RIDOM: comprehensive and public sequence database for identification of Mycobacterium species.

ral
ssBioMed CentBMC Infectious Diseases
Open AcceResearch article
RIDOM: Comprehensive and public sequence database for
identification of Mycobacterium species
Dag Harmsen*1, Stefan Dostal2, Andreas Roth3, Stefan Niemann4,
Jörg Rothgänger5, Michael Sammeth6, Jürgen Albert6, Matthias Frosch2 and
Elvira Richter4
Address: 1Institut für Hygiene, Universität Münster, Münster, Germany, 2Institut für Hygiene und Mikrobiologie, Universität Würzburg, Würzburg,
Germany, 3Institut für Mikrobiologie und Immunologie, Lungenklinik Heckeshorn, Berlin, Germany, 4Nationales Referenzzentrum für
Mykobakterien, Forschungszentrum Borstel, Borstel, Germany, 5Ridom GmbH, Würzburg, Germany and 6Lehrstuhl für Informatik II, Universität
Würzburg, Würzburg, Germany
Email: Dag Harmsen* - dharmsen@uni-muenster.de; Stefan Dostal - sdostal@web.de; Andreas Roth - mikromau@zedat.fu-berlin.de;
Stefan Niemann - sniemann@fz-borstel.de; Jörg Rothgänger - jrothgaenger@ridom.de; Michael Sammeth - micha@sammeth.net;
Jürgen Albert - albert@informatik.uni-wuerzburg.de; Matthias Frosch - mfrosch@hygiene.uni-wuerzburg.de; Elvira Richter - erichter@fz-
borstel.de
* Corresponding author
Abstract
Background: Molecular identification of Mycobacterium species has two primary advantages when
compared to phenotypic identification: rapid turn-around time and improved accuracy. The information
content of the 5' end of the 16S ribosomal RNA gene (16S rDNA) is sufficient for identification of most
bacterial species. However, reliable sequence-based identification is hampered by many faulty and some
missing sequence entries in publicly accessible databases.
Methods: In order to establish an improved 16S rDNA sequence database for the identification of clinical
and environmental isolates, we sequenced both strands of the 5' end of 16S rDNA (Escherichia coli
positions 54 to 510) from 199 mycobacterial culture collection isolates. All validly described species (n =
89; up to March 21, 2000) and nearly all published sequevar variants were included. If the 16S rDNA
sequences were not discriminatory, the internal transcribed spacer (ITS) region sequences (n = 84) were
also determined.
Results: Using 5'-16S rDNA sequencing a total of 64 different mycobacterial species (71.9%) could be
identified. With the additional input of the ITS sequence, a further 16 species or subspecies could be
differentiated. Only Mycobacterium tuberculosis complex species, M. marinum / M. ulcerans and the M. avium
subspecies could not be differentiated using 5'-16S rDNA or ITS sequencing. A total of 77 culture
collection strain sequences, exhibiting an overlap of at least 80% and identical by strain number to the
isolates used in this study, were found in the GenBank. Comparing these with our sequences revealed that
an average of 4.31 nucleotide differences (SD ± 0.57) were present.
Conclusions: The data from this analysis show that it is possible to differentiate most mycobacterial
species by sequence analysis of partial 16S rDNA. The high-quality sequences reported here, together with
ancillary information (e.g., taxonomic, medical), are available in a public database, which is currently being
Published: 11 November 2003
BMC Infectious Diseases 2003, 3:26
Received: 19 August 2003
Accepted: 11 November 2003
This article is available from: http://www.biomedcentral.com/1471-2334/3/26
© 2003 Harmsen et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 10
(page number not for citation purposes)
expanded in the RIDOM project http://www.ridom-rdna.de), for similarity searches.

BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/26
Background
Reliable microbial identification using conventional
methods often requires several techniques, such as the use
of colony morphology, gram staining, determination of
nutritional requirements and/or biochemical reactions.
Identification of mycobacteria at the species level using
conventional biochemical tests is laborious with a long
turn-around time, leading to significant delays in diagno-
sis and ambiguous results occur frequently. Other meth-
ods based on lipid analysis, such as high-performance
liquid chromatography, thin-layer chromatography and
gas-liquid chromatography are used only in a few clinical
laboratories [1–3]. Identification using molecular tech-
niques, on the other hand, provides two primary advan-
tages when compared to phenotypic identification: a
more rapid turn-around time and improved accuracy in
identification [4–6]. With assays based on molecular tech-
niques, the genetic targets vary, as does the method of tar-
get characterization. Three targets that have proven useful
in identification are the 16S ribosomal RNA (16S rDNA)
gene [7–10], the internal transcribed spacer (ITS) region
[11] and the hsp65 gene [12–14]. The main advantage of
16S rRNA gene analysis is that it can be applied in the
identification of all bacteria, even those which are dead or
are uncultivable [15,16]. The ITS region has a greater dis-
criminatory power than the 16S rDNA, but does not allow
the recognition and the reliable phylogenetic placement
of species not previously described. The most common
method of target characterization is amplification, fol-
lowed by either probe hybridization, restriction fragment
length polymorphism analysis, or sequencing. Although
sequence analysis requires more specialized equipment
than the other methods, this technology is becoming less
expensive and provides the highest level of resolution and
portability. Sequencing of the 16S RNA gene is therefore
regarded as the most suitable method for identification of
mycobacteria in the clinical laboratory setting [5,6,17].
Existing sequence databases and analytical tools (e.g., the
National Center for Biotechnology Information [NCBI]
GenBank and the Ribosomal Database Project [RDP]
[18,19]) are not optimal for accurate identification of
clinically relevant microorganisms. The deficiencies in the
contents of these databases include the presence of ragged
sequence ends (resulting in wrong 'best' matches in simi-
larity searches), faulty sequence entries (due to error-
prone sequencing techniques used earlier, e.g., reverse
transcriptase sequencing), absence of quality control of
sequence entries, noncharacterized entries, outdated
nomenclature, and the lack of type strains pertaining to
many clinically important microorganisms. Furthermore,
search results are not presented in a user-friendly manner.
Our ribosomal differentiation of microorganisms
Sequencing the entire 16S rDNA is not a practical method
for routine identification. However, the information con-
tent of the 5' end of the gene is sufficient for specific iden-
tification of most Mycobacterium species (i.e., only one
sequencing run) [7]. Therefore, in order to establish an
improved 16S rDNA reference sequence database for the
identification of clinical isolates, we sequenced both
strands of the 5'-16S rDNA (Escherichia coli position 54–
510) from 199 mycobacterial isolates. All validly
described species (n = 89; up to March 21, 2000) and
nearly all published sequevar variants were included. If
the 16S rDNA sequences were not discriminatory (i.e., dif-
ferent and unique), the ITS region sequences were also
determined. The ultimate goal of this study was to come
up with an algorithm for genetic differentiation of all
mycobacteria, using insertion element and gyrB PCRs in
addition to rRNA operon sequencing, when the latter tar-
get was not discriminatory enough.
(This study was presented in part at the 101st General
Meeting of the American Society for Microbiology,
Orlando, Florida, 20 to 24 May 2001.)
Methods
Bacterial strains and growth conditions
The strains investigated in this study are listed in Table 1
(see Additional file Table 1). Culture collection isolates,
including the type strains, were used in this analysis when
available. Most strains were cultivated on Löwenstein-
Jensen media at 28°C and 37°C. Mycobacterium haemo-
philum was cultivated on Löwenstein-Jensen media with
factor X strips (Becton Dickinson, Heidelberg, Germany),
whereas M. avium subsp. paratuberculosis was cultured on
Middlebrook-Cohn 7H10 agar with OADC and mycobac-
tin enrichment. M. genavense was grown in broth media
(BACTEC 13A medium, Becton Dickinson). For some iso-
lates, only DNA and no culture was available (see Table 1
footnotes, e.g., M. leprae or M. lepraemurium). All isolates
with missing sequence entries in public databases or with
sequence discrepancies detected by GenBank BLAST
searches were additionally identified using extensive con-
ventional biochemical methods [1]. At least two different
culture collection strains from these species were included
in this study.
In vitro amplification and DNA sequencing of the 16S
ribosomal RNA genes and its region
A loopful of bacterial cells for extraction of DNA was
washed with distilled water and incubated in 200 µl TE
buffer (Tris-HCl, 10 mM; EDTA, 1 mM; pH 7.0) for 30
min at 80°C. The DNA was extracted with N-cetyl-N-, N,
N-trimethylammoniumbromide (CTAB)/NaCl according
to the protocol of van Embden et al. [23]. The final DNAPage 2 of 10
(page number not for citation purposes)
(RIDOM) project attempts to overcome these problems
[20–22].
precipitate was suspended in 200 µl TE buffer and stored
frozen (-20°C) until PCR was performed. Two microliters

BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/26
of this suspension (approximately 10 ng of DNA) were
used for PCR amplifications. PCR was performed in a total
volume of 50 µl containing 200 µM deoxynucleoside tri-
phosphates (dATP, dCTP, dGTP, and dTTP), 10 pmol of
each primer, 5 µl of 10-fold concentrated PCR buffer (100
mM Tris-HCl; 500 mM KCl; 15 mM MgCl2; pH 8.3), and
1 U of AmpliTaq DNA polymerase (Applied Biosystems,
Weiterstadt, Germany). Thermal cycling reactions con-
sisted of an initial denaturation (80°C, 5 min) followed
by 28 cycles of denaturation (94°C, 45 s), annealing
(53°C for both 16S rDNA- and ITS-PCR, 1 min), and
extension (72°C, 90 s), with a single final extension
(72°C, 10 min). The broad-range primers 16S-27f (5'-
AGA GTT TGA TCM TGG CTC AG -3') and 16S-907r (5'-
CCG TCA ATT CMT TTR AGT TT -3') were used for 16S
ribosomal DNA PCR. The universal primers 16S-1511f
(5'- AAG TCG TAA CAA GGT ARC CG -3') and 23S-23r
(5'- TCG CCA AGG CAT CCA CC -3') were used for ampli-
fication of the ITS region. Identical or near-identical
primer binding sites have already been described by Lane
[24]. Reactions took place in a dedicated automated DNA
thermal cycler (GeneAmp 2400, Applied Biosystems). In
order to control for the presence of contaminating nucleic
acids, controls containing water in place of template
DNA, were run in parallel in each run. The amplicons
were sequenced using the BigDye Terminator V2.0 Ready
Reaction Cycle Sequencing Kit (Applied Biosystems). The
sequencing reaction required 2 µl of Premix, 5 pmol of
sequencing primer and 0.2 µg of the PCR product tem-
plate in a total volume of 10 µl. For sequencing 16S rDNA
either the primer 16S-27f or 16S-519r (5'- GWA TTA CCG
CGG CKG CTG -3') were used both with annealing tem-
perature of 53°C. For sequencing ITS either the primer
16S-1511f or 23S-23r was employed, with an annealing
temperatures of 55°C and 51°C, respectively. All
sequencing reactions were performed using the GeneAmp
2400 system with 25 cycles of denaturation (96°C, 10 s),
annealing (temperature depending on the sequencing
primer used, 5 s), and extension (60°C, 4 min). The
sequencing products were purified using the recom-
mended Centri-Sep Spin Columns (Princeton Separa-
tions, Adelphia, NJ), followed by preparation for running
onto the ABI Prism 377 or 310 Genetic Analyzer, in
accordance with the instructions of the manufacturer
(Applied Biosystems). The nucleotide sequences for both
DNA strands were determined. Ambiguities were rese-
quenced and at least 98% percent of the complete double-
stranded sequences of the 16S rDNA and ITS targets were
obtained.
Subcloning of PCR products
M. celatum isolates exhibit 16S rDNA interoperon varia-
bility. Furthermore, several fast growing mycobacteria
sequencing of PCR products of these isolates was there-
fore not possible. PCR products of these strains were sep-
arated on an agarose gel and the first band, larger than 200
bp, was cut and cleaned with the Jetsorb Gel Extraction kit
(Genomed, Bad Oeynhausen, Germany). The cleaned
DNA was subcloned in a plasmid vector with the TOPO
TA Cloning kit (Invitrogen, Carlsbad, CA) according to
the recommendations of the manufacturer. Transformed
Escherichia coli strains were cultured and crude DNA
extractions were performed by heating and centrifugation.
PCRs with M13 primers were run with an aliquot of the
supernatant and the PCR products of three subclones each
were sequenced as stated above.
Analysis of the ribosomal DNA sequences and statistical
analysis
The sequencing output from the ABI Prism Genetic Ana-
lysers was analysed using the Sequence Navigator version
1.0.1 computer software (Applied Biosystems). The
region from base positions 54 to 510 (corresponding to E.
coli 16S rDNA positions) for the 16S rDNA and the com-
plete ITS were further analysed. Sequences from primer
regions were not included in this analysis. The MegAlign
(version 3.11) component of the Lasergene program
(DNASTAR Inc., Madison, WI) was used for multiple
alignment and phylogenetic analysis. Multiple sequence
alignments were determined using the CLUSTAL W algo-
rithm. Spearman's rank correlation test, a non-parametric
measure of the degree of association between two numer-
ical values, was used to access the correlation between the
means of base differences (i.e., differences between Gen-
Bank and RIDOM sequence data) stratified by years and
the GenBank submission date. The StatView version 5.0
statistical software package was used to calculate the
Spearman's rank correlation (rs) and the significance of
association (SAS Institute Inc., Cary, NC).
RIDOM implementation
We have recently changed the uniform resource locator
(URL) of our RIDOM service (from http://www.ridom.de
to http://www.ridom-rdna.de) and substantially
improved the implementation. Main backend compo-
nents of RIDOM include the PHRED/PHRAP, FASTA and
CLUSTAL W programs that are embedded into Java Serv-
lets [25–27]. In order to view the sequence
chromatograms in the new "Trace Editor", client comput-
ers need to have a recent version of a standard WWW
browser (Netscape or IE version 4 or higher) and Sun's
Java Plug-in (1.2 or higher) installed.
Results
A total of 199 partial 16S rDNA (corresponding to E. coli
positions 54 to 510) and 84 complete ITS sequences fromPage 3 of 10
(page number not for citation purposes)
contain ITS operons which differ in length and/or base
composition (Table 1, explanatory footnotes). Direct
mycobacterial culture isolates were newly determined, for
the purpose of building up a high-quality reference

BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/26
sequence database. All validly described species and sub-
species (n = 89; up to March 21, 2000) were included. In
this study a valid publication of a new name or new
nomenclature combination refers to publications appear-
ing in the International Journal of Systematic Bacteriology
(IJSB) / International Journal of Systematic and Evolu-
tionary Microbiology (IJSEM, from January 2000), either
as an original article or in the Validation Lists regularly
appearing in this journal. The Validation Lists constitute
valid publication of new names and new combinations
that meet validation criteria and which have been previ-
ously published in journals other than IJSB and IJSEM.
Names not considered validly published should no longer
be used or should be used in quotation marks (e.g.
"Mycobacterium album") to indicate that the name has
not been validly published. One hundred sixty of the 199
isolates sequenced were obtained from culture collec-
tions. The remaining 39 strains were obtained for
sequencing from private collections (Table 1, footnotes).
Additionally, fifteen 16S rDNA and 19 ITS GenBank
entries were included in the subsequent analysis (Tables
1, footnotes). The 16S rDNA from many Mycobacterium
species had been previously published. In contrast, the ITS
sequence was generated from several species for the first
time.
Differentiation of Mycobacterium species based on rRNA
operon sequencing
A 16S rDNA phylogenetic tree was created that included
one representative strain of each sequence variant. Species
having identical sequences are shown in bold (Figure 1).
According to 5'-16S rDNA sequencing, 64 different myco-
bacterial species (71.9%) could be identified. With the
additional input of the ITS sequence, a further 16 species
or subspecies could be resolved. The groups that shared
identical partial 16S rDNA and which could be discrimi-
nated with the aid of their ITS sequences were: (i) Myco-
bacterium abscessus and M. chelonae sequevar I; (ii) M.
gastri and M. kansasii sequevars I & IV; (iii) M. fortuitum 3rd
biovariant (sorbitol +, sequevar II),M. farcinogenes and M.
senegalense; (iv) M. fortuitum 3rd biovariant (sorbitol -,
sequevar III) and M. porcinum; (v) M. fortuitum subsp.
acetamidolyticum and M. fortuitum subsp. fortuitum seque-
var I; (vi) M. peregrinum and M. septicum; (vii) M. murale
and M. tokaiense; and (viii) M. flavescens sequevar II and
M. novocastrense. Only the four Mycobacterium tuberculosis
complex species, M. marinum / M. ulcerans (Mul A) and
the three M. avium subspecies could not be differentiated
using 5'-16S rDNA or ITS sequencing.
16S rRNA gene variability of Mycobacterium species
Intraspecies rRNA gene heterogeneity was encountered in
the case of some mycobacterial species. Sequevar (sqv.)
labelled with a species name acronym and an Arabic cap-
ital letter (e.g., Mka A for M. kansasii ITS sequevar variant
A). 16S rDNA sequevars were designated with Roman
numerals (e.g., M. chelonae sqv. I for M. chelonae 5'-16S
rDNA variant I). The 16S rDNA sequevar designations for
M. kansasii are somewhat inconsistent (i.e., M. kansasii
sqv. I and sqv. IV as well as M. kansasii sqv. III and IV-2
have identical 5'-16S rDNA sequences) because the
sequence variants were initially determined by hsp65 anal-
ysis [11,13,29]. The following 16S rDNA sequence vari-
ants were observed: (i) M. avium sqv. I-II, (ii) M. chelonae
sqv. I-II, (iii) M. flavescens sqv. I-II, (iv) M. fortuitum sqv. I-
V, (v) M. gordonae sqv. I-V, (vi) M. intracellulare sqv. I-V,
(vii) M. kansasii sqv. I & IV, II, III & VI-2, V, VI-1 and VI-3,
(viii) M. lentiflavum sqv. I-II, (ix) M. parafortuitum sqv. I-II,
(x) M. simiae sqv. I-II, (xi) M. terrae sqv. I-III, and (xii) M.
xenopi sqv. I-III.
ITS micro-heterogeneity of the genus Mycobacterium
A total of 84 complete ITS sequences from mycobacterial
culture isolates were newly determined. We were not able
to obtain isolates of all published ITS sequence variants.
Therefore, for some M. avium, M. intracellulare, M. kansasii
and M. simiae variants our ITS analysis relied on a few
recently submitted GenBank entries (Table 1, explanatory
footnote). Furthermore, the ITS region was not studied in
the same detail as the 5'-16S rDNA. Nevertheless several
new sequence variants were observed. The following is a
detailed listing of the results: (i) M. avium Mav A-E, (ii)M.
chelonae Mche A-C, (iii) M. flavescens Mfla A-B, (iv) M. for-
tuitum Mfor A-D, (v) M. gordonae Mgo A-E, (vi) M. intrac-
ellulare MAC A-F, MAC H-L and Min A-D, (vii) M. kansasii
Mka A-F, (viii)M. peregrinum Mpe A-C, (ix) M. phlei
Mphle-A-B, (x) M. scrofulaceum Mscro A-B, (xi) M. simiae
Msi A-E, (xii) M. ulcerans Mul A-B, and (xiii) M. xenopi
Mxe A-C.
Comparison of RIDOM and GenBank mycobacterial 16S
rDNA sequences
Performing a similarity search with RIDOM sequences
against GenBank, we found sequences of 77 identical cul-
ture collection strains with a minimum overlap of 80%.
Comparing these entries in detail with our sequences, we
found an average of 4.31 nucleotide differences (SD ±
0.57). Using the Spearman's rank correlation test a
significant negative correlation between the means of base
differences stratified by years, and the submission date
was also found (with rs -0.56 and p < 0.0001; Figure 2).
Furthermore, seven out of the 160 sequenced culture col-
lection strains turned out to be "wrong" (4.4%), i.e., dif-
fered excessively from published sequence and
phenotypic data. These isolates were omitted from further
analysis (Table 1, footnotes).Page 4 of 10
(page number not for citation purposes)
designations were then chosen according to the nomen-
clature of Frothingham and Wilson [28]. ITS variants were

BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/26
5'-16S rDNA based phylogenetic tree of the genus Mycobacterium, including one representative strain of each sequence variant (sequevar = sqv.)Figure 1
5'-16S rDNA based phylogenetic tree of the genus Mycobacterium, including one representative strain of each sequence variant
(sequevar = sqv.). T designates the type strain of this species. Multiple sequence alignments were determined using the CLUS-
TAL W algorithm in the MegAlign component of the Lasergene program. The tree was rooted using Corynebacterium pseu-
dodiphtheriticum as the outgroup sequence. Sequences were all determined in our laboratory unless indicated by GenBank
accession number. Species having identical sequences are shown in bold. The numbers on the abscissa represent the percent
0
10.7
246810
M. gastri DSM 43505 T
M. kansasii DSM 44162 T (sqv. I) & M. kansasii DSM 43221 (sqv. IV)
M. kansasii Bo 10492/98 (sqv. V)
M. kansasii S221 (sqv. II)
M. kansasii Bo 539/99 (sqv. III) & M. kansasii Bo 5160/97 (sqv. VI-2)
M. kansasii DSM 44431 (sqv. VI-1)
M. kansasii Bo 8875/99 (sqv. VI-3)
M. haemophilum ATCC 29548 T
M. leprae AHI 1104/96
M. malmoense DSM 44163 T
M. bohemicum DSM 44277 T
M. avium subsp. avium DSM 43216 (sqv. II)
M. avium subsp. avium DSM 44156 T (sqv. I)
M. avium subsp. paratuberculosis DSM 44133 T (sqv. I)
M. avium subsp. silvaticum DSM 44175 T (sqv. I)
M. lepraemurium LRC
M. intracellulare S350 (sqv. IV)
M. intracellulare ATCC 35847 (sqv. II)
M. intracellulare ATCC 35772 (sqv. V)
M. intracellulare DSM 43223 T (sqv. I)
M. intracellulare ATCC 35770 (sqv. III)
M. conspicuum DSM 44136 T
M. szulgai DSM 44166 T
M. africanum ATCC 25420 T (subtype I) & M. africanum AHI 15/99 (subtype II)
M. bovis subsp. bovis ATCC 19210 T & M. bovis BCG DSM 43990
M. microti ATCC 19422 T
M. tuberculosis ATCC 27294 T
M. bovis subsp. caprae CIP105776 T
M. marinum DSM 44344 T
M. ulcerans ATCC 19423 T
M. asiaticum DSM 44297 T
M. gordonae Bo 11340/99 (sqv. III)
M. gordonae DSM 43212 (sqv. II)
M. gordonae DSM 44160 T (sqv. I)
M. gordonae Bo 10681/99 (sqv. IV)
M. gordonae Bo 9411/99 (sqv. V)
M. scrofulaceum DSM 43992 T
M. xenopi DSM 43995 T (sqv. I)
M. xenopi S91 (sqv. III)
M. xenopi S88 (sqv. II)
M. botniense ATCC 700701 T
M. heckeshornense Wue Tb0687/99
M. celatum DSM 44243 T
M. branderi ATCC 51789 T
M. shimoidei DSM 44152 T
M. triviale DSM 44153 T
M. nonchromogenicum DSM 44164 T
M. terrae S281 (sqv. III)
M. hiberniae DSM 44241 T
M. terrae DSM 43227 T (sqv. I)
M. terrae DSM 43541 (sqv. II)
M. chitae ATCC 19627 T
M. fallax DSM 44179 T
M. murale DSM 44340 T
M. tokaiense ATCC 27282 T
M. gadium DSM 44077 T
M. moriokaense DSM 44221 T
M. pulveris DSM 44222 T
M. thermoresistibile DSM 44167 T
M. agri ATCC 27406 T
M. confluentis DSM 44017 T
M. hassiacum DSM 44199 T
M. goodii ATCC 700504 T
M. smegmatis DSM 43756 T
M. madagascariense ATCC 49865 T
M. phlei DSM 43239 T
M. brumae DSM 44177 T
M. austroafricanum DSM 44191 T
M. vaccae DSM 43292 T
M. duvalii DSM 44244 T
M. flavescens DSM 43531 (sqv. II)
M. novocastrense DSM 44203 T
M. flavescens DSM 43991 T (sqv. I)
M. tusciae DSM 44338 T
M. heidelbergense ATCC 51253 T
M. simiae ATCC 15080 (sqv. II)
M. lentiflavum ATCC 51988 (sqv. II, [X80770])
M. lentiflavum DSM 44418 T (sqv. I)
M. interjectum ATCC 51457 T
M. simiae DSM 44165 T (sqv. I)
M. intermedium DSM 44049 T
M. genavense Wue Tb0268/96
M. triplex ATCC 700071 T
M. kubicae ATCC 700732 T [AF133902]
M. hodleri DSM 44183 T
M. cookii DSM 43922 T
M. poriferae ATCC 35087 T
M. mageritense CIP 104973 T
M. wolinskyi ATCC 700010 T
M. diernhoferi DSM 43524 T
M. neoaurum DSM 44074 T
M. farcinogenes DSM 43637 T
M. fortuitum 3rd biovariant ATCC 49403 (sqv. II, sorbitol + )
M. senegalense DSM 43656 T
M. mucogenicum 49650 T
M. fortuitum subsp. acetamidolyticum DSM 44220 T (sqv. I)
M. fortuitum subsp. fortuitum DSM 46621 T (sqv. I)
M. peregrinum ATCC 14467 T
M. septicum DSM 44393 T
M. alvei DSM 44176 T
M. fortuitum DSM 43075 (sqv. V)
M. fortuitum 3rd biovariant ATCC 49404 (sqv. III, sorbitol -)
M. porcinum DSM 44242 T
M. fortuitum S358 (sqv. IV)
M. aichiense DSM 44147 T
M. rhodesiae DSM 44223 T
M. sphagni ATCC 33027 T
M. komossense DSM 44078 T
M. abscessus DSM 44196 T
M. chelonae DSM 43804 T (sqv. I)
M. chelonae S385 (sqv. II)
M. immunogenum T [AJ011771]
M. chlorophenolicum DSM 43826 T
M. chubuense DSM 44219 T
M. aurum DSM 43999 T
M. obuense DSM 44075 T
M. parafortuitum S513 (sqv. II)
M. parafortuitum DSM 43528 T (sqv. I)
M. gilvum ATCC 43909 T
Corynebacterium pseudodiphtheriticum DSM 44287 TPage 5 of 10
(page number not for citation purposes)
distance between different isolates.