Germinability and Heat Resistance of Spores of Clostridium difficile Strains

Abstract

Out of 111 Clostridium difficile strains, 108 produced sporesin numbers of more than 105/ml and the remaining three did not produce any spores in brain heart infusion medium. The germination frequency in the medium without lysozyme varied widely from strain to strain, ranging from less than 10-8 to 100, and in 77 of the 108 strains the germination frequency was 10-5 or less. The spores, when treated with sodium thioglycollate and then inoculated into the medium containing lysozyme, germinated in all of the 108 strains at a frequency of 10-0.5 or more. The spores of two strains germinated at a frequency of more than 10-0.5 in all methods. Spores of C. difficile strains were fairly highly heat-resistant; D100c values ranged from 2.5 to 33.5 min.Clostridium difficile is a major cause of antibiotic-associated pseudomembranous colitis of man (1). Ionesco (3) investigated the conditions for germination of C. difficile spores and found that germination frequency of the spores was quite low, but moderately increased in the presence of lysozyme, and that complete germination of the spores required treatment of the spores with sodium thioglycollate and inoculation of the spores into a medium containing lysozyme.The present study was undertaken, with a large number of C. difficile strains, to establish the relationship between the germination of C. difficile spores and the addition of lysozyme with or without sodium thioglycollate treatment, and to determine the heat resistance of the spores.

Recovery of spores of Clostridium difficile altered by heat or alkali

“…dzficile spores with 0.1-0.3 N NaOH resulted in a remarkable decrease in the recovery rate of spores, although strains KZ 1628 and KZ 1648, which showed very low recovery rate even when untreated, did not show any decrease in spore recovery by alkalitreatment. Two different methods, the thioglycollate-lysozyme method (Ionesco, 1978;Nakamura et al, 1985) and taurocholate method (Raibaud et al, 1980;Wilson et a f . , 1982) have been reported to be effective in the recovery of C. dzficile spores.…”

“…In contrast, strains K Z 1628 and KZ 1648 had very low spore-recovery rates of < 0.01%. Spores were produced in Brain Heart Infusion broth (BHI ; BBL Microbiology System, Cockeysville, MD, USA) supplemented with Na2 H P 0 4 0.2% w/v (m-BHI) as described before (Nakamura et al, 1985). Briefly, 0.1 ml of an 18-h culture of each strain in liver broth was inoculated into 10 ml of m-BHI and incubated anaerobically at 37°C for 5 days.…”

“…Quantitative determinations of heat-or alkali-treated spores and vegetative cells were made with pour plates incorporating GS-BHI medium-BHI (Nissui, Tokyo, Japan) supplemented with glucose 0.8% w/v, soluble starch 1% w/v, L-cysteine-HClO.OS% w/v and agar 1.3% w/v as described by Nakamura et al (1985). A 0-1-ml amount of each 10-fold dilution of spore suspension in BHI broth suppplemented with L-cysteine-HC1 0.05% w/v was pipetted into 9-cm diameter petri dishes and mixed with 20 ml of GS-BHI agar.…”

“…Survival of C. dificile in an aerobic environment is possible because this obligate anaerobe can form aerotolerant spores; there are reports that C. dzficile can survive for > 5 months in aerobic conditions (Kim et al, 1981). Although it is well known that C. dificile spores cannot be effectively recovered in ordinary growth medium (Raibaud et al, 1980), their recovery is distinctly enhanced when they are treated with sodium thioglycollate and inoculated in lysozyme containing media (Ionesco, 1978 ;Nakamura et al, 1985). It has also been reported that addition of sodium taurocholate to media resulted in good spore recovery (Raibaud et al, 1980;Wilson et al, 1982) and sodium taurocholate-containing medium has been shown to be useful for detection of C. dificile spores, particularly when faecal specimens have not been handled optimally or when vegetative forms have lost viability after prolonged exposure to air (Buggy et al, 1983(Buggy et al, , 1985Wilson, 1983).…”

Effect of Various Sodium Taurocholate Preparations on the Recovery of Clostridium difficile Spores

“…This indicates that taurocholate method is more effective in recovery of C. difficile spores. As to the relationship between recovery of the spores and toxigenicity, although no relationship had been found in thioglycollate-lysozyme method (8), it was shown that toxigenic strains tended to recover more abundantly in the presence of sodium taurocholate.…”