Charcoal agar interference in antibiotic susceptibility testing of Bordetella pertussis: evaluation and alternative methodologies

Erythromycin-resistant Bordetella pertussis (ERBP) has increased sharply in China, posing significant challenges to clinical treatment. The lack of a standardized antimicrobial susceptibility testing (AST) system for B. pertussis makes it difficult to screen out effective antibiotics, which further aggravates the generation of drug-resistant strains and causes a vicious cycle. In this study, we modified a superior charcoal-free medium for antibiotic susceptibility testing, enabling more reliable evaluation of antibiotic efficacy. The minimum inhibitory concentrations (MICs) of erythromycin (E), trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin (LVF), and piperacillin (PIP) were determined using E-test strips on the agar plates with and without charcoal. The performance of these two media for AST was compared to establish a more accurate laboratory detection method. Subsequently, the superior charcoal-free medium was used to assess the antibiotic susceptibility of 98 B. pertussis strains isolated from clinical specimens after the COVID-19 pandemic, analyzing the sensitivity distribution of these first-line and alternative antibiotics. The MIC values of TMP-SMX and LVF were significantly higher on charcoal-containing agar plates than on charcoal-free medium. E-test results revealed that the MICs of B. pertussis strains to TMP-SMX and LVF had increased by approximately five gradient levels on the charcoal-containing agar compared to charcoal-free agar. Quality control (QC) testing demonstrated that the MICs of TMP/SMX and LVX fell outside the acceptable QC range due to adsorption by charcoal particles. Antimicrobial susceptibility of 98 B. pertussis clinical isolates showed that all strains were resistant to E with the MIC90 exceeded the maximum value (> 256 μg/mL). The overall MIC90 values for TMP-SMX, LVF and PIP were 1/0.75/<0.016 μg/mL on charcoal-containing agar and 0.023/0.008/<0.016 μg/mL on charcoal-free medium, respectively. Statistical analysis of violin plots confirmed that the MIC values of the second-line antibiotic TMP-SMX and LVF were significantly elevated on charcoal-containing agar. An accurate AST method can provide crucial guidance for optimal antibiotic selection in patients with B. pertussis infections. Our findings demonstrate that charcoal-free agar serves as an effective alternative medium for reliable AST of B. pertussis clinical isolates.