LightCycler-Based Differentiation of Mycobacterium abscessus and Mycobacterium chelonae

Abstract

In this study we introduce a rapid procedure to identify Mycobacterium abscessus (types I and II) and M. chelonae using LightCycler-based analysis of the hsp65 gene. Results from 36 clinical strains were compared with hsp65 gene restriction analysis and biochemical profiles of bacilli. As all three methods yielded identical results for each isolate, this procedure offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnosis of mycobacterial diseases.Among frequently isolated clinical strains of rapidly growing mycobacteria are Mycobacterium abscessus and Mycobacterium chelonae (2). Patients typically present with local abscesses, lymphadenitis, and orbital or pulmonary infections (10, 11). M. abscessus is also recovered from respiratory specimens of patients with cystic fibrosis (1, 4). The differences in antimicrobial susceptibilities between M. abscessus and M. chelonae demand an easy differentiation of these two closely related species (14). Restriction analysis of an hsp65 gene fragment has been used to this end for over 10 years (7,12,13). However, this method requires subsequent gel analysis of amplified DNA. Likewise, interpretation of fragment sizes is sometimes difficult because of the similarity of restriction patterns. Alternative diagnostic procedures include glycan analysis of the bacterial capsule using high-performance liquid chromatography, which is highly reliable but can be performed only in specialized laboratories (13). In this study, we developed a simple and rapid method based on LightCycler technology to distinguish M. abscessus and M. chelonae. We compared the biochemical profiles, the restriction patterns of the hsp65 gene, and partial sequences of the hsp65 gene of 36 clinical strains, including M. abscessus strain ATCC 19977 and M. chelonae strain ATCC 35752 as reference strains. All strains had previously been identified as belonging to the M. chelonae-M. abscessus group by 16S rRNA gene analysis as described previously (5).Biochemical profiling included testing for growth on 5% sodium chloride and growth on citrate as the sole carbon source as suggested by Yakrus and colleagues (13). Briefly, bacteria were precultured in liquid medium using mycobacterial growth indicator tubes (MGIT) (Becton Dickinson, Oxford, United Kingdom). Positive MGIT cultures were subcultured with 0.1 ml of a 1:10 dilution on Lowenstein-Jensen selective medium (L-J) containing 5% NaCl (Remel, Lenexa, Kans.). Utilization of sodium citrate was determined as described by Silcox et al. (8). Briefly, a basal medium was prepared by dissolving 2.4 g of (NH 4 ) 2 SO 4 (Merck, Darmstadt, Germany), 0.5 g of KH 2 PO 4 (Merck), and 0.5 g of MgSO 4 · 7H 2 O (Merck) in 950 ml of distilled water; the pH was adjusted to 7.0, and after adding 20 g of Noble agar (Difco Laboratories, Detroit, Mich.), the mixture was autoclaved. Sodium citrate (5.6 g) (J. T. Baker, Deventer, The Netherlands) was dissolved in 50 ml of distilled water, filter sterilized, and added aseptical...

Characterization of Rough and Smooth Morphotypes of Mycobacterium abscessus Isolates from Clinical Specimens

“…It might be useful to further differentiate M. abscessus strains to obtain more information about differences in epidemiology and virulence between the two subspecies. Differentiation can be achieved by analysis of hsp65 gene polymorphism (8,9). Subspecies differentiation could also serve as a surrogate marker for inducible clarithromycin resistance.…”

“…Fatal infections with M. abscessus have been reported, especially after lung transplantation (7). M. abscessus has been subdivided in type I and type II, which, together with Mycobacterium chelonae, share identical 16S rRNA genes but show differences within the hsp65 gene (8,9). Based on multilocus sequence analysis of hsp65, rpoB, secA, and the 16S-23S internal transcribed spacer (ITS) region, M. abscessus was further subdivided into three species, M. abscessus (sensu stricto), M. bolletii, and M. massiliense (10,11).…”

Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

“…Identification of mycobacterial infections earlier on certain occasions lacked specificity and sensitivity while em- [33][34][35][36][37][38][39][40]. Further, detection of antitubercular resistant isolates that were usually detected by broth dilution method have been replaced by RT PCR targeting mutant genes isoniazid (katG), rifampin (rpoB) and ethambutol (embB) from culture or clinical specimens [41][42][43][44][45].…”

Usefulness of Three-Channel Multiplex Real-Time PCR and Melting Curve Analysis for Simultaneous Detection and Identification of the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria

“…For 16S rRNA hypervariable region A, the newly designed primers were 5Ј-ACG GAA AGG TCT CTT CG-3Ј and 5Ј-GTC GTC GCC TTG GTA G-3Ј and the hybridization probe sequence was LightCycler Red 670-TTA GCG GTG TGG GAT GAG CCC CAT GTC TTG TGG TGG AAA GCG-fluorescein. The sets of primers for the 16S rRNA general region, the hsp65 region, and 16S rRNA hypervariable region A can be used to amplify a 1,026-bp fragment (3, 6), a 439-bp fragment (3,11,12,13,16), and a 218-bp fragment, respectively (14).The reaction mixture (20 l), containing 3.0 l of extracted nucleic acid, 2.0 l of LightCycler FastStart DNA Master Hybridization Probe (Roche, Penzerg, Germany), 0.5 l of each primer, 0.2 l of each probe, and 1.6 l of 25 mM MgCl 2 , was denatured initially for 10 min at 95°C and then treated for 3 s at 95°C, 10 s at 56°C, and 30 s at 72°C for 50 cycles with a LightCycler 2.0 (Roche). After 30 s at 95°C and 2 min at 38°C, the mixture was heated from 38°C to 80°C at a rate of 0.2°C/s.…”

“…For 16S rRNA hypervariable region A, the newly designed primers were 5Ј-ACG GAA AGG TCT CTT CG-3Ј and 5Ј-GTC GTC GCC TTG GTA G-3Ј and the hybridization probe sequence was LightCycler Red 670-TTA GCG GTG TGG GAT GAG CCC CAT GTC TTG TGG TGG AAA GCG-fluorescein. The sets of primers for the 16S rRNA general region, the hsp65 region, and 16S rRNA hypervariable region A can be used to amplify a 1,026-bp fragment (3, 6), a 439-bp fragment (3,11,12,13,16), and a 218-bp fragment, respectively (14).…”