- Email: [email protected]
‘Pygmaiobacter massiliensis’ sp. nov., a new bacterium isolated from the human gut of a Pygmy woman
Accepted Manuscript “Pygmaiobacter massiliensis” sp., nov., a new bacterium isolated from the human gut of a pygmy female Melhem Bilen, Maxime Descartes Mbogning, Frédéric Cadoret, Gregory Dubourg, Ziad Daoud, Pierre Edouard Fournier, Didier Raoult PII:
S2052-2975(16)30152-4
DOI:
10.1016/j.nmni.2016.12.015
Reference:
NMNI 282
To appear in:
New Microbes and New Infections
Received Date: 1 December 2016 Revised Date:
14 December 2016
Accepted Date: 14 December 2016
Please cite this article as: Bilen M, Mbogning MD, Cadoret F, Dubourg G, Daoud Z, Fournier PE, Raoult D, “Pygmaiobacter massiliensis” sp., nov., a new bacterium isolated from the human gut of a pygmy female, New Microbes and New Infections (2017), doi: 10.1016/j.nmni.2016.12.015. 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
Running title: Pygmaiobacter massiliensis
RI PT
“Pygmaiobacter massiliensis” sp., nov., a new bacterium isolated from the human gut of a pygmy female.
Melhem Bilen1,2, Maxime Descartes Mbogning1, Frédéric Cadoret1,Gregory Dubourg1,
M AN U
1
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Ziad Daoud2, Pierre Edouard Fournier1, and Didier Raoult1*
Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095,
Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, 27 Boulevard Jean Moulin, 13385, Marseille cedex 05, France 2
Clinical Microbiology Department, Faculty of Medicine and Medical sciences,
Corresponding author. E-mail address: [email protected]
EP
*
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University of Balamand, www.balamand.edu.lb, POBox:33, Amioun, Lebanon.
Keywords: Culturomics, Pygmaiobacter massiliensis, Emerging bacteria, Gut microbiota,
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Human microbiota.
ACCEPTED MANUSCRIPT
Abstract:
2
This study supports the main characteristics of a new genus “Pygmaiobacter” and a new species
3
“Pygmaiobacter massiliensis” strain Marseille-P3336 (CSUR P3336); that was isolated from a
4
stool sample of a healthy 47-year-old pygmy female.
5
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1
The Institut Fédératif de Recherche ethic committee, gave us an approval under the number the
7
number 09-022. Samples were collected for microbial content description as part of the human
8
microbiome description by culturomics [1].
9
First, stool sample was cultured in a blood culture bottle at 37°C after being diluted with
M AN U
SC
6
phosphate buffer saline (PBS). The culture bottle was supplemented with 5% sheep blood and
11
5% filtered rumen. Strain Marseille-P3336 colony was isolated at day 10 on 5% blood enriched
12
Columbia agar (bioMérieux, Marcy l'Etoile, France). The first identification trial of strain
13
Marseille-P3336 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
14
(MALDI-TOF-MS) using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany) failed
15
[2]. Thus, a 16S rRNA gene sequencing was performed in order to identify our strain based on
16
sequence similarities. Thus, 3130-XL sequencer (Applied Biosciences, Saint Aubin, France) was
17
used along with fD1-rP2 primers (Eurogentec, Seraing, Belgium) as previously reported [3].
18
Strain Marseille-P3336 exhibited a 92.38% sequence identity with Anaerofilum pentosovorans
19
(NR_029313.1), the phylogenetically closest species with standing nomenclature (Fig. 1).
20
Colonies were smoothwith a mean diameter of 0.1-0.5 mm. Bacterial cells were Gram positive
21
bacilli, catalase and oxidase negative with a mean diameter of 1.8µm.
22
Since strain Marseille-P3336 16S rRNA gene sequence diverges by more than 5% from the
23
closest species with standing in nomenclature, we propose the creation of the new genus named
24
“Pygmaiobacter” (Pyg. maio.bac’ter N.L. masc. gen. n, “Pygmaiobacter”, for Pygmy, the
AC C
EP
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10
ACCEPTED MANUSCRIPT
population from which the samples were collected [4]. Marseille-P3336 is the type strain of the
26
new species “Pygmaiobacter massiliensis” gen. nov., sp. nov. (mas.il.i.en’sis. L. gen. masc. adj.
27
“massiliensis” pertaining to Massilia, the antic name of the city of Marseille where this bacteria
28
has been discovered.
29
MALDI-TOF MS spectrum accession number. The MALDI-TOF-MS spectrum of P.
30
massiliensis is available online (http://www.mediterranee-
31
infection.com/article.php?laref=256&titre=urms-database)
32
Nucleotide sequence accession number. The 16S rRNA gene sequence was deposited in
33
Genbank under accession number LT631513.
34
Deposit in a culture collection. Strain Marseille-P3336 was deposited in the Collection de
35
Souches de l’Unité des Rickettsies (CSUR, WDCM 875) under number P3336.
36
Conflict of interest:
37
None to declare.
38
Funding sources:
39
This work was funded by Méditerrannée-Infection Foundation.
41
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EP
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40
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Figure legend
43
Figure 1: Phylogenetic tree that showin the position of “P. massiliensis” strain Marseille-P3336
44
within the phylogenetically closest species. CLUSTALW tool was used for sequence alignment
45
and phylogenetic inferences were generated using the Mega software by the maximum-likelihood
46
method. As for the bootstrap values obtained after 500 repeats, they are shown on the nodes.
47
Bootstrap scores of at least 90% were kept. The scale bar indicates a 2% nucleotide sequence
48
divergence.
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42
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ACCEPTED MANUSCRIPT
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Reference list
51
[1]
Lagier J-C, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut
53
microbiota. Clin Microbiol Rev 2015;28:237–64. doi:10.1128/CMR.00014-14.
54
[2]
RI PT
52
Seng P, Abat C, Rolain JM, Colson P, Lagier J-C, Gouriet F, et al. Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser
56
desorption ionization-time of flight mass spectrometry. Journal of Clinical Microbiology
57
2013;51:2182–94. doi:10.1128/JCM.00492-13. [3]
Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA
M AN U
58
SC
55
59
sequence analysis of a large collection of environmental and clinical unidentifiable
60
bacterial isolates. Journal of Clinical Microbiology 2000;38:3623–30.
61
[4]
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
63
prokaryotes. Int J Syst Evol Microbiol 2014;64:346–51. doi:10.1099/ijs.0.059774-0.
66
EP
65
AC C
64
TE D
62
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ACCEPTED MANUSCRIPT
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Figure 1
S2052-2975(16)30152-4
DOI:
10.1016/j.nmni.2016.12.015
Reference:
NMNI 282
To appear in:
New Microbes and New Infections
Received Date: 1 December 2016 Revised Date:
14 December 2016
Accepted Date: 14 December 2016
Please cite this article as: Bilen M, Mbogning MD, Cadoret F, Dubourg G, Daoud Z, Fournier PE, Raoult D, “Pygmaiobacter massiliensis” sp., nov., a new bacterium isolated from the human gut of a pygmy female, New Microbes and New Infections (2017), doi: 10.1016/j.nmni.2016.12.015. 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
Running title: Pygmaiobacter massiliensis
RI PT
“Pygmaiobacter massiliensis” sp., nov., a new bacterium isolated from the human gut of a pygmy female.
Melhem Bilen1,2, Maxime Descartes Mbogning1, Frédéric Cadoret1,Gregory Dubourg1,
M AN U
1
SC
Ziad Daoud2, Pierre Edouard Fournier1, and Didier Raoult1*
Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095,
Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, 27 Boulevard Jean Moulin, 13385, Marseille cedex 05, France 2
Clinical Microbiology Department, Faculty of Medicine and Medical sciences,
Corresponding author. E-mail address: [email protected]
EP
*
TE D
University of Balamand, www.balamand.edu.lb, POBox:33, Amioun, Lebanon.
Keywords: Culturomics, Pygmaiobacter massiliensis, Emerging bacteria, Gut microbiota,
AC C
Human microbiota.
ACCEPTED MANUSCRIPT
Abstract:
2
This study supports the main characteristics of a new genus “Pygmaiobacter” and a new species
3
“Pygmaiobacter massiliensis” strain Marseille-P3336 (CSUR P3336); that was isolated from a
4
stool sample of a healthy 47-year-old pygmy female.
5
RI PT
1
The Institut Fédératif de Recherche ethic committee, gave us an approval under the number the
7
number 09-022. Samples were collected for microbial content description as part of the human
8
microbiome description by culturomics [1].
9
First, stool sample was cultured in a blood culture bottle at 37°C after being diluted with
M AN U
SC
6
phosphate buffer saline (PBS). The culture bottle was supplemented with 5% sheep blood and
11
5% filtered rumen. Strain Marseille-P3336 colony was isolated at day 10 on 5% blood enriched
12
Columbia agar (bioMérieux, Marcy l'Etoile, France). The first identification trial of strain
13
Marseille-P3336 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
14
(MALDI-TOF-MS) using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany) failed
15
[2]. Thus, a 16S rRNA gene sequencing was performed in order to identify our strain based on
16
sequence similarities. Thus, 3130-XL sequencer (Applied Biosciences, Saint Aubin, France) was
17
used along with fD1-rP2 primers (Eurogentec, Seraing, Belgium) as previously reported [3].
18
Strain Marseille-P3336 exhibited a 92.38% sequence identity with Anaerofilum pentosovorans
19
(NR_029313.1), the phylogenetically closest species with standing nomenclature (Fig. 1).
20
Colonies were smoothwith a mean diameter of 0.1-0.5 mm. Bacterial cells were Gram positive
21
bacilli, catalase and oxidase negative with a mean diameter of 1.8µm.
22
Since strain Marseille-P3336 16S rRNA gene sequence diverges by more than 5% from the
23
closest species with standing in nomenclature, we propose the creation of the new genus named
24
“Pygmaiobacter” (Pyg. maio.bac’ter N.L. masc. gen. n, “Pygmaiobacter”, for Pygmy, the
AC C
EP
TE D
10
ACCEPTED MANUSCRIPT
population from which the samples were collected [4]. Marseille-P3336 is the type strain of the
26
new species “Pygmaiobacter massiliensis” gen. nov., sp. nov. (mas.il.i.en’sis. L. gen. masc. adj.
27
“massiliensis” pertaining to Massilia, the antic name of the city of Marseille where this bacteria
28
has been discovered.
29
MALDI-TOF MS spectrum accession number. The MALDI-TOF-MS spectrum of P.
30
massiliensis is available online (http://www.mediterranee-
31
infection.com/article.php?laref=256&titre=urms-database)
32
Nucleotide sequence accession number. The 16S rRNA gene sequence was deposited in
33
Genbank under accession number LT631513.
34
Deposit in a culture collection. Strain Marseille-P3336 was deposited in the Collection de
35
Souches de l’Unité des Rickettsies (CSUR, WDCM 875) under number P3336.
36
Conflict of interest:
37
None to declare.
38
Funding sources:
39
This work was funded by Méditerrannée-Infection Foundation.
41
SC
M AN U
TE D
EP
AC C
40
RI PT
25
ACCEPTED MANUSCRIPT
Figure legend
43
Figure 1: Phylogenetic tree that showin the position of “P. massiliensis” strain Marseille-P3336
44
within the phylogenetically closest species. CLUSTALW tool was used for sequence alignment
45
and phylogenetic inferences were generated using the Mega software by the maximum-likelihood
46
method. As for the bootstrap values obtained after 500 repeats, they are shown on the nodes.
47
Bootstrap scores of at least 90% were kept. The scale bar indicates a 2% nucleotide sequence
48
divergence.
SC
RI PT
42
AC C
EP
TE D
M AN U
49
ACCEPTED MANUSCRIPT
50
Reference list
51
[1]
Lagier J-C, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of culture in microbiology through the example of culturomics to study human gut
53
microbiota. Clin Microbiol Rev 2015;28:237–64. doi:10.1128/CMR.00014-14.
54
[2]
RI PT
52
Seng P, Abat C, Rolain JM, Colson P, Lagier J-C, Gouriet F, et al. Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser
56
desorption ionization-time of flight mass spectrometry. Journal of Clinical Microbiology
57
2013;51:2182–94. doi:10.1128/JCM.00492-13. [3]
Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA
M AN U
58
SC
55
59
sequence analysis of a large collection of environmental and clinical unidentifiable
60
bacterial isolates. Journal of Clinical Microbiology 2000;38:3623–30.
61
[4]
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
63
prokaryotes. Int J Syst Evol Microbiol 2014;64:346–51. doi:10.1099/ijs.0.059774-0.
66
EP
65
AC C
64
TE D
62
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
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Figure 1