JOURNAL OF CLINICAL MICROBIOLOGY, July 2009, p. 1985–1995 Vol. 47, No. 70095-1137/09/$08.00�0 doi:10.1128/JCM.01688-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cohort Study of Molecular Identification and Typing ofMycobacterium abscessus, Mycobacterium massiliense,

and Mycobacterium bolletii�

Adrian M. Zelazny,1,2* Jeremy M. Root,2 Yvonne R. Shea,1 Rhonda E. Colombo,2 Isdore C. Shamputa,3Frida Stock,1 Sean Conlan,4 Steven McNulty,5 Barbara A. Brown-Elliott,5 Richard J. Wallace, Jr.,5

Kenneth N. Olivier,2 Steven M. Holland,2 and Elizabeth P. Sampaio2,6

Microbiology Service, Department of Laboratory Medicine, Clinical Center,1 Immunopathogenesis Section,2 and Tuberculosis Research Section,3

Laboratory of Clinical Infectious Diseases, NIAID, and National Human Genome Research Institute,4 National Institutes ofHealth, Bethesda, Maryland; Department of Microbiology, The University of Texas Health Science Center, Tyler,

Texas5; and Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil6

Received 30 August 2008/Returned for modification 30 January 2009/Accepted 26 April 2009

Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease.Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Healthcare-associated outbreaks have recently been investigated by the use of molecular identification and typingtools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M.bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult andrelies on the sequencing of one or more housekeeping genes. We performed extensive molecular identificationand typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at theNIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB,hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted furthersequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonalgroups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB,hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliensetype strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, amultilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus andclosely related species. Molecular typing complements sequence-based identification and provides informationon prevalent clones with possible relevant clinical aspects.

Rapidly growing mycobacteria (RGM) are ubiquitous or-ganisms increasingly emerging as important human pathogens.Mycobacterium abscessus is commonly associated with woundinfections and abscess formation and is the most frequentRGM causing chronic lung disease, often in immunocompro-mised patients (15, 22, 24). M. abscessus is also notable for itsresistance to treatment and the poor clinical outcome of infec-tion with the organism (22, 24). Within the past decade, twonew species of mycobacteria closely related to M. abscessus, M.massiliense and M. bolletii, have been described (1, 3). Infor-mation on the pathogenic role of M. massiliense and M. bolletiiis still scant. Recent reports have described the isolation of M.massiliense from two patients in the United States (29) and onepatient in Italy (35) and, lately, the identification of M. mas-siliense and M. bolletii among South Korean isolates (18). BothM. massiliense and M. bolletii have also been linked to healthcare-associated outbreaks (8, 19, 37).

The species-level identification of RGM can provide the

first indication of antibiotic susceptibility and can suggestthe appropriate type of patient management. For example,M. abscessus is more resistant to many antibiotics both in vivoand in vitro than M. fortuitum and M. mucogenicum, but it isusually susceptible to amikacin and clarithromycin (6, 15, 24).M. massiliense was originally reported to be distinguishablefrom M. abscessus and related species by its susceptibility todoxycycline (3); however, resistant isolates have since been de-scribed (19, 37), suggesting that antibiotic susceptibility resultsmay not reliably differentiate among these closely related species.

Although 16S rRNA gene sequencing has been used for theidentification of nontuberculous mycobacteria (NTM), includ-ing RGM, it has limited value in distinguishing among someclosely related species (9, 14). Therefore, the use of severalother gene targets for the identification of mycobacteria hasbeen proposed (2, 5, 11, 23, 25, 31, 32, 39, 41). Discriminationamong M. abscessus, M. massiliense, and M. bolletii (which haveidentical 16S rRNA gene sequences) has proven to be difficult,with sequencing of different gene targets often providing con-flicting results. Among these gene targets, partial sequencingof rpoB has increasingly been used (1, 19, 29, 37).

Genotypic analysis of NTM has proven useful not only in theinvestigation of outbreaks and pseudo-outbreaks (38) but alsoin characterizing the molecular epidemiology of strains, and in

* Corresponding author. Mailing address: Microbiology Service, De-partment of Laboratory Medicine, National Institutes of Health, 10Center Dr., Bldg. 10/2C-385, Bethesda, MD 20892. Phone: (301) 496-6200. Fax: (301) 402-1886. E-mail: azelazny@mail.nih.gov.

� Published ahead of print on 6 May 2009.

1985

assessing clonal distribution and expansion (4, 7, 13, 17). Inparticular, molecular typing has recently been used for thecharacterization of health care-related outbreaks of M. massil-iense and M. bolletii (19, 37).

We sought to perform a thorough molecular investigation,including strain identification and typing, for a series of 42clinical isolates (CIs) of M. abscessus, M. massiliense, and M.bolleti from patients monitored in our institution between 1999and 2007. A retrospective patient chart review assessed demo-graphics, underlying conditions, and clinical history.

The 42 CIs and 3 type strains were subjected to multilocussequence analysis, including sequencing of rpoB, hsp65, secA,and the internally transcribed spacer (ITS) region. The relat-edness among the isolates was assessed by use of an automatedrepetitive-sequence-based PCR (rep-PCR) and pulsed-fieldgel electrophoresis (PFGE). This is the most extensive molec-ular characterization of non-outbreak-related isolates from pa-tients with M. abscessus, M. massiliense, and M. bolletii infec-tions.

MATERIALS AND METHODS

Bacterial strains. Forty-two CIs collected from 1999 to 2007 under appropri-ate clinical protocols with informed consent were included in this study. Theywere grown from sputum (n � 31), bronchoalveolar lavage fluid (n � 3), ab-scesses (n � 3), blood cultures (n � 2), skin (n � 2), or a lymph node (n � 1).The methods used for the identification of the CIs included assessments ofpigment production, growth rate, and colony characteristics, followed by mul-tilocus sequencing. Species identification was considered unambiguous onlywhen there was agreement in the species assignation by hsp65, rpoB, and secAgene sequencing analysis.

Type strains of M. abscessus (ATCC 19977), M. massiliense (CIP 108297), andM. bolletii (CIP 108541) were used. The bacterial strains were stored at �70°C inTween albumin broth (Remel, Lenexa, KS). Prior to use, the strains were sub-cultured onto Middlebrook 7H11 agar (Remel).

DNA isolation, PCRs, and sequencing. DNA was extracted from a 10-�lloopful of each mycobacterial colony by use of an UltraClean microbial DNAisolation kit (Mo Bio Laboratories, Solana Beach, CA), according to the man-ufacturer’s instructions. PCRs were carried out with Illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare, Buckinghamshire, United Kingdom). Unlessstated otherwise, the amplified PCR fragments were sequenced under contractwith the Laboratory of Molecular Technology in Frederick, MD.

Partial amplification of the hsp65 gene was performed with primers Tb11 (5�-ACCAAC GAT GGT GTG TCC AT-3�) and Tb12 (5�-CTT GTC GAA CCG CAT ACCCT-3�) (33) that were tailed with the M13 sequencing primer sites M13F (5�-GTAAAA CGA CGG CCA G-3�) and M13R (5�-CAG GAA ACA GCT ATG AC-3�),respectively. Sequencing of the 401-bp fragment was carried out at the NIH with tailprimers M13F and M13R, as described elsewhere (39).

Partial amplification of the rpoB sequence was carried out with the primer pairMyco-F (5�-GGC AAG GTC ACC CCG AAG GG-3�) and Myco-R (5�-AGCGGC TGC TGG GTG ATC ATC-3�) (2). A 711-bp sequence was derived fromthe amplicon by using the same primer pair and also sequencing primers Myco-seqF (5� GAA GGG TGA GAC CGA GCT GAC-3�) and MycoseqR (5�-GCTGGG TGA TCA TCG AGT ACG G-3�) (2).

Partial amplification of the secA1 gene was carried out with primers Mtu.For1(5�-GAC AGY GAG TGG ATG GGY CGS GTG CAC CG-3�) andMtu.Rev490 (5�-GCG GAC GAT GTA RTC CTT GTC SCG-3�) tailed with theM13F and M13R sequencing primer sites, respectively (39). Sequencing of a465-bp fragment was carried out with tail primers M13F and M13R, along withthe PCR primers Mtu.For and Mtu.Rev490.

Partial amplification and sequencing of the 16S-23S rRNA gene ITS region wasperformed with primers Sp1 (5�-ACC TCC TTT CTA AGG AGC ACC-3�) and Sp2(5�-GAT GCT CGC AAC CAC TAT CCA-3�), as described elsewhere (26).

Sequence and phylogenetic analysis. The Lasergene (version 5.51) program(DNAStar, Inc., Madison, WI) was used for sequence assembly. Multiple-se-quence alignment was carried out by the method with the CLUSTAL W (version2.0) program.

Phylogenetic analyses of rpoB and multiple concatenated sequences wereconstructed by using the PhyML (version 2.4.5) program and PHYLIP (phylog-

eny inference package, version 3.5c; J. Felsenstein, University of Washington,Seattle) (the method of Fitch in Dnapars software). A superdistance matrix(SDM) was generated from each gene’s distance matrix by using SDM software(10), followed by tree construction with the Fitch method. The resulting treeswere depicted by using the FigTree (version 1.1.2) program (Institute of Evolu-tionary Biology, University of Edinburgh). The reliability of each tree was as-sessed by the bootstrap method with a total of 1,000 bootstrapped iterations.

Mycobacterial typing. The DiversiLab Mycobacterium typing kit (BacterialBarcodes, Inc., a subsidiary of bioMerieux, Inc.) was used for rep-PCR typing ofthe mycobacterial isolates, according to the manufacturer’s instructions. Ampli-fied fragments of various sizes and intensities were separated and detected byusing a microfluidics chip with the Agilent 2100 bioanalyzer (Agilent Technol-ogies, Palo Alto, CA). Further analysis was performed with DiversiLab software(version 3.3), which uses the Pearson correlation coefficient to determine dis-tance matrices and the unweighted pair group method with arithmetic means tocreate dendrograms (16). The reports generated included dendrograms andvirtual gel images.

Analysis of large restriction fragment profiles by PFGE was carried out asdescribed previously (40) with few modifications. In brief, organisms cast intolow-melting-point agarose plugs were lysed with lysozyme, sodium dodecyl sul-fate, and proteinase K. The modified protocol included the addition to themycobacterial cultures of cycloserine (1 mg/ml) and ampicillin (0.1 mg/ml) andincubation for 24 h at 30°C just before the plugs were made. The genomic DNAcontained in the plugs was digested with the restriction endonuclease AseI andseparated by PFGE with a CHEF Mapper system (Bio-Rad Laboratories, Rich-mond, CA). The PFGE band patterns were analyzed with the BioNumerics(version 4.01) program (Applied Maths, Inc., Austin, TX).

Clinical data. A retrospective chart review was performed for 40 patientsidentified by the NIH Microbiology Laboratory as having had a positive culturefor M. abscessus, M. massiliense, or M. bolletii between January 1999 and De-cember 2007. Two patients whose isolates were included in the molecular portionof this study (one M. abscessus isolate and one M. massiliense isolate) wereexcluded from clinical analysis due to a lack of relevant clinical data. The studywas approved by the NIH Office of Human Subjects Research. Pertinent datafrom the date of the first positive M. abscessus, M. massiliense, or M. bolletiiculture at the NIH were accrued by examination of the medical record andelectronic laboratory database. The basic demographics, underlying comorbidi-ties, and clinical histories of the patients were recorded. The source of thepositive culture, other microbial pathogens isolated, and the type of mycobacte-rial infection (pulmonary, disseminated, or soft tissue infection and/or lymphad-enitis) were determined. For those patients with pulmonary isolates, the resultsof computed tomography of the chest performed at or near the time of thepositive culture were reviewed (20) by a single investigator (K.N.O.) blinded tothe microbiology data.

Descriptive and comparative statistics were performed with Prism (version4.0c) software (GraphPad Software, Inc., San Diego, CA). Comparison betweenthe M. abscessus and M. massiliense groups was performed by Fisher’s exact testfor categorical variables and Student’s t test or the Mann-Whitney test forcontinuous variables. Two-tailed P values were recorded, and significance was setat an alpha value of �0.05. The data for the cases of M. bolletii infection were notincluded in the comparative statistics, due to the small sample size.

RESULTS

Identification of CIs. Comparison of the hsp65 sequences ofthe 42 CIs and 3 type strains led to the identification of 27 CIsas M. abscessus, 12 CIs as M. massiliense, and 3 CIs as M.bolletii (Table 1). A similar analysis conducted by sequencingof the rpoB gene assigned the 42 CIs as follows: 33 M. abscessusisolates, 7 M. massiliense isolates, and 2 M. bolletii isolates(Table 1).

A phylogenetic tree of the partial rpoB gene sequences (Fig.1) separated a monophyletic cluster of M. abscessus from acluster that included M. massiliense and M. bolletii. The last twospecies were further separated from each other with a highbootstrap value. Among the isolates identified as M. abscessusby rpoB gene sequencing, comparison of the sequences of theCIs with the corresponding sequence of the type strain re-vealed 100% identity for 26 isolates. The sequences of seven

1986 ZELAZNY ET AL. J. CLIN. MICROBIOL.

other isolates showed 99.9% identity with the sequence of thetype strain; the lack of complete identity was due to a singlenucleotide difference: six CIs (group A in Fig. 1) exhibited aone-base substitution at position 187 (C to T), while CI1218showed a T-to-C transition at base 169.

None of the CIs identified as M. massiliense by rpoB genesequencing showed a perfect match with the correspondingtype strain. All of them had a two-base change that led to99.7% identity with the type strain. Five isolates differed fromthe type strain at base 152 (C to T) and base 343 (T to C)(group B in Fig. 1). CI74 showed substitutions at base 67 (T toG) and base 517 (G to A), while for CI72, the substitutionswere at position 343 (T to C) and position 490 (G to A).

The two CIs identified as M. bolletii by rpoB gene sequencingshowed one- and two-base differences with the type strain,respectively. CI78 and CI59 showed a G-to-C transversion atposition 13. In addition, CI78 had an additional C-to-T substi-tution at position 634.

Evaluation of a novel target, secA, for identification of M.abscessus group. To evaluate the performance of a novel gene

TABLE 1. Comparison of genes sequences from clinical isolatesand reference strains of M. abscessus, M. massiliense,

and M. bolletii

Organism

hsp65 rpoB secA

No. ofisolates

%Identity

No. ofisolates

%Identity

No. ofisolates

%Identity

M. abscessus 27 100 26 100 19 1007a 99.9 6 99.8

1 99.6

M. massiliense 12 100 7 99.7 7 1002 99.83 99.6

M. bolletii 1 100 1 99.9 2 1002b 99.8 1 99.7 2c 99.8

a Includes clinical isolates 71, 73, 76, 510, and 1210 identified as M. massilienseby secA and hsp65 sequencing.

b Includes clinical isolate 79 identified as M. abscessus by rpoB sequencing.c Includes clinical isolates 79 and 1214 identified as M. abscessus by rpoB

sequencing.

FIG. 1. Phylogenetic tree derived from rpoB sequences of 42 CIs and the type strains of M. abscessus, M. massiliense, and M. bolletti. The treewas constructed by using the PhyML (version 2.4.5) program and M. fortuitum as the outgroup. The numbers on the branches represent thepercentage of 1,000 bootstrap samples supporting the branch.

VOL. 47, 2009 MOLECULAR STUDY OF M. ABSCESSUS AND RELATED SPECIES 1987

target, the secA gene, recently reported to have been used forthe identification of mycobacteria (39), we amplified a 465-bpfragment (after subtraction of the primer sequences) for all CIsand type strains. The sequences of the amplified fragment werealigned and compared with those of the type strains of M.abscessus, M. massiliense, and M. bolletii. Results obtained bysecA analysis were compared with those generated by sequenc-ing of rpoB, a gene target increasingly used for discriminationamong these and other related species (1, 19, 29, 37).

By secA sequencing, 26 CIs were identified as M. abscessus(Table 1), all of which had been identified as such by hsp65 andrpoB gene sequencing. secA analysis identified 12 isolates as M.massiliense, 7 of which had been assigned to this species byrpoB and hsp65 gene sequencing (Table 1). The secA genesequences of the other five CIs (CI71, CI73, CI76, CI510,CI1210) showed 100% similarity to the secA gene of the M.massiliense type strain. However, they were identified as M.

abscessus by rpoB gene sequencing and as M. massiliense byhsp65 gene sequencing and showed a 100% match to the cor-responding type strains. Four CIs were identified as M. bolletiiby secA sequencing; two of them (CI78, CI59) were identifiedas such by rpoB and hsp65 gene sequencing.

Integrated phylogenetic analysis. We sought to integrate thephylogenetic analysis of the three housekeeping genes using agene concatenation approach and SDM approach (10). Themaximum-likelihood method (Fig. 2) and the distance-basedand maximum-parsimony methods (data not shown) were usedto construct trees from the concatenated gene alignments. Fiveisolates (CI71, CI73, CI76, CI510, CI1210) with conflictingspecies assignments by use of the individual phylogenetic treesformed a separate clade between the M. abscessus and the M.massiliense groups. Isolates CI79 and CI1214 also formed aseparate group closer to M. abscessus. This topology was sup-ported by the maximum-likelihood and distance-based trees,

FIG. 2. Phylogenetic tree derived from concatenated hsp65, rpoB, and secA sequences of 42 CIs and the type strains of M. abscessus, M.massiliense, and M. bolletti. The tree was constructed by using the PhyML (version 2.4.5) program and M. fortuitum as the outgroup. The numberson the branches represent the percentage of 1,000 bootstrap samples supporting the branch.

1988 ZELAZNY ET AL. J. CLIN. MICROBIOL.

while the maximum-parsimony method was inconclusive withregard to these seven isolates. Finally, an additional tree gen-erated by the SDM method (10) (Fig. 3) also supported thegrouping of the five isolates with ambiguous identities separatefrom the M. abscessus and M. massiliense groups.

In order to complement the results obtained by the inte-grated phylogenetic analysis of concatenated housekeepinggenes, we also sequenced the ITS region, a nonhousekeepinggene region previously used to discriminate between M. mas-siliense and M. abscessus (3). Sequencing of the ITS regionconfirmed the species assignments of all seven CIs previouslyidentified as M. massiliense by hsp65, rpoB, and secA genesequencing. The five CIs with ambiguous identities identifiedas M. massiliense by both secA and hsp65 sequencing but as M.abscessus by rpoB sequencing were identified as M. massilienseby sequencing of the ITS region. Each of them had a perfectmatch to the M. massiliense type strain, including a character-istic A-to-G transition and a C insertion (3).

Typing of M. abscessus, M. massiliense, and M. bolletii byrep-PCR. Following the species assignment of all 42 CIs, weassessed the relatedness among the clinical and referencestrains for each of the species. For that purpose, we chose acommercial high-throughput rep-PCR developed for molecu-lar typing of microorganisms (16) using a typing kit designedfor Mycobacterium spp. DNA was extracted from all clinicaland reference isolates and then subjected to fingerprinting byrep-PCR. The results of rep-PCR analysis of all CIs as well asthe corresponding type strains are shown in Fig. 4. Most M.abscessus isolates showed similar patterns and clustered to-gether with the M. abscessus type strain (group I in Fig. 4). Asecond group of M. abscessus isolates (n � 11) clustered to-gether as a clearly separate but less homogeneous group(group II). Finally, a separate branch was observed for twodistinct clinical M. abscessus isolates (group III).

Six CIs identified as M. massiliense by rpoB sequencing clus-tered together (group IV) and were separated from CI72.

FIG. 3. Phylogenetic tree derived from concatenated hsp65, rpoB, and secA sequences of 42 CIs and the type strains of M. abscessus, M.massiliense, and M. bolletti. The tree was constructed by using the SDM generated from each gene’s distance matrix with SDM software (10).

VOL. 47, 2009 MOLECULAR STUDY OF M. ABSCESSUS AND RELATED SPECIES 1989

1990

Interestingly, five CIs ambiguously identified as M. abscessus byrpoB sequencing but as M. massiliense by sequencing of thehsp65 and secA genes and the ITS region formed a distinctcluster that appeared as a separate branch in the tree that alsoincluded the M. massiliense type strain (group V).

Finally, the two CIs of M. bolletii showed similar patterns (groupVI) which were different from that of the M. bolletii type strain.

Typing of representative strains of M. abscessus, M. massil-iense, and M. bolletii by PFGE. In order to confirm the typingresults obtained by rep-PCR, 12 selected strains of M. absces-sus, M. massiliense, and M. bolletii were subjected to PFGEanalysis. The strains were compared for their relatedness bythe method of Tenover et al. (34), with minor modifications, asdescribed previously (40). As shown in Fig. 5, distinct patternswere observed for the type strains of M. abscessus, M. massil-iense, and M. bolletii. Both CIs of M. abscessus clustered to-gether with the type strain. The type strain of M. massiliensewas closely related (few band differences) to CIs 71, 76, 510,and 73, while several band differences were observed for thetype strain of M. massiliense compared to those observed forCI72 and CI75, suggesting that the last two isolates are unre-lated. M. bolletii CI59 showed a distinct pattern which wasdifferent from that of the M. bolletii type strain.

Clinical characteristics. Among the 40 patients evaluated,27 were classified as having M. abscessus infections, 11 wereclassified as having M. massiliense infections, and 2 were clas-sified as having M. bolletii infections by our intensive molecularapproach. The demographic and clinical characteristics of thepatients are summarized in Table 2.

The majority (89%) of patients infected with M. abscessusidentified their race as white, whereas 55% of the patients(odds ratio, 6.6; 95% confidence interval, 1.2 to 36.1; P � 0.05)infected with M. massiliense and 50% of the patients infectedwith M. bolletii identified their race as white. The median ageat the time of M. abscessus isolation was 54 years (range, 10 to79 years), whereas the median age at the time of M. massilienseisolation was 27 years (range, 14 to 55 years) (P � 0.05). Thelatter group had a bimodal age distribution, with six patientsbeing between the ages of 14 and 27 years and five patientsbeing between the ages of 47 and 55 years.

In our cohort, pulmonary infection was more common thandisseminated disease, soft tissue infection, or lymphadenitis,accounting for over 77% of total infections. All patients withpulmonary infection had abnormalities on chest imaging: 97%had nodules, 43% had cavities and nodules, and over 93% hadbronchiectasis. Bilateral disease was present in 90% and in-volved an average of five lobes (the lingula was counted as adistinct lobe). Among the patients with pulmonary infections,15 (48%) had a history of chronic pulmonary disease (i.e.,cystic fibrosis, primary ciliary dyskinesia, asthma, idiopathicbronchiectasis, etc.) that predated a diagnosis of pulmonaryNTM infection. An additional eight patients (all infected withM. abscessus) had a history of M. avium complex (MAC) pul-monary infection without having known lung disease precedingthe diagnosis of MAC infection (one patient had a prior MACinfection and preexisting lung disease). Interestingly, all 7 of thepatients with M. massiliense pulmonary infection had known lungdisease prior to their diagnosis of NTM infection, whereas only 8

FIG. 4. Rep-PCR-based dendrogram and virtual gel image fingerprints obtained from 42 CIs of M. abscessus, M. massiliense, and M. bolletii andthe corresponding type strains (ATCC 19977, CIP 108297, and CIP 108541, respectively). The DiversiLab system with the Mycobacterium sp.fingerprinting kit was used. Pearson’s correlation coefficient was used to create a pairwise percent similarity matrix, and the tree was generated bythe unweighted pair group method with arithmetic means (UPGMA). Bar, percent similarity among strains.

FIG. 5. Dendrogram of PFGE patterns of M. abscessus, M. massiliense, and M. bolletii isolates digested with AseI. The comparison was performed with theBioNumerics (version 4.01) program (Applied Maths, Inc.).

VOL. 47, 2009 MOLECULAR STUDY OF M. ABSCESSUS AND RELATED SPECIES 1991

(35%) of the 23 patients with M. abscessus infection carried adiagnosis of chronic pulmonary disease before they were diag-nosed with either MAC or M. abscessus infection (odds ratio,27.4; 95% confidence interval, 1.4 to 540.2; P � 0.01).

DISCUSSION

M. abscessus is a commonly isolated RGM. Recently, twonew related species, M. massiliense and M. bolletii, have beendescribed (1, 3). To investigate these new species, we per-

formed a multilocus sequencing approach for species-levelidentification. We also explored an alternative typing methodother than the lengthy and technically demanding PFGE byassessing strain relatedness with a rapid PCR-based commer-cial system using a protocol developed for Mycobacterium spp.Multilocus identification including a novel gene target (secA),and molecular typing gave us the opportunity to evaluate boththe clinical relevance of these distinctions and the performanceof different molecular identification and typing approaches.

Initial molecular identification by partial sequencing of the

TABLE 2. Patient demographics and clinical characteristics classified by organism

Clinical characteristicResult for the following organisma:

M. abscessus (n � 27) M. massiliense (n � 11) M. bolletii (n � 2)

Gender (no. �%� of patients)Male 8 (29.6) 5 (45.5) 1 (50)Female 19 (70.4) 6 (54.5) 1 (50)

Median (range) age (yr) 54 (10–79) 27 (14–55) 31 (14–48)

Median (range) BMI (kg/m2) 22.5 (14.5–30.6)b 24.4 (15.4–34.2) 17.7 (15.0–20.4)

Type of mycobacterial infection (no. �%� of patients)Pulmonary 23 (85.2) 7 (63.6) 1 (50.0)Disseminated 3 (11.1) 2 (18.2) 1 (50.0)Skin, soft tissue 1 (3.7) 1 (9.1) 0Lymphadenitis 0 1 (9.1) 0

Comorbidities (no. �%� of patients)Pulmonary

Prior pulmonary MAC infection 8 (29.6) 0 0Bronchiectasis (preceding NTM infection)c 8 (29.6) 6 (54.5) 0Asthma 3 (11.1) 2 (18.2) 0COPDd 1 (3.7) 1 (9.1) 0Cystic fibrosis 3 (11.1) 3 (27.3) 0Primary ciliary dyskinesia 0 1 (9.1) 0

ImmunodeficiencyIFN-e receptor deficiency 0 0 1 (50)IFN- autoantibody 1 (3.7) 1 (9.1) 0Monocytopenia 1 (3.7) 0 0Severe combined immunodeficiency 0 1 (9.1) 0Job syndrome 1 (3.7) 1 (9.1) 0HIVf infection 0 0 0Unclassified 3 (11.1) 0 0

Active systemic immunosuppression 3 (11.1) 0 1 (50)History of transplant 0 1 (9.1) 1 (50)Malignancy 3 (11.1) 2 (18.2) 1 (50)History of chemotherapy 2 (7.4) 2 (18.2) 1 (50)Diabetes 1 (5.9) 3 (27.3) 0Autoimmune disorder 2 (7.4) 2 (18.2) 0

Symptoms (no. �%� of patients)Fever 5 (18.5) 2 (18.2) 2 (100)Chills and/or night sweats 6 (22.2) 2 (18.2) 0Weight loss 7 (25.9) 3 (27.3) 1 (50)Fatigue 13 (48.2) 3 (27.3) 1 (50)Dyspnea 13 (48.2) 2 (18.2) 1 (50)Cough 22 (81.5) 6 (54.6) 1 (50)Hemoptysis 4 (14.8) 0 0Lymphadenopathy 2 (7.4) 3 (27.3) 2 (100)Wound or skin lesion 4 (14.8) 1 (9.1) 0

a Species classification is based on consensus agreement among all identification methods used.b BMI, body mass index. Body mass index data were missing for one subject infected with M. abscessus.c Specifically refers to documented bronchiectasis preceding diagnosis with an NTM infection.d COPD, chronic obstructive pulmonary disease.e IFN-, gamma interferon.f HIV, human immunodeficiency virus.

1992 ZELAZNY ET AL. J. CLIN. MICROBIOL.

hsp65 gene classified these strains as M. abscessus (n � 27), M.massiliense (n � 12), and M. bolletii (n � 3) (Table 1). All 27strains identified as M. abscessus and all 12 isolates identifiedas M. massiliense showed perfect matches to the correspondingtype strain, as reported previously (29). Two hsp65 sequevarsfor M. abscessus corresponding to type I and type II hsp65 byPCR-restriction fragment length polymorphism analysis havebeen described (12, 21, 25). Our hsp65 sequencing results in-dicate that all the M. abscessus CIs were the type I sequevarand all the M. massiliense CIs were the type II sequevar.

On the basis of previous reports suggesting that rpoB genesequencing may yield better discrimination among theseclosely related species, we partially sequenced the rpoB gene ofour CIs. The rpoB sequencing results matched those obtainedby hsp65 sequencing for most isolates; however, rpoB sequenc-ing identified more isolates (n � 33) as belonging to M.abscessus than did hsp65 sequencing (n � 27). While mostisolates identified as M. abscessus showed 100% identity to thetype strain, none of the isolates identified as M. massiliense orM. bolletti showed perfect matches to the corresponding typestrains. It is interesting to note that the sequences from neitherof two CIs of M. massiliense from the United States (29) andBrazil (37) reported recently matched the rpoB sequence of thetype strain with 100% identity. However, a perfect match to thesequence of the M. massiliense type strain was observed amongisolates in a recent outbreak of M. massiliense in South Korea(19).

In general, the RGM interspecies sequence divergence de-termined by partial sequencing of rpoB is 3%, and it has beenproposed that isolates with less than 1.7% sequence divergenceare considered to belong to the same species (2). All seven CIsidentified as M. massiliense by rpoB sequencing showed 99.7%similarity (0.3% divergence) to the type strain. The five CIsidentified as M. abscessus by rpoB sequencing and as M. mas-siliense by sequencing of the three different targets (see below)also exhibited less than 1.7% sequence divergence from thetype strain of M. abscessus (99.9% identity) and 3% diver-gence from the type strains of M. massiliense and M. bolletii(96.5% and 95.5% identities, respectively) by rpoB sequencing.

In order to resolve the discrepancies in the identification ofseven isolates, we performed an integrated phylogenetic anal-ysis of hsp65, rpoB, and secA using different methods. By suchanalyses, isolates CI79 and CI1214 were most closely related toM. abscessus. On the other hand, a group of five isolates witha perfect match to the M. abscessus type strain by rpoB se-quencing and the M. massiliense type strain by hsp65 and secAsequencing repeatedly emerged as an independent branch be-tween M. abscessus and M. massiliense in different phylogeneticanalyses. Sequencing of the nonhousekeeping ITS regionshowed a perfect match to the ITS region sequence of the M.massiliense type strain, with the characteristic A-to-G transi-tion and a C insertion being detected (3).

Multilocus sequencing combined with molecular typing pro-vides valuable information on the identities of and the clonalrelationship among bacterial isolates (30, 36). We used a rep-PCR method optimized for Mycobacterium spp. (16) which wasshown to be useful for the typing of M. chelonae, M. abscessus,and M. fortuitum (27, 28). Dendrograms and virtual gel imageswere generated for all isolates belonging to each of the threespecies (Fig. 4). Rep-PCR analysis grouped the large majority

of M. abscessus CIs into two major clusters (groups I and II;Fig. 4). Only two M. abscessus isolates (group III) showed adistinct pattern unrelated to these two major clusters. Mostisolates of M. massiliense clustered into a major group (groupIV) separated from one clinical isolate (CI72) and the typestrain. Interestingly, all five isolates (CI71, CI73, CI76, CI510,CI1210) that formed a separate clade between M. abscessusand M. massiliense (Fig. 2) clustered together with the M.massiliense type strain by rep-PCR.

PFGE analysis of representative strains confirmed the re-sults obtained by rep-PCR. Distinct PFGE patterns could beobserved for isolates of each of the species. Within each spe-cies, a comparable degree of relatedness was observed by bothtechniques. For example, the rep-PCR patterns of M. massil-iense showed three easily distinguishable groups (group V,CI72, and group VI). Similarly, PFGE clustered togethermembers of group IV, which exhibited few or no band differ-ences. On the other hand, very distinct patterns were observedwith CIs 72 and 75, with the latter isolate belonging to rep-PCR group V.

Combining the results of multilocus sequencing and molec-ular typing was very informative. CI79 and CI1214, which wereidentified as M. abscessus by rpoB sequencing but as M. bolletii(CI79) and M. abscessus (CI1214) by hsp65 sequencing and asM. bolletii (both CI79 and CI1214) by secA sequencing, showedthe rep-PCR pattern of M. abscessus group II and group I,respectively (Fig. 4). This supports the fact that both isolatesare in fact M. abscessus.

The five CIs that showed discordant results, M. abscessus byrpoB sequencing and M. massiliense by hsp65, secA, and ITSregion sequencing, clustered together with the type strain of M.massiliense (Fig. 4). PFGE analysis confirmed that these fiveisolates were highly related to the M. massiliense type strain(Fig. 5), supporting their identification as M. massiliense.

Our clinical data suggest possible differences between pa-tients who develop infections with M. abscessus and those whobecome infected with M. massiliense. However, the strengthand generalizability of this observation are limited by the smallcohort size and the retrospective nature of the study. A pro-spective investigation is needed to determine whether any dif-ferences in host factors, treatment response, and clinical out-come exist among patients infected with these species.

Our multilocus sequencing approach is useful for the mo-lecular identification of the newly described species M. massil-iense and M. bolletii, as some strains show equivocal resultswhen only one or a few gene targets are used. Partial sequenc-ing of rpoB alone did not reliably differentiate all the CIs.Interestingly, in a recent study Kim et al. reported on twoisolates which showed discordant results, M. massiliense byrpoB sequence analysis and M. abscessus by hsp65 sequenceanalysis, and identified them as M. massiliense on the basis ofthe additional analysis of sodA and the ITS region (18). Un-fortunately, that study did not include an integrated phyloge-netic analysis or molecular typing of the strains to assess therelatedness of the strains to each another or other clinical orreference strains.

We hypothesize that our five related CIs, CI71, CI73, CI76,CI510, and CI1210, that formed a separate clade between M.abscessus and M. massiliense (Fig. 2) but that were identified asM. massiliense by sequencing of the ITS region and that ap-

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peared to be related to the M. massiliense type strain by twoindependent typing methods are M. massiliense. On the basis ofthe molecular diversity, we cannot rule out the possibility ofdistinct taxa (i.e., at the subspecies level) within M. massiliense.Comparative genomic hybridizations with a mycobacterial chipwill be performed to address this issue. Beyond taxonomicconsiderations, we believe that it is important to the clinicalmicrobiologist to use a multilocus sequencing approach com-bined with typing techniques in order to identify such groupswithin the bacterial population. These groups likely share de-fined microbiologic, epidemiologic, and possibly virulence fea-tures.

In summary, a multilocus sequencing approach is requiredfor discrimination among the closely related species M. absces-sus, M. massiliense, and M. bolletii. Partial sequencing of rpoB(711 bp) alone does not reliably differentiate these three spe-cies. We suggest that these organisms first be identified by secAsequencing, a target routinely used in our laboratory for directdetection as well as the identification of all Mycobacterium spp.The identities of isolates identified as M. abscessus, M. massil-iense, or M. bolletii by secA sequencing are confirmed by se-quencing of additional gene targets. Sequence-based identifi-cation combined with molecular typing supports theidentification of isolates with ambiguous identities and pro-vides information on prevalent clones with potentially relevantclinical features. The molecular, biological, and clinical char-acteristics of these strains will help us to better understand andtreat severe infections due to RGM.

ACKNOWLEDGMENTS

We thank Frank G. Witebsky and Patrick R. Murray for criticallyreviewing the manuscript and Patricia Conville for the bionumericanalysis.

This research was supported by the Intramural Research Program ofthe NIH, the NIH Clinical Center, and the National Institute of Al-lergy and Infectious Diseases.

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