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.

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 de-pends 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 January 2001 and December 2006 in the Samsung Medical Center, Seoul, South Korea, according to the diagnostic criteria published by the American Thoracic Society (17). The 85 strains were initially identified as M. abscessus by PCR-restriction fragment length polymorphism (PRA) analysis of rpoB (12).
To obtain our VNTR targets, the entire genome of M. abscessus ATCC 19977 (GenBank accession no. CU458896.1) was examined using a tandem repeat finder program (http: //tandem.bu.edu/trf/trf.html). In target loci from the 1,496 repeats initially selected by the program, the 16 loci were selected according to the following criteria; repeat number of at least twice, sequence match of at least 85%, and a longer period size than 20 bp. Screening of the 16 selected loci was done against three reference strains, M. abscessus ATCC 19977, M. massiliense CIP108297, and M. bolletii CIP108541. During screening, two loci were identified that could discriminate among the members of the M. abscessus complex. The two pairs of primers, VNTR11 and Mab2, amplified the following regions of the M. abscessus ATCC 19977 genome: 2951442 to 2952583 (designated VNTR11) and 4058506 to 4058576 (designated VNTR23) ( Table 1). The DNA template was amplified in a 20-l volume using the two pairs of primers (1 M each primer). The amplification profile consisted of 10 min at 95°C, followed by 30 cycles, with 1 cycle consisting of 30 s at 95°C, 30 s at 63°C, and 45 s at 72°C. We extended the established method to 85 clinical strains independently identified by a multiple-target sequence analysis of rpoB, hsp65, gnd, and glpK as previously described (Table 1)  bolletii showed one pattern with two copies of VNTR23. Interestingly, none of the M. massiliense strains were positive for VNTR23 (Fig. 1).
For the easy and precise differentiation of M. abscessus species, duplex PCR with redesigned primers, Mab1 and Mab2, was developed to avoid amplification overlaps between the two loci (Fig. 2). Capillary electrophoresis (Agilent Technologies, Waldbronn, Germany) was applied to ensure accurate differentiation of amplicon size. The primer specificity of the two   This is the first study to demonstrate the precise identification of M. abscessus complex to the species level using VNTR targets. The assay not only accurately distinguished M. abscessus complex isolates from other mycobacterial species, including M. chelonae and M. fortuitum, but also discriminated among M. abscessus complex isolates to the species level.
An effective discrimination among closely related yet pathogenetically diverse members of M. abscessus complex will support a better diagnosis and treatment regimen as well as further our understanding of the epidemiology of these pathogens.
To extend these results, studies of the association between clinical outcomes, distinct pathogenesis, drug resistance, and VNTR profile using a greater number of VNTR targets are under way.