Accepted Manuscript
“Khelaifiella massiliensis”, “Niameybacter massiliensis” “Brachybacterium
massiliense” “Enterobacter timonensis”, “Massilibacillus massiliensis”, new bacterial species and genera isolated from the gut microbiota of healthy infants
Maryam Tidjani Alou, Frederic Cadoret, Souleymane Brah, Aldiouma Diallo, Cheikh Sokhna, Vicky Mehrej, Jean-Christophe Lagier, Pierre-Edouard Fournier, Didier Raoult
PII: S2052-2975(17)30015-X
DOI: 10.1016/j.nmni.2017.02.002 Reference: NMNI 303
To appear in: New Microbes and New Infections
Received Date: 17 January 2017 Accepted Date: 27 February 2017
Please cite this article as: Tidjani Alou M, Cadoret F, Brah S, Diallo A, Sokhna C, Mehrej V, Lagier J- C, Fournier P-E, Raoult D, “Khelaifiella massiliensis”, “Niameybacter massiliensis” “Brachybacterium massiliense” “Enterobacter timonensis”, “Massilibacillus massiliensis”, new bacterial species and genera isolated from the gut microbiota of healthy infants, New Microbes and New Infections (2017), doi:
10.1016/j.nmni.2017.02.002.
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“Khelaifiella massiliensis”, “Niameybacter massiliensis” “Brachybacterium massiliense”
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“Enterobacter timonensis”, “Massilibacillus massiliensis”, new bacterial species and 2
genera isolated from the gut microbiota of healthy infants.
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Maryam Tidjani Alou1, Frederic Cadoret1, Souleymane Brah2, Aldiouma Diallo3, Cheikh 4
Sokhna3, Vicky Mehrej1, Jean-Christophe Lagier1, Pierre-Edouard Fournier1 and Didier 5
Raoult1,3,4. 6
1Aix-Marseille Université, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, Institut 7
Hospitalo-Universitaire Méditerranée-Infection 19-21 Boulevard Jean Moulin, 13385, 8
Marseille cedex 05, France.
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2Hôpital National de Niamey, Niamey, Niger.
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3Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes IRD 198, CNRS 11
7278, Campus Commun UCAD-IRD of Hann, Dakar, Senegal.
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4Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz 13
University, Jeddah, Saudi Arabia 14
* Corresponding author: Pr. Didier Raoult ([email protected]) 15
Running title: New bacteria from the gut of healthy infants.
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Keywords: new species; gut microbiota; culturomics; taxono-genomic 17
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Abstract 1
Here are presented the main characteristics of “Khelaifiella massiliensis” strain Mt13T (=
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CSUR P1935, = DSM100591), “Niameybacter massiliensis” strain Mt14T (= CSUR P1909, = 3
DSM100592), “Brachybacterium massiliense” strain mt5T (= CSUR P2240 = DSM101766), 4
“Enterobacter timonensis” strain mt20T (= CSUR P2201 = DSM 101775) and 5
“Massilibacillus massiliensis” strain Marseille-P2411T (= CSUR P2411 = DSM102838), new 6
species isolated from the gut of healthy African infants.
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Extensive knowledge of the gut microbiota composition has become essential for the 8
understanding of many aspects of health and disease. For that purpose, stool samples were 9
collected from a 7-month old healthy girl with a weight-for-height z-score (WHZ) of 0.15 10
from Niger and a 38-month-old healthy girl with a WHZ score of -0.12 from Senegal. The 11
diversity of these samples was explored using the culturomics concept [1,2]. Oral consent for 12
this study was obtained from the child’s parents and the study received authorization from the 13
local ethics committee of the Institut Federatif de Recherche IFR48 under number 09-022. As 14
a part of this study, strains Mt13T and Mt14T were isolated in the sample from Niger while 15
strains mt20T, mt5T and Marseille-P2411T were isolated in the sample from Senegal.
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Growth conditions and strains phenotypic characteristics 17
Strain Mt13T was first isolated after a 3-day preincubation in an anaerobic blood culture bottle 18
supplemented with sheep blood and seeding on 5% sheep blood–enriched Columbia agar 19
(bioMérieux, Marcy l’Etoile, France) at 37°C in anaerobic atmosphere. Large greyish and 20
irregular colonies with a mean diameter of 8 mm. Cells were Gram-stain-positive bacilli with 21
a mean diameter of 0.67 µm and a mean length of 2.99 µm. Catalase and oxidase activities 22
were absent.
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Strain Mt14T was first isolated after a 7-day preincubation in an anaerobic blood culture bottle 24
supplemented with rumen and sheep blood and seeding on 5% sheep blood–enriched 25
Columbia agar at 37°C in anaerobic atmosphere. Small white colonies were obtained with a 26
mean diameter of 3 mm. These colonies were formed with Gram-stain-positive bacilli with a 27
mean diameter of 0.61 µm and a mean length of 4.47 µm.
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Strain MT5T and strain mt20T were first isolated after a 24-hour and 3-day aerobic 29
preincubation respectively in a liquid medium containing 37 g of Difco Marine Broth (Becton 30
Dickinson, Le Pont de Claix, France) per litre of sterile water at 37°C and on 5% sheep 31
blood–enriched Columbia agar in aerobic condition. Strain mt20T formed large brown 32
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colonies with a mean diameter of 8 mm. Cells were Gram-stain negative bacilli with a mean 33
diameter of 0.8 µm and a mean length of 1.3 µm. Strain mt20T exhibited oxidase and catalase 34
activities. As for strain mt21T, it formed white full, circular colonies with a mean diameter of 35
3 mm which were formed with Gram-stain-positive cooci. Cells had a mean diameter of 0.57 36
µm and exhibited catalase activity and no oxidase activity.
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After a 7-day preincubation in an anaerobic blood culture bottle supplemented with sheep 38
blood and rumen and seeding on 5% sheep blood enriched Colombia agar in anaerobic 39
atmosphere, strain Marseille-P2411T was isolated. Small translucent colonies with a white 40
center measuring a mean diameter of 2 mm were obtained. These Gram-stain-positive bacilli 41
had a mean diameter of 0.6 µm and a mean length of 3.65 µm. Catalase and oxidase activities 42
were not exhibited by strain Marseille-P2411T. 43
Strain identification 44
After a failed identification of all five strains using matrix-assisted laser 45
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex 46
spectrometer (Bruker Daltonics, Bremen, Germany) [3,4], the 16S rRNA gene was sequenced 47
using fD1 and rP2 primers [5] as well as the rpoB gene for strain mt20T [6]. Kim et al 48
suggested a 98.65% and a 95% similarity level threshold to define a new species and a new 49
genus respectively without performing DNA-DNA hybridization [7] using the 16S rRNA 50
gene while a 97.7% threshold was set for identification using the rpoB gene [6].
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The 16S rRNA gene of strain Mt13T (accession number LN850733) showed a 94.23%
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similarity level with Hathewaya histolytica strain ATCC 19401T (accession number 53
AB566416) [8]. This classifies strain Mt13T as a putative new genus within the family 54
Clostridiaceae (Figure 1) for which we suggest the name Khelaifiella (Khe.lai.fi.el’la N.L.
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fem. n. composed of Khelaifia, in honor of the microbiologist Saber Khelaifia and bacterium 56
mean rod) with the type species being “Khelaifiella massiliensis” (mas.si.li.en’sis; L. fem.
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adj. massiliensis for Massilia, the Roman name of Marseille, where strain Mt13T was first 58
isolated). Strain Mt13T is the type strain of the species “Khelaifiella massiliensis”.
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The 16S rRNA gene sequences of strain Mt14T (accession number LN850735) 60
revealed a 92.18% similarity level with Cellulosilyticum lentocellum strain RHM5T (accession 61
number X71851) [9] thus confirming strain Mt14T as a new genus within the family 62
Lachnospiraceae (Figure 2). The name Niameybacter (Nia.mey.bac’ter for Niamey, the 63
capital of Niger where the stool sample was collected and bacter for bacterium) is proposed 64
for this new genus as well the type species “Niameybacter massiliensis” (mas.si.li.en’sis; L.
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masc. adj. massiliensis for Massilia, the Roman name of Marseille, where strain Mt14T was 66
first isolated). Strain Mt14T is the type strain of the species “Niameybacter massiliensis”.
67
The 16S rRNA sequence of strain MT5T (accession number LN906631) presented a 68
98.18% similarity level with Brachybacterium saurashtrense strain JG 06T (accession number 69
EU937750) [10]. Strain MT5T was thus classified as a new species within the genus 70
Brachybacterium (Figure 3) for which we suggest the name “Brachybacterium massiliense”
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(mas.si.li.en’se; L. masc. neut. adj. massiliense for Massilia, the Roman name of Marseille, 72
where strain MT5T was first isolated). Strain MT5T is the type strain of the species 73
“Brachybacterium massiliense”.
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The 16S rRNA gene sequence of strain mt20T (accession number LN906632) showed 75
a 98.4% similarity level with Enterobacter cloacae strain ATCC 13047T (accession number 76
NR_102794) [7,8] thus classifying strain mt20T as a new species within the genus 77
Enterobacter (Figure 4). The rpoB gene sequence (accession number LN906633) showed a 78
95.12% similarity level with the rpoB gene of Enterobacter cloacae strain ATCC 13047T 79
(accession number AJ543726), thus confirming the status of strain mt20T as a putative new 80
species [6]. Strain mt20T is the type strain of “Enterobacter timonensis” sp. nov.
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(ti.mo.nen’sis. L. gen. masc. timonensis, of Timone, the name of the hospital where strain 82
mt20T was first isolated).
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The 16S rRNA gene sequence of strain Marseille-P2411 (accession number 84
LT161896) presented a similarity level of 94.2% with Anaerosinus glycerini strain DSM 5192 85
(GenBank accession number NR_025297) [11], which is the phylogenetically closest species 86
with a validly published name, was obtained (Figure 5). A new genus was thus created within 87
the family Veillonellaceae named “Massilibacillus” (mas.si’li, L., masc. adj., massili for 88
Massilia, the old Roman name for Marseille where the genus was first isolated; bacillus as a 89
reference to the rod shape of the cell). Strain Marseille-P2411T is the type species of 90
“Massilibacillus massiliensis” (mas.si.li.en’sis, L., masc. adj., massiliensis for Massilia, the 91
old Roman name for Marseille where strain Marseille-P2411T was first isolated).
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MALDI-TOF MS spectra 93
The MALDI-TOF MS spectrum of “Khelaifiella massiliensis”, “Niameybacter massiliensis”, 94
“Enterobacter massiliensis”, “Brachybacterium massiliense” and “Massilibacillus 95
massiliensis” are available at 96
http://www.mediterraneeinfection.com/article.php?laref=256&titre=urms-database.
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Nucleotide sequence accession number 98
The 16S rRNA sequences of strains Mt13T, Mt14T, MT5T and Marseille-P2411T are deposited 99
in the Genbank database under accession numbers LN850733, LN850735, LN906631 and 100
LT161896 respectively.
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The 16S rRNA and rpoB gene sequences for strain mt20T are also deposited in the Genbank 102
database under accession number LN906632 and LN906633 respectively.
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Deposit in a culture collection 104
Strains Mt13T, Mt14T, MT5T, mt20T and Marseille-P2411T were deposited in the Collection 105
de Souches de l’Unité des Rickettsies (CSUR, WDCM 875) under numbers P1935, P1909, 106
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P2240, P2201 and P2411 respectively. Strains Mt14T, MT5T, mt20T and Marseille-P2411T 107
were also deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen 108
GmbH (DSMZ) under numbers DSM100592, DSM101766, DSM101775 and DSM102838 109
respectively.
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Acknowledgement 111
This study was funded by the Fondation Méditerranée Infection.
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Conflict of Interest 113
None to declare.
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Figure legends 155
Figure 1. Phylogenetic tree showing position of “Khelaifiella massiliensis” strain Mt13T 156
relative to other phylogenetically close species with validly published name. Sequences were 157
aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum 158
likelihood method within MEGA software [12]. Numbers at nodes are percentages of 159
bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree.
160
Selenomonas sputigena was used as outgroup. Scale bar indicates 2% nucleotide sequence 161
divergence.
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Figure 2. Phylogenetic tree showing position of “Niameybacter massiliensis” strain Mt14T 163
relative to other phylogenetically close species with validly published name. Sequences were 164
aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum 165
likelihood method within MEGA software [12]. Numbers at nodes are percentages of 166
bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree.
167
Veillonella parvula was used as outgroup. Scale bar indicates 2% nucleotide sequence 168
divergence.
169
Figure 3. Phylogenetic tree showing position of “Brachybacterium massiliensis” strain MT5T 170
relative to other phylogenetically close species with validly published name. Sequences were 171
aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum 172
likelihood method within MEGA software [12]. Numbers at nodes are percentages of 173
bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree.
174
Dermabacter hominis was used as outgroup. Scale bar indicates 0.5 % nucleotide sequence 175
divergence.
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Figure 4. Phylogenetic tree showing position of “Enterobacter timonensis” strain mt20T 177
relative to other phylogenetically close species with validly published name. Sequences were 178
aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum 179
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likelihood method within MEGA software [12]. Numbers at nodes are percentages of 180
bootstrap values obtained by repeating analysis 500 times to generatemajority consensus tree.
181
Rosenbergiella nectarea was used as outgroup. Scale bar indicates 0.5% nucleotide sequence 182
divergence.
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Figure 5. Phylogenetic tree showing position of “Massilibacillus massiliensis” strain 184
Marseille-P2411T relative to other phylogenetically close species with validly published 185
name. Sequences were aligned using CLUSTALW, and phylogenetic inferences were 186
obtained using maximum likelihood method within MEGA software [12]. Numbers at nodes 187
are percentages of bootstrap values obtained by repeating analysis 500 times to generate 188
majority consensus tree. Thermosinus carboxydivorans was used as outgroup. Scale bar 189
indicates 1% nucleotide sequence divergence.
190
