IJSEM Papers in Press. Published December 8, 2014 as doi:10.1099/ijs.0.068395-0
1
Characterization of 17 strains belonging to the
2
Mycobacterium simiae complex and description of
3
Mycobacterium paraense sp. nov.
4 5 6 7 8 9 10 11 12 13
Ana R. Fusco da Costa,1 Tarcisio Fedrizzi,2 Maria L. Lopes,1 Monica Pecorari,3 Wana L. Oliveira da Costa,1 Elisabetta Giacobazzi,2 Jeann R. da Costa Bahia,1 Veronica De Sanctis,2 Karla V. Batista Lima,1 Roberto Bertorelli,2 Antonella Grottola,3 Anna Fabio,3 Alessandro Mariottini,4 Pamela Ferretti,2 Francesca Di Leva,2 Giulia Fregni Serpini,3 Sara Tagliazucchi,3 Fabio Rumpianesi,3 Olivier Jousson,2 Nicola Segata,2 Enrico Tortoli5.
14
1
15 16 17 18 19 20 21 22 23
Bacteriology and Mycology Section, Evandro Chagas Institute, Pará, Brazil.
2
NGS Facility, Laboratory of Biomolecular Sequence and Structure Analysis for Health, Centre for Integrative Biology, University of Trento, Italy 3
Microbiology and Virology Unit, Policlinico University Hospital, Modena, Italy
4
Genetic Unit, Careggi University Hospital, Florence, Italy
5
Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan, Italy
24 25
Running title: Mycobacterium paraense sp. nov.
26
New taxa: Actinobacteria
27 28 29 30 31
Correspondence: Enrico Tortoli [email protected]
32 33 34 35
The GenBank accession numbers for the Mycobacterium sequences determined in this study are
36
as follows: HM770870, KJ920347-8 and KJ948993-9006, for 16S rDNA gene; KJ586582-3,
37
KJ920347-8 and KJ949021-33, for ITS1; EU370529, HM775985, HM056136-38, HM056140-
38
3, HM056145, KJ949035-40 and KJ957808, for hsp65 gene, and KJ586587, KJ920346,
39
KJ949007-20 and KJ957807 for rpoB gene.
40
SUMMARY
41
Fourteen mycobacterial strains isolated from pulmonary samples of independent
42
patients in the state of Pará (Brazil), and three strains isolated in Italy, were
43
characterized using a polyphasic approach. Thorough genetic investigation, including
44
whole genome sequencing, demonstrated their belonging to the M. simiae complex with
45
most close relation to Mycobacterium interjectum. For 14 of them evidence emerged
46
supporting their inclusion in a previously unreported Mycobacterium species for which
47
the name Mycobacterium paraense sp. nov. is proposed. The new species is
48
characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies,
49
and HPLC mycolic acid profile different from other known mycobacteria. In different
50
genetic regions high sequence microheterogeneity was detected. The type strain is
51
IEC26 T (DSM46749T = CCUG66121T).
52
The mycobacteria related to Mycobacterium simiae constitute at present the largest
53
group, or complex, within the genus Mycobacterium. It actually includes 17 officially
54
recognized species (Tortoli et al., 2011).
55
Seventeen strains sharing phenotypic and genotypic characteristics consistent with this
56
complex were isolated, in many cases repeatedly (39 isolations in total) from 16 patients
57
with pulmonary symptoms. Most of such mycobacteria were found during the re-
58
identification, by means of DNA sequencing, of clinical isolates which, with PCR
59
Restriction Analysis (PRA), had either been previously identified as Mycobacterium
60
asiaticum or had presented unknown PRA-patterns (da Costa et al., 2010).
61
The genetic sequences of these strains showed a high degree of similarity, with the
62
Mycobacterium interjectum (Springer et al., 1993) being the most closely related
63
species. The polyphasic characterization of such strains leads us to uphold the
64
assignment of 14 of them to a novel species for which the name Mycobacterium
65
paraense is proposed.
66 67 68
Mycobacterial strains Thirty-six mycobacteria were isolated, between 1999 and 2010, at the Evandro Chagas
69
Institute from sputum samples of 14 patients resident in the state of Pará, Brazil. All the
70
patients presented respiratory symptomatology, such as chronic cough and chest pain,
71
persisting for two years or more. Some of them presented bronchiectasis that was
72
occasionally associated with hemoptysis. Three strains were isolated in Italy, two of
73
them (FI-07156 and FI-10043) were obtained from the same patient at a three year
74
interval.
75 76 77
Biochemical and cultural tests Major biochemical tests recommended for the speciation of mycobacteria were
78
performed as described previously (Kent & Kubica, 1985), including niacin
79
accumulation, nitrate reduction, Tween 80 hydrolysis (10 days), urease, β-glucosidase,
80
tellurite reduction, and catalase. The strains presented negative results for the majority
81
of the tests performed. Only catalase (thermostable and semiquantitative) tests were
82
uniformly positive while discordant results were obtained for Tween 80 hydrolysis. The
83
majority of the strains grew smooth, pale yellow, scotochromogenic colonies on
84
Löwenstein–Jensen in two weeks or more at 37 °C. Several strains however remained
85
unpigmented both in the light and in the dark. Growth was slower at 30 °C and inhibited
86
at 42 °C. Colonies were grown on media supplemented with p-nitrobenzoate (500 µg
87
ml-1) and thiacetazone (10 µg ml-1), not on those supplemented with hydroxylamine
88
(500 µg ml-1) and isoniazid (1 µg ml-1).
89
High performance liquid chromatography (HPLC) of cell wall mycolic acids.
90
Mycolic acids were analyzed as reported previously (Rhodes et al., 2005). In short,
91
colonies grown for 15 days at 37 °C on Middlebrook 7H11 agar were saponified with
92
KOH (25% in H2O), extracted with chloroform, derivatized according to the
93
manufacturer’s instructions (MIDI) and loaded onto an Agilent ChemStation HPLC
94
(Agilent Technologies). Mycolic acids were separated with a gradient of methanol and
95
2-propanol (starting ratio 75/25%, end ratio 95/5 %) and analyzed using the software
96
Sherlock Version Myco 1.0 and the database MICAG1 1.02. All strains, with the
97
exception of IEC1808 whose pattern was unique, were characterized by a late emerging
98
major cluster of peaks (between 7 and 9 minutes); first came a series of peaks
99
presenting very variable heights among the strains (Fig. 1). Such mycolic acid pattern
100
was clearly different from the one of M. interjectum (Tortoli et al., 1996). The system
101
identified such profiles as Mycobacterium simiae, with a low similarity index (100X per
184
base. Assembly was performed with SPAdes 3.0.0 software (Bankevich et al., 2012)
185
and whole-genome sequence comparison with Mauve software (Darling et al., 2010).
186
The analysis showed that two isolates (FI-07156 and FI-10043) presented overlapping
187
almost all SNPs (>99.9%), a level of similarity indicative of representing a single strain
188
(Goris et al., 2007) (Figure 5). The genome-wide similarities of IEC26 T, IEC33 and FI-
189
07156/FI-10043 were high enough (>99%) to support their belonging to a single species
190
as confirmed by the high sequence conservation and the very limited difference in the
191
gene content (Figure 5). The strain IEC1808 was in contrast more divergent and did not
192
belong to the same species as the other strains (Figure 5).
193
In conclusion the WGS fully confirmed the results of phylogenetic analysis based on
194
16S rRNA, hsp65, rpoB and ITS1 and revealed that the two strains isolated from the
195
same patient represented, despite several nucleotide diversities in the genetic regions
196
above, a single strain.
197 198
Description of Mycobacterium paraense sp. nov. Mycobacterium paraense
199
(pa.ra.en’se. N.L. neut. adj. paraense, of, or belonging to, Pará, isolated in the state of
200
Pará, Brazil). Cells are Gram-stain positive, non-motile, non-spore-forming, acid–
201
alcohol-fast bacilli. Unpigmented or light yellow colonies develop in two weeks or
202
more at temperature ranging between 25 and 37 °C both in light and dark. Among
203
biochemical features, niacin accumulation, nitrate reduction, urease, β-glucosidase and
204
tellurite reduction were negative; catalase only was uniformly positive. They are
205
therefore not suitable to differentiate the novel species from other slowly growing
206
scotochromogenic species. The HPLC profile of mycolic acids is characterized by one
207
late cluster of peaks. In 16S rRNA, hsp65, rpoB genes and in ITS1 the new species is
208
characterized by large microheterogeneity and is most closely related to M. interjectum
209
and, to lower extent, to other species belonging to the M. simiae complex. The type
210
strain is IEC26 T (DSM46749 T = CCUG66121 T); it was isolated from the sputum of a
211
patient from Parauapebas, Pará, Brazil.
212 213
REFERENCES
214 215
Adékambi, T., Colson, P. & Drancourt, M. (2003). rpoB-based identification of
216
nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 41,
217
5699-5708.
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Bankevich, A., Nurk, S., Antipov, D. & other authors (2012). SPAdes: a new
219
genome assembly algorithm and its applications to single-cell sequencing. J Comput
220
Biol 19, 455-477.
221
Clinical and Laboratory Standards Institute (2011). Susceptibility testing of
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mycobacteria, nocardiae and other aerobic actinomycetes; approved standard - Second
223
Edition. M24-A2. Wayne, PA: C.L.S.I.
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da Costa, A. R., Lopes, M. L., Furlaneto, I. P., de Sousa, M. S. & Lima, K. V.
225
(2010). Molecular identification of nontuberculous mycobacteria isolates in a Brazilian
226
mycobacteria reference laboratory. Diagn Microbiol Infect Dis 68, 390-394.
227
Darling, A. E., Mau, B. & Perna, N. T. (2010). progressiveMauve: multiple genome
228
alignment with gene gain, loss and rearrangement. PLoS ONE 5, e11147.
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Fanti, F., Tortoli, E., Hall, L., Roberts, G. D., Kroppenstedt, R. M., Dodi, I., Conti,
230
S., Polonelli, L. & Chezzi, C. (2004). Mycobacterium parmense sp. nov. Int J Syst Evol
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Microbiol 54, 1123-1127.
232
Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. &
233
Tiedje, J. M. (2007). DNA-DNA hybridization values and their relationship to whole-
234
genome sequence similarities. Int J Syst Evol Microbiol 57, 81-91.
235
Kent, P. T. & Kubica, G. P. (1985). Public health mycobacteriology. A guide for the
236
level III laboratory. Atlanta: U.S. Department of Health and Human Services.
237
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M.,
238
Bange, F. C. & Böttger, E. C. (1993). Genotypic identification of mycobacteria by
239
nucleic acid sequence determination: report of a 2-year experience in a clinical
240
laboratory. J Clin Microbiol 31, 2882-2889.
241
McNabb, A., Eisler, D., Adie, K., Amos, M., Rodrigues, M., Stephens, G., Black,
242
W. A. & Isaac-Renton, J. (2004). Assessment of partial sequencing of the 65-
243
kiloDalton heat shock protein gene (hsp65) for routine identification of mycobacterium
244
species isolated from clinical sources. J Clin Microbiol 42, 3000-3011.
245
Rhodes, M. W., Kator, H., McNabb, A. & other authors (2005). Mycobacterium
246
pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from
247
Chesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 55, 1139-
248
1147.
249
Roth, A., Fisher, M., Hamid, M. E., Michalke, S., Ludwig, W. & Mauch, H. (1998).
250
Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-
251
23S rRNA gene internal transcribed spacer sequences. J Clin Microbiol 36, 139-147.
252
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
253
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
254
Springer, B., Kirschner, P., Rost-Meyer, G., Schröder, K. H., Kroppenstedt, R. M.
255
& Böttger, E. C. (1993). Mycobacterium interjectum, a new species isolated from a
256
patient with chronic lymphadenitis. J Clin Microbiol 31, 3083-3089.
257
Sullivan, M. J., Petty, N. K. & Beatson, S. A. (2011). Easyfig: a genome comparison
258
visualizer. Bioinformatics 27, 1009-1010.
259
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6:
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Molecular Evolutionary Genetics analysis version 6.0. Mol Biol Evol 30, 2725-2729.
261
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTALW: improving the
262
sensitivity of progressive multiple sequence alignment through sequence weighting,
263
position specific gap penalties and weight matrix choice. Nucleic Acids Res 11, 4673-
264
4680.
265
Torkko, P., Suomalainen, S., Iiavanainen, E., Tortoli, E., Suutari, M., Seppänen, J.,
266
Paulin, L. & Katila, M. L. (2002). Mycobacterium palustre sp. nov., a potentially
267
pathogenic slow-growing mycobacterium isolated from veterinary and clinical
268
specimens, and Finnish stream water. Int J Syst Evol Microbiol 52, 1519-1525.
269
Tortoli, E., Bartoloni, A., Burrini, C., Colombrita, D., Mantella, A., Pinsi, G.,
270
Simonetti, M. T., Swierczynski, G. & Böttger, E. C. (1996). Characterization of an
271
isolate of the newly described species Mycobacterium interjectum. Zentralbl Bakteriol -
272
Int J Med Microbiol 283, 286-294.
273
Tortoli, E., Böttger, E. C., Fabio, A. & other authors (2011). Mycobacterium
274
europaeum sp. nov., a scotochromogenic species related to the Mycobacterium simiae
275
complex. Int J Syst Evol Microbiol 61, 1606-1611.
276 277
Table 1. Minimal inhibitory concentrations (µg/mL) of antimycobacterial drugs suitable for slowly growing mycobacteria.
DRUGS
Amikacin Ciprofloxacin Clarithromycin Doxycycline Ethambutol Linezolid Moxifloxacin Rifabutin Rifampicin Streptomycin Sulfamethoxazole a
not established
MIC IEC26T indicating resistance >32 8 >2 8 >16 2 >4 >16 >4 16 >16 16 >2 2 >2 0.25 >1 4 n.e.a 32 >38 >152
IEC30
IEC34
IEC24
IEC31
IEC27
IEC32
IEC28
IEC33
IEC29
FI-07156
16 16 2 >16 >16 16 2 1 >8 64 19
8 16 2 >16 >16 16 2 1 >8 >64 76
8 16 4 >16 >16 16 2 0.25 1 16 152
8 16 16 >16 16 16 2 0.5 >8 64 76
16 16 >64 >16 >16 32 2 1 >8 >64 38
8 8 2 >16 16 16 2 1 4 8 9.5
16 8 4 >16 >16 16 2 >8 >8 >64 9.5
8 16 2 >16 16 16 4 0.25 8 16 >152
8 8 1 >16 8 16 0.5 0.25 >8 4 4.75
8 >16 2 >16 >16 16 8 0.5 8 16 >152
Table 2. Combination of different sequevars in various strains. Strains
16S rRNA
hsp65
rpoB
ITS1
IEC23*
A
E
B
C
IEC24
A
B
A
A
IEC25
A
B
A
A
IEC26T
A
A
A
A
IEC27
A
A
A
A
IEC28
A
B
A
A
IEC29
A
A
A
A
IEC30
A
F
C
A
IEC31
B
B
D
D
IEC32
B
A
E
A
IEC33
B
C
F
B
IEC34
B
C
G
A
IEC1808*
C
D
H
E
IEC1883
B
D
I
A
FI-07156
A
A
J
B
FI-10043
B
A
K
F
FI-13041*
D
G
L
G
* strains that were not considered included in the new species
Figure 1. Representative mycolic acid pattern of M. interjectumT and strain IEC26T; LMMIS, low molecular mass internal standard, HMMIS, high molecular mass internal standard. Figure 2. Sequence heterogeneity presented in the 16S rRNA gene by different test strains (numbers indicate E. coli corresponding positions, dots indicate identities). Figure 3. Phylogenetic tree based on 16S rRNA sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.005 substitutions per nucleotide position. Figure 4. Phylogenetic tree based on concatenated sequences of 16S rRNA, ITS1, hsp65 and rpoB, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.005 substitutions per nucleotide position. Figure 5. Whole-genome comparison of five sequenced and assembled strains. a, genome alignment (Sullivan et al., 2011); b, pairwise SNPs concordance rates; c, SNPs based phylogenetic tree on the whole-genome alignments. Supplementary file 1. Phylogenetic trees based on hsp65 gene sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position. Supplementary file 2. Phylogenetic trees based on rpoB gene sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position. Supplementary file 3. Phylogenetic trees based on ITS1 sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position.
T
M. genavense ATCC51234 (NR029223)
80 69
T
M. triplex ATCC700071 (GQ153279) T
M. montefiorense ATCC BAA-256 (NR028808) T
M. florentinum DSM44852 (AJ616230) T
M. stomatepiae DSM45059 (HM022202)
94
T
M. sherrisii ATCC BAA-832 (AY353699) T
M. simiae ATCC25275 (GQ153280) 87
T
M. lentiflavum ATCC51985 (AF480583) T
M. heidelbergense DSM44471 (X70960) T
62
M. parmense CIP107385 (HM022201) T
M. europaeum DSM45397 (HM022196)
92
T
M. parascrofulaceum ATCC BAA-614 (GQ153273) T
M. kubicae ATCC700732 (HM022200)
83
T
M. palustre DSM44572 (NR028940)
78
M. saskatchewanense ATCC BAA-544T (AY208856) T
M. interjectum DSM44064 (X70961) 81
65
T
99 61
IEC26 , IEC23-25, IEC27-30, FI-07156
FI-13041
IEC31-34, IEC1883, FI-10043
IEC1808
62
T
M. intermedium DSM44049 (X67847)
M. triviale ATCC23292T (AY734994)
M. branderi ATCC51789T (AJ012756)
66 55 82
T
M. kyorinense DSM45166 (AB370111)
69
M. celatum DSM44243T (AJ536040)
M. cookii ATCC49103T (AF480598)
M. shimoidei ATCC27962T (AJ005005)
M. noviomagense DSM45145T (EU239955)
78
M. botniense ATCC700701T (NR028878)
99
M. heckeshornense DSM44428T (NR028759)
59 53
M. conspicuum DSM44136T (X88922)
M. asiaticum ATCC25276T (AF480595)
M. gordonae ATCC14470T (X52923)
74
T
M. tuberculosis ATCC27294 (FJ468345)
0.005
M. xenopi DSM43995T (AJ536033)
b. IEC1808
FI-10043
IEC26
92.4287%
IEC33
99.4860%
92.5168%
FI-07156
99.5500%
99.4815%
92.5450%
99.9983%
99.5490%
99.4818%
92.5461%
M. interjectumT
HMMIS
LMMIS
M. paraenseT
HMMIS
LMMIS
3
4
5
6
7
8
9 min
1
Characterization of 17 strains belonging to the
2
Mycobacterium simiae complex and description of
3
Mycobacterium paraense sp. nov.
4 5 6 7 8 9 10 11 12 13
Ana R. Fusco da Costa,1 Tarcisio Fedrizzi,2 Maria L. Lopes,1 Monica Pecorari,3 Wana L. Oliveira da Costa,1 Elisabetta Giacobazzi,2 Jeann R. da Costa Bahia,1 Veronica De Sanctis,2 Karla V. Batista Lima,1 Roberto Bertorelli,2 Antonella Grottola,3 Anna Fabio,3 Alessandro Mariottini,4 Pamela Ferretti,2 Francesca Di Leva,2 Giulia Fregni Serpini,3 Sara Tagliazucchi,3 Fabio Rumpianesi,3 Olivier Jousson,2 Nicola Segata,2 Enrico Tortoli5.
14
1
15 16 17 18 19 20 21 22 23
Bacteriology and Mycology Section, Evandro Chagas Institute, Pará, Brazil.
2
NGS Facility, Laboratory of Biomolecular Sequence and Structure Analysis for Health, Centre for Integrative Biology, University of Trento, Italy 3
Microbiology and Virology Unit, Policlinico University Hospital, Modena, Italy
4
Genetic Unit, Careggi University Hospital, Florence, Italy
5
Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan, Italy
24 25
Running title: Mycobacterium paraense sp. nov.
26
New taxa: Actinobacteria
27 28 29 30 31
Correspondence: Enrico Tortoli [email protected]
32 33 34 35
The GenBank accession numbers for the Mycobacterium sequences determined in this study are
36
as follows: HM770870, KJ920347-8 and KJ948993-9006, for 16S rDNA gene; KJ586582-3,
37
KJ920347-8 and KJ949021-33, for ITS1; EU370529, HM775985, HM056136-38, HM056140-
38
3, HM056145, KJ949035-40 and KJ957808, for hsp65 gene, and KJ586587, KJ920346,
39
KJ949007-20 and KJ957807 for rpoB gene.
40
SUMMARY
41
Fourteen mycobacterial strains isolated from pulmonary samples of independent
42
patients in the state of Pará (Brazil), and three strains isolated in Italy, were
43
characterized using a polyphasic approach. Thorough genetic investigation, including
44
whole genome sequencing, demonstrated their belonging to the M. simiae complex with
45
most close relation to Mycobacterium interjectum. For 14 of them evidence emerged
46
supporting their inclusion in a previously unreported Mycobacterium species for which
47
the name Mycobacterium paraense sp. nov. is proposed. The new species is
48
characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies,
49
and HPLC mycolic acid profile different from other known mycobacteria. In different
50
genetic regions high sequence microheterogeneity was detected. The type strain is
51
IEC26 T (DSM46749T = CCUG66121T).
52
The mycobacteria related to Mycobacterium simiae constitute at present the largest
53
group, or complex, within the genus Mycobacterium. It actually includes 17 officially
54
recognized species (Tortoli et al., 2011).
55
Seventeen strains sharing phenotypic and genotypic characteristics consistent with this
56
complex were isolated, in many cases repeatedly (39 isolations in total) from 16 patients
57
with pulmonary symptoms. Most of such mycobacteria were found during the re-
58
identification, by means of DNA sequencing, of clinical isolates which, with PCR
59
Restriction Analysis (PRA), had either been previously identified as Mycobacterium
60
asiaticum or had presented unknown PRA-patterns (da Costa et al., 2010).
61
The genetic sequences of these strains showed a high degree of similarity, with the
62
Mycobacterium interjectum (Springer et al., 1993) being the most closely related
63
species. The polyphasic characterization of such strains leads us to uphold the
64
assignment of 14 of them to a novel species for which the name Mycobacterium
65
paraense is proposed.
66 67 68
Mycobacterial strains Thirty-six mycobacteria were isolated, between 1999 and 2010, at the Evandro Chagas
69
Institute from sputum samples of 14 patients resident in the state of Pará, Brazil. All the
70
patients presented respiratory symptomatology, such as chronic cough and chest pain,
71
persisting for two years or more. Some of them presented bronchiectasis that was
72
occasionally associated with hemoptysis. Three strains were isolated in Italy, two of
73
them (FI-07156 and FI-10043) were obtained from the same patient at a three year
74
interval.
75 76 77
Biochemical and cultural tests Major biochemical tests recommended for the speciation of mycobacteria were
78
performed as described previously (Kent & Kubica, 1985), including niacin
79
accumulation, nitrate reduction, Tween 80 hydrolysis (10 days), urease, β-glucosidase,
80
tellurite reduction, and catalase. The strains presented negative results for the majority
81
of the tests performed. Only catalase (thermostable and semiquantitative) tests were
82
uniformly positive while discordant results were obtained for Tween 80 hydrolysis. The
83
majority of the strains grew smooth, pale yellow, scotochromogenic colonies on
84
Löwenstein–Jensen in two weeks or more at 37 °C. Several strains however remained
85
unpigmented both in the light and in the dark. Growth was slower at 30 °C and inhibited
86
at 42 °C. Colonies were grown on media supplemented with p-nitrobenzoate (500 µg
87
ml-1) and thiacetazone (10 µg ml-1), not on those supplemented with hydroxylamine
88
(500 µg ml-1) and isoniazid (1 µg ml-1).
89
High performance liquid chromatography (HPLC) of cell wall mycolic acids.
90
Mycolic acids were analyzed as reported previously (Rhodes et al., 2005). In short,
91
colonies grown for 15 days at 37 °C on Middlebrook 7H11 agar were saponified with
92
KOH (25% in H2O), extracted with chloroform, derivatized according to the
93
manufacturer’s instructions (MIDI) and loaded onto an Agilent ChemStation HPLC
94
(Agilent Technologies). Mycolic acids were separated with a gradient of methanol and
95
2-propanol (starting ratio 75/25%, end ratio 95/5 %) and analyzed using the software
96
Sherlock Version Myco 1.0 and the database MICAG1 1.02. All strains, with the
97
exception of IEC1808 whose pattern was unique, were characterized by a late emerging
98
major cluster of peaks (between 7 and 9 minutes); first came a series of peaks
99
presenting very variable heights among the strains (Fig. 1). Such mycolic acid pattern
100
was clearly different from the one of M. interjectum (Tortoli et al., 1996). The system
101
identified such profiles as Mycobacterium simiae, with a low similarity index (100X per
184
base. Assembly was performed with SPAdes 3.0.0 software (Bankevich et al., 2012)
185
and whole-genome sequence comparison with Mauve software (Darling et al., 2010).
186
The analysis showed that two isolates (FI-07156 and FI-10043) presented overlapping
187
almost all SNPs (>99.9%), a level of similarity indicative of representing a single strain
188
(Goris et al., 2007) (Figure 5). The genome-wide similarities of IEC26 T, IEC33 and FI-
189
07156/FI-10043 were high enough (>99%) to support their belonging to a single species
190
as confirmed by the high sequence conservation and the very limited difference in the
191
gene content (Figure 5). The strain IEC1808 was in contrast more divergent and did not
192
belong to the same species as the other strains (Figure 5).
193
In conclusion the WGS fully confirmed the results of phylogenetic analysis based on
194
16S rRNA, hsp65, rpoB and ITS1 and revealed that the two strains isolated from the
195
same patient represented, despite several nucleotide diversities in the genetic regions
196
above, a single strain.
197 198
Description of Mycobacterium paraense sp. nov. Mycobacterium paraense
199
(pa.ra.en’se. N.L. neut. adj. paraense, of, or belonging to, Pará, isolated in the state of
200
Pará, Brazil). Cells are Gram-stain positive, non-motile, non-spore-forming, acid–
201
alcohol-fast bacilli. Unpigmented or light yellow colonies develop in two weeks or
202
more at temperature ranging between 25 and 37 °C both in light and dark. Among
203
biochemical features, niacin accumulation, nitrate reduction, urease, β-glucosidase and
204
tellurite reduction were negative; catalase only was uniformly positive. They are
205
therefore not suitable to differentiate the novel species from other slowly growing
206
scotochromogenic species. The HPLC profile of mycolic acids is characterized by one
207
late cluster of peaks. In 16S rRNA, hsp65, rpoB genes and in ITS1 the new species is
208
characterized by large microheterogeneity and is most closely related to M. interjectum
209
and, to lower extent, to other species belonging to the M. simiae complex. The type
210
strain is IEC26 T (DSM46749 T = CCUG66121 T); it was isolated from the sputum of a
211
patient from Parauapebas, Pará, Brazil.
212 213
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214 215
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216
nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 41,
217
5699-5708.
218
Bankevich, A., Nurk, S., Antipov, D. & other authors (2012). SPAdes: a new
219
genome assembly algorithm and its applications to single-cell sequencing. J Comput
220
Biol 19, 455-477.
221
Clinical and Laboratory Standards Institute (2011). Susceptibility testing of
222
mycobacteria, nocardiae and other aerobic actinomycetes; approved standard - Second
223
Edition. M24-A2. Wayne, PA: C.L.S.I.
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da Costa, A. R., Lopes, M. L., Furlaneto, I. P., de Sousa, M. S. & Lima, K. V.
225
(2010). Molecular identification of nontuberculous mycobacteria isolates in a Brazilian
226
mycobacteria reference laboratory. Diagn Microbiol Infect Dis 68, 390-394.
227
Darling, A. E., Mau, B. & Perna, N. T. (2010). progressiveMauve: multiple genome
228
alignment with gene gain, loss and rearrangement. PLoS ONE 5, e11147.
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Fanti, F., Tortoli, E., Hall, L., Roberts, G. D., Kroppenstedt, R. M., Dodi, I., Conti,
230
S., Polonelli, L. & Chezzi, C. (2004). Mycobacterium parmense sp. nov. Int J Syst Evol
231
Microbiol 54, 1123-1127.
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Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. &
233
Tiedje, J. M. (2007). DNA-DNA hybridization values and their relationship to whole-
234
genome sequence similarities. Int J Syst Evol Microbiol 57, 81-91.
235
Kent, P. T. & Kubica, G. P. (1985). Public health mycobacteriology. A guide for the
236
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237
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M.,
238
Bange, F. C. & Böttger, E. C. (1993). Genotypic identification of mycobacteria by
239
nucleic acid sequence determination: report of a 2-year experience in a clinical
240
laboratory. J Clin Microbiol 31, 2882-2889.
241
McNabb, A., Eisler, D., Adie, K., Amos, M., Rodrigues, M., Stephens, G., Black,
242
W. A. & Isaac-Renton, J. (2004). Assessment of partial sequencing of the 65-
243
kiloDalton heat shock protein gene (hsp65) for routine identification of mycobacterium
244
species isolated from clinical sources. J Clin Microbiol 42, 3000-3011.
245
Rhodes, M. W., Kator, H., McNabb, A. & other authors (2005). Mycobacterium
246
pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from
247
Chesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 55, 1139-
248
1147.
249
Roth, A., Fisher, M., Hamid, M. E., Michalke, S., Ludwig, W. & Mauch, H. (1998).
250
Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-
251
23S rRNA gene internal transcribed spacer sequences. J Clin Microbiol 36, 139-147.
252
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
253
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
254
Springer, B., Kirschner, P., Rost-Meyer, G., Schröder, K. H., Kroppenstedt, R. M.
255
& Böttger, E. C. (1993). Mycobacterium interjectum, a new species isolated from a
256
patient with chronic lymphadenitis. J Clin Microbiol 31, 3083-3089.
257
Sullivan, M. J., Petty, N. K. & Beatson, S. A. (2011). Easyfig: a genome comparison
258
visualizer. Bioinformatics 27, 1009-1010.
259
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6:
260
Molecular Evolutionary Genetics analysis version 6.0. Mol Biol Evol 30, 2725-2729.
261
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTALW: improving the
262
sensitivity of progressive multiple sequence alignment through sequence weighting,
263
position specific gap penalties and weight matrix choice. Nucleic Acids Res 11, 4673-
264
4680.
265
Torkko, P., Suomalainen, S., Iiavanainen, E., Tortoli, E., Suutari, M., Seppänen, J.,
266
Paulin, L. & Katila, M. L. (2002). Mycobacterium palustre sp. nov., a potentially
267
pathogenic slow-growing mycobacterium isolated from veterinary and clinical
268
specimens, and Finnish stream water. Int J Syst Evol Microbiol 52, 1519-1525.
269
Tortoli, E., Bartoloni, A., Burrini, C., Colombrita, D., Mantella, A., Pinsi, G.,
270
Simonetti, M. T., Swierczynski, G. & Böttger, E. C. (1996). Characterization of an
271
isolate of the newly described species Mycobacterium interjectum. Zentralbl Bakteriol -
272
Int J Med Microbiol 283, 286-294.
273
Tortoli, E., Böttger, E. C., Fabio, A. & other authors (2011). Mycobacterium
274
europaeum sp. nov., a scotochromogenic species related to the Mycobacterium simiae
275
complex. Int J Syst Evol Microbiol 61, 1606-1611.
276 277
Table 1. Minimal inhibitory concentrations (µg/mL) of antimycobacterial drugs suitable for slowly growing mycobacteria.
DRUGS
Amikacin Ciprofloxacin Clarithromycin Doxycycline Ethambutol Linezolid Moxifloxacin Rifabutin Rifampicin Streptomycin Sulfamethoxazole a
not established
MIC IEC26T indicating resistance >32 8 >2 8 >16 2 >4 >16 >4 16 >16 16 >2 2 >2 0.25 >1 4 n.e.a 32 >38 >152
IEC30
IEC34
IEC24
IEC31
IEC27
IEC32
IEC28
IEC33
IEC29
FI-07156
16 16 2 >16 >16 16 2 1 >8 64 19
8 16 2 >16 >16 16 2 1 >8 >64 76
8 16 4 >16 >16 16 2 0.25 1 16 152
8 16 16 >16 16 16 2 0.5 >8 64 76
16 16 >64 >16 >16 32 2 1 >8 >64 38
8 8 2 >16 16 16 2 1 4 8 9.5
16 8 4 >16 >16 16 2 >8 >8 >64 9.5
8 16 2 >16 16 16 4 0.25 8 16 >152
8 8 1 >16 8 16 0.5 0.25 >8 4 4.75
8 >16 2 >16 >16 16 8 0.5 8 16 >152
Table 2. Combination of different sequevars in various strains. Strains
16S rRNA
hsp65
rpoB
ITS1
IEC23*
A
E
B
C
IEC24
A
B
A
A
IEC25
A
B
A
A
IEC26T
A
A
A
A
IEC27
A
A
A
A
IEC28
A
B
A
A
IEC29
A
A
A
A
IEC30
A
F
C
A
IEC31
B
B
D
D
IEC32
B
A
E
A
IEC33
B
C
F
B
IEC34
B
C
G
A
IEC1808*
C
D
H
E
IEC1883
B
D
I
A
FI-07156
A
A
J
B
FI-10043
B
A
K
F
FI-13041*
D
G
L
G
* strains that were not considered included in the new species
Figure 1. Representative mycolic acid pattern of M. interjectumT and strain IEC26T; LMMIS, low molecular mass internal standard, HMMIS, high molecular mass internal standard. Figure 2. Sequence heterogeneity presented in the 16S rRNA gene by different test strains (numbers indicate E. coli corresponding positions, dots indicate identities). Figure 3. Phylogenetic tree based on 16S rRNA sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.005 substitutions per nucleotide position. Figure 4. Phylogenetic tree based on concatenated sequences of 16S rRNA, ITS1, hsp65 and rpoB, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.005 substitutions per nucleotide position. Figure 5. Whole-genome comparison of five sequenced and assembled strains. a, genome alignment (Sullivan et al., 2011); b, pairwise SNPs concordance rates; c, SNPs based phylogenetic tree on the whole-genome alignments. Supplementary file 1. Phylogenetic trees based on hsp65 gene sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position. Supplementary file 2. Phylogenetic trees based on rpoB gene sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position. Supplementary file 3. Phylogenetic trees based on ITS1 sequences, constructed using the neighbor-joining method bootstrapped 1000 times. Bootstrap values are given at nodes. Bar, 0.01 substitutions per nucleotide position.
T
M. genavense ATCC51234 (NR029223)
80 69
T
M. triplex ATCC700071 (GQ153279) T
M. montefiorense ATCC BAA-256 (NR028808) T
M. florentinum DSM44852 (AJ616230) T
M. stomatepiae DSM45059 (HM022202)
94
T
M. sherrisii ATCC BAA-832 (AY353699) T
M. simiae ATCC25275 (GQ153280) 87
T
M. lentiflavum ATCC51985 (AF480583) T
M. heidelbergense DSM44471 (X70960) T
62
M. parmense CIP107385 (HM022201) T
M. europaeum DSM45397 (HM022196)
92
T
M. parascrofulaceum ATCC BAA-614 (GQ153273) T
M. kubicae ATCC700732 (HM022200)
83
T
M. palustre DSM44572 (NR028940)
78
M. saskatchewanense ATCC BAA-544T (AY208856) T
M. interjectum DSM44064 (X70961) 81
65
T
99 61
IEC26 , IEC23-25, IEC27-30, FI-07156
FI-13041
IEC31-34, IEC1883, FI-10043
IEC1808
62
T
M. intermedium DSM44049 (X67847)
M. triviale ATCC23292T (AY734994)
M. branderi ATCC51789T (AJ012756)
66 55 82
T
M. kyorinense DSM45166 (AB370111)
69
M. celatum DSM44243T (AJ536040)
M. cookii ATCC49103T (AF480598)
M. shimoidei ATCC27962T (AJ005005)
M. noviomagense DSM45145T (EU239955)
78
M. botniense ATCC700701T (NR028878)
99
M. heckeshornense DSM44428T (NR028759)
59 53
M. conspicuum DSM44136T (X88922)
M. asiaticum ATCC25276T (AF480595)
M. gordonae ATCC14470T (X52923)
74
T
M. tuberculosis ATCC27294 (FJ468345)
0.005
M. xenopi DSM43995T (AJ536033)
b. IEC1808
FI-10043
IEC26
92.4287%
IEC33
99.4860%
92.5168%
FI-07156
99.5500%
99.4815%
92.5450%
99.9983%
99.5490%
99.4818%
92.5461%
M. interjectumT
HMMIS
LMMIS
M. paraenseT
HMMIS
LMMIS
3
4
5
6
7
8
9 min
