title: Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.
abstract: A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.
authors.affiliation: Infectious Disease Section, Department of Medicine, Veterans Affairs Chicago Health Care System, Lakeside Division, and Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Sambol
authors.forename: M M
authors.initials: MM
authors.lastname: Merrigan
authors.forename: D
authors.initials: D
authors.lastname: Lyerly
authors.forename: D N
authors.initials: DN
authors.lastname: Gerding
authors.forename: S
authors.initials: S
authors.lastname: Johnson
mesh_terms: D000368:Aged; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity; D016360:Clostridioides difficile / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity*; D003470:Culture Media; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology*; D004768:Enterotoxins / Q000235:genetics* / Q000633:toxicity; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D007082:Ileum; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D000091402:Prohibitins; D011817:Rabbits; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; C000717331:PHB2 protein, human; D000091402:Prohibitins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D015247:Deoxyribonuclease HindIII