Oribacterium parvum, sp. nov. and Oribacterium asaccharolyticum sp. nov.
obligately anaerobic bacteria from the human oral cavity
Running title: Oribacterium parvum and O. asaccharolyticum sp. nov.
New Taxa, subsection Firmicutes
Maria V. Sizova1*, Paul A. Muller1, David Stancyk1, Nicolai S. Panikov1, Manolis
Mandalakis1#, Amanda Hazen1, Tine Hohmann1, Sebastian N. Doerfert1, William
Fowle1, Ashlee M. Earl2, Karen E. Nelson3, and Slava S. Epstein1*
1Department of Biology, Northeastern University, Boston, Massachusetts 02115
2The Broad Institute of MIT & Harvard, Cambridge, Massachusetts 02142
3J. Craig Venter Institute, Rockville, Maryland 20850
# Present address: Department of Chemistry, University of Crete, GR-71409
٭Corresponding author. Mailing address: Northeastern University, 360
Huntington Ave Boston, MA 02115. Phone: 617-373-3229, Fax: 617-373-3724,
E-mail: email@example.com; firstname.lastname@example.org*.†
*SEM photomicrographs of cells of strains ACB1T and ACB7T (Fig. S1) as well as a list of
selected proteins and their functions from strains ACB1T, ACB7T, ACB8 and other Oribacterium
spp. identified by RAST (Table S1) is available as supplementary material in IJSEM Online.
† The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains
ACB1T, ACB7T and ACB8 are HM120210-HM120212. The complete high quality draft genome
sequencing was performed as a part of the Human Microbiome Project by the Broad Institute of
Harvard and MIT and available at http://www.broadinstitute.org/ and by the J. Craig Venter
Institute. The GenBank/EMBL/DDBJ assembled genome sequence of strains ACB1T, ACB7T and
ACB8 are NZ_AFZC00000000, NZ_AFZD00000000 and NZ_AJZT00000000.
IJSEM Papers in Press. Published May 13, 2014 as doi:10.1099/ijs.0.060988-0
Three strictly anaerobic Gram-positive non-spore-forming rod-shaped motile bacteria
designated as Oribacterium sp. ACB1T, ACB7T and ACB8 were isolated from the
human subgingival dental plaque. All strains required yeast extract for growth. Strains
ACB1T and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin, and
raffinose; strain ACB7T grew weakly on sucrose only. The growth temperature range
was 30 - 42 oC with optimum at 37 oC. Major metabolic fermentation end products of
strain ACB1T were acetate and lactate; the only product of strains ACB7T and ACB8
was acetate. Major fatty acids of strain ACB1T were C14:0, C16:0, C16:1 ω7c DMA
(dimethyl aldehyde) and C18:1 ω7c DMA. Major fatty acids of strain ACB7T were C12:0,
C14:0, C16:0, C16:1 ω7c and C16:1 ω7c DMA. The hydrolysate of peptidoglycan
contained meso-diaminopimelic acid indicating peptidoglycan type A1γ. Genomic DNA
G + C content varied from 42 to 43.3% between strains. According to 16S rRNA gene
sequence phylogeny, strains ACB1T, ACB8 and ACB7T formed two separate branches
within the genus Oribacterium, with sequence similarity to type species of Oribacterium
sinus at 98.1-98.6%. Predicted DNA-DNA hybridization values between strains ACB1T,
ACB8, ACB7T and O. sinus were <70%. Based on distinct genotypic and phenotypic
characteristics, we suggest that strains ACB1T and ACB8, and strain ACB7T represent
two distinct species of the genus Oribacterium, for which the names Oribacterium
parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type
strains are strain ACB1T (= DSM 24637T; = HM-481T; =ATCC BAA-2638T) and strain
ACB7T (= DSM 24638T; = HM-482T; =ATCC BAA-2639T).
In this study we report the characterization of three strict anaerobic strains designated
as Oribacterium sp. ACB1T, ACB7T and ACB8 isolated from the subgingival plaque
obtained from a 25-year old African American female.
The study protocol was approved by the Institutional Review Board of Northeastern
University; informed consent was obtained from the subject. Novel isolates were
enriched on liquid basic anaerobic medium (BM) supplemented with L-cysteine-HCl as
a reducing agent and isolated in pure culture on agar-BM under anaerobic atmosphere
(2% H2, 1% CO2, 97% N2) (Sizova et al., 2012). The Human Oral Microbiome Database
(HOMD) classified isolated strains as members of oral taxon 108 (Chen et al., 2010).
According to the 16S rRNA gene sequence phylogeny, strains ACB1T, ACB7T and
ACB8 belong to the genus Oribacterium (Carlier et al., 2004) within the family
Lachnospiraceae (Rainey, 2009).
Colony morphology assessment was performed on Wilkins-Chalgren (WC) blood agar,
and TY (trypticase peptone-yeast extract) agar medium. All media were supplemented
with L-cysteine-HCl as a reducing agent (Sizova et al., 2012). Cell morphology was
observed with a Leica DMBL light microscope equipped with phase contrast. For
electron microscopy, cells grown in TY for 24 - 48 h were collected and were fixed as
described before (Ellis, 2006; Sizova et al., 2013) and observed with a Hitachi S4800
scanning electron microscope. Thin sections were stained with uranyl acetate and lead
citrate and observed with a JEOL JEM 1010 transmission electron microscope. The
Gram reaction was determined using Difco Gram stain kit. Resistance to various
antibiotics and bile was tested with Oxoid ’AN-IDENT’ and Remel susceptibility test
disks; zones less than 10 mm were considered as resistant. Oxidase, catalase and
nitrate reduction activities were tested with Remel reagents. Biochemical reactions and
individual carbon source utilization was assessed with API 20A tests and with cultures
grown on liquid medium with glucose, lactose, maltose, sucrose, cellobiose,
maltodextrin, raffinose or starch supplemented with yeast extract. The effect of
temperature was assessed with cultures grown on TY medium. All experiments were
conducted under anaerobic conditions; growth was scored as visible turbidity.
Fermentation products were determined by HPLC in acidified supernatant of cultures
grown on glucose - yeast extract and TY media before and after distillation (Agilent
1200 series HPLC; Poroshell 120 SB-C18 column 2.7 m, 3.0 × 100 mm with guard
column (Agilent Technologies); 10 μM H2SO4 was used as the mobile phase). Cell
biomass grown in TGY (trypticase peptone-glucose-yeast extract) for 48 h was used for
the whole-cell fatty acids and peptidoglycan analyses. Fatty acids were methylated,
extracted and analyzed by GC using the Sherlock Microbial Identification System at
Microbial ID, Inc. The peptidoglycan structure was analyzed in the hydrolysates (4N
HCl, 100 oC, 16 h) by the method of Rhuland et al. (1955) and by GC/MS analysis after
isolation of the peptidoglycan and its hydrolysis (Schumann, 2011) at the Identification
Service of the German Collection of Microorganisms and Cell Cultures. The 16S rRNA
gene sequence was compared with the gene sequences available from GenBank;
phylogenetic analysis was performed as described previously (Sizova et al., 2012).
Whole genome sequencing of strains ACB1T and ACB7T was carried out by the Broad
Institute of Harvard and MIT, and is available at http://www.broadinstitute.org/. The
whole genome of strain ACB8 was sequenced by the J. Craig Venter Institute. The
genome sequences of strains ACB1T, ACB7T and ACB8 were compared with available
genome sequences of other members of the genus Oribacterium. Individual coding
sequences were submitted to the RAST server (Aziz et al., 2008) for subsystem
annotations. DNA base content (mol% G + C) was calculated from the whole genome
sequences. DNA-DNA hybridization (DDH) values were predicted by the Genome-to-
Genome Distance (GGD) calculator 2.0, formula 3, and available online at
http://ggdc.dsmz.de/ (Auch et al., 2010a; Auch et al., 2010b; Meier-Kolthoff et al., 2013).
Cells of three strains were non-spore forming highly motile oval rods, sometimes
appearing swollen. Cells of strains ACB1T and ACB8 were 1.2 ± 0.4 μm long and 0.45 ±
0.09 μm wide; cells of strain ACB7T were 1.6 ± 0.6 μm long and 0.50 ± 0.08 μm wide
(Fig. 1, Fig. S1, Table 1). About 5% of strain ACB7T cells were curved rods up to 5-7 μm
long. Cells appeared Gram-variable after staining, but were structurally Gram-positive
(Fig. 1). Additionally, three to five RAST-annotated genes associated with synthesis of
teichoic and lipoteichoic acids were present in the genome (Table 2). Surprisingly, we
detected seven genes associated with synthesis of lipooligosaccharides (LOS) in strain
ACB1T, but not in strain ACB7T, ACB8 or other Oribacterium strains with available
genomes (Table 2). After 48 h incubation on TY agar plates at 37 oC strain ACB1T
formed beige round convex colonies 1 to 1.5 mm in diameter. Colonies of strain ACB7T
were round non pigmented 0.5 mm in diameter after 48 h and umbonate 2-3 mm in
diameter with irregular wavy edges after 168 h. All three strains were non-hemolytic.
After 1-2 days of incubation strains ACB1T, ACB7T and ACB8 produced a diffusible
black pigment on BM and TY liquid media supplemented with L-cysteine-HCl. The
pigment was visible as a grainy substance surrounding cells (Fig. S1b). Most cells of
strain ACB7T and some cells of strain ACB1T contained intracellular nanometer-sized
particles (Fig. 1) most likely ferrous sulfide (Sizova et al., 2013). Upon inspection of
genomes of strains ACB1T, ACB8 and ACB7T, we identified five to six genes putatively
annotated as encoding the cysteine desulfurase enzyme EC 22.214.171.124 (Table S1). The
activity of these genes likely explains the production of black precipitate, which is
presumably FeS (Frazzon & Dean, 2003; Mihara & Esaki, 2002). Similar particles were
previously observed in the cells of another member of the Lachnospiraceae family
Stomatobaculum longum which also contained a single gene encoding a cysteine
desulfurase enzyme in its genome (Sizova et al., 2013).
Isolated strains grew only under strict anaerobic conditions. Growth occurred at 30 to 42
oC, with optimum at 37 oC. Isolates ACB1T and ACB7T were susceptible to discs
containing 1 mg kanamycin, 5 µg vancomycin, 50 µg metronidazole, 2 units penicillin,
15 mg rifampicin and bile and resistant to 10 μg colistin. Catalase, oxidase and urease
activities were negative; nitrate reduction was not detected. Gelatin was not liquefied,
indole was not produced. Strain ACB7T hydrolyzed aesculin while strain ACB1T did not.
All strains were able to grow on yeast extract and Bacto proteose peptone No. 3 but not
on casamino acids or trypticase alone. Strain ACB1T produced acid on API 20A media
containing glucose, maltose and lactose, but not sucrose, arabinose, cellobiose,
mannose, melezitose, raffinose, rhamnose, trehalose, xylose, glycerol, mannitol, salicin
and sorbitol. In liquid medium supplemented with 0.5 – 2.0 g l-1 of yeast extract strains
ACB1T and ACB8 weakly fermented glucose, lactose, maltose, maltodextrin, and
raffinose but not cellobiose or starch; strain ACB8 grew weakly on sucrose. Strain
ACB7T did not produce acid on API 20A media, and did not grow on liquid medium with
any of the tested carbon sources with exception of weak growth on sucrose (i.e. OD 600
reached ~ 0.1 units after 7-10 days of incubation). No visible biomass was formed in
medium with 0.5 – 2.0 g l-1 of yeast extract only; poor growth was observed in liquid
medium with 1 g l-1 of yeast extract and 0.5 g l-1 of L-cysteine- HCl. Strains ACB1T and
ACB8 produced gas on TY or TGY liquid media. The major metabolic end products of
strain ACB1T were acetate and lactate. Acetate was the only end product of strains
ACB7T and ACB8.
The whole-cell hydrolysate of strain ACB7T contained meso-diaminopimelic acid (meso-
Dpm); meso-Dpm was also detected in strains ACB1T and ACB7T after isolation and
hydrolysis of peptidoglycan. The occurrence of meso-Dpm in isolated strains indicated
peptidoglycan type A1γ (or A1γ’; A31 or A32.1) according to http://www.peptidoglycan-
types.info. Six and seven RAST-annotated genes associated with diaminopimelic acid
(DAP) synthesis were present in the genome of strains ACB1T, ACB8 and ACB7T
respectively (Table 2).
Genomic DNA G + C content of strains ACB1T, ACB8 and ACB7T was 42.1, 42.0 and
43.3% respectively. Fatty acid methyl esters (FAME) profile showed that the strain
ACB1T contained C14:0 (18.6%), C16:0 (24.1%), C16:1 ω7c DMA (18.1%) and C18:1
ω7c DMA (6.1%) as major fatty acids; minor amounts of C14:0 DMA (4.8%), C15:0
(3.4%), C16:1 ω7c (3.3%), C16:0 DMA (3.7%), and trace amounts of C14:1 ω7c DMA
(1.65%), C15:0 anteiso (0.6%), C15:1 ω6c (0.3%), C16:0 aldehyde (1.1%), C16:1 ω9c
(0.2%), C16:1 ω5c (0.55%), C17:1 ω6c (0.4%), C17:0 (0.15%), C18:1 at 17.254 DMA
(0.3%), C18:0 (0.1%), and C18:1 ω9c DMA (0.1%). Strain ACB7T contained C12:0
(5.4%), C14:0 (22.4%), C16:0 (15.7%), C16:1 ω7c (8.5%) and C16:1 ω7c DMA (7.7%)
as major fatty acids; minor amounts of C10:0 DMA (4.0%), C14:0 DMA (3.3%), C15:0
(4.2%), C15:0 (3.1%), and trace amounts of C11:0 DMA (1.8%), C13:0 (1.3%), C14:1
ω7c DMA (0.85%), C15:0 anteiso (0.5%), C16:0 aldehyde (1.2%), C16:0 DMA (1.2%),
C18:1 ω9c (1.4%) C18:1 ω9c DMA (1.1%), C18:1 ω7c DMA (0.8%).
In whole genomes of strains ACB1T, ACB7T and ACB8 we found no predicted gene
sequences with recognizable homology to lipoquinones, mycolic acids or
lipopolysaccharides biosynthesis. Nine to thirteen RAST-annotated genes associated
with polyamines metabolism, and fifteen to nineteen genes associated with polar lipids
metabolism, were present in the genomes (Table 2).
The 16S rRNA gene-based phylogenetic tree showed that strains ACB1T, together with
ACB8, and ACB7T formed two separate branches within the genus Oribacterium (Fig.
2). The genus Oribacterium comprises a single named species O. sinus (Carlier et al.,
2004; Rainey, 2009). Other known strains (Table 2, 3, S1) were reported in GenBank
and HOMD (http://www.homd.org/index.php). Strains ACB1T and ACB8 were closely
related to each other with 99.7% of 16S rRNA gene sequence identity and shared 98.5-
98.6% homology with the type strain Oribacterium sinus AIP 354.02T (Carlier et al.,
2004) and 98.71-98.85% with O. sinus strain F0268 (Dewhirst et al., 2010). Strain
ACB7T shared 98.1% homology with the type strain and 98.7% with strain F0268 (Table
3). The values of 16S rRNA gene sequence similarities between strains ACB1T, ACB7T
and the type strain O. sinus AIP 354.02T were below the 98.7% - 98.65% cut-off values
proposed for species demarcation by Stackebrandt & Ebers (2006) and Kim et al.
(2014) respectively. The predicted value of DNA-DNA hybridization between strains
ACB1T and ACB8 was 98.2% (Table 3). Predicted DDH value between strains ACB1T
and ACB7T was 22.6% and between strains ACB8 and ACB7T was 23.1% - much less
than threshold of 70%, the widely accepted value of relatedness between different
species (Gevers et al., 2005; Tindall et al., 2010; Yarza et al., 2008). Predicted DDH
values suggested that strains ACB1T and ACB8 represent the same species, whereas
strain ACB7T belongs to a different species. Predicted values of DDH between strains
ACB1T, ACB8 and O. sinus F0268 and between strain ACB7T and O. sinus F0268 were
only 15.6% and 15.7%, respectively, clearly indicating three separate species (Table 3).
Pair-wise comparison of 16S rRNA gene sequence similarities with predicted DDH
values of six Oribacterium strains revealed that values less than 99.5% of 16S rRNA
gene sequence similarity corresponded to DDH values less then 22.3% (Table. 3). The
99.5% similarity value of 16S rRNA gene sequences between species within a genus is
higher than the reported range of 98.2 - 99.0% (Kim et al., 2014; Meier-Kolthoff et al.,
2013; Stackebrandt & Ebers, 2006; Yarza et al., 2008). However, there are genera like
Brucella, Burkholderia, Bacillus, Brevundimonas, Escherichia, Salmonella, Shigella, etc.
that contain separate species with 100% 16S rRNA gene sequence identity (Ash et al.,
1991; Fukushima et al., 2002; Gee et al., 2004; Gevers et al., 2005; Jaspers &
Tables 1 to 3 summarize physiological and genomic properties that can be used to
differentiate strains ACB1T, ACB7T, ACB8 from Oribacterium sinus and other members
of the genus. The type strain O. sinus (Carlier et al., 2004) was isolated from sinus pus
of a 6-year-old child with a bilateral maxillary sinusitis, while strains ACB1T, ACB7T and
ACB8 were enriched from a non-infectious subgingival plaque sampled from a generally
healthy 25-year-old adult. O. sinus cells were 0.8 - 1.0 µm wide compare to 0.45 - 0.5
μm wide cells of strains ACB1T, ACB8, and ACB7T. In contrast to O. sinus our three
strains did not produce indole. Strains ACB1T and ACB8 fermented lactose and maltose
while the type strain and strain ACB7T did not. O. sinus and strains ACB1T and ACB8
used glucose and raffinose as carbon sources, while strain ACB7T did not. The only
strain that hydrolyzed aesculin was strain ACB7T. Major metabolic end products of O.
sinus and strain ACB1T were acetate and lactate; strains ACB7T and ACB8 produced
FAME profile, spectrum of utilized carbon sources, DNA G+C content, metabolic end
products as well as the number of annotated genes responsible for biosynthesis of
teichoic and lipoteichoic acids, polar lipids, polyamines, DAP and lypooligosaccharides,
distinguish strains ACB1T, ACB8 and ACB7T from O. sinus.
On the basis of physiological, biochemical and molecular properties, we suggest that
strains described in this study represent two new species, for which we propose names
Oribacterium parvum, sp. nov. for strains ACB1T and ACB8 and Oribacterium
asaccharolyticum sp. nov for strain ACB7T.
Emended description of Oribacterium gen. nov. (Carlier et al., 2004).
Elongated ovoid rods, about 1.2–2.2 μm long and 0.45–1 μm wide, usually single, in
pairs or, occasionally, in short chains. Motile with laterally inserted flagella. Gram-
positive but may appear Gram-negative after staining. Strictly anaerobic. Does not form
spores. Weakly fermentative. Major metabolic end products are acetate and lactate or
acetate only. Major (>10%) fatty acids are C14:0, C16:0 and anteiso-C15:0 or C16:1
ω7c DMA. DNA G+C content is 42.1 – 43.3 mol%. Phylogenetically related to members
of the family ‘Lachnospiraceae’. The type species is Oribacterium sinus.
Description of Oribacterium parvum, sp. nov.
Oribacterium parvum (par’vum. L. neut. adj. parvum small, little ) Cells are Gram-
variable after staining but structurally Gram-positive, short motile ovoid rods, 1.2 × 0.45
μm wide, sometimes swollen, occurring single, in chains or aggregates. No spores are
formed. Strictly anaerobic. Colonies are round convex beige, 1.5 mm in diameter, non-
hemolytic on WC agar. Black pigment is produced in TY or TGY liquid medium
supplemented with L-cysteine-HCl. Yeast extract is required for growth on glucose,
lactose, maltose, maltodextrin and raffinose liquid media; gas is produced. Indole is not
produced. Gelatin is not liquefied. Aesculin is not hydrolyzed. Catalase, oxidase and
urease are negative. Nitrate is not reduced. The type strain is susceptible to kanamycin,
vancomycin, metronidazole, penicillin, rifampicin and bile and resistant to colistin. DNA
G + C content is 42.0 - 42.1 mol%. The major metabolic end products are acetate and
lactate. Growth temperature range is 30 - 42 oC. Major fatty acids are C14:0, C16:0,
C16:1 ω7c DMA and C18:1 ω7c DMA. The peptidoglycan type is A1γ. The type strain
ACB1T (=DSM 24637T, = HM-481T; =ATCC BAA-2638T) was isolated from the human
subgingival dental plaque. Habitat: human mouth.
Description of Oribacterium asaccharolyticum sp. nov.
Oribacterium asaccharolyticum (a.sac.cha.ro.ly′ti.cum. Gr. pref. a not; Gr. n. saccharon
sugar; N.L. neut. adj. lyticum able to lyse from Gr. adj. lytikos able to loose; N.L. neut.
adj. asaccharolyticum not digesting sugar).
Cells are Gram-variable after staining but structurally Gram-positive, motile rods 1.6 ×
0.5 μm, some cells up to 5 - 7 μm long, often swollen, occurring single, in pairs or
chains. No spores are formed. Strictly anaerobic. Colonies are non-hemolytic round non
pigmented, 0.5 mm in diameter after 48 h and umbonate 2-3 mm in diameter with
irregular wavy edges after 168 h. Growth is supported by yeast extract and Bacto
proteose peptone. Black pigment is produced in liquid medium supplemented with L-
cysteine-HCl; gas is not produced. The lowest concentrations of yeast extract and L-
cysteine-HCl required for visible growth is 1 g l-1 and 0.5 g l-1 respectively. In liquid
medium growth is not supported by glucose, maltose, lactose, cellobiose, maltodextrin,
raffinose, and starch; sucrose supports poor growth. Gelatin is not liquefied. Aesculin is
hydrolyzed. Catalase, oxidase and urease are negative. Nitrate is not reduced. The type
strain is susceptible to kanamycin, vancomycin, metronidazole, penicillin, rifampicin and
bile and resistant to colistin. DNA G + C content is 43.3 mol%. The major metabolic end
product is acetate. Growth temperature range is 30 - 42 oC. Major fatty acids are C12:0,
C14:0, C16:0, C16:1 ω7c and C16:1 ω7c DMA. The peptidoglycan type is A1γ. The
type strain ACB7T (=DSM 24638T, = HM-482T; =ATCC BAA-2639T) was isolated from
the human subgingival dental plaque. Habitat: human mouth.
This work was supported by NIH Grants 1RC1DE020707-01 and 3 R21 DE018026-
02S1 to SSE, 1U54 AI84844-01 to KEN, EF-1002148 to NP, EU-FP7 Marie Curie
International Fellowship PIOF-GA-2009-235470 to M.M., U54HG004969 to AME. We
thank Mr. M. Torralba for technical assistance.
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Fig.1. Transmission electron micrograph of Oribacterium parvum strain ACB1T (a) and
O. asaccharoliticum strain ACB7T (b). General morphology and gram-positive cell
structure of cell ultrathin section is shown. Bar 500 nm.
Fig. 2. Minimum-evolution phylogenetic tree based on 16S rRNA gene sequence
comparisons of Oribacterium parvum strains ACB1Tand ACB8, O. asaccharolyticum
strain ACB7T, and other members of the genus Oribacterium and the Lachnospiraceae
family. Bootstrap values > 50% calculated for 1000 subsets are shown at branch-points.
Bar, 0.02 substitutions per position.