Utility of Sequencing the erm (41) Gene in Isolates of Mycobacterium abscessus subsp. abscessus with Low and Intermediate Clarithromycin MICs

Abstract

bThe erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of >8 g/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of <8 g/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of >16 g/ml, including ATCC 19977 T , revealed 10 sequevars. The sequence in ATCC 19977 T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 g/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of <2 (80%), 4 (10%), and 8 (10%) g/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from <2 to <4 g/ml and that isolates with an MIC of 8 g/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.T hree closely related taxa of rapidly growing mycobacteria (RGM) with a controversial species/subspecies status, i.e., Mycobacterium abscessus subsp. abscessus, "M. abscessus subsp. massiliense," and M. abscessus subsp. bolletii, comprise the heterogeneous M. abscessus group (1, 2). A 2013 phylogenetic analysis based on genomic sequencing corroborated previous recommendations that the three taxa should be separated into three subspecies (3). Of these three subspecies, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a relatively rare subspecies in the United States, demonstrate the presence of inducible macrolide resistance conferred by a novel erm gene, erm(41) (4).In contrast, the erm(41) gene is generally present in isolates of M. abscessus subsp. massiliense but contains a large, 397-bp deletion (that includes position 28T) that results in a nonfunctional erm(41) gene, and thus isolates do not show inducible macrolide resistance (2).In 2009, Nash and colleagues described two strains (MAB30 and MC1028) of M. abscessus subsp. abscessus which contained a nonfunctional erm gene (4); the strains were not inducibly resistant to clarithromycin, and even after extended incubation, they demonstrated susceptible clarithromycin MICs. The loss of function of the erm genes was associated with a T-to-C substitution at position 28 of th...

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

“…Similarly, assignment of the T or C SNP at position 28 of erm(41) by our real-time SNP assay showed 100% concordance to erm(41) gene sequencing. In this study, the majority (93% [64/69]) of the isolates with full-length erm(41) had a T at position 28, and 7% (5/69) had a C. Thus, the rate of inactive C28 erm(41) in our study (7%) is lower than the recently reported rate of 18% of M. abscessus with an inactive C28 erm(41) sequevar (28).…”

“…Another explanation lies in the technical challenges of this test. In a recent article, Brown-Elliott et al (28) reported that some C28 isolates with a MIC of 8 upon extended incubation showed a MIC of Յ2 g/ml upon repeat testing. In the same study, the authors proposed to change clarithromycin susceptibility breakpoints from Յ2 g/ml to Յ4 g/ml.…”

“…Examples of these isolates are CI20-2, CI20-6, CI20-8, and CI20-10. In the absence of guidelines for phenotypic testing of slowly growing isolates of MAG, sequencing of the erm(41) gene as suggested by BrownElliott et al (28) could be particularly useful for predicting clarithromycin susceptibility patterns for these MAG strains.…”

Evaluation of the GenoType® NTM DR for Subspecies Identification and Determination of Drug Resistance in Clinical M. abscessus Isolates

“…Repeat pDST revealed resistance for all three isolates. This finding indicates that discrepancies between genetic and phenotypic susceptibility testing of M. abscessus often relate to problems of poor growth and difficulties in interpreting MICs, and not to errors in genetic assays [22].…”

“…This is particularly important when determining susceptibility for M. abscessus as resistance is not exclusively due to mutations, but may result from other mechanisms such as upregulated drug export systems or decreased cell wall permeability [26]. On the other hand, pDST interpretation can be difficult for rapidly-growing mycobacteria as poor growth can lead to falsely susceptible results [22]. In this case GenoType® NTM DR can help to improve the interpretation of pDST results by supporting or refuting them.…”