NOTE / NOTE
In vitro evaluation of the effect of nicotine,
cotinine, and caffeine on oral microorganisms
Karina Cogo, Michelle Franz Montan, Cristiane de Ca ´ssia Bergamaschi,
Eduardo D. Andrade, Pedro Luiz Rosalen, and Francisco Carlos Groppo
Abstract: The aim of this in vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of
some oral bacterial species. It also evaluated the ability of these bacteria to metabolize those substances. Single-species
biofilms of Streptococcus gordonii, Porphyromonas gingivalis, or Fusobacterium nucleatum and dual-species biofilms of
S. gordonii – F. nucleatum and F. nucleatum – P. gingivalis were grown on hydroxyapatite discs. Seven species were
studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces
naeslundii, and the species mentioned above. The viability of planktonic cells and biofilms was analyzed by susceptibility
tests and time-kill assays, respectively, against different concentrations of nicotine, cotinine, and caffeine. High-
performance liquid chromatography was performed to quantify nicotine, cotinine, and caffeine concentrations in the culture
media after the assays. Susceptibility tests and viability assays showed that nicotine, cotinine, and caffeine cannot reduce
or stimulate bacterial growth. High-performance liquid chromatography results showed that nicotine, cotinine, and caffeine
concentrations were not altered after bacteria exposure. These findings indicate that nicotine, cotinine, and caffeine, in the
concentrations used, cannot affect significantly the growth of these oral bacterial strains. Moreover, these species do not
seem to metabolize these substances.
Key words: biofilm, nicotine, cotinine, caffeine, bacterial growth.
Re ´sume ´ : Le but de cette e ´tude in vitro e ´tait d’e ´valuer les effets de la nicotine, de la cotinine et de la cafe ´ine sur la viabi-
lite ´ de quelques espe `ces de bacte ´ries orales. Nous avons aussi e ´value ´ la capacite ´ de ces bacte ´ries de me ´taboliser de ces
substances. Des biofilms monospe ´cifiques de Streptococcus gordonii, Porphyromonas gingivalis, et de Fusobacterium nu-
cleatum et des biofilms bispe ´cifiques de S. gordonii – F. nucleatum, et de F. nucleatum – P. gingivalis ont e ´te ´ cultive ´s sur
des disques d’hydroxyapatite. Sept espe `ces ont e ´te ´ e ´tudie ´es en cellules planctoniques, soit Streptococcus oralis, Streptococ-
cus mitis, Propionibacterium acnes, Actinomyces naeslundii et les espe `ces identifie ´es plus haut. La viabilite ´ des cellules
planctoniques et des biofilms a e ´te ´ analyse ´e par des tests de susceptibilite ´ et des essais cine ´tiques de le ´thalite ´, respective-
ment, en re ´ponse a ` diffe ´rentes concentrations de nicotine, de cotinine et de cafe ´ine. Une chromatographie liquide a ` haute
performance (HPLC) a e ´te ´ re ´alise ´e pour quantifier les concentrations de nicotine, de cotinine et dle cafe ´ine dans le milieu
de culture apre `s les tests. Les essais de susceptibilite ´ et de viabilite ´ ont de ´montre ´ que la nicotine, la cotinine et la cafe ´ine
ne pouvaient re ´duire ou stimuler la croissance bacte ´rienne. Les re ´sultats obtenus en HPLC ont de ´montre ´ que les concentra-
tions de nicotine, de cotinine et de cafe ´ine n’e ´taient pas affecte ´es par l’exposition aux bacte ´ries. Ces donne ´es indiquent
que la nicotine, la cotinine et la cafe ´ine, aux concentrations utilise ´es, ne peuvent affecter significativement la croissance
de ces souches de bacte ´ries orales. De plus, ces espe `ces ne semblent pas me ´taboliser ces substances.
Mots-cle ´s : biofilm, nicotine, cotinine, cafe ´ine, croissance bacte ´rienne.
[Traduit par la Re ´daction]
Subgingival accumulation of bacterial biofilm has been
considered an etiologic agent of periodontal diseases. Patho-
genic biofilm is known to produce cytotoxic substances re-
sulting in gingival inflammation (Socransky and Haffajee
Some bacterial strains are important colonizers of the den-
tal biofilm. Early colonizers of biofilm — Actinomyces nae-
slundii and members of the mitis group of streptococci
(Streptococcus oralis, Streptococcus gordonii, and Strepto-
coccus mitis) — promote further bacterial colonization and
support biofilm structure (Mager et al. 2003; Socransky and
Received 22 February 2008. Accepted 7 April 2008. Published
on the NRC Research Press Web site at cjm.nrc.ca on 28 May
K. Cogo,1M. Franz Montan, C.C. Bergamaschi,
E.D. Andrade, P.L. Rosalen, and F.C. Groppo. Department of
Physiological Sciences, Area of Pharmacology, Anesthesiology
and Therapeutics, Dentistry School of Piracicaba, State
University of Campinas (UNICAMP), Avenida Limeira, 901,
Piracicaba, Sao Paulo 13414-903, Brazil.
1Corresponding author (e-mail: email@example.com).
Can. J. Microbiol. 54: 501–508 (2008)doi:10.1139/W08-032
##2008 NRC Canada
Haffajee 2005). Fusobacterium nucleatum is the most preva-
lent of the gram-negative species in the biofilm at later
stages (Moore and Moore 1994) and has been considered a
possible pathogen in periodontal diseases (Socransky and
Haffajee 2002). Porphyromonas gingivalis has been well
recognized as a periodontopathogen, and it was reported to
be more prevalent in diseased sites of subjects with perio-
dontitis than in healthy sites of diseased subjects (Riviere et
al. 1996). Another species that colonizes subgingival biofilm
of healthy and periodontally diseased patients is Propioni-
bacterium acnes (Socransky and Haffajee 2002).
The use of tobacco is recognized as one of the most im-
portant risk factors responsible for the development and pro-
gression of periodontal diseases as well as for a further
reduction in the response to the periodontal therapy
(Johnson and Hill 2004).
The relationship between cigarette smoking and the sub-
gingival microbiota is not clear. Some studies have reported
no difference in the prevalence of subgingival species of
microorganisms between smokers and nonsmokers with
periodontitis (Bostrom et al. 2001; Van der Velden et al.
2003; Apatzidou et al. 2005; Salvi et al. 2005). However,
some authors showed that smoking increases the likelihood
of prevalence and proportions of certain periodontal patho-
gens (Zambon et al. 1996; Haffajee and Socransky 2001;
van Winkelhoff et al. 2001).
Tobacco smoke contains more than 4000 substances. Nic-
otine, which is one of the most important tobacco substan-
ces, has a short blood half-life (±2 h). Cotinine is the main
nicotine metabolite and has a longer blood half-life (±19 h)
Cigarette smoking is strongly associated with coffee
drinking. Epidemiological studies have shown that 86.4% of
smokers versus 77.2% of nonsmokers consume coffee,
which is rich in caffeine. Former smokers drink more coffee
than people who have never smoked but somewhat less than
smokers (Swanson et al. 1994).
Although nicotine and cotinine are known to have effects
in the oral cavity of smokers, especially on periodontal tis-
sues, nothing is known about the effects of caffeine on
periodontal diseases. In addition, very few in vitro studies
were found to evaluate the effects of nicotine, cotinine, and
caffeine on oral bacteria. The relationship between these
substances and the subgingival microbiota remains unclear.
The aim of the present study was to evaluate the effects
of nicotine, cotinine, and caffeine on the growth and viabil-
ity of planktonic cells and biofilms and to observe if these
microorganisms could degrade such substances.
The susceptibility of 7 oral bacteria species (S. oralis
PB182, S. mitis ATCC 903, S. gordonii ATCC 10558,
P. gingivalis 381, F. nucleatum ATCC 25586, P. acnes
ATCC 11827, and A. naeslundii I ATCC 12104) was exam-
ined by using the macrodilution broth test. Single- and dual-
P. gingivalis were analyzed by time-kill assays.
All bacteria were grown in brain heart infusion (BHI)
broth (Difco Co., Detroit, Michigan, USA). To cultivate
P. gingivalis, 5 mg/mL haemin and 1 mg/mL menadione
were added to BHI broth. Streptococci were grown in
microaerophilic conditions (10% CO2; Jouan IG150, Jouan,
France) at 37 8C. The other bacterial species were grown
under anaerobic conditions (10% CO2, 10% H2, and 80%
N2; MiniMacs Anaerobic Workstation, Don Whitley Scien-
tific, Shipley, UK) at 37 8C.
Susceptibility was assayed by using a macrodilution broth
test as previously described (Koneman et al. 1997). All the
substances tested were purchased from Sigma Chemical Co.
(Poole, UK). The test was carried out in tubes containing
5 mL of BHI broth. Nicotine and caffeine were diluted in
distilled and sterilized water, and cotinine in ethanol (0.8%
v/v). These substances were assayed at concentrations of
400, 100, 25, 6.25, 1.5, and 0.4 mg/mL.
All tubes received a standardized inoculum, containing
bacterial suspension prepared in sterile saline solution
(0.9% NaCl), and were adjusted with a spectrophotometer
to a cell density of 1 ? 108CFU/mL. An inoculum of
50 mL was added to each tube (final concentration of 1 ?
The tubes were then incubated at 37 8C in microaero-
philic conditions for 18 h for streptococci or under anaerobic
conditions for 48 h for the other bacteria. Bacterial viability
was assessed by the microbiological assay. Three tubes with
inoculum but without any of the tested substances were used
as positive control. Negative control tubes contained each
concentration of the substances without inocula. Three tubes
with vehicle (0.8% ethanol) and inocula were also used as
positive control. Samples from each tube were serially diluted
(10–2to 10–5) in saline solution and spirally plated (Spiral Pla-
ter System, Don Whitley Scientific) on BHI agar with 5% de-
fibrinated sheep blood. For P. gingivalis, 5 mg/mL haemin
and 1 mg/mL menadione were added to BHI agar. Petri dishes
were then incubated at 37 8C, in 10% CO2, for 18 h (strepto-
cocci), under anaerobic conditions for 48 h (other bacteria).
After incubation, colonies were counted to determine colony-
forming units (CFU) per millilitre.
The biofilm assay method was adapted from a previously
described method (Duarte et al. 2006). Single- and dual-
species biofilms of S. gordonii, P. gingivalis, F. nucleatum,
S. gordonii – F. nucleatum, P. gingivalis – F. nucleatum
were grown on the surface of sintered hydroxyapatite discs
(0.5 inch (1 inch = 25.4 mm) diameter calcium hydroxy-
apatite ceramic discs; Clarkson Calcium Phosphates, Wil-
liamsport, Pennsylvania, USA). These discs were previously
autoclaved and placed in a vertical position into the 50 mL
polystyrene tubes containing artificial saliva (Pratten et al.
1998), except for S. gordonii single-species biofilms.
Streptococcus gordonii single-species biofilms were grown
in BHI broth and 0.5% (m/v) sucrose. For P. gingivalis bio-
films, 5 mg/mL haemin and 1 mg/mL menadione were added
to the artificial saliva. The discs were immersed in artificial
saliva to promote salivary pellicle formation. A 10 mL vol-
ume of artificial saliva was added to each tube and inocu-
lated with the bacterial suspension. For single-species
biofilms, the inoculum was standardized to 108CFU/mL. In
dual-species biofilm, it was necessary to alter the inoculum
concentration for each species to provide proper biofilm
growth. For P. gingivalis – F. nucleatum biofilms, the inoc-
ulum concentration was 108and 105CFU/mL, respectively.
For S. gordonii – F. nucleatum biofilms, the inoculum was
105and 108CFU/mL, respectively. Each inoculum was
standardized by using a spectrophotometer assay. The tubes
were incubated for 24 h at 37 8C in 10% CO2 (for
502Can. J. Microbiol. Vol. 54, 2008
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S. gordonii single-species biofilm) or under anaerobic condi-
tions (for other biofilms). After this period, the artificial sal-
iva was replaced with BHI broth, which was renewed every
24 h. Again, haemin and menadione were added to BHI
broth to cultivate P. gingivalis biofilms. In mixed-culture
studies, colonies were differentiated by their Gram-stain re-
action. Biofilms were grown for 5 days (S. gordonii) or
7 days and after that were submitted to the viability assays.
The viability assays were carried out as previously de-
scribed by Duarte et al. (2006). The biofilms were trans-
ferredto their respective
nicotine, or cotinine at a concentration 400 mg/mL. The bio-
films transferred to 0.8% ethanol served as a positive control
viability agent (data not shown). The tubes were incubated
at 37 8C, in 10% CO2(for S. gordonii single-species bio-
film) or under anaerobic conditions for other biofilms). At
specific time intervals (0, 2, 5, 8, 18, 24, and 48 h), the
hydroxyapatite discs were removed from the tubes and
rinsed twice in 7.5 mL of sterile saline solution for 10 s
each. Each disc was transferred to a tube containing sterile
saline solution, and the biofilm was dispersed by sonication
(Vibra Cell 400w, Sonics and Materials Inc., Newtown,
Connecticut, USA) at 4 8C, with 5% amplitude, and 6 pulses
(9.9 s each pulse and at 5 s intervals). Biofilm suspensions
were plated on BHI agar with 5% defibrinated sheep blood
(plus haemin and menadione for P. gingivalis). Plates were
incubated at 37 8C for 48 h in microaerophilic conditions
(S. gordonii) or for 72 h in anaerobic conditions. After the
incubation period, the CFUs were quantified. In mixed-
culture studies, colonies were differentiated by their Gram-
stain reaction. The results of both microbiological tests
(macrodilution broth test or viability assay) were considered
statistically significant only when the bacterial increase or
decrease was 3.0 log10CFU/mL or greater from the initial
inoculum (Yagi and Zurenko 2003).
After the susceptibility tests and the biofilm assays, sam-
ples from the culture media were collected from the tubes
and stored at –80 8C to measure nicotine, cotinine, and caf-
feine concentrations by high-performance liquid chromatog-
raphy (HPLC). The liquid chromatography
consisted of an HPLC pump (Varian model 9012) and a
manual injection valve (Rheodyne 7125) equipped with a
50 mL loop (Varian Inc. Corp., Palo Alto, California). Anal-
ysis was performed on a C8 reversed-phase cartridge col-
umn (Symmetry cartridge, 150 mm ? 3.9 mm ? 5 mm,
I.D., Waters, Milford, Massachussetts, USA) with a C8 pre-
column (Symmetry cartridge, 150 mm ? 3.9 mm ? 5 mm,
I.D., Waters). Nicotine, cotinine, and caffeine were detected
by using a 9050 UV-VIS detector (Varian Inc. Corp.) and
the Star integrator software (Varian Inc. Corp.). All HPLC
analysis was performed by using a previously adapted and
validated method (Ceppa et al. 2000). Briefly, the samples
(800 mL) were previously filtered through 0.2 mm filters
(Millex JBR 610291; Millipore, Billerica, Massachusetts,
USA) and submitted to extraction by using 160 mL of
5 mol/L potassium hydroxideand 800 mL of dichloro-
methane. Samples were shaken at room temperature for
15 min and centrifuged (3000g) for 15 min at 4 8C. The
supernatant was discarded, and 0.7 mL of the inferior or-
ganic phase was collected. After evaporation to dryness at
ambient temperature, the residues were dissolved in 150 mL
of mobile phase (aqueous buffer – acetonitrile – methanol
(83:7:10, by volume)), and 2 mg/mL phenylimidazole was
added as an internal standard. The aqueous buffer consisted
of citric acid (9.9 g), sodium octane sulfonate (0.465 g),
potassium dihydrogen phosphate (6.435 g), and triethyl-
amine (6.5 mL) in 1 L of ultrapure water. The pH was ad-
justed to 3.4 and the flow rate was 1.0 mL/min. The
wavelength was set at 270 nm for nicotine and cotinine and
at 275 nm for caffeine. Total analysis time was 6 min.
Standard stock solutions of cotinine, nicotine, and caffeine
were diluted in their respective solvents, at concentrations
from 0.1 to 500 mg/mL, to perform the calibration curves.
All tests were performed in triplicate on at least in 2 sepa-
The number of CFU obtained in the susceptibility assays
and the concentration of each substance were analyzed by
using the Kruskal–Wallis rank-sum test. Statistical differen-
ces between control and concentration groups were deter-
mined by the Dunn’s test. Data obtained from biofilm
viability assays were performed by using the Mann–Whitney
U test. Substances concentrations obtained from HPLC anal-
ysis were evaluated with the Kruskal–Wallis test. Statistical
software (BioEstat version 4.0, Mamiraua/CNPq, Bele ´m,
PA, Brazil) was used to carry out the analysis. The signifi-
cant level was set at 5%.
The effects of nicotine, cotinine, and caffeine on the
growth of planktonic cells are shown in Fig. 1. The figure
summarizes the bacterial growth in log10CFU per millilitre
when exposed to different concentrations of the tested sub-
stances. The growth controls are represented as 0 mg/mL.
No statistically significant differences (Kruskal–Wallis, P >
0.05) were found among the groups.
The influence of these substances on bacterial single-
species biofilms is shown in Fig. 2. These substances did not
show any activity against single-species biofilms during the
48 h treatment period, since they were not able to reduce nor
increase the species viability more than 3.0 log10CFU/mL
(Mann Whitney, P > 0.05) according to Yagi and Zurenko
(2003). In dual-species biofilms, the viability of F. nuclea-
tum, P. gingivalis and S. gordonii was also not altered
(Mann Whitney, P > 0.05) in the presence of any of the
tested substances (Fig. 3).
Figures 4 and 5 show the concentration of nicotine, coti-
nine, and caffeine in the samples from the susceptibility
tests and biofilm assays, respectively. No statistically signif-
icant differences (Kruskal–Wallis, P > 0.05) were observed
in any of the assayed samples.
The present study showed that nicotine, cotinine, and caf-
feine did not alter the growth patterns of any of the bacterial
species tested. These findings are similar to those of
Teughels et al. (2005), who evaluated the bacterial viability
of Actinobacillus actinomycetemcomitans and P. gingivalis
when exposed to 10, 100, and 1000 mg/mL concentrations
of nicotine or cotinine for 0, 2, 4, and 6 h. The authors con-
cluded that neither nicotine nor cotinine significantly af-
fected bacterial viability.
There are some studies showing that these substances
could interfere on the viability of some bacterial species.
Pavia et al. (2000) showed that nicotine at concentrations
varying from 100 to 250 mg/mL can reduce the growth of a
broad spectrum of microorganisms, such as E. coli, Kleb-
Cogo et al.503
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Fig. 1. Growth (log10 CFU/mL) of bacterial strains tested after 18 h (streptococci) and 48 h (other bacteria) exposure to different concen-
trations of nicotine (A), cotinine (B), and caffeine (C).
504Can. J. Microbiol. Vol. 54, 2008
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Fig. 3. Growth curves for dual-species biofilms of Porphyromonas gingivalis – Fusobacterium nucleatum (A) and Streptococcus gordonii –
Fusobacterium nucleatum (B) exposed to nicotine, cotinine, and caffeine for 48 h.
Fig. 2. Growth curves for single-species biofilms of Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis ex-
posed to nicotine, cotinine, and caffeine for 48 h.
Cogo et al.505
##2008 NRC Canada
siella pneumoniae, Listeria monocytogenes, Cryptococcus
neoformans, and Candida albicans, but only slightly inhibits
or unaffects Staphylococcus aureus and Borrelia burgdoferi.
Viridans streptococci are also susceptible to nicotine, which
markedly reduces the bacterial cells viable counts. However,
these authors did not mention which species of viridans
streptococci were studied. We observed some species-
dependent effects but none of the effects significantly al-
tered the microorganism number.
Roberts and Cole (1979) reported a prolific growth of
Haemophilus influenzae in the presence of tobacco or nico-
tine added to a phosphate-buffered saline agar. This assay
was performed using a culture medium that poorly sup-
ported the growth of H. influenzae, suggesting that in a
nutrient-scarce condition, bacteria might use nicotine as a
nutrient source. In the present study, the assays were carried
out using rich culture media, which promoted proper bacte-
rial growth. Probably, bacterial growth stimulation by nico-
tine could be higher in a nutrient-poor environment.
Moreover, in a study by Roberts and Cole (1979), bacterial
growth was analyzed qualitatively through visual analyses
and not quantitatively (i,e., by calculating numbers of CFU
per millilitre or measuring the optical density) making a
comparison to this study difficult. Comparisons between our
results and those reported in previous studies was also diffi-
cult because of differences in methodology, nicotine concen-
trations, and species of microorganisms tested; most of them
were not from oral microbiota.
Keene and Johnson (1999) observed a biphasic dose-
dependent effect of nicotine on Streptococcus mutans
growth. Nicotine concentrations of 0.1 mol/L (16.22 mg/mL)
and 0.01 mol/L (1.622 mg/mL) inhibit growth of bacteria,
concentrations of 10–3mol/L (162.2 mg/mL) and 10–4mol/L
(16.22 mg/mL) stimulate it, while concentrations of 10–6mol/L
(162.2 ng/mL) and 10–7mol/L (16.2 ng/mL) reduce the num-
ber of viable cells. The present study did not support this nico-
tine dose-dependent profile.
The influence of caffeine on strains of E. coli growth was
investigated by Sandlie et al. (1980). Concentrations up to
8 mmol/L (1553 mg/mL) have little effect on the growth of
these bacteria, while in higher concentrations, the growth is
strongly decreased. A 50% inhibition of growth is found
Fig. 5. Concentration of nicotine, cotinine, and caffeine in the biofilm (single or dual species) cultures after the microbiological assays.
Fig. 4. Concentration of nicotine, cotinine, and caffeine in the planktonic cultures after the microbiological assays.
506Can. J. Microbiol. Vol. 54, 2008
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with a concentration of 20 mmol/L (3883 mg/mL). In the
present study, even at the highest concentration tested
(400 mg/mL), no significant reduction in bacterial growth
Some studies have reported that the concentration levels
of nicotine in saliva range from 70 to 1560 mg/mL
(Hoffmann and Adams 1981), with a mean level of approx-
imately 115 mg/mL (Dhar 2004). The mean levels of
cotinine reported in saliva range from 0.424 ng/mL to
3.6 mg/mL and in crevicular fluid from 2.5 to 15.0 mg/mL
(McGuire et al. 1989; Chen et al. 2001). In the present
study, the nicotine and part of the cotinine concentrations
were in agreement with the physiological levels of the pre-
vious studies, whereas some concentrations were higher
than those found in saliva and crevicular fluid. Nevertheless,
previous studies used high concentrations of these substan-
ces to induce cellular effects (Sayers et al. 1999; Teughels
et al. 2005). Concentrations of nicotine and cotinine used in
this study were adequate for the evaluation of their effects.
No investigation was found in the literature reporting caf-
feine concentrations on saliva or crevicular fluid.
In the present study, nicotine, cotinine, and caffeine con-
centrations were not altered after bacteria exposure. Prob-
ably, these bacterial species are not capable of degrading
these substances and, consequently, do not utilize them as a
source of energy under the conditions we have tested for
bacterial growth. In the environment, there are several
microorganisms that are able to degrade nicotine, contribu-
ting to the manufacturing process of tobacco by altering the
content of nicotine in final products (Brandsch 2006; Yuan
et al. 2006). These microbes could utilize nicotine as sour-
ces of carbon, nitrogen, and energy for their growth process
(Ruan et al. 2006).
In conclusion, nicotine, cotinine, and caffeine, in the con-
centrations tested, did not affect the growth of the oral bac-
teria strains evaluated. In addition, it appears that these
bacteria do not degrade these substances or utilize them as
an energy source.
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