Barbara A. Brown-Elliott

Clinical and Taxonomic Status of Pathogenic Nonpigmented or Late-Pigmenting Rapidly Growing Mycobacteria

SUMMARY The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing.

Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy

SUMMARY The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification.

A Novel Gene, erm(41), Confers Inducible Macrolide Resistance to Clinical Isolates of Mycobacterium abscessus but Is Absent from Mycobacterium chelonae

ABSTRACT Mycobacterium abscessus infections tend to respond poorly to macrolide-based chemotherapy, even though the organisms appear to be susceptible to clarithromycin. Circumstantial evidence suggested that at least some M. abscessus isolates might be inducibly resistant to macrolides. Thus, the purpose of this study was to investigate the macrolide phenotype of M. abscessus clinical isolates. Inducible resistance to clarithromycin (MIC > 32 μg/ml) was found for 7 of 10 clinical isolates of M. abscessus previously considered susceptible; the remaining 3 isolates were deemed to be susceptible (MIC ≤ 0.5 μg/ml). Inducible resistance was conferred by a novel erm gene, erm(41), which was present in all 10 isolates and in an isolate of Mycobacterium bolletii (M. abscessus type II). However, the erm(41) alleles were nonfunctional in the three susceptible M. abscessus isolates. No evidence of erm(41) was found in Mycobacterium chelonae, and an isolate of Mycobacterium massiliense appeared to be an erm(41) deletion mutant. Expression of erm(41) in M. abscessus conferred resistance to clarithromycin and erythromycin and the ketolide HMR3004. However, this species was found to be intrinsically resistant, independent of erm(41), to clindamycin, quinupristin (streptogramin B), and telithromycin. The ability to confer resistance to clindamycin and telithromycin, but not quinupristin, was demonstrated by expressing erm(41) in Maycobacterium smegmatis. Exposure of M. abscessus to the macrolide-lincosamide-streptogramin B-ketolide agents increased the levels of erm(41) mRNA 23- to 250-fold within 24 h. The inducible macrolide resistance phenotype of some M. abscessus isolates may explain the lack of efficacy of macrolide-based chemotherapy against this organism.

Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria

SUMMARY Within the past 10 years, treatment and diagnostic guidelines for nontuberculous mycobacteria have been recommended by the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA). Moreover, the Clinical and Laboratory Standards Institute (CLSI) has published and recently (in 2011) updated recommendations including suggested antimicrobial and susceptibility breakpoints. The CLSI has also recommended the broth microdilution method as the gold standard for laboratories performing antimicrobial susceptibility testing of nontuberculous mycobacteria. This article reviews the laboratory, diagnostic, and treatment guidelines together with established and probable drug resistance mechanisms of the nontuberculous mycobacteria.

Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo- outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy

PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.

Clinical experience with linezolid for the treatment of Nocardia infection

Linezolid is an oxazolidinone that has activity against most gram-positive bacteria, including in vitro activity against all Nocardia species and strains. We describe 6 clinical cases of nocardiosis that were successfully treated with linezolid. Two patients had underlying X-linked chronic granulomatous disease, and 2 patients were receiving chronic corticosteroid therapy. Four of 6 patients had disseminated disease, and 2 of these 4 patients had multiple brain abscesses. Four patients primarily received monotherapy; for the fifth patient, linezolid was added to a failing multiple-drug regimen, and, for the sixth patient, it was used as part of combination therapy. All 6 patients were successfully treated, although 1 patient had a presumed relapse of central nervous system infection after premature discontinuation of the drug. Linezolid appears to be an effective alternative for the treatment of nocardiosis.

Activities of Linezolid against Rapidly Growing Mycobacteria

ABSTRACT Linezolid is an oxazolidinone available as an oral drug which has activity against most gram-positive bacteria. However, few species of the genus Mycobacterium have been studied. We tested 249 clinical isolates and 10 reference strains of rapidly growing mycobacteria for susceptibility to linezolid by broth microdilution. Clinical species included the Mycobacterium fortuitum group (n = 74), M. abscessus (n= 98), M. chelonae (n = 50), M. mucogenicum (n = 10), and M. fortuitum third biovariant complex (10). The modal MIC for M. mucogenicum was 1.0 μg/ml, and the MIC at which 90% of the isolates tested are inhibited (MIC90) was 4 μg/ml; the modal MIC for the M. fortuitum group was 4 μg/ml, and the MIC90 was 16 μg/ml; the modal MIC for the M. fortuitum third biovariant complex was 4 μg/ml, and the MIC90 was 8 μg/ml; the modal MIC for M. chelonae was 8 μg/ml, and the MIC90 was 16 μg/ml; and the modal MIC for M. abscessus was 32 μg/ml, and the MIC90 was 64 μg/ml. Based on peak levels of linezolid in serum of 15 to 20 μg/ml, we propose the following broth MIC breakpoints for these species: susceptible, ≤ 8 μg/ml; moderately susceptible, 16 μg/ml; and resistant, ≥32 μg/ml). These studies demonstrate the excellent potential of linezolid for therapy of rapidly growing mycobacteria.

Cohort Study of Molecular Identification and Typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii

ABSTRACT Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease. Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Health care-associated outbreaks have recently been investigated by the use of molecular identification and typing tools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M. bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult and relies on the sequencing of one or more housekeeping genes. We performed extensive molecular identification and typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at the NIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB, hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and 2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted further sequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonal groups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB, hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliense type strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, a multilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus and closely related species. Molecular typing complements sequence-based identification and provides information on prevalent clones with possible relevant clinical aspects.

Comparison of the In Vitro Activity of the Glycylcycline Tigecycline (Formerly GAR-936) with Those of Tetracycline, Minocycline, and Doxycycline against Isolates of Nontuberculous Mycobacteria

ABSTRACT We compared the in vitro activity of the glycylcycline tigecycline (formerly GAR-936) with those of tetracycline, doxycycline, and minocycline by broth microdilution against 76 isolates belonging to seven species of rapidly growing mycobacteria (RGM) and 45 isolates belonging to five species of slowly growing nontuberculous mycobacteria (NTM). By using a resistance breakpoint of >4 μg/ml for tigecycline and >8 μg/ml for tetracycline, all RGM were highly susceptible to tigecycline, with inhibition of 50% of isolates at ≤0.12 μg/ml and inhibition of 90% of isolates at 0.25 μg/ml for Mycobacterium abscessus and inhibition of both 50 and 90% of isolates at ≤0.12 μg/ml for M. chelonae and the M. fortuitum group. The MICs of tigecycline were the same for tetracycline-resistant and -susceptible strains, and RGM isolates were 4- to 11-fold more susceptible to tigecycline than to the tetracyclines. In contrast, no slowly growing NTM were susceptible to tigecycline, and isolates of M. marinum and M. kansasii were less susceptible to this agent than to minocycline. This new antimicrobial offers exciting therapeutic potential for the RGM, especially for isolates of the M. chelonae-M. abscessus group, against which the activities of the currently available drugs are limited.

An Outbreak of Mycobacterium chelonae Infection Following Liposuction

Among 82 patients who underwent liposuction performed by a single practitioner in a 6-month period, 34 (41%) developed cutaneous abscesses. An organism identified as Mycobacterium chelonae by polymerase chain reaction restriction-enzyme analysis was recovered from cultures of samples from 12 of those patients. DNA large restriction-fragment pattern analysis by pulsed-field gel electrophoresis demonstrated that a strain of M. chelonae recovered from biofilm in the piped-water system in one of the physicians offices differed by only 2 restriction fragments from the 12 patient isolates, which differed from each other by 0 or 1 restriction fragment. A detailed retrospective cohort study that included interviews with former employees and statistical analysis of risk factors indicated that inadequate sterilization and rinsing of surgical equipment with tap water were likely sources of mycobacterial contamination. This is the first reported outbreak of nosocomial infection due to M. chelonae in which a source has been identified and the first to occur in association with liposuction in patients in the United States.