Repurposing Success: Real-World Validation of a Novel Cephalosporin Decontamination Protocol Using Chlorine Dioxide and Disc Diffusion Testing

Introduction: Beta-lactam manufacturing facilities face strict regulatory challenges to repurposing for non-beta-lactam production due to the high risk of cross contamination. This study introduces a novel decontamination protocol for cephalosporin facilities, combining chlorine dioxide (ClO 2 ) treatment with disc diffusion testing to verify the complete degradation and inactivation of beta-lactam residues in a real-world setting. Methods: Antimicrobial susceptibility discs (ASDs) contaminated with cephalosporins (Cefalexin, Cefaclor, Cefixime, Cefuroxime) were exposed to both aqueous (400 ppm for 24 h) and gaseous ClO 2 (5 mg/L for >5 h) in separate cycles, achieving a minimum cumulative exposure of 9600 ppm-hours in both. Post-treatment, replicates (4 = aqueous cycle, 5 = gaseous cycle) of each cephalosporin were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and microbial challenge testing to assess degradation and biological inactivation, respectively. Result: For all, LC-MS/MS confirmed cephalosporin degradation below the reporting limit (<0.02 μg/disc), while microbial challenge testing demonstrated no significant biological activity with <1 mm bacterial inhibition. Microbial challenge and LC-MS/MS results showed significant agreement (Cohen’s κ = 1.00) in both aqueous and gaseous ClO 2 cycles. Verification: Gaseous ClO 2 application at 9600 ppm-hours was applied to a 450 m 2 cephalosporin facility. Analysis confirmed that cephalosporin residues were degraded to levels below the acceptable limits (<1 μg/disc) with microbial challenge testing supporting the success of the cycle, with no significant biological activity remaining in the discs placed throughout the facility. Conclusion: This study presents the first successful protocol for cephalosporin facility decontamination using gaseous ClO 2 in a real-world setting.