A defined growth medium for Clostridium difficile

Karasawa, Tadahiro; Ikoma, Sayuri; Yamakawa, Kiyotaka; Nakamura, Shinichi

doi:10.1099/13500872-141-2-371

Cited by 153 publications

(163 citation statements)

References 9 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: 96%

“…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: 96%

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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“…The ODmax values of the three strains KZ 1630, KZ 1647 and KZ 1748 were 0.43 to 0.45 lower than those in was examined in CDMM lacking a single amino acid other than arginine; glycine and threonine were both deleted since they could substitute for each other (11). In the absence of histidine, glycine together with threonine, isoleucine or proline, 0Dmax values were reduced to 41-62% of those in CDMM and toxin production was also decreased; in the absence of isoleucine or proline, growth was not observed until after 36 hr of incubation.…”

Section: Methodsmentioning

confidence: 99%

“…In general, the contents and concentrations of nutrients are important for bacterial growth, which greatly influences the production of bacterial toxins. Therefore, in order to analyze toxin production by C. difficile in relation to growth, we developed a defined medium consisting of 11 amino acids, three vitamins, glucose and 11 minerals (11).…”

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confidence: 99%

Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium

Karasawa

Maegawa

Nojiri

et al. 1997

Microbiology and Immunology

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Twenty strains of Clostridium difficile were examined for the effect of arginine on toxin production in a defined medium. In three strains, the production of toxins A and B was greatly enhanced in the absence of arginine. These strains showed distinctively poorer growth in the absence of arginine in comparison with the remaining 17 strains, indicating that the presence of arginine is required for good growth among the three strains. From the present results, test strains were divided into two groups: a group in which arginine insufficiency caused distinctly poor growth and enhanced toxin production, and another group in which there was neither distinctly poor growth nor enhanced toxin production. The phenomenon is discussed in relation to the biosynthesis and catabolism of arginine.

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“…E. coli strain NovaBlue (Merck) was used as a recipient for all cloning procedures, and the donor strain CA434 (HB101 carrying R702) was used for conjugation of plasmids into C. difficile. C. difficile strain 630 (29) was cultured in TY broth without thioglycolate (34), C. difficile defined medium (35), or brain heart infusion broth and on brain heart infusion agar or blood agar supplemented with thiamphenicol (15 g/ml) where appropriate. Anhydrotetracycline (ATc; 20 -500 ng/ml) was used for induction of the P tet promoter in the C. difficile expression vectors described below.…”

Section: Methodsmentioning

confidence: 99%

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

Fagan

Fairweather

2011

Journal of Biological Chemistry

216

302

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Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of Gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic Gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.

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A defined growth medium for Clostridium difficile

Cited by 153 publications

References 9 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

Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

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