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. 2014 Jul 11;9(7):e102290.
doi: 10.1371/journal.pone.0102290. eCollection 2014.

Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis

Timothy J Gray  1 , Fanrong Kong  1 , Peter Jelfs  1 , Vitali Sintchenko  2 , Sharon C-A Chen  2
Affiliations

Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis

Timothy J Gray et al. PLoS One. .

Abstract

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S-23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Representative CGE electropherograms following PCR amplification of the 16S–23S rRNA internal transcribed spacer (ITS) for A) M. chelonae; B) M. abscessus; C) M. massiliense; D) M. fortuitum; and E) M. mucogenicum.
Peaks correlate with the ITS fragment length(s) which is shown above each peak. Panel A and D highlight the phenomenon of spurious double peaks which were less than 1 bp apart. Panel F shows the ITS-CGE electropherogram following the pre-extraction mix of M. abscessus and M. fortuitum demonstrating the typical peaks for each isolate.
Figure 2
Figure 2. Fragment length heterogeneity within the M. fortuitum complex (n = 64) following CGE of the PCR product of the 16S–23S internal transcribed spacer.
The number of isolates with each capillary gel electrophoresis type is indicated (n).
See this image and copyright information in PMC

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