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Diagnostic Microbiology and Infectious Disease 65 (2009) 358 – 364
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Mycobacteriology
Molecular identification of rapidly growing mycobacteria
isolates from pulmonary specimens of patients in the
State of Pará, Amazon region, Brazil
Ana Roberta Fusco da Costaa,b,⁎, Maria Luiza Lopesb , Sylvia Cardoso Leãoc ,
Maria Paula da Cruz Schneiderd , Maísa Silva de Sousaa , Philip Noel Suffyse ,
Tereza Cristina de Oliveira Corvelod , Karla Valéria Batista Limab
a
Núcleo de Medicina Tropical, Universidade Federal do Pará, Av. Generalíssimo Deodoro no. 92, Belém, Pará 66.055-240, Pará, Brazil
b
Seção de Bacteriologia e Micologia, Instituto Evandro Chagas, Pará 67030-000, Brazil
c
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil
d
Departamento de Genética, Universidade Federal do Pará, Pará, Brazil
e
Departamento de Medicina Tropical, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Received 1 May 2009; accepted 11 August 2009
Abstract
We isolated 44 strains of rapidly growing mycobacteria (RGM) from 19 patients with pulmonary infections assisted at the Instituto
Evandro Chagas (Pará, Brazil) from 2004 to 2007. Identification at the species level was performed by PCR restriction fragment length
polymorphism analysis (PRA) of a 441 bp hsp65 fragment and partial 16S rRNA, hsp65, and rpoB gene sequencing. Genotyping by PRA
yielded 3 digestion patterns: one identical to Mycobacterium abscessus type I (group I); another to M. abscessus type II, Mycobacterium
bolletii, and Mycobacterium massiliense (group II); and a third typical for Mycobacterium fortuitum type I (group III). When comparing
analysis of the 3 genes, more discrimination was obtained by rpoB gene sequence, which allowed good distinction between group I, II, and
III strains and subclassification of group II strains in SG IIa (M. bolletii) and SG IIb (M. massiliense). In this study, we show that the
description of new RGM species requires the establishment of standardized procedures for RGM identification and the alert of the clinician
about their involvement in pulmonary disease and the necessity of treatment for control and cure.
© 2009 Elsevier Inc. All rights reserved.
Keywords: Molecular identification; Rapidly growing mycobacteria; Pulmonary infections
1. Introduction
Rapidly growing mycobacteria (RGMs) belong to the
Runyon group IV, organisms that usually form colonies
within 7 days of incubation as opposed to slow-growing
mycobacteria, that is, Runyon groups I, II, and III and the
Mycobacterium tuberculosis complex, which require longer
⁎ Corresponding author. Seção de Bacteriologia e Micologia, Instituto
Evandro Chagas, BR 316, KM 7, Ananindeua, Pará 67030-000, Brazil. Tel.:
+55-91-3214-2116; fax: +55-91-3214-2114/Núcleo de Medicina Tropical,
Universidade Federal do Pará, Av. Generalíssimo Deodoro no. 92, Belém,
Pará 66.055-240, Brazil, Tel.: +55-91-3241-4681; fax: +55-3215-2354.
E-mail address: [email protected] (A.R.F. da Costa).
0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2009.08.003
incubation (Ringuet et al., 1999). The RGMs are ubiquitous
organisms and are frequently isolated from environmental
sources. They are increasingly encountered in clinical
microbiology laboratories and have emerged as significant
human pathogens, causing infections in healthy and
immunocompromised hosts. They can cause numerous
types of infections that include cutaneous disease, lymphadenitis, disseminated disease, and pulmonary infection
(Brown-Elliott and Wallace, 2002). However, the isolation
of RGM from a respiratory sample is insufficient evidence
for the presence of lung disease, which requires differentiation from contamination or colonization by clinical,
radiographic, and bacteriologic criteria (Griffith et al.,
2007). The RGM that commonly causes pulmonary disease
in humans includes species that belong to the Mycobac-
A.R.F. da Costa et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 358–364
terium fortuitum and Mycobacterium chelonae complexes
(Daley and Griffith, 2002). RGM identification at species
level is necessary because it provides the first indication
regarding the mycobacteria antibiotic susceptibility. Identification of these organisms by biochemical methods is not
always a straightforward procedure (Brown-Elliott and
Wallace, 2002), and genotyping has been shown to be an
attractive alternative. The most widely used method for
molecular identification of RGM is the polymerase chain
reaction (PCR) restriction fragment length polymorphism
analysis (PRA) of a 441 bp hsp65 fragment (Brunello et
al., 2001; Devallois et al., 1997; Simmon et al., 2007), but
small differences of band sizes and of patterns or even the
sharing of the same patterns between different species
reduced the accuracy of the identification procedure
(Runyon, 1959; Turenne et al., 2001). As an alternative,
clinical and reference laboratories worldwide have adopted
the 16S rRNA gene sequencing method as a routine
procedure for Mycobacterium and other infectious agent
identification, but the difficulty in isolate identification has
been reported in cases where sequence identities are ≥99%
(Tortoli, 2003). Sequencing of other genes for discrimination of RGM to the species level has been reported, and
characterization of the hsp65 gene seems more discriminative than that of the 16S rRNA gene (McNabb et al., 2004;
König et al., 2005; Petrini, 2006). In addition, sequencing
of the rpoB gene (and especially of the polymorphic region
V) is emerging as a preferential tool to identify mycobacterial taxa (Adékambi et al., 2003). This study describes our
finding on the clinical significance of infection with these
RGM and on the accuracy and discriminative power of
genotyping of 3 major genes for identification of RGM
associated with pulmonary infections in patients from the
State of Pará, the Amazon region of Brazil.
2. Materials and methods
2.1. Patients and Mycobacterium strains
From January 2004 to December 2007, a total of 44
RGM isolates from 19 patients (2 or more isolates/patient)
were recovered from sputum and included in this study.
Initial presumptive distinction of M. tuberculosis from
nontuberculous mycobacteria (NTM) was obtained by
culture in Lowenstein-Jensen medium containing p-nitrobenzoic acid (0.5 mg/mL) and direct observation of colony
aspect (morphology, pigmentation, and growth rate). All
identified patients manifested respiratory symptoms, and the
RGM isolates were considered to be significant, according
to the American Thoracic Society guidelines (Griffith et al.,
2007). These RGM strains were consecutively and
sporadically detected in the Bacteriology Section of the
Instituto Evandro Chagas (Belém, Pará, Brazil). The
medical records of 8 patients infected by species of M.
chelonae complex were available and contained information
regarding respiratory symptoms, history of treatment, and
359
results of the microscopic examination of sputum for acidfast bacilli (AFB).
2.2. Genotyping by PRA
All 44 RGM isolates of this study had already been
analyzed by PRA for variability within hsp65 gene. In brief,
a 441-bp fragment of the hsp65 gene was amplified with
primers TB11 and TB12 (Invitrogen, Brazil), as previously
described (Brasil, 2008; Telenti et al., 1993). A negative
control (water) and positive control (M. tuberculosis H37Rv
ATCC 27294 DNA) were included in every run. After
confirming the amplification of 441-bp PCR products, the
amplicons were digested with 10 U of BstEII (Biolabs, New
England) and HaeIII (Invitrogen, Sao Paulo, Brazil) in
separate reactions. The restriction fragments were separated
by electrophoresis in 8% polyacrylamide gel (Proquimios,
Brazil) containing 50- and 25-bp ladder molecular weight
markers (Invitrogen).
2.3. Sequencing
Sequencing of part of the 16S rRNA gene, hsp65 and
rpoB, was performed on 1 sample of each patient. The
amplification conditions were identical to those described by
previous publications (Adékambi et al., 2003; Kim et al.,
2005; Shin et al., 2006). After verification of PCR products
on agarose gel Seakem LE 1% (Cambrex, United Kingdom),
these were purified using the SNAP TM gel purification kit
(Invitrogen). The amplified products of the 16S rRNA gene
(499 bp in RGM), hsp65 (644 bp) and rpoB (711 in the M.
chelonae complex and 720 bp in the M. fortuitum complex),
were direct sequenced by using both forward and reverse
primers of each system using BigDye Terminator v3.1 cycle
sequencing kits (Applied Biosystems, Foster City, CA) and
analyzed on an ABI3130 sequencer (Applied Biosystems,
Tokyo, Japan).
2.4. Genotype analysis
The PRA patterns were analyzed by simple visual
comparison with molecular weight markers. PRA patterns
were interpreted using the published algorithms (Brunello
et al., 2001; Devallois et al., 1997; Telenti et al., 1993) and
the PRASITE (http://www.app.chuv.ch/prasite/index.html).
The partial 16S rRNA gene, hsp65 and rpoB, sequences
obtained were aligned with the closest relatives retrieved
from BLAST nucleotide database (http://www.ncbi.nlm.nih.
gov/BLAST/). The sequences of Mycobacterium abscessus,
Mycobacterium bolletii, M. chelonae, Mycobacterium massiliense, M. fortuitum, and Mycobacterium houstonense
retrieved from GenBank were used for comparison (accession number in parenthesis next to the species names). Only
423 bp (positions 414–836 in the hsp65 sequence of
M. tuberculosis, GenBank accession no. M15467) were
used for species differentiation by hsp65 analysis. This
shorter sequence was analyzed because of some RGM hsp65
sequences available in GenBank with smaller sizes
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compared with those obtained in this study. The 423 bp are a
segment within the of 441-bp hsp65 fragment, which was
used in differentiation of Mycobacterium spp. (Kim et al.,
2005). The sequences were aligned, and their similarities
were calculated using the multiple-alignment algorithm in
the Bioedit software (version 7.0.9; Tom Hall [http://www.
mbio.ncsu.edu/BioEdit/bioedit.html]). Dendrograms were
inferred from partial rpoB and hsp65 nucleotide sequences
obtained from RGM isolates in this study, and those of
RGM-type strains were retrieved from GenBank using
neighbor-joining method with Kimura's 2-parameter distance correction model in MEGA software (version 4.0;
Tamura, Dudley, Nei, and Kumar [http://www.megasoftware.net/]). Bootstrap analysis (1000 repeats) was applied
using the M. tuberculosis H37Rv ATCC 27294 sequences
(rpoB, U12205, and hsp65, M15467) as outgroup.
Sequences of the rpoB gene available in GenBank database
of M. abscessus, M. bolletii, and M. massiliense were also
analyzed in this study (Table 1). The DNA polymorphism
analyses were performed by DnaSP software (version 4.50.3;
[Faculty of Biology, University of Barcelona, Spain [http://
www.ub.es/dnasp]).
2.5. Nucleotide sequence accession number
The nucleotide sequences obtained in this study are
available in the GenBank database under accession numbers
FJ590454–FJ590472 (16S rRNA), FJ536235–FJ536253
(hsp65), and FJ590435–FJ590453 (rpoB).
3. Results
3.1. Patients and mycobacterial isolates
During the period of January of 2004 and December 2007,
44 isolates of RGM were detected among 19 of the 742
patients who had presented symptoms of pulmonary infection,
Table 1
Amino acids differences among M. abscessus, M. bolletii, and
M. massiliense in rpoB gene sequence
Speciesa
Codon (nucleotide positions)
and infection by Mycobacterium spp. had been diagnosed in
Bacteriology Section at the Evandro Chagas Institute,
representing 38% (19/50) of all patients with NTM pulmonary
infection. The patients infected by RGM were detected among
cases initially diagnosed as pulmonary tuberculosis.
In 8 patients, the average time between the first medical
attendance and diagnosis of NTM infection was 11 months.
The presence of symptoms such as productive cough, weight
loss, and thoracic pain was similar among these. Additional
symptoms and signs, as decreased lung volume and frank
hemoptysis, were found in 1 and 2 patients with M. bolletii
infection, respectively.
After failure of conventional treatment of tuberculosis and
NTM diagnosis, the treatment regimen adopted included the
administration of ethambutol, clarithromycin, and, in some
cases, amikacin. Two patients infected by M. abscessus were
treated for 6 months but remained with a positive AFB smear
test; however, they presented symptomatic improvement.
Three patients with M. massiliense infection presented
symptomatic improvement after therapy and converted
their sputum smear to negative after 1-month treatment,
although they still presented positive cultures. Three patients
with M. bolletii infection showed a more severe form of the
disease. These patients presented persistent positive AFB
smear and did not improve clinically after 6 months of
therapy with ethambutol and clarithromycin. No clinical
information was available for M. fortuitum-infected patients.
3.2. Identification by PRA
A total of 3 BstEII and HaeIII digestion patterns of PRA
were identified among the 44 isolates of 19 patients. Because
all isolates from the same patient had the same identification by
PRA, 1 isolate from each of the 19 patients was studied, and
they were assigned P01 to P19. The PRA patterns characterized 3 different groups: group I (G I): P01 to P02 (BstEII, 235/
210; HaeIII, 145/70/60/55) characteristic of M. abscessus type
I; group II (G II): P03 to P15 (BstEII, 235/210; HaeIII, 200/
70/55) common to M. abscessus type II, M. bolletii and
M. massiliense; and group III (G III): P16 to P19 (BstEII, 235/
115/85; HaeIII, 145/120/60) found in M. fortuitum type I.
3.3. Sequence analysis of part of 16S rRNA gene
954 (3017-9) 959 (3032-4) 963 (3044-6) 966 (3053-5)
M. abscessus GAG (E)
M. bolletii
GAC (D)
M. massiliense GAT (D)
GCC (A)
GAG (E)
GAG (E)
GCA (A)
GAG (E)
GAG (E)
ACC (T)
CAA (Q)
CAG (Q)
Nucleotide differences are highlighted in bold.
a
Analysis based on rpoB sequences from M. abscessus, M. bolletii,
and M. massiliense species obtained in this study and those from strains
described with the following GenBank accession numbers: M. abscessus
(AY262741, EU109292, CU458896, AY147164, EU597584, EU370230,
EU597582, EU597598, EU597595, EU597585, EU370229, EU597599,
EU591501), M. bolletii (EU109293, AY859692, EU220424, EU220423,
EU220422), and M. massiliense (EU109294, EU109296, AY593981,
EU254721, EU117207, EU109295, EU090065, EU090063, EU090066,
EU090064, EU031909, EU031907, EU031906, EU031905, EU031904,
EU009469, EU009468, EU009467, EU370524, EU031908, EU220421,
FJ194538, FJ194539).
The sequence of the 463-bp fragment of the 16S rRNA
gene of all 15 G I and G II strains was identical to the
sequence shared by M. abscessus (AF547892), M. chelonae
(AF547909), M. massiliense (AY593980), and M. bolletii
(AY859681). The 16S rRNA sequences of the 4 G III isolates
were identical to that of M. fortuitum (AY457066) and
99.1% similar to that of M. houstonense DQ987744. The
similarity between G I–II and G III was 97.4%.
3.4. Sequence analysis of part of hsp65 gene
Upon sequence analysis of the 423-bp fragment of
hsp65, some synonymous single-nucleotide polymorphisms
(SNPs) were observed between fragments obtained from
A.R.F. da Costa et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 358–364
isolates belonging to the same groups. Both G I isolates had
identical sequences to that of M. abscessus (AY458075).
Nucleotide diversity between G I (P01 and P02) and G II
(P03-P15) was represented by transitions T533C, C542T,
C602T, and C755T and by transversions T530G, G644C,
and C827G, and nucleotide divergence between isolates of
G I and G II varied from 1.2% to 1.5% and presented
bootstraps of 98% and 96%, respectively, as presented in the
dendrogram (Fig. 1A).
The isolates of G II showed nucleotide diversity varying
from 0.5% to 0.8%, due to transitions C602T and C755T and
by transversion G644C. The sequences shared by isolates
P03, P04, and P05, and that of P06 presenting the transition
T602C, were highly similar (99.5–99.7%) to that of M.
bolletii (AY859675). The sequence common to strains P07,
P08, P09, P10, P11, P12, P13, P14, and P15 was 99.7%
similar to that of M. massiliense (AY596465). The
transversion G827C was observed in all G II isolates when
compared with the reference sequences of M. bolletii
(AY859675) and M. massiliense (AY596465).
361
The members of G III (P16-P19) presented 100% of
similarity with M. fortuitum (DQ866789), and the next most
closely related species was M. houstonense (AY458077),
which shared 99.4% similarity.
3.5. Sequence analysis of part of rpoB gene
The analysis of the 711-bp fragment of rpoB showed that
the G I isolates had identical sequences to that of M.
abscessus (AY147164) and presented grouping with bootstrap value of 100% in the dendrogram (Fig. 1B). The
sequence divergence between G I and G II isolates ranged
from 3.6% to 4.4%.
In the dendrogram based on the partial rpoB sequences of
the G II isolates, 2 main branches were observed that
presented bootstrap values of 100% and 96% and were
composed of isolates P03 to P06 (SG IIa) and P07 to P15
(SG IIb) (Fig. 1B). Compared with the sequence of reference
strains, SG IIa isolates were 99.7% to 99.8% similar with M.
bolletii (AY859692); organisms of SG IIb showed 99.7% to
Fig. 1. Relationships between type strains and 19 isolates (P01–P19) inferred from partial hsp65 (A) and rpoB sequences (B). Dendrograms were constructed by
neighbor-joining method and Kimura's 2-parameter distance correction model. The support of each branch, as determined from 1000 bootstrap samples, is
indicated by the value at each node (as a percentage). M. tuberculosis H37Rv was used as outgroup. The scale bar represents 1% and 2% difference in hsp65 and
rpoB nucleotide sequences, respectively.
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100% similarity to M. massiliense (AY593981). Isolate P03
presented the transition G2605C when compared with M.
bolletii (AY859692). Sequence variability between SG IIa
and SG IIb ranged from 1.5% to 1.7%, whereas that observed
within SG IIa and SG IIb ranged, respectively, from 0.2% to
0.3% and 0.2% to 0.5%, in SG IIb. The sequence divergence
between M. massiliense (AY593981) and M. bolletii
(AY859692) was 1.6%.
G III isolates exhibited similarity ranging between 99.5%
and 100% with M. fortuitum (DQ866802). SNPs were
identified by sequence comparison of isolates with published
sequence of M. fortuitum (DQ866802). The SNPs G2744A,
G3013A and G2932C were found in the P17 isolate, while
the SNPs T2662C and G3013C were present in the P19
isolate. The G III isolate sequences were 98.7% to 99%
related with M. houstonense (AY147173), the latter presenting 99% similarity to the sequence of the M. fortuitum
reference strain (DQ8668802).
When comparing the rpoB sequence of G II isolates and
that M. abscessus (AY147164), SNPs were observed in 35
different loci, most being transitions and concentrated in the
region 3019 to 3070. When compared with the sequence of
the reference strain M. abscessus (AY147164), the transversion C2659G occurred in G II clinical isolates P08, P11, and
P14, and modifications in codons 954, 959, 963, and 966 of
isolates P03 to P15 also resulted in alterations in the
β-polymerase amino acids sequences (Table 1). Our data
further suggest that composition of codons 954 and 966
(transitional SNPs) allow the differentiation among
M. abscessus, M. bolletii, and M. massiliense (Table 1).
4. Discussion
In the present study, genotypic analysis was performed
to characterize isolates of RGM from 19 patients with
pulmonary disease. We identified the presence of 4
different species that belong to the M. chelonae and
M. fortuitum complexes, frequently observed as a cause of
pulmonary infections (Brown-Elliott and Wallace, 2002).
Information on the nature and frequency of NTM that
causes pulmonary infections is scarce in Brazil, because
notification of disease caused by such organisms is not
obligatory and not considered an epidemic emergency for
the sanitary authorities.
All patients infected by RGM were detected among cases
diagnosed as pulmonary tuberculosis, and it had already
been treated for six months with rifampicin, isoniazid and
pyrazinamide (RHZ) scheme and presented treatment failure
(data not shown). This treatment failure was due to sample
bias because our patient population was selected for
infection with RGM, known to present incomplete response
and failure of the traditional antituberculosis treatment
(Glassroth, 2008; Griffith et al., 2007). Normally, AFB
smear-positive patients who were suspicious of pulmonary
tuberculosis are treated without confirmation by culture,
according to the guidelines set by the National Tuberculosis
Control Program (Brasil, 2005). Even after diagnosis of
disease due to infection by RGM, 8 of the patients with
available clinical files were treated using the same treatment
schemes that included ethambutol, clarithromycin, and,
sometimes, amikacin. According to the American Thoracic
Society, the outcome of in vitro susceptibility testing for
many NTM species and isolates does not correlate well with
clinical response to antimycobacterial drugs. Data on
recommendations for routine in vitro susceptibility testing
of NTM isolates are limited, especially for RGM. Nonetheless, data showing susceptibility of RGM toward drugs such
as amikacin, cefoxitin, clarithromycin, ciprofloxacin, doxycycline, linezolid, sulfamethoxazole, and tobramycin should
be considered and should be the basis for choice of adequate
therapy schemes (Griffith et al., 2007).
3.6. Genetic diversity
Among the isolates studied, the number polymorphic sites
in 3 loci analyzed varied from 12 (16S rRNA) to 91 (rpoB),
and the number of alleles per locus varied from 2 (16S rRNA)
to 10 (rpoB) in all isolates. The somewhat low intraspecies
diversity observed presently could be due to the small
sample size, therefore, complicating a thorough quantitative
determination of variability (Table 2).
Table 2
Genetic diversity of the selected loci among RGM isolates from pulmonary specimens analyzed in this study
Locus
Fragment
length (bp)a
No alleles
Average genetic
diversity ± SD
No polymorphic
sites
No nucleotide substitutions/
nucleotide site
Average nucleotide
diversity ± SD
16S rRNAb
hsp65c
M. bolletii
M. fortuitum
rpoBd
M. bolletii
M. massiliense
M. fortuitum
499
624
624
624
711/720
711
711
720
2
8
2
4
10
3
3
3
0.351 ±
0.766 ±
0.500 ±
1.000 ±
0.930 ±
0.833 ±
0.750 ±
0.833 ±
12
46
1
5
91
2
3
4
2.40
7.37
0.16
0.80
12.63
0.28
0.42
0.55
0.008 ± 0.001
0.02575 ± 0.003
0.0008 ± 0.001
0.0043 ± 0.001
0.04620 ± 0.003
0.00164 ± 0.001
0.00211 ± 0.001
0.00301 ± 0.001
a
b
c
d
0.111
0.092
0.265
0.177
0.031
0.222
0.079
0.222
Values are the total numbers of sites, excluding gaps or missing data.
No intraspecies diversity was found in 16S rRNA of the RGM in this study.
Only M. bolletii and M. fortuitum showed intraspecies diversity in hsp65 sequence.
Variability was not found among rpoB sequences of M. abscessus isolates.
A.R.F. da Costa et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 358–364
In this study, the species observed in the largest number
of patients was M. massiliense (n = 9, 47.4%), followed by
M. bolletii (n = 4, 21.1%), M. fortuitum (n = 4, 21.1%),
and M. abscessus (n = 2, 10.5%). Reports on occurrence and
frequency of M. massiliense and M. bolletii associated to
pulmonary infections are rare (Adékambi et al., 2004, 2006;
Kim et al., 2007; Tortoli, 2003), whereas M. abscessus and
M. fortuitum are described as the most frequently occurring
RGM species in pulmonary infection (Brown-Elliott and
Wallace, 2002; Petrini, 2006). In Brazil, infections with M.
massiliense and M. bolletii were associated only with
medical invasive procedures (Cardoso et al., 2008; VianaNiero et al., 2008), and our study describes the first report of
pulmonary infection by this species in Brazil. We suspect
that the frequency of M. massiliense and M. bolletii has been
underestimated because of the fact that conventional
identification procedures and that based on PRA analysis
will not distinguish these species from M. abscessus.
The PRA analysis of the strains presented the patterns 235
and 210 bp in BstEII restriction and 200, 70, 55, and 50 bp in
HaeIII restriction in the case of 15 isolates; this pattern is
shared by M. bolletii, M. massiliense, and M. abscessus type
II, as described previously (Brasil, 2008; Viana-Niero et al.,
2008). Recent studies involving partial sequencing of rpoB
gene of isolates previously characterized as M. abscessus
type II by PRA-hsp65 revealed that these strains were
actually M. massiliense or M. bolletii and strongly suggest
the need to reanalyze the real contribution of these species as
emerging pathogens (Cardoso et al., 2008; Kim et al., 2008;
Viana-Niero et al., 2008).
Based on the polymorphism C644G, observed presently
and also in sequences available in GenBank, we propose the
inclusion into PRA, of the restriction enzymes Sm1I
(C↓TYRAG) or Sap1 (GCTCTTCN↓NNN), for differentiation between M. bolletii and M. massiliense isolates.
Although Sm1I yields fragments of 248 and 193 bp for M.
bolletii and no digestion for M. massiliense, Sap1 yields
245- and 196-bp fragments for M. massiliense (245/196) and
leaves the M. bolletii fragment uncut (data not shown).
Nucleotide sequence variations in the hsp65 gene observed
in clinical isolates of the 2 before mentioned subgroups
(M. bolletii and M. massiliense) were similar to “M. abscessus
variants” to that described in other studies (Kim et al., 2008;
König et al., 2005; Ringuet et al., 1999), except for the SNP
G827C, when compared with GenBank sequences of M.
abscessus, M. massiliense, and M. bolletii available in the
GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In
earlier studies, describing other isolates from Brazil and Korea,
the same SNP was observed (Kim et al., 2007; Viana-Niero et
al., 2008), although a geographic distribution of hsp65 alleles
could be one of the reasons for such observations. That
polymorphism is present in the complementary sequence of
reverse primer TB12 (Telenti et al., 1993).
Upon analysis of the 711-bp fragment of rpoB, a new
SNP (C2659G and T2659G) was described in 3 isolates
(P08, P11, and P14) of the M. chelonae complex. Other
363
eventual signature nucleotides were suggested by the
composition of the codons 954 and 966 of isolates of M.
abscessus, M. bolletii, and M. massiliense, reinforcing the
closer relationship of M. massiliense and M. bolletii. Despite
modifications in codons 954, 959, 963, and 966 from isolates
P03 to P15, substitutions appear in a nonconserved region
from the RNA polymerase RPB10 interaction site, according
to the Conserved Domain Database at National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Upon the use of the rpoB sequence for tree building, the
neighbor-joining–based bootstrap value of 88% obtained
presently supported the classification of SG IIa (M. bolletii)
and SG IIb (M. massiliense) organisms as separate (Fig. 1B),
the divergence among them varied from 1.5% to 1.7%.
Similar rpoB sequence divergence (1.6%) was observed
between the type strains of M. massiliense (AY593981) and
M. bolletii (AY859692). Viana-Niero et al. (2008) related
divergences of 2% between strains of M. massiliense and M.
bolletii from outbreak cases in Brazilian patients who
experienced different invasive procedures performed in 16
private hospitals and clinics in the city of Belém. When
compared with M. fortuitum isolates, M. houstonense
(AY147173) was the other closely related species, and
Adékambi et al. (2003) already described a 99% interspecific
similarity between the latter. When considering the criteria of
similarity in rpoB defined by Adékambi et al. (2003), we
verified that M. massiliense and M. bolletii represent a single
entity. These results had been concordant with that found in
the analysis of the hsp65 sequences, whose similarities
between the cited species had been greater than 99%.
Furthermore, it is important to recognize that M. massiliense
and M. bolletii were defined as new taxa based on assays that
included the following: 16S rRNA, hsp65, sodA, recA e rpoB
sequencing, DNA G + C content determination, phenotypic
characterization, and cellular fatty acid analysis and antibiotic
susceptibility testing, only for M. bolletii. However, in
accordance with the parameters recommended for ad hoc
committee for the reevaluation of the species definition in
bacteriology, DNA ± DNA hybridization should be considered
as molecular criteria for species delineation (Stackebrandt
et al., 2002). According to Tortoli (2003), this technique
represents the most important approach to a quantitative
definition of species and is the sole technique reflecting the
whole genomic complexity, elucidating of relationships
between closely related taxa, such as RGM species.
New RGM species have been identified recently, based
on molecular methods (Euzéby, 2008), and these seem to be
the best approach for that purpose. Usually, sequence
analysis comparing degrees of similarity are used for RGM
identification. However, intra- and interspecies limits were
still not established for distinction of these “new” and “old”
RGM including variants. This situation reveals the necessity
to establish minimal standards for accurate identification of
RGM species based on such methodology.
From our study, we concluded that the identification of
RGM involved in pulmonary infection to the species level is
364
A.R.F. da Costa et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 358–364
important for the prognosis of clinical severity and the
contribution of the infection to the disease development. This
was clearly demonstrated in the case of the 3 patients with
M. bolletii infections who developed more severe disease.
Although, at present, no well-defined therapeutic scheme is
available for treatment of lung disease due to M. abscessus,
M. bolletii, and M. massiliense, identification is the first step
for the recognition of RGM species related to the disease
symptoms and the basis for development of treatment
schemes that could lead to disease control and cure.
Acknowledgments
The authors thank Maurimélia Mesquita da Costa for
English language review and Emilyn Costa Conceição for
technical assistance.
This work was supported by Fundação de Amparo à
Pesquisa do Estado do Pará (FAPESPA, process no. 102/
2004), Instituto Evandro Chagas and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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