Characterization of the Synergistic Antibacterial Effect of Verapamil on Bacterial Isolates From Cancer Patients

This study evaluates the potential synergistic antibacterial effect of verapamil in combination with selected antibiotics against multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacterial isolates obtained from cancer patients.

• Study Samples Clinical specimens will be collected as part of routine diagnostic care from cancer patients.

Specimen types may include:

Blood Urine Respiratory samples Wound swabs Other relevant clinical specimens Only non-duplicate bacterial isolates will be included. Only isolates classified as MDR, XDR, or PDR will be selected for further analysis.

• Isolation of Bacterial Isolates Specimens will be cultured using standard microbiological techniques.

Culture media will include:

Blood agar MacConkey agar Plates will be incubated under appropriate conditions (temperature and atmosphere) according to standard laboratory protocols.

• Identification of Bacteria

Bacterial isolates will be identified using:

Colony morphology Gram staining Biochemical tests Where available, automated identification systems will be used. Identification procedures will follow standard microbiological guidelines.

• Antimicrobial Susceptibility Testing (AST)

AST will be performed according to:

Clinical and Laboratory Standards Institute (CLSI), 2025 guidelines

Methods include:

Kirby-Bauer disk diffusion method Automated susceptibility testing systems (if available) Results will be interpreted using CLSI breakpoints. Quality control will be ensured using standard reference strains.

• Preparation of Verapamil

Verapamil stock solution will be prepared:

Under aseptic conditions Using sterile distilled water or appropriate solvent Serial dilutions will be prepared to achieve required working concentrations.

• Determination of Minimum Inhibitory Concentrations (MICs)

MICs for:

Selected antibiotics Verapamil

Will be determined using:

Broth microdilution method

Procedures include:

Preparation of serial twofold dilutions Standardization of bacterial inoculum (e.g., 0.5 McFarland) Incubation under appropriate conditions MIC values will be recorded as the lowest concentration inhibiting visible growth.

• Assessment of Synergistic Activity

Synergy testing will be performed using:

Checkerboard assay

Interaction between verapamil and antibiotics will be evaluated by calculating:

Fractional Inhibitory Concentration Index (FICI)

Interpretation of FICI:

≤ 0.5 → Synergistic > 0.5 - 1 → Additive > 1 - 4 → Indifferent > 4 → Antagonistic

• Additional/Confirmatory Testing

Where applicable, additional methods may be used:

Disk diffusion-based combination testing These methods will support and validate synergy findings.

• Exploratory Analysis

If significant synergy is observed:

Further analysis may be conducted to investigate resistance mechanisms

This may include:

Evaluation of changes in resistance patterns Analysis of resistance-related gene expression (if feasible)

• Quality Control and Laboratory Standards

All procedures will follow:

Standard microbiological protocols Institutional biosafety regulations

Quality assurance measures will be applied to ensure:

Accuracy Reproducibility Reliability of results

Inclusion criteria

• Bacterial isolates from clinical samples submitted for the SECI laboratory for culture and sensitivity testing that are:
• MDR: resistant to one or more agent within three or more of antimicrobial classes
• XDR: resistant to one or more agent within all but two antimicrobial classes
• PDR: resistant to all agents within all antimicrobial classes.

Exclusion criteria

• Bacterial isolates from non-cancer patients.
• Contaminant or non-pathogenic isolates.
• Duplicate isolates from the same patient with identical antibiogram.
• Bacterial isolates that are neither MDR, XDR OR PDR.