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.

Keywords: Clostridium difficile; Clostridium difficile infection (CDI); anaerobe; germination; resistance; spores; sporulation.

FIGURE 1

Clostridium difficile vegetative cells are sensitive to ethanol, while spores are highly resistant to ethanol. (A) Survival of 630 spo0A, R20291 spo0A and 5325 spo0A vegetative cells grown to an OD₆₀₀ ∼1.0 in BHIS medium and (B) 630Δerm, R20291 and 5325 spores in 1X PBS after exposure to the indicated concentrations (v/v) of ethanol for 15 min. The means and standard error of the means for at least three biological replicates are shown; the limit of detection equals 20 CFU ml^-1 for vegetative cells and 10 CFU ml^-1 for spores and is denoted by a dashed line. Asterisks indicate a P value of <0.05 as determined by two-way ANOVA followed by a Dunnett’s multiple comparisons test to compare the condition to the corresponding control of individual strains.

FIGURE 2

Clostridium difficile 5325 vegetative cells exhibit greater resistance to solvents while R20291 spores are more sensitive to butanol. (A,C) Survival of 630 spo0A, R20291 spo0A and 5325 spo0A vegetative cells grown to an OD₆₀₀ ∼1.0 in BHIS medium after a 15 min exposure to the indicated concentrations (v/v) of (A) butanol and (C) chloroform. (B,D) Survival of 630Δerm, R20291 and 5325 spores in 1X PBS after a 15 min exposure to (B) butanol and (D) chloroform. The means and standard error of the means for at least three biological replicates are shown; the limit of detection is 10 CFU ml^-1 and is denoted by a dashed line. Asterisks indicate a P-value of <0.05 as determined by two-way ANOVA followed by a Dunnett’s multiple comparisons test to compare the condition to the corresponding control of individual strains.

FIGURE 3

Clostridium difficile 630 spo0A vegetative cells demonstrate greater resistance to oxidative stress. Survival of 630 spo0A, R20291 spo0A and 5325 spo0A vegetative cells grown to an OD₆₀₀ ∼1.0 in BHIS medium after exposure to (A) atmospheric oxygen after the indicated length of time (h) or (B) hydrogen peroxide (H₂O₂) for 15 min. The means and standard error of the means for at least three biological replicates are shown; the limit of detection is 10 CFU ml^-1 and is denoted by a dashed line. Asterisks indicate a P-value of <0.05 as determined by two-way ANOVA followed by a Dunnett’s multiple comparisons test to compare the condition to the corresponding control of individual strains. ND, not determined.

FIGURE 4

Clostridium difficile spores are resistant to high temperatures. Survival of (A) 630 spo0A, R20291 spo0A and 5325 spo0A vegetative cells grown to an OD₆₀₀ ∼1.0 in BHIS medium or (B) 630Δerm, R20291 and 5325 spores in 1X PBS after a 20 min exposure to the indicated temperature. The means and standard error of the means for at least three biological replicates are shown; the limit of detection is 10 CFU ml^-1 and is denoted by a dashed line. Asterisks indicate a P-value of <0.05 as determined by two-way ANOVA followed by a Dunnett’s multiple comparisons test to compare the condition to the corresponding control of individual strains.

FIGURE 5

Clostridium difficile vegetative cells and spores are resistant to sodium hypochlorite (household bleach) at a physiological pH. Survival of (A) 630 spo0A, R20291 spo0A and 5325 spo0A vegetative cells grown to an OD₆₀₀ ∼1.0 in BHIS medium or (B) 630Δerm, R20291 and 5325 spores suspended in BHIS medium after exposure to 400 mg l^-1, 1000 mg l^-1, 5000 mg l^-1, 8000 mg l^-1 free chlorine (FC) for 15 min. The means and standard error of the means for at least three biological replicates are shown; the limit of detection is 10 CFU ml^-1 and is denoted by a dashed line. Asterisks indicate a P-value of <0.05 as determined by two-way ANOVA followed by a Dunnett’s multiple comparisons test to compare the condition to the corresponding control of individual strains.