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Introduction

Chlorhexidine (CHX) is the most commonly used broad-spectrum antiseptic agent in oral hygiene. It is widely prescribed in different dental fields, especially in patients that cannot perform correctly mechanical biofilm control, due to physical/mental impairment, lack of motivation or xerostomia (1). Additionally, CHX has multiple applications in dentistry because of its substantivity. CHX is adsorbed by the oral mucosa, oral proteins, and onto hydroxyapatite of the dental surface (2). This adsorbed CHX is gradually released at effective doses that guarantee the persistence of its antibacterial activity. CHX is available commercially as mouthwash, gel and aerosol spray. For professional use slow-release disks or chips of CHX are available (1).

Mouthwashes containing CHX have been reported to be effective in oral hygiene regimens, as adjuvant in the management of gingivitis, periodontitis and peri-implant disease. Dental plaque is the etiological factor of gingivitis and periodontitis. Mouthwashes are an ideal vehicle to incorporate agents such as CHX that has demonstrated to have a positive effect preventing plaque accumulation and gingival inflammation (3). Commercially, these therapeutic agents are available in wide range of concentrations, or combined with secondary agents to enhance its clinical effect. In general, chlorhexidine mouthwashes can be classified as bactericidal or therapeutic when concentrations above 0,12% are used, or low-dose concentration mouthwashes for more diluted presentations (1). Figure 1 summarize some of the particular characteristics and indications (Figure 1.)

Robust scientific literature has established that patients with gingivitis, have significant reductions in plaque and gingivitis scores with mechanical oral hygiene and CHX mouthwash (4). Additionally, CHX can break up existing plaque, inhibit plaque regrowth and reduce gingival inflammation (4,5). In patients with inadequate self-performed plaque control during supportive periodontal therapy, a low CHX concentration mouth rinse (0.05%) combined with 0.05% cetyl-pyridinium chloride (an antiplaque agent) showed significant reductions of microbial loads in saliva and in the gingival sulcus (6). Specifically subgingival counts of Fusobacterium nucleatum and Prevotella intermedia decreased when compared to placebo (6). Adding fluoride to CHX mouthwash (0.1% sodium fluoride and 0.2% CHX) significantly decreased salivary S. mutans count after two weeks in 12-14 year-old students (7). The combination of these two chemotherapeutic are beneficial for certain high-risk groups for the prevention of caries and gingivitis. A meta-analysis evaluated the effect of chlorhexidine mouthwash as an adjunct to mechanical therapy in periodontal patients and found a slight reduction in probing depths and a slight effect on clinical attachment gain (9).

CHX may also be used in the treatment of halitosis by reducing halitosis-related bacteria. Mouthwashes containing low doses of CHX (0.05%), 0.05% cetylpiridinium chloride and 0.14% zinc lactate have shown to reduce organoleptic scores and volatile sulphur compounds (VSC) in patients with oral malodor (10, 11). The use of CHX (0.02%) mouth rinse alone, also produced a significant reduction in VSC and organoleptic scores (11,12). Similar results with CHX at a concentration of 0.12% were reported in combination with mechanical brushing (13,14). A mouth wash that combined zinc at 0.3%, and chlorhexidine at a low concentration of 0.025%, was efficient in removing VSC, suggesting a synergistic mode of action of these two components (15). In the treatment of halitosis, adjuvants, such as chlorhexidine mouthwashes do not substitute the medical management of the cause.

CHX has been recommended preoperatively and postoperative after oral surgery to avoid and associated complications. Before oral surgery, CHX is indicated for surgical hand antisepsis to reduce infection since there is evidence to reduce bacterial load on skin of the clinician and patient’s surgical site (18). As stated before, CHX controls biofilm, therefore it is used during the peri-operative period, before and after oral surgery procedures (1). CHX is suggested during healing phase, in cases where regular biofilm control procedures cannot be performed, since these would interfere with wound healing. The convenience of using CHX after oral surgery is due to its capacity of preventing recolonization of bacteria at the surgical site and reducing gingival signs of inflammation (19). The use of CHX on the day of surgery and several days after, has shown to prevent or reduce the occurrence of alveolitis after third molar extractions (20,21). CHX is also used as a pre-rinse to reduce aerosolization of microbes and viruses during dental procedures. In a recent study, CHX mouthwash has shown to be effective in reducing the SARS-CoV-2 viral load in saliva for a short-term period, two hours after using 15ml 0.12% CHX once (22).

Orthodontic patients have a high prevalence of gingivitis due to the presence of brackets, bands and other accessories that facilitate biofilm and calculus formation (23). Data indicate that the use of CHX mouthwash at 0.12% concentration in a short-term period of 3 months, in addition to regular oral hygiene habits, is effective in reducing plaque and gingivitis in adolescents undergoing orthodontic treatment (24). Also, a mouthwash of CHX at 0.2% and toothbrushing has proven to reduce significantly biofilm and gingivitis levels in adolescents wearing brackets (25,26). The use of CHX mouthwashes at 0.2% in patients with fixed orthodontic appliances led to a significant reduction in the level of Streptococcus mutans (27). Nonetheless, because of its adverse effects after continuous use, CHX at both concentrations 0.12% and 0.2% should not be indicated for long-term periods, and only should be considered for treating acute gingival inflammation.

CHX is normally used in concentrations ranging from 0.12% and 0.2%. Both concentrations have been reported as safe, and have a low level of tissue toxicity. Allergic reactions to CHX are not a common adverse event reported in the general population. Even though it has been pointed out as an efficacious compound with antibacterial activities, it use has been associated with staining of teeth and oral mucosa, and in some cases may be related with dysgeusia. Therefore, mouthwashes with concentrations of CHX at 0.12% and 0.2% are both recommended for short-term use. Lower doses of CHX (0.06%) provide a comparable effect in preventing biofilm formation and control of gingivitis when compared to formulations with higher CHX concentrations. The type of action is dose dependent, for instance, lower concentrations of CHX have been reported to have bacteriostatic properties, and higher concentrations a bactericidal effect (1). A clinical study examined the dental plaque and gingivitis inhibitory effects of low-dose 0.06% CHX preparations in comparison with CHX at 0.1%, an amine fluoride/stannous fluoride (ASF) solution and a water control as an adjunct to the daily mechanical oral-hygiene measures (28). After three months, participants using both concentrations of CHX had less plaque accumulation, suggesting that if maintaining clinical health is the goal, CHX at 0.06% is a good alternative to CHX at 0.1% since patients exhibit less teeth staining (29).

Even though CHX was introduced to the dental market almost seven decades ago, few studies have evaluated the antimicrobial and antifungal capacity of different concentrations of CHX mouthwashes. Therefore, the aim of this study, was to evaluate in vitro, the antibacterial and antifungal capacity of three commercially available mouthwashes, with different concentrations of CHX, 0.12%, 0.06%, and 0.03%.

Materials and methods

To test the antibacterial and antifungal capacity of three available mouthwashes with different concentrations of CHX, three molecules were selected from the same company (Stein Co, Cartago, Costa Rica). All the samples were blinded for the experimental operator, who only identified the solutions by its color. The commercial mouthwashes tested are shown in table 1.

The experimental method selected was the Kirby-Bauer method to evaluate the antibacterial effect of each compound by measuring the inhibitory effect of the bacteria or fungal strain exposed to the antiseptic solution. This method was employed selecting 4 different strains of ATCC cultures. Table 2 summarize the strains and the antibiotic or antifungal agent used as control.

Bacteria strains were seeded in individual blood agar plates, while Candida albicans ATCC® 90028™ (C. albicans) was seeded in Sabouraud dextrose agar (SDA). All strains were cultured for 24 hours. From each bacterial strains, two pure colonies were seeded in tryptic soy broth: and for C. albicans in yeast extract peptone dextrose agar. Broths were homogenize in continuous stirring under 100rpm, at 37ºC. Three consecutive washes with phosphate buffer solution (PBS) were performed, and colonies were adjust to a 0,5 standard McFarland concentration scale. Bacteria strains were seeded in Mueller-Hinton Agar; and C. Albicans in Mueller-Hinton with Methylene Blue and dextrose.

The antiseptic discs were prepared adding 20µL of each solution to standard clean paper discs, while standard antibiotic/antifungal discs were selected according to each strain. Each disc was placed 5 minutes after the strains were seeded, and each one kept a minimal distance of 20mm between each other. All samples were prepared by triplicate. After all the discs were placed, Petri dishes were placed in an incubator at 37ºC for 24 hours. The antimicrobial effectiveness was evaluated by measuring the zones of inhibition, using a millimetric rulers that must pass right in the middle of the disc (with a standard measure of 6mm).

Statistical analysis

The statistical analysis was done using R Software version 3,5. Shapiro-Wilk’s test was used to evaluate the normality of the data, and the Levene´s test to evaluate the homogeneity of variances. The differences between the zones of inhibition was evaluated by one-way ANOVA. Statistical significance was determined at p<0.05.

Results

For the Staphylococcus aureus subsp. Aureus ATCC® 25923™ (S. aureus) group (Figure 2) the inhibition zone between the groups showed statistical difference (F=399,6; p<0,001). Multiple comparison was evaluated with a Tukey post hoc test, showing that the antibiotic inhibition zone of the antibiotic disc was statistically different (p<0.001) when compared with the three mouthwashes. All the mouthwashes were different between each other, showing an inhibition zone proportional to the concentration of the CHX.

Enterococcus faecalis ATCC® 29212™ (E. faecalis) group showed significant statistical differences between the inhibition zones of all samples (F=73,54; p<0,001) (Figure 3). Multiple comparison was evaluated with a Tukey post hoc test, showing that the antibiotic inhibition zone of the antibiotic disc showed statistically significant differences (p<0.001). Mouthwashes containing 0,12% and 0,06% CHX didn’t showed significant statistical differences, but both groups were different than the 0,03% CHX mouthwash.

Escherichia coli ACCT® 8739™ (E. coli) group showed significant statistical differences between the inhibition zones of all samples (F=46.28; p<0,001) (Figure 4). Post hoc Tukey analysis showed significant statistical differences for the antibiotic disc, superior than all the mouthwashes, with no differences between each other (p>0.05).

Finally, antifungal effect in the C. albicans group showed also significant statistical differences between the inhibition zones of all samples (F=1041; p<0,001) (Figure 5). Post hoc Tukey analysis showed significant statistical differences within the groups. The inhibition zone of the antifungal disc was superior to all mouthwashes (p<0.001); followed by 0,12% CHX mouthwash that showed significant statistical differences (p<0.05). 0,06% and 0,03% CHX mouthwashes didn´t show significant statistical differences.

Discussion

In this study, the antibacterial and antifungal capacity of three commercially available mouthwashes with different concentrations of CHX (0.12%, 0.06%, and 0.03% respectively) was evaluated in vitro. All samples showed some degree of antibacterial and antifungal effect.

The anti-staphylococcal activity of CHX mouthwashes at concentrations of 0.2% and 0.12% had been observed previously in other studies (30, 31, 32, 33). In the present investigation, all the mouthwashes inhibited S. aureus growth in vitro. The three different concentrations showed an inhibition zone proportional to the concentration of CHX. Our results suggest that lower doses of CHX are useful as part of decontamination regimens to decolonize presence of staphylococcal activity in the oral cavity and oropharynx. Isolates of S. aureus have been obtained in subgingival and supragingival plaque of patients with periodontitis and/or periimplantitis. This is relevant, since the reduction in the number of S. aureus in the oral cavity, prior to surgical procedures, has been associated with a lower incidence of infective endocarditis and postoperative complications (32) in patients with respiratory infections.

The antimicrobial effect of 2% chlorhexidine against E. faecalis has been vastly reported in literature. In this study, the three concentrations of CHX mouthwashes analyzed were effective against E. faecalis, showing a lower effect than the control. Concentrations of 0,12% and 0,06% didn´t showed statistical differences, but both were statistically different when compared to 0,03% concentration . E. faecalis is a component of the normal oral microbiota. It is of interest in endodontic research, since it is one of the species mainly involved in failures of root canal treatment (34,35). E. faecalis can exhibit resistance against common disinfectants and endodontic irrigants (36,37). Further research must evaluate the clinical importance of using regularly low concentrations of CHX, against the E. faecalis community in oral cavity.

E. coli is a gram-negative bacteria, commonly found in the gut of humans, and most strains do not cause human disease. However, some strains produce toxins and other virulence factors, such as lipopolysaccharide, that may produce food poisoning if people swallow E. coli by eating contaminated foods, by drinking contaminated water, or by hand to mouth contact. The presence of E. coli has also been reported in subgingival biofilm of patients with periodontitis, and a significant higher prevalence was observed in diseased sites of periodontal patients compared to periodontally healthy subjects (38). Case reports and in vitro studies have associated gram-negative enteric rods to early dental implant failure and peri-implantitis (39,40,41). Our results indicate that the three concentrations of CHX mouthwashes displayed a similar inhibition zone to E. coli. It has to be considered that only a single strain of E. coli was used in this experiment. Therefore, this may pose a limitation in the interpretation of the results since there can be great genetic variation with different strains of the same specie.

The antimicrobial activity of chlorhexidine against C. albicans and other common non-albicans yeast species has been documented and advocate the use of CHX gluconate as an adjunct in the management of oral candidiasis (42,43,44). CHX at a concentration of 0.2% has shown a significant antifungal activity comparable to ketoconazole (45). Even a brief exposure to subtherapeutic concentrations of chlorhexidine (0.00125, 0.0025 and 0.005%) for thirty minutes, modulates germ tube formation of C. albicans isolates and may suppresses pathogenicity (45). In our study, the inhibition zone of 0,12% CHX mouthwash was superior to lower concentrations, 0,06% and 0,03%. However, all showed antifungal capacity. Such result is particularly important, considering vulnerable populations to fungal oral infections, such as removable prothesis users or immunocompromised patients. Low dose chlorhexidine mouthwash employed in these patients routinely may show important clinical benefits.

A meta-analysis that included publications evaluating CHX’s efficacy against bacterial plaque and gingivitis for six months, demonstrated superiority in those patients who included CHX mouthwashes in their oral hygiene routine (46). CHX at a concentration of 0.12% demonstrated statistically superior efficacy controlling dentobacterial plaque in all the publications evaluated (46). In cases of generalized gingivitis, CHX mouthwashes have shown greater efficacy than the use of gels (47). CHX rinsing protocols should be the first choice for those patients where daily oral hygiene is difficult (47). Rinsing for 60 seconds 2 times a day with 10 mL of chlorhexidine can inhibit the growth of dentobacterial plaque by 60%; and reduce gingivitis by 50-80% (4).

Even though we provide in vitro results, our findings are of relevance since all the species used in our experiment are microorganisms that may be present in dental plaque. Most studies on CHX’s control of dental plaque and clinical indices do not show differences between concentrations of 0.12% and 0.2%; however, at lower doses, fewer adverse effects have been reported. In our study, the three concentrations of CHX had antibacterial and antifungal capacity. Low dose regimens could therefore reduce side effects, such as tooth discoloration, taste disturbance, stain and mucosal erosions, produced by CHX, but maintain reasonable antibacterial and antifungal efficacy.

Although this research offers important evidence of the antiseptic effect of CHX mouthwashes, important limitations and perspectives must be discussed before considering clinical extrapolations. First, this in vitro study may not reflect the clinical scenario in humans, considering the existence of multispecies biofilm in both periodontal and dental tissues, so confirmatory clinical data will be needed. Second, the use of ATCC strains may behave different from planktonic bacteria obtained from different populations, thus further ex vivo studies using bacteria obtained from human patients is suggested. Finally, a comparison of the observed effect on single cultured bacteria in Petri dishes vs biofilm in vitro models will help to confirm the validity of these results.

Conclusions

The low dose and therapeutic concentrations of chlorhexidine in the mouthwashes showed a different degree of inhibitory effect against the bacterial and fungal strains tested, exhibiting a concentration related behavior in most of the cases.

Acknowledgements

Stein Laboratories, Costa Rica and University of Costa Rica project B9022.

Conflict of Interest

None.

Author contribution statement

Conceptualization and design: DCB, VEV.

Literature review: DCB, KRC.

Methodology and validation: DCB, VEV.

Formal analysis: DCB, KRC, VEV.

Investigation and data collection: VEV.

Resources: DCB, VEV.

Data analysis and interpretation: DCB, KRC, VEV.

Writing-original draft preparation: DCB, KRC.

Writing-review and editing: DCB, KRC, VEV.

Supervision: DCB, KRC, VEV.

Project administration: DCB, KRC, VEV.

Funding acquisition: DCB, KRC, VEV.

References:

Varoni E., Tarce M., Lodi G., Carrassi A. (2012) Chlorhexidine (CHX) in dentistry: state of the art. Minerva Stomatol ; 61 (9): 399-419.
Hjeljord L.G. Rolla G. Bonesvoll (1983) Chlorhexidine-protein interactions. J. Periodontal Res Suppl. 12: 11-16.
Van der Weijden F.A., Van der Sluijs E., Ciancio S.G., Slot D.E. (2015). Can Chemical Mouthwash Agents Achieve Plaque/Gingivitis Control? Dent Clin North Am; 59 (4): 799-829.
Van Strydonck D.A.C., Slot D.E., Van der Velden U., Van der Weij-den F. (2012). Effect of a chlorhexidine mouthrinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J Clin Periodontol ; 39: 1042-1055.
Löe H., Schiott C.R. (1970). The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodontal Res ; 5 (2): 79-83.
Escribano M., Herrera D., Morante S., Teughels W., Quirynen M., Sanz M. (2010). Efficacy of a low-concentration chlorhexidine mouth rinse in non-compliant periodontitis patients attending a supportive periodontal care programme: a randomized clinical trial. J Clin Periodontol. ; 37 (3): 266-75.
Sadat Sajadi, F., Moradi, M., Pardakhty, A., Yazdizadeh, R., & Madani, F. (2015). Effect of Fluoride, Chlorhexidine and Fluoride-chlorhexidine Mouthwashes on Salivary Streptococcus mutans Count and the Prevalence of Oral Side Effects. Journal of dental research, dental clinics, dental prospects, 9 (1), 49-52.
de Waal Y.C., Raghoebar G.M., Meijer H.J., Winkel E.G., van Winkelhoff A.J. (2015). Implant decontamination with 2% chlorhexidine during surgical peri-implantitis treatment: a randomized, double-blind, controlled trial. Clin Oral Implants Res ; 26 (9): 1015-23.
Luiz Fernando Noira Passos da Costa, Cristine da Silva Furtado Amaral, Davi da Silva Barbirato, Anna Thereza Thomé Leão, Mariana Fampa Fogacci (2017). Chlorhexidine mouthwash as an adjunct to mechanical therapy in chronic periodontitis: A meta-analysis, The Journal of the American Dental Association ; 148 (5): 308-318.
Winkel E.G., Roldán S., Van Winkelhoff A.J., Herrera D., Sanz M. (2003). Clinical effects of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinc-lactate on oral halitosis. A dual-center, double-blind placebo-controlled study. J Clin Periodontol ; 30 (4):300-6.
Fedorowicz Z., Aljufairi H., Nasser M., Outhouse T.L., Pedrazzi V. (2008). Mouthrinses for the treatment of halitosis. Cochrane Database Syst Rev; (4): CD006701.
van den Broek A.M., Feenstra L., de Baat C. (2008). A review of the current literature on management of halitosis. Oral Dis ; 14 (1): 30-9.
Bosy A., Kulkarni G.V., Rosenberg M., McCulloch C.A. (1994).Relationship of oral malodor to periodontitis: evidence ofindependence in discrete subpopulations. J Periodontol; 65: 37-46.
De Boever E.H., Loesche W.J. (1995). Assessing the contributionof anaerobic microflora of the tongue to oral malodor.J AmDent Assoc; 126: 1384-1393.
Thrane P.S., Young A., Jonski G., Rölla G. (2007). A new mouthrinse combining zinc and chlorhexidine in low concentrations provides superior efficacy against halitosis compared to existing formulations: a double-blind clinical study. J Clin Dent ; 18 (3): 82-6.
de Assis D.F., Prado M. do, Simão R.A. (2011) . Evaluation of the interaction between endodontic sealers and den-tin treated with different irrigant solutions. J Endod; 37:1550-2
Ferguson D.B., Marley J.T., Hartwell G.R. (2003). The effect of chlorhexidine gluconate as an endodontic irrigant onthe apical seal: long-term results. J Endod; 29: 91-4.
Tanner J., Dumville J.C., Norman G., Fortnam M. (2016). Surgical hand antisepsis to reduce surgical site infection. Cochrane Database Syst Rev; (1):CD004288.
Cortellini P., Labriola A., Zambelli R., Prato G.P., Nieri M., Tonetti M.S. (2008). Chlorhexidine with an anti discoloration system after periodontal flap surgery: a cross-over, randomized, triple-blind clinical trial. J Clin Periodontol; 35 (7): 614-20.
Caso A., Hung L.K., Beirne O.R. (2005). Prevention of alveolar osteitis with chlorhexidine: a meta-analytic review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod; 99 (2): 155-9.
Minguez-Serra M.P., Salort-Llorca C, Silvestre-Donat FJ (2009). Chlorhexidine in the prevention of dry socket: effectiveness of different dosage forms and regimens. Med Oral Patol Oral Cir Bucal ; 14 (9): e445-9.
Yoon, J. G., Yoon, J., Song, J. Y., Yoon, S. Y., Lim, C. S., Seong, H., Noh, J. Y., Cheong, H. J., & Kim, W. J. (2020). Clinical Significance of a High SARS-CoV-2 Viral Load in the Saliva. Journal of Korean medical science, 35 (20): e195.
Davies T.M., Shaw W.C., Worthington H.V., Addy M., Dummer P., Kingdon A. (1991). The effect of orthodontic treatment on plaque and gingivitis. Am J Orthod Dentofacial Orthop; 99 (2):155-6.
Anderson GB, Bowden J, Morrison EC, Caffesse RG (1997). Clinical effects of chlorhexidine mouthwashes on patients undergoing orthodontic treatment. Am J Orthod Dentofacial Orthop. 1997; 111 (6): 606-12.
Gehlen I., Netuschil L., Berg R., Reich E., Katsaros C. (2000). The influence of a 0.2% chlorhexidine mouthrinse on plaque regrowth in orthodontic patients. A randomized prospective study. Part I: clinical parameters. J Orofac Orthop; 61 (1): 54-62.
Ousehal L., Es-Said R., Hamdoune H., Elquars F., Khadija A. (2011). Evaluation of dental plaque control in patients wearing fixed orthodontic appliances: a clinical study. Int Orthod; 9 (1): 140-55.
Enita N., Dzemidzic V., Tiro A., Hadzic S. (2015). Antimicrobial activity of chlorhexidine in patients with fixed orthodontic appliances. Braz. J. Oral Sci; 10 (2): 79-82.
Claydon N., Smith S., Stiller S., Newcombe RG, Addy M (2002). A comparison of the plaque-inhibitory properties of stannous fluoride and low-concentration chlorhexidine mouthrinses. J Clin Periodontol ; 29 (12): 1072-7.
Hoffmann T., Bruhn G., Richter S. et al (2001). Clinical controlled study on plaque and gingivitis reduction under long-term use of low-dose chlorhexidine solutions in a population exhibiting good oral hygiene. Clin Oral Invest; 5: 89-95.
A.J. Smith, D. Morrison, Douglas Robertson, Michael K. Tang, Z Al-Doori (2003) Efficacy of oral hygiene products against MRSA and MSSA isolates. Journal of Antimicrobial Chemotherapy; 52 (4): 738-739.
Nascimento A.P., Tanomaru J.M., Matoba-Júnior F., Watanabe E., Tanomaru-Filho M., Ito I.Y. (2008). Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary Staphylococcus aureus. J Appl Oral Sci;16 (5): 336-9.
Albuquerque R.F. Jr., Head T.W., Mian H., Rodrigo A., Müller K., Sanches K., Ito I.Y. (2004). Reduction of salivary S. aureus and mutans group streptococci by a preprocedural chlorhexidine rinse and maximal inhibitory dilutions of chlorhexidine and cetylpyridinium. Quintessence Int; 35: 635-40.
Aghazadeh M., Eslami H., Kafil H., Aghazadeh Z., Behroozian A., Motahari P. (2016). A Comparison of antimicrobial effect of the mouthwash containing chlorhexidine, Cetylpyridinium chloride, and zinc lactate (Halita) and chlorhexidine against Pseudomonas aeruginosa and staphylococcus aureus: in vitro study. Scholars Bulletin. 2. 547-553.
Sundqvist G., Figdor D., Sjögren U. (1998). Microbiology analyses of teeth with endodontic treatment and the outcome of conservative retreatment. Oral Surg Oral Med Oral Pathol; 85: 86-93.
Neglia R., Ardizzoni A., Giardino L., Ambu E., Grazi S., Calignano S., et al (2008). Comparative in vitro and ex vivo studies on the bactericidal activity of Tetraclean, a new generation endodontic irrigant, and sodium hypochlorite. New Microbiol; 31: 57-65.
Ardizzoni A., Blasi E., Rimoldi C., Giardino L., Ambu E., Righi E., et al (20090. An in vitro and ex vivo study on two antibiotic-based endodontic irrigants: a challenge to sodium hypochlorite. New Microbiol; 32: 57-66.
Portenier I., Waltimo T.M.T., Ørstavik D., Haapasalo M. (2005). The susceptibility of starved, stationary phase and growing cells of Enterococcus faecalis to endodontic medicaments. J Endod ;31: 380-6.
Souto, Renata & Andrade, Arnaldo & Uzeda, Milton & Colombo, Ana. (2006). Prevalence of “non-oral” bacteria in subgingival biofilm of subjects with chronic periodontitis. Brazilian Journal of Microbiology - Brazilian Journal of Microbiology; 37: 208-215.
Ardila Medina, Carlos Martín, & Villa-Correa, Yesica Alejandra. (2015). Gram-Negative Enteric Rods Associated to Early Implant Failure and Peri-Implantitis: Case Report and Systematic Literature Review. International journal of odontostomatology, 9 (2): 329-336.
Leonhardt A., Dahlén G., Renvert S. (2203). Five-year clinical, microbiological, and radiological outcome following treatment of peri-implantitis in man. J Periodontol;7 4 (10): 1415-22.
Kuo, Hsien-Nan & Mei, Hsiang-I & Liu, Tung-Kuan & Liu, Tse Ying & Lo, Lun-Jou & Lin, Chun-Li. (2017). In Vitro Laser Treatment Platform Construction with Dental Implant Thread Surface on Bacterial Adhesion for Peri-Implantitis. BioMed Research International. 2017: 1-7.
Ellepola A.N., Samaranayake L.P. (2001). Adjunctive use of chlorhexidine in oral candidoses: a review. Oral Dis. 2001 Jan; 7 (1): 11-7.
Suci, P. A., & Tyler, B. J. (2002). Action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage Candida albicans biofilm. Antimicrobial agents and chemotherapy, 46 (11), 3522-3531.
Shino B., Peedikayil F.C., Jaiprakash S.R., Ahmed Bijapur G, Kottayi S., Jose D. (2016). Comparison of Antimicrobial Activity of Chlorhexidine, Coconut Oil, Probiotics, and Ketoconazole on Candida albicans Isolated in Children with Early Childhood Caries: An In Vitro Study. Scientifica; 2016: 1-5.
Ellepola A., N., B., Joseph B., J., Khan Z., U. (2012). Effects of Subtherapeutic Concentrations of Chlorhexidine Gluconate on Germ Tube Formation of Oral Candida. Med Princ Pract; 21:120-124.
Gunsolley J.C. (2006). A meta-analysis of six-month studies of antiplaque and antigingivitis agents. J Am Dent Assoc;137 (12): 1649-57.
Supranoto S.C., Slot D.E., Addy M., Van der Weijden G.A. (2015). The effect of chlorhexidine dentifrice or gel versus chlorhexidine mouthwash on plaque, gingivitis, bleeding and tooth discoloration: a systematic review. Int J Dent Hyg ; 13 (2): 83-92.