Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells

Abstract

Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands.

The alternative sigma factor σB plays a crucial role in adaptive strategies of Clostridium difficile during gut infection

“…We found that only the first dilutions of the R20291 strain grew in the presence of 0.4% or 0.1% of O 2 as compared to the growth of all dilutions of the 630Δ erm strain. This result suggesting that the R20291 strain was more sensitive to O 2 than the 630Δ erm strain is consistent with recent studies (Edwards et al ., ). In addition, the complemented strain in the R20291 genetic background showed higher tolerance to O 2 than the parental strain did at 0.1% and 0.4% of O 2 .…”

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis

“…A minimum of three biological replicates were performed for each growth curve shown. The pH of cultures and solutions was measured as previously described (Edwards et al, 2016b).…”

“…Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer (without dye) and mechanically disrupted as previously described (Edwards et al, 2016a;Nawrocki et al, 2016). The pH of the cultures were also measured at the time of harvest, as previously described (Edwards et al, 2016b). Protein concentrations were assessed using a micro BCA assay (Thermo Scientific) and 8 mg of whole cell protein loaded onto a 12% polyacrylamide gel.…”

CotL, a new morphogenetic spore coat protein of Clostridium difficile

“…Spores are also produced in the gut and cause both spreading into the environment and relapse (Deakin et al, 2012;Janoir et al, 2013). The resistance to extreme environmental conditions and chemical compounds, including oxygen, antibiotics and disinfectants, makes the spores difficult to eradicate (Rupnik et al, 2009;Edwards et al, 2016).…”