Growth of Clostridium difficile and production of toxins A and B in complex and defined media

Haslam, S. C.; Ketley, Julian M.; Mitchell, Tim; Stephen, J.; Burdon, D. W.; Candy, D. C. A.

doi:10.1099/00222615-21-4-293

Cited by 85 publications

(64 citation statements)

References 6 publications

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“…A moderate toxin-suppressing effect of glycine, isoleucine, leucine, methionine, threonine, tryptophan, and valine was observed. Interestingly, these amino acids together with cysteine and proline are required for maximum cell yield of C. difficile in defined media (7,10). Although these amino acids did not affect growth in PY, there was an inverse relationship between toxin production and the levels of these amino acids.…”

Section: Discussionmentioning

confidence: 95%

“…The onset of C. difficile growth in the large intestine, followed by C. difficile-associated diarrhea (CDAD), is thought to be caused by the reduction of protective colonic microbiota, especially by antibiotic treatment (14). Toxin production by C. difficile has been demonstrated to be dependent on the nutrient level of the growth medium (7,10,12,18,24,25). The type and amount of nutrients present have been shown to affect growth of C. difficile in a continuous culture system containing a complex microflora (23,26), suggesting that competition for nutrients in the colon plays a role in colonization by the pathogen and in the development of CDAD.…”

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confidence: 99%

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Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in Clostridium difficile

et al. 2000

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It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Åkerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n ‫؍‬ 6) and clinical isolates (n ‫؍‬ 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.

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Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 99%

Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in Clostridium difficile

et al. 2000

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“…Toxin production by strain KZ 1647 was examined in the biotin-limited defined medium supplemented with nine amino acids (g/L): alanine (0. defined medium [9]). Toxin levels were low compared to those in biotin-limited defined medium without the supplementation.…”

Section: Inhibition Of Toxin Production By Amino Acidsmentioning

confidence: 99%

“…Nine amino acids -tryptophan, methionine, valine, iso leucine, proline, leucine, cysteine, glycine and threonine [8] -from the 18 amino acids in the defined medium of Haslam et al [9] were selected for the development of a defined medium to achieve better toxin production. The first seven amino acids are essential for bacterial growth and the last two are growth-enhancing [6, lo].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions

Yamakawa¹,

Karasawa²,

Ohta³

et al. 1998

Journal of Medical Microbiology

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Production of toxins A and B by Clostridium difficile is enhanced in a defined medium with biotin-limited conditions. In the present study compounds inhibitory to enhanced toxin production by a C. dij$jjcile strain were examined. Increases in biotin concentration from 0.05 nM to 50 nM accelerated growth and inhibited enhanced toxin production. Asparagine, glutamic acid and glutamine (10 mM) showed an effect on growth and toxin production similar to that of biotin. Lysine (10 mM) suppressed growth and inhibited toxin production. Addition of these toxin-inhibitory compounds within an incubation period of 2 days inhibited the enhanced toxin production, but later addition showed only slight inhibition of toxin production. Amino acids contained in the defined medium under the biotin-limited conditions were actively utilised in the presence of the three toxin-inhibitory amino acids, but in the presence of lysine, amino-acid utilisation was suppressed. Different mechanisms of action of these toxin-inhibitory molecules, which may be divided into excess biotin, asparagine-glutamic acid-glutamine group, and lysine, are discussed.

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“…A commercially available latex reagent for toxin A does not detect this toxin (Lyerley & Wilkins, 1986) and enzyme-linked immunosorbent assay for toxin A (Laughon et al 1984) is not routinely performed in diagnostic microbiology laboratories. Moreover, the production of both toxins A and B depends on the composition of the growth medium in vitro and, presumably, on the nutritional status of the gut in vivo (Haslam et al 1986). In view of these considerations it seems likely that the isolation of C. difficile may be of more value than toxin detection in the diagnosis 356 T. V. RILEY AND OTHERS of disease associated with C. difficile, and that enrichment media should consequently be used to maximize isolation rates Levett, 1984).…”

Section: Introductionmentioning

confidence: 99%

Comparison of alcohol shock enrichment and selective enrichment for the isolation ofClostridium difficile

et al. 1987

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SUMMARYTwo enrichment methods were compared for their ability to recover Clostridium difficile from stool samples. One method used selective enrichment in an antibioticcontaining broth followed by detection with a latex particle agglutination (LPA) reagent. The other used enrichment in a non-selective broth following treatment of the specimen with alcohol. With clinical specimens enrichment culture was significantly more successful at detecting C. difficile than direct plating. Alcohol shock enrichment was twice as effective as direct culture, while selective broth enrichment was three times more effective. The use of LPA for screening selective enrichment broths for C. difficile should prove a cost-effective measure as only positive broths (about 20%) require subculture for confirmation.

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Growth of Clostridium difficile and production of toxins A and B in complex and defined media

Cited by 85 publications

References 6 publications

Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in Clostridium difficile

Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in Clostridium difficile

Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions

Comparison of alcohol shock enrichment and selective enrichment for the isolation ofClostridium difficile

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