Download rpoB gene sequence-based characterization of emerging non

International Journal of Systematic and Evolutionary Microbiology (2006), 56, 133–143
DOI 10.1099/ijs.0.63969-0
rpoB gene sequence-based characterization of
emerging non-tuberculous mycobacteria with
descriptions of Mycobacterium bolletii sp. nov.,
Mycobacterium phocaicum sp. nov. and
Mycobacterium aubagnense sp. nov.
Toı̈di Adékambi, Pierre Berger, Didier Raoult and Michel Drancourt
Correspondence
Michel Drancourt
Michel.Drancourt@medecine.
Unité des Rickettsies, CNRS UMR 6020 IFR 48, Faculté de Médecine, 27, Boulevard Jean
Moulin, Université de la Méditerranée and Assistance Publique-Hôpitaux de Marseille Timone,
Fédération de Microbiologie Clinique, Marseille, France
univ-mrs.fr
Over the past 10 years, 16S rRNA gene sequencing has contributed to the establishment of
more than 45 novel species of non-tuberculous mycobacteria and to the description of emerging
mycobacterial infections. Cumulative experience has indicated that this molecular tool
underestimates the diversity of this group and does not distinguish between all recognized
mycobacterial taxa. In order to improve the recognition of emerging rapidly growing mycobacteria
(RGM), rpoB gene sequencing has been developed. Our previous studies have shown that
an RGM isolate is a member of a novel species if it exhibits >3 % sequence divergence in the rpoB
gene from the type strains of established species. When applied to a collection of 59 clinical
RGM isolates, rpoB gene sequencing revealed nine novel isolates (15?3 %) whereas only two
isolates (3?4 %) were deemed to be novel by conventional 16S rRNA gene sequence analysis. A
polyphasic approach, including biochemical tests, antimicrobial susceptibility analyses, hsp65,
sodA and recA gene sequence analysis, DNA G+C content determination and cell-wall fatty acid
composition analysis, supported the evidence that these nine isolates represent three novel
species. Whereas Mycobacterium phocaicum sp. nov. (type strain N4T=CIP 108542T=CCUG
50185T) and Mycobacterium aubagnense sp. nov. (type strain U8T=CIP 108543T=CCUG
50186T; Mycobacterium mucogenicum group) were susceptible to most antibiotics,
Mycobacterium bolletii sp. nov. (type strain BDT=CIP 108541T=CCUG 50184T; Mycobacterium
chelonae–abscessus group) was resistant to the quinolones, tetracycline, macrolides and
imipenem. Only M. bolletii was resistant to clarithromycin. These data illustrate that rpoB gene
sequence-based identification is a powerful tool to characterize emerging RGM and mycobacterial
infections and provides valuable help in differentiating RGM at both the intra- and interspecies
level, thus contributing to a faster and more efficient diagnosis and epidemiological follow-up.
INTRODUCTION
Over the last 10 years, 16S rRNA gene sequence analysis
of non-tuberculous mycobacteria (NTM) has led to the
description of 45 novel species and has contributed to the
Abbreviations: MIC, minimum inhibitory concentrations; NTM, nontuberculous mycobacteria; RGM, rapidly growing mycobacteria.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA,
recA, hsp65 and sodA genes of Mycobacterium bolletii BDT,
Mycobacterium phocaicum N4T and Mycobacterium aubagnense U8T
are AY859681–AY859683, AY859687–AY859689, AY859675–
AY859677 and AY862403 and AY859706–AY859707, respectively.
A table detailing whole-cell fatty acid content and a figure showing the
hypervariable region of the rpoB gene are available as supplementary
material in IJSEM Online.
63969 G 2006 IUMS
description of novel clinical isolates. Infections caused by
rapidly growing mycobacteria (RGM) have received greater
clinical attention in recent years because of their increasing
incidence in AIDS patients (Smith et al., 2001; Wallace
et al., 1993, 1997) as well as in non-immunocompromised
patients (Brown-Elliott & Wallace, 2002). Many RGM are
ubiquitous in the environment and water has been shown to
be an important source of these opportunistic mycobacteria
(Covert et al., 1999; Dailloux et al., 1999; Wallace et al.,
1998). This fact is illustrated by reports of cases such as
Mycobacterium fortuitum furunculosis following footbaths
(Winthrop et al., 2002), a disseminated infection in a leukaemia patient (Kauppinen et al., 1999) and hypersensitivity
pneumonitis in automobile workers exposed to metalworking fluids (Wallace et al., 2002; Wilson et al., 2001). Likewise,
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133
T. Adékambi and others
contamination of hospital equipment and medication,
traced to the ubiquitous presence of these organisms in
tap water and their resistance to commonly used disinfectants (Tiwari et al., 2003; Wallace et al., 1998), was responsible for pseudo-outbreaks of infections associated with
surgical implants, health care-associated septicaemia and
lung disease following bronchoscopy (Anaissie et al., 2002;
Ashford et al., 1997; Ferguson et al., 2004; Lai et al., 1998;
Wallace et al., 1998). The most common types of infection
are skin and soft tissue infections, infections characterized
by slowly progressive granulomatous inflammation, lymphadenitis and skeletal, catheter-related, disseminated and
pulmonary infections (Brown-Elliott & Wallace, 2002;
Schinsky et al., 2004).
Increasing recovery of RGM from environmental and
clinical sources has prompted the development of laboratory methods to determine their taxonomic affiliation and
identify them precisely. Accurate identification of RGM
species is a key step towards the description of the emerging
opportunistic infections that they cause. Conventional
laboratory methods alone are unable to discriminate
between RGM because of their overlapping phenotypic
patterns (Conville & Witebsky, 1998; Springer et al., 1996).
Cell-wall fatty acid and mycolic acid composition analysis
contributed to the identification of novel mycobacterial
species (Butler & Kilburn, 1990; Munoz et al., 1997), but the
discriminatory power of the analyses was limited by profile
similarity among emerging RGM (Wilson et al., 2001). 16S
rRNA gene sequencing has been used as the first-line
method for the identification of unusual mycobacterial
isolates (Pauls et al., 2003; Tortoli et al., 2001) and for
further discrimination of emerging NTM (Böddinghaus
et al., 1990; Rogall et al., 1990). It has contributed greatly to
the delineation of novel species of the genus Mycobacterium
and to the description of new clinical forms due to known
NTM. However, 16S rRNA gene sequence-based description of novel mycobacterial taxa is still a matter of debate.
Ambiguous results can be obtained due to the possible
presence of two copies of the 16S rRNA gene with different
sequences in the same organism (Adékambi & Drancourt,
2004; Ninet et al., 1996; Reischl et al., 1998; Turenne et al.,
2001). Closely related RGM species, such as Mycobacterium
houstonense and Mycobacterium senegalense, cannot be discriminated by this molecular tool (Adékambi et al., 2003;
Adékambi & Drancourt, 2004).
Partial sequencing of PCR-amplified rpoB, the gene encoding the b-subunit of bacterial RNA polymerase, has been
developed as a suitable tool for the accurate identification of
RGM (Adékambi et al., 2003) and its usefulness for determining the taxonomy of this group of micro-organisms has
been demonstrated (Adékambi & Drancourt, 2004). In this
study, this new tool was applied to a collection of clinical
RGM isolates and 9/59 isolates from 52 patients were found to
exhibit three original rpoB sequences, indicative of novel
RGM species. A polyphasic investigation, including biochemical tests, antimicrobial susceptibility analyses, DNA
G+C content determination, cell-wall fatty acid composition
analyses and sequence-based analyses, confirmed that these
isolates were indeed prototype strains for emerging RGM of
clinical interest. The names Mycobacterium bolletii sp. nov.,
Mycobacterium phocaicum sp. nov. and Mycobacterium
aubagnense sp. nov. are proposed for these three novel RGM.
METHODS
Mycobacterial strains, genetic and phylogenetic analyses.
The nine isolates under study and their clinical sources are listed in
Table 1. In addition, we determined sequences for two RGM species:
‘Mycobacterium massiliense’ CIP 108297 and Mycobacterium alvei CIP
103464T. These newly determined sequences were added to those
of 20 species studied previously (Adékambi et al., 2003). DNA was
extracted from colonies grown on 5 % sheep blood agar using the
Fast-prep device and the FastDNA kit according to the manufacturer’s recommendations (BIO 101). Five molecular targets including
the 16S rRNA (Weisburg et al., 1991), hsp65 (Telenti et al., 1993),
Table 1. Laboratory and clinical information for the three novel RGM
Strain
M. bolletii
BDT (=CIP 108541T=CCUG 50184T)
E2
U2
U6
M. phocaicum
N4T (=CIP 108542T=CCUG 50185T)
N9
N30
M. aubagnense
U8T (=CIP 108543T=CCUG 50186T)
D5
134
Clinical source
GenBank accession number
DNA G+C content
(mol%)
16S rRNA
recA
hsp65
sodA
Sputum
Stomach aspirate
Sputum
Sputum
AY859681
AY859687
AY859675
AY862403
63±2
Bronchial aspirate
Sputum
Bronchial aspirate
AY859682
AY859688
AY859676
AY859706
65±1
Bronchial aspirate
Joint fluid
AY859683
AY859689
AY859677
AY859707
65±1
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Three novel rpoB-defined Mycobacterium species
sodA (Adékambi & Drancourt, 2004), recA (Blackwood et al.,
2000) and rpoB (Adékambi et al., 2003) genes were amplified and
sequenced. A partial rpoB sequence of 764 bp was amplified using
the primer pair Myco-F (59-GGCAAGGTCACCCCGAAGGG-39)
and Myco-R (59-AGCGGCTGCTGGGTGATCATC-39) (see Supplementary Fig. S1 in IJSEM Online) and a 723 bp sequence (apart
from 41 nt at both ends of the amplicon, corresponding to primer
binding sites) was derived from the amplicon by using the same
primer pair in both directions (Adékambi et al., 2003). Products of
sequencing reactions were recorded with an ABI Prism 3100 DNA
sequencer following the manufacturer’s standard protocol (Perkin
Elmer Applied Biosystems). The percentage similarity between the
sequences was determined using the CLUSTAL W program supported by
the PBIL website (http://npsa-pbil.ibcp.fr/cgi-bin/npsa). For phylogenetic analyses, sequences were trimmed so that they started and
finished at the same nucleotide position for all isolates. Multisequence alignment was performed by using the CLUSTAL_X program,
version 1.81 from the PHYLIP software package (Thompson et al.,
1997). A phylogenetic tree was obtained from DNA sequences by using
the neighbour-joining method with Kimura’s two-parameter (K2P)
distance correction model with 1000 bootstrap replications in the
MEGA version 2.1 software package (Kumar et al., 2001). The tree was
rooted using Mycobacterium tuberculosis and Mycobacterium leprae.
The sequences determined in this study have been deposited in
GenBank/EMBL/DDBJ (Table 1 and Fig. 1).
DNA G+C content determination and cellular fatty acid analysis. After culture on 5 % sheep blood agar for 3 days, DNA extrac-
tion, purification, degradation and determination of the DNA G+C
content by HPLC were performed as described by Mesbah et al.
(1989) except that a Waters 625 LC system with a Waters 486
Tenable Absorbance Detector and a Waters 746 Data Module
(Millipore) were used. Three determinations were performed. Total
fatty acid methyl esters were extracted and prepared by following
the instructions for the Microbial Identification System (Microbial
ID) and were analysed by GLC using a gas chromatograph (HP
6890A; Hewlett Packard). Identification of fatty acids for isolates
BDT, N4T and U8T was performed using the Microbial Identification
System with a library created using Sherlock Library Generation
System version 4.0 software (Sasser, 1990).
Phenotypic characterization of the isolates. Sputum and
bronchioalveolar specimens were decontaminated as previously
described (Kent & Kubica, 1985; Kubica et al., 1963). Half of the
sediment was frozen whilst the other half was inoculated into
BACTEC 9000MB broth according to the manufacturer’s instructions (BD Biosciences) after Ziehl–Neelsen staining. The joint fluid
specimen was inoculated directly into BACTEC 9000MB broth.
Mycobacteria were subcultured on Middlebrook 7H10 agar, eggbased Lowenstein–Jensen (LJ) slants (bioMérieux) and 5 % sheep
blood agar (bioMérieux) and cultures were inspected twice weekly
Fig. 1. Phylogenetic tree of the partial rpoB gene sequences of the three novel mycobacterial species and 21 RGM prepared
by using the neighbour-joining method and Kimura’s two-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 and M.
leprae were used as the outgroups. Bar, 2 % difference in nucleotide sequences.
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135
T. Adékambi and others
for the presence of colonies. We observed colony morphology, pigmentation and the ability of the isolate to grow at various temperatures (24, 30, 37 and 42 uC) on 5 % sheep blood agar, LJ slants,
Middlebrook 7H10 agar and LJ slants in the presence of 5 % NaCl.
We tested the activities of arylsulfatase and catalase, iron uptake
and degradation of p-aminosalicylic acid (Kent & Kubica, 1985;
Vincent et al., 2003). Additional biochemical tests were performed
by inoculation of API Coryne and API 20E strips (bioMérieux)
(Adékambi et al., 2004) according to the manufacturer’s instructions with an incubation time of 3 days at 30 uC under a highly
humidified atmosphere.
Antibiotic susceptibility testing. Mycobacterial suspensions of
the isolates were prepared by emulsifying colonies grown on 5 %
sheep blood agar slants into 5 ml sterile water to achieve a density
equal to a 1?0 McFarland turbidity standard by visual examination.
Suspensions were mixed vigorously on a vortex mixer for 20 s and
then inoculated on the entire surface of a 5 % sheep blood agar
plate. The minimum inhibitory concentrations (MIC) of rifampicin, ciprofloxacin, ofloxacin, sparfloxacin, doxycycline, minocycline,
erythromycin, clarithromycin, azithromycin, amikacin, penicillin,
amoxycillin, imipenem, cefotaxime, ceftriaxone, metronidazole, teicoplanin and vancomycin were determined by incubation with the
respective E-test (AB Biodisk) at 30 uC for 3 days. An additional
disk-diffusion method on 5 % sheep blood agar for 3 days at 30 uC
was used to determine the susceptibility to trimethoprim/sulfamethoxazole (1?25/23?75 mg), tobramycin (10 mg), amoxycillin/clavulanate (30 mg), pipemidic acid (30 mg), cefalotin (30 mg) and cefoxitin
(30 mg). The MIC of the antimicrobial agents tested was determined
according to the breakpoints recommended by the NCCLS (NCCLS
2002, 2003) and those proposed by Brown-Elliott & Wallace (2002).
Every test was done three times on three separate days in order to
ensure the reproducibility of results.
RESULTS
Mycobacterial strains
Of the nine isolates studied, three isolates (N4T, N30 and
U8T) were recovered from bronchial aspirate specimens,
four isolates (BDT, U2, U6 and N9) from sputum specimens,
one isolate (E2) from a stomach aspirate and one isolate
(D5) from joint fluid.
Genetic and phylogenetic analyses
We showed previously that RGM isolates belong to the
same species if they have <2 % partial rpoB gene sequence
divergence from an established species. Isolates can be
considered to belong to a novel species if they exhibit >3 %
rpoB sequence divergence from established species using the
partial 723 bp rpoB sequence (Adékambi et al., 2003, 2004).
When applying rpoB gene sequencing and the above criteria
to the first-line identification of 59 RGM clinical isolates
collected from 52 patients over a 7 year period, we found
that nine isolates (15?3 %) were not well established at the
species level. Four isolates, BDT, E2, U2 and U6, shared only
95?0 % sequence similarity with Mycobacterium abscessus
ATCC 19977T, three isolates, N4T, N9 and N30, shared
95?5 % sequence similarity with Mycobacterium mucogenicum ATCC 49650T, and two isolates, U8Tand D5, shared
90?1 % sequence similarity with M. mucogenicum ATCC
49650T due to 15 bp insertions corresponding to the amino
136
acids ANGAY inserted at position 141 (M. mucogenicum
ATCC 49650T partial rpoB gene amino-acid sequence
numbering). rpoB gene sequence variation was not observed
among isolates belonging to the same species. An rpoB
phylogenetic tree was created that included a representative
sequence for each of the nine isolates and 21 sequences from
established RGM species tested in our laboratory (Fig. 1).
These analyses suggested that strain BDT was recently
derived from M. abscessus and belongs to the Mycobacterium
chelonae–M. abscessus group. Strain N4T and U8T were
closely related to M. mucogenicum and belonged to the M.
mucogenicum group. A bootstrap value ¢75 % in the
neighbour-joining tree supported the fork separating strain
BDT, strain N4T and strain U8T from the closely related
species. Their lineages were clearly different from that
of closely related species and quite distant from other
recognized RGM.
Further sequence analyses of the 16S rRNA gene sequences
over 1483 bp showed that isolates BDT, E2, U2 and U6
shared 100 % sequence similarity with M. abscessus ATCC
19977T, isolates N4T, N9 and N30 shared 100 % sequence
similarity with M. mucogenicum ATCC 49650T and isolates
U8T and D5 shared 99?1 % sequence similarity with M.
mucogenicum ATCC 49650T (14 bp difference). Further
molecular characterization of the seven isolates for which
the 16S rRNA sequence exhibited 100 % similarity with the
corresponding type strain, but showed large differences in
the rpoB gene sequence, was done by analysis of the hsp65,
sodA and recA gene sequences. For the hsp65, sodA and recA
genes, strain BDT shared 98?2, 97?7 and 97?9 % similarity,
respectively, with M. abscessus. Strain N4T showed 98?8 %
similarity with M. mucogenicum ATCC 49650T in the hsp65
gene sequence, 98?3 % for the sodA gene and 98?4 % for
the recA gene. Thus, the isolates initially identified as M.
abscessus or as M. mucogenicum ATCC 49650T on the basis
of their phenotypic pattern and 16S rRNA gene sequencing
were unambiguously distinguished from closely related
species by rpoB, recA, sodA and hsp65 gene sequencing. The
one remaining novel strain, U8T, shared 96?0 % similarity
with M. mucogenicum ATCC 49650T in the hsp65 gene
sequence, 96?6 % in the sodA gene and 94?8 % in the recA
gene. Complete rpoB gene sequence analyses of the three
novel species revealed similar relationships to the partial
rpoB sequence analysis described previously (Adékambi
et al., 2003). Molecular analysis, including sequence analyses
of the 16S rRNA, hsp65, sodA, recA and rpoB genes, identified the nine isolates as representatives of three novel
mycobacterial species. No gene sequence variation was
observed among isolates belonging to the same species.
DNA G+C content and fatty acid analysis
The DNA G+C content was 63–65 mol% and the results
are summarized in Table 1. GLC analyses of the fatty acids of
isolates revealed the expected pattern diagnostic for members of the genus Mycobacterium. The profiles of strains
BDT, N4T and U8T contained straight-chain saturated and
unsaturated fatty acids including 16 : 0 (35?2, 15?7 and
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Three novel rpoB-defined Mycobacterium species
19?7 %, respectively), 18 : 1v9c (32?1, 23?9 and 20?4 %),
16 : 1v7c/15 : 0 iso (9?7, 10?7 and 9?8 %), 16 : 1v7c (3?8, 6?1
and 10?6 %), 14 : 0 (6?1, 4?9 and 6?5 %), 17 : 1v7c (0, 20?3
and 13?9 %), 18 : 2v6,9c/18 : 0 ante (6?9, 4?3 and 3?3 %).
Minor amounts of other fatty acids were also detected.
Tuberculostearic acid (TBSA; 10-methyl 18 : 0) was only
detected in isolate BDT (2?3 %). Details of the fatty acid
content are presented in Supplementary Table S1 in IJSEM
Online. Based on the pattern of fatty acids disclosed by GLC
analysis and using the MIDI fatty acid database, the
similarity of strain BDT and M. abscessus CIP 104536T was
93?3 %, strain N4T and M. mucogenicum ATCC 49650T
showed 90?9 % similarity and strain U8T and M. mucogenicum ATCC 49650T showed 93?7 % similarity. These results
suggest that the three strains were different from closely
related species.
Phenotypic characteristics and susceptibility
testing
Biochemical and susceptibility characteristics that differentiated the clinical isolates from closely related species are
summarized in Tables 2 and 3. Colonies of strains BDT, N4T
and U8T are non-pigmented and appear on 5 % sheep blood
agar, Middlebrook 7H10 agar and egg-based LJ slants in
2–5 days at temperatures between 24 and 37 uC. No growth
occurs at 42 uC. In the M. mucogenicum group, strain N4T
utilizes citrate as the sole carbon source, unlike strain U8T
and M. mucogenicum ATCC 49650T. All three strains tested
Table 2. Differentiating biochemical characteristics of the
three novel RGM and closely related Mycobacterium species
Strains: 1, M. bolletii BDT; 2, M. abscessus CIP 104536T; 3, M.
phocaicum N4T; 4, M. mucogenicum ATCC 49649; 5, M. mucogenicum ATCC 49650T; 6, M. aubagnense U8T. All species were able
to grow at 24, 30 and 37 uC, but not at 42 uC. All strains are nonpigmented and test negative for the following enzyme activities:
b-galactosidase, N-acetyl-b-glucosaminidase, urease, gelatinase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and
tryptophan desaminase. All species are positive for catalase and arylsulfatase (3 days) activity, are able to hydrolyse aesculin and can
degrade p-aminosalicylate. Production of H2S and indole and iron
uptake are negative. All strains are able to produce acetoin. +,
Positive; 2, negative; ±, intermediate.
Characteristic
1
2
3
4
5
6
Enzyme activity:
Alkaline phosphatase
a-Glucosidase
b-Glucuronidase
Nitrate reductase
Pyrazinamidase
Pyrrolidonyl arylamidase
Citrate utilization
Tolerance of 5 % NaCl
+
2
2
2
+
+
2
+
+
+
+
2
+
+
2
+
+
2
2
+
+
2
+
2
2
2
2
+
+
2
+
2
2
2
2
+
+
2
2
2
+
2
2
2
2
±
2
2
http://ijs.sgmjournals.org
positive for acetoin and aesculin activity and were negative
for urease activity. Strain N4T and M. mucogenicum ATCC
49650T exhibited positive activities for nitrate reductase and
pyrazinamidase, unlike strain U8T. Strains N4T and U8T
showed positive alkaline phosphatase activity, in contrast
with M. mucogenicum ATCC 49650T. Using the API Coryne
strip, none of the strains tested produced acid from Dglucose, D-ribose, D-xylose, D-mannitol, D-maltose, Dlactose, D-sucrose or glycogen in 3 days. Using the API
20E strip, none of the strains utilized D-glucose, D-mannitol,
inositol, D-sorbitol, L-rhamnose, D-sucrose, D-melibiose,
amygdalin or L-arabinose as sole carbon sources in 3 days.
Strain N4T, U8T and M. mucogenicum ATCC 49650T were
susceptible to imipenem, minocycline, doxycycline, clarithromycin, erythromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin and sparfloxacin. Strain N4T was
resistant to amoxycillin, amoxycillin/clavulanate and trimethoprim/sulfamethoxazole, in contrast to M. mucogenicum
ATCC 49650T. Strain U8T was susceptible to tobramycin,
unlike M. mucogenicum ATCC 49650T. Strain BDT and M.
abscessus have intermediate resistance to amikacin, but
strain BDT was resistant to clarithromycin, whereas M.
abscessus was susceptible. The antimicrobial susceptibility
profiles and the biochemical test patterns obtained for
strains BDT, N4T and U8T and the corresponding isolates
were identical.
DISCUSSION
16S rRNA gene sequencing has been used as the reference
method for identifying unusual mycobacterial isolates
(Pauls et al., 2003; Tortoli et al., 2001; Turenne et al.,
2001). This approach has contributed to the description of
45 novel NTM species over the last 10 years. However, the
investigation of large NTM collections by 16S rRNA gene
sequencing has not resolved the classification of all isolates.
The use of 16S rRNA gene sequencing for the identification
of clinical NTM has indicated that 37 % of such isolates
remain unclassified and suggests the need for complementary molecular tools for proper phylogenetic assignment
and accurate NTM identification (Pauls et al., 2003). The
common assumption that bacterial isolates are members of
the same species if they have fewer than 5–15 bp differences
in the 16S rRNA gene sequence (Fox et al., 1992) or if they
have 16S rRNA gene sequence similarity >97 % (Drancourt
et al., 2000) may not be applicable to mycobacteria, whose
members are much more closely related to each other
(Adékambi & Drancourt, 2004; Tortoli, 2003; Turenne
et al., 2001). Furthermore, 16S rRNA gene sequence-based
descriptions of novel mycobacterial taxa are still a matter of
debate because ambiguous results have been found due to
the presence of two copies of the 16S rRNA gene with
different sequences in the same organism (Adékambi &
Drancourt, 2004; Ninet et al., 1996; Reischl et al., 1998;
Turenne et al., 2001). Molecular tools including sodA (Zolg
& Philippi-Schulz, 1994), dnaJ (Takewaki et al., 1994), 32kDa protein-encoding gene (Soini & Viljanen, 1997), hsp65
(Ringuet et al., 1999) recA (Blackwood et al., 2000), 16S-23S
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T. Adékambi and others
Table 3. Antimicrobial
Mycobacterium species
susceptibility
of
the
three
novel
RGM
and
closely
related
Strains: 1, M. bolletii BDT; 2. M. abscessus CIP 104536T; 3, M. phocaicum N4T; 4, M. mucogenicum
ATCC 49649; 5, M. mucogenicum ATCC 49650T; 6, M. aubagnense U8T. Values are MIC (mg ml21).
AMC, Amoxycillin/clavulanate; TMP/SMZ, trimethoprim/sulfamethoxazole. +, Positive.
AMC (disc 20/10 mg)
Amikacin
Amoxycillin
Azithromycin
Cefalotin (disc 30 mg)
Cefinase test
Cefotaxime
Cefoxitin (disc 30 mg)
Ceftriaxone
Ciprofloxacin
Clarithromycin
Colistin (disc 50 mg)
Doxycycline
Erythromycin
Imipenem
Metronidazole
Minocycline
Ofloxacin
Penicillin
Pipemidic acid (disc 20 mg)
Rifampicin
Sparfloxacin
Teicoplanin
TMP/SMZ (disc 1?25/23?75 mg)
Tobramycin (disc 10 mg)
Vancomycin
1
2
3
4
5
6
>4
24
>256
>256
>32
+
>256
>32
>256
>32
>256
>2
>32
>256
>32
>256
>256
>32
>32
>16
>32
>32
>256
>8
>8
>256
>4
32
>256
>256
>32
+
>256
>32
>256
>32
1?5
>2
>32
>256
>32
>256
>256
>32
>32
>16
>32
>32
>256
>8
>8
>256
>4
1?5
64
1?5
>32
+
32
<2
>256
0?50
0?064
>2
1?5
3
0?50
>256
0?75
0?75
>32
>16
>32
0?25
>256
>8
>8
>256
<4
4
3
1?5
>32
+
48
<8
>256
0?25
0?125
>2
0?125
1?5
0?75
>256
0?50
3
>32
>16
>32
0?50
128
<2
>8
>256
<4
1
1?5
0?125
>32
+
12
<8
>256
0?125
0?032
>2
1?5
1
0?75
>256
1?5
0?75
>32
>16
>32
0?25
>256
<2
>8
>256
<4
3
0?125
2
>32
+
64
<8
>256
0?125
0?125
>2
1
1
0?75
>256
1?5
0?25
>32
>16
>32
0?125
64
<2
<4
>256
rRNA internal transcribed spacer (ITS) (Park et al., 2000),
DNA gyrase genes (Dauendorffer et al., 2003) and secA1
(Zelazny et al., 2005) sequence analyses have been proposed
as alternative tools for the molecular identification of NTM
isolates, including RGM. However, no criteria have been
validated for the delineation of species using these molecular tools. The rpoB gene is a single-copy gene encoding
the b-subunit of bacterial RNA polymerase and has been
previously used for the molecular identification of numerous bacterial genera, including Mycobacterium (Adékambi
et al., 2003; Kim et al., 1999; Lee et al., 2003). In this study,
when rpoB gene sequencing was applied to improve the
genetic identification of clinical RGM isolates, we obtained
9/59 clinical isolates (15?3 %) that exhibited >3 % rpoB
gene sequence divergence from the closest related species.
However, 7/9 isolates (77?8 %) shared 100 % similarity with
the corresponding 16S rRNA gene sequence. Similarly,
Blackwood et al. (2000) found that Mycobacterium peregrinum ATCC 14467T and M. peregrinum ATCC 23015,
which have complete 16S rRNA gene sequence identity,
diverged by 3?9 % in the 915 bp recA gene sequence;
the intraspecies variation of mycobacteria was <1?3 %.
138
Sequencing of the rpoB gene alone revealed that 15?3 % of
isolates were deemed to be novel, corresponding to three
novel species, whereas conventional 16S rRNA gene
sequencing identified only 3?4 % of the isolates as novel,
corresponding to one novel species. The intraspecies
similarity of the 915 bp recA gene ranges from 98?7 to
100 % (Blackwood et al., 2000). This additional finding
allowed us to consider the sequences which were deemed to
be novel as representing genuine novel species. Further
molecular analysis, including hsp65 and sodA gene sequence
analyses, confirmed that the nine isolates represented
Mycobacterium species that were distinct from known
Mycobacterium species. These data illustrate that rpoB
gene sequencing is a powerful tool for the characterization
of novel RGM species, as previously illustrated by the use of
the rpoB gene sequence for the delineation of ‘M. massiliense’
(Adékambi et al., 2004) and Mycobacterium cosmeticum
(Cooksey et al., 2004).
Previous studies have shown that the antibiotic susceptibility of RGM varies widely (Brown et al., 1992; Swenson
et al., 1985; Wallace et al., 1990, 1991; Woods et al., 2000).
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Three novel rpoB-defined Mycobacterium species
Large variations in antibiotic susceptibility to the currently
available anti-RGM antimicrobials among isolates that
apparently belong to the same RGM species has confirmed
the need for accurate identification at the species level (Yang
et al., 2003; Yakrus et al., 2001). Our results clearly demonstrate that species of the M. mucogenicum group were often
more susceptible to antibiotics than species of the M.
chelonae–abscessus group. Notable differences were found
for antibiotics such as the tetracyclines (minocycline and
doxycycline), the macrolides (erythromycin and azithromycin), the quinolones (ciprofloxacin, ofloxacin and sparfloxacin) and imipenem. Amikacin was effective against
almost all species. Our data showed that first- and thirdgeneration cephalosporins demonstrated poor activities
against members of each group whereas cefoxitin had
variable activity. Although the predictive value of in vitro
antibiotic susceptibility testing of RGM is uncertain, some
anti-RGM antimicrobials could be useful markers for differentiating the three novel species from closely related species.
Unlike M. mucogenicum ATCC 49650T, strain U8T was susceptible to tobramycin (MIC<4 mg ml21) and strain N4T
was resistant to amoxycillin (MIC >64 mg ml21), amoxycillin/clavulanate (MIC>4 mg ml21) and trimethoprim/
sulfamethoxazole (MIC>8 mg ml21). Strain BDT was resistant to clarithromycin (MIC >256 mg ml21) but M. abscessus
was susceptible, even though they shared 100 % similarity in
16S rRNA gene sequences. Recent antibiotic susceptibility
testing disturbingly showed that 21–36 % of M. abscessus
isolates were resistant to clarithromycin (Yang et al., 2003;
Yakrus et al., 2001), making the differentiation of strain BDT
and M. abscessus important. In another case, Mycobacterium
boenickei and Mycobacterium neworleansense, which share
99?9 % similarity in 16S rRNA gene sequences, differed in
their susceptibility to doxycycline and minocycline (Schinsky
et al., 2004). These data illustrate that accurate identification
of emerging RGM species can reveal the presence of large
variations in susceptibility to the currently available antiRGM antimicrobials.
The polyphasic approach we adopted showed clear-cut
differences between closely related species with identical 16S
rRNA gene sequences. Indeed, 16S rRNA gene sequence
identity alone is not a useful criterion for the identification
of mycobacteria. A review of the 13 novel mycobacterial
species described in the 2004 issues of International Journal
of Systematic and Evolutionary Microbiology indicated that
only 6/13 descriptions of novel species incorporated DNA–
DNA hybridization results, including only 4/9 novel species
which exhibited greater than 99 % 16S rRNA gene sequence
similarity with the most closely related species. Furthermore, DNA–DNA hybridization was performed by only
one of the nine research groups that proposed novel species
in 2004. These data indicate that the majority of novel
mycobacterial species are not described on the basis of
DNA–DNA hybridization, even when 16S rRNA gene
sequences are almost identical. In these cases, species were
described on the basis of one to three other gene sequences.
We performed sequence analysis for five different genes in
http://ijs.sgmjournals.org
the present study. These findings are in agreement with the
proposition of the ad hoc committee for re-evaluation of
the species definition to sequence five housekeeping genes
instead of DNA–DNA hybridization in order to describe
novel species (Stackebrandt et al., 2002). Due to the highly
conserved nature of the 16S rRNA gene, there is no linear
correlation between the DNA–DNA relatedness value and
16S rRNA gene sequence similarity for closely related
organisms (Grimont, 1988; Stackebrandt & Goebel, 1994).
For example, M. houstonense shared 100 % 16S rRNA gene
sequence similarity to M. senegalense but in DNA–DNA
hybridization studies, labelled genomic DNA from M.
houstonense was <65 % related to that of M. senegalense
(Schinsky et al., 2004). Using the rpoB gene as an alternative
tool, M. houstonense shared 97?0 % sequence similarity to M.
senegalense (Adékambi et al., 2003). This similarity value
fulfils the definition of a novel mycobacterial species
(Adékambi et al., 2003). Without DNA–DNA hybridization,
the rpoB gene has been used for the description of novel
mycobacterial species such as ‘M. massiliense’ and M.
cosmeticum (Adékambi et al., 2004, Cooksey et al., 2004).
Furthermore, recent studies with species of Bartonella,
Bosea and Afipia have shown a correlation between DNA–
DNA relatedness and rpoB gene sequence similarity for
closely related organisms and rpoB gene sequencing has been
proposed for the classification of isolates misidentified by
16S rRNA gene sequencing (Khamis et al., 2003; La Scola
et al., 2003).
A species diagnosis is essential for the management of
patients with RGM infections. For example, it is well known
that infections with M. abscessus in patients with cystic
fibrosis or with chronic meningitis are very difficult to treat
(Sanguinetti et al., 2001; Maniu et al., 2001). In some cases,
M. abscessus is essentially ineradicable with the possible
exception of a resection of the affected lobe of the lung
(Griffith et al., 1993; Wallace et al., 1997). Knowledge of the
characteristics of mycobacterial species is important for the
treatment and prognosis of patients with RGM. Thus, all
clinical isolates of RGM should be identified to the species
level. Phenotypic characterization, including pigmentation,
growth rate, biochemical test algorithms and 16S rRNA
gene sequencing, have been used in the identification of
Mycobacterium species since the earliest isolation of RGM in
clinical specimens. However, rpoB gene sequencing has
revealed advantages over both conventional testing and 16S
rRNA gene sequence analysis for identifying mycobacterial
species and detecting novel species. In keeping with previous
findings, it can generally be assumed that a novel species has
been detected if base pair discrepancies are found in the
variable regions of the rpoB gene and the organism has
distinct phenotypic properties (Adékambi et al., 2004).
Molecular identification of mycobacteria is a key step
towards the description of emerging RGM species and the
opportunistic infections they cause. The characteristics of
one of the novel species described in this paper, Mycobacterium bolletii sp. nov., also illustrate that accurate
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139
T. Adékambi and others
identification of RGM can be predictive of antibiotic susceptibility patterns of prime clinical value. The present data
indicate that selected partial rpoB gene sequencing could be
used as a first-line molecular tool for the accurate identification of emerging RGM in man.
Description of Mycobacterium bolletii sp. nov.
Mycobacterium bolletii (bol9let.i.i. N.L. gen. n. bolletii of
Bollet, to honour our deceased colleague Claude Bollet,
a famous clinical microbiologist and taxonomist).
The organisms are acid-fast and Gram-positive bacilli. Colonies are non-pigmented and appear on 5 % sheep blood
agar, Middlebrook 7H10 agar and egg-based LJ slants in
2–5 days at temperatures between 24 and 37 uC, optimally
at 30 uC. No growth occurs at 42 uC. This species is a
multidrug-resistant mycobacteria, notably to clarithromycin but with intermediate resistance to amikacin. It is
associated with chronic pneumonia. It is positive for 3-day
arylsulfatase activity, negative for iron uptake and urease
activity, does not grow on LJ containing 5 % NaCl at 35 uC
and does not utilize citrate, sorbitol or mannitol as sole
sources of carbon. It shares 100 % 16S rRNA and 95?6 %
rpoB gene sequence similarity with M. abscessus, the closest
species.
The type strain, BDT (=CIP 108541T=CCUG 50184T), was
recovered from sputum.
Description of Mycobacterium phocaicum
sp. nov.
Mycobacterium phocaicum [pho.cai9cum. L. neut. adj. phocaicum pertaining to Phocaea, a maritime town of lonia, a
colony of the Athenians, whose inhabitants fled to escape
from Persian domination and founded Massilia (Marseille),
which was the source of the type strain].
The organisms are acid-fast and Gram-positive bacilli.
Colonies are non-pigmented and appear on 5 % sheep blood
agar, Middlebrook 7H10 agar and egg-based LJ slants in
2–5 days at temperatures between 24 and 37 uC, optimally
at 30 uC. No growth occurs at 42 uC. This species is associated with chronic pneumonia. It is susceptible in vitro to
cefoxitin, imipenem, minocycline, doxycycline, clarithromycin, erythromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin and sparfloxacin and is resistant to
amoxycillin, amoxycillin/clavulanate and tobramycin. It is
positive for 3-day arylsulfatase, nitrate reductase and
pyrazinamidase activities and for the production of aesculin
and acetoin and negative for iron uptake and urease activity.
M. phocaicum differs from M. mucogenicum ATCC 49650T
by exhibiting positive activity for penicillinase and alkaline
phosphatase and by utilizing citrate as a sole source of
carbon. It shares 100 % 16S rRNA and 95?0 % rpoB gene
sequence similarity with M. mucogenicum ATCC 49650T, the
closest phylogenetic species.
140
The type strain, N4T (=CIP 108542T=CCUG 50185T), was
recovered from bronchial aspirate.
Description of Mycobacterium aubagnense
sp. nov.
Mycobacterium aubagnense (au.bag.nen9se. N.L. neut. adj.
aubagnense pertaining to Aubagne, the city from where the
first patient originated).
The organisms are acid-fast and Gram-positive bacilli. Colonies are non-pigmented and appear on 5 % sheep blood
agar, Middlebrook 7H10 agar and egg-based LJ slants in
2–5 days at temperatures between 24 and 37 uC, optimally at
30 uC. No growth occurs at 42 uC. This species is associated
with chronic pneumonia and sepsis. It is susceptible in vitro
to amoxycillin, amoxycillin/clavulanate, cefoxitin, imipenem, minocycline, doxycycline, clarithromycin, erythromycin, azithromycin, amikacin, tobramycin, ciprofloxacin,
ofloxacin, sparfloxacin and trimethoprim/sulfamethoxazole. It is positive for 3-day arylsulfatase activity and for the
production of aesculin and acetoin, but negative for iron
uptake and urease activity. Does not utilize citrate as a sole
source of carbon. M. aubagnense differs from M. mucogenicum ATCC 49650T by exhibiting positive alkaline phosphatase activity and no nitrate reductase or pyrazinamidase
activity. It shares 99?1 % 16S rRNA and 92?7 % rpoB gene
sequence similarity with M. mucogenicum ATCC 49650T, the
closest phylogenetic species.
The type strain, U8T (=CIP 108543T=CCUG 50186T), was
recovered from bronchial aspirate.
ACKNOWLEDGEMENTS
This work is dedicated to Dr Claude Bollet. We thank Christian de
Fontaine for technical assistance, Pierre Yves-Levy for providing
mycobacterial isolates and Esther Platt for expert reviewing of the
manuscript.
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