- Email: [email protected]
“Intestinimonas massiliensis” sp. nov, a new bacterium isolated from human gut
Accepted Manuscript “Intestinimonas massiliensis”sp. nov, a new bacterium isolated from the human gut Guillaume Durand, Pamela Afouda, Didier Raoult, Dr Grégory Dubourg PII:
S2052-2975(16)30104-4
DOI:
10.1016/j.nmni.2016.09.014
Reference:
NMNI 235
To appear in:
New Microbes and New Infections
Received Date: 21 September 2016 Accepted Date: 28 September 2016
Please cite this article as: Durand G, Afouda P, Raoult D, Dubourg G, “Intestinimonas massiliensis”sp. nov, a new bacterium isolated from the human gut, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.09.014. 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 “Intestinimonas massiliensis”sp. nov, a new bacterium isolated from the human gut Guillaume DURAND1,2, Pamela AFOUDA1, Didier RAOULT1-3 and Grégory DUBOURG1,2* 1
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS
7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté
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de médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France 2
Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de
Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, Assistance Publique-Hôpitaux
de Marseille, Marseille, France
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3
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Bactériologie-Hygiène–Virologie, University, Hospital Centre Timone,
* Corresponding author: Dr Grégory DUBOURG, URMITE UM63, CNRS7278, IRD198, INSERM1095, Faculté de Médecine, Aix Marseille Université, 27 boulevard Jean Moulin, 13385 Marseille cedex 5, France, tel + 33 (0)491 385 517, fax +33 (0)491 387 772,
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[email protected]
Running head: “Intestinimonas massiliensis” sp. nov.
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Keywords: “Intestinimonas massiliensis” sp. nov.; taxonogenomics; culturomics; gut microbiota, anaerobe.
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Abstract word count: 39 Article word count: 287 References: 5
Table/Figures: 1
ACCEPTED MANUSCRIPT ABSTRACT
2
Here we report the main features of the proposed new bacterial species “Intestinimonas massiliensis”
3
sp. nov. The type strain GD2T (CSUR P1930) was isolated from the gut microbiota of a healthy patient
4
using a culturomics approach combined with taxono-genomics.
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1
ACCEPTED MANUSCRIPT 5
As a part of our study of the Human Microbiome by culturomics (1), we isolated in the stool of a healthy French donor (28-year-old) the gram-negative rod and strictly anaerobe strain GD2T. The
7
written consent of the donor was obtained and the study was validated by the ethics committee of the
8
Federative Research Institute IFR48 under number 09-022. The stool was stored at -20 ° C for 10 days
9
and then inoculated on agar enriched with sheep blood (5%) and rumen fluid (5%) previously filter-
10
sterilized through a 0.2 µm pore filter (Thermo Fisher Scientific, Villebon sur Yvette, France). The
11
plates were then incubated under anaerobic condition into an anaerobic cabine for 72 hours. The
12
subculture of colonies using the same protocol allowed the isolation of the GD2T strain. The strain
13
GD2T could not be identified by matrix-assisted laser desorption-ionization time-of-flight screening
14
(score <1.7) using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany) (1–3).
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6
Colonies appeared white and regular with a mean diameter of 1-2 mm on blood agar-enriched
16
Colombia. “Intestinimonas massiliensis” is a non-motile Gram-negative rod with a mean diameter of
17
0.5µm and 1.8µm of length without spore-forming activity. Catalase and oxidase were also negative.
18
The 16S rRNA gene was completely sequenced as previously described (4). It shared 94.4% sequence
19
identity with Intestinimonas butyriciproducens DSM 26588T (NR_118554) .The bacterium was
20
therefore putatively classified as a new species belonging to the Intestinimonas genus. Because of the 16S identity percent lower than 98.65% with the species closest with a validly
EP
21
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15
published name standing in nomenclature (5), we propose the new strain “Intestinimonas massilliensis”
23
GD2T (mas.il. i.en'sis. L. gen. masc. n. massiliensis, of Massilia, the Latin name of Marseille where the
24
strain GD2T was first isolated) belonging to the genus Intestinimonas.
25
MALDI-TOF-MS spectrum accession number
26
The MALDI-TOF-MS spectrum of “Intestinimonas massiliensis” is available at http://mediterranee-
27
infection.com/article.php? laref=256&titre=urms-database.
28
(Last access 2016/09/15)
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22
ACCEPTED MANUSCRIPT Nucleotide sequence accession number
30
The 16S rRNA gene sequence was deposited in GenBank under accession number LN866996.
31
Deposit in a culture collection
32
Strain GD2T was deposited in the collection de Souches de l’Unités des Rickettsies (CSUR, WDCM
33
875) under number P1930.
34
Aknowledgement
35
This work was funded by Mediterrannée-Infection Foundation.
36
Conflict of interest
37
None declared.
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ACCEPTED MANUSCRIPT 38
References
39
1. Lagier J-C, Hugon P, Khelaifia S, Fournier P-E, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol
41
Rev. 2015 Jan;28(1):237–64.
42
RI PT
40
2. Lagier J-C, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin. Microbiol. Infect. 2012 Dec;18(12):1185–
44
93.
3. Seng P, Abat C, Rolain JM, Colson P, Lagier J-C, Gouriet F, et al. Identification of Rare
M AN U
45
SC
43
46
Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser
47
Desorption Ionization-Time of Flight Mass Spectrometry. J Clin Microbiol. 2013 Jul 1;51(7):2182–
48
94.
4. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA sequence
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49 50
analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin
51
Microbiol. 2000 Oct;38(10):3623–30.
5. Kim M, Oh H-S, Park S-C, Chun J. Towards a taxonomic coherence between average nucleotide
EP
52
identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst
54
Evol Microbiol. 2014 Feb;64(Pt 2):346–51.
55
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ACCEPTED MANUSCRIPT Figure legend Figure 1: Phylogenetic tree based on the 16S rRNA gene sequence showing the position of “Intestinimonas massiliensis” sp. nov., strain GD2T with other close relative species among the Firmicutes phylum. The EMBL database accession numbers are indicated in parentheses.
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Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained with kimura two parameter model using neighbor-joining method with 1000 bootstrap replicates,
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M AN U
SC
within MEGA6 software. The scale bar represents a 1% nucleotide sequence divergence.
S2052-2975(16)30104-4
DOI:
10.1016/j.nmni.2016.09.014
Reference:
NMNI 235
To appear in:
New Microbes and New Infections
Received Date: 21 September 2016 Accepted Date: 28 September 2016
Please cite this article as: Durand G, Afouda P, Raoult D, Dubourg G, “Intestinimonas massiliensis”sp. nov, a new bacterium isolated from the human gut, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.09.014. 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 “Intestinimonas massiliensis”sp. nov, a new bacterium isolated from the human gut Guillaume DURAND1,2, Pamela AFOUDA1, Didier RAOULT1-3 and Grégory DUBOURG1,2* 1
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS
7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté
RI PT
de médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France 2
Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de
Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, Assistance Publique-Hôpitaux
de Marseille, Marseille, France
M AN U
3
SC
Bactériologie-Hygiène–Virologie, University, Hospital Centre Timone,
* Corresponding author: Dr Grégory DUBOURG, URMITE UM63, CNRS7278, IRD198, INSERM1095, Faculté de Médecine, Aix Marseille Université, 27 boulevard Jean Moulin, 13385 Marseille cedex 5, France, tel + 33 (0)491 385 517, fax +33 (0)491 387 772,
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[email protected]
Running head: “Intestinimonas massiliensis” sp. nov.
EP
Keywords: “Intestinimonas massiliensis” sp. nov.; taxonogenomics; culturomics; gut microbiota, anaerobe.
AC C
Abstract word count: 39 Article word count: 287 References: 5
Table/Figures: 1
ACCEPTED MANUSCRIPT ABSTRACT
2
Here we report the main features of the proposed new bacterial species “Intestinimonas massiliensis”
3
sp. nov. The type strain GD2T (CSUR P1930) was isolated from the gut microbiota of a healthy patient
4
using a culturomics approach combined with taxono-genomics.
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT 5
As a part of our study of the Human Microbiome by culturomics (1), we isolated in the stool of a healthy French donor (28-year-old) the gram-negative rod and strictly anaerobe strain GD2T. The
7
written consent of the donor was obtained and the study was validated by the ethics committee of the
8
Federative Research Institute IFR48 under number 09-022. The stool was stored at -20 ° C for 10 days
9
and then inoculated on agar enriched with sheep blood (5%) and rumen fluid (5%) previously filter-
10
sterilized through a 0.2 µm pore filter (Thermo Fisher Scientific, Villebon sur Yvette, France). The
11
plates were then incubated under anaerobic condition into an anaerobic cabine for 72 hours. The
12
subculture of colonies using the same protocol allowed the isolation of the GD2T strain. The strain
13
GD2T could not be identified by matrix-assisted laser desorption-ionization time-of-flight screening
14
(score <1.7) using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany) (1–3).
M AN U
SC
RI PT
6
Colonies appeared white and regular with a mean diameter of 1-2 mm on blood agar-enriched
16
Colombia. “Intestinimonas massiliensis” is a non-motile Gram-negative rod with a mean diameter of
17
0.5µm and 1.8µm of length without spore-forming activity. Catalase and oxidase were also negative.
18
The 16S rRNA gene was completely sequenced as previously described (4). It shared 94.4% sequence
19
identity with Intestinimonas butyriciproducens DSM 26588T (NR_118554) .The bacterium was
20
therefore putatively classified as a new species belonging to the Intestinimonas genus. Because of the 16S identity percent lower than 98.65% with the species closest with a validly
EP
21
TE D
15
published name standing in nomenclature (5), we propose the new strain “Intestinimonas massilliensis”
23
GD2T (mas.il. i.en'sis. L. gen. masc. n. massiliensis, of Massilia, the Latin name of Marseille where the
24
strain GD2T was first isolated) belonging to the genus Intestinimonas.
25
MALDI-TOF-MS spectrum accession number
26
The MALDI-TOF-MS spectrum of “Intestinimonas massiliensis” is available at http://mediterranee-
27
infection.com/article.php? laref=256&titre=urms-database.
28
(Last access 2016/09/15)
AC C
22
ACCEPTED MANUSCRIPT Nucleotide sequence accession number
30
The 16S rRNA gene sequence was deposited in GenBank under accession number LN866996.
31
Deposit in a culture collection
32
Strain GD2T was deposited in the collection de Souches de l’Unités des Rickettsies (CSUR, WDCM
33
875) under number P1930.
34
Aknowledgement
35
This work was funded by Mediterrannée-Infection Foundation.
36
Conflict of interest
37
None declared.
AC C
EP
TE D
M AN U
SC
RI PT
29
ACCEPTED MANUSCRIPT 38
References
39
1. Lagier J-C, Hugon P, Khelaifia S, Fournier P-E, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol
41
Rev. 2015 Jan;28(1):237–64.
42
RI PT
40
2. Lagier J-C, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin. Microbiol. Infect. 2012 Dec;18(12):1185–
44
93.
3. Seng P, Abat C, Rolain JM, Colson P, Lagier J-C, Gouriet F, et al. Identification of Rare
M AN U
45
SC
43
46
Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser
47
Desorption Ionization-Time of Flight Mass Spectrometry. J Clin Microbiol. 2013 Jul 1;51(7):2182–
48
94.
4. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA sequence
TE D
49 50
analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin
51
Microbiol. 2000 Oct;38(10):3623–30.
5. Kim M, Oh H-S, Park S-C, Chun J. Towards a taxonomic coherence between average nucleotide
EP
52
identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst
54
Evol Microbiol. 2014 Feb;64(Pt 2):346–51.
55
AC C
53
ACCEPTED MANUSCRIPT Figure legend Figure 1: Phylogenetic tree based on the 16S rRNA gene sequence showing the position of “Intestinimonas massiliensis” sp. nov., strain GD2T with other close relative species among the Firmicutes phylum. The EMBL database accession numbers are indicated in parentheses.
RI PT
Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained with kimura two parameter model using neighbor-joining method with 1000 bootstrap replicates,
AC C
EP
TE D
M AN U
SC
within MEGA6 software. The scale bar represents a 1% nucleotide sequence divergence.