Efficient Differentiation of Mycobacterium abscessus Complex Isolates to the Species Level by a Novel PCR-Based Variable-Number Tandem-Repeat Assay

Abstract

A novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates. The assay accuracy was confirmed by a multiple-target sequence analysis. The duplex PCR assay is a one-step, reliable, and accurate assay for discriminating M. abscessus species.Mycobacterium abscessus belongs to a group of rapidly growing mycobacteria (RGM) that cause a wide spectrum of infections in humans (6,17). Recently, M. abscessus complex was divided into three closely related Mycobacterium species: M. abscessus (sensu stricto) (hereafter referred to as M. abscessus), M. massiliense, and M. bolletii (1, 4).M. abscessus is the most drug-resistant mycobacterial species known and exhibits unsatisfactory treatment response rates, especially for patients with pulmonary disease (6, 17). Inducible resistance of M. abscessus to clarithromycin has been suggested as an explanation for the lack of efficacy of clarithromycin-based treatments against the bacterium (14). In contrast, M. massiliense shows marked susceptibility to clarithromycin due to the absence of inducible resistance to macrolides (8). Therefore, treatment response rates to clarithromycin-based antibiotic therapy are higher in patients with M. massiliense than in those with M. abscessus lung disease (10). M. bolletii, a rare pathogen at present, has also been shown to be highly resistant to clarithromycin (3, 9, 11).Since recent studies suggest that antibiotic susceptibility and treatment outcomes differ significantly among these species, the species-level identification of M. abscessus complex isolates has been strongly recommended in order to determine the clinical significance and to assist in managing patients (8,10).Despite progress in our ability to identify mycobacterial species using molecular methods, M. abscessus complex isolates cannot be reliably identified at the species level by sequencing a single genetic locus such as rpoB or hsp65 (13). Currently, the species-level identification of M. abscessus complex solely depends on multiple sequencing analysis targeting several genes, including rpoB, hsp65, gnd, glpK, secA, and sodA (2, 13, 15). This process requires a number of additional, laborious steps (e.g., cloning) to obtain the sequences of the products, leading to expensive and complicated procedures that must be done in reference laboratories.Increasingly, variable-number tandem-repeat (VNTR) analyses have been used for the molecular typing of isolated mycobacterial strains as well as for mycobacterial species identification (7, 16). In this study, a novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates.A total of 85 clinical isolates were collected between

Investigation of the population structure of Mycobacterium abscessus complex strains using 17-locus variable number tandem repeat typing and the further distinction of Mycobacterium massiliense hsp65 genotypes

“…The Mycobacterium abscessus complex (M. abscessus sensu lato) contains rapidly growing mycobacteria that have been increasingly recognized as opportunistic human pathogens (Choi et al, 2011). The M. abscessus complex comprises ubiquitous environmental micro-organisms that are frequently associated with hospital-acquired outbreaks and pseudo-outbreaks (Brown-Elliott & Wallace, 2002;Duarte et al, 2009;Fisher & Gloster, 2005;Monego et al, 2011;Nunes et al, 2014).…”

Molecular Fingerprinting of Mycobacterium abscessus Strains in a Cohort of Pediatric Cystic Fibrosis Patients

“…The whole-genome sequence of M. abscessus was recently published (14), making it possible to design variable-number tandemrepeat (VNTR) typing schemes. One recent publication describes such an assay that can differentiate the individual members of the complex but not individual strains (4). In this study, we describe a variable-number tandem repeat (VNTR) typing scheme capable of strain discrimination within each species and compare it to an automated rep-PCR typing scheme (9).…”

Molecular Typing of Mycobacterium abscessus Based on Tandem-Repeat Polymorphism

“…Recently, Choi et al used two tandem-repeat loci to distinguish Mycobacterium abscessus from other mycobacterial species and discriminated Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii by different copy numbers of the two VNTR targets (7). In this article, we describe the development and evaluation of a new VNTR assay for the molecular typing of Mycobacterium abscessus strains.…”