Source of variation detected in ribotyping patterns of Haemophilus influenzae: comparison of traditional ribotyping, PCR-ribotyping and rDNA restriction analysis

Abstract

The pattern of EcoRI restriction fragments of chromosomal DNA that hybridize with a probe for genes encoding 16S and 23S rRNA is highly discriminatory for non-capsulate Haemophilus influenzae (NCHI). The source of variation detected by these probe-based ribotyping patterns was investigated by restriction analysis of rRNA operon (rrn) amplification products from nine representative strains. Digestion of rrn amplification products with EcoRI indicated one conserved EcoRI site within 16S rDNA and no EcoRI sites within the 16S-23S intergenic spacer region of the nine strains, and an EcoRI site at the 5' end of 23S rDNA from seven of the nine strains. Comparison of the EcoRI ribotyping patterns obtained with separate probes for 16S and 23S rDNA showed more variation with the 23S probe indicating variation in EcoRI sites downstream from the operon. Restriction analyses of 16S and 23S rDNA amplification products with AluI, HhaI, HaeIII and TaqI 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.