Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

Introduction

Investigating non-tuberculous mycobacteria in Iran recently succeeded in the characterization of two new species of rapidly growing scotochromogenic mycobacteria, i.e. Mycobacterium iranicum isolated in 2009 from the bronchoalveolar lavage of a 60-year-old female patient suffering from chronic pulmonary disease[1] and Mycobacterium celeriflavum isolated in 2010 from the sputum of a 44-year-old male suffering from chronic obstructive pulmonary disease[2]. We recently had the opportunity to investigate four clinical isolates made in Iran and we proved they were representative of one additional new species of non-tuberculous Mycobacterium that we named Mycobacterium ahvazicum. AFP-003T strain was isolated in 2009 from the sputum and bronchoalveolar lavage specimen of a 68-year-old Iranian female suffering from chronic pulmonary disease. Phenotypic and genetic investigations based on 16S rRNA and rpoB gene sequencing revealed that the AFP-003 strain was probably representative of a new species of non-tuberculous Mycobacterium in Iran. Following the isolation of strain AFP-003, three other strains exhibiting the very same phenotypic and genetic characters were isolated in Iran: strain AFP-004 was isolated in 2009 from a biopsy of diseased soft tissues in a 49-year-HIV-infected patient, strain MH1 was isolated in 2013 from sputum in a 60-year-old male patient and strain RW4 was isolated in 2015 from a bronchoalveolar lavage specimen in a 19-year-old patient suffering from asthma. Strain AFP-003 was then fully characterized as a type strain and then designated as strain AFP-003T.

Results

AFP-003T yielded smooth, yellow and scotochromogenic colonies after 3–4-weeks of incubation on Löwenstein-Jensen medium at a temperature between 33 °C and 42 °C, with an optimal growth at 37 °C; but it did not grow on Löwenstein-Jensen containing 5% NaCl. The observation of colonies by electron microscopy showed rod-shaped bacilli measuring 1.53 ± 0.32 µm long and 0.64 ± 0.07 µm large (Fig. 1). AFP-003T exhibited a heat-stable (68 °C) catalase but was negative for semi-quantitative catalase; and negative for urease activity, iron uptake, tellurite reduction, arylsulfatase activity after three days, niacin production, nitrate reduction, Tween hydrolysis and growth on MacConkey agar without crystal violet. These conventional phenotypic tests were not sufficient to differentiate AFP-003T from Mycobacterium lentiflavum (Table 1). However, AFP-003T reproducible matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) profile did not match any of the profiles entered in the Bruker database (version December, 2015, including M. lentiflavum), suggesting that AFP-003T was not identifiable as M. lentiflavum and could indeed be representative of a hitherto undescribed species of Mycobacterium.

Figure 1

Transmission electron microscopy of Mycobacterium ahvazicum strain AFP-003T. The scale bar represents 200 nm.

Table 1

Phenotypic characteristics of M. ahvazicum strain AFP-003T and related slowly growing mycobacteria species.

Characteristics	1	2	3	4	5
Growth at:
30 °C	−	+	+	+	+
37 °C	+	+	+	+	−
42 °C	+	−	+	+	−
Pigmentation	S/yellow	S/yellow	SP/yellow	N	N
Arysulfatase	−	−	+	+	+
Catalase (68 °C)	+	+	+	+	+
Catalase (>45 mm of foam)	−	−	+	−	−
Nitrate reduction	−	−	−	+	−
Tween 80 hydrolysis	−	−	−	−	−
Niacin production	−	−	V	−	−
Tellurite reduction	−	−	+	ND	+
Urease activity	−	−	+	+	−
Tolerance to NaCl (5%, w/v)	−	−	−	−	−
Growth on MacConkey agar without crystal violet	−	−	ND	ND	−

1, M. ahvazicum sp. nov.; 2, M. lentiflavum; 3, M. simiae; 4, M. triplex; 5, M. stomatepiae. S; scotochromogenic, SP; scotochromogenic or photochromogenic, N; nonchromogenic, ND; Not determined; −, negative; +, positive; V, variable. Data for species other than M. ahvazicum sp. nov are from Levi et al.[22], Pourahmad et al.[23] and Tortoli (2006).

AFP-003T was then shown to be in vitro susceptible to ciprofloxacin, clarithromycin and rifampicin (Table 2). Furthermore, Biolog® Phenotype MicroArray test showed that AFP-003T grew under other 14 inhibitory chemical conditions including minocycline, lincomycin, guanidine HCl, Niaproof Anionic Surfactant, vancomycin, tetrazolium violet, tetrazolium blue, nalidixic acid, lithium chloride, potassium tellurite, aztreonam, sodium butyrate and sodium bromate; and was able to metabolize eight carbon sources including α D-glucose, glucuronamide, methyl pyruvate, α-keto-glutaric, α-keto-butyric acid, acetoacetic acid, propionic acid and acetic acid (Table 3). The 16S rRNA gene sequence’s (GenBank accession: LT797535) highest similarity was of 98.1%, 97.8%, 97.5% and 97.4% with M. lentiflavum ATCC 51985, Mycobacterium simiae, Mycobacterium triplex and Mycobacterium sherrisii, respectively. Partial rpoB gene sequencing was previously shown to be a useful marker to delineate new Mycobacterium species[3] and we sequenced a 619-bp rpoB gene fragment in AFP-003T strain (GenBank accession: FR695853). This sequence’s highest similarity was of 96.43%, 95.55% and 94.95% with Mycobacterium florentinum DSM 44852, Mycobacterium stomatepiae DSM 45059 and Mycobacterium genavense FI-06288 respectively. These values being all below the 97% cut-off value previously proposed to delineate different species among Mycobacterium[3] enforced the suggestion that AFP-003T was representative of a new species belonging to the M. simiae complex, the largest complex in the genus Mycobacterium currently comprising 18 species[4,5] (Fig. 2).

Table 2

Minimum inhibitory concentration of selected antibiotics against two M. ahvazicum strains.

Drug	MIC range (g /L)		Interpretation*
	AFP-003T	AFP-004
Amikacin	8	32	I
Ciprofloxacin	2	1	S
Clarithromycin	1	4	S
Ethambutol	8	8	R
Rifampicin	0.05	0.05	S
Streptomycin	2	16	I

*I, intermediate; S, susceptible; R, resistant.

Table 3

Phenotype Microarray Biolog, Gen III Microplate profile of M. ahvazicum strain AFP-003T.

Position	Substrates	Activity
C 1	alpha-D-Glucose	+
C 12	D-Serine	+
D 12	Minocycline	+
E 10	Lincomycin	+
E 11	Guanidine HCl	+
E 12	Niaproof 4	+
F 6	Glucuronamide	+
F 10	Vancomycin	+
F 11	Tetrazolium Violet	+
F 12	TetrazoliumBlue	+
G 2	Methyl Pyruvate	+
G 6	alpha-Keto-Glutaric Acid	+
G 10	Nalidixic Acid	+
G 11	Lithium Chloride	+
G 12	Potassium Tellurite	+
H 5	alpha-Keto-Butyric Acid	+
H 6	Acetoacetic Acid	+
H 7	Propionic Acid	+
H 8	Acetic Acid	+
H 10	Aztreonam	+
H 11	Sodium Butyrate	+
H 12	Sodium Bromate	+
A 1	Negative Control
A 2	Dextrin
A 3	D-Maltose
A 4	D-Trehalose
A 5	D-Cellobiose
A 6	Gentiobiose
A 7	Sucrose
A 8	D-Turanose
A 9	Stachyose
A 10	D-Raffinose
A 11	alpha-D-Lactose
A 12	D-Melibiose
B 1	beta-Methyl-Dglucoside
B 2	D-Salicin
B 3	N-Acetyl-Dglucosamine
B 4	N-Acetyl-beta-Dmannosamine
B 5	N-Acetyl-D-galactosamine
B 6	N-Acetyl Neuraminic Acid
B 7	D-Mannose
B 8	D-Fructose
B 9	D-Galactose
B 10	1% NaCl
B 11	4% NaCl
B 12	8% NaCl
C 2	3-Methyl Glucose
C 3	D-Fucose
C 4	L-Fucose
C 5	L-Rhamnose
C 6	Inosine
C 7	D-Sorbitol
C 8	D-Mannitol
C 9	D-Arabitol
C 10	1% Sodium Lactate
C 11	Fusidic Acid
D 1	myo-Inositol
D 2	Glycerol
D 3	D-Glucose-6-PO4
D 4	D-Fructose-6-PO4
D 5	D-Aspartic Acid
D 6	D-Serine
D 7	Gelatin
D 8	Glycyl-L-Proline
D 9	L-Alanine
D 10	Troleandomycin
D 11	Rifamycin SV
E 1	L-Arginine
E 2	L-Aspartic Acid
E 3	L-Glutamic Acid
E 4	L-Histidine
E 5	L-Pyroglutamic Acid
E 6	L-Serine
E 7	Pectin
E 8	D-Galacturonic Acid
E 9	L-Galactonic Acid Lactone
F 1	D-Gluconic Acid
F 2	D-Glucuronic Acid
F 3	Mucic Acid
F 4	Quinic Acid
F 5	D-Saccharic AcidD-Saccharic Acid
F 7	p-Hydroxy-Phenylacetic Acid
F 8	D-Lactic Acid Methyl Ester
F 9	L-Lactic Acid
G 1	Citric Acid
G 3	D-Malic Acid
G 4	L-Malic Acid
G 5	Bromo-Succinic Acid
G 7	Tween 40
G 8	gama-Amino-Butryric Acid
G 9	alpha-Hydroxy-Butyric Acid
H 1	β-Hydroxy-D,Lbutyric Acid
H 2	Formic Acid
H 3	Positive Control
H 4	pH 6
H 9	pH 5

Figure 2

Phylogenetic tree based on the 16S rRNA gene sequence indicating the phylogenetic position of M. ahvazicum strain AFP-003T relative to other species of M. simiae and other mycobacteria species including Mycobacterium tuberculosis as an out group. Sequences were aligned using CLUSTLE W implemented on MEGA7[33]. The analysis involved 34 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1,233 positions in the final dataset. Phylogenetic inferences obtained using the maximum likelihood method based on the Tamura and Nei model (bootstrapped 1000 times). Bootstrap values >50% are given at nodes. Bar, 0.005 substitutions per nucleotide position.

We therefore decided to sequence the genome of AFP-003T strain. Genome sequencing yielded four scaffolds indicative of one 6,121,237-bp chromosome (66.24% GC content) without evidence for any extra-chromosomal replicon (Fig. 3). The genome of AFP-003T is smaller than that of Mycobacterium parascrofulaceum and M. triplex (6.564 Mb and 6.383 Mb, respectively) but larger than that of Mycobacterium interjectum, Mycobacterium genavense, M. sherrisii and M. simiae (5.848 Mb, 4.936 Mb, 5.687 Mb and 5.783 Mb, respectively); its GC% content is lower than that of M. parascrofulaceum, M. interjectum, M. genavense, M. sherrisii and M. triplex (68.45%, 106 67.91%, 66.92%, 66.92% and 66.6%, respectively) but higher than that of M. simiae (66.17%). The AFP-003T genome encodes for 5,704 proteins and 52 RNAs including 49 tRNA and one complete rRNA operon in agreement with its classification as slowly growing mycobacterium. A total of 4,869 genes (85.36%) were assigned with putative function (by COGs or by nr blast), whereas 88 genes (1.54%) were identified as ORFans. The remaining genes were annotated as hypothetical proteins without COG assignment (835 genes, 14.64%). A total of 2,617 proteins were found to be associated with the mobilome, including 194 phage proteins. Further genome analysis predicted two incomplete 23.3-Kb and 12.6-Kb prophage regions (Fig. 4). A total of 1,225 proteins were found to be associated with virulence, 95 proteins were associated with toxin/antitoxin systems and 11 genes encoded for bacteriocins while no gene was associated with the resistome. We identified a large number of genes assigned to COG functional categories for transport and metabolism of lipids (10.6%), secondary metabolites biosynthesis, transport and catabolism (6.8%), amino acid transport and metabolism (4.03%) and energy production and conversion (5.3%) (Table 4).

Figure 3

Graphical circular map of the chromosome of M. ahvazicum strain AFP-003T. From outside to the center: Genes on the forward strand colored by COG categories (only genes assigned to COG), genes on the reverse strand colored by COG categories (only gene assigned to COG), RNA genes (tRNAs green, rRNAs red), GC content and GC skew.

Figure 4

Genomic organization of two uncomplete prophage regions in the genome of M. ahvazicum strain AFP-003T.

Table 4

Number of genes associated in the M. ahvazicum strain AFP-003T genome with the 25 general COG functional categories. The total % is based on the total number of protein coding genes in the annotated genome.

Code	Value	% of total	Description
[J]	173	3.03	Translation
[A]	1	0.02	Rna processing and modification
[K]	173	3.03	Transcription
[L]	97	1.70	Replication, recombination and repair
[B]	0	0.00	Chromatin structure and dynamics
[D]	29	0.51	Cell cycle control, mitosis and meiosis
[Y]	0	0.00	Nuclear structure
[V]	116	2.03	Defense mechanisms
[T]	93	1.63	Signal transduction mechanisms
[M]	154	2.70	Cell wall/membrane biogenesis
[N]	15	0.26	Cell motility
[Z]	0	0.00	Cytoskeleton
[W]	4	0.07	Extracellular structures
[U]	24	0.42	Intracellular trafficking and secretion
[O]	120	2.10	Posttanslational modification, protein turnover,chaperones
[X]	22	0.39	Mobilome: prophages, transposons
[C]	298	5.22	Energy production and conversion
[G]	207	3.63	Carbohydrate transport and metabolism
[E]	230	4.03	Amino acid transport and metabolism
[F]	73	1.28	Nucleotide transport and metabolism
[H]	211	3.70	Coenzyme transport and metabolism
[I]	603	10.57	Lipid transport and metabolism
[P]	223	3.91	Inorganic ion transport and metabolism
[Q]	388	6.80	Secondary metabolites biosynthesis, transport and catabolism
[R]	503	8.82	General function prediction only
[S]	181	3.17	Function unknown

The genome of AFP-003T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases. M. ahvazicum genome exhibits an average nucleotide identity of 86% with M. genavense, 82% with M. simiae, 81% with M. interjectum, 72% with M. triplex, 69% with M. parascrofulaceum and 68% with M. sherrisii (Tables 5, 6). In silico DNA-DNA hybridization analysis yielded 36.45% ± 3.46% with M. triplex, 32.55% ± 3.46% with M. genavense, 26% ± 3.39% with M. sherresii, 25.8% ± 3.39% with M. simiae, 24.7% ± 3.39% with M. interjectum and 24.2% ± 3.39 with M. parascrofulaceum. Ori-Finder[6] was used to predict the origin of replication in the genome of strain AFP-003T. We found three OriC regions separated by the dnaA gene and located in scaffold 1 (218, 312 and 391 bp) (Supplementary File 1). The three predicted OriC region showed no homology sequence with those of the DoriC database7[7]. Contigs have been deposited (EBI accession number: FXEG02000000). Annotated genome is available at https://www.ebi.ac.uk/ena/data/view/PRJEB20293.

Table 5

Numbers of ortholog genes between genomes (upper right), average percentage similarity of nucleotides corresponding to orthologs between genomes (lower left) and number of ORFs per genome (bold); in selected M. simiae complex species including M. ahvazicum strain AFP-003T.

	M. genavense	M. ahvazicum	M. interjectum	M. simiae	M. triplex	M. sherrisii	M. parascrofulaceum
M. genavense	5375	3011	2417	2727	3036	2857	2646
M. ahvazicum	0.86	5758	3286	3585	3913	3672	3569
M. interjectum	0.81	0.81	5953	3002	3154	3008	3137
M. simiae	0.82	0.82	0.80	5533	3557	3502	3222
M. triplex	0.75	0.72	0.69	0.69	5988	3647	3404
M. sherrisii	0.68	0.68	0.67	0.71	0.68	5020	3299
M. parascrofulaceum	0.6	0.69	0.70	0.68	0.73	0.68	6456

Table 6

Comparison of M. ahvazicum AFP-003T with related mycobacteria species using GGDC, formula 2 (DDH estimates based on identities/HSP length.

Species	DDH with M. ahvazicum AFP003T (%)
M. triplex	36.45 ± 3.46
M. genavense	32.55 ± 3.46
M. sherresii	26 ± 3.39
M. simiae	25.8 ± 3.39
M. interjectum	24.7 ± 3.39
M. parascrofulaceum	24.2 ± 3.39

To better describe AFP003T, the mycolic acids were identified. The mass spectrometry analysis of Mycobacterium tuberculosis H37Rv strain (used as a positive control) showed the previously described mycolic acid pattern[8,9], including α- (C74-84), methoxy- (C80-90) and keto- (C80-89) forms. Strain AFP-003T showed two known mycolic acids subclasses, α- (C71-74) and α′- (C64-68) forms, representing 15% of relative intensity defining an original mycolic acid profile (Table 7, Fig. 5).

Table 7

Identified mycolic acids for strains AFP-003T and Mycobacterium tuberculosis H37Rv (control).

Mycolic acid subclass	Formula	Calculated [M − H]−	Mycobacterium ahvazicum AFP-003T			Mycobacterium tuberculosis H37Rv
			Measured [M − H]−	Error (ppm)	%a	Measured [M − H]−	Error (ppm)	%a
α-	C71H138O3	1038.05732	1038.06117	3.7	1.5
	C72H140O3	1052.07297	1052.07398	1.0	0.9
	C73H142O3	1066.08862	1066.08942	0.8	0.9
	C74H144O3	1080.10427	1080.10151	−2.6	0.4	1080.10569	1.3	1.0
	C75H146O3	1094.11992				1094.11679	−2.9	0.4
	C76H148O3	1108.13557				1108.13778	2.0	7.8
	C77H150O3	1122.15122				1122.15009	−1.0	1.5
	C78H152O3	1136.16687				1136.16903	1.9	25.0
	C79H154O3	1150.18252				1150.18015	−2.1	1.7
	C80H156O3	1164.19817				1164.19906	0.8	17.0
	C81H158O3	1178.21382				1178.21126	−2.2	0.8
	C82H160O3	1192.22947				1192.22827	−1.0	4.4
	C84H164O3	1220.26077				1220.25784	−2.4	1.1
α′-	C64H126O3	941.96342	941.96209	−1.4	1.5
	C66H130O3	969.99472	969.99218	−2.6	8.4
	C68H134O3	998.02602	998.02758	1.6	0.8
Keto-	C80H156O4	1180.19309				1180.19739	3.6	0.7
	C82H160O4	1208.22439
	C84H164O4	1236.25569				1236.26005	3.5	1.3
	C85H166O4	1250.27134				1250.27109	−0.2	1.8
	C86H168O4	1264.28699				1264.28423	−2.2	1.2
	C87H170O4	1278.30264				1278.30276	0.1	4.1
	C88H172O4	1292.31829				1292.31631	−1.5	0.3
	C89H174O4	1306.33394				1306.33735	2.6	0.4
Methoxy-	C80H158O4	1182.20874				1182.21141	2.3	0.3
	C81H160O4	1196.22439				1196.22575	1.1	1.1
	C82H162O4	1210.24004				1210.23864	−1.2	0.7
	C83H164O4	1224.25569				1224.25752	1.5	3.9
	C84H166O4	1238.27134				1238.27213	0.6	1.5
	C85H168O4	1252.28699				1252.29003	2.4	8.3
	C86H170O4	1266.30264				1266.30089	−1.4	2.5
	C87H172O4	1280.31829				1280.31691	−1.1	5.7
	C88H174O4	1294.33394				1294.33233	−1.2	3.8
	C89H176O4	1308.34959				1308.35587	4.8	0.8
	C90H178O4	1322.36524				1322.36553	0.2	0.6

aRelative intensity was calculated from the sum of detected monoisotopic peaks.

Figure 5

ESI-MS spectra of the [M − H]− mycolic acid ions. (A) Mycobacterium tuberculosis H37Rv (control), (B) Mycobacterium ahvazicum AFP-003T.

The unique phenotypic, genetic and genomic characteristics of AFP-003T strain all support the fact that it is representative of a hitherto undescribed species in the genus Mycobacterium. We named this new species Mycobacterium ahvazicum sp. nov., derived from the name Ahvaz, the city in the southwest of Iran where the strain AFP-003T (=JCM 18430) was discovered; and strain AFP-003T is the type strain of M. ahvazicum. The data here reported indicated that M. ahvazicum is another new species belonging to the large M. simiae complex in which 18 new species have been reported over the last fifty years. Interestingly, seven of these isolates have been isolated from sputum[4,5,10-14], five from cervical lymph nodes[15-19], one from blood[20], one from rhesus macaques[21], two from fishes[22,23], one from water[24] and one from an unknown human clinical source[25] (Table 8).

Table 8

Synopsis of the M. simiae complex species characterized since 1965.

Species	Isolation source	Clinical presentation	Isolation year	Characterisation year	Isolation site	Growth	Ref
M. simiae	Rhesus macaques	ND	ND	1965	Hungary	SGM	(21)
M. intermedium	Sputum	Pulmonary disease	ND	1993	Germany	SGM	(13)
M. interjectum	lymph node	Chronic lymphadenitis	ND	1993	Germany	SGM	(18)
M. genavense	Blood, bone marrow, livers, spleens, intestines, lymph nodes	Fever and diarrhea and experienced weight loss.	ND	1993	Geneva	SGM	(20)
M. triplex	Lymph node	ND	ND	1996	USA	SGM	(19)
M. lentiflavum	sputum, gastric juice, urine	Spondylodiscitis	1991–1993	1996	USA	SGM	(14)
M. heidelbergense	Cervical lymph nodes	Lymphadenitis	ND	1997	Germany	SGM	(15)
M. kubicae	Respiratory specimen	ND	1994–1997	2000	USA	SGM	(10)
M. palustre	Water		1993	2002	Finland	SGM	(24)
M. montefiorense	Moray eels	Granulomatous skin disease	2001	2003	USA	SGM	(22)
M. parmense	Cervical lymph node	Local swelling of submandibular	1999	2004	Italia	SGM	(16)
M. sherrisii	Clinical specimen	ND	1975	2004	USA	SGM	(25)
M. saskatchewanense	Sputum and pleural fluid	Bronchiectasis	2000	2004	Canada	SGM	(11)
M. parascrofulaceum	Sputum and bronchoscopy specimens	Symptoms of TB except for a dry cough	2002	2004	Canada	SGM	(12)
M. florentinum	Cervical lymph node	Lymphadenopathy	1993	2005	Italia	SGM	(17)
M. stomatepiae	Spleen tissue of Stomatepia mariae	Granulomatous lesions in spleen	ND	2008	London, UK	SGM	(23)
M. europaeum	Sputum (Human)	Cavitary pneumopathy	1995	2011	Italy, Florence	SGM	(5)
M. paraense	Sputum	Pulmonary symptoms	ND	2015	Brazil	SGM	(4)
M. ahvazicum	Sputum	Chronic Pulmonary disease	2009	2017	Iran	SGM	This work

SGM: Slowly Growing Mycobacteria, ND: No Data.

The discovery of M. ahvazicum is one more example illustrating that digging for mycobacteria in previously under-explored territories would reveal new species, as previously illustrated by our recent report of Mycobacterium massilipolyniensis in one remote island of the French Polynesian territories[26].

Methods

Phenotypic characterization

Biochemical tests were carried out using standard methods[27] and the minimal inhibitory concentration (MIC) of the major antimycobacterial agents was determined using the broth microdilution method[28].

Biolog Phenotype microarray

The ability of AFP-003T to metabolize 71 different carbon substrates and resist to 23 inhibitory chemicals was tested using Gen III Microplates Biolog® Phenotype MicroArray (Biolog Inc)[29]. AFP-003T was cultured at 37 °C on Middlebrook 7H10 agar medium supplemented with 10% (v/v) oleic acid/albumin/dextrose/catalase (OADC) (Becton Dickinson, Sparks, MD, USA) for 2 weeks. Colonies were gently taken with the wet swab off the agar plate culture and then rubbed against the wall of a dry glass tube. The cells were then suspended in IF-B (Biolog inoculating fluid recommended for strongly reducing and capsule producing bacteria, including Mycobacteria) and adjusted to 90% transmittance using a turbidimeter (Biolog Inc). Two plates (duplicate) were then inoculated and incubated in the OmniLog PM System (Biolog Inc.) at 37 °C for three days. The results were obtained as area under the curve (AUC) by Biolog’s parametric software.

Transmission Electron Microscopy

The size of the microorganisms was determined by transmission electron microscopy (Morgani 268D; Philips, Eindhoven, The Netherlands) after negative staining at an operating voltage of 60 kV.

Extraction and analysis of mycolic acids

AFP-003T and Mycobacterium tuberculosis H37Rv (used a positive control) were cultured on Middlebrook 7H10 agar medium supplemented with 10% 0ADC for three weeks. Mycolic acids were prepared as detailed previously with modifications[8,30]. At least six inoculation loops were collected from a culture plate and transferred into 2 mL of potassium hydroxide 9 M. Mycolic acids were hydrolyzed at 100 °C during 2 hours. Free mycolic acids were then extracted with 2 mL of chloroform at low pH by adding 3 mL of 6 N hydrochloric acid. The organic phase was collected and dried at 40 °C under a stream of nitrogen. Free mycolic acids were then dissolved in 100 µL of a methanol-chloroform mixture (50:50, v/v) and subjected to electrospray-mass spectrometry analysis after a 2000 fold dilution in methanol. Samples were analyzed in the Sensitivity Negative ionization mode using a Vion IMS QTof high resolution mass spectrometer (Waters, Guyancourt, France). Samples were infused at 10 µL/min after fluidics wash with a chloroform/methanol solution (50:50) and monitored from 500 to 2000 m/z during 2 minutes. Ionization parameters were set as follow: capillary voltage 2.5 kV, cone voltage 50 V, source and desolvation temperatures 120/650 °C. Mass calibration was adjusted automatically during analysis using a Leucine Enkephalin solution at 50 pg/µL (554.2620 m/z). Mass spectra between 900 and 1400 m/z were used for subsequent data interpretation. Mycolic acids were described according to previously detailed structures[31].

MALDI-TOF-MS

Using a sterile 200 µL tip, a small portion of a colony was picked on a Middlebrook 7H10 solid-medium and applied directly on a ground-steel MALDI target plate. Then, one µL of a matrix solution (saturated α-cyano-4- hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) (Bruker Daltonics) was used to over-lay the sample. After 5 minutes-drying at room temperature, the plate was loaded into the Microflex LT (Bruker Daltonics) mass spectrometer. Spectra were recorded following the parameters as previously described[32]. All signals with resolution ≥400 were automatically acquired using AutoXecute acquisition control in flexControl software version 3.0 and the identifications were obtained by MALDI Biotyper software version 3.0 with the Mycobacteria Library v2.0 database (version December, 2015).

Phylogenetic analysis

Phylogenetic and molecular evolutionary analyses based on the 16S rRNA gene sequence were inferred using the maximum likelihood method implemented on MEGA7[33], with the complete deletion option, based on the Tamura-Nei model for nucleotide sequences. Initial trees for the heuristic search were obtained automatically by applying the neighbor-joining and BIONJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. Statistical support for internal branches of the trees was evaluated by bootstrapping with 1000 iterations.

Genome sequencing

Total DNA of strain AFP-003T was extracted in two steps: A mechanical treatment was first performed by acid-washed (G4649-500g Sigma) glass beads using a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/sec) for 90 s. Then after a 2-hour lysozyme incubation at 37 °C, DNA was extracted on the EZ1 biorobot (Qiagen) with EZ1 DNA tissues kit. The elution volume was of 50 µL. gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 32.5 ng/µL. Genomic DNA was sequenced on the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the two applications: paired end and mate pair. Both strategies were barcoded to be mixed respectively with 11 other genomic projects prepared according to the Nextera XT 166 DNA sample prep kit (Illumina) and with 11 others projects according to the Nextera Mate 8 Pair sample prep kit (Illumina). To prepare the paired-end library, 1ng of gDNA was fragmented and amplified by limited PCR (12 cycles), introducing dual-index barcodes and sequencing adapters. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the libraries were normalized and pooled for sequencing on the MiSeq. Automated cluster generation and paired-end sequencing with dual indexed 2 × 250-bp reads were performed in a 9-hour run. Total information of 9.0 Gb was obtained from a 1,019 k/mm2 cluster density with a cluster passing quality control filters of 90.2% (17,374,744 passed filtered reads). Within this run, the index representation for AFP-003T was determined to be of 8.20%. The 1,424,260 paired end reads were trimmed and filtered according to the read qualities. The mate pair library was prepared with 1.5 µg of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The optimal size of obtained fragments was of 5.043 kb. No size selection was performed and 544 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with optima on a bi modal curve at 421 and 881 bp on the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA) and the final concentration library was measured at 16.97 nmol/L. The libraries were normalized at 2 nM, pooled with 11 other projects, denatured and diluted at 15 pM. Automated cluster generation and 2 × 250-bp sequencing run were performed in a 39-hour run. This library was loaded on two different flow cells. For each run, global information of 5.3 and 7.2 Gb was obtained respectively from a 559 and 765 K/mm2 cluster density with a cluster passing quality control filters of 96.3 and 94.7% (10,450,000 and 14,162,000 passed filter clustersfor each sequencing run). Within these runs, the index representation for AFP-003T was determined to be of 8.51 and 7.62%. The 888,760 and 1,079,096 paired-end reads. The three runs leaded to a total of 3,392,116 paired-end reads which were filtered according to the read qualities. The reads were assembled using the SPAdes software (http://bioinf.spbau.ru/spades)[34]. Contigs obtained were combined by use of SSPACE[35] assisted by manual finishing and GapFiller[36]. Open reading frames (ORFs) were predicted using Prodigal[37] with default parameters. The predicted ORFs were excluded if they spanned a sequencing gap region (containing N). The predicted bacterial protein sequences were searched against the GenBank database and the Clusters of Orthologous Groups (COGs) database using BLASTP (E value 1e-03, coverage 0.7 and 30% identity). If no hit was found, it searched against the NR database using BLASTP with an E value of 1e-03, coverage 0.7 and 30% identity. The tRNAs and rRNAs were predicted using the tRNA Scan-SE and RNAmmer tools, respectively[38,39]. SignalP and TMHMM were used to foresee the signal peptides and the number of transmembrane helices, respectively[40,41]. For each selected genome, complete genome sequence, proteome genome sequence and Orfeome genome sequence were retrieved from the FTP site of National Center for Biotechnology Information (NCBI). All proteomes were analyzed using proteinOrtho[42]. An annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes per cluster of orthologous groups of proteins (using the same method as for the genome annotation). The origin of replication was predicted using OriFinder[5,6] (http://tubic.tju.edu.cn/Ori-Finder/) and homology with other OriC regions was searched using blast algorithm in DoriC database7 (http://tubic.tju.edu.cn/doric/). The M. ahvazicum strain AFP-003T genome was further incorporated into in silico DNA-DNA hybridization (DDH)[43] with reference genomes selected based on 16S rRNA gene proximity; and DDH values were estimated using the GGDC version 2.0 online tool[44]. For AFP-003T genome comparison, we used the following species: of M. parascrofulaceum, M. triplex, M. interjectum, M. genavense, M. sherrisii and M. simiae.