- Email: [email protected]
Description of ‘Arabia massiliensis’ gen. nov., sp. nov., ‘Gordonibacter massiliensis’ sp. nov., and ‘Bacilliculturomica massiliensis’ gen. nov., sp. nov., isolated from a faecal specimen of a 50-year-old Saudi Bedouin woman
Accepted Manuscript Description of “Arabia massiliensis” gen. nov, sp. nov., “Gordonibacter massiliensis” sp. nov., and “Bacilliculturomica massiliensis” gen.nov, sp. nov., isolated from a fecal specimen of a 50 year-old Saudi Bedouin female Sory Ibrahima Traore, Esam Ibraheem Azhar, Muhammad Yasir, Fehmida Bibi, Pierre-Edouard Fournier, Asif Ahmad Jiman-Fatani, Jeremy Delerce, Frédéric Cadoret, Jean-Christophe Lagier, Didier Raoult PII:
S2052-2975(17)30047-1
DOI:
10.1016/j.nmni.2017.05.011
Reference:
NMNI 335
To appear in:
New Microbes and New Infections
Received Date: 18 April 2017 Revised Date:
18 May 2017
Accepted Date: 22 May 2017
Please cite this article as: Traore SI, Azhar EI, Yasir M, Bibi F, Fournier P-E, Jiman-Fatani AA, Delerce J, Cadoret F, Lagier J-C, Raoult D, Description of “Arabia massiliensis” gen. nov, sp. nov., “Gordonibacter massiliensis” sp. nov., and “Bacilliculturomica massiliensis” gen.nov, sp. nov., isolated from a fecal specimen of a 50 year-old Saudi Bedouin female, New Microbes and New Infections (2017), doi: 10.1016/j.nmni.2017.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Description of “Arabia massiliensis” gen. nov, sp. nov., “Gordonibacter massiliensis” sp. nov., and “Bacilliculturomica massiliensis” gen.nov, sp. nov., isolated from a fecal specimen of a 50 year-old Saudi Bedouin female
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Sory Ibrahima Traore1, Esam Ibraheem Azhar2,3, Muhammad Yasir2, Fehmida Bibi2, PierreEdouard Fournier1, Asif Ahmad Jiman-Fatani4, Jeremy Delerce1, Frédéric Cadoret1, JeanChristophe Lagier1 and Didier Raoult1,2*
Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, Institut
SC
1
M AN U
Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, 27 Boulevard Jean Moulin, 13385, Marseille cedex 05, France 2
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King Abdulaziz
TE D
3
University, Jeddah, Saudi Arabia
Department of Medical Microbiology and Parasitology, Faculty of Medicine, King
EP
4
AC C
Abdulaziz University, Jeddah, Saudi Arabia
* Corresponding author: Pr. Didier Raoult: [email protected]
Running head: “Arabia massiliensis”, “Gordonibacter massiliensis”, “Bacilliculturomica massiliensis” Keywords: “Arabia massiliensis”, “Gordonibacter massiliensis”,” Bacilliculturomica massiliensis”, gut microbiota.
ACCEPTED MANUSCRIPT Abstract:
2
We report here the main characteristics of “Arabia massiliensis” strain Marseille-P3078T gen.
3
nov, sp. nov, “Gordonibacter massiliensis” Marseille-P2775T sp. nov and “Bacilliculturomica
4
massiliensis” strain Marseille-P3303 gen.nov, sp. nov. The culturomics approach combined
5
with taxono-genomics was used to characterize those strains which were all isolated from a
6
fecal specimen of a 50-year-old Saudi Bedouin female.
7
SC
8 9
M AN U
10 11 12 13
18 19 20 21 22 23 24 25
EP
17
AC C
16
TE D
14 15
RI PT
1
ACCEPTED MANUSCRIPT Concerning the study of the human gut microbiota content, we isolated in 2016 using a
27
bacterial culturomics approach three bacteria that could not be identified by matrix-assisted
28
laser desorption-ionization (MALDI TOF) on a Microflex spectrometer (Bruker Daltonics,
29
Brenen, Germany) [1;2]. These strains were isolated from the stool sample of a 50-year-old
30
healthy Bedouin female living in the Jazan region of Saudi Arabia. This healthy individual
31
gave an informed consent. This study was performed in Saudi Arabia after approval from the
32
Ethical Committee of the King Abdulaziz University (Saudi Arabia), and the local ethic
33
committee of the IFR48 (Marseille, France) under the numbers 014-CEGMR-2-ETH-P and
34
09-022, respectively. All these three strains failed to be identified by MALDI TOF and their
35
16S rRNA gene were sequenced using fD1-rP2 primers as described previously using a 3130-
36
XL sequencer (Applied Biosciences, Saint Aubin, France) [3]
37
The stool sample was pre-incubated for 7 days at 37°C blood culture bottle (Becton Dickinson
38
Diagnostics, Le Pont-de-Claix, France) supplemented with 3 mL of rumen fluid filter-
39
sterilized through a 0.2 µm pore filter (Thermo Fisher Scientific, Villebon sur Yvette,
40
France). The strains Marseille-P3078 and Marseille-P2775 were isolated after an initial
41
growth of 48 hours on Columbia agar supplemented with 5% sheep blood at 37°C under strict
42
anaerobic conditions.
43
Colonies of strain Marseille-P3078 appeared beige, non-hemolytic, motile, non-spore
44
forming, with 0.5 mm size. The cells were Gram-negative, small rod-shaped ranging 1µm of
45
length and 0.7µm of diameter. The strain did not show oxidase activity but catalase positive
46
activity. The strain Marseille-P3078 had a 16S rRNA gene sequence identity of 94.59% with
47
Gordonibacter pamelae strain 7-10-1-b (NR_102934), the phylogenetically closest species
48
with standing in nomenclature (Figure 1). Gordonibacter pamelaeae strain 7-10-1-bT was
49
isolated from the colon of a patient suffering from acute Crohn’s disease. This isolate formed
50
small, pale-white, semi-translucent colonies on solid cultivation media. The strain was
AC C
EP
TE D
M AN U
SC
RI PT
26
ACCEPTED MANUSCRIPT catalase-positive, obligately anaerobic, non-spore-forming, Gram-stain-positive, short-
52
rods/coccobacilli [4]. This similarity < 95% leads us to putatively classify Marseille-P3078 as
53
a new member in the Eggerthellaceae family of the Firmicutes phylum [5]. Therefore we
54
propose the creation of the new genus “Arabia” (A.ra.bia NL gen fem, Arabia for the region
55
where sample were collected, Saudi Arabia). “Arabia massiliensis” is the type strain of the
56
new genus “Arabia”. Marseille-P3078 T is the type strain of the species “Arabia massiliensis”
57
(ma.ssi.lien’sis. L. adj. fem. Massiliensis for Massilia, the latine name of Marseille, France,
58
where the strain was isolated).
59
Strain Marseille-P2775 presents beige colonies, non-hemolytic, motile and non-spore
60
forming, with a 1 mm size. The cells were Gram-negative, rod-shaped, ranging 1.2µm of
61
length for 0.5µm of diameter. The strain did not show oxidase activity but catalase positive
62
activity. The 16S rRNA gene sequence shows an identity of 95% with Arabia massiliensis
63
and 97.19% with Gordonibacter urolithinfaciens strain CEBAS 1/15P (NR_134044), the
64
phylogenetically closest species with standing in nomenclature (Figure 2). Gordonibacter
65
urolithinfaciens strain CEBAS 1/15PT, capable of metabolizing ellagic acid (EA) to
66
urolithins, that was isolated from healthy human feces and characterized by determining
67
phenotypic, biochemical and molecular methods, was obligately anaerobic, non-spore-
68
forming, Gram-stain-positive, short-rods/coccobacilli [6]. This similarity < 98.65% leads us to
69
putatively classify Marseille-P2775 as a new member in the genus Gordonibacter, in the
70
Eggerthellaceae family of the Firmicutes phylum [5]. Therefore we propose the creation of
71
the new species “Gordonibacter massiliensis” (ma.ssi.lien’sis. L. adj. neut. Massiliensis to
72
Massilia, the antic name of Marseille, France, where the strain was isolated). Marseille-
73
P2775T is the type strain of the new species “Gordonibacter massiliensis”.
74
Strain Marseille-P3303 was isolated after an initial growth of 72 hours on Columbia agar
75
supplemented with 5% sheep blood at 37°C under strict anaerobic conditions, after the stool
AC C
EP
TE D
M AN U
SC
RI PT
51
ACCEPTED MANUSCRIPT specimen was pre-incubated for 30 days in an anaerobic blood culture bottle enriched with 37
77
grams/L of Difco Marine Broth (Becton Dickinson) à 37°C. It was then subcultured on 5%
78
sheep blood-enriched agar (bioMérieux, Marcy L’Etoile, France). The colonies appeared
79
ochre, non-hemolytic, non-motile, spore forming, with a 2 mm size. The cells were Gram-
80
positive, rod-shaped, ranging 2-5µm of length and 0.6µm of wide. The strain did not show
81
catalase or oxidase activity. The 16S rRNA gene sequence shows an identity of 92.52% with
82
Anaerovorax odorimutans strain NorPut1 (NR_028911), the phylogenetically closest species
83
with standing in nomenclature (Figure 2). The strictly anaerobic, gram-positive, non-spore-
84
forming bacterium strain NorPut1T ferments putrescine to acetate, butyrate, molecular
85
hydrogen and ammonia [7]. This similarity < 95% leads us to putatively classify Marseille-
86
P3303 as a new member in the Clostridiaceae family of Firmicutes [5]. Therefore we propose
87
the creation of the new genus “Bacciliculturomica” (Ba.ci.lli.cul.tu.ro.mi’ca L. gen. fem.
88
composed by Bacilli, rod shaped bacterium and Culturomica to refer at the “Culturomics”
89
laboratory, in Marseille, where the strain was isolated). “Bacciliculturomica massiliensis” is
90
the type strain of the new genus “Bacilliculturomica”. Marseille-P3303T is the type strain of
91
the species “Bacciliculturomica massiliensis” (ma.ssi.lien’sis L. adj. fem. Massiliensis to
92
Massilia, the antic name of Marseille, France, where the strain was isolated).
93
MALDI-TOF-MS spectra accession number
94
The MALDI-TOF-MS spectra of these species are available at http://mediterranee-
95
infection.com/article.php? laref=256&titre=urms-database.
96
Nucleotide sequence accession number
97
The 16S r RNA gene sequence was deposited in Genbank under accession numbers:
98
“Arabia massiliensis” strain Marseille-P3078T (LT598545), “Gordonibacter massiliensis”
99
Marseille-P2775T (LT558845) and “Bacilliculturomica massiliensis” strain Marseille-P3303T
100
AC C
EP
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76
(LT631507).
ACCEPTED MANUSCRIPT 101 102
Deposit in a culture collection.
104
The strains were deposited in the Collection de Souches de l’Unité des Rickettsies (CSUR,
105
WDCM 875) under numbers P3078 (“Arabia massiliensis” strain Marseille-P3078T), P2775
106
(“Gordonibacter
107
massiliensis” strain Marseille-P3303T)
108
Conflict of interest:
109
None to declare.
110
Funding sources:
111
This work was funded by Fondation Mediterrannée-Infection.
113
119 120 121 122 123 124 125
EP
118
AC C
117
TE D
114
116
and
P3303
M AN U
112
115
Marseille-P2775T)
(“Bacilliculturomica
SC
massiliensis”
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103
ACCEPTED MANUSCRIPT 126 127
Figure legend
129
Figure 1: Phylogenetic tree showing the positions of Arabia massiliensis strain Marseille-
130
P3078T relative to other phylogenetically-close neighbors. Sequences were aligned using
131
CLUSTALW, and phylogenetic inferences obtained with kimura two parameter models using
132
the maximum-likelihood method within the MEGA software. Numbers at the nodes are
133
percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a
134
majority consensus tree. The scale bar indicates a 1-2% nucleotide sequence divergence.
135
Figure 2: Phylogenetic tree showing the position of Gordonibacter massiliensis Marseille-
136
P2775T relative to other phylogenetically-close neighbors. Alignment and phylogenetic
137
inferences were done as described for Figure 1.
138
Figure 3: Phylogenetic tree showing the position of Bacilliculturomica massiliensis strain
139
Marseille-P3303T relative to other phylogenetically-close neighbors. Alignment and
140
phylogenetic inferences were done as described for Figure 1.
143 144 145 146 147 148 149 150 151
SC
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TE D
EP
142
AC C
141
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128
ACCEPTED MANUSCRIPT 152 153 154
References
155 (1) Lagier J C, Hugon P, Khelaifia S, Fournier P E, La S B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 2015; 28(1):237-264.
159 160 161
(2) Lagier J C, Khelaifia S, Alou M T, Ndongo S, Dione N, Hugon P et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol 2016; 1:16203.
162 163 164
(3) Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral J P, Raoult D. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 2000; 38(10):3623-3630.
165 166 167 168 169
(4) Wurdemann D, Tindall B J, Pukall R, Lunsdorf H, Strompl C, Namuth T et al. Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohn's disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov. Int J Syst Evol Microbiol 2009; 59(Pt 6):1405-1415.
170 171 172
(5) Kim M, Oh H S, Park S C, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64(Pt 2):346-351.
173 174 175
(6) Selma M V, Tomas-Barberan F A, Beltran D, Garcia-Villalba R, Espin J C. Gordonibacter urolithinfaciens sp. nov., a urolithin-producing bacterium isolated from the human gut. Int J Syst Evol Microbiol 2014; 64(Pt 7):2346-2352.
176 177 178 179 180
(7) Matthies C, Evers S, Ludwig W, Schink B. Anaerovorax odorimutans gen. nov., sp. nov., a putrescine-fermenting, strictly anaerobic bacterium. Int J Syst Evol Microbiol 2000; 50 Pt 4:1591-1594.
182
SC
M AN U
TE D
EP
AC C
181
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156 157 158
M AN U
SC
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ACCEPTED MANUSCRIPT
AC C
EP
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Figure1 :
ACCEPTED MANUSCRIPT Gordonibacter pamelaeae (NR 102934.1) Gordonibacter urolithinfaciens (NR 134044.1) 97
Gordonibacter massiliensis (LT558845) Paraeggerthella hongkongensis (NR 042825.1)
100
RI PT
Eggerthella sinensis (NR 042840.1) Eggerthella lenta (NR 074377.1)
Slackia heliotrinireducens (NR 074439.1)
Asaccharobacter celatus (NR 114402.1)
Adlercreutzia equolifaciens (NR 041609.1)
SC
100
M AN U
0.01
AC C
EP
TE D
Figure2 :
ACCEPTED MANUSCRIPT Bacilliculturomica massiliensis (LT631507) Anaerovorax odorimutans (NR 028911.1)
98
Eubacterium nodatum (NR 119311.1) Eubacterium minutum (NR 036949.1)
100 100
Aminicella lysinilytica (NR 134694.1)
Eubacterium sulci (NR 025289.1)
100
RI PT
Eubacterium infirmum (NR 118780.1)
Eubacterium saphenum (NR 026031.1)
Eubacterium brachy (NR 036993.1)
SC
Mogibacterium timidum (NR 036995.1)
Mogibacterium pumilum (NR 028608.1)
100
Mogibacterium neglectum (NR 027203.1)
100
M AN U
Mogibacterium diversum (NR 027191.1) Mogibacterium vescum (NR 024712.1)
0.01
AC C
EP
TE D
Figure3 :
S2052-2975(17)30047-1
DOI:
10.1016/j.nmni.2017.05.011
Reference:
NMNI 335
To appear in:
New Microbes and New Infections
Received Date: 18 April 2017 Revised Date:
18 May 2017
Accepted Date: 22 May 2017
Please cite this article as: Traore SI, Azhar EI, Yasir M, Bibi F, Fournier P-E, Jiman-Fatani AA, Delerce J, Cadoret F, Lagier J-C, Raoult D, Description of “Arabia massiliensis” gen. nov, sp. nov., “Gordonibacter massiliensis” sp. nov., and “Bacilliculturomica massiliensis” gen.nov, sp. nov., isolated from a fecal specimen of a 50 year-old Saudi Bedouin female, New Microbes and New Infections (2017), doi: 10.1016/j.nmni.2017.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Description of “Arabia massiliensis” gen. nov, sp. nov., “Gordonibacter massiliensis” sp. nov., and “Bacilliculturomica massiliensis” gen.nov, sp. nov., isolated from a fecal specimen of a 50 year-old Saudi Bedouin female
RI PT
Sory Ibrahima Traore1, Esam Ibraheem Azhar2,3, Muhammad Yasir2, Fehmida Bibi2, PierreEdouard Fournier1, Asif Ahmad Jiman-Fatani4, Jeremy Delerce1, Frédéric Cadoret1, JeanChristophe Lagier1 and Didier Raoult1,2*
Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, Institut
SC
1
M AN U
Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, 27 Boulevard Jean Moulin, 13385, Marseille cedex 05, France 2
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, King Abdulaziz
TE D
3
University, Jeddah, Saudi Arabia
Department of Medical Microbiology and Parasitology, Faculty of Medicine, King
EP
4
AC C
Abdulaziz University, Jeddah, Saudi Arabia
* Corresponding author: Pr. Didier Raoult: [email protected]
Running head: “Arabia massiliensis”, “Gordonibacter massiliensis”, “Bacilliculturomica massiliensis” Keywords: “Arabia massiliensis”, “Gordonibacter massiliensis”,” Bacilliculturomica massiliensis”, gut microbiota.
ACCEPTED MANUSCRIPT Abstract:
2
We report here the main characteristics of “Arabia massiliensis” strain Marseille-P3078T gen.
3
nov, sp. nov, “Gordonibacter massiliensis” Marseille-P2775T sp. nov and “Bacilliculturomica
4
massiliensis” strain Marseille-P3303 gen.nov, sp. nov. The culturomics approach combined
5
with taxono-genomics was used to characterize those strains which were all isolated from a
6
fecal specimen of a 50-year-old Saudi Bedouin female.
7
SC
8 9
M AN U
10 11 12 13
18 19 20 21 22 23 24 25
EP
17
AC C
16
TE D
14 15
RI PT
1
ACCEPTED MANUSCRIPT Concerning the study of the human gut microbiota content, we isolated in 2016 using a
27
bacterial culturomics approach three bacteria that could not be identified by matrix-assisted
28
laser desorption-ionization (MALDI TOF) on a Microflex spectrometer (Bruker Daltonics,
29
Brenen, Germany) [1;2]. These strains were isolated from the stool sample of a 50-year-old
30
healthy Bedouin female living in the Jazan region of Saudi Arabia. This healthy individual
31
gave an informed consent. This study was performed in Saudi Arabia after approval from the
32
Ethical Committee of the King Abdulaziz University (Saudi Arabia), and the local ethic
33
committee of the IFR48 (Marseille, France) under the numbers 014-CEGMR-2-ETH-P and
34
09-022, respectively. All these three strains failed to be identified by MALDI TOF and their
35
16S rRNA gene were sequenced using fD1-rP2 primers as described previously using a 3130-
36
XL sequencer (Applied Biosciences, Saint Aubin, France) [3]
37
The stool sample was pre-incubated for 7 days at 37°C blood culture bottle (Becton Dickinson
38
Diagnostics, Le Pont-de-Claix, France) supplemented with 3 mL of rumen fluid filter-
39
sterilized through a 0.2 µm pore filter (Thermo Fisher Scientific, Villebon sur Yvette,
40
France). The strains Marseille-P3078 and Marseille-P2775 were isolated after an initial
41
growth of 48 hours on Columbia agar supplemented with 5% sheep blood at 37°C under strict
42
anaerobic conditions.
43
Colonies of strain Marseille-P3078 appeared beige, non-hemolytic, motile, non-spore
44
forming, with 0.5 mm size. The cells were Gram-negative, small rod-shaped ranging 1µm of
45
length and 0.7µm of diameter. The strain did not show oxidase activity but catalase positive
46
activity. The strain Marseille-P3078 had a 16S rRNA gene sequence identity of 94.59% with
47
Gordonibacter pamelae strain 7-10-1-b (NR_102934), the phylogenetically closest species
48
with standing in nomenclature (Figure 1). Gordonibacter pamelaeae strain 7-10-1-bT was
49
isolated from the colon of a patient suffering from acute Crohn’s disease. This isolate formed
50
small, pale-white, semi-translucent colonies on solid cultivation media. The strain was
AC C
EP
TE D
M AN U
SC
RI PT
26
ACCEPTED MANUSCRIPT catalase-positive, obligately anaerobic, non-spore-forming, Gram-stain-positive, short-
52
rods/coccobacilli [4]. This similarity < 95% leads us to putatively classify Marseille-P3078 as
53
a new member in the Eggerthellaceae family of the Firmicutes phylum [5]. Therefore we
54
propose the creation of the new genus “Arabia” (A.ra.bia NL gen fem, Arabia for the region
55
where sample were collected, Saudi Arabia). “Arabia massiliensis” is the type strain of the
56
new genus “Arabia”. Marseille-P3078 T is the type strain of the species “Arabia massiliensis”
57
(ma.ssi.lien’sis. L. adj. fem. Massiliensis for Massilia, the latine name of Marseille, France,
58
where the strain was isolated).
59
Strain Marseille-P2775 presents beige colonies, non-hemolytic, motile and non-spore
60
forming, with a 1 mm size. The cells were Gram-negative, rod-shaped, ranging 1.2µm of
61
length for 0.5µm of diameter. The strain did not show oxidase activity but catalase positive
62
activity. The 16S rRNA gene sequence shows an identity of 95% with Arabia massiliensis
63
and 97.19% with Gordonibacter urolithinfaciens strain CEBAS 1/15P (NR_134044), the
64
phylogenetically closest species with standing in nomenclature (Figure 2). Gordonibacter
65
urolithinfaciens strain CEBAS 1/15PT, capable of metabolizing ellagic acid (EA) to
66
urolithins, that was isolated from healthy human feces and characterized by determining
67
phenotypic, biochemical and molecular methods, was obligately anaerobic, non-spore-
68
forming, Gram-stain-positive, short-rods/coccobacilli [6]. This similarity < 98.65% leads us to
69
putatively classify Marseille-P2775 as a new member in the genus Gordonibacter, in the
70
Eggerthellaceae family of the Firmicutes phylum [5]. Therefore we propose the creation of
71
the new species “Gordonibacter massiliensis” (ma.ssi.lien’sis. L. adj. neut. Massiliensis to
72
Massilia, the antic name of Marseille, France, where the strain was isolated). Marseille-
73
P2775T is the type strain of the new species “Gordonibacter massiliensis”.
74
Strain Marseille-P3303 was isolated after an initial growth of 72 hours on Columbia agar
75
supplemented with 5% sheep blood at 37°C under strict anaerobic conditions, after the stool
AC C
EP
TE D
M AN U
SC
RI PT
51
ACCEPTED MANUSCRIPT specimen was pre-incubated for 30 days in an anaerobic blood culture bottle enriched with 37
77
grams/L of Difco Marine Broth (Becton Dickinson) à 37°C. It was then subcultured on 5%
78
sheep blood-enriched agar (bioMérieux, Marcy L’Etoile, France). The colonies appeared
79
ochre, non-hemolytic, non-motile, spore forming, with a 2 mm size. The cells were Gram-
80
positive, rod-shaped, ranging 2-5µm of length and 0.6µm of wide. The strain did not show
81
catalase or oxidase activity. The 16S rRNA gene sequence shows an identity of 92.52% with
82
Anaerovorax odorimutans strain NorPut1 (NR_028911), the phylogenetically closest species
83
with standing in nomenclature (Figure 2). The strictly anaerobic, gram-positive, non-spore-
84
forming bacterium strain NorPut1T ferments putrescine to acetate, butyrate, molecular
85
hydrogen and ammonia [7]. This similarity < 95% leads us to putatively classify Marseille-
86
P3303 as a new member in the Clostridiaceae family of Firmicutes [5]. Therefore we propose
87
the creation of the new genus “Bacciliculturomica” (Ba.ci.lli.cul.tu.ro.mi’ca L. gen. fem.
88
composed by Bacilli, rod shaped bacterium and Culturomica to refer at the “Culturomics”
89
laboratory, in Marseille, where the strain was isolated). “Bacciliculturomica massiliensis” is
90
the type strain of the new genus “Bacilliculturomica”. Marseille-P3303T is the type strain of
91
the species “Bacciliculturomica massiliensis” (ma.ssi.lien’sis L. adj. fem. Massiliensis to
92
Massilia, the antic name of Marseille, France, where the strain was isolated).
93
MALDI-TOF-MS spectra accession number
94
The MALDI-TOF-MS spectra of these species are available at http://mediterranee-
95
infection.com/article.php? laref=256&titre=urms-database.
96
Nucleotide sequence accession number
97
The 16S r RNA gene sequence was deposited in Genbank under accession numbers:
98
“Arabia massiliensis” strain Marseille-P3078T (LT598545), “Gordonibacter massiliensis”
99
Marseille-P2775T (LT558845) and “Bacilliculturomica massiliensis” strain Marseille-P3303T
100
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(LT631507).
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Deposit in a culture collection.
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The strains were deposited in the Collection de Souches de l’Unité des Rickettsies (CSUR,
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WDCM 875) under numbers P3078 (“Arabia massiliensis” strain Marseille-P3078T), P2775
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(“Gordonibacter
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massiliensis” strain Marseille-P3303T)
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Conflict of interest:
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None to declare.
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Funding sources:
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This work was funded by Fondation Mediterrannée-Infection.
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and
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Marseille-P2775T)
(“Bacilliculturomica
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massiliensis”
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Figure legend
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Figure 1: Phylogenetic tree showing the positions of Arabia massiliensis strain Marseille-
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P3078T relative to other phylogenetically-close neighbors. Sequences were aligned using
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CLUSTALW, and phylogenetic inferences obtained with kimura two parameter models using
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the maximum-likelihood method within the MEGA software. Numbers at the nodes are
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percentages of bootstrap values obtained by repeating the analysis 1000 times to generate a
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majority consensus tree. The scale bar indicates a 1-2% nucleotide sequence divergence.
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Figure 2: Phylogenetic tree showing the position of Gordonibacter massiliensis Marseille-
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P2775T relative to other phylogenetically-close neighbors. Alignment and phylogenetic
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inferences were done as described for Figure 1.
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Figure 3: Phylogenetic tree showing the position of Bacilliculturomica massiliensis strain
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Marseille-P3303T relative to other phylogenetically-close neighbors. Alignment and
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phylogenetic inferences were done as described for Figure 1.
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References
155 (1) Lagier J C, Hugon P, Khelaifia S, Fournier P E, La S B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 2015; 28(1):237-264.
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(2) Lagier J C, Khelaifia S, Alou M T, Ndongo S, Dione N, Hugon P et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol 2016; 1:16203.
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(3) Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral J P, Raoult D. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 2000; 38(10):3623-3630.
165 166 167 168 169
(4) Wurdemann D, Tindall B J, Pukall R, Lunsdorf H, Strompl C, Namuth T et al. Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohn's disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov. Int J Syst Evol Microbiol 2009; 59(Pt 6):1405-1415.
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(5) Kim M, Oh H S, Park S C, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64(Pt 2):346-351.
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(6) Selma M V, Tomas-Barberan F A, Beltran D, Garcia-Villalba R, Espin J C. Gordonibacter urolithinfaciens sp. nov., a urolithin-producing bacterium isolated from the human gut. Int J Syst Evol Microbiol 2014; 64(Pt 7):2346-2352.
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(7) Matthies C, Evers S, Ludwig W, Schink B. Anaerovorax odorimutans gen. nov., sp. nov., a putrescine-fermenting, strictly anaerobic bacterium. Int J Syst Evol Microbiol 2000; 50 Pt 4:1591-1594.
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Figure1 :
ACCEPTED MANUSCRIPT Gordonibacter pamelaeae (NR 102934.1) Gordonibacter urolithinfaciens (NR 134044.1) 97
Gordonibacter massiliensis (LT558845) Paraeggerthella hongkongensis (NR 042825.1)
100
RI PT
Eggerthella sinensis (NR 042840.1) Eggerthella lenta (NR 074377.1)
Slackia heliotrinireducens (NR 074439.1)
Asaccharobacter celatus (NR 114402.1)
Adlercreutzia equolifaciens (NR 041609.1)
SC
100
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0.01
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Figure2 :
ACCEPTED MANUSCRIPT Bacilliculturomica massiliensis (LT631507) Anaerovorax odorimutans (NR 028911.1)
98
Eubacterium nodatum (NR 119311.1) Eubacterium minutum (NR 036949.1)
100 100
Aminicella lysinilytica (NR 134694.1)
Eubacterium sulci (NR 025289.1)
100
RI PT
Eubacterium infirmum (NR 118780.1)
Eubacterium saphenum (NR 026031.1)
Eubacterium brachy (NR 036993.1)
SC
Mogibacterium timidum (NR 036995.1)
Mogibacterium pumilum (NR 028608.1)
100
Mogibacterium neglectum (NR 027203.1)
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Mogibacterium diversum (NR 027191.1) Mogibacterium vescum (NR 024712.1)
0.01
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Figure3 :