Rebecca W. Wilson

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.

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.

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.

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections : a cooperative study from the international working group on mycobacterial taxonomy

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

Macrolide/Azalide Therapy for Nodular/Bronchiectatic Mycobacterium avium Complex Lung Disease

BACKGROUND There is no large study validating the appropriateness of current treatment guidelines for Mycobacterium avium complex (MAC) lung disease. This is a retrospective single-center review evaluating the efficacy of macrolide/azalide-containing regimens for nodular/bronchiectatic (NB) MAC lung disease. METHODS Patients were treated according to contemporary guidelines with evaluation of microbiologic responses. Macrolide susceptibility of MAC isolates was done at initiation of therapy, 6 to 12 months during therapy, and on the first microbiologic recurrence isolate. Microbiologic recurrence isolates also underwent genotyping for comparison with the original isolates. RESULTS One hundred eighty patients completed > 12 months of macrolide/azalide multidrug therapy. Sputum conversion to culture negative occurred in 154 of 180 patients (86%). There were no differences in response between clarithromycin or azithromycin regimens. Treatment regimen modification occurred more frequently with daily (24 of 30 [80%]) vs intermittent (2 of 180 [1%]) therapy (P = .0001). No patient developed macrolide resistance during treatment. Microbiologic recurrences during therapy occurred in 14% of patients: 73% with reinfection MAC isolates, 27% with true relapse isolates (P = .03). Overall, treatment success (ie, sputum conversion without true microbiologic relapse) was achieved in 84% of patients. Microbiologic recurrences occurred in 74 of 155 patients (48%) after completion of therapy: 75% reinfection isolates, 25% true relapse isolates. CONCLUSIONS Current guidelines for macrolide/azalide-based therapies for NB MAC lung disease result in favorable microbiologic outcomes for most patients without promotion of macrolide resistance. Intermittent therapy is effective and significantly better tolerated than daily therapy. Microbiologic recurrences during or after therapy are common and most often due to reinfection MAC genotypes.

In Vitro Activities of Linezolid against Multiple Nocardia Species

ABSTRACT Linezolid was tested by broth microdilution against 140 clinicalNocardia isolates belonging to seven species. The MIC at which 50% of the strains are inhibited (MIC50) and MIC90 for all species other than Nocardia farcinica were 2 and 4 μg/ml. Linezolid is the first antimicrobial agent demonstrated to be active against allNocardia species.

Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

ABSTRACT Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.

Mycobacterial contamination of metalworking fluids: involvement of a possible new taxon of rapidly growing mycobacteria

Contamination of air and metalworking fluid (MWF) systems with a rapidly growing mycobacterium (RGM) was detected in 1995 in a single manufacturing plant with recent cases of hypersensitivity pneumonitis (HP). Extensive environmental sampling was performed to determine the extent of the contamination and its variability over time. RGM were present in multiple indoor air samples, 100% of the central MWF storage tanks, and 75% of the freestanding cutting, drilling, and grinding machines. With one exception, contamination was limited to a recently introduced formulation (brand) of semisynthetic MWF used in 95% of the facilitys machining operations. In general, the mycobacterial counts were stable over time, with the degree of contamination ranging from 10(2)-10(7) colony forming units (CFU)/mL. A few systems were culture positive for the mycobacterium (> 10(1) CFU/mL), changed to culture negative (< 10(1) CFU/mL), then changed back to culture positive without explanation. Samples obtained from diluted (5%) but unused MWF, a replenishment line with 2% unused MWF, an MWF pasteurizer, city water, and deionized water were culture negative for this species of mycobacterium. Inoculation and growth studies demonstrated that this mycobacterium does not grow in liquid samples of 5% unused MWF. By molecular techniques, the mycobacterial isolates consisted of a single strain and represented a previously undescribed taxon closely related to Mycobacterium chelonae/abscessus. The relationship of this mycobacterium to the cases of HP is unknown.

Repeat Positive Cultures in Mycobacterium intracellulare Lung Disease after Macrolide Therapy Represent New Infections in Patients with Nodular Bronchiectasis

The genomic DNA patterns (genotypes) of 55 episodes of late positive sputum isolates, collected after >or=4 consecutive months of negative sputum cultures, in prospective macrolide treatment trials of Mycobacterium avium complex (MAC) lung disease were assessed by pulsed-field gel electrophoresis (PFGE). Having >or=2 cultures positive for MAC after completion of therapy was documented 23 times; of 20 episodes studied by PFGE, 17 (85%) represented new genotypes (i.e., new infections), and 87% occurred in patients with nodular bronchiectasis. With >or=2 positive cultures after therapy was stopped prematurely, 6 (86%) of 7 episodes were relapses. Single positive cultures after completion of therapy occurred 16 times; only 1 (6%) was predictive of a subsequent relapse. No late isolates were macrolide resistant. Thus, relapses of MAC lung disease with these macrolide regimens are unusual, and most infections after completing therapy resulted from new strains in patients with nodular bronchiectasis.

Sequence-Based Identification of Aerobic Actinomycetes

ABSTRACT We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the genera Streptomyces, Gordonia, and Tsukamurella and 10 taxa of Nocardia were studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65 hsp gene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing of Nocardia species revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences of Nocardia transvalensis, N. otitidiscaviarum, and N. nova isolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within the Nocardia spp. are unnamed at present. A major unresolved issue is the type strain of N. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.