strains were used as a quality control strains.

Bacterial DNA was extracted from colonies grown overnight on blood agar using the QIAamp DNA Mini Kit and the QIAcube instrument (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. PCR was performed for the detection of mec A gene in MRSA isolates, as previously described [27]; this is considered as the gold standard method for detection of methicillin resistance in S. aureus.

MRSA isolates were screened for hVISA using the glycopeptide resistance detection (GRD) E test (bioMérieux, France) and confirmed with the PAP/AUC method. The GRD E test was performed using 0.5 McFarland-adjusted inoculum swabbed onto Mueller–Hinton agar (MHA) containing 5% blood. The zone of inhibition was read at 24 and 48 h after incubation at 35 ± 2 °C [28]. The test isolate was considered positive for hVISA if the MIC of vancomycin or teicoplanin was 8 µg/ml. PAP-AUC analysis was performed as previously described [29]. The bacterial suspension was plated onto freshly prepared BHIA plates containing 0.5–8 μg/ml of vancomycin. Colonies were counted after 48 h of growth at 35 ± 2 °C. For calculation of AUC, viable counts were plotted against increasing concentration of vancomycin using the GraphPad Prism™ (v.7.0) software package. All PAP-AUC experiments were performed in duplicate. For vancomycin PAP analysis, the AUC ratio was calculated by dividing the AUC of the test strain by the AUC of the MU3 (hVISA) strain. The PAP-AUC ratio was interpreted as follows, < 0.9 as vancomycin-susceptible S. aureus (VSSA), ≥ 0.9 as hVISA phenotype, and > 1.3 as vancomycin-intermediate S. aureus (VISA). For each batch of the PAP-AUC experiment, the hVISA (MU3, ATCC 700698), VISA (MU50, ATCC 700699) and S. aureus ATCC 29213 (VSSA) were used as the reference, comparator and negative control strains, respectively.

Checkerboard synergy testing was performed by the microbroth dilution method, as previously described [30]. Vancomycin was tested at a concentration of 0.03–8 µg/ml. MHB containing 2% NaCl was used to perform in vitro synergy testing. In the checkerboard assay, vancomycin was combined with oxacillin in the concentration of 1–128 µg/ml for MRSA and hVISA. For MSSA, oxacillin was used at a concentration of 0.25–8 µg/ml. Microtiter plates were incubated at 37 ± 2 °C for 24 h. Growth control and sterility control were included in each test panel. The first non-turbid well in each row and column was used to calculate the fractional inhibitory concentration (FIC) index. An FIC index of ≤ 0.5 was defined as synergy, an FIC index of > 0.5 to 4.0 was defined as indifferent, and an FIC index of > 4.0 was defined as antagonistic.

The time-kill assay was performed in duplicate on all PAP-confirmed hVISA, MRSA, and MSSA isolates. Time-kill assays were performed according to previously published techniques [31]. An initial bacterial inoculum of approximately 5 × 10⁶ for each isolate was inoculated into CA-MHB containing 2% NaCl. Time-kill experiments were performed at half-MIC of oxacillin and vancomycin for the respective isolates. Each antibiotic alone, together with oxacillin in combination with vancomycin, were tested. The inoculum was diluted 1 in 100 using sterile saline. A volume of 100 µl was plated on the MHA plates at times of 0, 3, 6 and 24 h post-incubation in a shaker-incubator at 35 ± 2 °C. After 24 h of incubation (48 h for hVISA) isolates at 35 ± 2 °C, colonies were counted and results were expressed as log₁₀CFU/ml. Each batch of testing included sterility control and growth control (without any antibiotic). Synergy was defined as ≥ 2 log₁₀ decrease in CFU/ml for combination antibiotics in comparison to a single agent. Antagonism was defined as ≥ 2 log₁₀ increase in CFU/ml for the combination antibiotic in comparison to the most active single agent. Bactericidal activity was defined as a ≥ 3 log₁₀ CFU/ml reduction from baseline. Regrowth was defined as a ≥ 3 log₁₀ decrease in CFU/ml followed by a ≥ 2 log₁₀ increase in CFU/ml at 24 h.

This article does not contain any studies with human participants or animals performed by any of the authors. The study was approved by an institutional review board (IRB. Min. No. 10643 dated April 2017).