Validation of Spore-Forming Organisms Recovery from Peroxygen-Based Disinfectants

Neutralization method evaluation is a crucial preliminary step in biocidal agent evaluation and environmental monitoring (EM) programs. In the present study, four commercial peroxygen/Ag+-based sporicidal disinfectants in pharmaceutical industry were tested against 3 spore-forming microorganisms (Aspergillus brasiliensis, Bacillus subtilis subsp. spizizenii and Bacillus cereus). An in-house made neutralizing broth was used to dilute biocidal agents to 1:10 and 1:100 (v/v) dilutions. Disinfectant preparation, dilution and all other test conditions were performed under the laboratory conditions that simulated the working environment. Each of the neutralizer toxicity and efficiency was examined using the statistical comparison among three experimental groups viz: viability control, neutralizer toxicity and neutralizer efficacy. Different analysis criteria were investigated to compare the rate of success and failure of each replicate group for neutralizer efficacy (NE) and neutralizer toxicity (NT). Criteria of comparison were Harmonized Chapter USP<61>, USP<1227> of 3 independent replicates recovery in agar medium and finally Sutton initial recovery criteria of ≥0.75 was applied followed by Dunnett's Multiple Comparison Test in case of suspect failure. These criteria were in agreement; where Bacillus cereus gave the highest rate of failure in neutralization study. Disinfectants Mil and Pury were the hardest to neutralize and Bacillus species were sensitive to them in the neutralization procedure. Bixco disinfectant was the most successfully neutralized at 1:10 dilution with the 3 organisms. The in-house made chemical neutralizer was effective in microbial spores' recovery from residual sporicidal biocides at 1:100. Thus, it is suitable to be incorporated in both EM media and sanitization program validation.