rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria

Abstract

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from "Mycobacterium houstonense." Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.

FIG. 1.

Mean variability for successive windows of 60 nucleotide positions. Position 1 corresponds to the nucleotide number 205 bp upstream of the beginning of the rpoB sequence of M. tuberculosis. Horizontal arrows, variable regions I to V. MF and MR, PCR primer pair according to the work of Kim et al. (30); Myco-F and Myco-R, PCR primer pair in this study.

FIG. 2.

Percent homology of 20 RGM with M. fortuitum according to the 16S rRNA gene, a 306-bp rpoB sequence (30), a 723-bp rpoB sequence (present study), and the complete rpoB sequence.

FIG. 3.

Phylogenetic tree of the 723-bp rpoB regions of 63 RGM clinical isolates and 20 reference strains. The partial 723-bp rpoB sequences were aligned by using the multisequence alignment program Clustal X, version 1.81, in the PHYLIP software package (19, 61). Phylogenetic relationships were inferred using the neighbor-joining method and the Jukes Cantor parameter. The scale bar represents a 2% difference in nucleotide sequences. Bootstrap values (out of 100) are given at each node.