Mycobacterium canariasense sp. nov.
M. Soledad Jime ´nez,1M. Isolina Campos-Herrero,2Diana Garcı ´a,2
Marina Luquin,3Laura Herrera1and Marı ´a J. Garcı ´a4
M. Soledad Jime ´nez
1Laboratorio de Micobacterias, Servicio de Bacteriologı ´a, Centro Nacional de Microbiologı ´a,
Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
2Servicio de Microbiologı ´a, Hospital de Gran Canaria Dr Negrin, Las Palmas de Gran Canaria,
3Unitat de Microbiologı ´a, Departament de Genetica i de Microbiologı ´a, Universitat Auto ´noma de
Barcelona, Bellaterra, Barcelona, Spain
4Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Auto ´noma de
Madrid, Madrid, Spain
A novel rapidly growing, non-pigmented mycobacterium was isolated from blood samples
obtained from 17 patients with febrile syndrome. Bacterial growth occurred at 30 and 376C
on Lo ¨wenstein–Jensen medium and also on MacConkey agar without crystal violet. Strains
contained a- and a9-mycolates in their cell wall. Sequence analysis of the hsp65 and 16S rRNA
genes identified the isolates as rapidly growing mycobacteria. Sequences of both genes were
unique within the mycobacteria. DNA–DNA hybridization showed that the isolates had less
than 15% reassociation with 13 other recognized rapidly growing mycobacteria. The name
Mycobacterium canariasense sp. nov. is proposed for this novel opportunistic pathogen,
which is most closely related to Mycobacterium diernhoferi. The type strain is 502329T
(=CIP 107998T=CCUG 47953T).
Rapidly growing mycobacteria are ubiquitous environmen-
tal bacteria commonly found in water and soil (Brown-
Elliott & Wallace, 2002). Members of this group, especially
the three major species Mycobacterium fortuitum, Myco-
bacterium chelonae and Mycobacterium abscessus, have been
reported as aetiological agents of a variety of infections,
including bacteraemia and disseminated disease in patients
with long-term venous catheters (Raad et al., 1991). They
have alsobeeninvolvedin nosocomialoutbreaks orpseudo-
outbreaks related to contamination of hospital water
supplies and reagents (Ashford et al., 1997; Chadha et al.,
1998; LaBombardi et al., 2002). In this study, a novel
mycobacterium involved in nosocomial infection is des-
cribed; the name proposed for this species is Mycobacterium
canariasense sp. nov.
Duringthe period January2000 toSeptember2002,amyco-
bacterium that could not be identified by conventional
procedures was isolated from blood specimens of 17
patients with suspected nosocomial acquisition. Patients
were admitted to a tertiary care hospital in the Canary
Islands, Spain. Most of them (n=15) had a malignant
disease and all of them carried at that time a central venous
catheter. Mycobacteria were considered to be the cause of
the febrile syndrome in 12 cases. Ten patients were treated
with specific antibiotic therapy and the central catheter
was removed in nine of them. Catheters were left in two
patients, who later relapsed. The same mycobacterium
grew in their subsequent blood cultures. Most of the
patients recovered after treatment. However, two patients
died as a consequence of their disease status. All bacterial
isolates had homogeneous biochemical characteristics and
antimicrobial susceptibility patterns. The first isolate and
four other randomly selected strains were sent to the refer-
ence Laboratory of Mycobacteria (Centro Nacional de
Microbiologı ´a, Madrid, Spain) for identification. These five
isolates were examined for several phenotypic and geno-
typic characteristics and results were compared with those
displayed by other rapidly growing mycobacterial species.
Acid–alcohol-fastness, colony morphology and pigment
production, as well as the ability to grow at various
temperatures (22–45uC), on Lo ¨wenstein–Jensen medium
(LJ), in the presence of 5% NaCl and on MacConkey
agar without crystal violet (Kubica & David, 1980) were
examined. A total of 12 biochemical tests was performed
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and
hsp65 genes of Mycobacterium canariasense strain 502329Tare
AY255478 and AY255477, respectively.
Some characteristics of strain 502329T(Table A), TLC of methyl
mycolates of M. canariasense strains and related Mycobacterium
species (Fig. A), GC analysis of fatty acid methyl esters of strain
502329T(Fig. B), hybridization dot-blot assays (Fig. C) and DNA–DNA
relatedness between strain 502329Tand other Mycobacterium species
(Table B) are available as supplementary material in IJSEM Online.
02999 G 2004 IUMSPrinted in Great Britain 1729
International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1729–1734
sources and acid formation (Silcox et al., 1981; Tsukamura,
1967). Hydrolysis of seven different amides was also tested
(Vestal, 1975). Sensitivities to eight different antimyco-
bacterial drugs were tested by the proportion method in
LJ (Canetti et al., 1963). E-test strips on Mueller–Hinton
medium were used to determine sensitivity to 13 further
Bacterial cells were partially acid-fast rods that grew on LJ,
initially as non-pigmented colonies, but later developing a
more yellowish, smooth, moist and shiny appearance.
Growth on LJ occurred after 2–3 days at 30 and 37uC;
growth did not occur at the other temperatures tested. The
main phenotypic characteristics of isolates are indicated
in Table 1 (see supplementary material in IJSEM Online
for a complete set of data). All isolates of M. canariasense
exhibited identical characteristics. The novel species could
be differentiated from the M. chelonae group by single
carbon source testing, from M. fortuitum and M. diernhoferi
by the nitrate reduction test and from Mycobacterium
frederiksbergense and other related species by pigmentation
development (Table 1).
Fatty and mycolic acids were liberated from each strain
sample by saponification. These compounds were then
extracted with diethyl ether and treated with diazomethane
to obtain the methyl esters (Daffe ´ et al., 1983). Mycolic acid
methyl esters were studied by analytical one-dimensional
TLC using silica gel 60 TLC plates (Merck) as described
previously (Luquı ´n et al., 1991a). Fatty acid methyl esters
and methyl mycolate cleavage products were determined
by GC-MS (Luquin et al., 1991b).
TLC analysis of the mycolic acid methyl esters revealed that
the five M. canariasense strains contained a- and a9-
mycolates (see supplementary material available in IJSEM
Online). To date, this mycolate pattern has only been
described in M. chelonae and M. abscessus (see Table 1)
(Hinrikson & Pfyffer, 1994) and species within the genus
Tsukamurella (Hamid et al., 1993). This mycolate pattern
allows M. canariasense to be differentiated from related
Table 1. Phenotypic characteristics of M. canariasense and other rapidly growing mycobacteria
Species: 1, M. canariasense; 2, M. abscessus; 3, M. chelonae; 4, M. fortuitum; 5, M. mucogenicum; 6, M. senegalense; 7, M. immunogenum;
8, M. neoaurum; 9, M. diernhoferi; 10, M. frederiksbergense. 2, Less than 15% of isolates are positive; +, more than 85% of isolates are
positive; numbers in parentheses indicate percentages of positive strains; ND, not determined. Biochemical data corresponding to myco-
bacteria other than M. canariasense were taken from Schro ¨der et al. (1997), Springer et al. (1995), Wayne & Kubica (1986), Willumsen et al.
(2001) and Wilson et al. (2001).
Growth on 5% NaCl
Arylsulfatase (3 days)
Use as sole carbon source:
Acid production from:
Mycolic acid typeD
ND ND ND
I, IId, V, VIIdI, IId, V, VIId
I, IV, VII, IV, VI I, IV, VI
*MacConkey agar without crystal violet.
DMycolic acid type: I, a-mycolate; II, a9-mycolate; III, methoxy-mycolate; IV, keto-mycolate; V, epoxy-mycolate; VI, was esters; VII, v-1
dMay not be present in certain strains.
1730International Journal of Systematic and Evolutionary Microbiology 54
M. S. Jime ´nez and others
species such as Mycobacterium mucogenicum and M.
diernhoferi (see Table 1; Mun ˜oz et al., 1997). GC-MS
analysis of M. canariasense strains indicated the presence
of fatty acid methyl esters with 14–24 carbon atoms, of
which hexadecanoate (C16:0) and octadecenoate (C18:1)
were the most prominent. All strains also showed appreci-
derived from thermal cleavage of methyl mycolates was
tetracosanoate (C24:0; see supplementary material available
in IJSEM Online), which allows M. canariasense to be
differentiated from Tsukamurella. Species of this genus
released C20and C22esters from pyrolysis of mycolates, but
not C24(Goodfellow et al., 1978). Unlike M. canariasense,
strains of M. diernhoferi, M. frederiksbergense, M. muco-
genicum and Mycobacterium neoaurum revealed secondary
alcohols by GC-MS analysis; thus, the absence of these
compounds in M. canariasense strains enables them to be
clearly differentiated from these closely related species.
PCR was performed on DNA isolated from bacterial
LJ cultures using the boiling method and centrifugation.
A 439 bp region of the hsp65 gene was subjected to PCR
amplification followed by restriction analysis using the
primers and conditions described by Telenti et al. (1993).
Amplicons were also sequenced in triplicate according to
Ringuet et al. (1999) using the BigDye Terminator
sequencing kit and the ABI Prism 3700 automated
sequencer (Applied Biosystems). In addition, 1514 bp of
the 16S rRNA gene were sequenced from PCR amplicons
produced as described by Springer et al. (1995). Sequences
of hsp65 and 16S rRNA genes were aligned against pre-
viously described sequences from rapidly growing myco-
bacterial species, using EDITSEQ and MEGALIGN software
(Lasergene MegAlign; DNAstar) (Altschul et al., 1997).
Sequences were clustered using CLUSTAL W and weightings
The hsp65 gene of M. canariasense strains contained a
PCR-RFLP pattern that differed from those published
previously or compiled in the PRAsite database (http://
www.hospvd.ch:8005). Patterns consisted of two fragments
of 325 and 130 bp by BstEII digestion and three fragments
of 140, 90 and 80 bp by HaeIII digestion. The sequence of
the M. canariasense hypervariable region (Ringuet et al.,
1999) was significantly different from those of other closely
related rapidly growing species. Phylogenetic analysis of
441 bp of this gene demonstrated that M. diernhoferi and
M. mucogenicum are the closest non-pigmented relatives
(94?3 and 93?5% similarity, respectively), whereas M.
neoaurum and M. frederiksbergense were the most closely
related pigmented species (94?4 and 93?6% similarity,
respectively) (Fig. 1).
Sequences of the 16S rRNA gene were also identical in all
five novel isolates studied. A detailed comparison of the
16S rRNA gene sequence with sequences of other rapidly
growing mycobacterial species demonstrated 100% simi-
larity in the genus-specific regions, but revealed a significant
number of differences in helix 18 and the species-specific
(Kirschner et al., 1993). The complete sequence of the 16S
rRNA gene of M. canariasense shows substantial differences
when compared with those corresponding to other rapidly
growing mycobacteria. Fig. 2 shows a comparison of the
M. canariasense 16S rRNA gene sequence with 10 other
closely related mycobacteria; Tsukamurella paurometabola
was used as an outgroup. The dendrogram shows that
M. diernhoferi is the closest non-pigmented relative (99?2%
similarity) to M. canariasense, followed by M. mucogenicum
(97?0%) and, as seen previously with the hsp65 gene,
growing mycobacteria (98?7%). The similarity between
M. canariasense and T. paurometabola was 91?2%.
Total genomic DNA was purified from liquid bacterial
cultures, as described previously (Domenech et al., 1994).
DNA–DNA hybridization experiments were performed on
membrane filters, using a novel dot-blot-based procedure.
Genomic DNA from M. canariasense strain 502329T
Fig. 1. Phylogenetic position of M. canariasense among other closely related, rapidly growing mycobacterial species as
determined by hsp65 gene sequences using the CLUSTAL W method with weightings. M. tuberculosis was used as outgroup.
GenBank accession numbers are given in parentheses.
Mycobacterium canariasense sp. nov.
(300 ng) was labelled in vitro using the Megaprime DNA
labelling system (Amersham) and 25 mCi (925 kBq)
[a-32P]dCTP (Amersham). Portions (500 ng) of each
unlabelled DNA were dot-blotted and bound to nylon
membrane filters (Hybond-N+; Amersham). Hybridiza-
tion and washes were carried out as described previously
(Brown et al., 1999), except that ECL buffer (Amersham)
was used both for pre-hybridization and hybridization
steps. The relative binding ratios (expressed as percentages)
for each species were calculated from the counts of homo-
logous bound DNA, as measured using an Instant Imager
counter (Izasa Instruments). In addition, the blots were
stripped and rehybridized with a PCR amplicon from the
16S rRNA gene asthe newprobe, asdescribed by Domenech
et al. (1997). This procedure has several advantages com-
pared to previous methods (Keswani & Whitman, 2001),
mainly because the amount of DNA fixed in each dot can
be determined by measuring the amount of radioactivity
when using the 16S rRNA gene as the second probe. Due
to the high sequence conservation within mycobacterial
species inherent within this probe (Menendez et al., 2002),
the radioactivity level can be considered to be proportional
to the amount of target molecules on the filter. The number
of copies of the 16S rRNA gene was already known for the
mycobacterial species included in this work (Menendez
et al., 2002; M. J. Garcia, unpublished results). The levels of
DNA–DNA hybridization between M. canariasense strains
were greater than 90 %. On the other hand, the level of
reassociation between the M. canariasense type strain and 13
other related rapidly growing species corresponded to less
than 15%. These results strongly suggest that our isolates
represent a novel species (Lan & Reeves, 2001). Results of
DNA–DNA hybridization analysis are available as supple-
mentary material in IJSEM Online.
RFLP analysis of the 16S rRNA gene was also performed to
complement DNA–DNA hybridization data (Domenech
experimental procedure (Domenech et al., 1997) was used,
with the exception that ECL buffer (Amersham) was used
for both the pre-hybridization and hybridization steps.
Fig. 3 shows the RFLP patterns from BamHI-digested
Fig. 2. Phylogenetic position of M. canariasense among other related rapidly growing mycobacterial species as determined
by comparison of the 16S rRNA gene sequences. The tree was inferred using the CLUSTAL W method with weightings and
rooted using T. paurometabola as outgroup. GenBank accession numbers are given in parentheses.
1234567891011 12 13 kbp
Fig. 3. RFLP patterns of 16S rRNA genes from M. canaria-
sense strains and other rapidly growing mycobacterial species.
Patterns were obtained using BamHI as the restriction enzyme.
Lanes: 1, M. canariasense 502329T; 2, M. canariasense
513578; 3, M. canariasense 517062; 4, M. canariasense
552822; 5, M. abscessus ATCC 19977T; 6, M. fortuitum
subsp. fortuitum ATCC 6841T; 7, Mycobacterium goodii ATCC
700504T; 8, Mycobacterium mageritense ATCC 700351T;
9, M. mucogenicum ATCC 49650T; 10, Mycobacterium porci-
num CIPT 141460001=CIP 105392T; 11, Mycobacterium
smegmatis CIPT 141330010=CIP 104444T; 12, Mycobac-
terium wolinskyi ATCC 700009; 13, N. asteroides ATCC
3308. Fragment sizes (in kbp) are indicated on the left.
1732International Journal of Systematic and Evolutionary Microbiology 54
M. S. Jime ´nez and others
genomic DNAs from M. canariasense strains, eight other
rapidly growing mycobacterial species and Nocardia
asteroides. Patterns of different species were different, but
All species tested, with the exception of M. abscessus,
produced a pattern with two bands, indicating the presence
of two copies of the 16S rRNA gene. These results indicate
that M. canariasense belongs to the II-s mycobacterial class,
i.e. species with two rrn operons per genome and short helix
18 in the 16S rRNA gene coding region (Menendez et al.,
On the basis of biochemical characteristics, mycolic and
fatty acid patterns, PCR-RFLP of the hsp65 gene, sequences
of conserved genes and DNA–DNA hybridization, M.
canariasense is proposed as a novel rapidly growing
mycobacterium, most closely related to M. diernhoferi and
Description of Mycobacterium canariasense
Mycobacterium canariasense (ca.na.ri.a.sen9se. L. gen. adj.
canariasense referring to the Latin adjective of the Spanish
islands where all strains were isolated).
Cells are partially acid-fast rods. Visible growth appears in
2–3 days as smooth, moist, shiny, non-pigmented colonies
on Lo ¨wenstein–Jensen medium. Growth occurs at 30 and
37uC, but not at 22, 42 or 45uC. Grows on MacConkey
agar without crystal violet, but does not grow in the pres-
ence of 5% NaCl. Positive for arylsulfatase activity (3 days)
and Tween 80 hydrolysis. Produces a low level of heat-
stable catalase and is negative for reduction of nitrates.
These characteristics allow this novel species to be differ-
entiated from other closely related species such as M.
diernhoferi and M. mucogenicum. The inability to utilize
citrate as a single carbon source allows differentiation from
M. chelonae. Lipid composition of the cell wall is charac-
terized by the presence of a- and a9-mycolates, similar to
M. chelonae and M. abscessus. Results of DNA analyses such
as PCR-RFLP of the hsp65 gene, sequences of conserved
genes and DNA–DNA hybridization define M. canariasense
as a distinct genomic mycobacterial species most closely
related to M. diernhoferi and M. mucogenicum. The 16S
rRNA and hsp65 gene sequences of M. canariasense are
unique. This species belongs to the II-s mycobacterial class
according to the classification of Menendez et al. (2002).
The type strain is 502329T(=CIP 107998T=CCUG
We are grateful to Dr T. J. Bull for his helpful revision of the
manuscript. This work was supported by grants from Spanish
institutions (2002SGR-00099 from the Generalitat de Catalunya;
and 08.2/0009/2001 from the Comunidad Autonoma de Madrid) and
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