Fig 1.
Heat killing of Clostridium difficile spores exposed to chlorhexidine gluconate (CHG) and chlorhexidine free base (CHX) solutions.
The mean log10colony-forming unit (CFU) reduction of C. difficile spores exposed to CHG and CHX solutions at 80°C. After 5 minutes of exposure to CHG or CHX, heat killing increased as the concentration of chlorhexidine was increased. However, after 10 or 15 minutes of exposure to chlorhexidine at 80°C, similar reductions were achieved at each concentration. The means of the data from four experiments conducted are presented. Error bars indicate standard error.
Fig 2.
Enhancement of heat killing of Clostridium difficile spores exposed to alcoholic chlorhexidine gluconate (CHG) solutions.
The mean log10colony-forming unit (CFU) reduction of C. difficile spores exposed to 4% w/v CHG solution prepared in water or 70% ethanol. No killing of spores was observed in aqueous or alcoholic chlorhexidine solutions at 20°C. At 37°C, the presence of alcohol reduced the incubation time required to achieve an ~1 log10CFU reduction from 3 hours to 1 hour. At 55°C, alcohol boosted spore reductions from 1.5 log10CFU (aqueous) to 3log10CFU (alcoholic), and 3log10CFU (aqueous) to 5log10CFU (alcoholic) after 1 and 2 hours respectively. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.
Fig 3.
Comparison of heat killing of Clostridium difficile spores in chlorhexidine gluconate (CHG) solutions prepared with isopropanol or ethanol.
The mean log10colony-forming unit (CFU) reductions of C. difficile spores achieved after 1 or 3 hours of exposure to 0.04% or 4% w/v CHG prepared in water, 70% isopropanol, or 70% ethanol at 55°C. CHG solutions prepared with ethanol significantly enhanced heat killing of spores after 1 hour of incubation compared to CHG solutions prepared in either isopropanol or water (P <0.01 compared to isopropanol; P <0.001 compared to water). After 3 hours of incubation in 0.04% w/v CHG, both isopropanol and ethanol enhanced reduction of spores compared to aqueous CHG solution; however, at increased CHG concentrations (4% w/v), spores were completely eliminated by both aqueous and alcoholic preparations. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.
Fig 4.
The Impact of pH on heat killing of Clostridium difficile spores exposed to chlorhexidine gluconate (CHG).
The mean log10colony-forming unit (CFU) reductions of C. difficile spores achieved after 1 or 3 hours of incubation in pH altered aqueous and alcoholic CHG solutions (0.04% w/v). Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic CHG solutions (P <0.001 for each comparison to pH 4.0). In aqueous or alcoholic CHG solutions, increasing the pH to ≥9.5 enhanced heat killing of spores by ≥1log10CFU after 3 hours of incubation. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.