- Email: [email protected]
“Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract
Accepted Manuscript “Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract Khoudia Diop, Oleg Mediannikov, Didier Raoult, Florence Bretelle, Florence Fenollar, Pr PII:
S2052-2975(16)30102-0
DOI:
10.1016/j.nmni.2016.09.012
Reference:
NMNI 233
To appear in:
New Microbes and New Infections
Received Date: 4 September 2016 Revised Date:
16 September 2016
Accepted Date: 21 September 2016
Please cite this article as: Diop K, Mediannikov O, Raoult D, Bretelle F, Fenollar F, “Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.09.012. 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
“Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract
RI PT
Running title: “Vaginella massiliensis”
1
SC
Khoudia Diop1, Oleg Mediannikov1, Didier Raoult1,2, Florence Bretelle1,3 and Florence Fenollar1*
Aix Marseille Univ, Institut hospitalo-universitaire Méditerranée-infection, URMITE, UM63,
M AN U
CNRS 7278, IRD 198, Inserm U1095, Faculté de médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France Tel: +33 491 385 517, Fax: +33 491 387 772. 2
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
University, Jeddah, Saudi Arabia.
Department of Gynecology and Obstetrics, Gynépole, Hôpital Nord, Assistance Publique-
TE D
3
Hôpitaux de Marseille, AMU, Aix-Marseille Université.
EP
*Corresponding author: Pr Florence Fenollar
AC C
Email: [email protected]
Keywords: “Vaginella massiliensis”, vaginal flora, culturomics, human microbiota, Flavobacteriaceae
ACCEPTED MANUSCRIPT
1 2
Abstract We relate the main characteristics of “Vaginella massiliensis” strain Marseille P2517 (= DSM 102346 = CSUR P2517), a new member of the Flavobacteriaceae family. The strain
4
Marseille P2517 was cultivated in the vaginal swab of a healthy, 22-year-old, woman.
RI PT
3
AC C
EP
TE D
M AN U
SC
5
1
ACCEPTED MANUSCRIPT
As part of the study of the vaginal microbiota by the concept of “microbial culturomics”
7
[1], in February 2016, we isolated, from the vaginal sample of a healthy 22-year-old woman, a
8
bacterium that could not be identified using matrix-assisted laser desorption-ionization time-of-
9
flight (MALDI-TOF) mass spectrometry (MS) performed with a MicroFlex spectrometer (Bruker
10
Daltonics, Bremen, Germany) [2]. The study was authorized by the local ethics committee of the
11
IFR48 (Marseille, France) under the agreement 09-022; the patient gave also her written consent.
12
The initial growth of strain Marseille P2517 was obtained at 37°C under aerobic conditions
SC
RI PT
6
after 2 days of culture on Chocolat PolyViteX (PVX) agar (BD Diagnostics, Le Pont-de-Claix,
14
France) after 7 days of pre-incubation in a blood culture bottle (BD Diagnostics) enriched with 4
15
ml of rumen that was filter-sterilized through at 0.2µm pore filter (Thermo Fisher Scientific,
16
Villebon-sur-Yvette, France) and 3 ml of sheep blood (bioMérieux, Marcy l’Etoile, France).
17
Bacterial cells were Gram-negative rod-shaped bacilli, non-motile, and non-spore-forming with a
18
diameter ranging from 0.54 to 0.68 µm and a length ranging from 1.2 to 1.5 µm. Strain Marseille
19
P2517 exhibited positive oxydase activity but catalase was negative. After 2 days of incubation at
20
37°C under aerobic conditions on blood agar (bioMérieux), colonies were light yellow, opaque,
21
circular, and smooth with a diameter of 1.7 to 2 mm. Strain Marseille P2517 is obligate aerobe.
22
The 16S rRNA sequence was obtained after amplification using the universal primer pair
EP
TE D
M AN U
13
(fD1 and rp2) and a 3130-XL sequencer (Applied Biosciences, Saint-Aubin, France), as
24
previously reported [3]. 16S rRNA gene sequence-based identification of strain Marseille P2517
25
showed 93% of identity with Weeksella virosa DSM 16922 (GenBank accession number
26
NR_074495), the phylogenetically closest bacterium with a validly published name (Figure 1).
27
As this sequence was below the 95% threshold set by Stackebrandt and Elbers to define a new
28
genus without carrying out DNA-DNA hybridization [4], strain Marseille P2517 was classified as
AC C
23
2
ACCEPTED MANUSCRIPT
29
the representative strain of a new genus belonging to the family Flavobacteriaceae in the phylum
30
Bacteroidetes [5].
31
Weeksella virosa was first described in 1987 by Holmes et al. [6]. This bacterium is rodshaped, aerobic, Gram-negative, non-motile, and non-saccharolytic, exhibiting a positive activity
33
for catalase and oxidase [6]. Weeksella virosa is mostly isolated from the human urogenital tract.
34
RI PT
32
As well the strain Marseille P2517 presents a 16S rRNA divergence around 7% with its phylogenetically closest species [8], we propose that it may be the representative strain of a novel
36
genus preliminarily named here "Vaginella" (Va.gi.nel′la. L. fem. n. vagina, vagina, a part of the
37
female genital tract; L. dim. suff. -ella; N. L. dim. fem. n. Vaginella, a small vagina, referring to
38
the source of the isolation of the type strain). Strain Marseille P2517 is the type strain of the new
39
species “Vaginella massiliensis” (mas.si.li.en'sis. L. gen. adj. massiliensis, from Massilia, the
40
Latin name of Marseille, France, where the organism was first grown, identified and
41
characterized).
M AN U
TE D
42
SC
35
MALDI-TOF MS spectrum accession number. The MALDI-TOF MS spectrum of “Vaginella
44
massiliensis” is available at http://www.mediterranee-
45
infection.com/article.php?laref=256&titre=urms-database.
AC C
46
EP
43
47
Nucleotide sequence accession number. The 16S rRNA gene sequence was deposited in
48
EMBL-EBI under accession number LT223570.
49
3
ACCEPTED MANUSCRIPT
Deposit in culture collection. Strain Marseille P2517 was deposited in the collection “Deutsche
51
Sammlung von Mikroorganismen” (DSM) and in the “Collection de Souches de l’Unité des
52
Rickettsies” (CSUR, WDCM 875) under numbers 102346 and P2517, respectively.
AC C
EP
TE D
M AN U
SC
RI PT
50
4
ACCEPTED MANUSCRIPT
Acknowledgments
54
This research is funded by the Méditerranée-Infection Foundation.
AC C
EP
TE D
M AN U
SC
RI PT
53
5
ACCEPTED MANUSCRIPT
Figure 1. Phylogenetic tree highlighting the phylogenetic position of “Vaginella massiliensis”
56
strain Marseille P2517T relative to other species close members of Flavobacteriaceae. GenBank
57
accession numbers are indicated after the name in brackets. Sequences were aligned using
58
CLUSTALW, and phylogenetic inferences obtained using the approximately maximum-
59
likelihood method within the Mega6 Software. Numbers at the nodes are percentages of bootstrap
60
values obtained by repeating the analysis 500 times to generate a majority consensus tree. Only
61
the bootstraps score equal or greater than 95% were retained. The scale bar indicates a 2%
62
nucleotide sequence divergence.
63
AC C
EP
TE D
M AN U
SC
RI PT
55
6
ACCEPTED MANUSCRIPT
64
References
65
1.
66
Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota.
67
Clin Microbiol Rev. 2015;28:237-64.
68
2.
69
Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted
70
Laser Desorption Ionization-Time of Flight Mass Spectrometry. J Clin Microbiol. 2013;51:2182-
71
94.
72
3.
73
DNA sequence analysis of a large collection of environmental and clinical unidentifiable
74
bacterial isolates. J Clin Microbiol. 2000;38:3623–30.
75
4.
76
Microbiol Today. 2006;33:152-5.
77
5.
« Classification of bacteria ». http://www.bacterio.net/-classificationsz.html#Weeksella.
78
6.
Holmes B., Steigerwalt A. G., Weaver R. E. and Brenner Don J. Weeksella virosa gen.
79
nov., sp. nov. (Formerly Group IIf), Found in Human Clinical Specimens. System. Appl.
80
Microbial. 1986;8:185-190.
81
7.
82
Species Living Tree project: A 16S rRNA-based phylogenetic tree of all sequenced type strains.
83
Syst Appl Microbiol. 2008;31:241-250.
RI PT
Lagier J.C., Hugon P., Khelaifia S., Fournier P.E., La Scola B., Raoult D. The Rebirth of
M AN U
SC
Seng P., Abat C., Rolain J.M., Colson P., Lagier J.C., Gouriet F., et al. Identification of
Drancourt M., Bollet C., Carlioz A., Martelin R., Gayral J.P., Raoult D. 16S ribosomal
AC C
EP
TE D
Stackebrandt E., Ebers J. Taxonomic parameters revisited: tarnished gold standards.
Yarza P., Richter M., Peplies J., Euzeby J., Amann R., Schleifer K.H., et al. The All-
84
7
S2052-2975(16)30102-0
DOI:
10.1016/j.nmni.2016.09.012
Reference:
NMNI 233
To appear in:
New Microbes and New Infections
Received Date: 4 September 2016 Revised Date:
16 September 2016
Accepted Date: 21 September 2016
Please cite this article as: Diop K, Mediannikov O, Raoult D, Bretelle F, Fenollar F, “Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.09.012. 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
“Vaginella massiliensis” gen. nov., sp. nov., a new genus cultivated from human female genital tract
RI PT
Running title: “Vaginella massiliensis”
1
SC
Khoudia Diop1, Oleg Mediannikov1, Didier Raoult1,2, Florence Bretelle1,3 and Florence Fenollar1*
Aix Marseille Univ, Institut hospitalo-universitaire Méditerranée-infection, URMITE, UM63,
M AN U
CNRS 7278, IRD 198, Inserm U1095, Faculté de médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France Tel: +33 491 385 517, Fax: +33 491 387 772. 2
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
University, Jeddah, Saudi Arabia.
Department of Gynecology and Obstetrics, Gynépole, Hôpital Nord, Assistance Publique-
TE D
3
Hôpitaux de Marseille, AMU, Aix-Marseille Université.
EP
*Corresponding author: Pr Florence Fenollar
AC C
Email: [email protected]
Keywords: “Vaginella massiliensis”, vaginal flora, culturomics, human microbiota, Flavobacteriaceae
ACCEPTED MANUSCRIPT
1 2
Abstract We relate the main characteristics of “Vaginella massiliensis” strain Marseille P2517 (= DSM 102346 = CSUR P2517), a new member of the Flavobacteriaceae family. The strain
4
Marseille P2517 was cultivated in the vaginal swab of a healthy, 22-year-old, woman.
RI PT
3
AC C
EP
TE D
M AN U
SC
5
1
ACCEPTED MANUSCRIPT
As part of the study of the vaginal microbiota by the concept of “microbial culturomics”
7
[1], in February 2016, we isolated, from the vaginal sample of a healthy 22-year-old woman, a
8
bacterium that could not be identified using matrix-assisted laser desorption-ionization time-of-
9
flight (MALDI-TOF) mass spectrometry (MS) performed with a MicroFlex spectrometer (Bruker
10
Daltonics, Bremen, Germany) [2]. The study was authorized by the local ethics committee of the
11
IFR48 (Marseille, France) under the agreement 09-022; the patient gave also her written consent.
12
The initial growth of strain Marseille P2517 was obtained at 37°C under aerobic conditions
SC
RI PT
6
after 2 days of culture on Chocolat PolyViteX (PVX) agar (BD Diagnostics, Le Pont-de-Claix,
14
France) after 7 days of pre-incubation in a blood culture bottle (BD Diagnostics) enriched with 4
15
ml of rumen that was filter-sterilized through at 0.2µm pore filter (Thermo Fisher Scientific,
16
Villebon-sur-Yvette, France) and 3 ml of sheep blood (bioMérieux, Marcy l’Etoile, France).
17
Bacterial cells were Gram-negative rod-shaped bacilli, non-motile, and non-spore-forming with a
18
diameter ranging from 0.54 to 0.68 µm and a length ranging from 1.2 to 1.5 µm. Strain Marseille
19
P2517 exhibited positive oxydase activity but catalase was negative. After 2 days of incubation at
20
37°C under aerobic conditions on blood agar (bioMérieux), colonies were light yellow, opaque,
21
circular, and smooth with a diameter of 1.7 to 2 mm. Strain Marseille P2517 is obligate aerobe.
22
The 16S rRNA sequence was obtained after amplification using the universal primer pair
EP
TE D
M AN U
13
(fD1 and rp2) and a 3130-XL sequencer (Applied Biosciences, Saint-Aubin, France), as
24
previously reported [3]. 16S rRNA gene sequence-based identification of strain Marseille P2517
25
showed 93% of identity with Weeksella virosa DSM 16922 (GenBank accession number
26
NR_074495), the phylogenetically closest bacterium with a validly published name (Figure 1).
27
As this sequence was below the 95% threshold set by Stackebrandt and Elbers to define a new
28
genus without carrying out DNA-DNA hybridization [4], strain Marseille P2517 was classified as
AC C
23
2
ACCEPTED MANUSCRIPT
29
the representative strain of a new genus belonging to the family Flavobacteriaceae in the phylum
30
Bacteroidetes [5].
31
Weeksella virosa was first described in 1987 by Holmes et al. [6]. This bacterium is rodshaped, aerobic, Gram-negative, non-motile, and non-saccharolytic, exhibiting a positive activity
33
for catalase and oxidase [6]. Weeksella virosa is mostly isolated from the human urogenital tract.
34
RI PT
32
As well the strain Marseille P2517 presents a 16S rRNA divergence around 7% with its phylogenetically closest species [8], we propose that it may be the representative strain of a novel
36
genus preliminarily named here "Vaginella" (Va.gi.nel′la. L. fem. n. vagina, vagina, a part of the
37
female genital tract; L. dim. suff. -ella; N. L. dim. fem. n. Vaginella, a small vagina, referring to
38
the source of the isolation of the type strain). Strain Marseille P2517 is the type strain of the new
39
species “Vaginella massiliensis” (mas.si.li.en'sis. L. gen. adj. massiliensis, from Massilia, the
40
Latin name of Marseille, France, where the organism was first grown, identified and
41
characterized).
M AN U
TE D
42
SC
35
MALDI-TOF MS spectrum accession number. The MALDI-TOF MS spectrum of “Vaginella
44
massiliensis” is available at http://www.mediterranee-
45
infection.com/article.php?laref=256&titre=urms-database.
AC C
46
EP
43
47
Nucleotide sequence accession number. The 16S rRNA gene sequence was deposited in
48
EMBL-EBI under accession number LT223570.
49
3
ACCEPTED MANUSCRIPT
Deposit in culture collection. Strain Marseille P2517 was deposited in the collection “Deutsche
51
Sammlung von Mikroorganismen” (DSM) and in the “Collection de Souches de l’Unité des
52
Rickettsies” (CSUR, WDCM 875) under numbers 102346 and P2517, respectively.
AC C
EP
TE D
M AN U
SC
RI PT
50
4
ACCEPTED MANUSCRIPT
Acknowledgments
54
This research is funded by the Méditerranée-Infection Foundation.
AC C
EP
TE D
M AN U
SC
RI PT
53
5
ACCEPTED MANUSCRIPT
Figure 1. Phylogenetic tree highlighting the phylogenetic position of “Vaginella massiliensis”
56
strain Marseille P2517T relative to other species close members of Flavobacteriaceae. GenBank
57
accession numbers are indicated after the name in brackets. Sequences were aligned using
58
CLUSTALW, and phylogenetic inferences obtained using the approximately maximum-
59
likelihood method within the Mega6 Software. Numbers at the nodes are percentages of bootstrap
60
values obtained by repeating the analysis 500 times to generate a majority consensus tree. Only
61
the bootstraps score equal or greater than 95% were retained. The scale bar indicates a 2%
62
nucleotide sequence divergence.
63
AC C
EP
TE D
M AN U
SC
RI PT
55
6
ACCEPTED MANUSCRIPT
64
References
65
1.
66
Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota.
67
Clin Microbiol Rev. 2015;28:237-64.
68
2.
69
Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted
70
Laser Desorption Ionization-Time of Flight Mass Spectrometry. J Clin Microbiol. 2013;51:2182-
71
94.
72
3.
73
DNA sequence analysis of a large collection of environmental and clinical unidentifiable
74
bacterial isolates. J Clin Microbiol. 2000;38:3623–30.
75
4.
76
Microbiol Today. 2006;33:152-5.
77
5.
« Classification of bacteria ». http://www.bacterio.net/-classificationsz.html#Weeksella.
78
6.
Holmes B., Steigerwalt A. G., Weaver R. E. and Brenner Don J. Weeksella virosa gen.
79
nov., sp. nov. (Formerly Group IIf), Found in Human Clinical Specimens. System. Appl.
80
Microbial. 1986;8:185-190.
81
7.
82
Species Living Tree project: A 16S rRNA-based phylogenetic tree of all sequenced type strains.
83
Syst Appl Microbiol. 2008;31:241-250.
RI PT
Lagier J.C., Hugon P., Khelaifia S., Fournier P.E., La Scola B., Raoult D. The Rebirth of
M AN U
SC
Seng P., Abat C., Rolain J.M., Colson P., Lagier J.C., Gouriet F., et al. Identification of
Drancourt M., Bollet C., Carlioz A., Martelin R., Gayral J.P., Raoult D. 16S ribosomal
AC C
EP
TE D
Stackebrandt E., Ebers J. Taxonomic parameters revisited: tarnished gold standards.
Yarza P., Richter M., Peplies J., Euzeby J., Amann R., Schleifer K.H., et al. The All-
84
7