International Journal of Oral & Maxillofacial Pathology.

2014;5(1):21-26 ISSN 2231 – 2250

Available online at http://www.journalgateway.com or www.ijomp.org

Original Research

The Anaerobic Microflora: The Accused in Oral Malodour?

Nandita Shenoy, Junaid Ahmed, Arjun Kumar Tallada, Vidya Pai, Almas Binnal
Abstract
Background: Oral malodour is foul-smelling breath from the oral cavity and is due to metabolic
products of bacteria in the oral cavity but can also be caused by many other systemic diseases.
Oral malodour is believed to originate from foul-smelling gases, such as hydrogen sulfide
produced by oral bacteria. Aim: To identify hydrogen sulfide producing bacteria from tongue
biofilm and to investigate the relationship between bacterial flora and hydrogen sulphide levels in
the oral cavity. Materials and Methods: Oral malodour levels in 26 subjects (aged 16-69 years)
were assessed by portable volatile sulfide monitor Halimeter and gas chromatography. They were
accordingly divided into two groups: an odour group and a no/low odour group. Tongue coatings
were sampled and organisms were categorized. Results: Tongue coatings were spread onto
sheep blood agar (anaerobic) containing Casein enzymic hydrolase, Papaic digest of soyabean
meal, yeast extract, sodium chloride, L-cysteine, Hemin, and Agar at final pH (at 250C) 7.4±0.2.
The plates were incubated at 37 0C for 48-72hrs aerobically and anaerobically in anaerobic
chamber with mixed gas supply. Bacteria forming black or grey colonies were selected as
hydrogen sulphide-producing phenotypes. The numbers of total bacteria (P < 0.05) and hydrogen
sulphide-producing bacteria (P < 0.05) in the odour group were significantly larger than those in
the no/low odour group. Species of Veillonella, Prevotella, Bacteriods and Peptostreptococcus
were the predominant hydrogen sulphide-producing bacteria in odour group and low in no/low
odour group. Conclusion: These results suggest that an increase in the number of hydrogen
sulphide-producing bacteria in the tongue biofilm is responsible for oral malodour, although the
bacterial composition of tongue biofilm did not differ between the two groups.

Keywords: Oral malodour; Halimeter; Hydrogen sulphide; Oral malodor measurement; Volatile
sulphur compounds; Tongue coating; Tongue biofilm; Microorganisms.

Nandita Shenoy, Junaid Ahmed, Arjun Kumar Tallada, Vidya Pai, Almas Binnal. The Anaerobic Microflora:
The Accused in Oral Malodour? International Journal of Oral & Maxillofacial Pathology; 2014:5(1):21-26.
©International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta
Software Private Limited. All Rights Reserved.

Introduction offensive breath.8 The quantities of these

Oral malodour is an unpleasant or offensive molecules in the breath can be measured at
odour emanating from the breath. Even chair side with a portable sulphide monitor.9
though majority of oral malodour is of oral The organoleptic assessment is a subjective
origin, there are multiple other systemic measurement. It is a very good qualitative
causes that have to be addressed while we method, however not very precise
diagnose and treat this condition. 1-3 Halitosis concerning to quantity. Invitro studies have
has a multifactorial aetiology, but its main demonstrated that gram-negative anaerobic
cause is the decomposition of the organic bacteria are capable of producing volatile
material by microorganisms of the oral sulfur compounds from blood products. In
cavity. The principal components of oral particular Treponema denticola,
malodour are volatile sulphur compounds, Porphyromonas gingivalis, Prevotella
which are primarily hydrogen sulphide (H2S), intermedia, Bacteroides forsythus and
methyl mercaptan (CH3SH) and Dimethyl Fusobacterium can produce significant
sulphide, produced through the putrefaction amounts of hydrogen sulfide and
of proteins containing methionine or cysteine methylmercaptan from serum proteins,
by oral anaerobic gram-negative cysteine and methionine.9
microorganisms.4-6
Recent studies have indicated that the
Proteolytic activity in the mouth is an dorsal surface of the tongue may be the
important factor in the development of oral primary source of microbial putrefaction in
malodour.7 Putrefaction of proteins, mucins the mouth.10 Tongue coating is an important
and peptides by microorganisms that reside factor in the formation of oral malodour in
on the tongue and in dental plaque results in both periodontally diseased and healthy
the formation of volatile sulphur compounds, people. However, it is not known which
which are thought to be responsible for bacterial species in the tongue coating are

©2014 International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta Software Private Limited. All Rights Reserved
22 Junaid Ahmed, et al.
responsible for this odor production. It is
possible that malodourous species
colonizing the dorsal surface of the tongue
are the same as those found in subgingival
plaques. Indeed, studies suggest that the
flora on the tongue is similar to odor-
producing periodontal bacteria.11 This study
is designed to identify the hydrogen sulphide
producing bacteriae from tongue biofilm and
to determine the number and type of
hydrogen sulphide producing bacteria.
Materials and Methods
Twenty six subjects complaining of halitosis,
was selected for this study. Informed
consent was obtained from each subject.
Patients with any systemic cause of halitosis
were excluded. All patients confirmed that
they were not suffering from any disease
and did not receive medical treatment
(especially no antibiotics and/or
corticosteroids) within three months before
measurements. Patients with signs of
pharyngitis or acute/chronic tonsillitis were
also excluded. All patients received a letter
with instructions before the examinations.
Two days before their appointment, they had
to avoid the intake of garlic, onions and
spicy food. Twelve hours before the
measurements, they also had to refrain from
alcohol or coffee, and from smoking. On the
morning of the appointment, it was forbidden
to use chewing gums, mints, drops, scents
and mouth rinses. On the other hand, they
could perform normal oral hygiene (tooth
brushing) and have breakfast. All
measurements were recorded between 8:30
and 11:30 hours (before lunch) and at least
two hours after eating or drinking and tooth
brushing. On the first visit, an assessment of
oral malodour and observable tongue
coating, a clinical oral examination and
sampling of tongue biofilm, was performed
as described below.
Odor Assessment: We made quantitative
measurements of volatile sulfur compounds
using a portable sulfide monitor set at 1 part
per million full scale (Interscan Model RH17-
B Halimeter, Interscan Corporation).7,8 We
set the monitor to zero on ambient air before
each measurement. The test consists on
asking the patient to breathe deeply inspiring
the air by nostrils and expiring by mouth,
while the examiner sniffs the odour at a
distance of 20 cm, considering it unpleasant
or not in a scale of 0 to 5. A teflon tube
connected to a flexible drinking straw was
attached to the air inlet of the monitor. For
ISSN 2231 - 2250
each measurement, the straw was inserted
three centimetres into the mouth until the
lips touched a stent attached to the straw.
The participant lightly closed his or her lips
over the straw and the monitor collected a
sample of the ambient air from the mouth.
We took three separate readings of peak
volatile sulphur compound levels, instructing
the patient to close his/her mouth for 30-60
seconds before inserting the straw. We
calculated a mean value for all the readings
and used this value in the statistical
analysis. Based on the halimeter reading we
categorised them in to two groups, the odour
group (n=14) and the non-odour group
(n=12). Subjects with low halimeter reading
less than 100 ppb were considered as non-
odour group/control group. Subjects with
halimeter reading more than 300 ppb were
considered as odour group/study group.
The oral cavity was examined, paying
attention to caries, the level of oral hygiene
(plaque accumulation, gingival
inflammation), periodontal pockets (using a
manual periodontal probe), removable
appliances, dry mouth and tongue coating.
Thickness and extent of tongue coating was
estimated by the naked eye according to the
method of Nara.7 Both thickness and extent
of tongue coating were scored as 0, 1, 2 or
3, and then the thickness score and the
extent score were multiplied.
Sampling of Tongue Biofilm: In order to
collect tongue biofilm, an area of 1 cm2,
predetermined by a window made of
sterilized plain paper on the rear dorsal
surface of the tongue, was firmly scraped
with sterilized toothpicks. All samples were
immediately introduced into an anaerobic
chamber suspended in 1 ml of distilled 40
mm potassium phosphate buffer (PPB, pH
7.0) solution.
Culture Conditions: Specimen collected in
RCM broth was incubated at 37ºC for 48
hours. Subcultures were done on sheep
blood agar (anaerobic) containing Casein
enzymic hydrolase (15gms/litre), Papaic
digest of soyabean meal (5gms/litre), yeast
extract (5gms/litre), sodium chloride
(5gms/litre), L-cysteine (0.5gms/litre), Hemin
(0.005gms/litre), Agar (13.5gms/litre), final
pH (at 250C) 7.4±0.2. The plates were
incubated at 370C for 48-72hrs aerobically
and anaerobically in anaerobic chamber with
mixed gas supply (hydrogen and nitrogen-
Coy laboratories) (Figure 6 and 7). The
plates for anaerobic incubation also had a
ISSN 2231 – 2250
disc of metronidazole applied in the centre of
primary streak. Various biochemical
reactions and microbiological tests that are
used for identification of microorganism are
given in table 1.
Statistical Analysis: The data was coded
and analysed using SPSS version 11.5. The
level of statistical significance was kept at p
value of 0.05. An unpaired t-test was used
for analysis significance.
1. Colony Morphology Count
2. Gram Staining
3. Catalase Test
4. Pigment Production
5. Sensitivity To Metronidazole
6. Blackening of Lead Acetate Strips
Table 1: Biochemical reactions and
Microbiological tests that are used for
identification of microorganisms
Results
Our study subjects were within the age
range of 16 to 69 years with mean being 31
years. The 14 individuals were included in
odour group of which 10 (71.4%) were
males and 4 (28.6%) were females. Twelve
individuals were included in non-odour group
of which three (25%) were males and nine
(75%) were females. (Figure 1)
Bacteria forming black or grey colonies were
selected as H2S-producing phenotypes. The
numbers of total bacteria (P < 0.05) and
H2S-producing bacteria (P < 0.05) in the
odour group were significantly larger than
those in the no/low odour group. Species of
Veillonella, Prevotella, Bacteriods, and
Peptostreptococcus were the predominant
H2S-producing bacteria in odour group and
microorganisms like streptococcus mutans
and E.coli were low in odour group. (Figure
2) In addition, the number of black or grey
colonies in the odour group was significantly
higher than that in the no/ low odour group
(P < 0.05) The predominant microorganisms
that were cultured in the non-odour group
were streptococcus mitis, streptococcus
mutans, and staphylococcus aureus and
these microorganisms were significantly
higher in odour group when compared with
the odour group.(p<0.05) (Figure 3)
Tongue biofilm samples were obtained from
the same part of the tongue using a
standardized method, and there was
significant differences were noted in
observable tongue coating.(p< 0.05) (Figure
The Anaerobic Microflora: The Accused in... 23
4) In the odour group (n=14) tongue coating
was present in 13 (93%) patients and in the
non-odour group tongue coating was
present in 2 (13%) of the patients. This
indicates that the amounts of observable
tongue coating were higher among the
subjects in the odour group when compared
with non-odour group. On correlation with
the tongue coating and the microorganisms,
the predominant microorganisms that were
present in the individuals with high tongue
coating are Peptostreptococcus, Prevotella,
Velionella, Bacteriods.(Figure 5)
Discussion
The present study was conducted with the
aim to identify hydrogen sulphide (H2S)-
producing bacteria among tongue biofilm
microflora and to investigate the relationship
between bacterial flora and H2S levels in the
oral cavity. Oral malodour levels in 26
subjects were assessed by halimeter and
organoleptic scores. Based on these
assessments, subjects were divided into two
groups: an odour group and a no/low odour
group. Age and gender matching was done
between the study group and the control
group. In terms of clinical parameters, there
were no significant differences in number of
present teeth, number of teeth with
untreated caries, number of teeth with
probing depth more than 4 mm or oral
hygiene status between the two groups.
(Figure 1) There was a significant difference
in tongue coating score between the two
groups and individuals with coated tongues
had higher malodour scores than individuals
with non-coated tongues. In the present
study fourteen individuals were in the odour
group and tongue coating was present in 13
(93%) patients and in the non-odour group
tongue coating was present in 2 (13%) of the
patients. Similar results were observed in a
study done by Mager et al., (2003) 13 states
that Veillonella species was one of the
prominent bacteria in the tongue biofilm.
Identification of H2S-producing Bacteria
in Tongue Biofilm:
The H2S -producing bacteria isolated in this
study were identified using molecular
biological methods. Peptostreptococcus
micros, Veillonella parvula and Prevotella
intermedia species were the predominant
H2S-producing bacteria, followed by
Streptococcus sangius species, in the odour
and no/low odour groups (Figure 2, 3).
Veillonella is par accounted for over 15% of
total (H2S)-producing bacteria in each
sample. However, there were no significant
24 Junaid Ahmed, et al. ISSN 2231 - 2250

differences in the profiles of H2S -producing

bacteria between the two groups. Tyrell et 20
al., (2003)12 in his study also found that the 10
most common microorganisms isolated were Present
0
Peptostreptococcus, Eubacterium group,
Actinomyces species, Eikenella corrodens, Odour Non Absent
Veillonella species, Pigmented Prevotella odour
species. Eh De Boever et al., (1995)8 in their Tongue coating
study stated that the two known volatile-
sulfur compound producing organisms were Figure 4: The correlation between the
Fusobacterium species and Prevotella tongue coating and malodour scores.
intermedia.

Tongue coating
15
4
10
9 Streptococcus…
5 10
0 3 Staphylococcus…
Odour Non odour
group(14) group(12) veloinella

MALE FEMALE Peptostreptococcus

0 5 10
Figure 1: Gender variation among the study
and control group. Figure 5: The correlation between the
tongue coating and microbial load.

Streptococcus mitis 1
E.coli 2
Streptococcus… 2
Staphylococcus… 4
Bacteriods 6
Vellionella 8
Prevotella 10
peptostreptococcus 11

0 5 10 15

Figure 2: The different microorganisms Figure 6: The culture media used in the
involved in the study group. study.

Low Halimeter reading(less than

100ppb)

Peptostreptococcus

Staphylococcus…

Streptococcus…
0 5 10

Figure 3: The different microorganisms

Figure 7: The anaerobic chamber used for
involved in the control group.
culture.
ISSN 2231 – 2250
Hartley et al., (1996)15 also frequently
identified these bacterial species in both
odour and no/low odour groups and
Donaldson et al., (2005)16 reported that
Veillonella, Prevotella and Fusobacterium
species were found in both odour and no/
low odour groups, and that Vibrio species
and unidentifiable Gram-negative and Gram-
positive anaerobes were more commonly
found in the odour group. Loesche and
Kazor (2002)17 reported that 74% of total
cultivable bacteria of the tongue biofilm
could be Veillonella parvula, Actinomyces
odontolyticus, Streptococcus intermedius
and Clostridium. However, in all these
studies, the H2S-productivity of the bacteria
was not assessed. Thus, our study is the
first report to show that Veillonella,
Bactreiods and Prevotella are predominant
as H2S-producing bacteria in tongue biofilm
and are responsible for oral malodour when
they increase in number.
Tongue Coating and Oral Malodour
In the present study, Out of 26 individuals
who are complaining of halitosis, 48% (12
individuals) were because of tongue coating.
Quirynen et al., (1998)14 in their study
reported that individuals with coated tongue
showed significantly higher malodour scores
than individuals with non-coated tongue.
Microbial putrefaction on the tongue is an
important factor responsible for oral
malodour. Few studies reported correlations
between the degree of oral malodour and
the amount of observable tongue coating.
They also suggested that periodontal
disease can induce observable tongue
coating accumulation. The tongue biofilm
comprises not only micro-organisms but also
epithelial cells released from the oral
mucosa and leukocytes from periodontal
pockets. This indicates that the amounts of
observable tongue coating bear little
relationship to the microbial population
density on the tongue coating and it is only
the latter (microbial density) that relates to
hydrogen sulfide levels or oral malodour.
De Boever and Loesche (1998)8 in their
study observed tongue with deep fissures to
have higher number of bacterial colony as
compared to patients with smooth tongue
surfaces. Delanghe et al., (1998)18 found
that in 87% of the patients with oral
malodour, the cause was of oral nature. Of
these oral causes, 51% were because of
tongue coating, 17% a result of gingivitis,
15% a result of periodontitis and 17% a
result of combinations. In a study by Oho et
The Anaerobic Microflora: The Accused in... 25
al., (2001)19 it was stated that the amount of
tongue coating in patients complaining of
halitosis was significantly greater in the
halitosis-positive group compared to the
halitosis-negative group. Morita and Wang
(2001)20 investigated the relationship
between sulcular sulphide level and oral
malodour in subjects with periodontal
disease. The volume of tongue coating and
the percentile of sites with bleeding upon
probing were significantly associated with
oral malodour.
Conclusion
Microbial putrefaction on the tongue is an
important factor in the development of bad
breath. Volatile sulfur compounds, tongue
coating and deep fissures in the tongue are
all associated with oral malodour and,
therefore, appear to be important factors in
the halitosis process. The predominant H2S-
producing bacteria are mainly commensal
species of the oral cavity, such as Prevotella
intermedia, Veillonella and Actinomyces
species. The numbers of both H2S-
producing bacteria and total bacteria in the
tongue biofilm were higher in the odour
group, suggesting that for subjects with low
to intermediate levels of malodour an
increase in bacterial density in the tongue
biofilm is associated with oral malodour.
Author Affiliations
1.Dr.Nandita Shenoy, Associate Professor,
2.Dr.Junaid Ahmed, Professor and Head,
3.Dr.Arjun kumar Tallada, Post Graduate,
Department of Oral Medicine and Radiology,
Manipal College of Dental Sciences, Affiliated to
Manipal University, 4.Dr.Vidya Pai, Professor,
Department of Microbiology, Yenepoya Medical
College, Affiliated to Yenepoya University,
5.Dr.Almas Binnal, Assistant Professor,
Department of Oral Medicine and Radiology,
Manipal College of Dental sciences, Mangalore,
Affiliated to Manipal University, India.
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Source of Support: Nil, Conflict of Interest: None Declared.
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Corresponding Author
Dr. Junaid Ahmed,
Professor and Head,
Department of Oral Medicine and Radiology,
Manipal College of Dental Sciences,
Manipal University, Mangalore, India.
Email: junaid.ahmed@manipal.edu
Ph: +91 9901470120