|Year : 2020 | Volume
| Issue : 1 | Page : 3-8
Determination of the antibacterial activity of atorvastatin against periodontal pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in vitro study
Swetalin Das, Pushpa S Pudakalkatti, Ancia Vaz, Prabhdeep Kour, Sreeshma Padmanabhan
Department of Periodontics, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India
|Date of Submission||25-Jul-2019|
|Date of Acceptance||02-Dec-2019|
|Date of Web Publication||30-Apr-2020|
Dr. Swetalin Das
Department of Periodontics, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, R. S. No. 47A/2, Bauxite Road, Belagavi, Karnataka - 590 010
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of the present study was to determine the antimicrobial activity of pure atorvastatin drug against the primary periodontal pathogens. Materials and Methods: Both minimum inhibitory concentrations and minimum bactericidal concentrations were used to assess the antibacterial effect of atorvastatin against Actinobaillus actinomycetemcomitans and Prophyromonas gingivalis by serial dilution method and culture method, respectively. Results: In the present study, both A. actinomycetemcomitans and P. gingivalis were sensitive to pure atorvastatin drug. P. gingivalis was found to be more sensitive than A. actinomycetemcomitans in this study. Conclusions: Data suggest a potent antimicrobial activity of atorvastatin against both A. actinomycetemcomitans and P. gingivalis. Hence, atorvastatin can be prescribed as a dual action drug in patients suffering from both hyperlipidemia and periodontal disease.
Keywords: Aggregatibacter actinomycetemcomitans, atorvastatins, minimum bactericidal concentration, minimum inhibitory concentration, porphyromonas gingivalis
|How to cite this article:|
Das S, Pudakalkatti PS, Vaz A, Kour P, Padmanabhan S. Determination of the antibacterial activity of atorvastatin against periodontal pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in vitro study. J Interdiscip Dentistry 2020;10:3-8
|How to cite this URL:|
Das S, Pudakalkatti PS, Vaz A, Kour P, Padmanabhan S. Determination of the antibacterial activity of atorvastatin against periodontal pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in vitro study. J Interdiscip Dentistry [serial online] 2020 [cited 2022 Oct 5];10:3-8. Available from: https://www.jidonline.com/text.asp?2020/10/1/3/283534
| Clinical Relevance to Interdisciplinary Dentistry|| |
Clinical relevance- Antimicrobial agents and antibiotics are identified to cause antimicrobial resistance. Hence, it would be of great benificial to the patient, if a single drug could simultaneously take care of more than a single infection like periodontitis and other systemic disease.
| Introduction|| |
Periodontal diseases are inflammatory and destructive diseases of the dentogingival complex associated with specific periodontal pathogens that colonize the tooth surface, gingival margin, and subgingival environment. Periodontal diseases are oral bacterial diseases that affect 10%–15% of the world's population. Although about 600 bacterial species exist in the oral cavity, only 10–15 bacterial species are recognized as potential periodontal pathogens. Out of these, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are considered the important pathogens for the initiation and progression of destruction of tooth-supporting structures.
A. actinomycetemcomitans is a highly nonmotile, Gram-negative, facultative anaerobe. The virulence factor of this pathogen includes a number of potentially damaging metabolites including leukotoxin and cytolethal distending toxin and has a high capacity to invade the host cell.P. gingivalis is also a nonmotile, asaccharolytic, Gram-negative, obligate anaerobic rod that produces large array of virulence factors such as hemoglobin receptor protein, cysteine, and proteases., Many longitudinal and retrospective studies were able to show an increased risk of periodontal breakdown in A. actinomycetemcomitans- and P. gingivalis-positive sites and better posttreatment results in their absence.
As these bacteria can invade periodontal tissues, mechanical therapy alone is sometimes ineffective. In this case, adjunctive antimicrobial therapy along with appropriate mechanical therapy would be effective in the significant reduction of both A. actinomycetemcomitans and P. gingivalis. The use of antibiotics in periodontal therapy mainly focuses on pathogenic microbiota, the patient's response, and the choice of the appropriate drug.
Minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial drug that will inhibit the visible growth of an organism, and minimum bactericidal concentration (MBC) is defined as the lowest concentration of an antimicrobial drug that will prevent the growth of an organism after subculture onto antibiotic-free media.In vitro detection of MIC of a drug against pathogens serves as a guideline for its in vivo application. Clinicians use MIC scores to choose appropriate antibiotics for patients with specific infections and to identify an effective dose of the drug.
The MIC and MBC of various commonly used antimicrobials have been investigated against both A. actinomycetemcomitans and P. gingivalis.[Table 1] and [Table 2] represent the MIC and MBC values of some of the antibiotics against A. actinomycetemcomitans and P. gingivalis.,
|Table 1: Minimum inhibitory concentrations and minimum bactericidal concentrations (μg/ml) of Aggregatibacter actinomycetemcomitans strains|
Click here to view
|Table 2: Minimum inhibitory concentrations and minimum bactericidal concentrations (μg/ml) for Porphyromonas gingivalis strains|
Click here to view
Statins are drugs that lower the level of cholesterol in the blood by reducing the production of cholesterol by the liver, providing an important and effective approach for the treatment of hyperlipidemia by reducing the blood cholesterol levels. According to angiographic studies, these drugs reduce the progression and may induce the regression of atherosclerosis. Statins differ in terms of their chemical structures, pharmacokinetic profiles, and lipid-modifying efficacy. The chemical structures of statins govern their watersolubility, which in turn influences their absorption, distribution, metabolism and excretion. Statins are believed to increase bone formation by stimulating the production of bone morphogenetic protein-2 which may play an important role in periodontal bone and ligament growth. Hence, it could be possible that statins might be protective not only to act as cardiovascular disease protector but also could be effective against chronic periodontal diseases.
Statins also have a number of pleiotropic effects such as anti-inflammatory, immune modulatory, antioxidant, and anticarcinogenic properties. Besides their antibone resorptive property, they also exert anabolic effect on bone.
Classification of statins
Statins are divided into two groups, according to their structure, as follows:
- Type I – Lovastatin, pravastatin, and simvastatin
- Type II – Fluvastatin, cerivastatin, atorvastatin, and rosuvastatin.
The difference between Type 1 and Type 2 statins is the replacement of the butyryl group of Type 1 statins by the fluorophenyl group of Type 2 statins.
Statins can also be classified by the way they are manufactured. Some are derived from microorganisms through biotechnology. These are called fermentation derived or Type 1, for example, lovastatin, pravastatin, and simvastatin. [Figure 1] shows the molecular structure of statin.
Others are made through chemical synthesis (no living organisms involved). These are synthetic or Type 2 statins. This Type 2 statins are made through chemical synthesis, for example, fluvastatin, cerivastatin, atorvastatin, and rosuvastatin.
Atorvastatin is a Type 2 statin and is obtained by chemical synthesis. It is marketed in the form of tablets (10–80 mg) under the trade name LIPITOR. Atorvastatin wasfirstly synthesized in the year 1985 by Bruce Roth while working at Parke–Davis Warner–Lambert Company (now Pfizer). In the year 2008, Lipitor became the top-selling branded pharmaceutical in the world. [Figure 2] shows the molecular structure of atorvastatin.
Atorvastatin helps in lowering blood cholesterol levels, and it has also been reported to stabilize plaque and prevent stroke through its anti-inflammatory and other mechanisms. This drug is also used to prevent hypercholesterolemia and mixed dyslipidemia to reduce total cholesterol, low-density lipoprotein (LDL)-cholesterol, apo-B, triglycerides, and C-reactive protein levels. Studies investigating atorvastatin have shown that its treatment leads to significant reductions in the levels of pro-inflammatory cytokines (tumor necrosis factor, interleukin [IL]-1 IL-1, and IL-6). In another study, atorvastatin significantly decreased bone resorption markers, including levels of serum IL-6. It also decreased COX-2 expression within peripheral blood monocytes in patients with acute myocardial infarction and increased IL-10 level in a dose-dependent manner. Atorvastatin has also been used to inhibit metalloproteinases, osteoclastogenesis, and bone destruction and the expression of the receptor activator of nuclear factor-kappa B ligand.
The present scientific literature on atorvastatin revealed its good applicability in the field of periodontics as a local drug delivery agent due to its anti-inflammatory and bone regenerative properties. Systemic antimicrobial effects of statins have also been proved. MIC values of different statins were evaluated against various nonperiodontal pathogens. The result showed better antibacterial activity of atorvastatins on majority of the tested bacteria [Table 3].
|Table 3: Minimum inhibitory concentrations of different statins against few systemic bacteria|
Click here to view
Although atorvastatin's antimicrobial activity was proven on many systemic pathogenic bacteria, until date, no studies have been carried out on its activity against specific periodontal pathogens. Hence, the presentin vitro study was aimed to find out the MIC and MBC values of pure atorvastatin drug that can be safely and effectively administered as an antimicrobial agent on specific periodontal pathogens, P. gingivalis and A. actinomycetemcomitans.
| Materials and Methods|| |
This study was conducted at the Department of Periodontology and Microbiology of Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre.
Pure atorvastatin drug (powder form) was obtained from IPCA laboratories limited, Mumbai, Maharashtra, India. It was certified to be free from any form of bacteria, yeast, or mold by the manufacturer after microbial analysis.
Preparation of bacterial suspension
From the maintained frozen stock cultures of A. actinomycetemcomitans and P. gingivalis, small quantity of cells was recovered and subcultured. Brain–heart infusion (BHI) broth was used as the culture medium to support the growth of bacteria. This culture was transferred into tubes containing 2 ml of the BHI medium to get culture suspension of A. actinomycetemcomitans and P. gingivalis. The selected test bacterial strains were adjusted for 0.5 McFarland turbidity standards to check the MIC of simvastatin against them.
Determination of minimum inhibitory concentrations
In this study, serial tube dilution technique was followed. Stock solution was prepared by dissolving the pure drug in dimethyl sulfoxide at a concentration of 10 mg/ml. Nine dilutions of each drug was done with BHI for MIC. In the initial tube, 20 μl of drug was added into the 380 μl of BHI broth. For dilutions, 200 μl of BHI broth was added into the next nine tubes separately. Then, from the initial tube, 200 μl was transferred to the first tube containing 200 μl of BHI broth. This was considered 10-1 dilution. From 10-1 diluted tube, 200 μl was transferred to the second tube to make 10-2 dilution. The serial dilution was repeated up to 10-9 dilution for each drug. From the maintained stock cultures of required organisms, 5 μl was taken and added into 2 ml of BHI broth. In each serially diluted tube, 200 μl of the above culture suspension was added. The tubes were incubated in an anaerobic jar at 37°C for 24 h and observed for turbidity, which indicates the growth of the organisms. The turbidity in each tube was compared with a positive control, which contained only the pure bacterial culture. The minimum concentration of the drug in the tube, which does not show any turbidity, was considered as MIC of the drug for that particular test organism.
Determination of minimum bactericidal concentrations
From the MIC dilution tubes, first, 3 or 5 tubes (which were sensitive in MIC) were spread across the center of a blood agar plate and allowed to dry for 20 min. After the plates had dried, a sterile spreading rod was used to evenly disperse the inoculum over the entire surface of the plate, which was then incubated at 37°C for 24 h, and the colonies were counted on the next day. The MBC was carried out to observe the bactericidal effect of atorvastatin against A. actinomycetemcomitans and P. gingivalis. If there was no growth of microorganism, then the drug was known to have bactericidal effect.
The MBC was recorded as the lowest concentration of the drug that prevents the growth of the bacteria, which reduces the viability of the initial bacterial inoculum.
| Results|| |
In the present study, both A. actinomycetemcomitans and P. gingivalis were sensitive to pure atorvastatin drug. The MIC values of P. gingivalis were sensitive until 0.8 μg/ml dilution and that of A. actinomycetemcomitans were sensitive until 12.5 μg/ml. P. gingivalis was found to be more sensitive than A. actinomycetemcomitans.
The MBC value of P. gingivalis showed bactericidal effect until 12.5 μg/ml dilution and of A. actinomycetemcomitans showed bactericidal effect until 50 μg/ml dilution. Atorvastatin showed greater bactericidal activity against P. gingivalis than A. actinomycetemcomitans [Table 4].
|Table 4: Minimum inhibitory concentration and minimum bactericidal concentration values of atorvastatins against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis|
Click here to view
| Discussion|| |
Periodontitis is a microbial disease characterized by an inflammatory breakdown of the tooth-supporting structures. The most desirable outcome of periodontal therapy is regeneration of the periodontal tissues lost as a result of disease. The need to achieve greater periodontal regeneration requires the use of an agent, which not only inhibits resorption of the alveolar bone but also stimulates new bone formation. Bisphosphonates such as alendronate are commonly used group of drugs which inhibit bone resorption by blocking the mevalonate pathway. However, bisphosphonates do not have the ability of new bone formation. Another widely used class of drugs is that of statins such as simvastatin and atorvastatin. These drugs have been shown to inhibit bone resorption and stimulate new bone formation.
Statins conduct their action by blocking a step in the body's production of cholesterol. Cholesterol is a natural product of the liver; sometimes, the liver produces excess of cholesterol. These drugs block the enzyme linked to the liver's cholesterol production, HMG-CoA reductase, hence, inhibiting the liver's ability to produce LDL. This leads to an increase in the number of LDL receptors on the surface of liver cells, resulting in more cholesterol being removed from the bloodstream and a reduction in risk for high cholesterol-related diseases. Statins have been proved to lower LDL levels from 18% to 55% and to raise high-density lipoprotein levels from 5% to 15%. The reaction catalyzed by HMG-CoA reductase and inhibited by statin is the conversion of HMG-CoA to a compound called mevalonate via an intermediate. Thus, statin inhibits the mevalonate pathway and consequently cholesterol synthesis. This reduction in mevalonate pathway intermediates with a subsequent inhibition of prenylation by statins and is responsible for a large proportion of the pleiotropic effects of these drugs, such as improving endothelial function, immunomodulation, antioxidant activity, and treatment of malignancies.
Most frequently employed antimicrobial drugs to treat periodontal disease are used at dosages ranging from 100 to 500 mg, twice or thrice a day. In contrast, the recommended usual dose of atorvastatin is 10–80 mg once a day for its hypolipidemic activity.
Fajardo et al. studied the effect of atorvastatin 20 mg on bone loss prevention in patients with chronic periodontitis. Results of their controlled double-blind study suggested that periodontal disease conditions showed significant improvement in atorvastatin group compared to placebo. In addition, significant improvements were observed in cholesterol levels and LDL levels.
Subramanian et al. in their study compared dose of atorvastatin, 10 mg and 80 mg, to test whether high doses of atorvastatin treatment would result in a reduction in periodontal inflammation as assessed by F-fluorodeoxyglucose positron emission computed tomography. They found that high dose of atorvastatin (80 mg) reduces periodontal inflammation.
Emani et al. conducted a study to evaluate the MIC value of simvastatin against Aa and Pg. P. gingivalis was sensitive until 2 μg/ml dilution and showed resistance to further dilution by illuminating its MIC. However, for A. actinomycetemcomitans, the performed dilutions could not show any visible growth of the organism; hence, MIC value was considered to be <1 μg/ml. A. actinomycetemcomitans was found to be more sensitive than P. gingivalis, showing its susceptibility to the last dilution (1 μg/ml) set for the study. However, in our study, P. gingivalis was found to be more sensitive to atorvastatin as compared to that of A. actinomycetemcomitans.
Masadeh et al. revealed that statins can induce variable degrees of antibacterial activity with atorvastatins, simvastatins, and rosuvastatins. Methicillin-resistant Staphylococcus aureus, methicillin-sensitive S. aureus, vancomycin-susceptible Enterococci, vancomycin-resistant Enterococci, Acinetobacter baumanni, Staphylococcus epidermidis, and Enterobacter aerogenes were more sensitive to both atorvastatin and simvastatins compared to rosuvastatins. On the other hand, Escherichia coli, Proteus mirabilis, and Enterobacter cloacae were more sensitive to atorvastatin as compared to simvastatin and rosuvastatin.
It was found that bacterial HMG-CoA is 10,000 times weaker than the enzyme found in eukaryotes. Thus, it is unlikely to attribute hypolipidemic mechanism of action (i.e., inhibition of HMG CoA reductase) of statins to their antibacterial activity. According to Chaudhry et al., the antimicrobial action of statins can be explained by their property of increasing bacterial clearance from the infected site or by promoting the apoptosis of microbial cells. Moreover, atorvastatin is hydrophobic in nature, which explains its antibacterial action, where it distresses the bacterial membrane causing its death. However, the exact mechanism of action needs further research.
Within the limitations of the present study, the lowest concentration of atorvastatin was proven to be effective for both A. actinomycetemcomitans and P. gingivalis. Further studies intended to: (a) investigate the susceptibility of other periodontal pathogens to atorvastatin, (b) study the safety of using atorvastatin in nonhyperlipidemic patients to treat periodontitis, (c) compare the efficacy of atorvastatin with other traditionally prescribed antimicrobials used for periodontal therapy, (d) evaluate the effect of atorvastatin in different formulations with different concentrations, and (e) to determine atorvastatin concentrationin vitro in gingival crevicular fluid and serum samples are required to know the ideal dosage and formulation for their antimicrobial activity in the treatment of periodontitis.
| Conclusions|| |
The presentin vitro MIC and MBC study showed good antibacterial activity of the atorvastatin. Although MIC value is considered the gold standard for determining the susceptibility of organisms to the given drug, the growth of microorganismsin vitro is exponential, whereas the growth in vivo can be very slow to none. Furthermore, future studies are recommended to elucidate the mechanism by which atorvastatins are inducing their antibacterial effects.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Popova C, Dosseva-Panova V, Panov V. Microbiology of periodontal diseases. A review. Biotechnol Biotechnol Equip 2013;27:3754-9.
Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006;42:80-7.
Raja M, Ummer F, Dhivakar CP. Aggregatibacter actinomycetemcomitans – A tooth killer? J Clin Diagn Res 2014;8:ZE13-6.
Garlet GP, Martins W Jr., Ferreira BR, Milanezi CM, Silva JS. Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease. J Periodontal Res 2003;38:210-7.
How KY, Song KP, Chan KG. Porphyromonas gingivalis
: An overview of periodontopathic pathogen below the gum line. Front Microbiol 2016;7:53.
Nakayama M, Ohara N. Molecular mechanisms of Porphyromonas gingivalis
-host cell interaction on periodontal diseases. Jpn Dent Sci Rev 2017;53:134-40.
Pavicić MJ, van Winkelhoff AJ, de Graaff J. Synergistic effects between amoxicillin, metronidazole, and the hydroxymetabolite of metronidazole against Actinobacillus actinomycetemcomitans
. Antimicrob Agents Chemother 1991;35:961-6.
Larsen T. Susceptibility of Porphyromonas gingivalis
in biofilms to amoxicillin, doxycycline and metronidazole. Oral Microbiol Immunol 2002;17:267-71.
Nissen SE, Nicholls SJ, Sipahi I, Libby P, Raichlen JS, Ballantyne CM, et al
. Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: The asteroid trial. JAMA 2006;295:1556-65.
Grover HS, Luthra S, Maroo S, Maroo N. The pleotropic role of statins: Could it be the imminent host modulation agent in periodontics? Dent Res J (Isfahan) 2013;10:143-8.
Saver BG, Hujoel PP, Cunha-Cruz J, Maupomé G. Are statins associated with decreased tooth loss in chronic periodontitis? J Clin Periodontol 2007;34:214-9.
Cunha-Cruz J, Saver B, Maupome G, Hujoel PP. Statin use and tooth loss in chronic periodontitis patients. J Periodontol 2006;77:1061-6.
Garrett IR, Gutierrez G, Mundy GR. Statins and bone formation. Curr Pharm Des 2001;7:715-36.
Rohilla A. Atorvastatin pleiotropism: Role in cardioprotection. Int J Pharm Biol Arch 2011;2:813-8.
Bakker-Arkema RG, Davidson MH, Goldstein RJ, Davignon J, Isaacsohn JL, Weiss SR, et al
. Efficacy and safety of a new HMG-CoA reductase inhibitor, atorvastatin, in patients with hypertriglyceridemia. JAMA 1996;275:128-33.
Ozaki K, Kubo T, Imaki R, Shinagawa H, Fukaya H, Ohtaki K, et al
. The anti-atherosclerotic effects of lipid lowering with atorvastatin in patients with hypercholesterolemia. J Atheroscler Thromb 2006;13:216-9.
Castro PF, Miranda R, Verdejo HE, Greig D, Gabrielli LA, Alcaino H, et al
. Pleiotropic effects of atorvastatin in heart failure: Role in oxidative stress, inflammation, endothelial function, and exercise capacity. J Heart Lung Transplant 2008;27:435-41.
Riad A, Du J, Stiehl S, Westermann D, Mohr Z, Sobirey M, et al
. Low-dose treatment with atorvastatin leads to anti-oxidative and anti-inflammatory effects in diabetes mellitus. Eur J Pharmacol 2007;569:204-11.
Masadeh M, Mhaidat N, Alzoubi K, Al-Azzam S, Alnasser Z. Antibacterial activity of statins: A comparative study of atorvastatin, simvastatin, and rosuvastatin. Ann Clin Microbiol Antimicrob 2012;11:13.
Shetty S, Shetty MK. Role of statins in periodontitis – An overview. Int J Adv Res 2014;2:763-5.
Suresh S. Statins in periodontal regeneration-the current scenario. Indian J Dent Adv 2012;4:808-14.
Fajardo ME, Rocha ML, Sánchez-Marin FJ, Espinosa-Chávez EJ. Effect of atorvastatin on chronic periodontitis: A randomized pilot study. J Clin Periodontol 2010;37:1016-22.
Subramanian S, Emami H, Vucic E, Singh P, Vijayakumar J, Fifer KM, et al
. High-dose atorvastatin reduces periodontal inflammation: A novel pleiotropic effect of statins. J Am Coll Cardiol 2013;62:2382-91.
Emani S, Gunjiganur GV, Mehta DS. Determination of the antibacterial activity of simvastatin against periodontal pathogens, Porphyromonas gingivalis
and Aggregatibacter actinomycetemcomitans
: Anin vitro
study. Contemp Clin Dent 2014;5:377-82.
] [Full text]
Jerwood S, Cohen J. Unexpected antimicrobial effect of statins. J Antimicrob Chemother 2008;61:362-4.
Chaudhry MZ, Wang JH, Blankson S, Redmond HP. Statin (cerivastatin) protects mice against sepsis-related death via reduced proinflammatory cytokines and enhanced bacterial clearance. Surg Infect (Larchmt) 2008;9:183-94.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]