Why is bacillus subtilis resistant to disinfectants

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Poole, K. The strains of B. We concluded that there are intrinsic differences in the susceptibility to chlorhexidine between the groups, but the molecular mechanisms are unknown. The minimum inhibitory or bactericidal concentrations of disinfectants other than chlorhexidine may also need to be clarified in the B. Bacillus cereus , Bacillus subtilis , Chlorhexidine , Susceptibility.

Chlorhexidine is one of the most widely used biguanides for antiseptic purposes, such as skin surface preparation and intravascular catheter maintenance.

Chlorhexidine inhibits bacterial growth by disrupting the structure of the cell membrane, leading to the leakage of cellular contents [1].

Chlorhexidine gluconate CHG or chlorhexidine acetate is generally used because of the low solubility of chlorhexidine base. Although the antiseptic activity of CHG has been validated in gram-positive and gram-negative bacteria and enveloped viruses, its efficacy against mycobacteria, nonenveloped viruses, fungi, and bacterial endospores is limited [2]. In spore-forming bacteria, chlorhexidine acts as a sporestatic agent rather than a sporicidal agent [3] [4] [5] [6].

Because CHG is generally ineffective against bacterial endospores, less attention has been paid to the differences in the action of CHG in the genus Bacillus. The efficacy of CHG against bacterial endospores was mainly examined using B. The bactericidal or inhibitory concentration of chlorhexidine in Bacillus spp. Bacillus consists of spore-forming, facultative anaerobic or aerobic gram-positive bacilli, and Bacillus spp. The genus Bacillus comprises many species ranging from pathogenicity to animals, including Bacillus cereus , to harmless species, including Bacillus subtilis.

Among them, the harmful species are classified into the cereus group, which comprises B. Several studies examined the activity of chlorhexidine against B. This study aimed to evaluate the susceptibility of the B. In this study, we divided bacterial strains into two groups, the cereus group or subtilis group. The cereus group consists of representative strains of B. The subtilis group consists of the B. The bacteriostatic and bactericidal concentrations of CHG were compared among Bacillus spp.

The decrease in bacterial counts under bacteriostatic concentrations was assessed by a time-kill assay using representative strains. The Bacillus strains employed in this study are listed in Table 1. The strains included 3 representative or genome strains of B. Table 1. In the table, descriptive statistics are provided for the minimum inhibitory concentration MIC and minimum bactericidal concentration MBC of CHG, and the disk diffusion test was performed using polymyxin B and colistin.

For representative strains of other cereus groups, B. In this study, the counts of living bacterial cells were measured as the average colony-forming units CFUs on three MH agar plates. CHG was serially diluted with sterilized water in each concentration and used in sensitivity studies.

The minimum inhibitory concentration MIC of CHG was assessed by the agar dilution method, referring to the method for dilution antimicrobial susceptibility tests of clinical and laboratory standard institute [21].

Each strain was seeded onto a standard nutrient agar plate. After incubation at room temperature for 30 min, nine volumes of inactivation solution were added to the mixture to inactivate CHG. These experiments were repeated at least twice to confirm the results. The modified time-kill test was performed using eight Bacillus isolates to evaluate the changes of bacterial cell populations under bacteriostatic concentrations of chlorhexidine.

The recovered mixture was seeded onto MH agar plates. The survival of bacterial cells was evaluated in comparison to the initial cell number. The experiments were repeated at least twice to confirm the results for each tested strain. Microscopic imaging was performed during incubation with several concentrations of CHG. Overnight cultures of B. One loop of the incubation mixture was placed on a slide glass, which was stained using the standard Gram staining method. After staining, the slides were examined using the oil immersion objective of an optical microscope BX51 with DP73, Olympus, Tokyo, Japan.

The disk diffusion test was repeated at least two times, and the average diameter was calculated. It should be noted that the MICs of S. In both groups, the sporicidal concentration was nearly identical to the practically used concentration. The time-kill test was used to examine the survival ratio of Bacillus spp.

There were no significant differences in effects between the B. Figure 1. The time before the addition of chlorhexidine was set as time zero. The bracket on the bars indicates the standard error S. In total, eight bacterial strains were examined; B. Cellular damage induced by CHG was assessed in both B. Damaged cells and debris were observed in B. Meanwhile, cellular damage and debris were observed in B. This morphological observation supported the differences in the susceptibility to CHG between the B.

In a preliminary study, other strains belonging to the cereus group, B. Other strains belonging to the subtilis group, B. Figure 2. Microscopic observation of B. Figure 3. Disk diffusion test of colistin and polymixin B. There are wide divergencies within this general classification. Thus, i spores of Bacillus subtilis are less susceptible to biocides than those of Clostridium difficile: ii Mycobacterium chelonae strains may show high resistance to glutaraldehyde and M. The mechanisms involved in biocide resistance to biocides are becoming better understood.

Intrinsic resistance intrinsic insusceptibility is found with bacterial spores, mycobacteria and Gram-negative bacteria. This resistance might, in some instances, be associated with constitutive degradative enzymes but in reality is more closely linked to cellular impermeability.

The coats s and, to some extent, the cortex in spores, the arabinogalactan and possibly other components of the mycobacterial cell wall and the outer membrane of Gram-negative bacteria limit the concentration of active biocide that can reach the target site s in these bacterial cells.