The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Clostridium difficile causes intestinal infections that range from mild self-curing diarrhea to pseudomembraneus colitis.

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Genetic variants of Clostridium difficile have been reported with increasing frequency, but their true incidence is unknown. C. difficile strains have been classified into variant toxinotypes according to variations in the pathogenicity locus encoding the major virulence factors, toxins A and B. Some strains produce an additional toxin, binary toxin CDT. This survey of clinical isolates (153) from patients in a single hospital set out to ascertain the distribution of variant toxinotypes and strains possessing binary toxin genes. A PCR-RFLP-based method of toxinotyping identified 123 (80 . 4 %) isolates as toxinotype 0, 13 (8 . 5 %) strains as non-toxigenic and 17 (11 . 1 %) as belonging to variant toxinotypes. Binary toxin genes were amplified by PCR in nine strains (5 . 8 %), all of which were variant toxinotypes. Toxin variants of C. difficile are pathogenic and commonly isolated and need to be considered when evaluating new diagnostic testing strategies for C. difficile disease.