No abstract

Pseudomembranous colitis, a severe diarrheal disease, has been linked to the administration of antibiotics and to two toxins produced by Clostridium difficile. Eighty-two strains of C. difficile isolated from humans and hamsters were assayed for the presence of plasmid DNA. Agarose gel electrophoresis of Sarkosyl-lysed cells indicated that 18% of the strains contained from one to four plasmids. The plasmid DNA in these strains ranged in molecular weight from 2.7 x 106 to 60 x

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure. Key words: Clostridium difficile, polymerase chain reaction, bacterial toxins.

Toxin A of Clastr~dium d~~cile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. dl~ci~e chromosomal DNA was developed which allowed for the construction of a genomic library in d gtll. Approx. 35 000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, d cd19. Verification of the immunological identity of the isolated toxin A gene fragment in A cdl9 was determined by affinity purifying toxin A antibodies specific for 1 cdl9 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in 1 cdl9 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. d@cile. The peptide coded for by the toxin A gene fragment in 1 cdl9 was not cytotoxic for 3T3 mammalian tissue culture cells.

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleriafowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.