NICOTINE: ITS STIMULATING AND INHIBITORY EFFECTS ON ORAL MICROORGANISMS AUBREY E. DUBOIS*1, ZACHARY C. BENNETT*1, UMARA KHALID2, ARIBA KHALID1, REED A. MEECE1, GABRIELLE J. DIFIORE3, AND RICHARD L. GREGORY4 1DEPARTMENT OF BIOLOGY, SCHOOL OF SCIENCE, PURDUE UNIVERSITY, INDIANAPOLIS, IN; 2DEPARTMENT OF PSYCHOLOGY, SCHOOL OF SCIENCE, PURDUE UNIVERSITY, INDIANAPOLIS, IN; 3DEPARTMENT OF KINESIOLOGY, SCHOOL OF PHYSICAL EDUCATION AND TOURISM MANAGEMENT, INDIANA UNIVERSITY, INDIANAPOLIS, IN; 4DEPARTMENT OF ORAL BIOLOGY, SCHOOL OF DENTISTRY, INDIANA UNIVERSITY, INDIANAPOLIS, IN *A.E.D. AND Z.C.B. ARE JOINT FIRST AUTHORS OF THIS WORK.

MANUSCRIPT RECEIVED 15 SEPTEMBER, 2014; ACCEPTED 30 OCTOBER, 2014

Copyright 2014, Fine Focus all rights reserved 64 • FINE FOCUS, VOL. 1 ABSTRACT Tobacco users are much more susceptible to dental caries and periodontal diseases than non- tobacco users. Research suggests that this increased susceptibility may be due in part to nicotine, a primary active component of tobacco. Five bacterial species and one yeast species commonly found in the human oral cavity, Lactobacillus casei, viscosus, Actinomyces naeslundii, dentocariosa, Enterococcus faecalis, and Candida albicans respectively, were utilized to investigate if any correlation existed between CORRESPONDING exposure to various concentrations of nicotine ranging from 0 to 32 mg/ml and the growth of AUTHOR each microorganism. The minimum inhibitory concentration (MIC), minimum bioflm inhibitory Richard L. Gregory concentration (MBIC), and planktonic growth were [email protected] measured. The MIC was determined to be 16 mg/ ml for all organisms except E. faecalis, which had an MIC of 32 mg/ml. Nicotine had a varying effect on planktonic growth across the different species. KEYWORDS A distinct upward trend in bioflm formation was found in A. viscosus, L. casei, E. faecalis, • dental caries and C. albicans through 8 mg/ml. Nicotine also • enhanced R. dentocariosa bioflm formation in • bioflm all concentrations through 8 mg/ml but was most • nicotine enhanced at 1 mg/ml. Alternatively, A. naeslundii • tobacco exhibited a complete downward trend through 32 • Lactobacillus casei mg/ml. The MBIC was found to be 16 mg/ml in all • Actinomyces viscosus organisms studied. These fndings further support • Actinomyces naeslundii research suggesting that the increased susceptibility • Rothia dentocariosa to oral health diseases experienced by tobacco • Enterococcus faecalis users may be caused in part by an upregulation in • Candida albicans bioflm formation of these oral pathogens. INTRODUCTION Periodontal disease is a condition that in which the gums pull away from the causes infammation of the gums including teeth and form pockets where dental plaque diseases such as gingivitis, in which begins to grow. This infection can lead to gums become swollen and bleed easily. both severe tissue and bone damage due Periodontitis, another form of periodontal to the increased exposure to previously disease, is an advanced form of gingivitis protected areas. Periodontal disease is also BACTERIAL HOST INTERACTIONS • 65 closely linked with dental caries. The lacks adequate strength to adhere to tooth occurrence of both periodontal disease enamel, however, when co-cultured with and dental caries can be associated with Streptococcus mutans, L. casei gains the several factors. Some of the most common ability to synthesize glucans thus improving risk factors include smoking, diabetes, tooth enamel adhesion. Because of this, L. autoimmune disorders, genetics, and obesity casei along with S. mutans have been found (14). The use of tobacco products remains to be the most prevalent leading to the largest risk factor for periodontal dental caries (19). disease. Environmental tobacco smoke has been associated with the increased ACTINOMYCES VISCOSUS likelihood of caries formation in children Actinomyces viscosus is a facultative (2). Recent studies in this laboratory anaerobic, Gram-positive bacterium that is demonstrated increased bioflm formation often isolated from the oral cavity, especially of S. mutans with increasing nicotine from subgingival plaque and along the teeth concentrations (20). The average nicotine and gums. A. viscosus is rod-shaped and content in a single cigarette has been found flamentous. Distinct characteristics of the in amounts up to 2 mg (12). A recent study flamentous shape include branching with tested over 40 smokeless tobacco products, swollen, rounded, or clavate ends (7). A. such as snuff and chew, and it was viscosus is a severely cariogenic bacterium discovered that they contained anywhere that acts as an agent in promoting and from 3.6-25.3 mg of nicotine per gram of initializing carious lesions (15). Studies have smokeless tobacco (33). Nicotine contents shown that the bacterium has adhesins of cigars have been found to vary greatly, which allow it to bind to complementary ranging from 5.9 mg per cigar to 335.2 mg receptors on a substrate. A. viscosus binds to per cigar (18). Studies have demonstrated salivary acidic proline-rich proteins (PRPs) that when smoking cigarettes, an average adsorbed onto the apatitic surfaces of the of approximately 1 mg of nicotine per tooth. Cryptic segments exposed in adsorbed cigarette is absorbed by the body, however, molecules are identifed by the adhesins, the amount of nicotine within oral bioflm which allow A. viscosus to effciently attach of smokers has not yet been explored (5). to teeth while planktonically suspended in This study used the following opportunistic saliva (1). pathogens, commonly found in the human oral cavity, to investigate possible effects on ACTINOMYCES NAESLUNDII growth and bioflm formation. Actinomyces naeslundii is a Gram-positive bacterium characterized by its rod shape LACTOBACILLUS CASEI and prominent fmbriae that allow it to Lactobacillus casei is a Gram-positive, bind to the tooth surface. A. naeslundii is facultative anaerobic bacterium. They are facultatively anaerobic, nonmotile, non- nonmotile and have a rod shape. Like other spore forming, and commonly grows in the strains of lactobacilli, L. casei produces a oral cavity. (6) A. naeslundii is also a major signifcant amount of lactic acid, allowing it component of dental plaque and it is known to remain viable under various pH levels. to cause dental caries, root canal infections, Poor diet, weak tooth enamel, previous and periodontal disease (27). It is one of the plaque buildup, and acidic conditions specifc species of the genus Actinomyces contribute to an optimal environment capable of causing the chronic bacterial for L. casei to thrive (22). L. casei alone infection, actinomycosis, 66 • FINE FOCUS, VOL. 1 in humans (8). A. naeslundii plays a role carbohydrates, and a number of amino in actinomycosis by initiating infections acids. It is also able to withstand extremely in carious teeth, as well as causing basic environments, high salt concentrations, infections in places where the mucosal desiccation, and the presence of many integrity of the tooth is compromised (32). (17). These attributes allow These infections are the precursor of the E. faecalis to thrive in deep root canals. actinomycosis, however, the bacterium Additionally, scientists recently discovered itself exhibits very low pathogenic E. faecalis has the ability to suppress properties. These properties are enhanced lymphocyte function as well as produce by the presence of microorganisms such as lytic enzymes which aid in destruction of species of Prevotella, Staphylococcus, and gingival tissues (9). Streptococcus (35). CANDIDA ALBICANS ROTHIA DENTOCARIOSA Candida albicans is a ubiquitous Rothia dentocariosa is a facultative anaerobic, pleomorphic fungus that colonizes the Gram-positive, pleomorphic bacterium that gastrointestinal, epithelial, and mucosal is commonly found in either flamentous tissues of over 70% of the human or coccoid forms (24). It is common in population (26). Whether C. albicans takes the oral cavity, and has been associated on hyphal, pseudohyphal, or yeast form with many types of bacterial infections. is dependent upon environmental factors Although R. dentocariosa was frst isolated such as temperature, pH, and presence in dental caries, endocarditis is the most of serum. C. albicans is an opportunistic common infection caused by the organism. pathogen that is often the cause of infection Cases have also been reported associating in immunocompromised patients, such R. dentocariosa with intrauterine fetal death as those with HIV/AIDS or indwelling (16,25,28). R. dentocariosa is commonly medical devices. Imbalances in normal associated with dental caries and periodontal body fora due to and steroid disease. Studies have demonstrated the use have also been shown to provoke the ability of R. dentocariosa to induce the overgrowth of Candida, causing candidiasis release of TNF-a by macrophages, thereby (21). Oral candidiasis, commonly known as eliciting infammatory responses within the thrush, can exacerbate already existing oral gingiva (36). diseases. C. albicans has also been found within dental caries and is a contributor ENTEROCOCCUS FAECALIS to periodontal disease (26). Many proteins Enterococcus faecalis is a facultative within saliva have been found to promote anaerobic, Gram-positive cocci bacterium adherence of C. albicans to gingival tissues that is typically found in pairs or short (10). Additionally, tissues already colonized chains. The most common locations for by streptococcal strains greatly enhance the this microorganism in humans are the ability of C. albicans to colonize the oral gastrointestinal tract, the vagina, and the cavity (10). oral cavity. E. faecalis is considered to be the most prevalent bacterium found in root canal infections (23). It is able to resist nutritional deprivation in part due to its ability to utilize a number of energy sources including malate, citrate, lactate, BACTERIAL HOST INTERACTIONS • 67 MATERIALS AND METHODS MICROBIAL STRAINS AND plate along with 10 μl of an overnight MEDIA culture of the respective organism. The bacterial microtiter plates, excluding A. One yeast and fve bacterial strains naeslundii, were incubated at 37°C in 5% were utilized for this investigation. All CO2 for 24 hours and C. albicans and A. microorganisms were obtained from neaslundii were incubated under the same the American Type Culture Collection conditions for 48 hours. After incubation, (ATCC), Manassas, VA. The bacterial the absorbance of each well in the plates strains used were: Lactobacillus casei was measured in a spectrophotometer (ATCC 393), Rothia dentocariosa (ATCC (SpectraMax 190; Molecular Devices, Inc., 17931), Enterococcus faecalis (ATCC 29212), Sunnyvale, CA) at an optical absorbance of Actinomyces naeslundii (ATCC 12104) and 595 nm (OD595) to determine the minimum Actinomyces viscosus (ATCC 43146). The inhibitory concentration (MIC). (20) To yeast strain used was Candida albicans quantify the planktonic growth, 120μl (ATCC 10231). Four of the bacterial strains aliquots of planktonic culture fuid was were grown in tryptic soy broth + 1% removed from each well into corresponding sucrose (TSBS; Difco Laboratories, Detroit, wells in a fresh 96-well microtiter plate MI), while Actinomyces naeslundii was and the absorbances determined in a grown in brain heart infusion broth (BHI; spectrophotometer at 595 nm. Absorbance Difco). All bacteria were incubated at 37°C values greater than .05 units of the control in 5% CO2 for 24 or 48 hours. C. albicans would be considered a signifcant change. was grown in yeast peptone dextrose Anything equal or less to .05 absorbance broth (YPD; Difco) at 30°C in 5% CO2 for units would be considered insignifcant. 48 hours. The design of the experiment is consistent with other studies that have MINIMUM BIOFILM INHIBITORY been both published and conducted in this laboratory (20). CONCENTRATION Ninety six-well plates of the organisms in MINIMUM INHIBITORY varying concentrations of nicotine were CONCENTRATION AND prepared as described above. Following incubation, the remaining planktonic PLANKTONIC GROWTH culture fuid was disposed of and 200μl QUANTIFICATION of 10% formaldehyde (Fisher Scientifc Co., Nicotine dilutions were prepared using Pittsburg, PA) was added to each well for a 1 g/ml nicotine stock solution (Sigma 30 minutes at room temperature. The plate Chemical Co., St. Louis, MO) in TSBS or was then washed in deionized (DI) water BHI for the bacteria and in YPD for the before adding 200μl of 0.3% crystal violet yeast. An initial dilution of 32 mg/ml (Sigma) to each well. The plate was allowed of nicotine was prepared and a 1:2 serial to incubate for an additional 30 minutes dilution series was made through 0.25 mg/ and then washed in DI water. 200μL of ml. Additionally, a control was prepared isopropanol (Fisher) was then added to each containing only 200 μl of media with no well to allow extraction of the crystal violet nicotine. Diluted aliquots of 190μl were from the bioflm cells. Following a one-hour transferred into a sterile 96-well microtiter incubation in isopropanol at 68 • FINE FOCUS, VOL. 1 room temperature, the optical density of STATISTICAL ANALYSES the bioflm was recorded at 490 nm. Using All experiments were carried out in this method, both R. dentocariosa and A. quadruplicate wells at least three times. naeslundii were unable to form tight bioflms Means, standard deviations, and statistical in their 96-well plates. Therefore, instead of signifcance were calculated using Excel. utilizing formaldehyde, crystal violet and Student t-tests were used to determine p isopropanol, the bioflm of R. dentocariosa values, which were considered signifcant if and A. naeslundii were resuspended in 200μl P<0.05. Data was transferred to SigmaPlot of a 0.9% saline solution and the optical 12.0 to graph and further analyze results. density was measured at 595 nm.

Table 1 Minimum inhibitory concentration (MIC) and minimum bioflm inhibitory concentration (MBIC) of nicotine on microorganisms

Organisms MIC (mg/ml) MBIC (mg/ml) L. casei 16 16 A. viscosus 16 16 A. naeslundii 16 16 R. dentocariosa 16 16 E. faecalis 32 16 C. albicans 16 16

Minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) of RESULTS nicotine on microorganisms Table 1 indicates the MIC and MBIC of each decreased at this concentration of nicotine. A. microorganism tested. All organisms had naeslundii growth decreased through 8 mg/ an MIC of 16 mg/ml of nicotine except ml and demonstrated complete inhibition E. faecalis, which had an MIC of 32 mg/ beginning at 16 mg/ml. Additionally, all ml of nicotine. Similarly, the MBIC of all organisms displayed a decrease in total organisms tested was 16 mg/ml of nicotine. growth at 32 mg/ml of nicotine.

The total growth of the organisms in each Fig. 2 denotes the planktonic growth of each nicotine concentration is presented in Fig. 1. organism in nicotine. Decreased growth This data represents the total of bioflm and occurred at 32 mg/ml of nicotine in all planktonic growth. L. casei, A. viscosus, R. organisms tested. Additionally, L. casei, A. dentocariosa, and C. albicans had an increase viscosus, and R. dentocariosa demonstrated in growth through 8 mg/ml of nicotine. E. statistically signifcant inhibition of faecalis growth increased through 16 mg/ planktonic growth at 16 mg/ml. An increase ml but growth of all other organisms was in growth occurred at 8 mg/ml of BACTERIAL HOST INTERACTIONS • 69

A. Lactobacillus casei B. Actinomyces viscosus 0.800 1.000

0.800 0.600 0.600 0.400 0.400 0.200 0.200 Optical Density at 595 nm Optical Density at 595 nm 0.000 0.000 0 0.25 0.5 124816 32 0 0.25 0.5 124816 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

C. Actinomyces naeslundii D. Rothia dentocariosa 1.000 1.000

0.800 0.800

0.600 0.600

0.400 0.400

0.200 0.200

Optical Density at 595 nm 0.000 Optical Density at 595 nm 0.000 0 0.25 0.5 1 2 4 8 16 32 0 0.250.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

E. Enterococcus faecalis F. 1.000 Candida albicans 0.700 0.600 0.800 0.500 0.600 0.400 0.300 0.400 0.200 0.200 0.100

Optical Density at 595 nm 0.000 Optical Density at 595 nm 0.000 0 0.250.5 1 2 4 8 16 32 0 0.25 0.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

Fig. 1: Total growth of nicotine-treated microorganisms in quadruplicate wells. After incubating with various concentrations of nicotine the OD was measured at 595 nm. The OD value is represented as (mean + SD). Each experiment was repeated at least three times. Asterisks indicate statistical signifcance (P< 0.05) compared to the 0 mg/ml nicotine control. 70 • FINE FOCUS, VOL. 1

A. Lactobacillus casei B. Actinomyces viscosus 0.200 0.300 0.250 0.150 0.200 0.100 0.150 0.100 0.050 0.050 Optical Density at 595 nm Optical Density at 595 nm 0.000 0.000 0 0.25 0.5 124816 32 0 0.25 0.5 124816 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

C. Actinomyces naeslundii D. Rothia dentocariosa 0.050 0.250

0.040 0.200

0.030 0.150

0.020 0.100

0.010 0.050 Optical Density at 595 nm 0.000 Optical Density at 595 nm 0.000 0 0.25 0.5 1 2 4 8 16 32 0 0.250.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

E. Enterococcus faecalis F. Candida albicans 0.070 0.025

0.060 0.020 0.050 0.040 0.015 0.030 0.010 0.020 0.005 0.010 Optical Density at 595 nm Optical Density at 595 nm 0.000 0.000 0 0.250.5 1 2 4 8 16 32 0 0.25 0.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

Fig. 2: Planktonic growth of nicotine-treated microorganisms in quadruplicate wells. After incubating with various concentrations of nicotine for 48 hours, the supernatant was removed and the OD of the supernatant was measured at 595 nm. The OD value is represented as (mean + SD). Each experiment was repeated at least three times. Asterisks indicate statistical signifcance (P< 0.05) compared to the 0 mg/ml nicotine control. BACTERIAL HOST INTERACTIONS • 71

A. Lactobacillus casei B. Actinomyces viscosus 1.500 1.250 4.000

1.000 3.000 0.750 2.000 0.500

0.250 1.000 Optical Density at 490 nm Optical Density at 490 nm 0.000 0.000 0 0.25 0.5 124816 32 0 0.25 0.5 124816 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

C. Actinomyces naeslundii D. Rothia dentocariosa 1.000 0.250

0.800 0.200

0.600 0.150

0.400 0.100

0.200 0.050

Optical Density at 490 nm 0.000 Optical Density at 490 nm 0.000 0 0.25 0.5 1 2 4 8 16 32 0 0.250.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

E. Enterococcus faecalis F. Candida albicans 0.300 1.000

0.250 0.800 0.200 0.600 0.150 0.400 0.100 0.050 0.200 Optical Density at 490 nm Optical Density at 490 nm 0.000 0.000 0 0.250.5 1 2 4 8 16 32 0 0.25 0.5 1 2 4 8 16 32 Nicotine Concentration (mg/ml) Nicotine Concentration (mg/ml)

Fig. 3: Bioflm formation of nicotine-treated microorganisms in quadruplicate wells. After incubating with various concentrations of nicotine for 24 or 48 hours, the bioflm was either stained with crystal violet or suspended in saline and the OD was assessed at 490 nm or 595 nm, respectively. The OD value is represented as (mean + SD). Each experiment was repeated at least three times. Asterisks indicate statistical signifcance (P< 0.05) compared to the 0 mg/ml nicotine control. 72 • FINE FOCUS, VOL. 1

Table 1 indicates the MIC and MBIC of each increase in growth occurred at 8 mg/ml of microorganism tested. All organisms had nicotine for A. viscosus and R. dentocariosa. an MIC of 16 mg/ml of nicotine except Moreover, E. faecalis planktonic growth E. faecalis, which had an MIC of 32 mg/ was enhanced at 8 mg/ml and 16 mg/ml of ml of nicotine. Similarly, the MBIC of all nicotine. Signifcant inhibition was observed organisms tested was 16 mg/ml of nicotine. in 1 mg/ml of nicotine for A. viscosus and for 4, 8, and 32 mg/ml of nicotine in C. albicans. The total growth of the organisms in each A. naeslundii along with other bacteria in this nicotine concentration is presented in Fig. 1. study depict a high sensitivity to nicotine. This data represents the total of bioflm and The planktonic readings varied between planktonic growth. L. casei, A. viscosus, R. trials, which made interpretations of dentocariosa, and C. albicans had an increase planktonic response to nicotine inconsistent in growth through 8 mg/ml of nicotine. E. at times and diffcult to interpret. faecalis growth increased through 16 mg/ ml but growth of all other organisms was Bioflm formation of the organisms in each decreased at this concentration of nicotine. A. nicotine concentration is indicated in Fig. 3. naeslundii growth decreased through 8 mg/ An upward trend in bioflm growth through ml and demonstrated complete inhibition 8 mg/ml of nicotine was observed for L. beginning at 16 mg/ml. Additionally, all casei, A. viscosus, E. faecalis, and C. albicans. organisms displayed a decrease in total R. dentocariosa exhibited the most growth growth at 32 mg/ml of nicotine. at 1 mg/ml of nicotine but also had increased growth in 0.25, 2, and 8 mg/ml. Inhibition Fig. 2 denotes the planktonic growth of each of bioflm formation for all organisms was organism in nicotine. Decreased growth indicated at 16 and 32 mg/ml of nicotine. occurred at 32 mg/ml of nicotine in all Additionally, A. naeslundii demonstrated organisms tested. Additionally, L. casei, A. statistically signifcant inhibition beginning viscosus, and R. dentocariosa demonstrated at 1 mg/ml and continuing through 32 mg/ml statistically signifcant inhibition of of nicotine. planktonic growth at 16 mg/ml. An DISCUSSION Signifcant inhibition occurred with all demonstrated stimulatory effects by nicotine tested organisms in nicotine concentrations on Streptococcus gordonii (34). Another of 16 and 32 mg/ml except E. faecalis, study investigating nicotine effects on providing a MIC of 32 mg/ml for E. faecalis oral microorganisms like Actinobacillus and a MIC of 16 mg/ml for the remaining actinomycetemcomitans, Porphymonas organisms. Similar studies demonstrate gingivalis, Fusobacterium nucleatum and S. other oral microorganisms, most notably gordonii demonstrated no effect could be S. mutans, also have a MIC of 16 mg/ml. observed in concentrations less than 1 mg/ (7)(20) It is possible that the MIC exists at ml (13). The research conducted with these nicotine concentrations between 8 and 16 microorganisms is consistent with this study mg/ml but further studies are required to and demonstrates signifcant variations in determine the validity of this supposition. activity at higher concentrations of nicotine. Research by Huang et al. (2014) also Alternatively, Pavia et al. (2000) were able BACTERIAL HOST INTERACTIONS • 73 to demonstrate that nicotine concentrations its perceived preference to grow as a bioflm between 100 and 250 ug/ml reduced growth rather than as a planktonic yeast phase. As of Escherichia coli, Listeria monocytogenes, Fig. 3 indicates, a notable upward trend in Candida albicans, Klebsiella pneumoniae and the bioflm formation of L. casei, A. viscosus, Cryptococcus neoformans (27). A. naeslundii, E. faecalis and C. albicans was demonstrated through 8 mg/ml of nicotine. All concentrations of nicotine exerted some Bioflm growth was most notably enhanced inhibition of planktonic growth, particularly in 8 mg/ml of nicotine in the aforementioned at 16 and 32 mg/ml, when compared to the organisms. R. dentocariosa presents a non-nicotine-treated controls for both L. casei unique demonstration of enhancement in and C. albicans. Previous research suggests concentrations of nicotine ranging from that planktonic bacteria are more susceptible 0.25 to 8 mg/ml but was most prominently to various chemicals and antibiotics and so enhanced at 1 mg/ml. In addition, studies have it is possible that the presence of nicotine shown that an increase in adhesion to surfaces induced this effect on the microorganisms occurs over time when C. albican, S. mutans, studied (4). No reports were found on and S. pneumonaiae are exposed to cigarette planktonic yeasts relating to chemical smoke condensate (CSC). (4)(29) CSC is a susceptibility, but it is plausible that similar crude aqueous extract of tobacco and contains effects as those observed in planktonic approximately 2.4% nicotine by weight bacteria might also exist in yeast. All species along with many other chemical components displayed planktonic inhibition at 32 mg/ of tobacco. It is plausible that one of the ml. Furthermore, at concentrations of 16 mg/ attributing factors to the increase in adhesion ml both R. dentocariosa and L. casei were is due to the nicotine contained within the signifcantly inhibited. C. albicans growth CSC, which would thus account for the was also inhibited in nicotine concentrations results observed in this study. Additionally, of 4 and 8 mg/ml. L. casei, A. viscosus, R. Antunes et al. 2012 (3) found that oxidative dentocariosa, E. faecalis, and C. albicans stress caused increased bioflm formation of demonstrated no statistically signifcant P. aeruginosa. This study identifed hydrogen differences in non-nicotine treated planktonic peroxide as one causative agent of oxidative organisms compared to those treated with stress in cigarette smoke. Other research nicotine concentrations up to 2 mg/ml. A. has indicated nicotine can also act as an viscosus, R. dentocariosa and E. faecalis oxidative stressor, which might account for had planktonic growth enhancement in the increased bioflm formation seen in many concentrations of 16, 8, and 8 through 16 mg/ of the nicotine concentrations used in the ml, respectively. All OD values measured current study (11). The MBIC of nicotine for from the planktonic growth were much all organisms studied was determined to be lower than OD values recorded from bioflm 16 mg/ml as indicated in Fig. 3. As with the growth. Visual analysis of the microtiter plates MIC, it is possible the MBIC also exists at also signifed greater bioflm growth when some concentration between 8 and 16 mg/ml compared to planktonic growth. It is known of nicotine, but further studies are needed to that bacteria prefer existing in bioflms rather explore this probability. than as unicellular planktonic cells and it has been suggested that nearly 90% of bacteria In this present study, concentrations at exist as bioflms in nature. (31) The present which enhanced growth was demonstrated study further supports this theory as well as correspond to average nicotine extends the observation to C. albicans due to concentrations found in many of the tobacco 74 • FINE FOCUS, VOL. 1 products discussed previously. It is believed oral fora of a smoker. Additionally, because that the fndings of this study support the microbes often exhibit different behaviors hypothesis that the increased risk of oral when interacting with multiple species, health issues faced by many tobacco users it would be benefcial to conduct a study is caused in part by the stimulation of which assesses the bioflm formation of oral microorganism bioflm formation by multiple bacterial species in the mouth. nicotine and other tobacco components. This would more closely simulate an in vivo environment in an attempt to simulate This study can be used to provide dental caries, oral bioflm, periodontal disease confrmation that further research on the and other diseases present in the mouth. relationship between oral microorganisms Furthermore, investigating the types of and nicotine is needed. Under similar interactions which occur between nicotine, growth conditions, the organisms could proline-rich proteins, and bacteria would be exposed to nicotine intermittently allow for a more thorough understanding of throughout a specifed length of time, much the specifc mechanisms which cause bioflm like how a smoker smokes periodically enhancement in the presence of nicotine. throughout the day. This study emulates the nicotine exposure which occurs to the

ACKNOWLEDGEMENTS This study was fnancially supported by the Center for Research and Learning, Indiana University Purdue University at Indianapolis through a Multidisciplinary Undergraduate Research Institute (MURI) grant. Assistance and advice provided by mentors Drs. Richard Gregory, Fengyu Song, Angela Bruzzaniti, and Jack Windsor was greatly appreciated. Further thanks are also extended to Ruijie Huang, Grace Gomez, and Landon Howell for additional support. The authors declare no conficts of interest exist. REFERENCES 1. Abozoid, S., Peretz, A., Nasser, W., & Zarfn, Y. 2013. 5. Benowitz, N.L. & Jacob, P., III. 1984. Daily intake of Rare infection-- prolonged Actinomyces naeslundii nicotine during cigarette smoking. Clin. Pharmacol. bacteremia caused by severe caries. Harefuah 153:379- Therap. 35:499-504. 80, 435. 6. Brook, I. 2008. Actinomycosis: diagnosis and 2. Aligne, C.A., Moss, M.E., Auinger, P., & Weitzman, management. South. Med. J. 101:1019-23. M. 2003. Association of pediatric dental caries with passive smoking. J. Am. Med. Assoc. 289:1258- 1264. 7. Brook, I. 2003. Microbiology and management of endodontic infections in children. J. Clin. Ped. Dent. 3. Antunes, M.B., Chi, J.J., Goldstein-Daruech, N., Palmer, 28:13-7. J.N., Zhu, J., & Cohen, N.A. 2012. Molecular basis of tobacco-induced bacterial bioflms: an in vitro study. 8. Brown, J., Georg, L. K., & Waters, L. C. 1969. Otolaryngology- Head and Neck Surgery 147:876-884. Laboratory identifcation of Rothia dentocariosa and its occurrence in human clinical materials. Appl. 4. Baboni F.B., Barp D., Izidoro A.C., Samaranayake L.P., Microbiol. 17:150-156. & Rosa E.A. 2009. Enhancement of Candida albicans virulence after exposition to cigarette mainstream 9. Cannon, R.D., & Chaffn, W.L. 1999. Oral colonization by smoke. Mycopathologia 14:227–235. Candida albicans. Crit. Rev. Oral Biol. Med. 10:359-383. BACTERIAL HOST INTERACTIONS • 75

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