Rapidly Growing Mycobacteria Species Research Articles

Clinical and laboratory aspects of the diagnosis and management of cutaneous and subcutaneous infections caused by rapidly growing mycobacteria

Rapidly growing mycobacteria (RGM) are known to cause pulmonary, extra-pulmonary, systemic/disseminated, and cutaneous and subcutaneous infections. The erroneous detection of RGM that is based solely on microscopy, solid and liquid cultures, Bactec systems, and species-specific polymerase chain reaction (PCR) may produce misleading results. Thus, inappropriate therapeutic measures may be used in dermatologic settings, leading to increased numbers of skin deformity cases or recurrent infections. Molecular tools such as the sequence analyses of 16S rRNA, rpoB and hsp65 or PCR restriction enzyme analyses, and the alternate gene sequencing of the superoxide dismutase (SOD) gene, dnaJ, the 16S-23S rRNA internal transcribed spacers (ITS), secA, recA1, dnaK, and the 32-kDa protein gene have shown promising results in the detection of RGM species. PCR restriction enzyme analyses (PRA) work better than conventional methods at identifying species that are closely related. Recently introduced molecular tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), pyrosequencing, DNA chip technology, and Beacon probes-combined PCR probes have shown comparable results in the detection of various species of RGM. Closely related RGM species (e.g., Mycobacterium fortuitum, M. chelonae, and M. abscessus) must be clearly differentiated using accurate molecular techniques because their therapeutic responses are species-specific. Hence, this paper reviews the following aspects of RGM: (i) its sources, predisposing factors, clinical manifestations, and concomitant fungal infections; (ii) the risks of misdiagnoses in the management of RGM infections in dermatological settings; (iii) the diagnoses and outcomes of treatment responses in common and uncommon infections in immunocompromised and immunocompetent patients; (iv) conventional versus current molecular methods for the detection of RGM; (v) the basic principles of a promising MALDI-TOF MS, sampling protocol for cutaneous or subcutaneous lesions and its potential for the precise differentiation of M. fortuitum, M. chelonae, and M. abscessus; and (vi) improvements in RGM infection management as described in the recent 2011 Clinical and Laboratory Standards Institute (CLSI) guidelines, including interpretation criteria of molecular methods and antimicrobial drug panels and their break points [minimum inhibitory concentrations (MICs)], which have been highlighted for the initiation of antimicrobial therapy.

Pulmonary Disease Caused by Rapidly Growing Mycobacteria: A Retrospective Study of 44 Cases in Japan

Background: The features of pulmonary disease caused by rapidly growing mycobacteria (RGM) have not been sufficiently documented. Objectives: To establish these features, we retrospectively evaluated 44 patients. Methods: We screened respiratory isolates at the National Toneyama Hospital (Osaka, Japan) between 2003 and 2007. Diagnosis was based on the latest guidelines of the American Thoracic Society. The patients were classified into 3 types according to their radiographic findings: fibrocavitary, nodular bronchiectatic and unclassified variant. Results: We obtained 1,348 nontuberculous mycobacteria respiratory isolates from 1,187 patients, including 119 RGM isolates from 100 patients. Forty-four of these 100 patients were definitively diagnosed with respiratory disease due to RGM. The most common pathogen was Mycobacteriumabscessus, which accounted for 65.9% of cases, followed by Mycobacterium fortuitum at 20.5%. There was a statistically significant difference in smoking history between patients infected with these 4 RGM species (excluding those with an unknown smoking history; p = 0.039). The overall evaluation of radiographic findings revealed 18.2% as fibrocavitary, 43.2% as nodular bronchiectatic and 38.6% as unclassified variants in these 44 patients. There was a significant difference in radiographic findings between the 4 RGM species (p = 0.002). There was also a significant difference in radiographic findings between M. abscessus and M. fortuitum infected patients (p = 0.022). Conclusions: Patients with M. abscessus seem to have less of a smoking history and more frequent nodular bronchiectatic radiographic patterns than patients with M. fortuitum. In contrast, fibrocavitary patterns might be more frequent with M. fortuitum infection.

Phenotypic and molecular characterization of quinolone resistance in Mycobacterium abscessus subsp. bolletii recovered from postsurgical infections

Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) were reported in many Brazilian states (2032 notified cases) from 2004 to 2010. Most of the confirmed cases were mainly associated with Mycobacterium massiliense (recently renamed as Mycobacterium abscessus subsp. bolletii) BRA100 clone, recovered from patients who had undergone invasive procedures in which medical instruments had not been properly sterilized and/or disinfected. Since quinolones have been an option for the treatment of general RGM infections and have been suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of all generations of quinolones for clinical and reference RGM by broth microdilution, and analysed the peptide sequences of the quinolone resistance determining regions (QRDRs) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty-four isolates of M. abscessus subsp. bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDRs, including the Ala-83 in GyrA, and Arg-447 and Asp-464 in GyrB, described as being responsible for an intrinsic low level of resistance to quinolones in mycobacteria. However, other RGM species showed distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

Rapidly Growing Mycobacteria Infection in Patients with Cancer

Rapidly growing mycobacteria (RGM) have been associated with various clinical syndromes in immunocompetent and immunocompromised hosts. The risk factors and outcomes of RGM infection in patients with cancer have not been clearly defined. Data were derived from 2 distinct sources. Demographic and clinical data were collected for all patients with cancer at Memorial Sloan-Kettering Cancer Center with a culture positive for RGM from January 1999 through December 2008. We also reviewed the literature for studies describing RGM infection in patients with cancer. During the 10-year period, 28 patients with cancer at Memorial Sloan-Kettering Cancer Center had cultures positive for RGM. Most cases occurred in patients with solid tumors and were confined to the lung. A review of the literature identified 313 additional patients with cancer and RGM infection. Combining our series data with cases from the literature, we defined 3 distinct syndromes: pulmonary disease, which occurred in 158 patients (47%); bloodstream infection, occurring in 151 patients (45%); and disseminated infection involving at least 1 end organ, affecting 26 persons (8%). The syndromes differed by age of onset, underlying cancer, main RGM species, and outcome. Persons with bloodstream infection typically were young and had an excellent outcome; those with disseminated infection were older, had pronounced immunosuppression, and had a very poor prognosis. RGM infections in patients with cancer comprise 3 distinct disorders with different risk factors, predominant mycobacterial species, and prognoses. In turn, the approach to management, including number and duration of antimycobacterial drugs, may be fundamentally different for various patients with cancer who receive a diagnosis of RGM infection.

Molecular identification of rapidly growing mycobacteria isolates from pulmonary specimens of patients in the State of Pará, Amazon region, Brazil

We isolated 44 strains of rapidly growing mycobacteria (RGM) from 19 patients with pulmonary infections assisted at the Instituto Evandro Chagas (Pará, Brazil) from 2004 to 2007. Identification at the species level was performed by PCR restriction fragment length polymorphism analysis (PRA) of a 441 bp hsp65 fragment and partial 16S rRNA, hsp65, and rpoB gene sequencing. Genotyping by PRA yielded 3 digestion patterns: one identical to Mycobacterium abscessus type I (group I); another to M. abscessus type II, Mycobacterium bolletii, and Mycobacterium massiliense (group II); and a third typical for Mycobacterium fortuitum type I (group III). When comparing analysis of the 3 genes, more discrimination was obtained by rpoB gene sequence, which allowed good distinction between group I, II, and III strains and subclassification of group II strains in SG IIa ( M. bolletii) and SG IIb ( M. massiliense). In this study, we show that the description of new RGM species requires the establishment of standardized procedures for RGM identification and the alert of the clinician about their involvement in pulmonary disease and the necessity of treatment for control and cure.

Sensibilidad a los antimicrobianos de micobacterias de crecimiento rápido mediante el método E-test

Rapidly growing mycobacteria (RGM) are considered opportunistic pathogens. An increasing number of post traumatic or surgical infections are caused by these microorganisms.To determine the antimicrobial susceptibility of RGM using the E-test method.A total of 54 isolates of RGM was obtained from several clinical samples and selected for this study Strains were identified to the species level by phenotypic and biochemical characteristics, PCR-restriction enzyme analysis of the hsp65 gene (PRA) and sequencing of the 16S rRNA. Susceptibility was investigated by E-test to amikacin, cefoxitin, ciprofioxacin, clarithromycin, imipenem, quinupristin/dalfopristin, linezolid and tigecycline.Twelve different species of RGM were identified: Mycobacterium fortuitum (23 strains), M chelonae (11), M abscessus (10), Msenegalense (2), Malvei (1), Mbrumae (1), Mmageritense (1), mucogenicum (1), M neoaurum (1), Mperegrinum (1), M septicum (1) y M smegmatis (1). All the strains were inhibited by low concentrations of amikacin and tigecycline. Susceptibility to cefoxitin, fluoroquinolones, clarithromycin, imipenem and linezolid was variable. All but two strains were resistant to quinupristin/ dalfopristin.Due to the uneven antimicrobial susceptibility of different species of RGM, an antimicrobial susceptibility test is mandatory for these microorganisms. The E-test method is well suited to determine minimum inhibitory concentrations.

Assessment of in vitro susceptibility to antimicrobials of rapidly growing mycobacteria by E-test

Rapidly growing mycobacteria (RGM) are considered opportunistic pathogens. An increasing number of post traumatic or surgical infections are caused by these microorganisms. To determine the antimicrobial susceptibility of RGM using the E-test method. A total of 54 isolates of RGM was obtained from several clinical samples and selected for this study Strains were identified to the species level by phenotypic and biochemical characteristics, PCR-restriction enzyme analysis of the hsp65 gene (PRA) and sequencing of the 16S rRNA. Susceptibility was investigated by E-test to amikacin, cefoxitin, ciprofioxacin, clarithromycin, imipenem, quinupristin/dalfopristin, linezolid and tigecycline. Twelve different species of RGM were identified: Mycobacterium fortuitum (23 strains), M chelonae (11), M abscessus (10), Msenegalense (2), Malvei (1), Mbrumae (1), Mmageritense (1), mucogenicum (1), M neoaurum (1), Mperegrinum (1), M septicum (1) y M smegmatis (1). All the strains were inhibited by low concentrations of amikacin and tigecycline. Susceptibility to cefoxitin, fluoroquinolones, clarithromycin, imipenem and linezolid was variable. All but two strains were resistant to quinupristin/ dalfopristin. Due to the uneven antimicrobial susceptibility of different species of RGM, an antimicrobial susceptibility test is mandatory for these microorganisms. The E-test method is well suited to determine minimum inhibitory concentrations.

Rapidly Growing Mycobacteria

We analyzed clinical and microbiologic features of 115 cases involving rapidly growing mycobacteria (RGM) isolated at the University of Texas M.D. Anderson Cancer Center, Houston (2000-2005) and identified by 16S ribosomal RNA gene sequencing analysis. At least 15 RGM species were included: Mycobacterium abscessus (43 strains [37.4%]), Mycobacterium fortuitum complex (33 strains [28.7%]), and Mycobacterium mucogenicum (28 strains [24.3%]) most common, accounting for 90.4%. Most M abscessus (32/43) were isolated from respiratory sources, whereas most M mucogenicum (24/28) were from blood cultures. Antimicrobial susceptibility tests showed that M abscessus was the most resistant species; M mucogenicum was most susceptible. From blood and catheter sources, 46 strains (40.0%) were isolated; 44 represented bacteremia or catheter-related infections. These infections typically manifested high fever (mean temperature, 38.9 degrees C), with a high number of RGM colonies cultured. All infections resolved with catheter removal and antibiotic therapy. Six strains (M abscessus and M fortuitum only) were from skin, soft tissue, and wound infections. There were 59 strains from respiratory sources, and 28 of these represented definitive to probable infections. Prior lung injuries and coisolation of other pathogenic organisms were common. Overall, 78 RGM strains (67.8%) caused true to probable infections without direct deaths.

Native valve endocarditis caused by rapidly growing mycobacteria: Case report and review of the literature

Rapidly growing mycobacteria (RGM) are included in group IV of Runyon's classification for mycobacteria [1]. Out of the more than 40 species of RGM, only few have clinical relevance. Those with the...

Method for Rapid Identification and Differentiation of the Species of the Mycobacterium chelonae Complex Based on 16S-23S rRNA Gene Internal Transcribed Spacer PCR-Restriction Analysis

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA)

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.

Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing

The current classification of non-pigmented and late-pigmenting rapidly growing mycobacteria (RGM) capable of producing disease in humans and animals consists primarily of three groups, the Mycobacterium fortuitum group, the Mycobacterium chelonae-abscessus group and the Mycobacterium smegmatis group. Since 1995, eight emerging species have been tentatively assigned to these groups on the basis of their phenotypic characters and 16S rRNA gene sequence, resulting in confusing taxonomy. In order to assess further taxonomic relationships among RGM, complete sequences of the 16S rRNA gene (1483-1489 bp), rpoB (3486-3495 bp) and recA (1041-1056 bp) and partial sequences of hsp65 (420 bp) and sodA (441 bp) were determined in 19 species of RGM. Phylogenetic trees based upon each gene sequence, those based on the combined dataset of the five gene sequences and one based on the combined dataset of the rpoB and recA gene sequences were then compared using the neighbour-joining, maximum-parsimony and maximum-likelihood methods after using the incongruence length difference test. Combined datasets of the five gene sequences comprising nearly 7000 bp and of the rpoB+recA gene sequences comprising nearly 4600 bp distinguished six phylogenetic groups, the M. chelonae-abscessus group, the Mycobacterium mucogenicum group, the M. fortuitum group, the Mycobacterium mageritense group, the Mycobacterium wolinskyi group and the M. smegmatis group, respectively comprising four, three, eight, one, one and two species. The two protein-encoding genes rpoB and recA improved meaningfully the bootstrap values at the nodes of the different groups. The species M. mucogenicum, M. mageritense and M. wolinskyi formed new groups separated from the M. chelonae-abscessus, M. fortuitum and M. smegmatis groups, respectively. The M. mucogenicum group was well delineated, in contrast to the M. mageritense and M. wolinskyi groups. For phylogenetic organizations derived from the hsp65 and sodA gene sequences, the bootstrap values at the nodes of a few clusters were <70 %. In contrast, phylogenetic organizations obtained from the 16S rRNA, rpoB and recA genes were globally similar to that inferred from combined datasets, indicating that the rpoB and recA genes appeared to be useful tools in addition to the 16S rRNA gene for the investigation of evolutionary relationships among RGM species. Moreover, rpoB gene sequence analysis yielded bootstrap values higher than those observed with recA and 16S rRNA genes. Also, molecular signatures in the rpoB and 16S rRNA genes of the M. mucogenicum group showed that it was a sister group of the M. chelonae-abscessus group. In this group, M. mucogenicum ATCC 49650(T) was clearly distinguished from M. mucogenicum ATCC 49649 with regard to analysis of the five gene sequences. This was in agreement with phenotypic and biochemical characteristics and suggested that these strains are representatives of two closely related, albeit distinct species.

High prevalence of antimicrobial resistance in rapidly growing mycobacteria in Taiwan.

An increasing number of clinical isolations of rapidly growing mycobacteria (RGM) at the National Taiwan University Hospital were noted from 1992 to 2001. Broth microdilution MICs of 15 antimicrobial agents were determined for 200 clinical isolates of RGM, including the Mycobacterium fortuitum group (69 isolates), M. chelonae (39 isolates), and M. abscessus (92 isolates). Our results showed that the resistance rates of these isolates to the currently available agents were remarkably high. Amikacin was active against nearly all RGM isolates. Clarithromycin was usually active against M. abscessus (79% susceptibility) and the M. fortuitum group (65% susceptibility). The majority of M. fortuitum group isolates were susceptible to ciprofloxacin (62%) and imipenem (61%). The susceptibilities to other conventional anti-RGM agents of these isolates were poor but differed markedly by species. The newer fluoroquinolones (levofloxacin, moxifloxacin, and gatifloxacin) and meropenem showed better in vitro activities against the M. fortuitum group isolates than against the other two species of RGM. Linezolid had fairly good activity against these RGM isolates, particularly against M. chelonae isolates (82% susceptible). Telithromycin had poor activity against these RGM isolates (the MICs at which 50% of the isolates tested are inhibited [MIC(50)s] were 32 to 64 microg/ml, and the MIC(90)s were >64 microg/ml).

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.

Hsp65 sequencing for identification of rapidly growing mycobacteria.

Partial sequencing of the hsp65 gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175-178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium peregrinum, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment. hsp65 sequencing unambiguously differentiated M. chelonae and M. abscessus, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.

Biofilm formation by the rapidly growing mycobacterial species Mycobacterium fortuitum

Rapidly growing mycobacteria (RGM) are found in soil and diverse aquatic environments. Two species, Mycobacterium fortuitum and Mycobacterium chelonae, are associated with disease and are difficult to eradicate. Biofilm formation may be a contributing factor to their mode of transmission and their resistance to antimicrobial agents. We investigated the ability of the RGM species M. fortuitum to colonise surfaces using a modified Robbins device. M. fortuitum formed dense biofilms within 48 h. The high numbers of sessile organisms recovered and the swiftness of colonisation suggest that M. fortuitum readily forms biofilms. These results suggest a novel mechanism for mycobacteria in evading antimicrobial treatment and also indicate that biofilms should be considered possible sites for mycobacterial contamination.

Biofilm formation by the rapidly growing mycobacterial species Mycobacterium fortuitum.

Rapidly growing mycobacteria (RGM) are found in soil and diverse aquatic environments. Two species, Mycobacterium fortuitum and Mycobacterium chelonae, are associated with disease and are difficult to eradicate. Biofilm formation may be a contributing factor to their mode of transmission and their resistance to antimicrobial agents. We investigated the ability of the RGM species M. fortuitum to colonise surfaces using a modified Robbins device. M. fortuitum formed dense biofilms within 48 h. The high numbers of sessile organisms recovered and the swiftness of colonisation suggest that M. fortuitum readily forms biofilms. These results suggest a novel mechanism for mycobacteria in evading antimicrobial treatment and also indicate that biofilms should be considered possible sites for mycobacterial contamination.

PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria

A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone. Single unique patterns were observed with HaeIII and/or BstEII for Mycobacterium fortuitum (100%), M. chelonae (94%), M. abscessus (96%), M. smegmatis (100%), M. mucogenicum (formerly the M. chelonae-like organism) (100%), and the sorbitol-negative third biovariant of M. fortuitum (100%). Evidence is presented in support of two subgroups within M. peregrinum, M. smegmatis, and the unnamed third biovariant of M. fortuitum (sorbitol positive and sorbitol negative). PCR-based technology provides a rapid, accurate system for the identification of clinically important species of RGM which should be particularly useful for reference laboratories.