Journal of Oral Microbiology

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Effect of nicotine on biofilm formation of

Streptococcus mutans isolates from smoking and
non-smoking subjects

Nasreen F. El-Ezmerli & Richard L. Gregory

To cite this article: Nasreen F. El-Ezmerli & Richard L. Gregory (2019) Effect of nicotine on biofilm
formation of Streptococcus�mutans isolates from smoking and non-smoking subjects, Journal of
Oral Microbiology, 11:1, 1662275, DOI: 10.1080/20002297.2019.1662275

To link to this article: https://doi.org/10.1080/20002297.2019.1662275

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JOURNAL OF ORAL MICROBIOLOGY
2019, VOL. 11, 1662275
https://doi.org/10.1080/20002297.2019.1662275

Effect of nicotine on biofilm formation of Streptococcus mutans isolates from

smoking and non-smoking subjects
Nasreen F. El-Ezmerlia and Richard L. Gregoryb
a
Department of Operative and Preventive Dentistry, School of Dentistry, Indiana University, Indianapolis, IN, USA; bDepartment of
Biomedical and Applied Sciences, School of Dentistry, Indiana University, Indianapolis, IN, USA

ABSTRACT ARTICLE HISTORY

Objectives: To investigate effects of nicotine on biofilm formation of Streptococcus mutans Received 4 April 2019
isolates from oral washes of smoker and non-smoker human subjects. Revised 23 August 2019
Materials and methods: This study was conducted using 60 S. mutans isolates with three Accepted 27 August 2019
S. mutans isolates collected from oral washes of ten smoking subjects and ten from non-
KEYWORDS
smoking subjects. Biofilm was formed by culturing each S. mutans strain (10 μl) in 190 μl of
Biofilm; tooth decay;
TSB supplemented with 1% sucrose (TSBS) containing 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and Streptococcus mutans;
32.0 mg/ml of nicotine for 24 h in 5% CO2 at 37°C in 96 well microtiter plates. The absorbance nicotine
values of biofilm were measured at 490 nm in a microplate spectrophotometer.
Results: There was a significant effect (p-value < 0.05) of nicotine concentrations and
smoking on the growth of biofilm, planktonic cells, and total absorbance, for all strains of
S. mutans. Isolates from smokers had significantly more biofilm at 0–16 mg/ml of nicotine
compared to those from non-smokers (p-value < 0.0001).
Conclusion: S. mutans smoker isolates are more affected by high nicotine concentrations
than non-smoker isolates.
Clinical Relevance: The use of nicotine products increases the growth of S. mutans and may
place tobacco users at risk for dental decay.

Tooth decay is a complex dieto-bacterial disease with formation, microorganism adherence, growth and
an association of social, behavioral and biological fac- maturation of the bacterial microcolonies, and lastly
tors [1]. This complex disease is considered an infec- detachment to form a new biofilm [4]. In the first
tious disease, which develops over time involving stage, if the tooth surface is clean, salivary molecules
a complex interaction of oral microflora, specifically can adsorb to hydroxyapatite on enamel tooth surfaces
Streptococcus mutans, dietary carbohydrates, and by electrostatic interactions forming the acquired
a susceptible tooth surface [2]. It has been well defined enamel pellicle. Initial microorganism adherence is
that S. mutans and tooth decay are closely related, the second stage that occurs when early colonizing
especially that S. mutans can adapt very well in bacteria attach to salivary acquired pellicle through
a high carbohydrate environment under acidic condi- a weak reversible attachment in the absence of sucrose
tions. S. mutans has the ability to metabolize sugars utilizing specific receptors and ligands [4]. S. mutans
forming organic acids that bathe tooth surfaces caus- has an important role in initial sucrose independent
ing its progressive mineral loss. It thrives in specific adherence involving a bacterial surface protein adhe-
oral conditions with unique characteristics [3]. sin called antigen I/II that interacts specifically with
Adherence of S. mutans to hard tooth structures is a high molecular weight salivary agglutinin glycopro-
considered one of the major characteristics that enable tein (SAG) found in the acquired enamel pellicle [5,6].
it to proliferate and microcolonize establishing The third stage involves formation of an extracellular
a mature cariogenic biofilm. Numerous cariogenic polysaccharide matrix and establishment of cariogenic
factors of S. mutans are involved in its ability to adhere biofilm attached to tooth surfaces which is contributed
and aggregate to form cariogenic biofilms including by an important cariogenic factor of S. mutans known
initial sucrose-independent adherence in which anti- as sucrose-dependent adherence involving Gtfs and
gen I/II is involved, and sucrose-dependent adherence Gbps [7,8]. S. mutans-associated Gtfs primarily pro-
based on the function of glucosyltransferases (Gtfs) duce both water soluble and insoluble glucans by
and other glucan-binding proteins (Gbp) [3]. Oral metabolizing sucrose to glucose and fructose and sub-
microbial biofilm (dental plaque) formation involves sequently polymerizing glucose to an extracellular
four stages including salivary acquired pellicle adhesive insoluble glucan that binds bacterial cells

CONTACT Richard L. Gregory [email protected] Department of Biomedical and Applied Sciences, School of Dentistry, Indiana University,
Indianapolis, IN, USA
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 N. F. EL-EZMERLI AND R. L. GREGORY

together through Gbp and Gtf receptors and adhere determined. Biofilm formation and metabolism of all
the cells to the enamel tooth surface [8]. It was seven S. mutans strains increased in a dose-dependent
observed that deletion of the Gtfs genes remarkably manner up to 16.0 mg/ml of nicotine. Planktonic cell
decreases the cariogenic potential of S. mutans strains growth exhibited the highest values between 2, 4 and
[9]. The synthesis of extracellular glucan enhances the 8 mg/ml of nicotine. Because of these significant
adherence of S. mutans through a cell to cell interac- effects of nicotine on S. mutans, it is possible that
tion where streptococcal Gtfs bind to glucan that suc- there may be a difference in the manner that
cessively adheres cells to smooth tooth surfaces [10]. S. mutans responds to nicotine in smokers. To date
Additionally, S. mutans synthesizes Gbps that have there is no information on the effect of nicotine on the
a significant role in establishing a mature biofilm by biofilm formation of S. mutans isolates from smokers.
adhering bacteria to the extracellular glucan. An Therefore, we proposed the use of an in vitro model to
in vitro study by Lynch et al. indicated that engineered better understand the effects of nicotine on biofilm
S. mutans with deleted Gbps genes affected the adher- formation of S. mutans isolates from smokers and
ence and aggregation of these organisms resulting in non- smoking subjects.
a decrease in the biofilm mass and change in its archi-
tecture [11].
Tobacco use is a behavioral risk factor that Materials and methods
adversely affects oral health and is directly linked to Bacterial strains and media
common life threatening diseases such as cancer, and
cardiovascular and respiratory diseases [12–14]. The Ten oral washes collected from smoking subjects and ten
oral cavity is the first place in the human body to get oral washes from non-smoking subjects were used in this
exposed to either chewing tobacco or tobacco smoke study. Three S. mutans isolates were cultured from each
and its chemical components. Therefore, tobacco not oral wash. Therefore, a total of 30 presumptive S. mutans
only affects systemic organs but it also has a significant smoker isolates and 30 S. mutans non-smoker isolates
influence on periodontal and other oral tissues [15] as were collected. The oral washes were collected as part of
well as oral microorganisms. Nicotine is one of the a large multicenter NIH-funded microbiome grant
major active ingredients of cigarette smoke [16]. This (HL098960) and were obtained under appropriate IRB
active chemical has a toxic effect on alveolar bone and approval (IRB number 1,401,371,742). Age, race, gender,
clinical attachment loss [17]. The exact effects of nico- smoking history and number of pack years history were
tine associated with tooth decay has not been fully obtained from each subject. The oral wash samples were
investigated. However, a study conducted on 824 stored at −80°C until used. Selective agar plates (MSSB;
male Mexican truck drivers found a remarkable asso- Mitis Salivarius Sucrose Bacitracin; Anaerobic Systems,
ciation between tobacco use and dental caries experi- Inc., Morgan Hill, CA) were used for culturing the oral
ence. Drivers who smoked more than 10 cigarettes/day wash samples in 5% CO2 at 37°C as an initial isolation
had twice as many carious lesions than non-smokers step, and three different colonies representing S. mutans
[18]. In an Italian military population it was deter- from each oral wash sample were selected and grown on
mined that heavy smokers had double the number of different MSSB plates. The isolates were subcultured in
decayed teeth than a general population [19]. Another tryptic soy broth (TSB, Acumedia, Baltimore, MA) for
study investigated the in vitro effect of cigarette smoke 24 h in 5% CO2 at 37°C. The isolates were stored in TSB
on the growth of S. mutans and Streptococcus sangui- with 20% glycerol at −80°C until used. Mannitol and
nis. They concluded that nicotine has a dose- raffinose carbohydrate fermentation assays were used to
dependent effect on the growth of S. mutans; since as confirm the identity of the subcultured S. mutans isolates
the nicotine concentration in the cigarettes increased [24]. A total of 34 S. mutans isolates were confirmed
there was an increase in S. mutans growth [20]. In an (11 from smokers and 23 from non-smokers) from
in vivo study, it was reported that nicotine treated rats a total of 60 non-confirmed S. mutans isolates
had a significant increase in S. mutans growth and (30 from smokers and 30 from non-smokers). Nicotine
developed more caries lesions than in nicotine from Sigma-Aldrich (St. Louis, MO) was used.
untreated rats [21]. Recently, we determined that nico-
tine stimulates S. mutans planktonic cell Gtf and Gbp
Biofilm formation
expression as a mechanism to increase planktonic cell
attachment to biofilm matrix leading to an increased Overnight cultures of each S. mutans strain (10 μl repre-
number of cells in the biofilm [22]. This may explain senting approximately 106 bacteria) grown in TSB were
the development of more carious lesions in smokers. incubated with 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and
In another study from this laboratory, seven S. mutans 32.0 mg/ml of nicotine in TSB containing 1% sucrose
strains were treated with different nicotine concentra- (TSBS; 190 μl) for 24 h at 37°C in 5% CO2 in sterile 96 well
tions (ranging from 0–16 mg/ml) [23]. Biofilm forma- microtiter plates (Fisher Scientific, Newark, DE). The
tion, and metabolic activity of the strains were total absorbance of each sterile 96 well microtiter plate
 JOURNAL OF ORAL MICROBIOLOGY 3

was measured at 595 nm in a microplate spectrophot- Results

ometer (SpectraMax 190; Molecular Devices, SunnyVale,
Due to non-normality of the data, a rank transforma-
CA) to assess the total bacterial growth (planktonic +
tion was used on the data prior to the analysis.
biofilm cells). One hundred and twenty μl of planktonic
A two-way ANOVA with a random effect for the
cells of each sterile 96 well microtiter platewas transferred
multiple experiments was used for the analysis.
to other microtiterplates and the planktonic cell absor-
There were significant effects of both nicotine con-
bance was determined at 595 nm. The biofilm remaining
centrations and smoking on the growth of biofilm,
in each sterile 96 well microtiter platewas washed twice
planktonic cells, and total absorbance, for all strains
with deionized water, fixed with 200 μl of 10% formalde-
of S. mutans (p < 0.0001; Figure 1–3). For biofilm,
hyde (Sigma) for 30 min at room temperature, and
there was a significant interaction of nicotine con-
washed three times with dionized water. Two hundred
centrations and smoking for S. mutans smoker strains
μl of 0.05% crystal violet was used to stain biofilm cells for
(Figure 3). For planktonic and total absorbance, there
30 min. The wells were washed three times and 200 μl of
was a significant interaction of nicotine concentration
isopropanol (Fisher, Pittsburg, PA) added for 60 min to
and non-smoking S. mutans isolates (p < 0.0001;
extract the crystal violet from the biofilm cells. The
Figures 1 and 2, respectively). Biofilm formation of
absorbance values were measured at 490 nm.
smoker isolates had dose-dependent effects up to
8.0 mg/ml. Isolates from smokers had significantly
more biofilm at 0–16 mg/ml nicotine compared to
Statistical methods
those from non-smokers (p < 0.0001). Non-smoker
Each of the 34 confirmed S. mutans strains was tested isolates had significantly more total absorbance at all
three times in quadruplicate. Summary statistics nicotine concentrations compared to smokers
(mean, standard deviation, standard error, range) of (p < 0.0001; Figure 1). There were significant differ-
the absorbance values (total absorbance, planktonic ences of the planktonic cells between smoker and
and biofilm) were calculated for each of the strains. non–smoker isolates (Figure 2).
The effects of nicotine concentration, smoker vs. There were significant differences between biofilm
non-smoker S. mutans strain, and their interaction formation of smoker isolates at 1, 4, and 8 mg/ml of
on biofilm formation were analyzed using ANOVA. nicotine and the zero nicotine concentration (Figure 3).
The ANOVA included fixed effects for the two fac- Significant differences between biofilm formation of
tors and their interaction and a random effect of non-smoker isolates and the zero nicotine concentra-
absorbance values were examined. A transformation tion were observed at the 4, 8, 16, and 32 mg/ml con-
of the data (e.g. natural logarithm) was necessary to centrations. There were significant differences between
satisfy the ANOVA assumptions. biofilm formation of smoker and non-smoker isolates

Streptococcus mutans total growth of isolates from

smokers and non-smokers at different nicotine concentrations

0.600
Smokers
Non-Smokers *#
*#
0.500 *#
*# *# *#
Absorbance 595 nm

0.400 #
* * *#
*
*
0.300

0.200

*#
0.100
*
*

0.000
0 0.25 0.5 1 2 4 8 16 32

Nicotine Concentration (mg/ml)

Figure 1. Asterisks indicate significant differences between total growth of S. mutans isolates (smokers/non-smokers) at
different nicotine concentrations and the zero nicotine control. # indicate significant differences between total growth of
S. mutans isolates of smokers/non-smokers at different nicotine concentrations.
4 N. F. EL-EZMERLI AND R. L. GREGORY

Figure 2. Asterisks indicate significant differences between S. mutans planktonic growth (smokers/non-smokers) at different
nicotine concentrations and the zero nicotine control. # indicate significant differences between S. mutans planktonic growth of
smokers/non-smokers at different nicotine concentrations.

Streptococcus mutans biofilm formation of isolates from

smokers and non-smokers at different nicotine concentrations

1.000
Smokers *#
Non-Smokers
*#
0.800
Absorbance 490 nm

*# #
0.600
#
# #

0.400 *
*
*#

*
0.200 *
*

0.000
0 0.25 0.5 1 2 4 8 16 32

Nicotine Concentration (mg/ml)

Figure 3. Asterisks indicate significant differences between S. mutans biofilm formation (smoker/non-smoker) at different
nicotine concentrations and the zero nicotine control. # indicate significant differences between S. mutans biofilm formation
of isolates from smokers and non-smokers at different nicotine concentrations.

at 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mg/ml nicotine con- (negative) for the 0.25 mg/ml nicotine concentration
centrations (Figure 1). For total absorbance and plank- for one of the three experiments. Several other correla-
tonic measurements there were significant differences tions indicated some relationship although they did not
between smoker and non-smoker isolates at all nicotine reach statistical significance.
concentrations (Figures 1 and 2). There was no signifi-
cant relationship between the number of pack years
Discussion
smoked (Tables 1 and 2) and biofilm formation of
S. mutans isolates at all nicotine concentrations. To determine the effect of smoking history and the
However, this correlation was statistically significant addition of nicotine on the formation of S. mutans
 JOURNAL OF ORAL MICROBIOLOGY 5

Table 1. Average Demographic Factors of Smoking and Non- nicotine had an antibacterial effect on both smoking
Smoking Human Subjects. and non-smoking isolates at high concentrations
Demographic Non-Smokers n = 10 (16–32 mg/ml). Furthermore, a recent study indi-
Factors Smokers n = 10
Gender F=1 F=4
cated that adding 1.0 mg/ml nicotine to S. mutans
M=9 M=6 biofilm cultures increases the production of lactate by
Average Age 40.5 years old 42.0 years old two folds compared to S. mutans biofilm cultures
Race White = 1 White = 5
African American = 9 African American = 5 with zero nicotine [25,26]. The results of the present
Average: study demonstrated that there was a significant dif-
Pack Years (8.5 cigarette per day/20)
x 34.3 year smoking ference in biofilm formation between smoker and
history = 14.5 pack years non-smoker isolates at almost all nicotine concentra-
tions. There was a more significant increase in bio-
film formation of smoker isolates at 1, 4, 8, 16 and
Table 2. Individual Demographic Factors of Oral Washes from
32 mg/ml compared to biofilm at the zero nicotine
Smoking and Non-Smoking Human Subjects.
concentration. In this study, it was clear that
Pack
IUPUI- ID # Race Sex Age Smoker Smoking History Years S. mutans isolates from smokers are more influenced
891084OR01 W M 52 No by high nicotine concentrations (up to 16 mg/ml)
891080OR02 W M 32 No than non-smokers. In addition, this study indicated
891086OR01 AA F 38 No
891090OR01 AA M 37 No that planktonic cell growth was greater in non-
891087OR01 AA M 40 No smoking isolates at all nicotine concentrations com-
891085OR01 W M 22 Yes ½ PPD~4 years 3 years
891091OR01 W M 52 No pared to the planktonic cell growth of smoker isolates
891088OR01 AA F 42 No at the same nicotine concentrations. The possible
891089OR01 W M 35 No
891092OR01 W F 56 No mechanism of nicotine on enhancement of biofilm
005017OR01 AA M 54 Yes 1 PPD~36 years 36 years growth of S. mutans strains tested in the present
005022OR01 AA M 43 Yes 3 cig 6.3 years
PD~42 years
study can be explained by a recent study that demon-
005016OR01 AA F 46 No strated the effect of nicotine on the expression of
005009OR01 AA M 53 Yes 1 PPD~32 years 32 years Gbps and Gtfs genes [22]. Interestingly enough, it
005010OR01 AA F 48 Yes 8 cig 12 years
PD~30 years was found that nicotine up-regulates the expression
005011OR01 AA M 51 Yes 1 PPD~37 years 37 years of Gbps and Gtfs genes of S. mutans planktonic cells
005020OR01 AA M 53 Yes 15 cig 25.5 years
PD~34 years and down-regulates Gbps and Gtfs of S. mutans bio-
005021OR01 AA M 57 Yes ½ PPD~43 years 22.5 years film cells [22]. Thus, an increase of planktonic cell
005024OR01 AA M 58 Yes 1 PPD~41 years 41 years
005025OR01 AA M 59 Yes ½ PPD~44 years 22 years attachment to biofilm results in increased growth of
biofilm. In this study, there was not a significant
relationship between the number of pack years
biofilm, planktonic cells, and total growth in vitro, smoked and biofilm formation of S. mutans isolates
S. mutans isolates from smokers and non-smokers at all nicotine concentrations. The present study
were compared in this study. To date, this is the hypothesized that nicotine produces significant dif-
first study that compares the effect of nicotine on ferences in biofilm formation between smoker and
both smoker and non-smoker isolates. In this study, non-smoker S. mutans isolates. According to the
nicotine enhanced biofilm growth in both S. mutans study results, this hypothesis was confirmed. The
smoker and non-smoker isolates. Biofilm formation rationale for this hypothesis was derived from pre-
increased in a dose-dependent manner up to 8.0 mg/ liminary data indicating that S. mutans can become
ml nicotine in both smoking and non-smoking oral tolerant to increased nicotine concentrations and this
strains. Furthermore, smoker isolates, when incu- tolerance appears to be stable (unpublished data).
bated with most of the nicotine concentrations, pro- This may allow smoker isolates to be able to respond
duced significantly more biofilm compared to the more vigorously to higher nicotine concentrations
non-smoker isolates. However, the total growth of than non-smoker isolates. This preliminary study
the non-smoking isolates was significantly more suggested that S. mutans becomes adapted with stable
than smoker isolates at several nicotine concentra- resistance at high nicotine concentrations by some
tions. This is consistent with a previous in vitro type of mutation and possible stable upregulation of
study from this laboratory reporting that biofilm for- antigen I/II after it had been passed at least three
mation and metabolism of S. mutans increased in times on 0 mg/ml nicotine. The use of nicotine pro-
a dose-dependent manner up to 16.0 mg/ml of nico- ducts increases the growth of S. mutans and may
tine [23]. Planktonic cell growth was highest between place tobacco users at risk for dental decay [27].
2, 4 and 8 mg/ml nicotine. The majority of isolates Results of this study suggest that there is more
had MIC values of 16.0 mg/ml nicotine, MBC of increased dental caries in smokers than non-
32.0 mg/ml nicotine, and MBIC of 16.0 mg/ml nico- smokers because of the significant increase of biofilm
tine [23]. This previous study also indicated that formation in the S. mutans smoker isolates compared
6 N. F. EL-EZMERLI AND R. L. GREGORY

to non-smoker S. mutans isolates. Further investiga- antigen I/II family adhesins for human or bacterial
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[9] Tsumori H, Kuramitsu H. The role of the
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Disclosure statement Streptococcus mutans biofilm architecture. FEMS
Author Nasreen Farouk El-ezmerli declares that she has no Microbiol Lett. 2007;268:158–165.
conflict of interest. Author Richard L Gregory has no conflict [12] Akaji EA, Folaranmi N. Tobacco use and oral health
of interest. This manuscript was derived from Dr. El- of inmates in a Nigerian prison. Niger J Clin Pract.
ezmerli’s MSD thesis and has not been published elsewhere. 2013;16:473–477.
[13] Chockalingam K, Vedhachalam C, Rangasamy S, et al.
Prevalence of tobacco use in urban, semi urban and
rural areas in and around Chennai city, India. PLoS
Ethical approval
One. 2013;8:e76005.
This article does not contain any studies with human [14] Johnson NB, Hayes LD, Brown K, et al. CDC National
participants or animals performed by any of the authors. Health Report: leading causes of morbidity and mor-
However, the oral washes were collected as part of a large tality and associated behavioral risk and protective
multicenter NIH-funded microbiome grant (HL098960) factors–USA, 2005-2013. MMWR Surveill Summ.
and were obtained under appropriate IRB approval (IRB 2014;63(Suppl 4):3–27.
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Funding [16] Jacob P 3rd, Yu L, Shulgin AT, et al. Minor tobacco
alkaloids as biomarkers for tobacco use: comparison
The work was supported by Indiana University School of of users of cigarettes, smokeless tobacco, cigars, and
Dentistry in Indiana, U.S.A. pipes. Am J Public Health. 1999;89:731–736.
[17] Al-Habashneh R, Al-Omari MA, Taani DQ. Smoking
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Informed consent periodontal diseases from a teaching hospital clinic.
For this type of study, formal consent is not required. Int J Dent Hyg. 2009;7:55–61.
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