Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Abstract

Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40-72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Keywords: Bacillus; Disinfectant; Pharmaceutical Compounding; Purification; Sporulation.

Fig. 1

Spore resistance to NaOCl on glass (A, B) and stainless steel (C, D) carriers. Bacillus spores used in this assay were purified with density-centrifugation. Blue and orange dashed lines indicate the log reduction of 3 and 5, respectively. Data are presented as the mean of result from two independent assays with triplicated samples and error bars indicate standard deviation from the mean.

Fig. 2

Effects of maturation on spore resistance to NaOCl. Spores used in this assay were kept at 4°C for 0, 7, 14, 21, and 28 days. Contact time with NaOCl was 10 min. Black box indicates the log reduction values that <3 (left; NaOCl:1500 ppm) or >5 (right; NaOCl: 5000 ppm).

Fig. 3

Effects of heat treatment on the spore purity and resistance to NaOCl. Spores used in this assay were treated at different temperatures (65°C, 70°C, and 80°C) for 5, 10, or 20 min. Blue and orange dashed lines indicate the log reduction of 3 and 5, respectively. Data are presented as the mean of results from two assays with triplicated samples.

Fig. 4

Effects of lysozyme treatment on spore resistance to NaOCl. Spores used in this assay were treated with different concentrations of lysozyme (1, 10, and 100 µg/mL) at 4°C for 1 hr. Contact time with NaOCl was 10 min. Blue and orange dashed lines indicate the log reduction of 3 and 5, respectively. Data are presented as the mean of results from two assays with triplicated samples and error bars indicate standard deviation from the mean.

Fig. 5

Effects of sonication treatment on spore resistance to NaOCl. Spores used in this assay were treated with/without sonication (37 kHz) for 5 min at room temperature. Contact time with NaOCl was 10 min. Blue and orange dashed lines indicate the log reduction of 3 and 5, respectively. All experiments were performed in two assays with triplicated samples. Data are presented as the mean and the error bars indicate standard deviation from the mean.