Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains

Abstract

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A3, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A3, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A3, toxin B+ strains. These results may suggest that toxin A3, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum. z

Toxin A–Negative, Toxin B–PositiveClostridium difficileInfection Diagnosed by Polymerase Chain Reaction

“…5,8 Two or 3 pairs of primers (NK1, NK2, NK3, NK9, NK11, A1C, A2C, A3C, A2N, A3N, A4N) for each have been reported across this deletion region that can identify TcdA variation by the presence and size of their amplicons. 9 Among these primers, we found 7 sites ( Figure IB) with mutations in NK2-NK3 and A4N in the public genome that may cause a false negative result for toxin A"B + strains when these regions are used for primers. We did not find mutations in any other primer regions.…”

Genetic markers for Clostridium difficile lineages linked to hypervirulence

“…Similarity coefficients were calculated with Pearson correlation and dendrograms were created with Bionumerics software version 6.01 (Applied Maths) using curve-based clustering. The presence of the genes tcdA, tcdB and cdtA, and possible deletions in the tcdC gene, were detected by PCR as described elsewhere and were used to study genetic similarity (Alonso et al, 1999;Kato et al, 1999;Spigaglia & Mastrantonio, 2002).…”

Prevalence and Genetic Characterization of Toxin A Variant Strains of Clostridium difficile among Adults and Children with Diarrhea in France

“…Recently, toxin A variant strains have been reported which fail to produce detectable toxin A by enzyme immunoassay (EIA) yet produce toxin B (1,7,9,19,20,22,23,32). Two types of toxin A-negative strains are well characterized: strain 1470 (reference strain of serogroup F from the classification of Delmée et al [14]) and strain 8864, described by Borriello et al and Lyerly et al (7,23).…”