Short Signature rpoB Gene Sequence to Differentiate Species in Mycobacterium abscessus Group

Abstract

Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that consist of three closely related species: M. abscessus (Ma), M. bolletii (Mb), and M. massiliense (Mm). Differentiation of these species can be difficult but is increasingly requested owing to recent infectious outbreaks and their differential drug resistance. We developed a novel and rapid pyrosequencing method using short signature sequences (35 to 45 bp) at a hypervariable site in the rpoB gene to differentiate the three MAG species, along with M. chelonae (Mc), and M. immunogenum (Mi). This method was evaluated using 111 M. chelonae-abscessus complex (MCAC) isolates, including six reference strains. All isolates were successfully differentiated to the species level (69 Ma, four Mb, six Mm, 23 Mc, and nine Mi). The species identifications by this method had 100% agreement with Sanger sequencing as well as an in-silico rpoB typing method. This short signature sequencing (SSS) method is rapid (6 to 7 h), accurately differentiates MAG species, and is useful for informing antimicrobial therapy decision. IMPORTANCE Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that include three species: M. abscessus, M. massiliense, and M. bolletii. These species are among the leading causes of nontuberculosis mycobacteria infections in humans but difficult to differentiate using commonly used methods. The differences of drug resistance among the species shape the treatment regimens and make it significant for them to be differentiated accurately and quickly. We developed and evaluated a novel short signature sequencing (SSS) method utilizing a gene called rpoB to differentiate the three MAG species, as well as other two species (M. chelonae and M. immunogenum). The identification results had 100% agreement with both the reference method of Sanger sequencing and rpoB typing method via a computer-simulated analysis. This SSS method was accurate and quick (6 to 7 h) for species differentiation, which will benefit patient care. The technology used for this method is affordable and easy to operate.

Keywords: M. chelonae-abscessus complex; Mycobacterium abscessus group (MAG); rpoB gene; short signature sequencing (SSS); species differentiation.

FIG 1

Typical pyrogram-generated signature rpoB sequences of the five species cited from their alignments to the reference sequence (ref) from M. abscessus strain CIP 104536 (T); –, indicates the same nucleotide as that in the reference sequence. Bolded font in the reference sequence indicates variable nucleotides.

FIG 2

In-silico analysis of typical short pyrogram signature sequences (35 bp) for other organisms in Mycobacterium chelonae-abscessus complex (MCAC) (33–38). –, indicates the same nucleotide as that in M. abscessus.