1
Survey of 150 strains belonging to the Mycobacterium terrae complex and description of Mycobacterium engbaekii sp. nov., Mycobacterium heraklionense sp. nov. and Mycobacterium longobardum sp. nov. Tortoli Enrico,1 Gitti Zoe,2 Klenk Hans-Peter,3 Lauria Stefania,4 Mannino Roberta,4 Mantegani Paola,1 Mariottini Alessandro,5 Ioannis Neonakis.2 1 Emerging Bacterial Pathogens, San Raffaele Scientific Institute, Milan, Italy 2 Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University Hospital of Heraklion, Heraklion, Greece 3 DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 4 Microbiology and Virology Laboratory, Careggi University Hospital, Florence, Italy 5 Cytogenetics and Genetics Unit, Careggi University Hospital, Florence, Italy Corresponding author: Enrico Tortoli, Emerging Bacterial Pathogens, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy; E-mail: [email protected] Running title: Mycobacterium terrae complex Keywords: Mycobacterium terrae complex, Mycobacterium engbaekii, Mycobacterium heraklionense, Mycobacterium longobardum, sp. nov. The GenBank accession number of the almost complete 16S rRNA sequences of M. engbaekii, M. heraklionense and M. longobardum are AF480577, GU084182 and JN571166 respectively. In the 16S rRNA gene the accession numbers of the different sequevars are JN571167-9 for M. arupense, JN571172 for M. engbaekii, JN571173 for M. hiberniae, JN571176-9 for M. kumamotonense, JN571171 for M. nonchromogenicum, JN571174-5 for M. senuense, JN571180-2 for M. terrae and JN571183-5 for other unassigned strains belonging to the M. terrae complex. In the hsp65 gene the accession numbers of the different sequevars are FJ263631 and JN571186-90 for M. arupense, JN571194-7 for M. engbaekii, JN571191-2 for M. heraklionense, JN571198 for M. hiberniae, JN571202-3 for M. kumamotonense, JN571199 for M. longobardum, JN571193 for M. nonchromogenicum, JN571200-1 for M. senuense, JN571204-7 for M. terrae and JN571208-14 for other unassigned strains belonging to the M. terrae complex. In the rpoB gene the accession numbers of the different sequevars are JN571215-29 for M. arupense, JN571242-5 for M. engbaekii, JN571230-6 for M. heraklionense, JN571246 for M. hiberniae, JN571251-58 and JN571290 for M. kumamotonense, JN571247-8 for M. longobardum, JN571237-41 for M. nonchromogenicum, JN571249-50 for M. senuense, JN571261-6 for M. terrae and JN571170 and JN571267-73 for other unassigned strains belonging to the M. terrae complex.
IJSEM Papers in Press. Published March 23, 2012 as doi:10.1099/ijs.0.038737-0
2
A thorough phenotypic and genotypic analysis of 150 strains belonging to the 1
Mycobacterium terrae complex resulted in the identification of a number of previously 2
unreported sequevars (sqvs.) within the species known to belong to the complex. In the 3
species Mycobacterium arupense were detected 3 sqvs. in the 16S rRNA gene, 6 sqvs. in 4
the hsp65 gene and 15 sqvs. in the rpoB gene; in Mycobacterium senuense 2 sqvs. were 5
present in each of the three genetic regions; in Mycobacterium kumamotonense 4, 2 and 9 6
sqvs. respectively and in M. terrae 3, 4 and 6 sqvs. respectively were found. The 7
extraneousness of Mycobacterium triviale with the M. terrae complex was confirmed. 8
The limited utility of biochemical tests and of mycolic acids analyses for the 9
differentiation of the members of M. terrae complex was confirmed as well. The survey 10
allowed furthermore to recognize three previously undescribed species characterized by 11
unique sequences in 16S rRNA, hsp65 and rpoB genes. Mycobacterium engbaekii sp. 12
nov., a species proposed 40 years ago but never validly published, is characterized by 13
pink photochromogenic pigmentation and rapid growth; phylogenetically it is related to 14
Mycobacterium hiberniae. The type strains of this species, of which eight strains were 15
investigated, is ATCC27353=DSM45694. A cluster of 24 strains was the basis for the 16
description of Mycobacterium heraklionense sp. nov. which has intermediate growth rate 17
and is unpigmented, the nitrate reductase activity is typically strong. Closely related to M. 18
arupense in the 16S rRNA gene, M. heraklionense is clearly differentiable from the latter 19
species in the other genetic regions. The type strain is NCTC13432=LMG2473= 20
CECT7509. Mycobacterium longobardum sp. nov., represented in the study from seven 21
strains, is characterized by a unique phylogenetic location within the M. terrae complex, 22
evidently divergent from any other species. The type strain is DSM45394=CCUG58460. 23
3
The Mycobacterium terrae complex (MTC) is a group, within the genus Mycobacterium, 24
created in the 1970s to gather Mycobacterium nonchromogenicum, Mycobacterium terrae 25
and Mycobacterium triviale, three species not differentiable with methods (biochemical 26
and cultural tests) available at that time. M. nonchromogenicum described in 1965 27
(Tsukamura, 1965), M. terrae described in 1966 (Wayne, 1966) and M. triviale described 28
in 1970 (Kubica et al., 1970) share important cultural features including intermediate 29
growth rate (from 5 to 15 days are required to develop clearly visible colonies from 30
diluted inocula on solid media) and lack of pigmentation. 31
In the early 1990s, the detection of a unique genetic signature: the presence of a two-32
nucleotide insertion in the helix 18 of the 16S rRNA gene (Kirschner et al., 33
1993,Springer et al., 1996), in comparison to other slow growing mycobacteria, 34
confirmed the consistence of the MTC. The signature above remains nowadays the most 35
reliable mark for the attribution of mycobacteria to MTC. At the same time the presence, 36
in M. triviale likewise in rapid growers, of an helix 18 14-nucleotide shorter, 37
unquestionably demonstrated the unrelatedness to this species to the complex. 38
A gap of more than 20 years separates recognition of the classical members of MTC from 39
the description of a new species related to such a group, Mycobacterium hiberniae 40
(Kazda et al., 1993). This novel mycobacterium is characterized by a unique phenotypic 41
feature; the pink pigmentation of the colonies, but the major role in its differentiation was 42
played by the genetic analysis that was, at that time, beginning to impose itself. The 43
boom years of MTC start however in 2006 with three new species described (Cloud et al., 44
2006, Masaki et al., 2006, Mun et al., 2008). 45
4
The identification at species level, within the members of the MTC still remains, almost 46
50 years later, problematic. As well as biochemical and cultural tests (Wayne & Kubica, 47
1986), the analysis of cell wall lipids revealed poorly discriminative. And, more recently, 48
DNA-probes specific for the MTC species have not been implemented by any of the 49
commercial hybridization methods; probably as a consequence of the limited interest 50
aroused by organisms grossly labeled, since the beginning, as non pathogenic. 51
Unexpectedly, even the identification by means of genetic sequencing remains elusive as 52
in public domain databases hundreds of sequences related to MTC are crowded without, 53
or with unreliable, species allocation. 54
The aim of this study was to investigate the phylogenetic and taxonomic structure of the 55
MTC on a large number of isolates and to make available in GenBank a panel of species-56
specific genetic sequences characterized by certain labels. 57
58
Materials and methods 59
60
Strains 61
For this study, all the strains assigned to the MTC on the basis of the routine 62
identification performed in our laboratory as from the year 1996 onward were 63
investigated. All such strains (no. 156) had been grown from clinical specimens and 64
stored frozen at -80°C. While the large majority of them had been isolated either in 65
Careggi Hospital laboratory or in other Italian hospitals, 27 strains had been obtained 66
from laboratories of other countries. After thawing, each strain was grown on 67
Middlebrook 7H11 medium at 37°C. The type strains of the species “M. engbaekii” 68
5
(ATCC27353), M. hiberniae (ATCC49874), Mycobacterium kumamotonense 69
(DSM45093), Mycobacterium senuense (DSM44999) and M. terrae (CIP104321) were 70
also included in the study; other reference strains investigated were M. 71
nonchromogenicum (PI140330001), and M. triviale (PI141030004). 72
73
Genetic sequencing 74
Three different regions were chosen for genetic characterization, the genes coding for the 75
16S rRNA (16S), for the 65 kDa heath shock protein (hsp65) and for the β subunit of the 76
RNA polymerase (rpoB). Of the 16S, according to a previously reported procedure 77
(Reischl et al., 1998), a trait including 479 bp was sequenced starting from the 78
Escherichia coli-corresponding position 28. The almost complete 16S sequence was 79
determined for the strains for which the sp. nov. status is proposed here. 80
Three hundred and ninety nine bp were sequenced in the hypervariable trait of hsp65 81
(McNabb et al., 2004), starting from the Mycobacterium tuberculosis-homologous 82
position 443. 83
In the rpoB the stretch recently proposed for the differentiation of rapidly growing 84
mycobacteria (Adékambi et al., 2003) was investigated; the length of the nucleotide 85
sequence (starting at the Mycobacterium smegmatis-corresponding position 2,554) ranged 86
from 711 to 726 bp in different strains. 87
In all the regions above both the forward and reverse strands were determined using Big 88
Dye terminator chemistry and an AB3730 DNA sequencer (Applied Biosystems). 89
For the designation of sequevars (sqvs.) for which the assignation to a species was 90
possible, the first three letters (capitalized) of the species name were used followed, for 91
6
16S, by a small letter (a, b, . . .), for hsp65, by a number (1, 2, . . .) and, for rpoB, by a 92
Roman numeral (i, ii, . . .). 93
Sequences of protein-coding genes (hsp65, 133 codons; rpoB, 237-242 codons) were also 94
translated to the amino acid residues composition to distinguish silent mutations from the 95
ones affecting the protein structure. 96
97
Phylogenetic analysis 98
The phylogenetic analysis was conducted according to the neighbour-joining method 99
(Saitou & Nei, 1987) under the total gap removal and Kimura’s two-parameter 100
substitution model (Kimura, 1980), and was evaluated by bootstrap analysis based on 101
1,000 replicates using the MEGA software version 5 {Tamura, 2011 38699 /id}. The 102
trees were rooted using, as outgroup, Mycobacterium tuberculosis whose respective 103
sequences retrieved from GenBank had been added previously to various alignments. 104
The phylogenetic reconstruction based on 16S included 22 different sqvs. detected among 105
the strains investigated in this study. The sequences, downloaded from GenBank, of the 106
most closely related slowly- and rapidly-growing mycobacteria were added as well. 107
To improve the robustness of the tree (Devulder et al., 2005,Mignard & Flandrois, 108
2008,Stackebrandt et al., 2002) the sequences of the three genetic regions were 109
concatenated in a single filament including a number of nucleotides ranging, in different 110
strains, from 1,589 to 1,604 (16S, 479; hsp65, 399; rpoB, 711-726). In the investigation 111
concerning the concatenated sequences all the combinations (no. 75) of 16S, hsp65 and 112
rpoB sqvs. detected in our strains were included. 113
114
7
Lipid investigations 115
High performance liquid chromatography (HPLC) of cell-wall mycolic acids was carried 116
out on all the strains after esterification to bromophenacyl esters as reported (C.D.C., 117
1996). 118
119
Biochemical and cultural tests 120
For all the strains, nitrate reduction, growth rate and pigmentation of colonies were 121
investigated. For the strains considered to represent new species a number of randomly 122
selected strains (10 of M. heraklionense, 5 of M. engbaekii and 2 of M. longobardum) 123
were investigated with a wider panel of tests (Table 3) according to the standard 124
procedures (Kent & Kubica, 1985). 125
126
Susceptibility testing 127
The susceptibility testing was performed on randomly selected strains belonging to the 128
new species proposed here (4 of M. heraklionense, 4 of M. engbaekii and 2 of M. 129
longobardum). The minimal inhibitory concentrations (MIC) of drugs selected for their 130
activity on slowly growing mycobacteria were determined using commercially available 131
microdilution plates (SLOMYCO, VersaTREK) following the CLSI recommendations 132
{C.L.S.I., 2011 58541 /id} 133
134
Results 135
Genetic sequencing 136
8
The alignment of the 22 sqvs. detected in the 16S of the 156 strains investigated added to 137
the existing evidence that M. triviale does not belong to the MTC. The helix 18 of the 138
16S was in fact, in the six strains of M. triviale present in our panel, 14 nucleotides 139
shorter than in remaining strains. Such a feature, not only excludes M. triviale from MTC 140
but even places this species within rapid growers. These six strains were therefore 141
excluded from the study. 142
Six of the 21 remaining 16S sqvs. turned out to overlap the sequences of the type strains 143
of the six species known to belong to the MTC and this allowed to assign them to M. 144
arupense, M. nonchromogenicum, M. hiberniae, M. senuense, M. terrae and M. 145
kumamotonense. Another sqvs. was 100% identical to the sequence of the not officially 146
recognized species “Mycobacterium engbaekii”. Only nine of the remaining 16 sqvs. 147
were found in GenBank database, five of them had been previously deposed by one of us, 148
while the species assignation of two others was either lacking or incorrect. The pairwise 149
matrix of distances (Table S1) allowed the identification of four clusters of sqvs. which 150
were assigned to the species M. arupense, M. senuense, M. kumamotonense and M. terrae 151
on the basis of the inclusion of the sequences of the respective type strains. Within such 152
clusters the intraspecies variability was <1%. One sqv. shared by two strains (FI-153
07105/FI-11038) differed, for one nucleotide only, from the not officially recognized 154
“Mycobacterium paraterrae” (Lee et al., 2010). The three sqvs. remaining (NEW1, GN-155
9188, FI-09379), differed from any type strain and did not fit any cluster. 156
Thirty sqvs. were found in the hsp65. With the similarity matrix (Table S2) eight clusters 157
were recognized in this region, which were characterized by pairwise distances <3% (3 158
exceptions with values up to 3.36%). Five such clusters were immediately attributed to 159
9
the species M. arupense, M. senuense, M. kumamotonense, M. terrae and “M. engbaekii”, 160
because of the inclusion of the sequences of the respective type strains; they all were 161
detected in strains assigned to the same species by the 16S sequence. One of the clusters 162
remaining (NEW2, including 2 sqvs.), although clearly separated from the cluster of M. 163
arupense, had been detected in strains assigned, on the basis of 16S, to the latter species. 164
The other two clusters (NEW3 and NEW4) included, two and three, orphan (unassigned 165
to any known species) sqvs. respectively. Interestingly two (FI-07105 and FI-11038) of 166
the three sqvs. included in NEW4 belonged to the strains presenting, in 16S, close 167
similarity with “M. paraterrae” but clearly differed from the latter in the hsp65 region. In 168
the species M. nonchromogenicum and M. hiberniae one single sequevar was present. 169
Three sqvs. (NEW1, GN-9188 and FI-05196) differed from any type strain and did not fit 170
any of the clusters above. Interestingly the sequevar NEW1 was detected in the strains 171
classified as NEW1 also on the basis of the 16S. 172
As expected, a large variability was detected in the rpoB fragment, with the presence of 173
58 sqvs. The pairwise matrix of distances (data not shown) included ten clusters with 174
internal variability below the limit (3%) proposed for rpoB region (Adékambi & 175
Drancourt, 2004) in the large majority of cases (5 exceptions with values up to 5.3%). For 176
this gene, the presence in GenBank of only one MTC sequence overlapping to ours 177
(furthermore not assigned to any known species), did not allow us to attribute any 178
sequevar to a species; the attribution was therefore inferred from that achieved, for each 179
strain, on the basis of the 16S and hsp65 sqvs. Following this approach it was possible to 180
classify one cluster within each of the species: M. arupense (10 sqvs.), M. 181
nonchromogenicum (5 sqvs.), “M. engbaekii” (4 sqvs.), M. senuense (2 sqvs.), M. 182
10
kumamotonense (9 sqvs.) and M. terrae (6 sqvs.). Of the remaining four clusters, one 183
(NEW1, with 2 sqvs.) was detected in strains classified as NEW1 on the basis of 16S and 184
hsp65 sequences; and one (NEW2, with 7 sqvs.) was detected in strains classified as 185
NEW2 on the basis of hsp65 sequence). One cluster (NEW5, with 3 sqvs.) was detected 186
in strains assigned to the species M. arupense on the basis of 16S and hsp65 sequences, it 187
was however very distant from the rpoB sqvs. of the strains presenting in 16S, and hsp65 188
too, the typical sqvs. of this species. The last cluster (NEW6, with 2 sqvs.) remained 189
orphan. The sqvs. FI-06258, FI-07105/FI-09015/FI-09399, FI-09379, FI-05396/FI-190
06246/FI-05196 and FI-11038 did not fit any cluster. Interestingly the five cases in which 191
the intraspecies variability exceeded 3% were detected within the cluster of M. terrae 192
where the sqv. obtained from the type strain (CIP104321) clearly differed from all the 193
others. 194
Of the sqvs. we detected, apart from the ones of the type strains and the ones previously 195
deposed by some of us, only a limited number was already present in GenBank. As 196
regard to 16S, two of the five sqv. present were not assigned to any species while the 197
label of the remaining three was correct in two cases and incorrect in one. For what 198
concern the hsp65, there were three sqvs. correctly assigned, two lacking any species 199
attribution and three mislabeled; furthermore, in this region, our sequence of the type 200
strain of M. senuense presented one mismatch in comparison to the one present in the 201
database. For rpoB sqvs. only two, both assigned to the species M. terrae, were present in 202
GenBank although not identified. In this region too one type strain (M. terrae) presented 203
one nucleotide discordance. 204
11
The combinations of 16S, hsp65 and rpoB sqvs. detected in the strains investigated here 205
are reported in Table S3. 206
207
Phylogenetic analysis 208
The tree constructed using the sequences of 16S revealed a clear separation of the strains 209
included in the MTC from both the slow and the rapid growers (Fig. 1). The strains 210
investigated here were distributed among two major branches including, the first, M. 211
senuense, M. terrae and M. kumamotonense and, the second, M. arupense, M. 212
nonchromogenicum, M. hiberniae, “M. engbaekii”, and the clusters NEW1 and NEW2. 213
The branch including M. triviale appeared very distant from the species of MTC. 214
The phylogenetic tree inferred from hsp65 was fully in agreement with the one obtained 215
from 16S sequences (Fig. S1) while in the one based on the rpoB a polyphyletic 216
distribution of different species emerged (Fig. S2). 217
The phylogenetic analysis conducted on the concatenated sequences of the 75 different 218
combinations detected in the 137 MTC strains which was possible to assign to a species 219
produced a quite robust dendrogram characterized by a high percentage of nodes, 220
including the early ones, with very high bootstrap values (Fig. 2). Different species-221
specific clusters were clearly separated although belonging to two major groupings: the 222
first including M. arupense, NEW2, M. nonchromogenicum, M. hiberniae, “M. 223
engbaekii” and NEW1; the second comprising M. senuense, M. terrae and M. 224
kumamotonense. 225
12
The analysis of translated nucleotide sequences revealed a large number of synonymous 226
mutations with, in the hsp65 region, only 12 different amino acid sequences being coded 227
by 31 different sqvs. and, in rpoB, only 27 by 59 sqvs. 228
229
HPLC of cell wall mycolic acids 230
The HPLC of cell wall mycolic acids performed on the whole initial group of 156 strains, 231
revealed two different types of pattern (Fig. 3). The motif shared by the large majority of 232
the strains was characterized by the presence of two, clearly distinct, clusters of peaks, 233
with the first one, including three major peaks, starting to elute after 3 min, and the 234
second, including three minor peaks, eluting about 3 min later. The other pattern was 235
presented only by the six strains of M. triviale subsequently excluded from the study; in 236
this species only a late emerging cluster of peaks was present (Fig. 3D). 237
Because of the common motif shared by all the members of the MTC, with only a limited 238
variability in the relative height in the major peaks of the first cluster we tried to correlate 239
their profile to the different species. If the lower peaks were not taken into account, 240
almost all the strains presented three major peaks in the first clusters. The most common 241
motif was characterized by a highest central peak followed by the first and the third in 242
decreasing order (Fig. 3A). This pattern was presented by the strains belonging to the 243
species M. nonchromogenicum and to the group NEW1. It was furthermore shared by 244
70% of the strains of M. arupense and was also represented in about half of the strains of 245
“M. engbaekii” and of the group NEW2. Less frequent, but scattered among various 246
groupings, was the motif in which the third peak was higher than the first with the second 247
being the highest. Unique to the species M. kumamotonense, although not presented by 248
13
all such strains, was a pattern characterized by three peaks almost equally high with the 249
height slightly rising from the first to the third (Fig. 3B). Equally unique, to the species 250
M. terrae, was the profile presenting in the first cluster four major peaks instead of three 251
(Fig. 3C). 252
253
Biochemical and cultural tests 254
The nitrate test was selected as the ability of reducing nitrate to nitrite is classically 255
considered the sole biochemical feature suitable to discriminate within the, otherwise 256
phenotypically homogeneous, species of the MTC (Wayne & Kubica, 1986). The nitrate 257
reductase was possessed by the strains of the of M. kumamotonense; it was absent in M. 258
arupense and M. terrae (Table S4). 259
The colonies of the strains investigated were buff and prevalently smooth; an exception 260
was represented by M. hiberniae and “M. engbaekii” which presented pink pigmentation 261
(Table S4). 262
At 37°C the growth on solid media, from standardized inocula, became distinctly visible, 263
on average, after 7-14 days, faster growth was occasionally observed, while M. senuense 264
grew typically slowly (Table S4). 265
266
Discussion 267
Several obvious conclusions seem to emerge from the results of genotypic and 268
phenotypic investigations carried on a large number of strains belonging to the MTC. 269
i. The legitimacy of the species of the MTC officially recognized at present is clearly 270
confirmed. 271
14
ii. The group we temporarily named NEW1 includes strains evidently different from any 272
other species of the MTC in all the three genetic regions investigated; for it (of which 273
seven strains were characterized in this study) the status of new species is proposed with 274
the name Mycobacterium longobardum. 275
iii. The strains included so far in the group NEW2, which had been initially assigned, on 276
the basis of the analysis of the 16S sequence, to the species M. arupense, are actually 277
clearly distant from the latter in the regions hsp65 and rpoB, and represent a previously 278
unreported species for which the name Mycobacterium heraklionense is proposed; of this 279
23 strains were characterized in this study. 280
iv. The seven strains investigated here and assigned to the not officially recognized 281
species “M. engbaekii” support the revival of M. engbaekii sp. nov. The name M. 282
engbaekii was proposed in 1972 for 15 rapidly growing strains presenting pink 283
pigmentation (Korsak & Boisvert, 1972); although the type strain of “M. engbaekii” was 284
deposed in the American Type Culture Collection the sp. nov. description did not follow 285
and it sank into oblivion. 286
v. Within the species M. arupense two variants can be clearly distinguished on the basis 287
of the rpoB sequence; sufficient evidence has not emerged so far to justify the elevation 288
of this new variant (indicated in this study as NEW6) to species rank, despite the 289
divergence, clearly over the proposed cut-off, in the rpoB region. 290
vi. Surprisingly, the recently described species M. arupense and M. kumamotonense are, 291
by far, the most frequently isolated members of the MTC. One of the reference strains 292
investigated here (M. nonchromogenicum PI140330001) turned out to be M. arupense, 293
which supports the hypothesis that the latter species and M. kumamotonense were 294
15
identified in the past (and, at times, at present too) as M. nonchromogenicum and M. 295
terrae respectively. In our study the isolations of M. nonchromogenicum and M. terrae 296
were not as frequent as generally acknowledged; which brings up for discussion the 297
widespread conviction considering M. nonchromogenicum the only member of MTC 298
potentially responsible for disease (Tsukamura et al., 1983). 299
vii. A number of strains have been detected presenting in 16S, hsp65 and rpoB either 300
conflicting sqvs. of MTC species (3 cases) or unreported sqvs. not related to other, 301
official or proposed, MTC species (10 cases; data not shown). Such strains (Table S5) 302
need further characterization and have not been included in the phylogenetic 303
investigations. 304
viii. Questions are raised by the type strain of M. terrae. While in the 16S its sequence 305
clusters with the ones of number of strains (Table S1) in hsp65 (Table S2) and, even more 306
evidently in rpoB, it clearly diverges from the others. A question remains so far 307
unanswered. Is the type strain of M. terrae a rare organism, poorly representative of the 308
species? Are, on the contrary, the other strains assigned here to M. terrae actually 309
members of a still undescribed new species? With the latter option implying that M. 310
terrae is so rare that no one was detected in our long-term study. 311
ix. A hoary, unresolved, problem is that of the lawfulness of describing a new species 312
based on a single isolate. It is not our aim to deal with this topic here; nevertheless in this 313
study a dozen unique strains emerged as potentially exploitable for the description of the 314
same number of new species! 315
316
Bacterial strains of the proposed new species 317
16
Seven strains of M. longobardum were isolated in two Italian hospitals between years 318
2006 and 2009 from the sputum of six Italian, and one Lebanese, patient. Microscopy 319
was negative and growth was obtained on solid media only (despite in four cases also 320
liquid media had been inoculated). In no case was the strain considered responsible for 321
disease. 322
Twenty three strains of M. heraklionense had been isolated in Greece (no. 10), in Italy 323
(no. 7) and in India (no. 6). The Greek strains were grown from one outpatient and nine 324
hospitalized patients in the island of Crete between 2002 and 2003. The Italian strains 325
were isolated between 2005 and 2011 in five different hospitals. No information is 326
available for the Indian strains apart from that they had been isolated before the year 327
2005. 328
Of the seven strains of M. engbaekii six had been isolated in Italy, in three different 329
hospitals between 1998 and 2011, and one in Guadeloupe. 330
Clinical and epidemiological information available for part of the strains above are 331
reported in Table 1. 332
333
Genetic sequences of the proposed new species 334
In the 16S, M. arupense was the officially recognized species most closely related, to M. 335
longobardum from which however 25 bp out 1,488 differed (similarity 98.3). M. 336
longobardum presented the best resemblance in the hsp65 (11 mismatches in 399 bp, 337
similarity 97.2%) with M. kumamotonense. 338
17
As far as M. heraklionense is concerned, it was characterized by high similarity with M. 339
arupense both in the 16S (only 7 discrepancies out of 1,427 bp, similarity 99.5%) and in 340
the hsp65 (99.2% similarity, 3 mismatches out of 399 bp). 341
For the three proposed new mycobacteria, the species most closely related in the rpoB 342
presented very low similarity (<94%), this value is however affected by the limited 343
coverage of the database of this genetic region. The comparison of their sequences with 344
those determined in this study for the species belonging to the MTC revealed, for each of 345
them, a clear divergence with the closer species; M. heraklionense differed 2.9% from M. 346
nonchromogenicum; M. longobardum differed 3.6% from M. heraklionense and M. 347
engbaekii differed 4.2% from M. heraklionense. 348
349
PCR restriction analysis (PRA) of the proposed new species 350
PRA patterns (Telenti et al., 1993) inferred on the basis of restriction sites present in the 351
hsp65 sequences were different for each of the three potential sp. nov. (Table 2); two 352
biotypes were detected for M. engbaekii. The PRA patterns above were unique and 353
suitable to differentiate M. engbaekii, M. heraklionense and M. longobardum from any 354
other known Mycobacterium species (http://app.chuv.ch/prasite/). 355
356
Phylogenetic analysis of the proposed new species 357
In the phylogenetic reconstructions based on 16S, hsp65 and rpoB the three proposed 358
new species belonged to a sub-branch of the MTC including also M. arupense, M. 359
nonchromogenicum and M. hiberniae; with M. kumamotonense, M. senuense and M. 360
terrae located on a different branch. The tree emerging from the concatenated sequences 361
18
clearly showed M. longobardum separated from the others species; M. heraklionense 362
closer to M. nonchromogenicum and M. engbaekii closer to M. hiberniae (Fig. 2). 363
364
HPLC of cell wall mycolic acids of the proposed new species 365
The low discriminating power, within the MTC, of HPLC of cell wall mycolic acids was 366
confirmed also by the strains belonging to the species M. engbaekii, M. heraklionense 367
and M. longobardum which presented the typical profile characterized by an early major 368
and a late minor clusters of peaks (Fig. 3). 369
370
Biochemical and cultural tests and susceptibility testing results of the proposed new 371
species 372
The major differences, revealed by biochemical and cultural tests (Table 3) among the 373
three species, concerned morphology and pigmentation of the colonies (rough pink 374
scotochromogenic in M. engbaekii, rough and unpigmented in M. longobardum and 375
smooth unpigmented in M. heraklionense), the Tween 80 hydrolysis (negative in M. 376
longobardum only), the arylsulfatase at three days (positive in M. longobardum only) and 377
the β-glucosidase (positive in M. heraklionense only). With respect to other known 378
members of the MTC the discriminative power of biochemical and cultural tests was, as 379
expected, very limited (data not shown) such confirming the perception which lead to 380
propose, about 40 years ago, the inclusion in a complex. 381
The susceptibility testing revealed susceptibility of the three species to clarithromycin 382
and resistance to quinolones with the activity of other molecules being variable (Table 4). 383
384
19
Description of M. engbaekii sp. nov. 385
M. engbaekii sp. nov. (eng.ba.e’ki.i N.L. gen. m. engbaekii of Engbaek, to honor of the 386
Danish mycobacteriologist H.C. Engbaek). 387
Cells are typically acid fast, rod-shaped, with some coccoid forms, but without branches 388
or aerial hyphae; spores are not produced. Mature growth is obtained in solid media at 389
temperatures between 25 and 37 °C in less than 10 days. Colonies grow rough and 390
unpigmented in the dark but after light exposure develop a pink pigmentation. The 391
organism is positive for nitrate reductase, Tween 80 hydrolysis and tellurite reduction, 392
and is negative for niacin accumulation, arylsulfatase at 3 days, urease and β-glucosidase. 393
Catalase at 68 °C is present and more than 45 mm foam in the semi-quantitative test are 394
produced. Isolates are susceptible to amikacin, clarithromycin, ethambutol, linezolid and 395
rifabutin, and resistant to doxycycline and sulfamethoxazole.. Mycolic acids produce an 396
early major and a late minor cluster of peaks. The gene sequences are unique in the 16S, 397
in the hsp65 (where 4 sqvs. are present) and in rpoB (4 sqvs.). Phylogenetically the 398
species is included in the MTC more closely related to M. hiberniae. The type strain is 399
ATCC27353=DSM45694. 400
401
Description of M. heraklionense sp. nov. 402
M. heraklionense sp. nov. (he.ra.kli.on.en’se N.L. adj. n. heraklionense from Heraklion 403
the city in Crete island where many such strains were isolated). 404
Cells are typically acid fast, rod-shaped, with some coccoid forms, but without branches 405
or aerial hyphae; spores are not produced. Mature growth is obtained in solid media at 406
temperatures between 25° and 37°C in 5 to 12 days. Colonies grow smooth and 407
20
unpigmented both in the dark and after light exposure. The organism is positive for 408
nitrate reductase, Tween 80 hydrolysis and β-glucosidase, and is negative for niacin 409
accumulation, and arylsulfatase at 3 days, tellurite reduction, and urease. Catalase at 68°C 410
is present and more than 45 mm foam in the semi-quantitative test are produced. Isolates 411
are susceptible to clarithromycin and resistant to quinolones, rifampicin, 412
sulfamethoxazole and doxycycline. Mycolic acids produce an early major and a late 413
minor cluster of peaks. The gene sequences are unique in the 16S, where two sqvs. 414
closely related to M. arupense are present, in the hsp65 (3 sqvs.), where best resembles 415
M. nonchromogenicum, and in rpoB (7 sqvs.), equally divergent from M. arupense and 416
M. nonchromogenicum. Phylogenetically the species is included in the MTC more closely 417
related to M. nonchromogenicum. The type strain is GN-1T=10803T (NCTC13432= 418
LMG24735=CECT7509). 419
420
Description of M. longobardum sp. nov. M. longobardum sp. nov. (lon.go’bar.dum N.L. 421
adj. n. from Lombardy the region were the strains were isolated). 422
Cells are typically acid fast, rod-shaped, with some coccoid forms, but without branches 423
or aerial hyphae; spores are not produced. Mature growth is obtained in solid media at 424
temperatures between 25° and 37°C in 7 to 14 days. Colonies grow rough and 425
unpigmented both in the dark and after light exposure. The organism is positive for 426
nitrate reductase and arylsulfatase at 3 days, and is negative for niacin accumulation, 427
Tween 80 hydrolysis, tellurite reduction, urease and β-glucosidase. Catalase at 68 °C is 428
present and more than 45 mm foam in the semi-quantitative test are produced. Isolates are 429
susceptible to sulfamethoxazole and clarithromycin, and resistant to quinolones, linezolid 430
21
and streptomycin. Mycolic acids produce an early major and a late minor cluster of 431
peaks. The gene sequences are unique in the 16S, in the hsp65 and in rpoB (where 2 sqvs. 432
are present). Phylogenetically the species is included in the MTC clearly separated from 433
other species. The type strain is FI-07034T (DSM45394=CCUG58460). 434
435
Acknowledgement 436
437
We thank Antonella Grottola (Laboratory of Microbiology and Virology, Modena 438
University Hospital, Modena, Italy) for her valuable comments on the manuscript. 439
We are grateful to Meg Lafferty for reviewing the language of the manuscript. 440
441
References 442
443
Adékambi, T., Colson, P., & Drancourt, M. (2003). rpoB-based identification of 444
nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 41, 445
5699-5708. 446
Adékambi, T. & Drancourt, M. (2004). Dissection of phylogenetic relationships among 447
19 rapidly growing Mycobacterium species by 16S rRNA. hsp65, sodA, recA, and rpoB 448
gene sequencing. Int J Syst Evol Microbiol 54, 2095-2105. 449
C.D.C. (1996). Standardized method for HPLC identification of mycobacteria. U.S. 450
Department of Health and Human Services, Public Health Service, Atlanta. 451
22
Cloud, J.L., Meyer, J.J., Pounder, J.I., Jost, K.C., Jr., Sweeney, A., Carrol, K.C., & 452
Woods, G.L. (2006). Mycobacterium arupense sp. nov., a non-chromogenic bacterium 453
isolated from clinical specimens. Int J Syst Evol Microbiol 56, 1413-1418. 454
C.L.S.I. (2011). Susceptibility testing of mycobacteria, nocardiae and other aerobic 455
actinomycetes; approved standard - Second Edition. C.L.S.I., Wayne. 456
457 Devulder, G., Perouse, d.M., & Flandrois, J.P. (2005). A multigene approach to 458
phylogenetic analysis using the genus Mycobacterium as a model. Int J Syst Evol 459
Microbiol 55, 293-302. 460
Kent, P.T. and Kubica, G.P. (1985). Public health mycobacteriology. A guide for the 461
level III laboratory. U.S. Department of Health and Human Services, Atlanta. 462
Kimura, M. (1980). A simple method for estimating evolutionary rates of base 463
substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111-464
120. 465
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M., Bange, 466
F.C., & Böttger, E.C. (1993). Genotypic identification of mycobacteria by nucleic acid 467
sequence determination: report of a 2-year experience in a clinical laboratory. J Clin 468
Microbiol 31, 2882-2889. 469
Korsak, T. & Boisvert, H. (1972). Mycobactéries a pigment rose. Ann Inst Pasteur 122, 470
31-41. 471
23
Kubica, G.P., Silcox, V.A., Kilburn, J.O., Smithwick, R.W., Beam, R.E., Jones, 472
W.D., & Stottmeier, K.D. (1970). Differential identification of mycobacteria. VI. 473
Mycobacterium triviale Kubica sp. nov. Int J Syst Bacteriol 20, 161-174. 474
Lee, H., Lee, S.A., Lee, I.K., Yu, H.K., Park, Y.G., Jeong, J., Lee, S.H., Kim, S.R., 475
Hyun, J.W. & other authors. (2010). Mycobacterium paraterrae sp. nov. recovered 476
from a clinical specimen: novel chromogenic slow growing mycobacteria related to 477
Mycobacterium terrae complex. Microbiol Immunol 54, 46-53. 478
Masaki, T., Ohkusu, K., Hata, H., Fujiwara, N., Iihara, H., Yamada-Noda, M., 479
Nhung, P.H.H.M., Asano, Y., Kawamura, Y., & Ezaki, T. (2006). Mycobacterium 480
kumamotonense sp. nov. recovered from clinical specimen and the first isolation report of 481
Mycobacterium arupense in Japan: novel slowly growing, nonchromogenic clinical 482
isolates relaed to Mycobacterium terrae complex. Microbiol Immunol 50, 889-897. 483
McNabb, A., Eisler, D., Adie, K., Amos, M., Rodrigues, M., Stephens, G., Black, 484
W.A., & Isaac-Renton, J. (2004). Assessment of partial sequencing of the 65-kiloDalton 485
heat shock protein gene (hsp65) for routine identification of mycobacterium species 486
isolated from clinical sources. J Clin Microbiol 42, 3000-3011. 487
Mignard, S. & Flandrois, J.P. (2008). A seven-gene, multilocus, genus-wide approach 488
to the phylogeny of mycobacteria using supertrees. Int J Syst Evol Microbiol 58, 1432-489
1441. 490
Mun, H.S., Park, J.H., Kim, H., Yu, H.K., Park, Y.G., Cha, C.Y., Kook, Y.H., & 491
Kim, B.J. (2008). Mycobacterium senuense sp. nov., a slowly growing non-chromogenic 492
24
species closely related to the Mycobacterium terrae complex. Int J Syst Evol Microbiol 493
58, 641-646. 494
N.C.C.L.S. (2003). Susceptibility testing for mycobacteria, nocardiae and other aerobic 495
actinomycetes; approved standard M24-A. N.C.C.L.S., Wayne, Pa. 496
Reischl, U., Emler, S., Horak, Z., Kaustova, J., Kroppenstedt, R.M., Lehn, N., & 497
Naumann, L. (1998). Mycobacterium bohemicum sp. nov., a new slow-growing 498
scotochromogenic mycobacterium. Int J Syst Bacteriol 48, 1349-1355. 499
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for 500
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425. 501
Springer, B., Stockman, L., Teschner, K., Roberts, G.D., & Böttger, E.C. (1996). 502
Two-laboratory collaborative study on identification of mycobacteria: molecular versus 503
phenotypic methods. J Clin Microbiol 34, 296-303. 504
Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, P.A., Kampfer, P., 505
Maiden, M.C.J., Nesme, X., Rossellò-Mora, R., Swings, J. & other authors. (2002). 506
Report of the ad hoc committee for the re-evaluation of the species definition in 507
bacteriology. Int J Syst Evol Microbiol 52, 1043-1047. 508
Tamura, K., Peterson, D., Peterson, N., Steker, G., Nei, M., & Kumar, S. (2011). 509
MEGA5: molecular evolutionary genetics analysis using maximum likelihood, 510
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731-2739. 511
25
Telenti, A., Marchesi, F., Balz, M., Bally, F., Böttger, E.C., & Bodmer, T. (1993). 512
Rapid identification of mycobacteria to the species level by polymerase chain reaction 513
and restriction enzyme analysis. J Clin Microbiol 31, 175-178. 514
Tsukamura, M. (1965). A group of mycobacteria from soil sources resembling 515
nonphotochromogens (group 3). A description of Mycobacterium nonchromogenicum. 516
Med Biol 71, 110-113. 517
Tsukamura, M., Kita, N., Otsuka, W., & Shimoide, H. (1983). A study of the 518
taxonomy of the Mycobacterium nonchromogenicum complex and report of six cases of 519
lung infection due to Mycobacterium nonchromogenicum. Microbiol Immunol 27, 219-520
236. 521
Wayne, L.G. (1966). Classification of mycobacteria. III. Species within Group III. Am 522
Rev Respir Dis 93, 919-928. 523
Wayne, L.G. & Kubica, G.P. (1986). Family Mycobacteriaceae CHESTER 1897, 63AL, 524
p. 1435-1457. In: P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), 525
Bergey's manual of systematic bacteriology. The Williams & Wilkins Co., Baltimore. 526
527
528
529 530 531
26
Table 1. Epidemiological, microbiological and clinical characteristics of 6 strains of M. engbaekii 16 strains of M. heraklionense and 7 strains of M. longobardum.
Speciesa Strain Age Sex Specimen type Microscopy Culture (pos/done)
Disease Sitee Year Sequevars (16S/hsp65/rpoB)
E FI-04007 74 M Gastric washing Neg NA NA GL 1998 a/2/iii E FI-98002 NA M NA NA NA NA MI 1998 a/1/ii E FI-06007 62 M Urine Neg 1/3 NA VI 2006 a/3/iii E FI-98058 NA M NA NA NA NA TR 1998 a/1/i E FI-98001 NA M NA NA NA NA MI 1998 a/1/iv E HSR-11012 75 M Bronchial aspirate Neg 1/1 NA VI 2011 a/1/ii H GN01T 74 M NA Neg 1/1 Renal failure, heart failure H 2002 a/2/ii H GN02 76 M NA Neg 1/1 Myelodisplastic syndrome H 2003 a/2/ii H GN04 83 M NA Neg 1/1 COPDc H 2003 a/2/ii H GN05 42 M NA Neg 1/1 Tuberculosis H 2003 a/2/ii H GN06 70 M NA Neg 1/1 Lung cancer H 2003 a/2/ii H GN08 59 M NA Neg 1/1 Non-Hodgkin lymphoma H 2003 a/2/ii H GN09 35 M NA Neg 1/1 Rheumatoid arthritis H 2003 a/2/ii H GN10 82 M NA Neg 1/1 Pulmonary fibrosis H 2003 a/2/ii H GN12 77 M NA Neg 1/1 Lung cancer H 2003 a/2/ii H FI-10248 61 M NA NA NA NA AN 2010 a/1/iii H HRS-11013 82 M Bronchial lavage Neg 1/2 NA NO 2011 a/1/iii H FI-06009 62 M Sputum Neg 1/3 NA NO 2006 a/1/i H FI-06255 NA F Sputum NA NA NA CO 2007 a/2/v H FI-08098 NA M NA Neg 1/1 NA MI 2008 a/1/iii H FI-08101 NA M NA NA NA NA MI2 2008 a/1/ii H FI-05158 74 F Sputum Neg 1/2 NA NO 2005 a/1/iii L FI-09110 71 M NA Neg 1/2b TB VA 2009 a/1/ii L FI-07054 65 M NA Neg 1/3b COPDc VA 2006 a/1/ii L FI-06254 78 M NA Neg 1/1b Pneumonia VA 2006 a/1/i L FI-09059 51 M NA Neg 1/9b Bronchitis VA 2008 a/1/i L FI-07034T 72 F NA Neg 1/4 Broncho-pneumonitis VA1 2006 a/1/i L FI-07020 76 M NA Neg 1/2 Lung cancer VA1 2006 a/1/ii L FI-07089 39 F NA Neg 2/3 Suspected TB in LESd patient VA1 2006 a/1/i
a E, M. engbaekii; H, M. heraklionense; L, M. longobardum b positive in liquid- but negative in solid-medium c chronic obstructive pulmonary disease d systemic lupus erythematosus e H, Greece (Heraklion); GL, Guadeloupe; other acronyms, different Italian cities.
27
Table 2. Patterns detected by PCR restriction analysis with enzymes BstEII and HaeIII in the strains for
which the status of sp. nov. is proposed.
Sequevars Restriction patterns
BstEII HaeIII
ENG1 289-96 124-58-54
ENG2-3-4 304-96 118-87-58
HER1-2 210-96-94 118-87-77
LON1 210-190 118-112-69
28
Table 3. Results of biochemical and cultural tests investigated for three proposed new species.
a photochromogenic
Test M. engbaekii M. heraklionense M. longobardum
Growth rate rapid intermediate intermediate
Growth at 25°C positive positive positive
Growth at 45°C variable negative negative
Pigmentation pinka absent absent
Colony morphology rough smooth rough
Niacin accumulation negative negative negative
Nitrate reduction negative positive positive
Catalase 68°C positive positive positive
Catalase >45 mm positive positive positive
Tween 80 hydrolysis variable positive negative
Tellurite reduction positive negative negative
Arylsulfatase 3 day negative negative positive
Urea hydrolysis negative negative negative
β-glucosidase positive positive negative
Tolerance of
MacConkey negative negative variable
p-nitrobenzoic acid positive positive positive
Thiophene carboxylic acid positive positive positive
Tiacetazone positive positive positive
Hydroxylamine positive variable positive
Isoniazid positive positive positive
Oleate positive negative positive
29
Table 4. Minimal inhibitory concentrations of several strains of M. engbaekii, M. heraklionense and M.
longobardum to antimycobacterial drugs.
a I, intermediate; R, resistant, S, susceptible.
Drug M. engbaekii M. heraklionense M. longobardum
MIC Interpretationa MIC Interpretationa MIC Interpretationa
Ciprofloxacin 2 I ≥16 R 16 R
Moxifloxacin 2-4 R >8 R ≥8 R
Linezolid ≤1 S 4-16 I/S 64 R
Rifampicin 4 I ≥8 R 4-8 I/R
Rifabutin ≤0.25 S ≤0.25 S 0.5 I
Sulfamethoxazole ≥76 R >152 R 5-9.5 S
Doxycycline 16 R ≥16 R 4 I
Ethambutol ≤0.5 S 2 S/I 4 I
Clarithromycin 1 S 1 S 2 S
Amikacin 2-8 S 1-32 S/I 32 I
Streptomycin 2-8 S/I 2->64 R/S 32 R
30
Table S3. Combinations of 16S, hsp65 and rpoB sqvs. detected in the strains investigated.
Genetic region Combinations number Species 16S hsp65 rpoB
ARUa ARU1 ARUi 7 ARU ARUa ARU1 ARUii 9 ARU ARUa ARU1 ARUiii 4 ARU ARUa ARU1 ARUix 1 ARU ARUa ARU1 ARUvi 2 ARU ARUa ARU1 ARUvii 1 ARU ARUa ARU1 ARUviii 4 ARU ARUa ARU1 ARUx 3 ARU ARUa ARU1 ARUxii 1 ARU ARUa ARU1 ARUxiii 2 ARU ARUa ARU1 ARUxiv 1 ARU ARUa ARU2 ARUi 2 ARU ARUa ARU2 ARUii 2 ARU ARUa ARU2 ARUiv 1 ARU ARUa ARU2 ARUviii 2 ARU ARUa ARU2 ARUx 1 ARU ARUa ARU2 ARUxv 1 ARU ARUa ARU3 ARUiv 1 ARU ARUa ARU3 ARUvi 1 ARU ARUa ARU4 ARUi 1 ARU ARUa ARU4 ARUv 1 ARU ARUa ARU5 ARUviii 1 ARU ARUb ARU6 ARUxi 1 ARU ARUc ARU1 ARUx 1 ARU
ENGa ENG1 ENGi 2 ENG ENGa ENG1 ENGii 2 ENG ENGa ENG1 ENGiv 1 ENG ENGa ENG2 ENGiii 1 ENG ENGa ENG3 ENGiii 1 ENG
HERa HER1 HERi 6 HER HERa HER1 HERii 1 HER HERa HER1 HERiii 4 HER HERa HER1 HERiv 1 HER HERa HER2 HERii 11 HER HERa HER2 HERv 1 HER
HIBa HIB1 HIBi 1 HIB
KUMa KUM1 KUMi 1 KUM KUMa KUM1 KUMv 1 KUM KUMa KUM1 KUMvii 5 KUM KUMa KUM1 KUMx 1 KUM KUMa KUM2 KUMi 1 KUM KUMa KUM2 KUMviii 1 KUM KUMb KUM1 KUMi 3 KUM
31
KUMb KUM1 KUMv 1 KUM KUMb KUM1 KUMviii 2 KUM KUMb KUM1 kUMiii 1 KUM KUMb KUM2 KUMii 3 KUM KUMb KUM2 kUMiii 1 KUM KUMb KUM2 kUMiv 1 KUM KUMb KUM2 KUMv 1 KUM KUMc KUM1 kUMix 1 KUM KUMc KUM2 KUMi 1 KUM KUMc KUM2 kUMii 3 KUM KUMc KUM2 kUMiii 1 KUM KUMc KUM2 kUMiv 1 KUM KUMc KUM2 KUMvi 1 KUM KUMc KUM2 KUMvii 1 KUM KUMd KUM1 KUMviii 1 KUM KUMd KUM2 KUMii 1 KUM
LONa LON1 LONi 4 LON LONa LON1 LONii 3 LON
NONa NON1 NONi 3 NON NONa NON1 NONii 3 NON NONa NON1 NONiii 1 NON NONa NON1 NONiv 1 NON NONa NON1 NONv 1 NON
SENa SEN1 SENi 1 SEN SENb SEN2 sENii 1 SEN
TERa TER1 TERi 1 TER TERa TER2 TERii 1 TER TERa TER2 TERiv 1 TER TERa TER3 TERiii 1 TER TERa TER4 TERvi 1 TER TERb TER4 TERv 1 TER
32
Table S4. Phenotypic features detected in different species of M. terrae complex.
Species Number of
strains
Nitrate
reductase
(% positive)
Pigmentation
(% positive)
Growth rate
M. arupense 51 negative (4%) none (0%) intermediate
M. engbaekii 7 positive (43%) pink (85%) rapid
M. hiberniae 1 negative (0%) pink (100%) intermediate
M. kumamotonense 34 positive (55%) none (6%) intermediate
M. nonchromogenicum 9 variable (55%) none (0%) intermediate
M. senuense 2 variable (50%) none (0%) slow
M. terrae 6 negative (0%) none (0%) intermediate
33
Table S5. Strains presenting conflicting or new sqvs, not assigned to any of the species of the M. terrae
complex.
Genetic region Combinations number 16S hsp65 rpoB
FI-07105 FI-07105 FI-07105 1 FI-07105 FI-11038 FI-11038 1 FI-09379 FI-09379 FI-09379 1 FI-09015 FI-09015 ARUx 1
ARUa ARU1 FI-07105 2 ARUa ARU1 HERvi 1 HERa FI-05196 FI-05196 1 HERa ENG4 FI-05196 2
FI-10193 FI-10193 FI-10193 1 KUMa FI-06258 FI-09100 1 KUMa KUM1 HERvii 1 TERc FI-06258 FI-06258 1 TERc FI-06258 FI-09100 1
34
Figure 1. Phylogenetic tree based on 16S rRNA sequences constructed using the neighbour-joining method
(bootstrapped 1,000 times). Bootstrap values >50% are given at nodes. Bar, 0.005 substitutions per
nucleotide position.
Figure 2. Phylogenetic tree based on concatenated sequences of 16S rRNA, hsp65 and rpoB constructed
using the neighbour-joining method, every combination of sequevars detected in the strains investigated was
included. Bootstrap was replicated 1,000 times; only values >50% are given at nodes. Bar, 0.01 substitutions
per nucleotide position.
Figure 3. Most frequently detected HPLC profiles of mycolic acids. A, pattern presented by M.
nonchromogenicum, M. longobardum, most M. arupense (70%) but occasionally found also within other
species of the M. terrae complex; B, pattern prevalent in the species M. kumamotonense; C, most frequent
profile of the species M. terrae; D, pattern of M. triviale; HMMIS, high molecular mass internal standard.
Figure S1. Phylogenetic tree based on hsp65 sequevars constructed using the neighbour-joining method
(bootstrapped 1,000 times). Bootstrap values >50% are given at nodes. Bar, 0.01 substitutions per nucleotide
position.
Figure S2. Phylogenetic tree based on rpoB sequevars constructed using the neighbour-joining method
(bootstrapped 1,000 times). Bootstrap values >50% are given at nodes. Bar, 0.02 substitutions per nucleotide
position.
Top Related