Molecular screening of Clostridium difficile toxins A and B genetic determinants and identification of mutant strains

McMillin, David E.

doi:10.1016/0378-1097(91)90258-c

Cited by 11 publications

(20 citation statements)

References 4 publications

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“…22,23 This suggests that simultaneous expression of the two toxins is due to the presence of both genes, although the unusual toxin A-negative, toxin B-positive strain was reported recently to have a whole toxin B gene and an incomplete toxin A gene.24*25 The correlation between the levels of toxin A and toxin B produced also indicates that expression of both toxin genes may be regulated in the defined medium in the same manner as in the complex medium.…”

Section: Discussionmentioning

confidence: 99%

Toxin production by Clostridium difficile in a defined medium with limited amino acids

Yamakawa

Kamiya

Xiao

et al. 1994

Journal of Medical Microbiology

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Summary. Basal defined medium (BDM) containing vitamins, minerals and seven amino acids-(/L) tryptophan 0.1 g, methionine 0-2 g, valine 0.3 g, isoleucine 0.3 g, proline 0.3 g, leucine 0.4 g and cysteine 0.5 g-which appeared to be essential for good growth of Clostridium dzficile was prepared. Addition of glycine 0.2 g/L and threonine 0-4 g/L to BDM produced better growth of strain VPI 10463, and this defined medium was designated minimum amino acid-defined medium (MADM). Production of toxins A and B by strain VPI 10463 in 6 x MADM containing (/L) tryptophan 0.6 g, methionine 1.2 g, valine 1.8 g, isoleucine 1.8 g, proline 1.8 g, leucine 2.4 g, cysteine 0.5 g, glycine 0.2 g and threonine 0.4 g, was much greater than in MADM. Toxin production by 20 C. dzficile strains was examined in two defined media-6 x MADM and complete amino acid-defined medium (CADM) containing 18 amino acids-and one complex medium, modified brain heart infusion medium (m-BHI). Simultaneous production of toxins A and B by all test strains was demonstrated in m-BHI and the two defined media. It was also shown that 6 x MADM was generally better than CADM and as effective as m-BHI for stimulating toxin production by 13 strains. This defined medium would be useful for studies on the physiology, metabolism and pathogenicity of C. dzficile.

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Section: Discussionmentioning

confidence: 99%

Toxin production by Clostridium difficile in a defined medium with limited amino acids

Yamakawa

Kamiya

Xiao

et al. 1994

Journal of Medical Microbiology

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“…A total of 203 N-terminal amino acids of toxins A and B were compared. Sixty-four percent of the residues were homologous, demonstrating the relatedness of toxins A and B. McMillin et al (191) used the polymerase chain reaction and fluorescent tags to demonstrate that 37 toxic strains of C. difficile normally had the genetic composition for toxins A and B and 10 nontoxic strains did not contain detectable toxin determinants. One strain, however, was found to contain the genetic composition for toxins A and B but was not cytotoxic under the conditions tested.…”

Section: Introductionmentioning

confidence: 99%

Clostridium difficile: clinical disease and diagnosis

1993

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Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.

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“…To our knowledge there has been only one other report of a strain producing a shorter toxin A protein and this strain was not investigated thoroughly at the molecular level (McMillin et al, 1991). Modification of the PAGE-blotting protocol enabled a better separation of the variant toxins from toxin A of the nonvariant toxinotype 0 strain, and also achieved differentiation of the two truncated toxin A molecules.…”

Section: Discussionmentioning

confidence: 99%

Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants

Blake¹,

Mitsikosta²,

Metcalfe³

2004

Journal of Medical Microbiology

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Clostridium difficile is a major nosocomial pathogen and a causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. PCR analysis of the toxin A and B genes of this bacterium has revealed 20 variant types (toxinotypes I-XX), many of which can cause human disease. Strains comprising the 15 toxin A-positive, toxin B-positive toxinotypes are not usually differentiated from non-variant strains by routine laboratories that do not utilize PCR tests. Consequently, the toxins from these variant strains have not been investigated thoroughly. The present studies revealed that toxin A-positive (A+B+) strains representing 12 variant toxinotypes all express considerably lower levels of toxin A and are less cytotoxic in vitro than non-variant strain VPI 10463. Truncated forms of toxin A were detected by immunoblotting in toxinotype VI and VII strains and these toxins were differentiated from each other and from toxin A of the non-variant strain. A further novel finding was the ability of toxin A-positive (A+B+) strains of toxinotypes IX, XIV and XV to exhibit an alternative Clostridium sordellii-like cytopathic effect on Vero cells, characterized by marked cell clumping. A rapid and simple method for toxin A removal from culture filtrates was developed. This enabled confirmation that the abnormal cytotoxicity observed for these strains is due to an altered toxin B, as has been found in toxin A-negative (AÀB+) strains. These findings indicate the potential for differentiation of certain toxin A-positive (A+B+) toxinotypes without the need for PCR techniques.

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Molecular screening of Clostridium difficile toxins A and B genetic determinants and identification of mutant strains

Cited by 11 publications

References 4 publications

Toxin production by Clostridium difficile in a defined medium with limited amino acids

Toxin production by Clostridium difficile in a defined medium with limited amino acids

Clostridium difficile: clinical disease and diagnosis

Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants

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