Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

spores to radiation or other stressors. spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents

Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms

<p>Abstract</p> <p>Background</p> <p>Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against <it>Clostridium difficile </it>spores, but also safe, rapid, and automated.</p> <p>Methods</p> <p>The Tru-D™ Rapid Room Disinfection device is a mobile, fully-automated room decontamination technology that utilizes ultraviolet-C irradiation to kill pathogens. We examined the efficacy of environmental disinfection using the Tru-D device in the laboratory and in rooms of hospitalized patients. Cultures for <it>C. difficile</it>, methicillin-resistant <it>Staphylococcus aureus </it>(MRSA), and vancomycin-resistant <it>Enterococcus </it>(VRE) were collected from commonly touched surfaces before and after use of Tru-D.</p> <p>Results</p> <p>On inoculated surfaces, application of Tru-D at a reflected dose of 22,000 μWs/cm²for ~45 minutes consistently reduced recovery of <it>C. difficile </it>spores and MRSA by >2-3 log₁₀colony forming units (CFU)/cm²and of VRE by >3-4 log₁₀CFU/cm². Similar killing of MRSA and VRE was achieved in ~20 minutes at a reflected dose of 12,000 μWs/cm², but killing of <it>C. difficile </it>spores was reduced. Disinfection of hospital rooms with Tru-D reduced the frequency of positive MRSA and VRE cultures by 93% and of <it>C. difficile </it>cultures by 80%. After routine hospital cleaning of the rooms of MRSA carriers, 18% of sites under the edges of bedside tables (i.e., a frequently touched site not easily amenable to manual application of disinfectant) were contaminated with MRSA, versus 0% after Tru-D (<it>P </it>< 0.001). The system required <5 minutes to set up and did not require continuous monitoring.</p> <p>Conclusions</p> <p>The Tru-D Rapid Room Disinfection device is a novel, automated, and efficient environmental disinfection technology that significantly reduces <it>C. difficile</it>, VRE and MRSA contamination on commonly touched hospital surfaces.</p

Effective and Reduced-Cost Modified Selective Medium for Isolation of Clostridium difficile▿

Both for epidemiologic studies and for diagnostic testing, there is a need for effective, economical, and readily available selective media for the culture of Clostridium difficile. We have developed a reduced-cost substitute for cycloserine-cefoxitin-fructose agar (CCFA), which is an effective but expensive selective medium for C. difficile. The modified medium, called C. difficile brucella agar (CDBA), includes an enriched brucella base as a substitute for proteose peptone no. 2, and the concentration of sodium taurocholate has been reduced from 0.1% to 0.05%. To compare the sensitivities and selectivities of CDBA and CCFA, cultures for C. difficile were performed using stool samples from patients with C. difficile-associated disease. CDBA was as sensitive as CCFA for the recovery of C. difficile, with a similar frequency of breakthrough growth of stool microflora (25% versus 31%, respectively). A liquid formulation of the modified medium, termed C. difficile brucella broth (CDBB), stimulated rapid germination and outgrowth of C. difficile spores, at a rate comparable to that in cycloserine-cefoxitin-fructose broth. Our results suggest that CDBA and CDBB are sensitive, selective, and reduced-cost media for the recovery of C. difficile from stool samples

Activate to eradicate: inhibition of Clostridium difficile spore outgrowth by the synergistic effects of osmotic activation and nisin.

BACKGROUND: Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores. PRINCIPAL FINDINGS: Exposure to germination solution triggered ~1 log(10) colony forming units (CFU) of spores to germinate, and heat activation increased the spores that germinated to >2.5 log(10)CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log(10)CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity. CONCLUSIONS: These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and effectively reduce the burden of C.difficile spores on skin and environmental surfaces

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

Sorting through the wealth of options: comparative evaluation of two ultraviolet disinfection systems.

Environmental surfaces play an important role in the transmission of healthcare-associated pathogens. Because environmental cleaning is often suboptimal, there is a growing demand for safe, rapid, and automated disinfection technologies, which has lead to a wealth of novel disinfection options available on the market. Specifically, automated ultraviolet-C (UV-C) devices have grown in number due to the documented efficacy of UV-C for reducing healthcare-acquired pathogens in hospital rooms. Here, we assessed and compared the impact of pathogen concentration, organic load, distance, and radiant dose on the killing efficacy of two analogous UV-C devices.The devices performed equivalently for each impact factor assessed. Irradiation delivered for 41 minutes at 4 feet from the devices consistently reduced C. difficile spores by ∼ 3 log10CFU/cm2, MRSA by>4 log10CFU/cm2, and VRE by >5 log10CFU/cm2. Pathogen concentration did not significantly impact the killing efficacy of the devices. However, both a light and heavy organic load had a significant negative impacted on the killing efficacy of the devices. Additionally, increasing the distance to 10 feet from the devices reduced the killing efficacy to ≤3 log10CFU/cm2 for MRSA and VRE and <2 log10CFU/cm2 for C.difficile spores. Delivery of reduced timed doses of irradiation particularly impacted the ability of the devices to kill C. difficile spores. MRSA and VRE were reduced by >3 log10CFU/cm2 after only 10 minutes of irradiation, while C. difficile spores required 40 minutes of irradiation to achieve a similar reduction.The UV-C devices were equally effective for killing C. difficile spores, MRSA, and VRE. While neither device would be recommended as a stand-alone disinfection procedure, either device would be a useful adjunctive measure to routine cleaning in healthcare facilities

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

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

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