Heat killing of <i>Clostridium difficile</i> spores exposed to chlorhexidine gluconate (CHG) and chlorhexidine free base (CHX) solutions.

<p>The mean log₁₀colony-forming unit (CFU) reduction of <i>C</i>. <i>difficile</i> 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.</p

Synergistic effects of nisin and osmotically activated <i>C. difficile</i> spores.

<p>The log₁₀CFU of unactivated and heat activated spores that were recovered from solutions after 30 minutes of incubation under ambient conditions. Spore suspension were either plated before heating or after heating at 80°C for 5 minutes (kills germinated but not dormant spores). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054740#pone-0054740-g003" target="_blank">Figure 3A–C</a> shows solutions containing solutes that readily permeate plasma membranes (ammonium hydroxide, TRIS HCl, and glycerol). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054740#pone-0054740-g003" target="_blank">Figure 3D</a> shows solutions containing sucrose which does not permeate plasma membranes. Spores were confirmed as dormant or germinated by phase contrast microscopy and DPA release. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.</p

Formulation of <i>Clostridium difficile</i> spore germination solution consisting of amino acids, minerals and taurocholic acid prepared in sterile deionized water.

<p>Formulation of <i>Clostridium difficile</i> spore germination solution consisting of amino acids, minerals and taurocholic acid prepared in sterile deionized water.</p

Comparison of heat killing of <i>Clostridium difficile</i> spores in chlorhexidine gluconate (CHG) solutions prepared with isopropanol or ethanol.

<p>The mean log₁₀colony-forming unit (CFU) reductions of <i>C</i>. <i>difficile</i> 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 (<i>P</i> <0.01 compared to isopropanol; <i>P</i> <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.</p

The Impact of pH on heat killing of <i>Clostridium difficile</i> spores exposed to chlorhexidine gluconate (CHG).

<p>The mean log₁₀colony-forming unit (CFU) reductions of <i>C</i>. <i>difficile</i> 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 (<i>P</i> <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.</p

Induced Sporicidal Activity of Chlorhexidine against <i>Clostridium difficile</i> Spores under Altered Physical and Chemical Conditions

<div><p>Background</p><p>Chlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with <i>Clostridium difficile</i>, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore’s protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against <i>C</i>. <i>difficile</i> spores.</p><p>Principal Findings</p><p><i>C</i>. <i>difficile</i> spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%–0.0004% w/v chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions.</p><p>Conclusions</p><p>Physical and chemical conditions that alter the protective barriers of <i>C</i>. <i>difficile</i> spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore.</p></div

Optimal doses of ultraviolet-C radiation for killing germinating versus dormant <i>Clostridium difficile</i> spores, MRSA, and VRE.

<p>Mean reduction (log₁₀ colony-forming units) in recovery of <i>C. difficile</i> spores from an epidemic NAP1/BI strain, a PFGE type USA 300 strain of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), and a vanB-type strain of vancomycin-resistant <i>Enterococcus</i> (VRE) from laboratory bench top surfaces after the use of a UV-C device at reflected doses ranging from 5,000 to 20,000 µWs/cm². <i>C. difficile</i> spores were incubated in either water or germination solution for 5 minutes before exposure to UV-C radiation. Spores were confirmed as dormant or germinated by phase contrast microscopy.</p

Germination in unactivated and heat activated <i>C. difficile</i> spores.

<p>The log₁₀CFU of unactivated and heat activated spores that germinated in the comprehensive germination medium (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054740#pone-0054740-t001" target="_blank">Table 1</a>) after 30 minutes of incubation under ambient conditions. Spores were confirmed as dormant or germinated by phase contrast microscopy, heat susceptibility (80°C for 5 minutes) and a modified Wirtz-Conklin stain. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.</p

Enhancement of heat killing of <i>Clostridium difficile</i> spores exposed to alcoholic chlorhexidine gluconate (CHG) solutions.

<p>The mean log₁₀colony-forming unit (CFU) reduction of <i>C</i>. <i>difficile</i> 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.</p

Inhibition of germinated <i>C. difficile</i> spores by nisin.

<p>The log₁₀CFU of unactivated and heat activated spores that were recovered from solutions after 30 minutes of incubation under ambient conditions. Spore suspension were either plated before heating or after heating at 80°C for 5 minutes (kills germinated but not dormant spores). Spores were confirmed as dormant or germinated by phase contrast microscopy and DPA release. The means of the data from experiments conducted in triplicate are presented. Error bars indicate standard error.</p