Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.

Abstract

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Full text

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JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2011, p. 491–499
0095-1137/11/$12.00doi:10.1128/JCM.01274-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 2
Multilocus Sequence Analysis and rpoB Sequencing of
Mycobacterium abscessus (Sensu Lato) Strains?
Edouard Macheras,1,3Anne-Laure Roux,2,3Sylvaine Bastian,4Sylvia Cardoso Lea ˜o,5Moises Palaci,6
Vale ´rie Sivadon-Tardy,1,3Cristina Gutierrez,7Elvira Richter,8Sabine Ru ¨sch-Gerdes,8Gaby Pfyffer,9
Thomas Bodmer,10Emmanuelle Cambau,11Jean-Louis Gaillard,1,2,3and Beate Heym1,3*
Service de Microbiologie–Hygie `ne, Ho ˆpital Ambroise Pare ´, Assistance Publique, Ho ˆpitaux de Paris (AP-HP), Boulogne-Billancourt,
France1; Laboratoire de Microbiologie, Ho ˆpital Raymond Poincare ´, AP-HP, Garches, France2; EA 3647, Universite ´ de Versailles
Saint-Quentin-en-Yvelines, Garches, France3; Centre National de Re ´fe ´rence des Mycobacte ´ries et de la Re ´sistance des
Mycobacte ´ries Aux Antituberculeux, Laboratoire de Bacte ´riologie–Hygie `ne, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, Paris, France4;
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brazil5; Nucleo de
Doenc ¸as Infecciosas, Universidade Federal do Espírito Santo, Espírito Santo, Brazil6; FIND, Geneva, Switzerland7;
National Reference Center for Mycobacteria, Forschungszentrum Borstel, Borstel, Germany8; Institut fu ¨r
Medizinische Mikrobiologie, Zentrum fu ¨r LaborMedizin, Luzerner Kantonsspital, Luzern, Switzerland9;
Institut fu ¨r Infektionskrankheiten, Universita ¨t Bern, Bern, Switzerland10; and Centre National de
Re ´fe ´rence des Mycobacte ´ries et de la Re ´sistance des Mycobacte ´ries Aux Antituberculeux,
Laboratoire Associe ´, Bacte ´riologie-Virologie-Hygie `ne, Groupe
Hospitalier Saint Louis–Lariboisie `re, Paris, France11
Received 23 June 2010/Returned for modification 2 September 2010/Accepted 12 October 2010
Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus
sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However,
recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some
authors have questioned their current taxonomic classification. We studied here a large collection of M.
abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA).
The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya,
glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP
104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T) and 120 isolates recovered between 1997 and
2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three
main clusters, each comprising the type strain of one species. However, divergence values between the M.
massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters
were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also
showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a
bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii
branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect.
Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense
clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%)
between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated
sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates
for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense
MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In
conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii
should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB
sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification
of the two proposed M. abscessus subspecies.
Mycobacterium abscessus is a rapidly growing mycobacterium
(RGM) that causes a wide spectrum of disease in humans,
including chronic lung disease, skin and soft-tissue disease, and
disseminated disease (12, 14, 53). M. abscessus lung disease
mostly develops in subjects with underlying lung disorders
(e.g., cystic fibrosis [CF] or prior mycobacterial infection) or
Lady Windermere syndrome (10, 19, 29, 35, 40, 41, 45, 46). M.
abscessus is also a leading cause of sporadic and epidemic cases
of skin and soft-tissue RGM infections after surgery or follow-
ing the use of contaminated syringes and needles (21, 22, 39).
Several large outbreaks of skin and soft-tissue infection have
been reported following the injection of adrenal cortex extract,
mesotherapy, tattooing, and piercing (6, 8, 15, 24, 52, 58).
The Mycobacterium massiliense and Mycobacterium bolletii
species were characterized in the early 2000s, and both are
closely related to M. abscessus (100% identity of 16S rRNA
sequences) (2, 5). Therefore, M. abscessus (now M. abscessus
* Corresponding author. Mailing address: Service de Microbiologie-
Hygie `ne, Ho ˆpital Ambroise Pare ´, Faculte ´ de Me ´decine Paris, Ile de
France-Ouest, Universite ´ de Versailles–Saint Quentin en Yvelines, 9
avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France.
Phone: 33 (0) 1 49 09 44 21. Fax: 33 (0) 1 49 09 59 21. E-mail:
beate.heym@apr.aphp.fr.
?Published ahead of print on 24 November 2010.
491

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sensu lato) is comprised of three species: M. abscessus sensu
stricto (for simplicity, M. abscessus sensu stricto here will be
referred to as M. abscessus), M. massiliense, and M. bolletii.
Although information about the pathogenic effects of M. mas-
siliense and M. bolletii in humans still is scarce, several recent
studies report that they cause a spectrum of diseases similar to
those associated with M. abscessus. However, there may be
some differences between the three species. Zelazny et al. have
reported that M. massiliense is more frequently present in the
respiratory tract of younger patients with preexisting lung dis-
ease than M. abscessus (59). Differences also have been re-
ported in the susceptibility patterns of the three species (13, 14,
54, 55). For example, M. massiliense was reported to be sus-
ceptible to doxycycline, whereas M. abscessus and M. bolletii
are not (5), although this finding has not been confirmed by
others (32, 52).
M. massiliense and M. bolletii were characterized as new
species distinct from M. abscessus on the basis of their rpoB
sequences (?3% sequence divergence) (2, 5). Partial rpoB
sequencing is now the gold standard for the molecular identi-
fication of the three species (3, 4, 16, 38, 48). However, recent
studies have highlighted the inaccuracy of single-target se-
quencing, including rpoB sequencing, for distinguishing be-
tween M. abscessus, M. massiliense, and M. bolletii (18, 31, 36,
59). Zelazny et al. found that the partial sequencing of rpoB,
hsp65, and secA led to inconsistent results in 7 of 42 clinical
isolates; most of these seven isolates had an M. abscessus rpoB
sequence and M. massiliense hsp65 and secA sequences, and
they clustered with the M. massiliense type strain in repetitive
sequence-based PCR (rep-PCR) and pulsed-field gel electro-
phoresis (PFGE) (59). We reported similar data in a study of
a panel of 59 clinical isolates by the partial sequencing of rpoB,
hsp65, and sodA (36). Target genes yielded discordant results
in 15 isolates, which had interspecific composite patterns (e.g.,
isolates with a rpoB sequence 100% identical to the M. absces-
sus type sequence and an hsp65 sequence 100% identical to the
M. massiliense type sequence). The identification of these iso-
lates was substantially improved by the partial sequencing of
five housekeeping gene sequences, indicating the value of a
multilocus sequencing approach (26, 36).
A panel of M. abscessus, M. massiliense, and M. bolletii
strains recently has been studied by biochemical tests, high-
performance liquid chromatography (HPLC), drug susceptibil-
ity testing, PCR restriction enzyme analysis of the hsp65 gene
(PRA-hsp65), rpoB and hsp65 gene sequencing, the restriction
fragment length polymorphism (RFLP) analysis of the 16S
rRNA gene, and DNA-DNA hybridization (34). The clinical
isolates studied and the type strains could not be separated,
and DNA-DNA hybridization showed more than 70% inter-
strain relatedness. The authors thus proposed a revision of the
taxonomic status of M. abscessus, M. massiliense, and M.
bolletii, whereby the three species are in fact a single species
(M. abscessus) and two subspecies (M. abscessus subsp. absces-
sus and M. abscessus subsp. massiliense) (34).
Multilocus sequence analysis (MLSA) is a phylogenetic
analysis of multiple internal fragments of genes that are ubiq-
uitous to the studied taxon, present as a single copy within the
genome, and are not subject to selective pressure (27). MLSA
defines an isolate by the sequences obtained from the internal
fragments of several housekeeping genes. The usual approach
in bacterial taxonomy is to concatenate the sequences of sev-
eral (typically six to eight) housekeeping genes. The concate-
nated sequences then are used to assess clustering patterns
among large numbers of strains within a genus or part of a
genus (17, 23, 27, 28). This approach has been used success-
fully to delineate microbial species (i.e., well-resolved clusters)
within various taxonomic groups, including groups of highly
recombinant bacteria, like Neisseria spp. (37). Conversely,
MLSA also allows the assignment of unknown strains to spe-
cies clusters, and this can be performed via the internet, open-
ing the way to electronic taxonomy (11).
In the present study, we developed an MLSA scheme and
applied it to a large collection of M. abscessus sensu lato
strains. The data obtained with this approach were compared
to those obtained by rpoB sequencing to (i) help clarify the
taxonomic status of M. abscessus, M. massiliense, and M. bolletii
and (ii) evaluate the accuracy of rpoB as a molecular identifi-
cation target.
MATERIALS AND METHODS
Bacterial strains and culture conditions. We studied 120 isolates of M. ab-
scessus sensu lato recovered from France (n ? 97), Germany (n ? 6), Switzerland
(n ? 7), and Brazil (n ? 10) between 1997 and 2007. One hundred nineteen of
the isolates were from clinical samples, and one was from the environment
(isolated from a sewer in Brazil). The sample origin was known for 90 of the
clinical isolates: 79 (87.8%) were from respiratory samples (57 CF subjects, 22
non-CF subjects) and 11 (12.2%) were from other samples (9 from skin and soft
tissue, 1 from pericardium, and 1 from a hip prosthesis). All of the clinical
isolates were from unrelated cases. One clinical isolate was from a nonsporadic
case, having also been recovered during a recent outbreak of M. massiliense skin
and soft-tissue disease after a laparoscopic procedure in Brazil (15, 33). The type
strains M. abscessus CIP 104536T(ATCC 19977T), M. massiliense (CIP 108297T),
and M. bolletii (CIP 108541T) also were included in the strain collection. Bacte-
rial strains were stored at ?70°C using cryopreservation beads and were grown
on sheep blood agar at 37°C for 4 days prior to use.
rpoB sequencing and rpoB-based identification. Mycobacterial DNA was ex-
tracted using Tris-EDTA, lysozyme, and proteinase K as described previously
(36). A 940-bp fragment of the rpoB gene was amplified by PCR using AmpliTaq
gold polymerase (Applied Biosystems, Courtaboeuf, France) with the primers
MYCOF1 and MYCOR2 (Table 1). Dideoxy sequencing was carried out on both
strands using a BigDye Terminator cycle sequencing kit (Applied Biosystems)
with the same primers. Sequencing products were purified by gel filtration (Bio-
gel P100; Bio-Rad, Marnes-la-Coquette, France) and were run on a 3700 DNA
analyzer (Applied Biosystems). The rpoB sequences trimmed to 752 bp (3) were
compared to the corresponding rpoB sequences from the reference type strains
(http://www.ncbi.nlm.nih.gov/).
PCR amplification and sequencing of 10 housekeeping genes in addition to
rpoB. Fragments from 10 housekeeping genes were amplified using the sets of
primers shown in Table 1: argH (argininosuccinate lyase), cya (adenylate cy-
clase), gdhA (glutamate dehydrogenase), glpK (glycerol kinase), gnd (6-phospho-
gluconate deshydrogenase), murC (UDP N-acetylmuramate-L-Ala ligase), pgm
(phosphoglucomutase), pknA (serine/threonine protein kinase), pta (phosphate
acetyltransferase), and purH (phoshoribosylaminoimiazolcarboxylase ATPase
subunit). The genes gdhA, glpK, murC, pknA, pta, and purH have been widely
used in multilocus sequence typing (MLST) schemes developed for Gram-pos-
itive bacteria (e.g., Streptococcus spp., Enterococcus spp., Staphylococcus spp.,
and Bacillus spp.) (7, 37, 49). The gene products for argH, cya, gnd, and pgm have
been used previously in the multilocus enzyme electrophoresis (MLEE) analysis
of mycobacteria (20, 56, 57, 60). Amplification was performed using 25 ?l of
ReddyMix PCR master mix (Thermo Fisher Scientific Inc.) and 1 ?l of each
primer (10 pmol). The dideoxy sequencing of the amplified gene fragments was
carried out on both strands with the Big Dye Terminator cycle sequencing kit
(Applied Biosystems), using the same primers as those for amplification. Se-
quencing products were purified and analyzed with an ABI 3700 DNA analyzer
as described above. The sequences were aligned and trimmed to defined start
and end positions using BioEdit version 7.0.5.3 (25). Gene sequences from the
three type strains were used as reference species sequences. Reference M. ab-
scessus sequences of the 10 housekeeping genes were obtained from the whole-
492MACHERAS ET AL.J. CLIN. MICROBIOL.

Page 3

genome sequence of M. abscessus CIP 104536T(http://www.ncbi.nlm.nih.gov)
(accession number NC_010397) (43). We recently determined the sequences of
the argH, cya, glpK, gnd, and murC genes in the reference strains M. bolletii CIP
108541Tand M. massiliense CIP 108297T(36).
Phylogenetic analyses. Unrooted individual gene trees and trees obtained
using concatenated sequences were generated using the neighbor-joining method
with 1,000 bootstrap replications. Trees were drawn to scale, with branch lengths
representing the inferred evolutionary distances. The evolutionary distances
were computed using the maximum composite likelihood method (51), and the
data units were the number of base substitutions per site. Codon positions were
in frame, and there was a total of 4,071 bp in the final data set. The neighbor-
joining method and MEGA 4 software were used for the phylogenetic analysis of
the sequence data (50).
Nucleotide sequence accession numbers. Sequences of the pgm, pta, and purH
gene fragments from the reference strains M. bolletii CIP 108541Tand M. mas-
siliense CIP 108297Twere determined in this study and submitted to the National
Center for Biotechnology Information (NCBI) website (accession numbers
HM371394, HM371395, and HM371396 for M. bolletii and HM371391,
HM371392, and HM371393, for M. massiliense).
RESULTS
Selection of eight housekeeping genes eligible for MLSA.
Ten sets of primers (Table 1) were designed to amplify and
sequence internal fragments of 10 housekeeping genes on a
first panel, including 10 clinical isolates and the three M. ab-
scessus sensu lato type strains. Fragments of the expected size
were amplified from 100% of the strains for eight genes: argH,
cya, glpK, gnd, murC, pgm, pta, and purH. The gdhA and pknA
genes were eliminated from the subsequent MLSA study: in
several strains, the amplification of gdhA did not yield frag-
ments of the expected size, and the sequencing of the ampli-
fication products showed phage sequences; for pknA, we re-
peatedly obtained nonspecific amplification products even with
the use of different primer pairs.
The eight selected housekeeping genes (argH, cya, glpK, gnd,
murC, pgm, pta, and purH) are scattered throughout the ge-
nome of M. abscessus CIP 104536Tand are at least 76 kb away
from each other (42).
Polymorphism of the genes included in the MLSA scheme.
Internal fragments from the eight housekeeping genes selected
for MLSA were amplified, and their nucleotide sequences
were determined for the 120 isolates and the three type strains.
Fragment sizes varied from 480 to 549 bp, with G?C contents
of between 62.4 and 68.2% (Table 2). Polymorphic sites for
each gene fragment were least frequent in pta (n ? 24) and
most frequent in argH (n ? 43). The glpK and murC fragments
had the lowest number of alleles (n ? 16), and the cya and pgm
fragments had the highest number of alleles (n ? 25). All
differences between alleles were due to point mutations; nei-
ther deletions nor insertions were observed. The ratio between
homologous and nonhomologous substitutions was between
0.0029 (cya) and 0.1971 (pgm) with a mean of 0.0769; thus,
most of the mutations were silent and did not generate nucle-
otide substitutions (Table 2). The maximum sequence diver-
gence between the 123 strains was greatest for argH (5.42%)
and lowest for pgm (2.02%); divergence exceeded 3% for four
genes: argH, cya, gnd, and murC.
TABLE 1. Primers used for PCR and sequencing
GenePrimer name Primer sequence
Amplified
fragment (bp)
Tm(°C)
Reference
or source
argHARGHF
ARGHSR1
5?-GACGAGGGCGACAGCTTC-3?
5?-GTGCGCGAGCAGATGATG-3?
629 60
58
36
cya ACF
ACSR1
5?-GTGAAGCGGGCCAAGAAG-3?
5?-AACTGGGAGGCCAGGAGC-3?
647 58
60
36
gdhAGDHAF
GDHASR1
5?-GTCAGTGCCCCGATCGCT-3?
5?-GGCTCTCGGAGTACGTCGA-3?
58260
60
This study
glpK GLPKSF1
GLPKSFR2
5?-AATCTCACCGGCGGTGTC-3?
5?-GGACAGACCCACGATGGC-3?
609 58
60
36
gnd GNDF
GNDSR1
5?-GTGACGTCGGAGTGGTTGG-3?
5?-CTTCGCCTCAGGTCAGCTC-3?
634 62
62
36
murC MURCSF1
MURCSR2
5?-CGGACGAAAGCGACGGCT-3?
5?-CCAAAACCCTGCTGAGCC-3?
60760
58
36
pgmPGMSF1
PGMSR2
5?-CCATTTGAACCCGACCGG-3?
5?-GTGCCAACGAGATCCTGCG-3?
59660
66
This study
pknAPKNAF
PKNASR1
5?-CAGGTGGACCTCGGACATG-3?
5?-AACCAGGCGCCCACCATC-3?
49362
60
This study
ptaPTASF1
PTASR2
5?-GATCGGGCGTCATGCCCT-3?
5?-ACGAGGCACTGCTCTCCC-3?
720 60
66
This study
purH PURHSF1
PURHSR2
5?-CGGAGGCTTCACCCTGGA-3?
5?-CAGGCCACCGCTGATCTG-3?
634 64
60
This study
rpoBMYCOF1
MYCOR2
5?-TCCGATGAGGTGCTGGCAGA-3?
5?-ACTTGATGGTCAACAGCTCC-3?
940 68
68
This study
VOL. 49, 2011 MLSA OF M. ABSCESSUS SENSU LATO493

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rpoB tree and polymorphism of rpoB sequences. The tree
built from the 123 rpoB sequences showed three distinct clus-
ters, each comprising the type strain of one species (Fig. 1).
According to Ade ´kambi et al. (2), these three clusters could be
equated with the species clusters M. abscessus (59 isolates), M.
bolletii (24 isolates), and M. massiliense (37 isolates). The mean
sequence divergence within each cluster was extremely low,
ranging from 0.36 to 0.41%. The mean divergence between the
M. abscessus and M. bolletii clusters was 4.38%, with all values
exceeding 3% (extremes were 3.59 to 4.65%), divergence be-
tween the M. abscessus and M. massiliense clusters was 3.40%,
with some values below 3% (extremes were 2.66 to 3.59%), and
that between M. massiliense and M. bolletii was only 1.68%, and
all values were below 3% (extremes were 1.33 to 2.13%). The
divergence values between type strain sequences were consis-
tent with these differences: 4.12% between M. abscessus CIP
104536Tand M. massiliense CIP 108297T, 3.32% between M.
abscessus CIP 104536Tand M. bolletii CIP 108541T, and 1.46%
between M. massiliense CIP 108297Tand M. bolletii CIP
108541T(Table 2).
Single-gene trees obtained with each of the MLSA genes. A
single-gene tree was built from the sequences for each of the
eight genes included in the final MLSA scheme. The trees built
using argH, cya, gnd, murC, pta, and purH sequences showed
three main branches, each carrying the type strain of one
species except for the purH tree (M. massiliense and M. bolletii
type strain sequences on the same branch) (Fig. 2). Bootstrap
values for the argH and murC genes were ?80%. However,
there was no tree in which each of the three clusters was made
up of strains assigned to a single species by rpoB sequencing
(Fig. 2). The resolution obtained with the glpK and pgm se-
quences was very poor, and this was consistent with the very
low polymorphism in these genes (as described above).
The divergence between type strain sequences was highest
with argH and gnd and lowest with glpK and pgm. With the
argH type sequences, divergence was 4.58% between M. ab-
scessus and M. massiliense and 3.54% between M. abscessus and
M. bolletii (3.54%), but it was only 2.92% between M. massil-
iense and M. bolletii. With the gnd type sequences, divergences
were 4.58% between M. abscessus and M. bolletii, 3.54% be-
tween M. massiliense and M. bolletii, and only 2.29% between
M. abscessus and M. massiliense (Table 2).
Trees obtained with concatenated sequences of the eight
MLSA genes. Figure 3A shows the tree obtained for the 123
strains by concatenating the sequences of the eight housekeep-
ing gene fragments (4,071bp). This tree shows three principle
clusters, each containing the sequence of only one of the three
type strains; it contains, as expected, the M. abscessus, M.
TABLE 2. Genes studiedc
Gene(s)
Size of
analyzed
fragment (bp)
%G?C
No. of
polymorphic
sites
No. of
alleles
dN/dS
Maximum
% of nt
divergencea
% of nt divergence between type strain sequencesb
MabsT versus
MmasT
MabsT versus
MbolT
MbolT versus
MmasT
argH
cya
glpK
gnd
murC
pgm
pta
purH
All MLSA genes
rpoB
480
510
534
480
537
495
486
549
66.8
68.2
62.7
65.2
68.6
62.2
66.1
65.6
65.7
65.6
40
31
25
33
39
29
20
31
19
25
16
20
16
25
20
21
79
22
0.1529
0.0029
0.0380
0.0307
0.1259
0.1900
0.0110
0.0502
ND
0.0827
5.47
3.62
2.48
4.73
4.44
2.02
2.74
2.98
2.85
4.79
4.58
1.96
1.69
2.29
2.05
1.41
1.85
1.82
2.19
3.32
3.54
1.96
1.31
4.58
3.54
1.41
1.65
2.00
2.43
4.12
2.92
2.75
0.94
3.54
3.35
0.81
2.26
0.18
2.01
1.46
4,071
752
248
43
aMaximum nucleotide divergence found between the 123 strains studied.
bM. abscessus CIP 104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T.
cND, not done; dN/dS, ratio of the rate of nonsynonymous substitutions (dN) to the rate of synonymous substitutions (dS); MabsT, M. abscessus type strain sequence;
MbolT, M. bolletii type strain sequence; MmasT, M. massiliense type strain sequence.
FIG. 1. Tree constructed from partial rpoB gene sequences. The
tree for all studied strains (n ? 123) was generated using the neighbor-
joining method. Bootstrap support values (%) are indicated for each
node. Species assignment of clinical isolates are according to the cri-
teria in Ade ´kambi et al. (2): f, M. abscessus; ●, M. massiliense; Œ, M.
bolletii; numbers in parentheses are the numbers of isolates if there are
two or more. Type strains:*, M. abscessus CIP 104536T;**, M. mas-
siliense CIP 108297T;***, M. bolletii CIP 108541T.
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massiliense, and M. bolletii clusters. Apart from a few excep-
tions, the M. abscessus cluster is compact, with a bootstrap
value of 100%. The respective bootstrap values for the M.
massiliense and M. bolletii branches are much lower at 71 and
65%, respectively. The M. massiliense cluster is more dis-
persed, with some sequences situated close to the other clus-
ters. One of these sequences is from the type strain CIP
108297T, which is not closely related to most of the other
sequences in the cluster. The M. bolletii cluster is more com-
pact, but some of its sequences are close to the main branch
point.
The mean nucleotide divergence values were 0.45% (range,
0 to 1.65%) within the M. abscessus cluster, 0.72% (0 to 1.25%)
within the M. massiliense cluster, and 0.64% (0 to 2.06%)
within the M. bolletii cluster. The mean divergence values be-
tween clusters were 2.17% (range, 1.13 to 2.58%) between the
M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%)
between the M. abscessus and M. bolletii clusters, and 2.28%
(0.86 to 2.68%) between the M. massiliense and M. bolletii
clusters. Divergence values for the MLSA-concatenated se-
quences from type strains were 2.19% between M. abscessus
and M. massiliense, 2.43% between M. abscessus and M. bolletii,
and 2.01% between M. massiliense and M. bolletii.
Effect of adding the rpoB sequence to the final MLSA
scheme. We studied the effect of adding the rpoB sequence to
the concatenated sequences of the eight MLSA genes. The
tree obtained (Fig. 3B, MLSA ? rpoB tree) was very similar to
the tree constructed from the MLSA gene sequences only
(MLSA tree). However, the bootstrap value for the M. massil-
iense branch was even lower (24 versus 71%). With a few
exceptions (see isolates 12 and 63), the distribution of isolates
into the three groups was very similar in the two trees (Fig. 3A
and B).
Discrepancies between MLSA data and rpoB-based identi-
fication. We compared the MLSA and rpoB sequences from
each of the 120 isolates using the type strain sequences for
reference. The results of this analysis were consistent for 110
isolates and inconsistent for 10 isolates (Table 3). The most
frequent inconsistency (n ? 7; isolates 18, 23, 46, 97, 103, 107,
and 134) was an MSLA sequence with 99.14 to 99.75% identity
to the M. massiliense type strain (versus ?98.03% identity to
the M. abscessus and M. bolletii type strains) and an rpoB
sequence similar to that of the M. abscessus type strain. Other
inconsistencies were an MLSA sequence similar to that of the
M. bolletii type strain, an rpoB sequence similar to that of the
M. abscessus type strain (isolate 144), an MLSA sequence
similar to that of the M. abscessus type strain, and an rpoB
sequence similar to that of the M. massiliense type strain (iso-
lates 71 and 121). Four of the isolates with an MLSA sequence
similar to that of M. massiliense and an rpoB sequence similar
to that of M. abscessus clustered together on the M. massiliense
FIG. 2. Trees constructed from the sequences of the eight individual genes included in the final MLSA scheme. The trees for all studied strains
(n ? 123) were generated using the neighbor-joining method. Bootstrap support values (%) at each of the nodes are indicated only for trees
showing well-defined clusters. Species assignment of clinical isolates according to the criteria of Ade ´kambi et al. (3): f, M. abscessus; ●, M.
massiliense; Œ, M. bolletii. Type strains:*, M. abscessus CIP 104536T;**, M. massiliense CIP 108297T;***, M. bolletii CIP 108541T.
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496 MACHERAS ET AL.J. CLIN. MICROBIOL.

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branch of the MLSA tree (Fig. 3, isolates 46, 97, 103, and 107).
These four isolates shared identical rpoB sequences.
DISCUSSION
Both the molecular identification and the taxonomy of M.
abscessus, M. massiliense, and M. bolletii currently are based
upon the partial sequencing of rpoB (2, 3, 16). However, sev-
eral studies recently have questioned the effectiveness of se-
quencing only the rpoB gene for the molecular identification of
these species (36, 59). More recently, doubts have been raised
about the taxonomic classification of M. massiliense and M.
bolletii (34). For the first time for this group of species, we used
an MLSA scheme with the aim of resolving these issues. We
compared the results obtained from using this scheme to those
obtained from the partial sequencing of rpoB.
The study of our collection of isolates with the chosen
MLSA scheme (eight housekeeping gene sequences, 4,071 bp)
clearly shows the existence of three principal groups, with each
containing the type strain of one of the three species (M.
abscessus CIP 104536T, M. massiliense CIP 108297T, and M.
bolletii CIP 108541T). At first sight, these groups can be termed
M. abscessus, M. massiliense, and M. bolletii. However, although
the M. abscessus branch is robust, the M. bolletii and M. mas-
siliense branches have relatively low bootstrap values (65 and
71%, respectively) and a diffuse appearance, particularly in the
M. massiliense branch. Furthermore, the current M. massiliense
type strain (CIP 108297T) does not cluster closely with the
more tightly grouped isolates in the M. massiliense branch.
Sequence analysis of the fragment of the rpoB gene, de-
scribed by Ade ´kambi as the gold standard for the molecular
diagnosis of RGM infection (3), also was very informative. Our
study is the first to use this approach with a large and diverse
collection of isolates. We found that the isolates can indeed
be divided into three distinct groups, each clustered around
one of the three type strains. However, the divergence between
the groups casts doubts about the extent to which these groups
are different species according to the criteria of Ade ´kambi (i.e.,
?3% rpoB sequence divergence between two RGM species)
(2, 5). This was most marked for the M. massiliense and M.
bolletii groups and for the M. massiliense and M. bolletii type
strains. The most different rpoB sequences in the M. massil-
iense and M. bolletii groups strains diverged by only 2.13%, and
the divergence between the rpoB sequences of the M. massil-
iense and M. bolletii type strains (not reported by Ade ´kambi et
al. [2, 5]) was only 1.46%. Therefore, according to the criteria
of Ade ´kambi et al., M. massiliense and M. bolletii do not con-
stitute different species (3).
The divergence of rpoB between M. massiliense/M. bolletii
and M. abscessus similarly raises doubts about whether M.
massiliense and M. bolletii are species distinct from M. absces-
sus. The divergence of the rpoB sequences between the M.
abscessus and M. massiliense type strains and between the M.
abscessus and M. bolletii type strains were more than 3% (4.12
and 3.32%, respectively). However, in our collection, the di-
vergence threshold of 3% was exceeded between the M. ab-
scessus and M. bolletii groups (values from 3.59 to 4.65%) but
not between the M. abscessus and M. massiliense groups (values
from 2.66 to 3.59%). As M. massiliense and M. bolletii appear
not to be entirely separate species, we compared the M. mas-
siliense/M. bolletii group to the M. abscessus group: the diver-
gence values were between 2.66 and 4.79%. Therefore, the
combined M. massiliense/M. bolletii group and the M. abscessus
group are not entirely separate species. These findings indicate
the need for the complete revision of the current taxonomic
classification of M. abscessus sensu lato; as recently proposed
FIG. 3. Trees constructed from concatenated sequences. (A) Concatenated MLSA sequences. (B) Concatenated MLSA ? rpoB sequences. The
trees for all studied strains (n ? 123) were generated by using the neighbor-joining method. Bootstrap support values (%) are indicated for each
node. Each isolate is indicated by its number in our collection. CIP type strains also are indicated. Boxes indicate isolates with discordant
rpoB-based identification (see Table 3 for further details). Note that isolates 12 and 63 are located on the M. bolletii branch of the MLSA tree
(A) and on the M. massiliense branch of the MLSA ? rpoB tree (B).
TABLE 3. Isolates with discordant concatenated MLSA sequence and rpoB sequencec
Isolate
no.
% of nt identity with type strain sequence
MLSA sequencea
rpoB sequence
MabsTMbolT MmasTMabsTMbolTMmasT
18
23
46b
97b
103b
107b
134
144
71
121
98.03
97.89
97.69
97.69
97.69
97.67
97.84
97.96
98.85
99.04
97.99
97.62
97.86
97.86
97.86
97.84
97.57
99.31
97.89
97.35
99.75
99.14
99.19
99.19
99.19
99.16
99.34
98.01
98.18
97.94
100
100
99.34
99.34
99.34
99.34
99.73
99.87
96.68
96.68
95.88
95.88
96.28
96.28
96.28
96.28
96.14
96.01
98.54
98.54
96.68
96.68
97.34
97.34
97.34
97.34
96.81
96.68
99.73
99.73
aConcatenated MLSA gene sequences (4,071 bp).
bAlso see Fig. 3.
cMabsT, M. abscessus type strain sequence; MbolT, M. bolletii type strain sequence; MmasT, M. massiliense type strain sequence. Boldface numbers indicate
discordant species identification by either MLSA or rpoB analysis for a given isolate.
VOL. 49, 2011MLSA OF M. ABSCESSUS SENSU LATO497

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by Leao et al. (34), M. abscessus, M. massiliense, and M. bolletii
should comprise a single species (M. abscessus).
The other objective of our study was to evaluate the use of
rpoB gene sequencing for the molecular identification of iso-
lates and discrimination between the different groups of M.
abscessus sensu lato. Several recent studies have questioned
the reliability of approaches based solely on rpoB sequencing
(31, 34, 47, 48). This is because of the possible horizontal
transfer of the rpoB gene between the different groups of M.
abscessus sensu lato, especially from the M. abscessus group to
the M. massiliense group. Our comparative analysis of the data
for rpoB and MLSA confirmed that such horizontal transfer
has occurred: almost 10% of the isolates studied had discor-
dant MLSA and rpoB sequences. The most frequent situation
was isolates having an rpoB sequence belonging to the M.
abscessus group and an MLSA sequence belonging to the M.
massiliense group. Thus, rpoB sequencing can identify the re-
vised M. abscessus species (formerly M. abscessus sensu lato)
but cannot discriminate 100% between M. abscessus, M. mas-
siliense, and M. bolletii. We recently have shown that other
potential targets for the molecular identification of RGM
strains, such as hsp65 and sodA, also can be horizontally trans-
ferred within the M. abscessus sensu lato groups (36). We also
found evidence of lateral transfer events involving the recently
proposed target secA (unpublished data). If it were useful to
distinguish between the M. abscessus, M. massiliense, and M.
bolletii groups, because of clinical or epidemiological features
specific to the infectious agent, for example, it would be valu-
able to identify other targets or combinations of targets to
overcome the problem of horizontal gene transfer between
these groups. Our team currently is addressing this issue.
This study provides information relevant to the phylogeny of
M. abscessus sensu lato. We show the existence of three groups,
M. abscessus, M. bolletii, and M. massiliense. Of these, the M.
massiliense group seems to have emerged most recently. Our
findings also reveal substantial horizontal gene transfer be-
tween the three groups, with a particularly marked flow of rpoB
from M. abscessus to M. massiliense. Some transfer from M.
abscessus to M. massiliense may be recent, as a small subgroup
of isolates in the M. massiliense group (obtained from respira-
tory samples in France and Brazil) had an rpoB sequence that
was almost 100% identical to the M. abscessus CIP 104536T
sequence. Although the three groups are not entirely separate
species, our data suggest that each group evolves in its own
way, maintaining a certain cohesion, and that genetic material
is exchanged between the groups, most likely via phages (30,
43, 44). The fact that they exchange genetic material indicates
that they share similar biotopes, but their distinctness indicates
a degree of specialization of each group. However, almost all
of the isolates that we tested were from clinical samples; few
M. abscessus sensu lato isolates from the environment are
available (see below). We therefore cannot totally exclude
sampling bias toward human pathogenic strains.
The analysis of the M. abscessus CIP 104536Tgenome by our
group has improved our understanding of the microorganism’s
natural lifestyle. Genes in the M. abscessus CIP 104536Tge-
nome also are found in bacteria living in soil or aquatic envi-
ronments, probably in close contact with plants. However, M.
abscessus also contains a large number of genes known to be
involved in intracellular parasitism, suggesting that it may have
evolved to escape free-living amoebas (1, 5, 9); this would
explain why M. abscessus is rarely isolated from soil or water,
although there is general agreement that it lives in such envi-
ronments. We have launched an extensive investigation of the
presence of RGM in the water treatment systems in the Paris
region, a region where respiratory infections involving M. ab-
scessus are particularly prevalent in at-risk populations. Our
MLSA approach will be valuable for comparing the population
structures of M. abscessus sensu lato isolates obtained in this
survey to those of clinical strains.
ACKNOWLEDGMENTS
We warmly thank Erick Denamur (INSERM U722, Paris, France)
for helpful discussions and Cristianne Kayoko Matsumoto for the
characterization of the Brazilian isolates.
We thank the association Vaincre la Mucoviscidose for the financial
support of this work.
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