Pathogenesis and toxins

In vitro growth characteristics and volatile sulfur compound

production of Solobacterium moorei
Abish S. Stephen
b,
*
, Declan P. Naughton
b
, Robert L. Pizzey
a
, David J. Bradshaw
a
,
Gary R. Burnett
a
a
GlaxoSmithKline Consumer Healthcare, St. Georges Avenue, Weybridge KT13 ODE, United Kingdom
b
Faculty of Science, Engineering and Computing, Kingston University, Penrhyn Road, Kingston-upon-Thames KT1 2EE, United Kingdom
a r t i c l e i n f o
Article history:
Received 8 November 2013
Received in revised form
13 January 2014
Accepted 15 January 2014
Available online 31 January 2014
Keywords:
Halitosis
Microbial ecology
Microbiology
Plaque
Volatile sulfur compounds
Hydrogen sulde
a b s t r a c t
Solobacterium moorei has recently been implicated as a causative agent of halitosis. In vitro experiments
to evaluate the role of S. moorei in halitosis have, however, been complicated by a paucity of information
on the ideal conditions for culturing this organism. This work aimed to optimize a liquid culture medium
for S. moorei, and to determine the growth-curve of the organism. Further, the ability of S. moorei to
generate volatile sulfur compounds was investigated and compared quantitatively to other oral anaer-
obes by an optimized head-space gas chromatography method. Serum-supplementation of standard
liquid growth media gave greater growth of S. moorei than non-supplemented broths, with the best
medium found to be serum-supplemented tryptone soya broth. S. moorei was able to metabolize cysteine
directly to hydrogen sulde, but was unable to produce methanethiol from methionine. S. moorei pro-
duced 2e3 times more hydrogen sulde (normalized for colony forming units) than Porphyromonas
gingivalis and Veillonella dispar, but considerably less than Fusobacterium nucleatum. The study has
identied reliable growth conditions for culture of S. moorei, which were employed to show that
S. moorei has the requisite biochemistry consistent with a potential role in halitosis.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Solobacterium moorei (formerly Bulleidia) is a Gram-positive,
non-sporulating, anaerobic bacillus that has been associated with
bacteremia [2,14], septicemia [5], wound-infection [22] and peri-
odontal disease [4,17]. In addition, studies have reported a high
degree of correlation between the presence of S. moorei on the
tongue dorsum and intra-oral halitosis with sample sizes of 13 [9]
and 57 [8] and the authors maintain the fact that other studies did
not show a strong correlation [10,21] was due to differences in
participant selection criteria, indicating a closer association of
S. moorei with severe halitosis. A recent study conducted on a larger
cohort of individuals with and without halitosis (n 193) also
detected a positive correlation between the presence of S. moorei in
the oral samples (tongue and saliva) and a variety of factors asso-
ciated with halitosis including organoleptic score, concentration of
hydrogen sulde in the breath and tongue coating [20].
Little is known of the extent to which S. moorei contributes to
generating the elevated hydrogen sulde and methanethiol con-
centrations observed in halitotic breath, partly due to poor in vitro
cultivability of this organismin standard broth media [22]. An early
study had observed black colonies of S. moorei grown on lead ac-
etate-supplemented agar, implying sulde production [8]. A recent
investigation [19] seeking to characterize the volatile sulfur com-
pound (VSC) production of S. moorei from a variety of complex
substrates was consistent with these initial observations and
conrmed the role of b-galactosidases in VSC production, as orig-
inally postulated [8]. However, neither study provided a clear
characterization of the specic sulfur-species produced by S. moorei
fromthe substrates tested, mainly due to the use of the non-specic
Halimeter as a sulde detector in the latter study [19]. Additionally,
the biochemically complex broth media with simple amino acid
supplements used to culture and study the VSC production of
S. moorei, whilst providing indirect evidence on the likely sulfur-
species being produced, did not establish a clear inference. This is
particularly relevant because the main VSCs involved in oral mal-
odor, namely hydrogen sulde and methanethiol are thought to
have widely different toxicological and physiological effects on
human oral tissue [23].
* Corresponding author. Research Centre for Clinical & Diagnostic Oral Sciences,
Blizard Building, Queen Marys School of Medicine & Dentistry, 4 Newark Street,
London E1 2AT, United Kingdom. Tel.: 44 (0) 20 7882 7157; fax: 44 (0) 20 7882
2191.
E-mail address: a.s.stephen@qmul.ac.uk (A.S. Stephen).
Contents lists available at ScienceDirect
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1075-9964/$ e see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anaerobe.2014.01.007
Anaerobe 26 (2014) 53e57
In the present study, a broth formulation that yielded rapid
growth of S. moorei, allowing bacterial harvest is described. This
broth culture was used to evaluate quantitatively the VSC-
production of S. moorei from cysteine and methionine, alone and
in combination, by headspace analysis using gas chromatography
(GC). The VSC production by S. moorei was compared with VSCs
generated by reference strains of the periodontal pathogen Por-
phyromonas gingivalis and other oral anaerobes Fusobacterium
nucleatum, Veillonella dispar and Prevotella melaninogenica that do
not showa clear association with halitosis, as reported for S. moorei
[8].
2. Methods
A stock culture of Solobacterium moorei (DSM 22971) was
maintained on Columbia blood agar (CBA; 5% v/v Horse blood;
bioMrieux, Basingstoke, UK) under anaerobic conditions (80% N
2
;
10% H
2
; 10% CO
2
) at 37

C, except when sub-culturing, which was
carried out aseptically at room temperature on the bench. Stocks of
F. nucleatum (NCTC 10562), P. gingivalis (ATCC 53978), P. melanino-
genica (NCTC 9336) and V. dispar (NCTC 11831) were also main-
tained as above.
2.1. Preparation and evaluation of broth media
Qualitative growth evaluation by colony morphology and light
microscopy was carried out for the following culture media: brain
heart infusion (BHI, Oxoid, Basingstoke, UK); tryptone soya broth
(TSB, Oxoid, Basingstoke, UK); fastidious anaerobe broth (FAB;
LabM, Bury, UK) at 3.7% w/v, 3.0% w/v and 3.0% w/v respectively,
with or without supplementation. The supplements were rabbit
serum (Sigma code: R7136) or horse plasma obtained by centri-
fuging debrinated horse blood (Oxoid; SR0050) at 2000 g for
10 min at 20

C. Base broth media were sterilized by autoclaving at
121

C for 20 min, and all supplements were then added at 5% v/v
aseptically after cooling to 45

C. All media were then pre-reduced
in an anaerobic atmosphere for at least 24 h before being inoculated
by swabbing approximately 20 S. moorei colonies aseptically from
72 h CBA stock cultures into each broth. Broths were monitored for
growth up to 10 days by spread-plating 100 mL broth samples on
pre-reduced CBA and incubating anaerobically.
2.2. Determination of growth curve
S. moorei colonies from 3 day CBA cultures were suspended in
0.1% w/v peptone water (0.85% salt; Oxoid) to OD
550
of 0.05 and
diluted 100-fold. 1 mL of this diluted suspension was used to
inoculate 200 mL of sterile TSB (3% w/v) supplemented with sterile
rabbit serum (5% v/v) on the bench, and incubated anaerobically at
37

C. The culture was monitored regularly by retrieving 1 mL
samples in the anaerobic environment before decimally diluting in
0.1% peptone water (0.85% salt). Viable counts were carried out
using a modication of the surface viable count method [13],
whereby duplicate 10 mL spots from an appropriate decimal dilu-
tion in 0.1% peptone water (0.85% salt; Oxoid) were streamed down
the agar plate in individual tracks (Fig. 4) to facilitate rapid drying
and prevent spreading or coalescing of subsequently growing col-
onies. The dilution that yielded the closest to 100 colonies was
counted.
2.3. Preparation of bacteria for headspace analysis
S. moorei was cultured in 200 mL of 3% w/v TSB (Oxoid) in
deionized water supplemented with 5% v/v rabbit serum (Sigma)
and was inoculated on the bench by swabbing (approx. 10
6
bacteria) from a three-day stock culture on CBA (bioMrieux)
before incubating anaerobically at 37

C. After 24 h, this culture was

centrifuged (2000 g, 20

C, 15 min) and the pellets washed twice in
phosphate buffered saline (PBS; pH 7.4). A dilution series of
S. moorei in PBS was prepared and a standard curve relating OD
550
and the number of viable bacteria was determined by track-plate
counting as described above. Anaerobic cultures of F. nucleatum, P.
gingivalis, P. melaninogenica and V. dispar in BHI broth (3.7% w/v)
were harvested after 48 h and bacterial suspensions in PBS were
prepared as described for S. moorei.
Bacterial suspensions (0.5 mL) of varying OD
550
were added to
10 mL glass headspace vials, followed by 0.5 mL of substrate, after
which the vials were immediately sealed under aerobic conditions.
Substrate stock solutions were either 0.5% w/v L-cysteine, 0.5% w/v
L-methionine or 0.5% w/v L-cysteine 0.5% w/v L-methionine for
S. moorei and 0.5% w/v L-cysteine for other bacterial suspensions,
prepared in deionized water. Vials were prepared in triplicate for
each bacterial suspension, and were incubated at 37

C for 1 h with
moderate shaking before adding 1 mL of absolute ethanol to pre-
vent further VSC development. The experiment was controlled by
incubating the bacterial suspension or the substrate solutions alone
in triplicate vials.
2.4. Gas chromatographic method
The gas chromatograph (Agilent 6890N), equipped with a
sulfur-specic ame photometric detector was calibrated with
standards generated from permeation tubes for H
2
S and meth-
anethiol by a gas standard generator (Kin-tek 491M). The head-
space of each vial was sampled manually using a gas-tight syringe
after thermally stabilizing the vials for 3 min at 80

C, and 250 mL of
each headspace sample was introduced into a Chromosil-330
packed column via a sulnert-treated sampling valve. The column
was maintained at 60

C throughout, with constant ow of helium
carrier gas at 45 mL/min.
3. Results
Growth was observed in all the broths supplemented with
rabbit serum or horse plasma by visual observation of turbidity
after 18 h incubation, with serum- and plasma-supplemented TSB
and BHI broth showing better growth than equivalent supple-
mented FAB medium, up to 48 h. Growth was conrmed by Gram-
staining and spread-plating of undiluted broth samples. All non-
supplemented broths showed poor growth in terms of visible
turbidity observed up to 48 h compared to their serum- and
plasma-supplemented counterparts, with plating on CBA con-
rming little change to the number of colonies observed. However,
there were slight differences in the numbers of colonies observed
from spread-plating non-supplemented broth culture samples at
24 h, which ranked as TSB > BHI > FAB, although all showed
negligible increases in the number of colonies observed up to 10
days, with the same trend still evident after 10 days incubation.
Using a minimalist approach, serum-supplemented broths were
chosen above plasma-supplemented broths, as there was no
observed difference in the number of colonies, following spread-
plating. The plasma used in this work was obtained from de-
brinated horse blood, and therefore the differences between this
plasma and the serum would be limited to the presence of coagu-
lationfactors not removedby the debrinationprocess. Bothserum-
and plasma-supplemented FAB media were then eliminated as
options, owing to a lower observable number of colonies, compared
to equivalent BHI or TSB media. A pilot growth experiment using
serum-supplemented BHI and TSB, showed TSB to have marginally
better growth in the log-phase than BHI (8.5 vs 8.0 log
10
CFU/mL
A.S. Stephen et al. / Anaerobe 26 (2014) 53e57 54
after 24 h). Although BHI supplemented with serum could be used
for culturing S. moorei, TSB is biochemically simpler, while showing
similar levels of growth to BHI, both in visible turbidity and CFU/mL.
Thus, serum-supplemented TSB was chosen as the standard culture
medium for further experiments with S. moorei.
Determination of the growth curve of S. moorei in rabbit serum-
supplemented TSB revealed a classical growth response with
readily recognizable growth phases (Fig. 1). This included a short
lag-phase up to 4 h then a log-phase achieving steady state at 24 h.
The steady state or the stationary phase was held till 36 h culmi-
nating in a decline or death-phase where a sharp decrease in the
number of colonies was observed. The best harvest of this culture
was obtained at 24 h, even showing good recovery of bacteria after
aerobically processing the culture for harvest.
The H
2
S concentration in the headspace of S. moorei suspensions
incubated with cysteine solution showed a doseeresponse with OD
(and thus with numbers of viable bacteria) (Fig. 2); no VSCs were
detected in the headspace of S. moorei suspensions incubated with
methionine solution. When cysteine and methionine were incu-
bated together with S. moorei, methanethiol concentrations
detected in the headspace of the vials were not different to control
vials containing the substrate solutions without S. moorei suspen-
sions. However, H
2
S concentrations observed were comparable to
H
2
S detected when S. moorei was incubated with cysteine on its
own (data not shown). Comparison of S. moorei with data obtained
for F. nucleatum, P. gingivalis, P. melaninogenica and V. dispar
revealed that equivalent colony forming units (CFU) of S. moorei in
PBS suspensions produced 2- and 3-fold higher H
2
S than V. dispar or
P. gingivalis, respectively. However, the F. nucleatum and
P. melaninogenica suspensions produced about 20 and 5-fold higher
amounts of detectable H
2
S from cysteine, respectively, than
S. moorei in aerobic conditions (Fig. 3).
4. Discussion
The observed growth of S. moorei in both horse plasma- and
rabbit serum-supplemented broth media reects its growth char-
acteristics on blood agar and its clinical association with wound
infection and bacteremia [12,14,22]. S. moorei colonies grown
anaerobically on CBA did not show any visible hemolysis up to 7-
days indicating that red blood cells per se are not a requirement
for the growth of S. moorei. This in turn, suggests that when
S. moorei is observed in bacteremia, wound infection or in
periodontal disease [17], it might not lyse erythrocytes and it is
likely that it utilizes serum proteins as an energy source. These may
be factors common to mammalian serum as no obvious preference
was shown to either rabbit- or horse-derived supplements in this
study. However, while the serum used to cultivate S. moorei in this
study contained appreciable amounts of hemoglobin, and in a
previous study [19], a broth formulation with hemin, yeast extract
and Tween-80 was used to grow this bacterium, our attempts to
culture this bacterium with just hemin and yeast extract supple-
mented TSB were unsuccessful, pointing to the possibility of a
factor analogous to Tween-80, rather than hemin or hemoglobin
present in serum stimulating the growth of S. moorei.
These observations may provide clues to the role of S. moorei in
the oral cavity of individuals with halitosis. It is possible that
S. moorei utilizes similar substrates in the oral cavity particularly via
gingival-crevicular uid, which is closely related to serum [11], and
this may also relate to S. moorei association with bacteremia and
wound infections. Also, the reported association of S. moorei with
fusobacteria in both oral and non-oral pathologies is notable, as it
suggests probable bacterial species with which S. moorei could be in
close association in the oral biolm of individuals with halitosis
[18,22]. S. moorei may help modulate VSC production of the oral
biolm in association with fusobacteria, as in planktonic cultures,
fusobacteria are among the most prolic VSC producing species
found in the oral biolm as suggested by data in the present study
(Fig. 3) and others [16,3].
The detection of H
2
S in a doseeresponse to OD in the headspace
of S. moorei suspensions incubated with cysteine (Fig. 2) corrobo-
rates previous observations [19,8]. However, owing to the
biochemical complexity of the broth media used in the former
study [19], it is unclear if the doseeresponse observed for cysteine
(and the lack of dose response for methionine) is a consequence of
the relative sensitivities of the Halimeter to hydrogen sulde and
methanthiol [7]. In addition, the non-specic nature of Halimeter
precludes denitive identication of the measured sulde as H
2
S.
We believe the present study is however, the rst demonstration
that conrms H
2
S as the sulfur-species produced by S. moorei from
cysteine.
No methanethiol was detected in the headspace of S. moorei
suspensions incubated with methionine either alone or in the
presence of cysteine, suggesting the absence of methionine
Fig. 1. Growth of S. moorei in 3% w/v TSB supplemented with 5% v/v rabbit serum.
Error bars represent standard errors from duplicate runs.
Fig. 2. Graph showing hydrogen sulde production of S. moorei with cysteine substrate
under aerobic conditions as a function of bacterial concentration. Error bars are
standard errors from triplicate vials at each absorbance.
A.S. Stephen et al. / Anaerobe 26 (2014) 53e57 55
degradation. The H
2
S concentrations observed in control vials were
comparable to incubation with cysteine on its own, suggesting that
methionine does not have an effect on cysteine degradation by this
bacterium.
Comparison of H
2
S production by S. moorei with other oral an-
aerobes revealed that under aerobic conditions equivalent CFU of
S. moorei can produce about 2 and 3-fold higher amounts of H
2
S
than the periodontal pathogen P. gingivalis or the common oral
anaerobe V. dispar, respectively. This may be because S. moorei is
able to tolerate aerobic conditions better than P. gingivalis or
V. dispar. Equivalent numbers of F. nucleatum and P. melaninogenica
produced considerably higher amounts of H
2
S in aerobic headspace
conditions suggesting that these organisms may have better
tolerance to aerobic conditions. Although there have been studies
measuring the VSC production capacities of the organisms tested in
this study, they involved incubating the organisms in anaerobic
conditions [15,16]. The viable counts (data not shown) performed
for the comparison of the different bacteria after aerobic processing
of cultures indicated that there are sufcient numbers of organisms
of each species to utilize the free-cysteine substrate. Anaerobic
species such as F. nucleatum and P. melaninogenica are commonly
reported to be present in a variety of oral niches including the
tongue and gingiva of orally healthy individuals [1,6,9]. The pro-
duction of VSCs in aerobic conditions may help in lowering the
redox potential of the microenvironment in which organisms less
tolerant of aerobic conditions can survive. A possible implication of
this hypothesis would be the involvement of non-pathogenic bac-
terial species present in the oral microbiota in disease processes
indirectly, by increasing the survival rates of potentially pathogenic
anaerobic oral bacteria in otherwise unfavorable conditions.
In conclusion, we cultured and determined the VSC producing
capability of the clinically-relevant, Gram-positive bacterium, S.
moorei, nding that it can produce H
2
S from L-cysteine but was
unable to degrade L-methionine. However, S. moorei was found to
be a moderate producer of H
2
S compared with other oral microbes
such as F. nucleatum and P. melaninogenica in the conditions of the
experiment. Quantitative data on the relative numbers of the
various VSC-producing species present in the different oral niches
of individuals with halitosis may help in assessing if S. moorei is
present in the oral cavity in sufcient numbers to contribute
signicantly to the malodorous breath of individuals with halitosis.
Conict of interest statement
The authors declare no conict of interest.
Acknowledgments
This work was fully nanced by GlaxoSmithKline.
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