New Rapid Scheme for Distinguishing the Subspecies of the Mycobacterium abscessus Group and Identifying Mycobacterium massiliense Isolates with Inducible Clarithromycin Resistance

Abstract

e Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977 T genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group. Rapidly growing mycobacteria (RGM) are ubiquitous environmental microorganisms and a significant cause of human disease (1). The prevalence of lung disease due to RGM is increasing and in many areas of the United States exceeds that of Mycobacterium tuberculosis (2). Within the RGM, the Mycobacterium abscessus group is a prominent cause of lung infections in patients with cystic fibrosis and chronic pulmonary disease (bronchiectasis, nodules, and cavitations) and of posttraumatic and postsurgical infections (1, 3). Infections with organisms in the M. abscessus group are difficult to treat, due to both natural broad-spectrum resistance and acquired resistance, with various antibiotic susceptibility patterns being observed among isolates (4). M. abscessus sensu lato, or the M. abscessus group, was recently divided into three closely related taxa, i.e., M. abscessus sensu stricto (hereinafter referred to as M. abscessus), Mycobacterium massiliense, and Mycobacterium bolletii. M. massiliense has been recognized increasingly as an emerging pathogen causing postsurgical wound infection outbreaks (5), and recently it was identified as a cause of respiratory outbreaks in two cystic fibrosis centers, with evidence of transmission between patients (6, 7).The taxonomic status of the M. abscessus group remains controversial. M. massiliense and M. bolletii were initially proposed as new species mainly on the basis of divergence of their rpoB sequences. However, further studies showed that these organisms could not be separated by biochemical tests and mycolic acid pattern analysis (8) and showed less genomic divergence than would be exp...

Development of a Peptide Nucleic Acid-Based Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation among Mycobacterium abscessus Strains

“…Because M. massiliense differs from M. abscessus in its susceptibility to clarithromycin, distinguishing between the two organisms has clinical importance (6, 7). Furthermore, phylogenetic separation between three types of M. massiliense has also been reported (4,8), highlighting the epidemiological importance of their separation (7,9,10).Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone (11,12). A PNA probe-based real-time PCR assay has been developed for the diagnosis of mycobacterial infections, particularly for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) in clinical specimens (13,14 T , and M. xenopi ATCC 19250 T ), were used in this study.…”

“…Because M. massiliense differs from M. abscessus in its susceptibility to clarithromycin, distinguishing between the two organisms has clinical importance (6, 7). Furthermore, phylogenetic separation between three types of M. massiliense has also been reported (4,8), highlighting the epidemiological importance of their separation (7,9,10).…”

“…Recently, it was reported that M. massiliense can be further subdivided into three genotypes (here referred to as types I, II-1, and II-2) based on analyses of the hsp65 sequence (4,5). Because M. massiliense differs from M. abscessus in its susceptibility to clarithromycin, distinguishing between the two organisms has clinical importance (6,7). Furthermore, phylogenetic separation between three types of M. massiliense has also been reported (4,8), highlighting the epidemiological importance of their separation (7,9,10).…”

New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group

“…Multiple genes, including rpoB, hsp65, secA, and others (17)(18)(19)(20)(21)(22), as well as PCR-based assays (10,23) have been evaluated as tools to discriminate between the closely related subspecies of the MAG. Since a truncated erm(41) gene was described as a hallmark of M. massiliense, size differences in PCR-amplified erm(41) PCR products were proposed as a simple method to differentiate M. massiliense from M. abscessus and M. bolletii (10).…”

Rapid Detection of Acquired and Inducible Clarithromycin Resistance in Mycobacterium abscessus Group by a Simple Real-Time PCR Assay

“…Prior studies described the application of conventional and probe-based real-time PCR for detection of functional erm(41) genes (7,10). The SYBR green-based real-time PCR assay described here provides a simpler alternative for detecting clarithromycin resistance in the M. abscessus group.…”