TYPING OF CLOSTRIDIUM-DIFFICILE STRAINS BY PCR-AMPLIFICATION OF VARIABLE-LENGTH 16S-23S RDNA SPACER REGIONS

To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. difficile strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable in vivo, since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing C. difficile typing methods; it has universal applicability, it is objective and is moderately rapid.