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Previous Article | Next Article 
Clinical Microbiology Reviews, April 2003, p. 319-354, Vol. 16, No. 2
0893-8512/03/$08.00+0 DOI: 10.1128/CMR.16.2.319-354.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Regional Reference Center for Mycobacteria, Microbiological and Virological Laboratory, Careggi Hospital, 50134 Florence, Italy
SUMMARY INTRODUCTION 16S rRNA Gene 65-kDa Heat Shock Protein Gene Other Molecular Targets of Diagnostic Interest Mycolic Acid Analysis Classification PIGMENTED SLOW GROWERS M. bohemicum M. celatum M. conspicuum M. doricum M. heckeshornense M. interjectum M. intermedium M. kubicae M. lentiflavum M. palustre M. tusciae NONPIGMENTED SLOW GROWERS M. branderi ''M. canettii'' M. genavense M. heidelbergense M. lacus M. shottsii M. triplex PIGMENTED RAPID GROWERS M. elephantis M. hassiacum M. novocastrense NONPIGMENTED RAPID GROWERS M. abscessus M. alvei M. brumae M. confluentis M. goodii M. holsaticum M. immunogenum M. mageritense M. mucogenicum M. peregrinum M. septicum M. wolinskyi SOLELY ENVIRONMENTAL SPECIES M. botniense M. chlorophenolicum M. cookii M. frederiksbergense M. hiberniae M. hodleri M. madagascariense M. murale M. vanbaalenii ''VIRTUAL'' MYCOBACTERIA ''M. visibilis'' NEW SUBSPECIES M. avium subsp. avium M. avium subsp. paratuberculosis M. avium subsp. silvaticum M. avium subsp. hominissuis M. bovis subsp. caprae UNIDENTIFIED MYCOBACTERIA CONCLUSIONS Isolation Source and Phenotypic Characters Phylogenetic Investigation Molecular Microheterogeneity within 16S rDNA Phenotype-Genotype Relations Limitations of Susceptibility Testing Significant or Not Significant? That's the Problem Most Frequent Pathologies M. scrofulaceum, Organism in Decline? Suggested Minimal Standards for the Description of New Taxa What Is the Impact of Expanded Taxonomy on Routine Mycobacteriology Laboratory Practice? Final Remarks ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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The rationale of genotypic taxonomy is linked to the detection, within the genome, of highly conserved regions harboring hypervariable sequences in which species-specific deletions, insertions, or replacements of single nucleotides are present. The gene encoding the 16S rRNA has been for many years, and still is, the primary target of molecular taxonomic studies, with several other genomic regions playing a minor role. Although the role of genetics has been preeminent in the recent advancement of mycobacterial taxonomy, an important contribution has also been made by chemotaxonomic investigations. This approach achieved excellent results with mycobacteria, thanks to the presence in their cell wall of an unusually heavy lipid burden which includes unique molecules such as mycolic acids. As a consequence of these two developments, the number of mycobacterial species has greatly increased in the last decade.
Important baseline articles concerning the nontuberculous mycobacteria exist. They include the milestone review by Wolinsky (175), properly updated 13 years later by Wayne and Sramek (169). Interestingly, 1992, the year the latter review was published, definitively sanctioned the success of the genotypic approach to mycobacterial taxonomy. The present paper, which aims to continue on the track of the above-mentioned authoritative reviews, focuses on the mycobacterial species described in the 1990s, with particular emphasis on those in which molecular and lipid analyses played an important role.
Like every single-stranded nucleotide sequence, the 16S rRNA folds up into a secondary structure (Fig. 1) characterized by loops (or helices). Helices 8, 9, 10, and 11 fall within region A, while helix 18 is in region B. The sequences of such helices present, in addition to nucleotide substitutions scattered all over the hypervariable regions, additional regions of variability that, being shared by clusters of species within the genus Mycobacterium, are of particular interest, mainly from the phylogenetic point of view (Fig. 2). Helix 10 may be extended by an insertion of one cytosine at the E. coli homologous position 184. Mycobacteria with such an insertion belong to a separate branch within the rapid growers and are known as "thermotolerant rapid growers," even though only some of them are able to grow at temperatures over 37°C. More extensive is the insertion at position 455 in helix 18. The number of nucleotides involved ranges from 8 in M. scrofulaceum to 12 in the majority of slow growers and even 14 in M. terrae and a few other species, which therefore appear phylogenetically related. A short helix (i.e., without any insertion) is thought to be the original condition.
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For identification purposes, the sequence of region A is usually sufficient. Region B may be considered confirmatory since in several cases its sequence is shared by a number of species. While slow growers contain one copy of the 16S rRNA gene (4), rapid growers, except for M. chelonae and M. abscessus, have two copies.
No cutoff exists for the minimal number of nucleotide differences indicating distinct taxa. The 5- to 15-base diversity in the 16S rRNA gene, proposed for other microorganisms (44), is not applicable to the genus Mycobacterium, whose members are more closely related to each other. Closely related species may differ only by a few bases or, in the presence of clear phenotypic differences, not differ at all. In contrast, several unequivocally defined species exist presenting genetic heterogeneity that, for various sequevars of M. avium complex (MAC), may involve differences of up to 7 bp.
Genetic sequencing, which is cumbersome (75) and often subject to interpretation errors when done manually, is nowadays made easier and reproducible due to the improved chemistry (e.g., sequenase and dye terminators) and sequencing methods and to the availability of fully automatic instrumentation. Furthermore, despite what is still believed, it has also become very inexpensive.
that are a major component of the conspicuous lipid content of the mycobacterial cell wall. They differ in the number of carbon atoms, ranging from 60 to 90, and in the presence of different functional groups. The mycolic acid pattern of the cell wall generally varies with the species. Thus, mycolic acid analyses can be a useful tool for mycobacterial identification. Two direct approaches may be used for the analysis of mycolic acids: thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A third method, gas-liquid chromatography (GLC), in which mycolic acids are investigated in the form of their cleavage products, is also used.
TLC allows the differentiation of esters of seven known mycolic acid types: type i or -mycolates, type ii or '-mycolates, type iii or methoxy-mycolates, type iv or keto-mycolates, type v or epoxy-mycolates, type vi or wax esters, and type vii or 
-1-methoxy-mycolates. Type i mycolate is present in all mycobacterial species detected so far. Type vii is present in only a few species of nonpigmented rapidly growing mycobacteria (M. senegalense and M. alvei and several strains of M. porcinum, M. fortuitum, and M. peregrinum) (86). The other types are variously distributed among different species; most mycobacteria contain no more than two or three mycolic acid types. Given the limited number of mycolic acid types, many patterns are shared by more than one species.
Once mycolic acids have been liberated from whole cells, they are extracted as mycolates by means of methyl esterification (12). They are then spotted on silica gel sheets (stationary phase), where they are separated by the capillary progression of elution solvents (mobile phase). The identification of separated compounds, which look like spots, is determined by their position and is generally performed by comparison with spots from reference strains run in parallel with the sample.
In HPLC analysis, the mycolic acids are separated on the basis of their polarity and the carbon chain length, with the more polar and shorter eluting first. In contrast to TLC, the identification of eluted compounds is unimportant; the arrangement of major peaks in the chromatogram, the position on the basis of retention times, and the height in comparison with other peaks are the only pieces of information needed. Each species is characterized by a pattern with a particular number, position, and height of peaks. The visual comparison of HPLC chromatograms with ones of known species is still the most reliable identification procedure.
To obtain an HPLC chromatogram, the mycolic acids are liberated from whole cells by means of saponification and extracted with chloroform. They are then derivatized to UV-adsorbing bromophenacyl esters (17, 20). The extract is injected in the column, where it is eluted by the mobile phase, a gradient of methanol and methylene chloride. The eluted fractions are quantified by UV absorption, using a detector set to 254 to 260 nm. More recently, improved sensitivity (16 to 26 times greater) has been achieved by replacing the UV absorption detection technique with one based on fluorescence absorption. Although a different derivatization technique is required, which utilizes fluorescent labeling compounds (coumarins), the chromatograms are fully comparable with the ones obtained by standard UV-HPLC.
With GLC, not only mycolic acids but the whole lipid component of the cell wall is analyzed (53). At operating column temperatures, ranging from 275 to 300°C, the pyrolysis of mycolic acids occurs with consequent splitting into saturated methyl esters 22, 24, or 26 carbon atoms long (C22:0, C24:0, and C26:0). Along with the above mycolic acid cleavage products, saturated and unsaturated fatty acids are eluted, including exadecanoic (C16:0), octadecenoic (C18:1) octadecanoic (C18:0), and tuberculostearic (10-methyloctadecanoic.) acids and secondary alcohols, stemming from wax esters. The distribution of the GLC products varies qualitatively and quantitatively in different species. Different concentrations of tuberculostearic acid are present in different mycobacteria, with the exception of M. gordonae. The most frequently detected compounds include saturated and unsaturated 16- and 18-carbon fatty acids and, among mycolic acid cleavage products, the C24:0 methyl ester. For the identification of single species, the most important compounds include methyl-branched fatty acids, alcohols, and C22:0, C24:0, and C26:0 mycolic acid cleavage products. Many species contain common peaks of various heights; characteristic peaks, even those of poor height, are however more important for identification. As with TLC, species do exist with indistinguishable profiles.
The fatty acids, once extracted into methyl esters from whole cells by means of methanolysis, are subjected to trifluoroacetylation, which improves alcohol detection. They are then injected into the gas chromatograph, where they are driven to the temperature-controlled column by the carrier gas. The quantity of the various eluted products is determined by means of a flame ionization detector and the identification, achieved on the basis of retention times, may be confirmed by mass spectrometry.
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Phenotypic features. M. bohemicum is a rod-shaped acid-fast bacillus which grows slowly at temperatures ranging from 25 to 40°C. The colonies are small (1 to 2 mm in diameter), smooth, and scotochromogenic, while the majority of biochemical tests are negative (Table 1). Its lipid component is characterized by an uncommon TLC pattern (Table 2), shared only by M. hassiacum and M. mucogenicum. Both GLC (Table 3) and HPLC profiles overlap with, or are hardly differentiable from, those of MAC and M. scrofulaceum (148). Interestingly, one strain, out of a group isolated from the environment, presented an HPLC chromatotype characterized by a single cluster, clearly different from all other strains isolated so far (140).
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A large group of M. bohemicum strains have been recently investigated (140); one of them was isolated from a generalized cutaneous infection in an elderly woman, three were isolated from the sputa of three different patients, one was isolated from the mesenteric lymph nodes of a goat, and five were isolated from stream water. The origin of one additional clinical isolate is unknown. Coisolation of M. bohemicum and M. palustre, both regarded as clinically insignificant, has also been reported (139). From such a survey, unusually rich for a species recognized less than 4 years ago, two major observations emerge: M. bohemicum should be regarded as a potential pathogen, and natural water is one of its reservoirs. The virulence of the species also seems confirmed: following intravenous infection of syngeneic gamma interferon-deficient and BALB/c mice with M. bohemicum, also the latter, nonimmunocompromised animals developed splenomegaly and granulomatous liver lesions (36).
Type strain: DSM 44277T. EMBL 16S rDNA sequence accession number: U84502.
Phenotypic features. M. celatum is acid fast and predominantly rod shaped. It grows in 20 to 35 days, between 33 and 42°C, forming small, smooth, colonies described as unpigmented, but the majority of the strains are pale yellow and scotochromogenic. Polymorphic colonies have also been reported (39). The most important distinctive biochemical test seems to be the positive arylsulfatase activity (Table 1).
In TLC, M. celatum has a mycolate set indistinguishable from those of a number of both slowly and rapidly growing species (Table 2); in contrast, the GLC pattern is uncommon (Table 3). The close similarity of the M. celatum and M. xenopi HPLC profiles (151) resulted, in the years preceding the sp. nov. description, in the inclusion of M. celatum in a group named "M. xenopi-like." However, a careful assessment of chromatograms allows the easy differentiation of the two species due to the earlier retention times of the peaks of the second cluster characterizing M. celatum.
A unique feature of M. celatum is the consistent resistance to rifampin, with very high MICs (Table 4) (151).
Genotypic features. The PRA analysis of the hsp65 gene divided the strains investigated in the sp. nov. description into two groups, characterized by seven and six bands. This grouping was confirmed by the patterns obtained by multilocus enzyme electrophoresis and by the sequencing of the 16S rDNA (19). A 10-base mismatch between the two different sequevars (named type 1 and type 2) was detected. A third variant (type 3) was subsequently described on the basis of eight strains from AIDS patients. The 16S rDNA sequence of type 3 differs by 7 bases from that of type 1 and by 17 bases from that of type 2 (16). The mismatches are outside the hypervariable regions A and B.
Picardeau et al. (104) have recently described a new insertion sequence, IS1407, in M. celatum. Three or four copies of IS1407 are present in identical genomic positions in M. celatum types 1 and 3, while it is absent in type 2. Furthermore, the same authors uncovered the presence of a single pulsed-field gel electrophoresis (PFGE) pattern among all type 1 and type 3 isolates and polymorphic patterns among type 2 isolates (104). The fact that M. celatum type 2 lacks IS1407, differs by 10 and 17 bp in 16S rDNA sequences from types 1 and 3, and has very different PFGE genotype patterns model seems to support the notion that it is (or may be) a different species (104). Interestingly two strains of M. celatum have been detected that contain two copies of the 16S rRNA gene; they have different sequences, of type 1 and type 3, respectively (109, 158).
The presence of only a single nucleotide mismatch with the short stretch of M. tuberculosis complex 16S rRNA targeted by the GenProbe DNA probe (AccuProbe Mycobacterium tuberculosis complex; GenProbe, San Diego, Calif.) is responsible for the cross-hybridization with M. celatum type 1 (18). This cross-reactivity, usually moderate, is detectable only when the stringency of the hybridization conditions is lower than recommended (AccuProbe Mycobacterium tuberculosis complex, package insert). On the basis of the sequence identity of types 1 and 3 in hypervariable region A, a cross-reactivity, although not documented so far, can be assumed for type 3 too. Phylogenetically, M. celatum has genetic markers of slow growers, most closely related to M. branderi and M. xenopi, with which it forms a branch that diverged early in the evolution of the slowlygrowing species (Fig. 3).
Clinical and epidemiological features. Several independent clinical strains, subsequently recognized as being identical to M. celatum (four of type 1 and one of type 2), had been phenotypically investigated in Finland before the sp. nov. description; they were isolated between 1972 and 1990 without being assigned to any species (11).
Numerous clinically significant isolations have been reported in AIDS patients, presenting as disseminated infection in 10 patients (7, 37, 48, 105, 151, 178), limited to the lungs in 5 cases (48), and exclusively extrapulmonary in one case of penile infection (29). In contrast, in nonimmunocompromised patients, only one case of fatal pulmonary disease in an elderly woman (21) and one of childhood lymphadenopathy (G. Haase, H. Skopnik, S. Bätge, and E. C. Böttger, Letter, Lancet 344:1020-1021, 1994) have been reported.
Although many clinical isolates of M. celatum are not clinically significant, the pathogenicity of the species seems, higher than that of the majority of nontuberculous mycobacteria, particularly in AIDS patients. Such a hypothesis also seems confirmed by the experimental inoculation of M. celatum into BALB/c and syngeneic gamma interferon-deficient mice. It gave rise to splenomegaly and granulomatous liver lesions in both immunocompetent and immunodeficient animals, demonstrating the high virulence of the strain (36).
Isolation from the environment has not been reported so far.
Type strains: ATCC 51131T (type 1), ATCC 51130T (type 2) and NCTC 12882T (type 3). EMBL 16S rDNA sequence accession numbers: L08169 (type 1), L08170 (type 2), and Z46664 (type 3).
Phenotypic features. The mycobacterial cells are coccobacillary and acid fast. Growth on solid media is slow and requires, at temperatures from 22 to 31°C, at least 3 weeks. Growth at 37°C occurs only in liquid media. A pale yellow scotochromogenic pigment is present, and the majority of biochemical traits are negative (Table 1).
The TLC mycolic acid pattern is shared with other slow growers including MAC and M. terrae (Table 2). The GLC fatty acid set is unique (Table 3). Similarity, again to M. terrae, is detected by HPLC, with both profiles presenting two clusters of peaks, with the early cluster being major and the late cluster being minor. The profiles are nevertheless distinguishable since the peaks of M. conspicuum present slightly later retention times (129).
Although the species is not very resistant in vitro (Table 4), both infected patients remained culture positive until death, despite treatment with multiple antibiotics.
Genotypic features. The 16S rDNA of M. conspicuum is, from the genetic point of view, characteristic of a slow grower; M. conspicuum occupies a phylogenetic position close to M. asiaticum (Fig. 2 and 3).
Clinical and epidemiological features. Both patients from whom M. conspicuum was isolated had severe immune impairment. Nevertheless, the virulence of the species does not seem lower than that of other opportunistic agents like MAC and M. genavense, especially since the disseminated mycobacterial infection was probably a major cause of death in both subjects. The virulence in vivo of M. conspicuum seems to be confirmed by the development of splenomegaly and granulomatous liver lesions not only in syngeneic gamma interferon-deficient mice but also in BALB/c mice following intravenous infection (36).
Although only two strains of M. conspicuum have been detected so far, there is the possibility of underisolation due to the low growth temperature required for the isolation of M. conspicuum.
Type strain: DSM: 44136T. EMBL 16S rDNA sequence accession number: X88922.
Phenotypic features. M. doricum is scotochromogenic and forms smooth yellow colonies in about 2 weeks at temperatures ranging from 25 to 37°C. Only urea and nitrates, among the most frequently used biochemical tests, are positive (Table 1).
The mycolic acid composition is not common among scotochromogens (Table 2), while the GLC pattern is similar to that of M. chlorophenolicum (Table 3). The HPLC profile is unique, characterized by an early major cluster of peaks followed by a minor one (153).
Antimicrobial susceptibility testing, performed in liquid medium, revealed good activity of first-line antituberculous drugs (Table 4).
Genotypic features. The nucleotide sequence of the 16S rRNA gene is characterized by a short helix 18 and a single cytosine insertion in helix 10; the combination of such features is considered the genetic signature of thermotolerant rapid growers, the group to which M. doricum presents the closest phylogenetic relatedness (Fig. 2 and 4). Among the slow growers not genetically related to M. simiae, M. doricum is at present the only species to present a short helix 18.
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Type strain: DSM 44339T, CIP 106867T. EMBL 16S rDNA sequence accession number: AF264700.
Phenotypic features. The growth of M. heckeshornense is scanty on Middlebrook agar medium and even poorer on egg medium. Growth is obtained after at least 4-weeks at 30 to 45°C. Many phenotypic features of M. heckeshornense are hardly distinguishable from those of M. xenopi: the colonies are small, scotochromogenic, and smooth, and the major biochemical tests are negative (Table 1).
The combination of mycolic acids by TLC is very common among mycobacteria (Table 2), while the fatty acid profile revealed by GLC are close to those of Mycobacterium botniense (Table 3), a recently described new species related to M. xenopi (141). The HPLC pattern (Fig. 5) is practically overlapping with those of M. xenopi and M. botniense. Therefore, there is a high risk of misidentification as M. xenopi.
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Genotypic features. M. xenopi is the species most closely related in the phylogenetic tree constructed on the basis of the 16S rDNA (Fig. 3). The relatedness is confirmed, although with higher sequence divergence, by comparison of the ITS regions. One strain resembling M. xenopi and containing two different copies of the 16S rDNA, corresponding to M. xenopi and M. heckeshornense respectively, has been reported (158).
Clinical and epidemiological features. The criteria required to demonstrate the pathogenicity of a nontuberculous mycobacterium (165) seem clearly fulfilled from in case that originated the sp. nov. description. Other isolations, although not documented in the literature, are inferred from the presence in the GenBank database of two identical 16S rDNA sequences. The first belongs to a strain from a patient with pulmonary disorders, and the second sequence came from a strain tentatively named "Mycobacterium sydneyiensis" (sic) (113).
Type strain: DSM 44428T. GenBank 16S rDNA sequence accession number: AF174290.
Phenotypic features. M. interjectum is a slowly growing (at 31 to 37°C) acid-fast coccobacillus that forms yellowish scotochromogenic colonies mostly resembling, in biochemical and cultural features, M. scrofulaceum (Table 1). A culture presenting both yellow and white colonies of M. interjectum at the same time has also been reported without any interconvertibility among the two chromatic variants detected (147).
The TLC pattern of mycolic acids is similar to those of MAC and M. scrofulaceum (Table 2). The pattern of fatty acids revealed by GLC is also close to the latter species (Table 3). Interestingly, two different HPLC profiles of mycolic acids may be obtained for M. interjectum; the first and most frequent grossly resembles those of MAC and M. scrofulaceum (143), while the other, which is unique, has a large cluster of peaks emerging during the second half of the elution (147). Available data concerning antimicrobial susceptibility show the species to be quite resistant to all antituberculous drugs (Table 4).
Genotypic features. The identity of the whole hypervariable region B of the 16S rDNA demonstrates the close genetic relatedness of M. interjectum to M. simiae and other "intermediate" species (Fig. 2 and 3).
Clinical and epidemiological features. Cervical lymphadenitis in childhood is the most frequent pathology attributable to M. interjectum. In addiction to the case which allowed the recognition of the species, four further similar cases have been reported (30, 84, 119). Two more cases of chronic lung disease (38, 84) and a repeated isolation from urine (143) should also be added. Only two isolations from AIDS patients have been reported (51, 147). The clinical significance of these isolations is, however, questionable. Well documented, although unpublished, is a case of pulmonary cavitary disease diagnosed following double bronchoscopic isolation of M. interjectum; in this case the patient was successfully treated for 5 months with clarithromycin.
No doubt exists about the potential pathogenicity of the species, as confirmed also by the investigation of virulence in vivo, carried out by experimental infection of BALB/c and syngeneic gamma interferon-deficient mice, which gave rise to splenomegaly and granulomatous liver lesions in both animals (36).
Type strain: DSM 44064T. EMBL 16S rDNA sequence accession number: X70961.
Phenotypic features. The development of recognizable colonies on egg-based media requires at least 2 weeks at 25 to 41°C. Although the species has been described as photochromogenic, its yellow pigmentation seems enhanced rather than induced by light exposure. The coccobacillary cell morphology and the complex of cultural and biochemical traits (positive arylsulfatase, urease, and Tween 80 hydrolysis [Table 1]) result in this species being hard to distinguish from M. asiaticum.
The mycolate investigation, carried out using TLC, reveals a pattern overlapping that of a large group of species including M. simiae, M. malmoense, and M. genavense (Table 2). The GLC profile of fatty acids, pyrolysis products, and alcohols is easily distinguishable from those of other slow growers (Table 3). The unique HPLC pattern of mycolic acid bromophenacyl esters is characterized by a single, large cluster of peaks emerging in the second part of elution (147).
The only strain detected so far is resistant to isoniazid and streptomycin (Table 4).
Genotypic features. The nucleotide sequence of hypervariable region B, in the 16S rDNA, overlapping those of M. simiae and related slow growers, is characterized by a short helix 18. This feature, emphasized also by the name (the Latin word "intermedius," likewise "interjectus" indicating another mycobacterial species, means "halfway"), is responsible for the phylogenetic position of the species, intermediate between rapid and slow growers, close to M. simiae (Fig. 2 and 3). M. intermedium is the most ancient species to diverge from the slow growers, typically characterized by a long helix 18.
Clinical and epidemiological features. No further isolation has been reported so far from patients or from the environment. The clinical significance of M. intermedium appears questionable. The repeated isolation and the presence of acid-fast bacilli in smears from sputum are not sufficient to demonstrate its pathogenic role in the only case reported so far. Furthermore, experimental infection with M. intermedium showed that it was pathogenic for gamma interferon-deficient mice but not for immunocompetent BALB/c mice (36).
Type strain: DSM 44049T. EMBL 16S rDNA sequence accession number: X67847.
Phenotypic features. The cells are strongly acid fast and are rod shaped and frequently bent. The colonies, which are yellow, smooth, and domed, are scotochromogenic and grow slowly between 28 and 37°C. Among the biochemical features, only semiquantitative catalase, clearly over 45 mm, stands out from other, mostly negative, tests (Table 1).
The TLC investigation of mycolic acids of M. kubicae reveals a common pattern among mycobacteria, including M. tuberculosis, while nothing is known about the GLC fatty acid composition. The well-documented HPLC pattern (41) is characterized by a single cluster of peaks grossly resembling the profile of M. asiaticum, hence the M. asiaticum-like appellation previously indicating such strains.
Genotypic features. Because of the sequence identity of the 16S rDNA hypervariable region B, M. kubicae clusters with the steadily increasing group of M. simiae-related slow growers (Fig. 2 and 3).
Clinical and epidemiological features. The only clinical information concerning 11 of 15 strains on which the description of M. kubicae is based is that the source was respiratory (sputum or bronchial aspirate). Thus no hypothesis may be made, at present, about its potential pathogenicity.
Type strains: ATCC 700732T, CIP 106428T. GenBank 16S rDNA sequence accession number: AF133902.
Phenotypic features. M. lentiflavum is a scotochromogenic slow grower (3 to 4 weeks is needed, on average, at 22 to 37°C) whose most common biochemical tests are negative (Table 1). The cells are acid fast and coccobacillary, while the colonies appear pale yellow, tiny and smooth.
The TLC pattern of mycolic acids reveals the presence of -, '-, and keto-mycolates, a pattern identical to those of M. simiae, M. malmoense, M. genavense, and M. intermedium (Table 2). The GLC profile does not present discriminative compounds (Table 3), while the HPLC chromatotype presents a three-clustered pattern very similar to the one of M. simiae; the two species are distinguishable but not easily (152).
The little available information about the susceptibility to antimycobacterial drugs suggests a high-level resistance to the majority of them (Table 4).
Genotypic features. The genetic sequence of M. lentiflavum presents, within the hypervariable region B of 16S rDNA, 100% sequence homology to M. simiae and related mycobacteria, with which it phylogenetically clusters (Fig. 2 and 3). Two sequevars differing in two nucleotides within the 5' end of 16S rDNA are present (http://www.ridom.de/static/download/dign_mycoba.pdf).
The PRA of hsp65 revealed, among the 22 isolates investigated at the time of the sp. nov. description, three different patterns, none of which overlapped with those of any previously evaluated mycobacterial species (130).
Clinical and epidemiological features. Although the majority of M. lentiflavum isolates appear to lack clinical significance (47), several pathologies related to this species have been reported. Among the strains involved in the sp. nov. description, the one isolated from a vertebral disk biopsy specimen (130) was most probably the agent of spondylodiscitis from which the patient suffered and whose symptoms, following treatment with multiple antituberculous agents, markedly improved. Other pathologies were reported later: four cases of cervical lymphadenitis (22, 57, 144), one case of cavitary pulmonary disease (144), one disseminated infection in a woman undergoing steroid therapy (67a), and, in AIDS patients, again one disseminated infection (98) and a hepatic abscess (144).
No environmental isolation has been reported so far; however, PCR products similar to those of M. lentiflavum have been obtained from soil (91), and six strains have been detected presenting hsp65 restriction profiles typical of M. lentiflavum, although differing results were obtained from 16S rDNA and ITS sequences (81).
Experimental investigation revealed a low-level virulence for M. lentiflavum, which was not able to cause persistent infection in BALB/c or gamma interferon-deficient mice (36).
Type strain: ATCC 51985. EMBL 16S rDNA sequence accession number: X80769.
Phenotypic features. M. palustre grows in 3 to 4 weeks at temperatures ranging from 22 to 42°C (optimum, 37°C) and forms smooth, yellow, scotochromogenic colonies. Tween 80 hydrolysis and urease are positive (Table 1).
No TLC mycolic acid investigation has been performed. The GLC fatty acid pattern is unique among mycobacteria (Table 2). The HPLC profile of M. palustre presents peaks arranged in a single cluster; such peaks, although corresponding to the ones of M. kubicae (41), differ from them in height (139).
Susceptibility testing, performed only on the isolate of human origin, revealed that all antituberculous drugs except isoniazid had low MICs, as did quinolones and macrolides (152).
Genotypic features. The sharing, within the 16S rDNA sequence of the hypervariable region B with M. simiae-related slowly growing mycobacteria determines the clustering of M. palustre with the latter group, close to M. kubicae (Fig. 2 and 3). All strains are characterized by a unique nucleotide sequence in the ITS region (139).
M. palustre strains yield different results when tested with two commercially available DNA probes. With AccuProbe, they hybridize with MAC-specific probe but not with the ones specific for M. avium and M. intracellulare (152). With INNO LiPA Mycobacteria (Innogenetics) they hybridize only with the genus Mycobacterium-specific probe, being negative with the others including those specific for the M. avium-intracellulare-scrofulaceum grouping and for the single species M. avium and M. intracellulare (150, 152).
Clinical and epidemiological features. The involvement in a case of childhood lymphadenopathy places M. palustre among potentially pathogenic species. There remains uncertainty concerning the significance of lymphatic infections in animals, given that M. palustre was grown from apparently healthy slaughtered pigs. Natural waters may well represent the environmental reservoir of the new species
Type strains: ATCC BAA-377T, DSM 44572T. EMBL 16S rDNA sequence accession number: AJ308603.
Phenotypic features. The cells of M. tusciae are rod shaped and acid fast; the growth is visible, on egg medium, in about 1 month at temperatures ranging from 25 to 32°C. Growth at 37°C is achieved only in liquid media or on Middlebrook agar. The colonies are rough and scotochromogenic and are characterized by a very elevated center surrounded by a flat and uneven fringe. All major biochemical tests are positive (Table 1).
The mycolic acid composition of the M. tusciae cell wall is one of the most common among slow-growers. It is shared, among others, by MAC, M. xenopi, and M. scrofulaceum (Table 2). The problems associated with distinguishing M. tusciae from M. scrofulaceum are present also in GLC analysis (Table 3), while the HPLC pattern, characterized by an early major cluster of peaks, differs from those of any species reported so far (149).
The species is characterized in vitro by moderate susceptibility to antituberculous drugs (Table 4).
Genotypic features. The 16S rDNA sequence has the distinctive features of thermotolerant rapid growers, i.e., a short helix 18 and a single cytosine insertion in helix 10. Nevertheless, in the phylogenetic tree, M. tusciae, although clustering with rapid growers, lies on a branch far from thermotolerant rapid growers and even farther from M. simiae-related organisms, with which it shares growth rate and a short helix 18 (Fig. 2 and 4).
Clinical and epidemiological features. Two strains of M. tusciae were isolated from potable water. Since the mouth is the usual route for agents of such childhood pathologies, the water may well represent a source for lymphonodal cervical infections. The isolation from a sterile site supports the clinical relevance of M. tusciae, especially considering that the slight immunosuppression of the child from whom it was grown (as a result of treatment with low-dose steroids) does not seem to have played a major role in this case.
Type strain: DSM 44338T. EMBL 16S rDNA sequence accession number: AF058299.
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Phenotypic features. The species is nonchromogenic, grows slowly, and forms smooth, often umbonate colonies after incubation at temperatures ranging from 25 to 45°C. Biochemical tests, except for arylsulfatase, are negative (Table 1).
The mycolic acid composition (TLC) is very common among slowly growing mycobacteria (MAC, M. scrofulaceum, etc., [Table 2]), while the profile of GLC fatty acids and the HPLC pattern are not distinguishable from those of M. celatum (Table 3) (17).
Like M. celatum, M. branderi is resistant to rifamycins and to isoniazid (Table 4).
Genotypic features. The 16S rRNA gene contains a long helix 18, made even longer by a one-nucleotide insertion. The most closely related species is M. celatum (Fig. 2 and 3).
Clinical and epidemiological features. The potential pathogenicity of M. branderi, hypothesized at the time of sp. nov. description, was confirmed by a subsequent report (174) describing the isolation of this species from the hand of a woman with ulcerative tenosynovitis. The isolation of M. branderi from bronchoalveolar lavage fluid of an elderly patient with pneumonic infiltrates, reported in the same paper, was, in contrast, considered not significant since a MAC isolate had also been grown from the sputum of the subject. Infection with M. branderi gives rise to splenomegaly and granulomatous liver lesions in both BALB/c and syngeneic gamma interferon-deficient mice, thus demonstrating virulence for immunocompetent animals as well (36).
Type strain: ATTC 51789T. EMBL 16S rDNA sequence accession number: X82234.
Phenotypic features. Although all the commonly investigated phenotypic tests (Table 1), including niacin accumulation, and the antimicrobial susceptibility pattern are compatible with M. tuberculosis (Table 4), its growth is much faster since as the colonies are clearly visible within 6 days. Serial subcultures of the strains reveal a consistent conversion of colonies from smooth to rough with a 1:500 rate and without any reversion from rough to smooth. The above colony conversion is accompanied by the loss of lipooligosaccharides.
No difference in the composition of cell wall mycolic acids (TLC) (Table 2) and fatty acids (GLC) (Table 3) exists between M. canettii and M. tuberculosis. The HPLC pattern too, unreported so far, is indistinguishable from that of M. tuberculosis (Fig. 6).
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Clinical and epidemiological features. An additional isolation has been reported in Switzerland from an AIDS patient with mesenteric tuberculosis who had worked for more than 20 years in Africa (102). Experimentally infected guinea pigs revealed tuberculous lesions and severe loss of body fat at autopsy. Such lesions were much more disseminated in animals inoculated with the smooth variant than in controls infected with M. tuberculosis H37Rv, demonstrating a greater virulence of the former. The shift from smooth to rough colonies is higher in vivo than in vitro, suggesting that the rough variant is better adapted to the in vivo conditions.
On the basis of the experimental data, the Canetti variant seems to be the most divergent member of the M. tuberculosis complex; nevertheless, it is still uncertain whether it represents a new species. van Soolingen et al. (159) suggest that the M. tuberculosis complex should be regarded as a single species, including several subspecies, and propose M. tuberculosis subsp. Canetti as name of the novel taxon.
The lack of other reports concerning isolates of M. canettii may well be due to misdiagnosis attributable to the extremely unusual colony morphology as well as its rarity. Animals are considered the most probable reservoir at present.
Type strain: CIPT 140010059T. EMBL 16S rDNA sequence accession numbers: AJ007315 (hypervariable region A), AJ007316 (hypervariable region B).
Phenotypic features. The most striking phenotypic feature of M. genavense is the extremely slow growth, obtained almost exclusively in liquid media. The large majority of strains were isolated with the radiometric Bactec 460TB instrumentation (Becton Dickinson); other liquid media were subsequently found suitable to support the growth. Scanty growth may be obtained after prolonged incubation on solid media such as Middlebrook 7H11 enriched with 2 µg of mycobactin J per ml or on Middlebrook 7H10 supplemented with 10% human blood (87).
The cell morphology is coccobacillary, and the colonies require 1 to 3 months to become visible. They are initially transparent but may become creamy and white with age. A wide range of temperatures, from 25 to 42°C, with preference for the higher ones, support growth. Because of the difficulty in obtaining enough biomass, very little is known about the phenotypic features of this species. Catalase (both semiquantitative and 68°C) and urease are positive, while niacin, nitrate, Tween 80 hydrolysis, and arylsulfatase are negative (Table 1).
Important features for the presumptive identification of M. genavense are the clear enhancement of growth achieved in acidified (pH 6.2) broth and the inhibition of growth, like M. tuberculosis (155) but unlike other nontuberculous mycobacteria, by p-nitro--acetylamino-ß-hydroxypropiophenone (131).
Knowledge concerning M. genavense lipid patterns is limited to HPLC analysis, which reveals a close similarity to M. simiae. The patterns of the two species are characterized by three almost overlapping late clusters of peaks (27).
Little is known about the antimicrobial susceptibility of M. genavense (Table 4). A pattern overlapping that of MAC has been hypothesized (101), but several clinical and microbiological data seem to suggest a lower resistance level, with only isoniazid and ethambutol being clearly inactive (9, 155).
Genotypic features. From the molecular point of view, M. genavense is characterized, within the 16S rDNA, by a sequence in hypervariable region B identical to the one of M. simiae and related mycobacteria (Fig. 2 and 3).
Clinical and epidemiological features. In the years preceding the introduction of highly effective antiretroviral treatments, M. genavense was the second most frequently isolated species, after M. avium, responsible for disseminated infections in AIDS patients. Most of these patients present with fever, diarrhea, and weight loss (101). M. genavense was isolated mainly from blood but also from various biopsy specimens (lymph node, spleen, and duodenal mucosa). Cases published before 1998 have been reviewed by Tortoli et al. (146). More recently, 20 additional isolations from AIDS patients have been reported, including cutaneous (S. Fournier and V. Vincent, Letter, Ann. Intern. Med. 128:409, 1998) and genital (160) infections.
Only four M. genavense infections have been reported in HIV-seronegative patients. Two were disseminated, in an immunosuppressed woman (6) and in a leukemic patient (79), while the other two were represented by lymphadenopathy in a child (82) and a soft tissue infection in an immunodeficient, HIV-negative patient (80a). In two cases, M. genavense DNA was detected using PCR in asymptomatic subjects (33). Noteworthy is the finding of M. genavense in hospital tap water; this is a possible link to the disseminated infections in HIV-infected patients (64).
M. genavense is the most common mycobacterial infection in birds (106), which to date are the only identified potential reservoir. Les frequent are infections in other animals, including a disseminated infection in an immunodeficient cat (67).
The many examples of clinical documentation, unusual for nontuberculous mycobacteria, show clearly that M. genavense is pathogenic, at least for HIV-infected persons and for birds. It may be responsible for localized or disseminated lesions, and death is not an exceptional outcome.
A parallel intravenous injection of M. genavense into immunocompetent and gamma interferon-deficient mice produced persistent infection of the liver and spleen only in the latter animals, suggesting that the virulence of the species is not sufficiently high for it to survive and spread in the presence of gamma interferon-activated macrophages (35). However, its persistence in gamma interferon-deficient mice closely resembles the disseminated infection of M. genavense in immunodeficient patients.
Type strain: ATCC 51234T. EMBL 16S rDNA sequence accession number: X60070.
Phenotypic features. Growth on egg medium is problematic. Colonies are small, nonpigmented, and smooth. Growth occurs at 30 to 37°C, not before the week 4 of incubation; it may, however, be observed earlier on agar and liquid Middlebrook media. Tween 80 hydrolysis and urease are positive (Table 1).
The mycolic acid pattern is identical to those of M. simiae, M. malmoense, and M. intermedium (Table 2). The GLC (Table 3) and HPLC patterns practically overlap with those of M. malmoense (55).
Unusually for a nontuberculous mycobacterium, the type strain of M. heidelbergense is susceptible to isoniazid in addition to other major antituberculous drugs (Table 4).
Genotypic features. The similarity, at the phenotypic level, to M. malmoense is not confirmed when the 16S rDNA is observed. M. heidelbergense presents complete homology of hypervariable region B to M. simiae and consequently a close phylogenetic relatedness to the latter species (Fig. 2 and 3). The PRA of hsp65 also is suitable to clearly differentiate M. heidelbergense from M. malmoense.
Clinical and epidemiological features. Like other strains isolated from lymph nodes, which usually require surgical, seldom repeated, intervention, M. heidelbergense must be considered pathogenic, and confirmation of its clinical significance comes from an isolate from a patient with a tumor-mimicking pulmonary infection (103). In contrast to the strain from which the species was described, the susceptibility pattern of the latter was characterized by resistance to all drugs tested but ciprofloxacin, a discrepancy that cannot be attributed to the different testing techniques which were used. Six other independent M. heidelbergense isolations, probably not clinically significant, have been reported: four from sputum, one from gastric fluid, and one from urine (55).
Intravenous inoculation of M. heidelbergense into immunocompetent (BALB/c) and gamma interferon-deficient mice gave rise to splenomegaly and granulomatous liver lesions only in the latter, demonstrating that gamma interferon is decisive for the outcome of infections with this mycobacterium (36).
Type strain: ATCC 51253T. EMBL 16S rDNA sequence accession number: X70960.
Phenotypic features. The strain, which is microscopically characterized by large bacilli with prominent beading, grows in 2 to 3 weeks at 25 to 42°C. The colonies are small, nonpigmented and dry. Among classical biochemical tests, only nitrate reduction and urease are positive (Table 1).
The lipidic pattern, which was not investigated by TLC and GLC, is characterized in HPLC by a single cluster of peaks practically overlapping that of M. gastri (156).
M. lacus is susceptible, in addition to antitubercular drugs, to various molecules including aminoglycosides, quinolones, and macrolides (Table 4).
Genotypic features. The sequences of the 16S rDNA and the ITS region are unique (Fig. 2). M. malmoense and M. marinum are the most closely related species (Fig. 3). The pattern obtained by PRA of hsp65 is distinct from every one reported previously.
Clinical and epidemiological features. The isolation from a tissue presenting caseating granulomas unquestionably supports the pathogenic role of the organism. Since the infection was probably contracted during of a trauma while the patient was swimming in a lake, the environment may well represent the natural reservoir of M. lacus.
Type strains: ATCC BAA-323T, DSM 44577T. GenBank 16S rDNA and ITS sequence accession number: AF406783.
Phenotypic features. Acid-fast coccobacilli form dysgonic, flat, rough, unpigmented colonies after 4 to 6 weeks of incubation at 23°C, while no growth is obtained at temperatures over 30°C; less frequently, smooth colonies develop. Almost all the most frequently investigated biochemical traits are negative, apart from urease and niacin accumulation (Table 1). The latter is an extremely rare feature among nontuberculous mycobacteria, except for M. simiae, in which the production of niacin may be present, although only inconsistently.
HPLC analysis, the only lipid investigation performed so far, revealed a single cluster of peaks, grossly resembling the one characterizing the M. tuberculosis complex but easily distinguishable from it by the clearly lower retention time of each peak (111).
M. shottsii is susceptible to major antituberculous drugs, with isoniazid being the only exception (Table 4).
Genotypic features. The 16S rDNA presents the structure typical of slow growers (Fig. 2 and 3).
Clinical and epidemiological features. The presence, in all the infected fish, of granulomatous lesions clearly demonstrates the pathogenicity of the species for striped bass; nothing is known about the potential pathogenicity for other fish and humans. Seawater seems a plausible candidate as the natural reservoir.
Type strains: ATCC 700981T, NCTC 13215T. GenBank 16S rDNA sequence accession number: AY005147.
Phenotypic features. The species, whose cells are short and acid fast, grows slowly, forming smooth unpigmented colonies after 2 or 3 weeks at 37°C. All major biochemical tests except Tween 80 hydrolysis are positive (Table 1).
