The pattern of RI restriction fragments of chromosomal DNA that hybridise with a probe for genes encoding 16S and 23S rRNA is highly discriminatory for non-capsulate (NCHI). The source of variation detected by these probe-based ribotyping patterns was investigated by restriction analysis of rRNA operon () amplification products from nine representative strains. Digestion of amplification products with RI indicated one conserved RI site within 16S rDNA and no RI sites within the 16S-23S intergenic spacer region of the nine strains, and an RI site at the 5' end of 23S rDNA from seven of the nine strains. Comparison of the RI ribotyping patterns obtained with separate probes for 16S and 23S rDNA showed more variation with the 23S probe indicating variation in RI sites downstream from the operon. Restriction analyses of 16S and 23S rDNA amplification products with I, I, III and I divided the nine ‘traditional’ ribotypes into a maximum of three and eight groups, respectively. Similar analyses of the 16S-23S intergenic regions (PCR-ribotyping) failed to distinguish any of the nine representative strains. Therefore, there is probably insufficient variation within the operon for it to form a good target for PCR-based typing methods. In contrast, ‘traditional’ ribotyping with cDNA from 16S plus 23S rRNA detects restriction site differences in the sequences flanking the operon, which show considerably more variation between strains. ‘Traditional’ ribotyping should therefore remain the standard for characterising NCHI in epidemiological investigations.


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