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J Clin Microbiol. Sep 2009; 47(9): 2691–2698.
Published online Jul 1, 2009. doi:  10.1128/JCM.00808-09
PMCID: PMC2738059

Characterization of Mycobacteria from a Major Brazilian Outbreak Suggests that Revision of the Taxonomic Status of Members of the Mycobacterium chelonae-M. abscessus Group Is Needed [down-pointing small open triangle]

Abstract

An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both the Mycobacterium massiliense and the M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the M. chelonae-M. abscessus group was carried out. The M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of hsp65 (PRA-hsp65), rpoB, and hsp65 gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains from M. chelonae and M. immunogenum was supported by the results of PRA-hsp65, DDH, and RFLP-16S rRNA and by the rpoB and hsp65 phylogenetic trees. Taken together, these results led to the proposition that M. abscessus, M. massiliense, and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and these two subspecies can be distinguished by two different PRA-hsp65 patterns, which differ by a single HaeIII band, and by differences in their rpoB (3.4%) and hsp65 (1.3%) sequences.

Since 2004, a series of localized skin and soft tissue infections caused by rapidly growing mycobacteria have occurred in Brazil in patients who have undergone invasive procedures, such as laparoscopic, arthroscopic, plastic surgery, or cosmetic interventions (12, 16, 23, 37). In 4 years, more than 2,000 cases were officially reported to Brazilian federal authorities, who consider this problem an epidemiological emergency (5). Almost all isolates studied so far have belonged to the Mycobacterium chelonae-M. abscessus group (39). The majority of them were identified as members of two recently described emerging pathogens, Mycobacterium massiliense (3) and Mycobacterium bolletii (1), both of which belong to the Mycobacterium chelonae-M. abscessus group.

All five members of the Mycobacterium chelonae-M. abscessus group, M. chelonae, M. abscessus (21), Mycobacterium immunogenum (40), M. massiliense (3), and M. bolletii (1), are nearly indistinguishable phenotypically. Common features include growth in less than 7 days, the absence of pigmentation, better growth at 30°C than at 35°C, a positive 3-day arylsulfatase test result, a negative nitrate reductase test result, and a negative iron uptake test result (41). Two biochemical tests, sodium chloride tolerance and the utilization of citrate, are useful in distinguishing the five members (1, 3, 40, 41).

Antimicrobial susceptibility can also be used to differentiate the members of the M. chelonae-M. abscessus group. M. abscessus is generally susceptible to cefoxitin (MIC < 16 μg/ml) and M. chelonae is resistant (MIC > 128 μg/ml). Otherwise, M. abscessus is resistant to tobramycin (MIC > 16 μg/ml) and M. chelonae is susceptible (MIC < 4 μg/ml) (9). M. immunogenum is resistant to both drugs (40). M. massiliense was initially reported to be susceptible to doxycycline (3), but clinical isolates with intermediate susceptibility and also resistance to this drug have also been described (30, 37). M. bolletii was described to be a highly resistant species, and clarithromycin was among the drugs to which it was resistant (1). However, the results displayed by these tests can vary between strains, as has been shown for M. massiliense (3, 30). Lipid analysis by high-performance liquid chromatography (HPLC) produces very similar mycolic acid patterns for M. chelonae, M. abscessus, and M. immunogenum and thus has limited discriminatory power (10). The HPLC profiles of M. massiliense and M. bolletii have not yet been reported.

Genotypic characterization of the group has shown that the 16S rRNA sequences of M. chelonae and M. abscessus differ by only 4 bp, while they have identical hypervariable region A sequences (9). Both M. massiliense and M. bolletii show 100% 16S rRNA sequence similarity to the 16S rRNA sequence of M. abscessus (1, 3). Finally, the M. immunogenum 16S rRNA sequence differs from the corresponding sequences of M. abscessus and M. chelonae by 8 bp and 10 bp, respectively.

PCR-restriction enzyme analysis (PRA) of a 441-bp fragment of the 65-kDa heat shock protein-encoding gene, hsp65 (PRA-hsp65), has thus far yielded five different patterns within the members of the M. chelonae-M. abscessus group: M. chelonae has a single pattern (pattern 1), while M. abscessus and M. immunogenum have two patterns each (patterns 1 and 2) (13, 29, 40). M. massiliense and M. bolletii have identical PRA-hsp65 patterns, and these in turn, are identical to pattern 2 of M. abscessus (1, 3). The M. abscessus PRA-hsp65 pattern 2 was also found in 152 isolates from the Brazilian outbreak mentioned above (data not shown).

The sequence of the rpoB gene has been demonstrated to be a powerful tool for the delineation of bacterial species (4) and was applied to the identification of rapidly growing mycobacteria, including members of the M. chelonae-M. abscessus group (2). The degree of interspecies variation found in the rpoB sequence is variable. Nevertheless, a 3% cutoff value can generally be applied to the mycobacteria, but some exceptions to that rule have been described. Analysis of interspecies variations in the rpoB gene sequence showed that the members of the Mycobacterium avium complex have a range of 4.5 to 5.7% sequence divergence (7) compared to the rpoB sequence of the closely related species M. marinum and M. ulcerans, whose rpoB sequences showed 0.4% divergence from each other (4). On the other hand, M. mucogenicum and M. abscessus have shown wider internal heterogeneity in the rpoB gene sequence, with the latter having up to 4.3% internal species variation (2).

The papers describing M. massiliense and M. bolletii have indicated that a specific sequence of the rpoB gene is a main characteristic in these species (1, 3). Consequently, sequence analysis of the rpoB gene as well as of the hsp65 gene, which we used in a previous study, led to the identification of several isolates from surgical patients as M. massiliense and other isolates from mesotherapy patients as M. bolletii because of the highest degrees of similarity of their rpoB and hsp65 gene sequences (37). Several unclear findings, however, emerged from that analysis. High degrees of similarity of the rpoB sequences of surgical isolates to the rpoB sequences of both M. massiliense (99.72%) and M. bolletii (98.45%) were detected. Moreover, the rpoB sequences of isolates from patients undergoing mesotherapy showed 100% similarity to the rpoB sequence of M. bolletii and 98.54% similarity to that of M. massiliense. According to the 3% cutoff that has been proposed (2), all isolates could therefore be identified either as M. massiliense or as M. bolletii on the basis of their rpoB gene sequences.

Similar results were observed with the hsp65 sequences. The hsp65 sequences of surgical isolates showed 100% and 99.25% similarities to the hsp65 sequences of M. massiliense and M. bolletii, respectively. Again, the hsp65 sequences of isolates from patients who had undergone mesotherapy displayed 100% similarity to the hsp65 sequence of M. bolletii and 99.24% similarity to the hsp65 sequence of M. massiliense. For hsp65, the value of 97% similarity is suggested by McNabb et al. (25) as the limit for the separation of species.

These inconclusive results prompted us to undertake an extensive phenotypic and genotypic characterization of the isolates from the Brazilian outbreaks. We applied a wide set of procedures, including DNA-DNA hybridization (DDH), which is so far considered the “gold standard” for the delineation of bacterial species (4, 31). Selected isolates from the outbreaks were compared to each other and also to reference strains representative of the five members of the M. chelonae-M. abscessus group. We showed that the DNA-DNA relatedness clearly identifies M. massiliense and M. bolletii, together with our clinical isolates, as M. abscessus, indicating that all of these isolates make up a single genomic species. Finally, taking into account the internal variability detected in the sequence of M. abscessus, we propose the description of two subspecies within this species.

MATERIALS AND METHODS

Mycobacterial strains.

Six clinical isolates and five reference strains were included in this study. Three isolates from the state of Para in Brazil were previously identified as M. massiliense (two were from the laparoscopic surgery outbreak [referred to as isolates B5 and B31 in this report] and one was from an abscess that formed after an intramuscular injection [referred to as isolate B67 in this report]), and three isolates from the state of Para were previously identified as M. bolletii and were from patients who had undergone mesotherapy (37). The latter three isolates are referred to as B60, B61, and B66 in this report. The type strains of the five species M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii were included as reference strains. The M. fortuitum type strain (ATCC 6841) was also used as an outgroup and a control in some experiments. Bacteria were cultivated in Mueller-Hinton liquid or solid medium; Middlebrook 7H9 broth supplemented with albumin, dextrose, and catalase; or Middlebrook 7H10 solid medium supplemented with oleic acid, albumin, dextrose, and catalase. Frozen stocks were prepared.

Phenotypic identification. (i) Biochemical tests.

The isolates were evaluated for a panel of biochemical and cultural features by standard procedures (18). The tests included analysis of the growth rate; growth at 25°C and 45°C; pigment production; colony morphology; growth on MacConkey agar; biochemical tests (nitrate reduction; Tween hydrolysis; tellurite reduction; and niacin, catalase, beta-glucosidase, arylsulfatase, and urease tests); and tolerance to thiophene-carboxylic acid, tiacetazone, p-nitrobenzoic acid, isoniazid, hydroxylamine, oleate, NaCl, and sodium citrate.

(ii) HPLC.

HPLC of cell wall mycolic acids, which may be a valid aid in the identification of mycobacterial species, was carried out as reported before (12).

(iii) Susceptibility testing.

MICs were determined by the microdilution method, according to the recommendations of the CLSI (formerly NCCLS) (27), with commercially available microplates (Sensititer RGMYCO; Trek Diagnostic Systems Inc., Cleveland, OH).

Genotypic identification. (i) DNA isolation.

Total genomic DNA was purified from liquid bacterial cultures as described previously (36).

(ii) PRA-hsp65.

The band patterns produced by PRA-hsp65 were obtained as described by Telenti et al. (33) and were compared to those in the PRASITE database (http://app.chuv.ch/prasite/index.html).

(iii) DNA sequencing and phylogenetic analysis.

The hsp65 and rpoB gene sequences were obtained and analyzed as described by Viana-Niero et al. (37). The corresponding sequences from the type strains included in this study were also retrieved from the GenBank database for comparative purposes. The alignment of the rpoB and hsp65 gene sequences was performed with ClustalX (version 2.0) software (22). Two separate phylogenetic trees were constructed for both genes by using M. tuberculosis strain H37Rv (ATCC 27294) as the outgroup. The evolutionary history was generated by the neighbor-joining method. The evolutionary distances were computed by use of the Kimura two-parameter model and bootstrap analysis with 1,000 replications. Phylogenetic analysis was performed with MEGA (version 4) software (32).

DNA-DNA similarity.

The total genomic relationships of DNAs were investigated for the six clinical isolates and the corresponding type strains of M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii. M. fortuitumT DNA was also analyzed so that DNA from a non-closely related fast-growing mycobacterium was included. DDH experiments were performed on membrane filters by a dot blot-based procedure, as described previously (17). Genomic DNAs from M. massiliense CIP 108297T and M. bolletii CIP 108541T were radioactively labeled in vitro and were used as probes to hybridize with 500 ng of each unlabeled DNA. Unlabeled DNAs were dot blotted and bound to nylon membranes (Hybond-N+; Amersham). The relative binding ratios (expressed as percentages) were calculated from the counts of bound homologous DNA, as measured with a Typhon Trio apparatus (GE Healthcare). The amount of DNA fixed in each dot was calculated by measuring the amount of radioactivity when the 16S rRNA gene was used as a second probe (17).

RFLP of 16S rRNA gene.

RFLP analysis of the 16S rRNA gene was also performed. This procedure allowed the identification of the number of rrn operons per genome carried by the mycobacteria (15). A previously described experimental procedure (14) was used, with the exception that ECL buffer (Amersham) was used for both the prehybridization and the hybridization steps.

RESULTS

Phenotypic identification.

The biochemical and culture tests turned out to be poorly discriminative (Table (Table1).1). Most of the clinical isolates presented almost overlapping results with the closely related rapid growers (M. abscessus, M. chelonae, M. immunogenum). The few discrepancies within the strains belonging to the species M. massiliense and M. bolletii may well be imputable to biological variability. The results of HPLC were also poorly informative. All the species investigated here were, in fact, characterized by a single pattern, with only minor differences being detected among the species (Fig. (Fig.11).

FIG. 1.
Comparison of representative HPLC mycolic acid patterns of members of the M. chelonae-M. abscessus group. HMWIS, high-molecular-weight internal standard.
TABLE 1.
Comparison of phenotypic and biochemical characteristics of isolates and strains

Although the majority of the test results confirmed the susceptibility patterns available in the literature for the reference strains (3, 9, 30, 40), several clear differences emerged (Table (Table2).2). The reference strain of M. massiliense, as well as two of three M. massiliense clinical strains (strains B67 and B31), turned out to be susceptible to amikacin (MIC range, 8 to 16 μg/ml for all except for one strain, strain B5; Table Table1)1) while in the species nova description, a MIC of 48 μg/ml has been reported (3). In contrast, there was a discrepancy in the MICs of the tetracyclines, with all of our strains, including the M. massiliense reference strain, which has been reported to have a MIC of 4 μg/ml (3), being resistant (MIC > 32 μg/ml). The results obtained with the commercial microplate, which included minocycline, were, in fact, confirmed by the doxycycline test, which was performed by the same technique with the prepared antibiotic. Even more striking were the discrepancies concerning M. bolletii, whose species nova description (1) emphasized its resistance to clarithromycin (MIC > 256 μg/ml). In contrast, all of our strains, including the M. bolletii reference strain, were susceptible (MIC range, <0.12 to 1 μg/ml).

TABLE 2.
Antimicrobial susceptibility results for isolates and strains included in this study

Genotypic identification.

Initially, the two methods that are the more frequently applied to the identification of mycobacterial species were used, i.e., PRA-hsp65 gene and DNA sequencing of the two selected genome targets, namely, the rpoB and hsp65 genes.

On the basis of the previously defined PRA-hsp65 patterns (13, 37), the PRA-hsp65 type 2 pattern of M. abscessus was shared by all six of our clinical isolates and by the M. massiliense and M. bolletii type strains (data not shown). The other type strains showed the expected patterns, according to the information in the PRASITE database (http://app.chuv.ch/prasite/index.html).

rpoB and hsp65 sequencing.

The sequences of region V of the rpoB gene were compared. The rpoB gene sequences of the Brazilian isolates were found to be almost identical to those of the M. massilienseT and M. bolletiiT strains from GenBank (greater than 98.5%) and were found to be closely related to the rpoB sequence of M. abscessusT (96.5% and 95.6%, respectively).

The rpoB sequences were translated into amino acid sequences for further comparison. Most of the nucleotide polymorphisms detected represented silent substitutions, with no amino acid change in the corresponding protein sequences being detected. The exceptions were the E/D, A/Q, A/E, and T/L substitutions in the M. abscessusT rpoB sequence and the V/I and N/D substitutions in the M. chelonaeT rpoB sequence (data not shown). In both cases, substitutions appeared in a nonconserved region from the RNA polymerase RPB10 interaction site, according to the Conserved Domain Database at NCBI (24). Comparisons of the sequences from the hsp65 gene indicated results similar to those corresponding to rpoB gene sequences, with even higher percentages of similarities being detected. The hsp65 sequence (401 bp) from the M. abscessus type strain showed 98.5 and 98.7% similarities to the corresponding sequences from M. massiliense and M. bolletii, respectively. A higher similarity index (99.2%) between the corresponding sequences from M. massiliense and M. bolletii was observed. Again, the hsp65 gene sequences of the Brazilian isolates were found to be almost identical to the M. massilienseT and M. bolletiiT hsp65 sequences (greater than 99%) and closely related to the M. abscessusT hsp65 sequence (98% similarity). All nucleotide polymorphisms detected represented silent substitutions.

The close relationship detected by comparison of the nucleotide sequences of both the rpoB and hsp65 genes is shown in the phylogenetic trees (Fig. 2a and b, respectively).

FIG. 2.
Phylogenetic tree based on rpoB (a) and hsp65 (b) gene sequences, showing the relationships between Brazilian clinical isolates (isolates B5, B31, B60, B61, B66, and B67) and other type strains of selected rapidly growing mycobacteria. GenBank accession ...

DDH.

Due to the high degree of similarity shown by our selected isolates of M. massiliense, M. bolletii, and M. abscessus, total DNA-DNA similarities were determined, because DDH is considered the reference method for the delimitation of bacterial species (4, 31). The results are summarized in Table Table3.3. The levels of DDH obtained by using M. massilienseT and M. bolletiiT as probes with DNA from M. abscessusT and all six clinical isolates were greater than 70%, indicating that they all belong to a single species. On the contrary, the level of hybridization with DNA from other species, including M. chelonaeT and M. immunogenumT, was less than 44%, showing a clear differentiation at the genomic level between these closely related species.

TABLE 3.
DNA-DNA genomic pairing between M. massilienseT and M. bolletiiT and other species, including Brazilian clinical isolates (B67, B31, B5, B60, B61, and B66)

RFLP of 16S rRNA gene.

RFLP analysis with 16S rRNA as the probe was used to characterize the mycobacteria. According to the patterns obtained, all strains except M. fortuitum and M. immunogenum have a single rrn operon per genome (Fig. (Fig.3).3). The patterns for M. fortuitum and M. immunogenum are in agreement with the corresponding data published previously (15, 40).

FIG. 3.
RFLP-16S rRNA patterns of mycobacterial DNAs digested with the restriction enzymes BamHI (a) and PstI (b). Lanes 1 to 11, isolates 1 to 11, respectively, as described in footnote a of Table Table1;1; lane 12, M. fortuitum ATCC 6841.

BamHI-digested DNAs showed a single band pattern for all strains except M. fortuitum and M. immunogenum. Compared to the bands from M. massiliense, M. bolletii, and M. abscessus, the single band was slightly smaller for isolates B67, B31, B60, and B66 and in these isolates was in a position similar to that of the smaller single band shown by M. chelonae (Fig. (Fig.3a).3a). These close patterns were distinguished by using PstI as second restriction enzyme.

The RFLP patterns of PstI-digested DNAs showed two bands that were in identical positions for all six Brazilian isolates, as well as M. massiliense, M. bolletii, and M. abscessus (Fig. (Fig.3b).3b). On the contrary, PstI-digested DNA from M. chelonae showed a different two-band pattern. These results showed that the Brazilian isolates, together with M. massiliense and M. bolletii, share RFLP-16S rRNA patterns with M. abscessus. It has been shown that the RFLP-16S rRNA patterns are species specific within rapidly growing mycobacteria (8, 14, 17, 26), and therefore, they should all be considered the species M. abscessus, which is clearly differentiated from M. chelonae and M. immunogenum according to their RFLP-16S rRNA patterns.

DISCUSSION

The increasing availability of gene sequences has had a tremendous influence on the taxonomy of bacteria, particularly that of the mycobacteria, with great increases in newly described species occurring every year (34). Some of the newly described species are considered emerging pathogens due to their relationships to clinical outbreaks (20, 29, 37). Besides the 16S rRNA gene, another conserved gene, rpoB, has widely contributed to the characterization of the species in the genus Mycobacterium (4). The degree of intraspecific rpoB sequence similarity has been estimated to range from 98.2 to 100% (4), with a few exceptions, such as the >4.3% intraspecies sequence divergence in the species M. abscessus (2).

Members of the M. chelonae-M. abscessus group are ubiquitous environmental organisms frequently associated with nosocomial outbreaks and pseudo-outbreaks (6, 20, 28, 29, 37, 38). Five related species with different associations with infection have been described within that group, making the proper identification of members of this complex important for both therapeutic management and epidemiological studies. The two species most recently incorporated into the group are M. massiliense and M. bolletii. Remarkably, the separation of these species was established mainly on the basis of their different rpoB gene sequences (1, 3). In the original description of these species, important differences in antimicrobial susceptibilities were also reported and were considered to support their separation.

In the recent outbreak of infections related to surgical and cosmetic procedures occurring in Brazil, the isolates were initially identified as M. massiliense (isolates from surgical cases) and M. bolletii (isolates from mesotherapy cases) (37). However, such identifications appeared to be inconclusive, with the isolates showing several characteristics that would allow them to be assigned to either one of the two species. In order to clarify their identities, a subset of six clinical isolates was extensively analyzed and compared to type strains not only of M. massiliense and M. bolletii but also of all other members of the M. chelonae-M. abscessus group. The analysis undertaken included both phenotypic and genotypic characterization.

M. massilienseT, M. bolletiiT, M. abscessusT, and the Brazilian clinical isolates could not be clearly distinguished on the basis of their phenotypic characteristics. Biochemical tests produced almost indistinguishable patterns (Table (Table1).1). HPLC analysis, which is well known to be unable to differentiate M. abscessus, M. chelonae, and M. immunogenum (10, 40), turned out to be of no use with M. bolletii and M. massiliense (Fig. (Fig.1),1), whose profiles had not been investigated before. Even more confusing were the antimicrobial susceptibility data, especially with regard to clarithromycin. In the species nova description by Adekambi et al. (1), who used a nonapproved CLSI method, a MIC of >256 μg/ml was reported for M. bolletii, while the same type strain, as well as all other clinical isolates of the same species, were fully susceptible on the basis of our findings. In the only other study (19) in which the susceptibility of M. bolletii has been investigated so far, again, resistance to clarithromycin was detected, although the MIC range (8 to 16 μg/ml) was very different from our MIC range and that of Adekambi et al. (1).

M. abscessusT could be separated from M. massilienseT and M. bolletiiT by use of the PRA-hsp65 pattern. The first species showed the type 1 pattern of M. abscessus, while the latter two shared the type 2 pattern of M. abscessus (1, 3, 37), with the difference between the two patterns being limited to a single HaeIII band. All the Brazilian clinical isolates under study also had the type 2 pattern of M. abscessus.

Genetic sequencing of the rpoB region, as proposed by Adekambi et al. (2) for the separation of rapidly growing mycobacteria, confirmed the high degree of heterogeneity within clinical isolates of M. abscessus, which was also reported by the same authors. Our results showed that the similarities in the rpoB gene sequences place M. abscessus, M. bolletii, and M. massiliense with the Brazilian isolates on the same branch, in which all their degrees of identity are within the range of M. abscessus intraspecies variability (Fig. (Fig.2a).2a). These results are consistent with the similarities found in a different conserved DNA target, the hsp65 gene (Fig. (Fig.2b),2b), whose use has also been proposed to be effective for the identification of mycobacteria (25).

The global DNA-DNA pairing values still remain the gold standard for the delineation of bacterial species (4, 9, 31, 42). Therefore, with the aim of clarifying the differences found, experiments were performed to analyze the total genomic relationships of the DNAs from the Brazilian clinical isolates and the type strains of the five species belonging to the M. chelonae-M. abscessus group. In the experimental design, the DNAs from M. massiliense and M. bolletii were used as probes. The DDH results revealed the clear species separation of M. chelonae and M. immunogenum from M. massiliense and M. bolletii, with the DDH values being less than 44% (Table (Table3).3). On the contrary, M. massiliense and M. bolletii could not be differentiated from each other or from M. abscessus, as the total DDH values were greater than 70% (Table (Table3).3). High hybridization values were also observed when the total DNAs of M. massiliense and M. bolletii were compared to the DNAs from the Brazilian clinical isolates (Table (Table3).3). According to these results, the two recently described species as well as the Brazilian clinical isolates appear to be members of the species M. abscessus.

M. abscessus as well as M. chelonae, unlike the large majority of rapidly growing mycobacteria, carry a single rrn operon per genome (15). RFLP analysis of the 16S rRNA genes also revealed in M. massiliense, M. bolletii, and the subset of Brazilian clinical isolates (Fig. (Fig.3)3) the presence of a single rrn operon per genome, similar to the findings for M. abscessus. Moreover, their RFLP patterns, which have been characterized as being species specific within rapidly growing mycobacteria (8, 15, 17, 26), were identical to the RFLP pattern of M. abscessus and different from the patterns of the other members of the M. chelonae-M. abscessus group (Fig. (Fig.33).

All results presented above indicate that the Brazilian M. abscessus, M. bolletii, and M. massiliense isolates belong to a single species. Since their description, “M. massiliense” and “M. bolletii” have been recognized to cause infectious diseases worldwide (11, 19, 20, 30, 35, 37). According to our results, from a public health perspective, the species responsible for all those cases should actually be assigned to the species M. abscessus.

The internal variability emerging from our findings and other previous data (2, 6, 28, 38) for the species M. abscessus suggests a new arrangement for the M. chelonae-M. abscessus group, in which only three species, M. chelonae, M. immunogenum, and M. abscessus, would be present, with M. abscessus being split into two subspecies, namely, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. For the latter subspecies, including the isolates previously identified as “M. massiliense” and “M. bolletii,” the name “massiliense” was chosen as it appeared first in the literature.

The two subspecies of M. abscessus can be distinguished genotypically by their PRA pattern type (the M. abscessus type 1 pattern for M. abscessus subsp. abscessus and the M. abscessus type 2 pattern for M. abscessus subsp. massiliense) and by differences in the rpoB and hsp65 sequences. Thus, the 711-bp rpoB sequence and the 401-bp hsp65 sequence from M. abscessus subsp. abscessus show similarities of less than 96.6% and 98.7%, respectively, with the sequences of M. abscessus subsp. massiliense (which includes the species formerly named “M. bolletii”).

Acknowledgments

This study received financial support from the Foundation for Research Support of the state of Sao Paulo (FAPESP, grant 06/1533-9) and from the European Commission (grant HEALTH-F3-2008-200999).

A. Leyva provided English editing of the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 July 2009.

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