Development of Resazurin-Based Assay for Rapid Evaluation of Sodium Hypochlorite Tolerance in Salmonella

Sodium hypochlorite (NaClO) is frequently utilized in food processing. More than 90% of Salmonella spp. isolates from poultry supply chains exhibited tolerance to NaClO, with MIC values exceeding 256 mg/L. Exposure to NaClO disinfection may lead to the emergence of bacterial tolerance to chlorine, which is frequently associated with antibiotic cross-resistance. This work employed a resazurin-based assay for rapid evaluation of the NaClO chlorine tolerance of Salmonella. The results were compared to the broth microdilution method for assessing bacterial tolerance. At the initial inoculum of 10⁷ CFU/mL, NaClO tolerance was successfully identified via colorimetry within 2 h. Notably, the fluorescence-based evaluation yielded significant results even sooner, showing a marked increase in intensity within 1 h of resazurin incubation. Even with an inoculum of 10⁵ CFU/mL, the resazurin-based method determines NaClO tolerance in just 6 h. Conversely, traditional broth microdilution requires an overnight culture to manifest sufficient turbidity for optical density monitoring. Furthermore, the broth microdilution method revealed NaClO tolerance (MIC > 256 mg/L) in 1.6% (1/64) of the Salmonella isolates. The modified resazurin assay, by contrast, detected tolerance in 6.3% (4/64) of isolates. The reference that differentiates between resistant and sensitive strains was 3.2 × 10⁵ RFU. When the strains exhibited an MIC value of 256 mg/L, the fluorescence intensity varied from around 1.2 × 10⁵ to 4 × 10⁵ RFU, reflecting inactivation effects at practical chlorine concentrations. This methodology is recognized as a rapid, high-throughput, and quantitative screening approach for assessing bacterial chlorine resistance.