JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2006, p. 3493–3498 0095-1137/06/$08.00⫹0 doi:10.1128/JCM.00724-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Mycobacterium barrassiae sp. nov., a Mycobacterium moriokaense Group Species Associated with Chronic Pneumonia Toı¨di Ade´kambi, Didier Raoult, and Michel Drancourt* Unite´ des Rickettsies, CNRS UMR 6020 IFR 48, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e, and Assistance Publique-Ho ˆpitaux de Marseille Timone Fe´de´ration de Microbiologie Clinique Marseille, France Received 5 April 2006/Returned for modification 16 June 2006/Accepted 25 July 2006
Three identical isolates of new rapidly growing mycobacteria (RGM) were recovered from the bronchial aspirate and sputum from a 49-year-old woman presenting with lung lesions. The case met the American Thoracic Society criteria for the diagnosis of nontuberculous mycobacterial infection. The three isolates grew in 3 days at 24 to 42°C. The 16S rRNA gene sequence analysis indicated that the sequences of the isolates were identical and shared 99.7% and 98.1% similarities with those of Mycobacterium moriokaense and Mycobacterium gadium, respectively. Partial 723-bp rpoB sequence analyses indicated that the sequences of the isolates shared 95.8% and 92.3% similarities with those of M. moriokaense and M. gadium, respectively. Polyphasic identification (including biochemical tests; antimicrobial susceptibility profiling; and hsp65, recA, and sodA gene sequence analyses, as well as GⴙC content determination and cell wall fatty acid composition analysis) supported the evidence that these isolates were representative of a new species. Phylogenetic analyses confirmed the close relationships of the isolates with M. moriokaense and the defined M. moriokaense group. These isolates were susceptible to the antimicrobials currently recommended for the treatment of RGM infections. These isolates differed from M. moriokaense by their susceptibility to vancomycin. We propose the name Mycobacterium barrassiae sp. nov. for this new species. The type strain is N7T (CIP 108545T and CCUG 50398T). It has become apparent that the bacterial genus Mycobacterium is more diverse than was previously realized and includes an increasing number of emerging pathogens, as depicted by 16S rRNA gene sequencing (36). Among the rapidly growing mycobacteria (RGM), the species most commonly recovered from patients belong to the Mycobacterium chelonae-Mycobacterium abscessus, Mycobacterium mucogenicum, Mycobacterium fortuitum, Mycobacterium mageritense, Mycobacterium wolinskyi, and Mycobacterium smegmatis groups (1, 2, 3, 4, 5, 6, 7, 10, 11, 31, 43, 44). Rarely encountered species include Mycobacterium novocastrense, which is responsible for cutaneous infections (33); Mycobacterium hodleri, which causes opportunistic infections in the course of rheumatoid arthritis (40); Mycobacterium neoaurum, which was isolated from a patient with catheter-related bacteremia (46); Mycobacterium flavescens, which caused a disseminated infection in a probable case of chronic granulomatous disease (8); and Mycobacterium thermoresistible, which was responsible for an infection following kneereplacement surgery (20). Other species were isolated from human samples, such as Mycobacterium hassiacum from urine (32); Mycobacterium elephantis from the sputum and granulomatous tissue of an axillary lymph node (26, 37, 39); Mycobacterium doricum from cerebrospinal fluid (38); Mycobacterium brumae from sputum and a case of catheter-related bacteremia (21, 22); Mycobacterium brisbanense from the antral sinus (31); Mycobacterium confluentis from sputum (17); Mycobacterium holsaticum from sputum, urine, and gastric fluid (28); Mycobacterium cosmeticum from a footbath drain and a granuloma-
A 49-year-old woman was examined for a lung lesion. She presented with fever and cough. She had no history of tuberculosis or smoking. A chest radiograph revealed a lesion in the lower lobe of the right lung. The diagnosis for the patient did not benefit from a computed tomography scan. Microscopic examination of the bronchial lavage fluid after Ziehl-Nielsen staining disclosed acid-fast bacilli. The patient was treated with a combination of rifampin, isoniazid, and pyrazinamide for 1 week, without improvement. Microbiological investigations of the bronchial aspirate revealed the presence of a nonidentified mycobacterium, designated strain N7T, which was found to be the most closely related to thermotolerant RGM. Two further sputum specimens were positive for acid-fast bacilli and yielded mycobacterial isolates further shown to be identical to strain N7T. The patient was then placed on a three-drug regimen comprising amikacin, clarithromycin, and ciprofloxacin. Her condition improved after 1 week. Follow-up after the patient had completed the 3-month treatment regimen disclosed negative sputum by direct examination and culture, and she was clinically well, with unremarkable X rays.
* Corresponding author. Mailing address: Unite´ des Rickettsies, Faculte´ de Me´decine, 27, Universite´ de la Me´diterrane´e, Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Phone: (33).04.91.32.43.75. Fax: (33).04.91.38.77.72. E-mail:
[email protected] .fr.
Phenotypic characterization of the isolates. One bronchial aspirate specimen and two sputum specimens were decontaminated, as described previously (16, 18). Half of the sediment was frozen, while the other half was stained with Ziehl-Neelsen stain and inoculated into BACTEC 9000MB broth, according to
tous subdermal lesion in a female patient who was undergoing mesotherapy (12); and Mycobacterium canariasense from the blood of a patient with febrile syndrome (15). We report here on an additional novel RGM species isolated from respiratory specimens. CASE REPORT
MATERIALS AND METHODS
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FIG. 1. Phylogenetic tree of the 16S rRNA gene sequences of 29 RGM and M. farcinogenes (SGM) prepared by using the NJ method and the K2P distance correction model. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (as a percentage). M. tuberculosis and M. leprae were used as the outgroups. The scale bar represents a 0.5% difference in nucleotide sequences.
the manufacturer’s instructions (BD Biosciences, Sparks, Md.). The mycobacteria were subcultured on Middlebrook 7H10 agar, egg-based Lowenstein-Jensen (LJ) slants (BioMe´rieux, La Balme-les-Grottes, France), and 5% sheep blood agar (bioMe´rieux); and the cultures were inspected for the presence of colonies twice weekly. Colony morphology, pigmentation, and the ability of the isolate to grow at various temperatures (24, 30, 37, and 42°C) on various media (5% sheep blood agar, LJ slants, Middlebrook 7H10 agar, and LJ slants in the presence of 5% sodium chloride) were observed. The isolates were tested for arylsulfatase and catalase activities, iron uptake, and the degradation of p-aminosalicylic acid (16, 41). Additional biochemical tests were performed by inoculation of API Coryne and API 20E strips, according to the manufacturer’s instructions (BioMe´rieux), with an incubation time of 7 days at 30°C under a highly humidified atmosphere.
Antibiotic susceptibility testing. Mycobacterial suspensions of the isolate were prepared by emulsifying colonies grown on 5% sheep blood agar slants into 5 ml sterile water to achieve a density equal to a 1.0 McFarland turbidity standard by visual examination. The suspensions were mixed vigorously on a vortex mixer for 20s and then inoculated onto the entire surface of a 5% sheep blood agar plate. The MICs of rifampin, ciprofloxacin, ofloxacin, sparfloxacin, doxycycline, minocycline, erythromycin, clarithomycin, azithromycin, amikacin, penicillin, amoxicillin, imipenem, cefotaxime, ceftriaxone, metronidazole, teicoplanin, and vancomycin were determined by incubation with the respective Etest (AB Biodisk, Solna, Sweden) at 30°C for 3 days (47). For all drugs, the MIC was recorded as the point of intersection between the zone edge and the Etest strip. Since the breakpoints for determining the susceptibility of RGM by the Etest method have not been standardized or approved by the Clinical and Laboratory Standards
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FIG. 2. Phylogenetic tree of the partial rpoB gene sequences of 29 RGM and M. farcinogenes (SGM) prepared by using the NJ method and the K2P distance correction model. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (as a percentage). M. tuberculosis and M. leprae were used as the outgroups. The scale bar represents a 2% difference in nucleotide sequences.
Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards [NCCLS]), the breakpoints for susceptibility used were those of the CLSI for broth microdilution interpretive criteria (NCCLS M100-S12 and M24-A) (24, 25) and those proposed by Brown-Elliott and Wallace (11). The disk diffusion method was used to determine the susceptibility to cephalothin (30 g), as described previously (43). Every test was done three times on three separate days in order to ensure the reproducibility of results.
Genetic and phylogenic analyses. Genetic and phylogenetic analyses were performed with the three isolates described here, along with 29 RGM (1, 3, 4, 5) and Mycobacterium farcinogenes, a slowly growing mycobacterium (SGM), as reported in Fig. 1 and Fig. 2. DNA was extracted from colonies grown on 5% sheep blood agar by using the Fast-prep device and the FastDNA kit, according to the recommendations of the manufacturer (Bio 101, Inc., Carlsbad, Calif.). We amplified and sequenced the 16S rRNA (45), hsp65 (29, 34), recA (9), sodA
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(2), and rpoB (1) genes. A 764-bp partial sequence of rpoB was amplified with primer pair Myco-F (5⬘-GGCAAGGTCACCCCGAAGGG-3⬘) and Myco-R (5⬘AGCGGCTGCTGGGTGATCATC-3⬘), and a 723-bp sequence (except for the 41 nucleotides at both ends of the amplicon, corresponding to the primer binding sites) was derived from that amplicon by using the same primer pair in both directions (1). The products of the sequencing reactions were recorded with an ABI Prism 3100 DNA sequencer by following the standard protocol of the supplier (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The percentages of similarity between the sequences were determined by using the Clustal W program, supported by the PBIL website (http://npsa-pbil.ibcp.fr/cgi-bin/npsa). For phylogenetic analyses, the sequences were trimmed in order to start and finish at the same nucleotide position for all the isolates. Multisequence alignment was performed by using the Clustal X program, version 1.81, in the PHYLIP software package (35). The phylogenetic tree was determined from the DNA sequences by using the parsimony, maximum-likelihood, and neighborjoining (NJ) methods with Kimura’s two-parameter (K2P) distance correction model with 1,000 bootstrap replications in the MEGA, version 2.1, software package (19) and was rooted by using Mycobacterium tuberculosis and Mycobacterium leprae. GⴙC content determination and cellular fatty acid analysis. After 3 days of culture on 5% sheep blood agar, DNA extraction, purification, degradation, and G⫹C content determination by high-pressure liquid chromatography were performed as described by Mesbah et al. (23), except that a Waters 625 LC system with a Waters 486 tenable absorbance detector and a Water 746 data module (Millipore, Saint Quentin en Yvelines, France) were used. Three determinations were done. Total fatty acid methyl esters were extracted and prepared by following the instructions of the Microbial Identification System (MIDI; Microbial ID Inc., Newark, Del.) and were analyzed by gas-liquid chromatography with a gas chromatograph (model HP 6890A; Hewlett-Packard, Waldbronn, Germany). Identification of the fatty acids of strain N7T was performed by using the Microbial Identification System with the library created with Sherlock Library Generation System, version 4.0, software (30). Nucleotide sequence accession number. The sequences of the following genes of M. barrassiae N7T (CIP 108545T and CCUG 50398T) determined in this study have been put into GenBank database under the indicated accession numbers: 16S rRNA, AY859685; rpoB, AY859696; hsp65, AY859679; and recA, DQ473309.
RESULTS AND DISCUSSION The patient described here presented with a clinical history of pulmonary infection. The patient did not have any underlying diseases when the pulmonary disease with isolate N7T was diagnosed. The patient displayed radiographic evidence of pulmonary disease that could not be attributed to other causes and that was associated with the repeated isolation of the same strain from three different pulmonary tissue specimens. This case met the American Thoracic Society criteria for the diagnosis of nontuberculous mycobacterial disease (42). The absence of an alternative pathogenic agent supports the potential clinical relevance of this novel species. We previously showed, using the partial 723-bp rpoB sequence (1, 4), that different RGM isolates belonged to different species if they exhibited ⬎3% rpoB sequence divergence. Isolate N7T shared only 95.8% and 92.3% rpoB gene sequence similarities with its closest relatives, M. moriokaense and M. gadium, respectively. These data thus suggested that isolate N7T is representative of a new mycobacterial species. Recently, the 723-bp rpoB fragment was used to characterize several new RGM species, including Mycobacterium massiliense, Mycobacterium bolletii, Mycobacterium phocaicum, Mycobacterium aubagnense, Mycobacterium conceptionense, and Mycobacterium jacuzzii (3, 4, 5, 27). A 16S rRNA gene sequence-derived phylogenetic tree that included the sequences of isolate N7T and 29 established RGM species defined a M. moriokaense group comprising M. moriokaense and isolate N7T with a boot-
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strap value of 71% (Fig. 1). An rpoB gene-derived tree confirmed this representation with a 99% bootstrap value, which supported the fork separating isolate N7T from M. moriokaense, giving much confidence to this analysis (Fig. 2). The parsimony and maximum-likelihood methods confirmed that isolate N7T clustered with M. moriokaense. Their lineages were clearly different from those of closely related species and quite distant from those of other recognized RGM. Further sequence analyses of the 16S rRNA over 1,483 bp showed that the sequence of isolate N7T shared 99.7% similarity with that of M. moriokaense (5-bp difference) and 98.1% similarity with that of M. gadium (28-bp difference). Likewise, the sequence of the hsp65 gene of isolate N7T had 98.1% and 94.1% similarities with those of M. moriokaense and M. gadium, respectively; and the sequence of the N7T recA gene had 95.6% and 90.3% similarities with those of M. moriokaense and M. gadium, respectively. Thus, molecular analyses, which included sequence analyses of the 16S rRNA, hsp65, sodA, recA, and rpoB genes, identified isolate N7T as a unique Mycobacterium species that did not correspond to any previously described Mycobacterium species. Blackwood et al. (9) showed that the intraspecies similarities of the recA gene sequences based on the analysis of 915 bp ranged from 98.7 to 100%. This additional finding allowed us to consider isolate N7T to be a representative of a new species within the genus Mycobacterium. We have used the sodA gene for the description of five new RGM species (3, 4, 5). However, this gene could not be amplified in isolate N7T, although it was amplified in M. moriokaense, a closely related species which differed from the isolate N7T by only 5 bp in the 16S rRNA gene sequence. A tentative sodA-specific primer pair used in the description of Mycobacterium mageritense (14) failed to amplify the sequence of isolate N7T. In addition, of the 97 mycobacterial strains studied by Devulder et al. (13), 15 could not be amplified by using the sodA gene. In all these cases, the amplification primers are supposed to be specific to the genus Mycobacterium. More strikingly, over 540 bp of the sodA gene, M. gadium had only a 3-bp difference (99.6% similarity) with the sequence of M. massiliense, which is quite distinct from M. gadium (Fig. 1 and Fig. 2), whereas the similarity within these two species is 84.2% when the partial rpoB gene is used (1). For these reasons, we do not recommend the use of sodA gene sequence analysis to describe a new RGM species. Further characterization included a G⫹C content value of 64% ⫾ 2% and a fatty acid profile diagnostic for members of the genus Mycobacterium. Isolate N7T was composed of straight-chain saturated and unsaturated fatty acids, including 16:0 (24.7%), 18:1 9c (8.5%), 16:1 7c/15:0 ISO (7.8%), 14:0 (6.4%), and TBSA 10Me18:0 (6.6%; where Me is methyl), and was characterized by large amounts of 17:1 6c (18.8%) and 19:0 cyclo 9c (20.1%). Minor amounts of other fatty acids were also detected. Details of the fatty acids contents are available in Table 1. Based on the fatty acid patterns disclosed by gas-liquid chromatography analysis and by use of the MIDI fatty acid database, the similarity of isolate N7T and M. moriokaense was 75.3%. These results suggest that isolate N7T is different from the closely related species. The biochemical and susceptibility characteristics that differentiated the isolates from the closely related species are
MYCOBACTERIUM BARRASSIAE SP. NOV.
VOL. 44, 2006 TABLE 1. Whole-cell fatty acids composition of M. barrassiae CIP 108545T (isolate N7T) Fatty acid
TABLE 3. Antimicrobial susceptibility test results for M. barrassiae and M. moriokaense
Composition (%)
10:0 ............................................................................................... 0.2 12:0 ............................................................................................... 0.3 Unknown 13.766 ......................................................................... 0.2 14:0 ............................................................................................... 6.4 15:0 ............................................................................................... 0.3 16:1 9c........................................................................................ 0.9 16:1 7c/15:0 ISO ....................................................................... 7.8 16:0 ...............................................................................................24.7 17:1 9c ISO ............................................................................... 0.5 17:1 6c........................................................................................18.8 18:1 9c........................................................................................ 8.5 18:0 ............................................................................................... 3.5 TBSA 10Me18:0.......................................................................... 6.6 Unknown 18.555 ......................................................................... 0.3 19:0 cyclo 9c..............................................................................20.1 20:0 ............................................................................................... 0.4 Unidentified 16.332..................................................................... 0.3 Unidentified 17.892..................................................................... 0.3
summarized in Tables 2 and 3, respectively. Isolate N7T and M. moriokaense exhibited positive activity for urease and negative activity for esculin, which are contrary to the results for the M. fortuitum, M. chelonae-M. abscessus, and M. mucogenicum group species (4). They were susceptible to imipenem, minocycline, doxycycline, clarithromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin, and sparfloxacin. Isolate N7T differed from M. moriokaense by its susceptibility to vancomycin. Iso-
TABLE 2. Comparison of biochemical characteristics of M. barrassiae and M. moriokaense Characteristic
Growth at 42°C Pigmentation Arysulfatase (3 days) Catalase 5% NaCl tolerance Degradation of p-aminosalicylate Iron uptake Nitrate reductase Pyrazinamidase Pyrrolidonylarylamidase Alkaline phosphatase -Galactosidase ␣-Glucosidase Esculin Urease Citrate Acetoin production -Lactamase production Cephalothin disk inhibitiona Utilization of the following as a sole carbon source: D-Mannitol Inositol D-Sorbitol L-Rhamnose L-Arabinose
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M. barrassiae CIP 108545T (N7T)
M. moriokaense CIP 105393T
⫹ ⫺ ⫺ ⫹ ⫹ ⫹
⫹ ⫺ ⫺ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫾ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺
⫹ ⫹ ⫹ ⫾ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺
⫺ ⫹ ⫹ ⫺ ⫹
⫺ ⫹ ⫺ ⫹ ⫹
a A 30-g cephalothin disk was used; the diameter of the zone of inhibition was ⬎6 mm.
MIC (g/ml) Antibiotic
M. barrassiae CIP 108545T (N7T)
M. moriokaense CIP 105393T
Penicillin Amoxicillin Ceftriaxone Cefotaxime Imipenem Doxycycline Minocycline Clarithromycin Erythromycin Azithromycin Amikacin Ciprofloxacin Ofloxacin Sparfloxacin Rifampin Metronidazole Teicoplanin Vancomycin
⬎32 ⬎256 ⬎256 ⬎256 1 0.032 0.125 1.5 ⬎256 0.25 0.25 0.016 0.032 0.032 ⬎32 ⬎256 ⬎256 8
⬎32 ⬎256 ⬎256 ⬎256 0.032 0.016 0.50 2 ⬎256 2 1.5 0.064 0.125 0.016 ⬎32 ⬎256 ⬎256 ⬎256
late N7T and M. moriokaense were resistant to penicillin and amoxicillin and exhibited positive -lactamase activity. Description of Mycobacterium barrassiae sp. nov. Mycobacterium barrassiae (N.L. gen. n. bar⬘ras.si.ae, of Barrassi, to honor Lina Barrassi, a technician in the Unite´ des Rickettsies, for her many contributions to the isolation of intra-amoebal bacteria). The organisms are acid-fast and gram-positive bacilli. Colonies are nonpigmented and appear on 5% sheep blood agar, Middelbrook 7H10 agar, and egg-based LJ slants in 3 to 6 days at temperatures between 25 and 42°C. This species is associated with chronic pneumonia. It is susceptible in vitro to imipenem, minocycline, doxycycline, clarithromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin, and sparfloxacin and is resistant to penicillin, amoxicillin, and erythromycin. It is positive for -lactamase, nitrate reductase, pyrazimidase, urease, iron uptake, and acetoin production activities; is negative for 3-day arylsulfatase and phosphatase alkaline; and uses inositol, sorbitol, and L-arabinose but not citrate and L-rhamnose as sole sources of carbon. Furthermore, M. barrassiae differed from M. moriokaense by susceptibility to vancomycin, the lack of -galactosidase and ␣-glucosidase activities, and the assimilation of sorbitol but not L-rhamnose as sole carbon sources. It shares 99.7% 16S rRNA and 95.9% rpoB gene sequence similarities with M. moriokaense, the nearest species. The type strain, which was recovered from a bronchial aspirate is strain N7T (CIP 108545T and CCUG 50398T). ACKNOWLEDGMENTS We thank Christian de Fontaine for technical assistance and Esther Platt for expert review of the manuscript. REFERENCES 1. Ade´kambi, T., P. Colson, and M. Drancourt. 2003. rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J. Clin. Microbiol. 41:5699–5708. 2. Ade´kambi, T., and M. Drancourt. 2004. Dissection of phylogenic relationships among nineteen rapidly growing mycobacterium species by 16S rRNA,
3498
3.
4.
5.
6. 7. 8. 9. 10.
11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24.
25.
´ KAMBI ET AL. ADE
hsp65, sodA, recA, and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54:2095–2105. Ade´kambi, T., M. Reynaud-Gaubert, G. Greub, M. J. Gevaudan, B. La Scola, D. Raoult, and M. Drancourt. 2004. Amoebal co-culture of Mycobacterium massiliense sp. nov. from the sputum of a patient with hemoptoic pneumonia. J. Clin. Microbiol. 42:5493–5501. Ade´kambi, T., P. Berger, D. Raoult, and M. Drancourt. 2006. rpoB genesequence-based characterization of emerging non-tuberculous mycobacteria with description of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int. J. Syst. Evol. Microbiol. 56:133–143. Ade´kambi, T., A. Stein, J. Carvajal, D. Raoult, and M. Drancourt. 2006. Description of Mycobacterium conceptionense sp. nov., a Mycobacterium fortuitum group organism isolated from a post-traumatic osteitis. J. Clin. Microbiol. 44:1268–1273. Ade´kambi, T., C. Foucault, B. La Scola, and M. Drancourt.. Report of two fatal cases of Mycobacterium mucogenicum central nervous system infection in immunocompetent patients. J. Clin. Microbiol. 44:837–840. Ade´kambi, T., and M. Drancourt. 2006. Isolation of Mycobacterium septicum from the sputum of patient suffering from hemoptoic pneumonia. Res. Microbiol. 157:466–470. Allen, D. M., and H. H. Chang. 1993. Disseminated Mycobacterium flavescens in a probable case of chronic granulomatous disease. J. Infect. 26:83–86. Blackwood, K. S., C. He, J. Gunton, C. Y. Turenne, J. Wolfe, and A. M. Kabani. 2000. Evaluation of recA sequences for identification of Mycobacterium species. J. Clin. Microbiol. 38:2846–2852. Brown, B. A., B. Springer, V. A. Steingrube, R. W. Wilson, G. E. Pfyffer, M. J. Garcia, M. C. Menedez, B. Rodrigez-Salgado, K. C. Jost, Jr., S. H. Chiu, G. O. Onyi, E. C. Bo ¨ttger, and R. J. Wallace, Jr. 1999. 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. Int. J. Syst. Bacteriol. 49:1493–1511. Brown-Elliott, B. A., and R. J. Wallace, Jr. 2002. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin. Microbiol. Rev. 15:716–746. Cooksey, R. C., J. H. de Waard, M. A. Yakrus, I. Rivera, M. Chopite, S. R. Toney, G. P. Morlock, and W. R. Butler. 2004. Mycobacterium cosmeticum sp. nov., a novel rapidly growing species isolated from a cosmetic infection and from a nail salon. Int. J. Syst. Evol. Microbiol. 54:2385–2391. Devulder, G., M. Perouse de Montclos, and J. P. Flandrois. 2005. A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. Int. J. Syst. Evol. Microbiol. 55:293–302. Domenech, P., M. S. Jimenez, M. C. Menendez, T. J. Bull, S. Samper, A. Manrique, and M. J. Garcia. 1997. Mycobacterium mageritense sp. nov. Int. J. Syst. Bacteriol. 47:535–540. Jimenez, M. S., M. I. Campos-Herrero, D. Garcia, M. Luquin, L. Herrera, and M. J. Garcia. 2004. Mycobacterium canariasense sp. nov. Int. J. Syst. Evol. Microbiol. 54:1729–1734. Kent, P. T., and G. P. Kubica. 1985. Public Health Mycobacteriology: a guide for the level III laboratory. U.S. Department of Health and Human Services publication no. (CDC) 86-8230. Centers for Disease Control, Atlanta, Ga. Kirschner, P., A. Teske, K. H. Schro ¨der, R. M. Kroppenstedt, J. Wolters, and E. C. Bo ¨ttger. 1992. Mycobacterium confluentis sp. nov. Int. J. Syst. Bacteriol. 42:257–262. Kubica, G. P., W. E. Dye, M. L. Cohn, and G. Middlebrook. 1963. Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am. Rev. Respir. Dis. 87:775–779. Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245. LaBombardi, V. J., L. Shastry, and H. Tischler. 2005. Mycobacterium thermoresistibile infection following knee-replacement surgery. J. Clin. Microbiol. 43:5393–5394. Lee, S. A., I. I. Raad, J. A. Adachi, and X. Y. Han. 2004. Catheter-related bloodstream infection caused by Mycobacterium brumae. J. Clin. Microbiol. 42:5429–5431. Luquin, M., V. Ausina, V. Le´vy-Fre´bault, M. A. Lane´elle, F. Belda, M. Garcia-Barcelo, G. Prats, and M. Daffe´. 1993. Mycobacterium brumae sp. nov., a rapidly growing, nonphotochromogenic mycobacterium. Int. J. Syst. Bacteriol. 43:405–413. Mesbah, M., U. Premachandran, and W. B. Whitman. 1989. Precise measurement of the G⫹C content of deoxyribonucleic acid by high-performance liquid chromatography. Int. J. Syst. Bacteriol. 39:159–167. National Committee for Clinical Laboratory Standards. 2003. Susceptibility testing of Mycobacteria, Nocardia, and other aerobic actinomycetes. Approved standard M24-A. National Committee for Clinical Laboratory Standards, Wayne, Pa. National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicrobial susceptibility testing. Twelfth informational supplement, M100-S12. National Committee for Clinical Laboratory Standards, Wayne, Pa.
J. CLIN. MICROBIOL. 26. Potters, D., M. Seghers, G. Muyldermans, D. Pierard, A. Naessens, and S. Lauwers. 2003. Recovery of Mycobacterium elephantis from sputum of a patient in Belgium. J. Clin. Microbiol. 41:1344. 27. Rahav, G., S. Pitlik, M. Scheflan, Z. Amitai, A. Lavy, M. Blech, N. Keller, Y. Davidson, and A. Zlotkin. An outbreak of Mycobacterium jacuzii infection following insertion of breast implants. Clin. Infect. Dis., in press. 28. Richter, E., S. Niemann, F. O. Gloeckner, G. E. Pfyffer, and S. RuschGerdes. 2002. Mycobacterium holsaticum sp. nov. Int. J. Syst. Evol. Microbiol. 52:1991–1996. 29. Ringuet, H., C. Akoua-Koffi, S. Honore, A. Varnerot, V. Vincent, P. Berche, J. L. Gaillard, and C. Pierre-Audigier. 1999. hsp65 sequencing for identification of rapidly growing mycobacteria. J. Clin. Microbiol. 37:852–857. 30. Sasser, M. 1990. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI technical note. MIDI, Newark, Del. 31. Schinsky, M. F., R. E. Morey, A. G. Steigerwalt, M. P. Douglas, R. W. Wilson, M. M. Floyd, W. R. Butler, M. I. Daneshvar, B. A. Brown-Elliott, R. J. Wallace, Jr., M. M. McNeil, D. J. Brenner, and J. M. Brown. 2004. Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates. Int. J. Syst. Evol. Microbiol. 54:1653–1667. 32. Schro ¨der, K. H., L. Naumann, R. M. Kroppenstedt, and U. Reischl. 1997. Mycobacterium hassiacum sp. nov., a new rapidly growing thermophilic mycobacterium. Int. J. Syst. Bacteriol. 47:86–91. 33. Shojaei, H., M. Goodfellow, J. G. Magee, R. Freeman, F. K. Gould, and C. G. Brignall. 1997. Mycobacterium novocastrense sp. nov., a rapidly growing photochromogenic mycobacterium. Int. J. Syst. Bacteriol. 47:1205–1207. 34. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bo¨ttger, and T. Bodmer. 1993. Rapidly growing mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175–178. 35. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882. 36. Tortoli, E. 2003. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin. Microbiol. Rev. 16:319–354. 37. Tortoli, E., L. Rindi, A. Bartoloni, C. Garzelli, A. Mantella, G. Mazzarelli, P. Piccoli, and C. Scarparo. 2003. Mycobacterium elephantis: not an exceptional finding in clinical specimens. Eur. J. Clin. Microbiol. Infect. Dis. 22:427–430. 38. Tortoli, E., C. Piersimoni, R. M. Kroppenstedt, J. I. Montoya-Burgos, U. Reischl, A. Giacometti, and S. Emler. 2001. Mycobacterium doricum sp. nov. Int. J. Syst. Evol. Microbiol. 51:2007–2012. 39. Turenne, C., P. Chedore, J. Wolfe, F. Jamieson, K. May, and A. Kabani. 2002. Phenotypic and molecular characterization of clinical isolates of Mycobacterium elephantis from human specimens. J. Clin. Microbiol. 40: 1230–1236. 40. van der Heijden, I. M., B. Wilbrink, L. M. Schouls, J. D. van Embden, F. C. Breedveld, and P. P. Tak. 1999. Detection of mycobacteria in joint samples from patients with arthritis using a genus-specific polymerase chain reaction and sequence analysis. Rheumatology 38:547–553. 41. Vincent, V., B. A. Brown-Elliot, K. C. Jost, Jr., and R. J. Wallace, Jr. 2003. Mycobacterium: phenotic and genotypic identification, p. 560–584. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, D.C. 42. Wallace, R. J., Jr., J. Glassroth, D. E. Griffith, K. N. Olivier, J. L. Cook, and F. Gordin. 1997. American Thoracic Society: diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am. J. Respir. Crit. Care Med. 156:S1–S15. 43. Wallace, R. J., Jr., V. A. Silcox, M. Tsukamura, B. A. Brown, J. O. Kilburn, W. R. Butler, and G. Onyi. 1993. Clinical significance, biochemical features, and susceptibility patterns of sporadic isolates of the Mycobacterium chelonaelike organism. J. Clin. Microbiol. 31:3231–3239. 44. Wallace, R. J., Jr., B. A. Brown, V. A. Silcox, M. Tsukamura, D. R. Nash, L. C. Steele, V. A. Steingrube, J. Smith, G. Sumter, Y. S. Zhang, and Z. Blacklock. 1991. Clinical disease, drug susceptibility, and biochemical patterns of the unnamed third biovariant complex of Mycobacterium fortuitum. J. Infect. Dis. 163:598–603. 45. Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697– 703. 46. Woo, P. C., H. W. Tsoi, K. W. Leung, P. N. Lum, A. S. Leung, C. H. Ma, K. M. Kam, and K. Y. Yuen. 2000. Identification of Mycobacterium neoaurum isolated from a neutropenic patient with catheter-related bacteremia by 16S rRNA sequencing. J. Clin. Microbiol. 38:3515–3517. 47. Woods, G. L., J. S. Bergmann, F. G. Witebsky, G. A. Fahle, B. Boulet, M. Plaunt, B. A. Brown, R. J. Wallace, Jr., and A. Wanger. 2000. Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. J. Clin. Microbiol. 38:656–661.
