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1 Regional Reference Center for Mycobacteria, Microbiology and Virology Laboratory, Careggi Hospital, 50134 Florence, Italy
2 Department of Experimental Pathology, Medical Biotechnologies, Infectivology and Epidemiology, University of Pisa, 56127 Pisa, Italy
3 Department of Preventive Medicine, Autonoma University of Madrid, 28029 Madrid, Spain
4 Microbiology and Virology Laboratory, S. Camillo-Forlanini Hospitals, 00149 Rome, Italy
5 Department of Public Health, University of Florence, 50134 Florence, Italy
6 German Collection of Microorganisms and Cell Cultures, 38124 Braunschweig, Germany
7 Clinical Laboratory, Campo di Marte Hospital, 55100 Lucca, Italy
8 Genetics and Cytogenetics Unit, Careggi Hospital, 50134 Florence, Italy
9 Microbiological and Virological Serum-immunology Laboratory, Careggi Hospital, 50134 Florence, Italy
10 Department of Specialized and Experimental Clinical Medicine, Microbiology Division, University of Bologna, 40138 Bologna, Italy
11 Regional Reference Center for Mycobacteria, S. Bortolo Hospital, 36100 Vicenza, Italy
Correspondence
Enrico Tortoli
e.tortoli{at}libero.it
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Published online ahead of print on 16 February 2004 as DOI 10.1099/ijs.0.02777-0.
The GenBank/EMBL/DDBJ accession number for the 16S rDNA and ITS regions sequence of strain FI-01069T is AJ548480.
Alignment of the ITS sequevars of MAC, PRA patterns and a table containing the fatty acid content are available as supplementary material in IJSEM Online.
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). Furthermore, a number of unnamed mycobacteria not belonging to any of these taxa (Frothingham & Wilson, 1994), sometimes referred to as M. avium–M. intracellulare cluster X (MAIX) (Viljanen et al., 1993), are included within the MAC.
The need to create a group that includes closely related, nevertheless different, organisms emerged long before the boom of genetic-based taxonomy, because of the scant or near impossibility of differentiation by means of cultural and biochemical tests; in fact, at that time, the only universally accepted characteristic that discriminated M. avium from M. intracellulare was the virulence of the latter in chickens (Runyon, 1967; Anz et al., 1970).
During the 1990s, genetic studies confirmed the relatedness of the organisms included in the MAC, although they revealed an intraspecies variability not present in other mycobacterial taxa (Frothingham & Wilson, 1993).
Apart from M. avium subsp. paratuberculosis, made easily recognizable by its mycobactin dependence and extremely slow growth, the differentiation of other members of the MAC remained unfeasible in diagnostic laboratories until the commercialization of DNA probes. The AccuProbe (Gen-Probe) allows easy and accurate differentiation between M. avium and M. intracellulare (Saito et al., 1989) and their clear distinction from unnamed MAIX (Viljanen et al., 1993). More recently, similar results became available using the INNO LiPA Mycobacteria (Innogenetics) reverse hybridization test (LiPA) (Tortoli et al., 2001), which, in a novel formulation (Tortoli et al., 2003), also allowed the differentiation of a subgroup [sequevar (sqv.)] of MAIX, so far tentatively assigned to the species M. intracellulare.
Our attention was first drawn to a number of strains identified by AccuProbe as M. intracellulare and by the first LiPA test as belonging to the M. avium–M. intracellulare–Mycobacterium scrofulaceum group (MAIS), but different from M. avium, M. intracellulare and M. scrofulaceum. An in-depth investigation of 12 such strains revealed both phenotypic and genotypic features that suggested their distinction from M. intracellulare and their belonging to a previously unrecognized species, within MAC, for which we propose the name Mycobacterium chimaera sp. nov.
Bacterial strains
The bacterial strains FI-01069T, FI-01129, FI-99018, FI-02109, FI-02038, FI-02197, FI-02110, FI-02126, FI-01022, FI-02211, FI-02212 and FI-03048 were independent (Table 1). They had been isolated over a 5 year period (1999–2003) from different patients in five Italian hospitals. Different media were used in various laboratories for culturing of the strains, including Middlebrook 7H9 agar, Lowenstein–Jensen medium and radiometric broth. All but one had been grown from respiratory specimens, and a single isolate had been obtained for six of them; the others (FI-01069T, FI-99018, FI-02110, FI-02126, FI-02210 and FI-02211) had been excreted repeatedly by patients.
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-glucosidase, Tween 80 hydrolysis, 3-day arylsulfatase, urease, tellurite reduction, 68 °C and semiquantitative catalase, growth rate, pigmentation, growth at 25, 37 and 45 °C, MacConkey growth; and tolerance to NaCl, thiophene carboxylic hydrazide, tiacetazone, p-nitrobenzoic acid, hydroxylamine, oleate and isoniazid) were tested using standard procedures described previously (Kent & Kubica, 1985).
TLC of mycolic acids
The mycolic acids present in the cell wall, extracted by means of methyl esterification, were separated by two-dimensional TLC on silica gel (Minnikin et al., 1984). Identification of the spots was made by comparison with those from reference strains. Two strains, FI-01069T and FI-02038, were chosen for this test.
Analysis of fatty acids
Fatty acid methyl esters were obtained from 40 mg (wet weight) of cells by saponification, methylation and extraction as described before (Miller, 1982). The methyl ester mixtures were separated by a gas chromatograph (model 5898A, Hewlett Packard) controlled by MIS software (Microbial ID). Peaks were integrated automatically and fatty acid names and percentages were determined using the Microbial Identification standard software package (Sasser, 1990). Strains FI-01069T, FI-01129 and FI-02038 were chosen for fatty acid analyses.
HPLC of cell wall mycolic acids
The standard procedure of the Centers for Disease Control and Prevention (CDC, 1996) was used for the extraction and derivatization to bromophenacyl esters of mycolic acids. Separation was obtained with a 126 model System Gold (Beckman) HPLC instrumentation by using a gradient of methanol and methylene chloride with an Ultrasphere-XL column (Beckman). Chromatograms were compared visually with those of our laboratory library.
DNA probe hybridization
For each strain, DNA hybridization was attempted with AccuProbe M. avium, AccuProbe M. intracellulare and AccuProbe MAC (Kiehn & Edwards, 1987), with both formulations of LiPA [the first (Tortoli et al., 2001) and the more recent one] and with a further reverse hybridization test GenoType Mycobacteria (HAIN). The tests were performed according to the recommendations of the manufacturers.
Genetic sequencing and phylogenetic analysis
The full-length 16S rRNA gene and the 16S–23S rDNA internal transcribed spacer (ITS) were amplified using primers and PCR protocols described previously (Kirschner et al., 1993; Roth et al., 1998). Sequencing of PCR products was carried out with an automated apparatus (ALFexpress DNA sequencer; Pharmacia Biotech) using the Thermo Sequenase fluorescent labelled primer cycle-sequencing kit with 7-deaza-dGTP and the Thermo Sequenase Cy5 dye terminator kit (Amersham Pharmacia Biotech). The sequences of the 16S rDNA and the ITS were compared with those present in the GenBank and Ridom (Harmsen et al., 1999) databases; ambiguities in published databases were considered as identities. The complete ITS sequence was aligned with all known such sequences of MAC using the CLUSTAL W program [EMBL-European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/)]. The maximum-likelihood method was used for the construction of the phylogenetic tree (Felsenstein, 1993; PHYLIP). The tree was rooted with the ITS sequence of Mycobacterium tuberculosis as the outgroup. Tree branches were reproduced by performing 100 bootstrap replicates.
RFLP and insertion element investigations
The presence of IS1245 was investigated by means of a RFLP-based assay using PvuII restriction enzyme (van Soolingen et al., 1998), in which the generated DNA fragments were separated electrophoretically on an agarose gel, blotted onto a nylon filter and tested by a peroxidase-labelled IS1245 probe. Other insertion elements characterizing various taxa included in MAC, IS900 (Green et al., 1989) and IS901 (Kunze et al., 1991), were investigated by PCR using previously reported primers (Sanderson, 1993; Kunze et al., 1992; Ahrens et al., 1995).
PCR restriction enzyme pattern analysis (PRA)
PRA was carried out as described previously (Telenti et al., 1993). Briefly, primers Tb11 and Tb12 were used in a 50 µl reaction volume, containing 1·5 mM MgCl2; PCR conditions were the same as described by Telenti et al. (1993). Fifteen microlitres of the hsp65 gene amplified product (441 bp) was digested with the restriction enzymes BstEII and HaeIII and separated electrophoretically on 4 % NuSieve agarose gels. Digestion patterns were compared to the PRASITE database for identification purposes (http://www.hospvd.ch:8005).
Antimicrobial susceptibility testing
Minimal inhibitory concentrations (MIC) were investigated on Middlebrook 7H11 agar plates using twofold dilutions of the following antibiotics: amikacin, ciprofloxacin, clarithromycin, ethambutol, rifampicin and streptomycin (Table 2).
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-glucosidase, Tween 80 hydrolysis, 3-day arylsulfatase, urease, semiquantitative catalase, NaCl tolerance and MacConkey growth, and positive for 68 °C catalase, tolerated thiophene carboxylic hydrazide, tiacetazone and isoniazid and grew at 25 and 37 °C but not at 45 °C. Variability was detected for the remaining tests. The colonies, which were smooth, were unpigmented.
Antimicrobial susceptibility results
The susceptibility pattern was rather variable, with only ethambutol being ineffective on all the strains. The MICs were very inhomogeneous, quite low in four strains and high in the others (Table 2
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Lipid analysis results
The TLC revealed the presence of 
-mycolates, ketomycolates and wax ester mycolates, which is the pattern shared by MAC and many other mycobacterial species (Luquín et al., 1991).
On the basis of the pattern of fatty acids disclosed by GLC analysis, the MIDI system identified our strains as best resembling the species M. intracellulare. The similarity to the M. intracellulare pattern stored in the MIDI fatty acid database was, however, very close to 0·5, which is considered the lower limit for strains belonging to the same species. Details of the fatty acid contents are available as supplementary data, in IJSEM Online.
The mycolic acid HPLC pattern was characterized by two clusters of peaks: an early major one and a second one emerging 156 s later (Fig. 1). Only one of the isolates (FI-02110) presented a third minor cluster of peaks just before the second one.
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Genetic sequences
Identical nucleotide sequences of the 16S rDNA and the 16S–23S rDNA ITS characterized our 12 strains. The 16S rDNA presented a single nucleotide mismatch (T
C) compared with M. intracellulare (GenBank accession no. AJ536036) at nucleotide position 403. In the Ridom database, there were only 442 bases of the 5' end of the 16S rDNA present; full identity emerged to M. intracellulare sqv. v (ATCC 35772). The sequence of the ITS was identical to that of sqv. MAC-A (accession no. L07847) (Frothingham & Wilson, 1993), which is characteristic of several MAC organisms other than M. avium and M. intracellulare. A supplementary figure showing the complete ITS alignment of known MAC sequevars is available in IJSEM Online.
Phylogenetic analysis (Fig. 2) shows three major branches in which M. avium, M. intracellulare and most of MAC sequevars are clustered. However, nine sequevars, at present included in MAC, form two minor clusters and four isolated branches; among the latter lies sqv. MAC-A, which is, phylogenetically, the most distant both from M. avium and M. intracellulare.
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Insertion sequences
None of the insertion elements (IS900, IS901 or IS1245) characterizing the various taxa included in the MAC was detectable in any of the strains.
Clinical significance of the isolates
Four of the strains isolated only once did not fulfil the criteria of the American Thoracic Society for clinical significance (Wallace et al., 1990); one had been isolated from urine, while the others had been grown from patients with chronic obstructive pulmonary disease, for which the mycobacterial origin could not be demonstrated (Table 1). The other seven strains had been isolated from patients (four male and three female), 56 to 82 years old, whose cases were considered clinically significant. None of them was immunodeficient; two had pulmonary cavitations and haemoptysis, while the others presented pulmonary abscesses, chronic obstructive pulmonary disease and bronchiectasis (Table 1). One such patient (FI-02210), hospitalized in the intensive care unit because of acute respiratory failure, survived only a few days; the others were treated with multiple anti-mycobacterial drugs after which three of them recovered and have so far not relapsed. In all such cases no other plausible cause for the pulmonary disease was detected.
The aggregation, within the MAC, of strains that, although not identical, shared major traits arose from the impossibility of their differentiation by means of biochemical tests. While the commercialization of AccuProbe seemed to have resolved, at the level of routine diagnostic purposes, the dilemma of the distinction of M. avium from M. intracellulare and of both from MAIX, questions again arose with the subsequent introduction of LiPA, mainly because of the presence of strains with discrepant hybridization characteristics (Makinen et al., 2002; Suffys et al., 2001; Mijs et al., 2002b; Miller et al., 2000; Scarparo et al., 2001; Tortoli et al., 2001). In fact, strains do exist that are identified by AccuProbe as M. intracellulare while they are located by LiPA within the MAIS group, however, excluding their belonging to the species M. avium, M. intracellulare or M. scrofulaceum.
As the two commercial DNA probes are characterized by different targets, the 16S rRNA gene in the AccuProbe and the ITS in the LiPA, the above results only reflect the combination of the genetic sequences present in adjacent regions in such organisms. In fact, all the strains characterized here combine sqv. v of M. intracellulare in the 16S rDNA and sqv. MAC-A in the ITS; a genetic ‘mosaic’ reported for the first time by Frothingham et al. (1993). The result achieved with GenoType, whose target is represented by the 23S rDNA, suggests that the sequence in this region is compatible with M. intracellulare.
The nucleotide sequences of 16S rDNA and ITS have confirmed the relatedness of the organisms included in the MAC, but, at the same time, revealed an unusual heterogeneity within it (Kirschner et al., 1993; Roth et al., 1998). Two and five sequevars of the 16S rDNA are present in M. avium and M. intracellulare (Ridom), respectively. With regard to the ITS, seven sequevars are known for M. avium, four for M. intracellulare and 23 for other members of the complex (Mijs et al., 2002a) (in the latter case, a further cause of confusion is represented by two sets of three distinct sequevars, which received identical names, MAC-J, MAC-K and MAC-L, from independent researchers). The ITS sequevars of the species M. avium and M. intracellulare are characterized by close relatedness, while the heterogeneity is very high among strains unassigned to such taxa, an observation that led repeatedly to the hypothesis of the existence of other species within this group (Frothingham & Wilson, 1993; Wayne et al., 1996; Wayne & Sramek, 1992).
The nucleotide sequences of the 16S rDNA of the strains investigated here present only one nucleotide mismatch compared with the most frequent sequevar (sqv. i) of M. intracellulare, but they are very distant from this species with regards to the ITS (sqv. MIN-A is the closest, with 20 mismatches, while 21 mismatches are present in each of the others).
Given the existence of mycobacterial species that differ by only one base in the 16S rDNA or that are even identical in this gene (e. g. Mycobacterium kansasii and Mycobacterium gastri; Kirschner et al., 1993), nothing seems to contradict the hypothesis that the strains investigated here are members of a novel taxon. In contrast, there is evidence to suggest that the strains investigated do not belong to the species M. intracellulare, towards which very poor relatedness exists at the ITS level. Moreover, the variations in both officially recognized species of MAC are very limited; 1 to 3 bases in M. avium and 1 to 2 in M. intracellulare.
When the whole stretch of the rDNA operon, including both 16S and ITS, is considered, the maximum number of mismatches is four among various sequevars of M. avium and seven for those of M. intracellulare. In contrast, MAC-A differs from the first species by, at least, 20 nt and from the second by 21 or more. Furthermore, within this region, various genetic markers distinguish MAC-A from any other variant, such as the replacement of adenosine at position 149 with thymidine, that of guanosine at position 234 with adenosine and the deletion at position 230. Finally, in the phylogenetic tree (Fig. 2), MAC-A occupies a separate branch, far from other MAIX and further from M. avium and M. intracellulare.
The HPLC pattern of mycolic acid is very consistent among the strains included in the MAC, which are typically characterized by three clusters of peaks. The first cluster is the main one and includes four major peaks, while the others, which emerge later and close to each other, present three major peaks each. The relative heights of the peaks in the second and third cluster may vary with M. avium and M. intracellulare presenting, in most cases, lower peaks in the third and in the second cluster, respectively (Butler et al., 1992). In contrast, the strains investigated here presented only the first and the third of such clusters (Fig. 1). The uniqueness of this phenotypic feature emerged also from the careful retrospective revision of over 300 HPLC profiles of MAC present in our laboratory file, which revealed only three such patterns. In only one of them (FI-99018), genetic sequencing revealed sqv. v and sqv. MAC-A in 16S rDNA and ITS, respectively; however, this was the only one which confirmed, once the HPLC was repeated, the typical two-cluster profile.
It seems conceivable that the GLC results, characterized by low similarity to typical M. intracellulare, are also somehow a confirmation of the peculiar lipid structure of the strains investigated here, but more strains have to be analysed before one can draw a final conclusion.
Several insertion elements characterize different taxa of MAC; IS900 is specific for M. avium subsp. paratuberculosis (Green et al., 1989), IS901 is specific for the bird-type M. avium (Kunze et al., 1991), while the number of elements of IS1245 varies (Ritacco et al., 1998). None of the above transposons was present in our strains. In contrast, two strains presenting DNA probe reactivity identical to ours, investigated in the recent paper describing M. avium subsp. hominissuis (Mijs et al., 2002a), presented multiple bands of IS1245 in RFLP analysis.
The PRA pattern characteristic of M. intracellulare PRA type I does not exclude the possibility that the strains investigated here belong to a different species, as previously reported for other strains (Leclerc et al., 2000).
There are a number of papers that emphasize the feature of drug resistance of MAC (Inderlied et al., 1993); among our strains, along with some quite resistant, four were highly susceptible, except for ethambutol, to almost all antibiotics usually tested against mycobacteria.
Of importance is that the proposed species has been isolated, so far, only from humans (De Smet et al., 1995; Frothingham & Wilson, 1994; Mijs et al., 2002a), usually elderly males with pulmonary disorders, but never from AIDS patients (De Smet et al., 1995; Frothingham & Wilson, 1994). The high frequency of cases in which it is clinically significant seems to suggest a virulence greater than other MAC organisms.
Description of Mycobacterium chimaera sp. nov.
Mycobacterium chimaera sp. nov. (chi.maer'a. L. n. chimaera the chimaera, the mythological being made up of parts of three different animals, referring to the apparent mix of genetic features characterizing the strains).
Slowly-growing, unpigmented mycobacterium characterized by acid-fast, non-motile and non-spore forming coccobacilli. AccuProbe and GenoType assign M. chimaera to the species M. intracellulare; LiPA has recently developed a specific probe, probably to comply with AccuProbe, which binds it to M. intracellulare. A unique taxonomic position emerges from genetic sequencing of both 16S rDNA and ITS, with the first being considered, at present, a sequevar of M. intracellulare and, the second, a sequevar of several MAC, other than M. avium and M. intracellulare. The typical HPLC profile easily differentiates M. chimaera from any other MAC. Additional signals may be provided by the anti-mycobacterial susceptibility pattern, characterized by full resistance to ethambutol, by the almost exclusive isolation from respiratory samples and by the frequent involvement in pulmonary disease of elderly people.
The type strain of M. chimaera is FI-01069T (=CIP 107892T=DSM 44623T). Strains FI-02038 and FI-01129 were deposited in the DSMZ as DSM 44621 and DSM 44622, respectively.
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ACKNOWLEDGEMENTS |
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J. van Ingen, M. J. Boeree, K. Kosters, A. Wieland, E. Tortoli, P. N. R. Dekhuijzen, and D. van Soolingen Proposal to elevate Mycobacterium avium complex ITS sequevar MAC-Q to Mycobacterium vulneris sp. nov. Int J Syst Evol Microbiol, September 1, 2009; 59(9): 2277 - 2282. [Abstract] [Full Text] [PDF]
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 S. Foongladda, S. Pholwat, B. Eampokalap, P. Kiratisin, and R. Sutthent Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth J. Mol. Diagn., January 1, 2009; 11(1): 42 - 48. [Abstract] [Full Text] [PDF]
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 I. Ben Salah, T. Adekambi, D. Raoult, and M. Drancourt rpoB sequence-based identification of Mycobacterium avium complex species Microbiology, December 1, 2008; 154(12): 3715 - 3723. [Abstract] [Full Text] [PDF]
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D. Bang, T. Herlin, M. Stegger, A. B. Andersen, P. Torkko, E. Tortoli, and V. O. Thomsen Mycobacterium arosiense sp. nov., a slowly growing, scotochromogenic species causing osteomyelitis in an immunocompromised child Int J Syst Evol Microbiol, October 1, 2008; 58(10): 2398 - 2402. [Abstract] [Full Text] [PDF]
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 J. E. Stout, G. W. Hopkins, J. R. McDonald, A. Quinn, C. D. Hamilton, L. B. Reller, and R. Frothingham Association between 16S-23S Internal Transcribed Spacer Sequence Groups of Mycobacterium avium Complex and Pulmonary Disease J. Clin. Microbiol., August 1, 2008; 46(8): 2790 - 2793. [Abstract] [Full Text] [PDF]
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O. Esparcia, F. Navarro, M. Quer, and P. Coll Lymphadenopathy Caused by Mycobacterium colombiense J. Clin. Microbiol., May 1, 2008; 46(5): 1885 - 1887. [Abstract] [Full Text] [PDF]
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 L. B. Gadkowski and J. E. Stout Cavitary Pulmonary Disease Clin. Microbiol. Rev., April 1, 2008; 21(2): 305 - 333. [Abstract] [Full Text] [PDF]
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C. Y. Turenne, R. Wallace Jr., and M. A. Behr Mycobacterium avium in the Postgenomic Era Clin. Microbiol. Rev., April 1, 2007; 20(2): 205 - 229. [Abstract] [Full Text] [PDF]
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M. I. Murcia, E. Tortoli, M. C. Menendez, E. Palenque, and M. J. Garcia Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant. Int J Syst Evol Microbiol, September 1, 2006; 56(Pt 9): 2049 - 2054. [Abstract] [Full Text] [PDF]
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C. Russo, E. Tortoli, and D. Menichella Evaluation of the New GenoType Mycobacterium Assay for Identification of Mycobacterial Species J. Clin. Microbiol., February 1, 2006; 44(2): 334 - 339. [Abstract] [Full Text] [PDF]
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C. Y. Turenne, M. Semret, D. V. Cousins, D. M. Collins, and M. A. Behr Sequencing of hsp65 Distinguishes among Subsets of the Mycobacterium avium Complex J. Clin. Microbiol., February 1, 2006; 44(2): 433 - 440. [Abstract] [Full Text] [PDF]
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A. McNabb, K. Adie, M. Rodrigues, W. A. Black, and J. Isaac-Renton Direct Identification of Mycobacteria in Primary Liquid Detection Media by Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene J. Clin. Microbiol., January 1, 2006; 44(1): 60 - 66. [Abstract] [Full Text] [PDF]
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M. Sanguinetti, L. Novarese, B. Posteraro, S. Ranno, E. De Carolis, G. Pecorini, B. Lucignano, F. Ardito, and G. Fadda Use of Microelectronic Array Technology for Rapid Identification of Clinically Relevant Mycobacteria J. Clin. Microbiol., December 1, 2005; 43(12): 6189 - 6193. [Abstract] [Full Text] [PDF]
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M. Drancourt and D. Raoult Sequence-Based Identification of New Bacteria: a Proposition for Creation of an Orphan Bacterium Repository J. Clin. Microbiol., September 1, 2005; 43(9): 4311 - 4315. [Full Text] [PDF]
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M. Semret, D. C. Alexander, C. Y. Turenne, P. de Haas, P. Overduin, D. van Soolingen, D. Cousins, and M. A. Behr Genomic Polymorphisms for Mycobacterium avium subsp. paratuberculosis Diagnostics J. Clin. Microbiol., August 1, 2005; 43(8): 3704 - 3712. [Abstract] [Full Text] [PDF]
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J. L. Cloud, K. C. Carroll, S. Cohen, C. M. Anderson, and G. L. Woods Interpretive Criteria for Use of AccuProbe for Identification of Mycobacterium avium Complex Directly from 7H9 Broth Cultures J. Clin. Microbiol., July 1, 2005; 43(7): 3474 - 3478. [Abstract] [Full Text] [PDF]
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L. Lebrun, F.-X. Weill, L. Lafendi, F. Houriez, F. Casanova, M. C. Gutierrez, D. Ingrand, P. Lagrange, V. Vincent, and J. L. Herrmann Use of the INNO-LiPA-MYCOBACTERIA Assay (Version 2) for Identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum Complex Isolates J. Clin. Microbiol., June 1, 2005; 43(6): 2567 - 2574. [Abstract] [Full Text] [PDF]
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COPD, Chronic obstructive pulmonary disease.
