Selection of Resistance to Clarithromycin in Mycobacterium abscessus Subspecies.

Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.

Mycobacterium abscessus: a new antibiotic nightmare.

The intrinsic and acquired resistance of Mycobacterium abscessus to commonly used antibiotics limits the chemotherapeutic options for infections caused by these mycobacteria. Intrinsic resistance is attributed to a combination of the permeability barrier of the complex multilayer cell envelope, drug export systems, antibiotic targets with low affinity and enzymes that neutralize antibiotics in the cytoplasm. To date, acquired resistance has only been observed for aminoglycosides and macrolides, which is conferred by mutations affecting the genes encoding the antibiotic targets (rrs and rrl, respectively). Here we summarize previous and recent findings on the resistance of M. abscessus to antibiotics in light of what has been discovered for other mycobacteria. Since we can now distinguish three groups of strains belonging to M. abscessus (M. abscessus sensu stricto, Mycobacterium massiliense and Mycobacterium bolletii), studies on antibiotic susceptibility and resistance should be considered according to this new classification. This review raises the profile of this important pathogen and highlights the work needed to decipher the molecular events responsible for its extensive chemotherapeutic resistance.

Genetic analysis of new 16S rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus.

OBJECTIVES: We studied the development and fitness cost of 2-deoxystreptamine aminoglycoside resistance of Mycobacterium abscessus. METHODS: Spontaneous 2-deoxystreptamine aminoglycoside-resistant mutants were selected and the frequency of their appearance was determined. The 3' part of the rrs gene was sequenced to characterize mutations. Additionally, we determined the MICs of aminoglycoside drugs for the different mutants obtained. The dominance/recessivity traits of the different mutations were examined and we explored the potential cost conferred by the mutations selected in vitro on the fitness of these isolates compared with the wild-type strain. RESULTS: The in vitro mutation rate for 2-deoxystreptamine aminoglycoside resistance was ∼10(-7) mutations/cell division. In addition to the known rrs A→G substitution at position 1408 (Escherichia coli numbering), which confers kanamycin resistance (Kan(R)), three new substitutions in rrs were identified in M. abscessus Kan(R) mutants, i.e. T→A at 1406, C→T at 1409 and G→T at 1491. Heterodiploids carrying genomic mutations T→A at 1406 and A→G at 1408 with the wild-type rrs gene carried by the pNBV1 vector showed a resistant phenotype. In contrast, heterodiploids carrying genomic mutations C→T at 1409 and G→T at 1491 with the wild-type rrs gene carried by the pNBV1 vector had a susceptible phenotype. No burden on fitness was observed for the different mutations. CONCLUSION: Mutations in the rrs gene that confer high-level 2-deoxystreptamine aminoglycoside resistance on M. abscessus differ in their dominance/recessivity traits and have no biological cost under our experimental conditions.

Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response.

Mycobacterium abscessus is considered to be the most virulent of the rapidly growing mycobacteria. Generation of bacterial gene knockout mutants has been a useful tool for studying factors that contribute to virulence of pathogenic bacteria. Until recently, the optimal genetic approach to generation of M. abscessus gene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, a M. abscessus mutant was generated in which the gene mmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, the mmpL4B deletion mutant had lost sliding motility and the ability to form biofilm, but acquired the ability to replicate in human macrophages and stimulate macrophage Toll-like receptor 2. This study demonstrates that deletion of a gene associated with expression of a cell-wall lipid can result in acquisition of an immunostimulatory, invasive bacterial phenotype and has important implications for the study of M. abscessus pathogenesis at the cellular level.

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HßD2 gene expression and IL-8 release.

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human ß-defensin 2 (HßD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HßD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HßD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.

Laboratory maintenance of Mycobacterium abscessus.

Mycobacterium abscessus, is an emerging, rapidly growing pathogen, with the ability to cause chronic lung disease. This unit covers background information and laboratory maintenance procedures for this bacterium, including growth in liquid and on solid medium. It also contains recommendations concerning long-term strain storage. M. abscessus is a Biosafety Level 2 organism, and the required safety measures are also discussed.