Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases

Abstract

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

FIG. 1

Algorithm of RFLP patterns of 28 slowly growing mycobacterial species and 4 Mycobacterium spp. of uncertain taxonomic status from PCR-amplified 16S-23S rDNA spacer sequences (547 strains). PCR products and restriction fragments are designated by molecular sizes in base pairs. HaeIII species-specific patterns are highlighted by boxes. CfoI patterns A to D are as follows: A, 126 to 144 and 91 to 96 (digest size varies depending on the PCR product size); B, 129 to 146 and 83; C, 126, 63, and 30; D, 160 and 62. DdeI patterns A-E are as follows: A, 120 and 90; B, 120 and 80; C, 120 and 70; D, 120 and 100; E, 214. TaqI pattern A is 155 and 70; 0, no restriction. Type strains were assigned to genotype I if more than one pattern occurred in a species. Genotypes Ia and Ib or IIa and IIb indicate that the strains are genetically very similar but new RFLP genotypes have occurred after loss or acquisition of one HaeIII restriction site due to allelic microheterogeneity. M. leprae and M. kansasii III RFLP patterns were deduced from nucleotide sequence accession no. X56657 (EMBL) and the M. kansasii genotype III sequence published by Alcaide et al. (1). For details concerning AvaII, HinfI, and MspI patterns and descriptions of Mycobacterium spp., see the text.

FIG. 2

Algorithm of RFLP patterns of 21 rapidly growing mycobacterial species and one rapidly growing Mycobacterium sp. of unknown taxonomic status from PCR-amplified 16S-23S rDNA spacer sequences. Details are given in the legend to Fig. 1.

FIG. 3

Gel electrophoresis and HaeIII RFLP patterns of slowly growing mycobacteria from PCR-amplified 16S-23S rDNA spacer sequences (the upper panel shows PCR products without restriction). The molecular sizes of the fragments are given in Fig. 1. The patterns are displayed in order of increasing size of the biggest fragment. M, molecular size marker (100-bp ladder). MAIS, M. avium-M. intracellulare-M. scrofulaceum.

FIG. 4

Gel electrophoresis and HaeIII RFLP patterns of M. fortuitum (lanes 1 to 8) and M. peregrinum (lanes 9 to 11) from PCR-amplified 16S-23S rDNA spacer sequences (the upper panel shows PCR products without restriction). The patterns are described in the legend to Fig. 2. M, molecular size marker (100-bp ladder).

FIG. 5

Sequence stability and microheterogeneity of 16S-23S rDNA spacer sequences in conserved and more variable regions with relevance for the cleaving action of HaeIII (GGCC) and CfoI (GCGC). Sequences not found in this study but published elsewhere are included (4, 8, 9). The respective sequevar designations are shown in brackets, and the number of strains sequenced for this study are shown in parentheses. Sequevars combined in one line exhibit base substitutions located in other regions of the spacer that are not displayed. Of Mav A to E and Min A to C, only Mav A or B and Min A were found. The sequevar Mgo B was not found among seven M. gordonae isolates examined.