Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-Mycobacterium abscessus group isolates.
ABSTRACT Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species (M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M. abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n = 1), 51.2% (n = 65), 46.5% (n = 59), and 1.6% (n = 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M. massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin had a point mutation at the adenine at position 2058 (A(2058)) or 2059 (A(2059)) in the peptidyltransferase region of the 23S rRNA gene.
- [Show abstract] [Hide abstract]
ABSTRACT: Bacteria of the Mycobacterium abscessus group are the second most common pathogens responsible for lung disease caused by nontuberculous mycobacteria in Korea. There is still a lack of studies investigating the genetic mechanisms involved in M. abscessus resistance to antibiotics other than clarithromycin. This study investigated the characteristics of drug resistance exhibited by M. abscessus clinical isolates from Korea. We performed drug susceptibility testing for a total of 404 M. abscessus clinical strains. Subspecies were differentiated by molecular biological methods and examined for mutations in drug resistance-related genes. Of the 404 strains examined, 202 (50.00%), 199 (49.26%), and 3 (0.74%) strains were identified as M. abscessus, M. massiliense, and M. bolletii, respectively. Of the 152 clarithromycin-resistant strains, 6 possessed rrl mutations, while 4 of the 30 amikacin-resistant strains contained rrs mutations, and 5 of the 114 quinolone-resistant strains had gyr mutations. All mutant strains had high minimal inhibitory concentration values for the antibiotics. Our results showed the distribution of the strains with mutations in drug resistance-related genes was low in the M. abscessus group. Furthermore, we performed drug susceptibility testing and sequence analyses to determine the characteristics of these genes in the M. abscessus group.Annals of Laboratory Medicine 01/2014; 34(1):31-7. · 1.48 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Mycobacterium massiliense which is recognized as a separate species from M. abscessus is little known regarding its clinical patterns and the response to treatment. We present a case of a localized cutaneous infection due to M. massiliense of the sole associated with acupuncture. M. massiliense was identified via polymerase chain reaction-hybridization analysis. We treated the patient with single-drug therapy consisting of clarithromycin for 4 months and the patient showed a significant response to this treatment.Annals of Dermatology 02/2014; 26(1):92-5. · 0.61 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Mycobacterium abscessus and M. massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time-consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and two M. bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S–23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group.Diagnostic microbiology and infectious disease 01/2014; · 2.45 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2008, p. 3384–3390
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 10
Proportions of Mycobacterium massiliense and Mycobacterium bolletii
Strains among Korean Mycobacterium chelonae-Mycobacterium abscessus
Hee-Youn Kim,1Yoonwon Kook,2Yeo-Jun Yun,1Chan Geun Park,1Nam Yong Lee,3Tae Sun Shim,4
Bum-Joon Kim,1and Yoon-Hoh Kook1*
Department of Microbiology, Cancer Research Institute, Institute of Endemic Diseases, SNUMRC, Seoul National University College of
Medicine, and Clinical Research Institute, Seoul National University Hospital, Seoul 110-799,1Life Science and Technology,
Underwood International College, Yonsei University, Seoul,2Department of Laboratory Medicine, Samsung Medical Center,
Sungkyunkwan University School of Medicine, Suwon,3and Division of Pulmonary and
Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center,
Seoul,4Republic of Korea
Received 15 February 2008/Returned for modification 23 April 2008/Accepted 19 August 2008
Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from
two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative
sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species
(M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains
tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M.
abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n ? 1), 51.2% (n ? 65), 46.5% (n ? 59), and 1.6%
(n ? 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis
and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional
analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified
by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M.
massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus
and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin
had a point mutation at the adenine at position 2058 (A2058) or 2059 (A2059) in the peptidyltransferase region
of the 23S rRNA gene.
Rapidly growing mycobacteria (RGM), which include mem-
bers of the Mycobacterium fortuitum complex, the M. chelonae
complex, and the M. smegmatis complex, are defined as myco-
bacteria that grow and form visible colonies on solid agar
media within 7 days. Because of their low virulence, they usu-
ally cause respiratory tract or disseminated infections in per-
sons with predisposing factors or who are immunosuppressed
(27, 29, 32). However, recently, infections in immunocompe-
tent persons have been reported more frequently. These cases
are soft-tissue infections associated with trauma and injection
and epidemics or pseudoepidemics that have occurred in hos-
pitals (6, 12). Ninety-five percent of soft-tissue RGM infections
are caused by members of the M. chelonae complex (7), which
comprises M. chelonae, M. abscessus, and M. immunogenum.
However, the situation is complex for the clinical microbiolo-
gist who must rapidly and correctly identify isolates because
there are reports of heterogeneity in M. abscessus isolates. Two
M. abscessus groups (groups I and II) were reported on the
basis of hsp65 PCR-restriction fragment length polymorphism
(RFLP) (11) and sequence analysis (21) of hsp65. High hetero-
geneity of M. abscessus isolates was also shown by rpoB analysis
(2). Meanwhile, new RGM species were reported: M. massil-
iense (4) and M. bolletii (1). These were very closely related to
M. abscessus and M. chelonae but showed different susceptibil-
ity to clarithromycin, and their pathogenic potentials were
shown by infections in immunocompetent and immunocom-
promised hosts (13, 19).
RGM infections are also common in South Korea (24, 27,
34). M. abscessus is the most frequently isolated nontubercu-
lous mycobacterium (NTM) in South Korea, second to the M.
avium complex (20), in contrast to other countries such as the
United States, Japan, and Sweden (9, 28, 39). In addition,
there was an epidemic associated with intramuscular injection
of an antimicrobial agent, which seemed to be caused by M.
abscessus, in 2005 at Icheon, South Korea. However, the caus-
ative microorganism was finally identified as M. massiliense
based on rpoB and hsp65 sequence analysis (19). Thus, because
of the close relationship between M. abscessus and two recently
reported species, M. massiliense and M. bolletii, we speculated
that these were not newly found in the mycobacterial popula-
tions but rather had been collectively identified as M. abscessus
or M. chelonae-M. abscessus group species in previous studies.
In the present study, we investigated several issues related to
the heterogeneity of the M. chelonae-M. abscessus group. One
is the isolation rates of four closely related species, M. chelo-
nae, M. abscessus, M. massiliense, and M. bolletii in Korean
clinical isolates. The others are colony morphology and sus-
ceptibility to clarithromycin. The first might be related to vir-
* Corresponding author. Mailing address: Department of Microbi-
ology, Seoul National University College of Medicine, 28 Yongon-
dong, Chongno-gu, Seoul 110-799, South Korea. Phone: (82) 2-740-
8306. Fax: (82) 2-743-0881. E-mail: firstname.lastname@example.org.
?Published ahead of print on 27 August 2008.
ulence (8, 14), and the last is important for treating M. absces-
sus infection (7). We reidentified a group of M. abscessus
isolates (Asan Collection) that had been previously isolated
and identified by hsp65 PCR restriction analysis (PRA) and
prospectively identified another group of RGM isolates (Sam-
sung Collection), which were mainly isolated from respiratory
specimens collected over 2 years, using hsp65 and rpoB se-
quence analysis (19, 38).
MATERIALS AND METHODS
Bacterial strains. Two different groups of RGM isolates obtained from two
hospitals in Seoul, South Korea, were used. One group (42 strains of M. absces-
sus), which had been previously isolated from patients with respiratory infection
and identified by hsp65 PRA, was provided by Tae Sun Shim, Asan Medical
Center, South Korea. These isolates were used for reidentification. The other
group comprised 102 RGM strains isolated from 99 patients with respiratory
infection and was provided by Nam Yong Lee, Samsung Medical Center. Isolates
in the Samsung Collection were collected from June 2005, just after recognition
of an injection-associated M. massiliense epidemic (19), until May 2007. The type
strains of M. abscessus (ATCC 19977), M. massiliense (CIP 108297), and M.
bolletii (CIP 108541) were used for comparison. RGM isolates cultivated on
Ogawa media were subcultured on blood agar plates at 37°C and 5% CO2for 4
days to observe colony morphology and purity and then used for species iden-
tification based on hsp65 PRA (11) and PCR sequencing of rpoB (19) and hsp65
DNA extraction and PCR. Total DNAs were extracted from cultured colonies
using the bead beater-phenol extraction method (17) and used as templates for
PCR. The following primer pairs were used: 285 (5?-GAG AGT TTG ATC CTG
GCT CAG-3?) and 244 (5?-CCC ACT GCT GCC TCC CGT AG-3?) for 16S
rRNA gene (351 bp) (36), RGMF (5?-GAC GAC ATC GAC CAC TTC GG-3?)
and RGMR (5?-GGG GTC TCG ATC GGG CAC AT-3?) for rpoB PCR (365
bp) (19), and Tb11 (5?-ACC AAC GAT GGT GTG TCC AT-3?) and Tb12
(5?-CTT GTC GAA CCG CAT ACC CT-3?) for hsp65 (441 bp) (38). Template
DNA (ca. 50 ng) and 20 pmol of each primer were added to a PCR mixture tube
(AccuPower PCR PreMix; Bioneer, Daejeon, South Korea) containing 1 unit of
Taq DNA polymerase, 250 ?M of deoxynucleotide triphosphate, 10 mM Tris-
HCl (pH 8.3), 10 mM KCl, 1.5 mM MgCl2, and gel loading dye. The final volume
was adjusted to 20 ?l with distilled water, and the reaction mixture was then
amplified as previously described (19, 36, 38) using a model 9700 Thermocycler
(Perkin-Elmer Cetus). Sequencing of sodA and the 16S-23S rRNA gene internal
transcribed spacer (16S-23S ITS) and 23S rRNA gene sequencing were per-
formed to resolve discrepancies in identification by rpoB and hsp65 analysis and
to identify mutations associated with clarithromycin resistance, respectively (3,
hsp65 PRA. PRA was performed as previously described (11). Briefly, 10 ?l of
the amplified PCR products was transferred to fresh microcentrifuge tubes and
digested with HaeIII (TaKaRa, Shiga, Japan) or BstEII (Promega, Madison, WI)
according to the supplier’s instructions. Following digestion, the mixtures were
electrophoresed on 3% agarose gel (at 100 V for 30 min). The DNA bands were
visualized by ethidium bromide staining and photographed. For those strains that
did not show clear PRA results, sizing of the DNA fragments on the basis of their
determined hsp65 sequences was performed by MapDraw (version 3.14;
DNASTAR, Madison, WI) and compared to the previous report (11).
Nucleotide sequencing. PCR products were purified using Qiaex II gel extrac-
tion kits (Qiagen, Hilden, Germany) and were sequenced directly using forward
and reverse primers with an Applied Biosystems automated sequencer (model
377) and BigDye Terminator cycle sequencing kits (Perkin-Elmer Applied Bio-
systems, Warrington, United Kingdom). Both strands were sequenced as a cross-
Sequence analysis. Determined partial 16S rRNA gene, rpoB, and hsp65
sequences (306 bp, 306 bp, and 401 bp, respectively) were aligned using the
ClustalW algorithm in MEGA4 (37). Phylogenetic trees were inferred from
partial rpoB and hsp65 nucleotide sequences of Korean isolates in this study, and
those of RGM type strains were retrieved from GenBank using the neighbor-
joining method in MEGA4 (37) and median network in SplitsTree4 (15).
Susceptibility testing for clarithromycin. MICs of clarithromycin were deter-
mined by the broth dilution method in microtiter plates (16) with slight modifi-
cation. Nine randomly selected strains of M. abscessus, nine strains of M. mas-
siliense, and two M. bolletii isolates were used for clarithromycin susceptibility
testing. M. abscessus ATCC 19977T, M. massiliense CIP 108297T, and M. bolletii
CIP 108541T, whose susceptibilities to clarithromycin are known, were used as
controls. Briefly, pure cultured colonies were transferred to 7H9 broth (Becton
Dickinson, Piscataway, NJ) with 0.02% Tween 80 (Junsei Chemical, Tokyo,
Japan) and 0.2% glycerol (Sigma) in a tube and vigorously vortex mixed to a
density equivalent of 0.5 on the McFarland scale. Bacterial suspensions (150 ?l)
were transferred to the wells of microtitration plates, which contained clarithro-
mycin (Boryung, Seoul, South Korea) (150 ?l) solubilized in 20% dimethyl
sulfoxide and twofold serially diluted with 7H9 broth from 256 to 0.06 ?g/ml. The
plates were incubated at 37°C for 72 h. Bacterial growth was observed, and
the MICs were determined. The susceptibility was determined according to the
breakpoints recommended by the Clinical and Laboratory Standards Institute
Separately, the 23S rRNA gene sequences of these 20 isolates and 3 type
strains were analyzed (25) to observe any point mutation at the adenine at
position 2058 (A2058) or at A2059in the peptidyltransferase region of the 23S
Nucleotide sequence accession numbers. Determined rpoB and hsp65 se-
quences that were different from reference strains have been deposited in
GenBank under accession no. EU732712 to EU732723.
Identification by hsp65 PRA. Of the 42 reidentified M.
abscessus strains (Asan Collection), 28 (66.7%) and 14 strains
(33.3%) belonged to M. abscessus groups I and II, respectively
(Table 1). Of the 102 strains in the Samsung Collection, 85
strains (83.3%) belonged to the M. chelonae-M. abscessus
group, and these included 37 (36.2%) strains of M. abscessus
group I, 47 strains (46.1%) of M. abscessus group II, and 1
strain (1.0%) of M. chelonae (Table 1). In addition, M. fortui-
tum (nine strains, 8.8%), M. peregrinum (four strains, 3.9%),
and M. porcinum (three strains, 2.9%) were identified among
the remaining mycobacteria.
Identification by gene sequence analysis. Species identifica-
tion of the RGM strains was accomplished by BLAST search
to measure the similarities and infer the constructed phyloge-
netic trees. The M. chelonae-M. abscessus group (42 Asan and
85 Samsung strains) identified by hsp65 PRA were separated
into four different species (M. chelonae, M. abscessus, M. mas-
siliense, and M. bolletii) (Table 2). Based on the rpoB sequence
analysis, all of the M. abscessus group I strains (65 strains) were
identified as M. abscessus. Their rpoB sequence similarities to
the reference sequences were 99.3 to 100%. The 61 M. absces-
sus group II strains were identified as M. massiliense (59
strains) and M. bolletii (2 strains). These strains showed rpoB
sequences identical to those of M. massiliense and M. bolletii,
respectively, but relatively low (97.3 to 98.7%) sequence sim-
ilarity to that of the M. abscessus reference strain. Interestingly,
two strains (1.4%) showed discordant results between rpoB
and hsp65 sequence analysis. These strains were M. massiliense
based on the rpoB sequences but were M. abscessus based on
TABLE 1. Distribution of M. chelonae-M. abscessus group isolates
identified by hsp65 PCR restriction analysis in this study
Species and group
No. (%) of strains in
Total no. of
VOL. 46, 2008ISOLATION RATE OF M. MASSILIENSE AND M. BOLLETII 3385
hsp65 sequences analysis. However, gene analysis of sodA (441
bp) and the 16S-23S ITS (216 bp) indicated that these two
strains were M. massiliense (similarity of 100%) (Table 2).
The other 17 mycobacterial strains were identified as M.
fortuitum (7 strains), M. peregrinum (4 strains), M. porcinum (2
strains), M. senegalense (1 strain), and M. mageritense (1 strain)
by rpoB and hsp65 sequence analysis. However, two strains
could not be identified exactly. One of these two strains was
genetically close to M. septicum (rpoB, 98.7%; hsp65, 98.3%),
and one was close to M. peregrinum (rpoB, 98.7%; hsp65,
98.3%). By additional 16S rRNA gene and ITS sequence anal-
ysis these were finally identified as M. septicum and M. pereg-
rinum (Table 2).
The relationships between tested RGM isolates and the type
strains of M. chelonae, M. abscessus, M. immunogenum, M.
massiliense, and M. bolletii are shown by the rpoB and hsp65
trees (Fig. 1). The M. chelonae-M. abscessus group formed
distinct clusters in both trees, showing their high similarity in
terms of rpoB and hsp65 sequences. All the M. abscessus group
I and II strains were separated from M. chelonae and M. im-
munogenum by long branches and formed two clusters (groups
I and II in Fig. 1). The M. abscessus type strain and isolates
formed group I, whereas M. massiliense and M. bolletii type
strains and isolates formed group II. All the strains of hsp65
PRA groups I and II belonged to groups I and II in both trees.
The bootstrap values supporting their relationships were high
(87 and 98% for each tree).
Colony morphology. Subcultured colonies of 85 M. chelo-
nae-M. abscessus group isolates (Samsung Collection) were
analyzed on blood agar plates. In general, isolates with rough
colonies (52 strains, 61.2%) were more frequently isolated than
those with smooth colonies (24 strains, 28.2%). The propor-
tions of smooth colonies in M. abscessus and M. massiliense
isolates were similar (27.1% and 28.2%, respectively). Most of
the isolates showed one of the two types. However, mixed
colonies of seven strains (8.2%) of M. abscessus from seven
different patients were observed (Table 3).
Susceptibility to clarithromycin. MICs for randomly se-
lected M. abscessus (9 strains), M. massiliense (9 strains), and
M. bolletii isolates (2 strains) were compared with those for
their type strains. MICs for the 10 M. abscessus strains ranged
from 0.25 to 64 ?g/ml. Four susceptible, four resistant, and two
intermediate strains were determined on the basis of CLSI
criteria (10). Three M. bolletii strains were resistant to cla-
rithromycin (MICs, 8 to 16 ?g/ml). Interestingly, M. massil-
iense strains were either markedly susceptible (MICs, 0.125 to
0.5 ?g/ml) or highly resistant (MICs, ?256 ?g/ml) to clarithro-
mycin. There was no intermediate group as for M. abscessus. In
addition, three highly resistant M. massiliense strains had a
point mutation at the adenine at position 2058 (A2058) or 2059
(A2059) in the peptidyltransferase region of the 23S rRNA
gene. However, those mutations were not found in the four
clarithromycin-resistant isolates of M. abscessus and two M.
bolletii isolates tested in this study (Table 4).
Recently, reports on the rapidly growing mycobacterial in-
fections in various clinical situations have greatly increased.
Among the RGM species, the M. chelonae-M. abscessus group
is the most common causative agent of soft-tissue infections
(7). Though there have been continuous taxonomic changes of
M. chelonae and M. abscessus, M. abscessus is not considered a
subspecies of M. chelonae (26) but rather a separate species
(22). However, another genotype analysis, such as hsp65 PRA
(11), and sequence analysis of hsp65 (21) and rpoB (2) again
revealed genetic heterogeneity in the M. abscessus population.
Furthermore, making things more complex, M. massiliense and
M. bolletii were newly reported and are very closely related to
M. abscessus (1, 4). Human infections and intracellular sur-
vival, which showed their pathogenic potential, were reported
(1, 4, 40).
When we used sequence analysis to identify mycobacteria,
we also found several RGM isolates that were closely related
but could not be identified as M. abscessus or M. chelonae. In
addition, after the identification of the causative agent in an
epidemic associated with intramuscular injection (19), the pos-
sibility that M. massiliense and M. bolletii could have been
identified as M. abscessus was raised. By confirming that M.
abscessus hsp65 PRA group II strains are M. massiliense by
sequence analysis, we could in part simplify the heterogeneous
structure of the M. abscessus population in this study. M. mas-
siliense accounted for a large proportion of clinical RGM iso-
lates. Nearly one-half of M. abscessus isolates could not be
properly identified prior to the different phenotype and geno-
type reports on M. massiliense and M. bolletii as new species.
The rate of isolation of M. massiliense in South Korea is much
higher than that in a recent report (33). It was not evaluated by
a nationwide surveillance study. However, because the two
groups of clinical isolates used in this study were collected from
two different hospitals (Asan and Samsung) where the NTM
infections are mainly reported (18, 20, 23), our results may in
part represent the proportions of M. chelonae, M. abscessus, M.
massiliense, and M. bolletii isolates in South Korea.
TABLE 2. Comparison of identification results for M. chelonae-M. abscessus group isolates using hsp65 PCR-RFLP and
sequence analysis of rpoB and hsp65
Species and group by
Result by PCR sequencing of:
Species No. (%)Species No. (%)
M. abscessus group I
M. abscessus group II
3386 KIM ET AL.J. CLIN. MICROBIOL.
In order to manage infectious diseases, it is important to
correctly identify the causative agents and to know their sus-
ceptibility to antimicrobial agents since different treatment
regimens are required for each infectious disease. Although
several unique phenotypes that can differentiate M. massiliense
and M. bolletii from M. abscessus and M. chelonae have been
identified (1, 4, 22), they are not easy to characterize in routine
clinical laboratories. In general, molecular methods are pre-
FIG. 1. Relationships between the type strains (●) of M. chelonae, M. immunogenum, M. abscessus, M. massiliense, and M. bolletii and 127 Korean M.
chelonae-M. abscessus group isolates (Asan, Asan Collection; RGM, Samsung Collection) inferred from partial rpoB (A) and hsp65 sequences (B). Trees
were constructed by the neighbor-joining (NJ) method (left) and median network analysis (right). Most of the isolates, which were identified as M.
whereas group II comprises M. massiliense and M. bolletii type strains and isolates identified as such. Numbers in parentheses stand for isolates with
identical sequences. In both NJ trees, M. tuberculosis (rpoB, AF057454; hsp65, AY299144) was used as the out-group to root the tree and the bootstrap
values presented at corresponding branches were determined from 1,000 replications; those less than 50% are not indicated.
VOL. 46, 2008ISOLATION RATE OF M. MASSILIENSE AND M. BOLLETII3387
ferred over phenotype analysis. For genotype identification of
these causative agents, PCR or PCR-linked methods have
been used. Although PRA is a rather simple and rapid method,
PCR sequencing is more useful for species identification, as
shown above. Since the early 1990s, genotype analysis, espe-
cially PCR sequencing, has greatly contributed to the classifi-
cation and identification of mycobacterial species. Nucleotide
sequence information for the 16S rRNA genes has been widely
used to find and define new species of mycobacteria (31, 35).
However, 16S rRNA gene sequence analysis has critical limi-
tations in that the pathogenic M. kansasii cannot be distin-
guished from nonpathogenic M. gastri. In the first report on M.
massiliense, because its 16S rRNA gene sequence was identical
to that of M. abscessus, other gene sequences (hsp65, rpoB, and
16S-23S ITS, etc.) were also presented to demonstrate the
genotype difference between the two species. Thus, we did not
apply 16S rRNA gene sequence analysis in the early stage of
Interestingly, two strains showed discordant results in the
rpoB and hsp65 analysis. Two M. massiliense strains based on
rpoB analysis were identified as M. abscessus by hsp65 analysis
as described in a previous report (33). However, additional
gene (sodA and 16S-23S ITS) analyses indicated that these two
strains were M. massiliense. In such situations, although these
strains may represent a very small proportion (1.4%), comple-
mentation with other gene analyses may be useful for precise
identification. Either sodA or the 16S-23S ITS may be suitable
for this purpose because these are frequently used and there
are abundant sequence data.
Aside from the precise identification of M. abscessus and its
related species, what led to our interest in these four species
was their susceptibility to clarithromycin, which is administered
orally. Among the many antimicrobial susceptibility profiles,
an especially low MIC of clarithromycin (0.125 ?g/ml) was
observed for M. massiliense (4, 19). Based on the susceptibility
testing, clarithromycin was used to treat patients who suffered
injection-associated abscess formation without any combina-
tion (19). In addition, M. bolletii was reported to be naturally
resistant to clarithromycin (1). Although we tested only 23
strains and more-comprehensive studies are needed, we ob-
served some interesting results. Some of our results were con-
cordant with previous reports in that seven M. massiliense
strains, which were in the susceptible range of CLSI criteria,
showed very low MICs (0.125 to 0.5 ?g/ml). In addition, al-
though only three strains were used, all M. bolletii strains were
resistant to clarithromycin (MICs, 8 to 16 ?g/ml). However,
unlike the three highly resistant (MICs ? 256 ?g/ml) M. mas-
siliense strains that had a mutation (A2058or A2059) in the 23S
rRNA gene, there was no mutation in resistant M. abscessus
and M. bolletii strains. Furthermore, there was an “intermedi-
ate” group among M. abscessus strains. These results suggest
that there are other mechanisms besides 23S rRNA gene mu-
tation that confer clarithromycin resistance in these strains.
Reports of a broad range of MICs (0.125 to 64 ?g/ml) and of
clarithromycin-resistant M. abscessus (41, 42) might have been
brought about, in part, by such heterogeneity in the M. absces-
sus population, which comprises three strains with different
susceptibilities to clarithromycin. Because we randomly se-
lected strains and analyzed only two strains of M. bolletii in this
study, we cannot comprehensively know the exact clarithromy-
cin resistance rate of these three species at this moment. In
addition, we cannot generalize that all M. bolletii strains are
naturally resistant to clarithromycin. Thus, because the suscep-
tibility depends on each strain, every isolate should be sub-
jected to susceptibility testing.
The pathogenicity of M. massiliense was carefully described
in an earlier report (4). We also suggest two lines of evidence
that M. massiliense is pathogenic. First, 46 M. massiliense iso-
lates of the Samsung Collection were collected from 99 pa-
tients over almost 2 years. There were 19 patients who gave
repeated culture-positive results (from two to seven times).
Although detailed information on these patients is not avail-
able and is out of the scope of this study, repeated positive
cultures from respiratory specimens fulfill the basic criteria for
diagnosis of NTM infection (5). Second, repeated incision and
drainage could not eradicate M. massiliense in patients in the
“injection-associated epidemic” (19). This reminds us of the
intra-amoebal survival and growth of M. massiliense. If this
mycobacterium can survive in tissue macrophages, simple
TABLE 3. Morphological variation of colonies observed among 85
M. chelonae-M. abscessus group isolates
No. (%) of strains
TABLE 4. Susceptibility of M. chelonae, M. abscessus, and
M. bolletii isolates to clarithromycin
Species (n) Strain
M. abscessus (10) ATCC19977T
M. bolletii (3) CIP 108541T
aS, susceptible; R, resistant; I, intermediate.
3388KIM ET AL.J. CLIN. MICROBIOL.
curettage may not be sufficient to eradicate it. Intracellular
survival and growth can play an important role, as in tubercu-
In conclusion, we evaluated the proportion of M. chelonae,
M. abscessus, M. massiliense, and M. bolletii strains among
Korean M. chelonae-M. abscessus group isolates by PCR se-
quencing and compared these results to hsp65 PCR-RFLP
results. M. massiliense and M. bolletii accounted for the M.
abscessus group II strains identified by hsp65 PRA and ac-
counted for 46.5% and 1.6% of the M. chelonae-M. abscessus
group strains analyzed, respectively. Repeated positive cul-
tures from the patient’s respiratory infection specimens sug-
gest that there are considerable numbers of patients with M.
massiliense pulmonary infection, which may be diagnosed as M.
abscessus (or M. chelonae-M. abscessus group) infection, in
This work was supported by a 2007 grant from the SNUH Research
fund (04-2007-012-0). H.-Y. Kim, Y.-J. Yun, and C. G. Park were
supported by the second stage of the Brain Korea 21 Project.
1. Ade ´kambi, T., P. Berger, D. Raoult, and M. Drancourt. 2006. rpoB gene
sequence-based characterization of emerging non-tuberculous mycobacteria
with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocai-
cum sp. nov. and Mycobacterium aubagnense sp. nov. Int. J. Syst. Evol.
2. Ade ´kambi, T., P. Colson, and M. Drancourt. 2003. rpoB-based identification
of nonpigmented and late-pigmenting rapidly growing mycobacteria. J. Clin.
3. Ade ´kambi, T., and M. Drancourt. 2004. Dissection of phylogenetic relation-
ships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65,
sodA, recA and rpoB gene sequencing. Int. J. Syst. Evol. Microbiol. 54:2095–
4. Ade ´kambi, T., M. Reynaud-Gaubert, G. Greub, M. J. Gevaudan, B. La Scola,
D. Raoult, and M. Drancourt. 2004. Amoebal coculture of “Mycobacterium
massiliense” sp. nov. from the sputum of a patient with hemoptoic pneumo-
nia. J. Clin. Microbiol. 42:5493–5501.
5. American Thoracic Society. 1997. Diagnosis and treatment of disease caused by
nontuberculous mycobacteria. Am. J. Respir. Crit. Care Med. 156:S1–S25.
6. Brantley, J. S., A. L. Readinger, and E. S. Morris. 2006. Cutaneous infection
with Mycobacterium abscessus in a child. Pediatr. Dermatol. 23:128–131.
7. Brown-Elliott, B. A., and R. J. Wallace, Jr. 2002. Clinical and taxonomic
status of pathogenic nonpigmented or late-pigmenting rapidly growing my-
cobacteria. Clin. Microbiol. Rev. 15:716–746.
8. Catherinot, E., J. Clarissou, G. Etienne, F. Ripoll, J. F. Emile, M. Daffe ´, C.
Perronne, C. Soudais, J. L. Gaillard, and M. Rottman. 2007. Hypervirulence
of a rough variant of the Mycobacterium abscessus type strain. Infect. Immun.
9. Centers for Disease Control and Prevention. 1999. Nontuberculous myco-
bacteria reported to the Public Health Laboratory Information System by
state public health laboratories in the United States, 1993–1996. NTM Re-
port 1-51. Centers for Disease Control and Prevention, Atlanta, GA.
10. Clinical and Laboratory Standards Institute. 2003. Susceptibility testing of
Mycobacteria, Nocardia, and other aerobic actinomycetes. Approved standard
M24-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
11. Devallois, A., K. S. Goh, and N. Rastogi. 1997. Rapid identification of
mycobacteria to species level by PCR-restriction fragment length polymor-
phism analysis of the hsp65 gene and proposition of an algorithm to differ-
entiate 34 mycobacterial species. J. Clin. Microbiol. 35:2969–2973.
12. Dytoc, M. T., L. Honish, C. Shandro, P. T. Ting, L. Chui, L. Fiorillo, J.
Robinson, A. Fanning, G. Predy, and R. P. Rennie. 2005. Clinical, microbi-
ological, and epidemiological findings of an outbreak of Mycobacterium
abscessus hand-and-foot disease. Diagn. Microbiol. Infect. Dis. 53:39–45.
13. Freudenberger, R. S., and S. M. Simafranca. 2006. Cutaneous infection with
rapidly-growing mycobacterial infection following heart transplant: a case
report and review of the literature. Transplant. Proc. 38:1526–1529.
14. Howard, S. T., E. Rhoades, J. Recht, X. Pang, A. Alsup, R. Kolter, C. R.
Lyons, and T. F. Byrd. 2006. Spontaneous reversion of Mycobacterium ab-
scessus from a smooth to a rough morphotype is associated with reduced
expression of glycopeptidolipid and reacquisition of an invasive phenotype.
15. Huson, D. H., and D. Bryant. 2006. Application of phylogenetic networks in
evolutionary studies. Mol. Biol. Evol. 23:254–267.
16. Inderlied, C. B., and G. E. Pfyffer. 2003. Susceptibility test methods: myco-
bacteria, p. 1149–1177. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A.
Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed.
ASM Press, Washington, DC.
17. Kim, B. J., S. H. Lee, M. A. Lyu, S. J. Kim, G. H. Bai, G. T. Chae, E. C. Kim,
C. Y. Cha, and Y. H. Kook. 1999. Identification of mycobacterial species by
comparative sequence analysis of the RNA polymerase gene (rpoB). J. Clin.
18. Kim, E. K., T. S. Shim, C.-M. Lim, S. D. Lee, Y. Koh, W. S. Kim, W. D. Kim,
and D. S. Kim. 2003. Clinical manifestations of pulmonary infection due to
rapidly growing nontuberculous mycobacteria. Tuberc. Respir. Dis. 54:283–
19. Kim, H. Y., Y. J. Yun, C. G. Park, D. H. Lee, Y. K. Cho, B. J. Park, S. I. Joo,
E. C. Kim, Y. J. Hur, B. J. Kim, and Y. H. Kook. 2007. Outbreak of
Mycobacterium massiliense infection associated with intramuscular injections.
J. Clin. Microbiol. 45:3127–3130.
20. Koh, W. J., O. J. Kwon, K. Jeon, T. S. Kim, K. S. Lee, Y. K. Park, and G. H.
Bai. 2006. Clinical significance of nontuberculous mycobacteria isolated
from respiratory specimens in Korea. Chest 129:341–348.
21. Ko ¨nig, B., I. Tammer, V. Sollich, and W. Ko ¨nig. 2005. Intra- and interpatient
variability of the hsp65 and 16S–23S intergenic gene region in Mycobacterium
abscessus strains from patients with cystic fibrosis. J. Clin. Microbiol. 43:
22. Kusunoki, S., and T. Ezaki. 1992. Proposal of Mycobacterium peregrinum sp.
nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus
(Kubica et al.) to species status: Mycobacterium abscessus comb. nov. Int. J.
Syst. Bacteriol. 42:240–245.
23. Lee, H. W., M.-N. Kim, T. S. Shim, G. H. Bai, and C. H. Pai. 2002. Nontu-
berculous mycobacterial pulmonary infection in immunocompetent patients.
Tuberc. Respir. Dis. 53:173–182.
24. Lee, W. J., T. W. Kim, K. B. Shur, B. J. Kim, Y. H. Kook, J. H. Lee, and J. K.
Park. 2000. Sporotrichoid dermatosis caused by Mycobacterium abscessus
from a public bath. J. Dermatol. 27:264–268.
25. Meier, A., P. Kirschner, B. Springer, V. A. Steingrube, B. A. Brown, R. J.
Wallace, Jr., and E. C. Bo ¨ttger. 1994. Identification of mutations in 23S
rRNA gene of clarithromycin-resistant Mycobacterium intracellulare. Antimi-
crob. Agents. Chemother. 38:381–384.
26. Metchock, B., F. S. Nolte, and R. J. Wallace, Jr. 1999. Mycobacterium, p.
399–437. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. ASM Press,
27. Oh, W. S., K. S. Ko, J. H. Song, M. Y. Lee, S. Y. Ryu, S. Taek, K. T. Kwon,
J. H. Lee, K. R. Peck, and N. Y. Lee. 2005. Catheter-associated bacteremia
by Mycobacterium senegalense in Korea. BMC Infect. Dis. 5:107.
28. Petrini, B. 2006. Non-tuberculous mycobacterial infections. Scand. J. Infect.
29. Pulcini, C., E. Vandenbussche, I. Podglajen, W. Sougakoff, C. Truffot-Per-
not, A. Buu-Hoï, E. Varon, and J. L. Mainardi. 2006. Hip prosthesis infection
due to Mycobacterium wolinskyi. J. Clin. Microbiol. 44:3463–3464.
30. Roth, A., U. Reischl, A. Streubel, L. Naumann, R. M. Kroppenstedt, M.
Habicht, M. Fischer, and H. Mauch. 2000. Novel diagnostic algorithm for
identification of mycobacteria using genus-specific amplification of the 16S–
23S rRNA gene spacer and restriction endonucleases. J. Clin. Microbiol.
31. Schro ¨der, K. H., L. Naumann, R. M. Kroppenstedt, and U. Reischl. 1997.
Mycobacterium hassiacum sp. nov., a new rapidly growing thermophilic my-
cobacterium. Int. J. Syst. Bacteriol. 47:86–91.
32. Sermet-Gaudelus, I., M. Le Bourgeois, C. Pierre-Audigier, C. Offredo, D.
Guillemot, S. Halley, C. Akoua-Koffi, V. Vincent, V. Sivadon-Tardy, A. Fer-
roni, P. Berche, P. Scheinmann, G. Lenoir, and J. L. Gaillard. 2003. Myco-
bacterium abscessus and children with cystic fibrosis. Emerg. Infect. Dis.
33. Simmon, K. E., J. I. Pounder, J. N. Greene, F. Walsh, C. M. Anderson, S.
Cohen, and C. A. Petti. 2007. Identification of an emerging pathogen, My-
cobacterium massiliense, by rpoB sequencing of clinical isolates collected in
the United States. J. Clin. Microbiol. 45:1978–1980.
34. Song, J. Y., J. W. Sohn, H. W. Jeong, H. J. Cheong, W. J. Kim, and M. J. Kim.
2006. An outbreak of post-acupuncture cutaneous infection due to Myco-
bacterium abscessus. BMC Infect. Dis. 6:6.
35. Springer, B., E. C. Bo ¨ttger, P. Kirschner, and R. J. Wallace, Jr. 1995.
Phylogeny of the Mycobacterium chelonae-like organism based on partial
sequencing of the 16S rRNA gene and proposal of Mycobacterium muco-
genicum sp. nov. Int. J. Syst. Bacteriol. 45:262–267.
36. Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Bo ¨ttger.
1996. Two-laboratory collaborative study on identification of mycobacteria:
molecular versus phenotypic methods. J. Clin. Microbiol. 34:296–303.
37. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol.
38. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bo ¨ttger, and T. Bodmer. 1993.
Rapid identification of mycobacteria to the species level by polymerase chain
reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175–178.
VOL. 46, 2008ISOLATION RATE OF M. MASSILIENSE AND M. BOLLETII3389
39. Tsukamura, M., N. Kita, H. Shimoide, H. Arakawa, and A. Kuze. 1988.
Studies on the epidemiology of nontuberculous mycobacteriosis in Japan.
Am. Rev. Respir. Dis. 137:1280–1284.
40. Viana-Niero, C., K. V. Lima, M. L. Lopes, M. C. Rabello, L. R. Marsola, V. C.
Brilhante, A. M. Durham, and S. C. Lea ˜o. 2008. Molecular characterization
of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected
from outbreaks of infections after laparoscopic surgeries and cosmetic pro-
cedures. J. Clin. Microbiol. 46:850–855.
41. Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang, P. Sander, G. O. Onyi,
and E. C. Bo ¨ttger. 1996. Genetic basis for clarithromycin resistance among
isolates of Mycobacterium chelonae and Mycobacterium abscessus. Antimi-
crob. Agents. Chemother. 40:1676–1681.
42. Yang, S. C., P. R. Hsueh, H. C. Lai, L. J. Teng, L. M. Huang, J. M. Chen,
S. K. Wang, D. C. Shie, S. W. Ho, and K. T. Luh. 2003. High prevalence of
antimicrobial resistance in rapidly growing mycobacteria in Taiwan. Antimi-
crob. Agents Chemother. 47:1958–1962.
3390 KIM ET AL.J. CLIN. MICROBIOL.