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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 3  |  Page : 128-132

Antimicrobial efficacy of copper nanoparticles against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in-vitro study


Department of Periodontology, MGV’s KBH Dental College and Hospital, Nashik, Maharashtra, India

Date of Submission27-Apr-2021
Date of Decision12-Jul-2021
Date of Acceptance15-Aug-2021
Date of Web Publication18-Oct-2021

Correspondence Address:
Shraddha Rajendra Shimpi
Shimpi, MGV’s KBH Dental College and Hospital, Mumbai-Agra Road, Panchavati, Nashik 422003, Maharashtra.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_84_21

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  Abstract 

Background: Plaque accumulation on teeth surfaces and prosthetic or orthodontic appliances present a serious challenge for the maintenance of oral health. Copper nanoparticles (NPs) can be incorporated into coatings and applied to restorative materials to prevent plaque formation and the progression of periodontal diseases. Objective: The aim of this article is to evaluate the antimicrobial efficacy of copper NPs against selected periodontal pathogens (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of copper NPs were determined using broth dilution assay. Copper NPs (size: 30—50 nm, purity: 99.9%) were used for the study. Results: Both P. gingivalis and A. actinomycetemcomitans were sensitive to copper NPs. Nano-copper had a bactericidal effect against P. gingivalis at a concentration of 0.8 μg/mL and a bacteriostatic effect against the bacterium at a concentration of 0.4 μg/mL. For A. actinomycetemcomitans, nano-copper had a bactericidal effect at a concentration of 3.12 μg/mL and a bacteriostatic effect at a 1.6 μg/mL concentration. Conclusion: Nano-copper exhibits an antibacterial effect against periodontal pathogens. Future studies are needed to explore the applicability of these copper-based antimicrobial agents in clinical settings.

Keywords: A. actinomycetemcomitans, antimicrobial, copper nanoparticles, MBC, MIC, P. gingivalis


How to cite this article:
Mahale SA, Shimpi SR, Sethi KS, Chaudhari DD, Kadam PS, Katkurwar AA. Antimicrobial efficacy of copper nanoparticles against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in-vitro study. Sci Dent J 2021;5:128-32

How to cite this URL:
Mahale SA, Shimpi SR, Sethi KS, Chaudhari DD, Kadam PS, Katkurwar AA. Antimicrobial efficacy of copper nanoparticles against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: An in-vitro study. Sci Dent J [serial online] 2021 [cited 2021 Nov 27];5:128-32. Available from: https://www.scidentj.com/text.asp?2021/5/3/128/328430




  Background Top


Dental biofilms (i.e., plaque), which consist of a complex community of bacteria and fungi, cause oral infections, such as dental caries, periodontitis, and other dental diseases.[1] It has a tendency to accumulate on the surfaces of the teeth, restorations, prosthesis and orthodontic appliances, surgical dressings, and suture materials.[1] The biofilm confers drug resistance and allows microorganisms to escape the host defense mechanism.[2]

Nanotechnology has emerged as a promising field for the management of oral diseases due to antimicrobial properties of nanoparticles (NPs), which have been employed as coatings for implants and suture materials and as root canal irrigants.[3] Copper and its derivatives are the oldest known antimicrobial agents used in traditional medicine; it is a trace element in most organisms containing copper proteins, such as tyrosinases, catechol oxidases, and hemocyanins. Thus it is an essential element for the metabolism in animal and plant cells.[4]

According to the American Environmental Protection Agency (EPA), copper is the only metal registered as having antimicrobial properties.[5] No microorganisms can survive on copper surfaces. Bacteria are killed within minutes on copper surfaces or copper alloys containing at least 60% copper, with copper killing capacity of 99.9% for most pathogens within 2 h of the contact period. This killing activity takes place at a rate of at least 7—8 h of time interval.[5],[6] Copper NPs exhibit a powerful broad antimicrobial spectrum, which is effective against wound-related pathogens, such as bacteria, fungi, and viruses.[7],[8]

Nano-copper also possesses anti-inflammatory properties.[8],[9] The copper-containing coating can be applied to restorative materials, where it kills bacteria, yeasts, and viruses by the process known as “contact killing.„ [10] The affinity of copper for amines and carboxylic groups in cell walls is thought to explain copper-induced damage of the bacterial cell structure.[11],[12] The nanometric dimensions of copper particles enhance its ability to penetrate into the cell, and the oxidation of copper to Cu+ leads to the formation of reactive oxygen species that induces DNA damage.[11],[12]

Copper NPs can be easily mixed into textiles, films, and molded plastic products, and their antibacterial and antiviral properties make them effective as disinfectants in industrial and commercial (e.g., water treatment plants and food processing) and medical (e.g., wound healing ointments and bandages) fields.[8],[13] In light of the advantages and effects of nano-copper as reported in the previous studies, the present in-vitro study aimed to evaluate the antimicrobial efficacy of copper NPs against the periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.


  Materials and Methods Top


Material procured

Pure forms of copper NPs (size: 30—50 nm; purity: 99.9%) (Nano Research Lab, Jharkhand, India) were used in the present study. To determine the antibacterial efficacy of the copper NPs against the periodontal pathogenic strains P. gingivalis (ATCC 33277) and A. actinomycetemcomitans (ATCC 43718), minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) tests were conducted.

MIC and MBC tests

The double dilution method was used for the determination of antibacterial activity. Nutrient broth (HIMEDIA M210-500G, Mumbai, India) and brain heart infusion broth (Maratha Mandal’s NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka) were prepared and added to test tubes. Stock solutions of copper NPs were prepared in dimethyl sulfoxide at a concentration of 10 mg/mL, followed by two-fold dilution at concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4, and 0.2 μg/mL (adjusted to 1× 108 CFU/mL, 0.5 McFarland’s standard).[14] To determine the MIC, nine dilutions of each drug (copper NPs) were made using thioglycollate broth. In the initial tube, 20 μL of the drug (copper NPs) was added to 380 μL of thioglycollate broth. In subsequent dilutions, 200 μL of thioglycollate broth was added to another nine tubes separately. Then, from the initial tube, 200 μL was transferred to the first tube containing 200 μL of thioglycollate broth. This was considered as 10—1 dilution. From the 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 (copper NPs).

From maintained stock cultures of A. actinomycetemcomitans and P. gingivalis, 5 μL was taken and added to 2 mL of thioglycollate broth. In each serially diluted tube, 200 μL of the above culture suspension was added. The tubes were then incubated for 48—72 h in an anaerobic atmosphere at 37°C. Turbidity was monitored visually both before and after incubation. For A. actinomycetemcomitans, a facultative anaerobe, tubes were incubated at 37°C for 48—72 h in a carbon dioxide atmosphere. For P. gingivalis, a strict anaerobe, tubes were incubated in an anerobic atmosphere for 48—72 h.

MBC was then checked to determine whether the drug (copper NPs), at the MIC, had a bacteriostatic or bactericidal effect against the test organisms. To determine the MBC, three to five tubes were plated which were initially sensitive to the MIC test and then incubated for 24 h. The number of colonies was counted on the next day.


  Results Top


After 24 h of incubation, turbidity was checked in all the test tubes containing inocula of P. gingivalis and A. actinomycetemcomitans, indicating bacterial growth. The results revealed that P. gingivalis was sensitive to copper NPs up to 0.8 μg/mL dilution [Figure 1]. Similarly, A. actinomycetemcomitans was sensitive to copper NPs up to 3.12 μg/mL dilution [Figure 2]. Thus, MIC of nano-copper for P. gingivalis and A. actinomycetemcomitans was 0.8 and 3.12 μg/mL, respectively.
Figure 1: MIC of copper nanoparticles against P. gingivalis by broth dilution assay

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Figure 2: MIC of copper nanoparticles against A. actinomycetemcomitans by broth dilution assay

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In terms of the MBC, a bacteriostatic effect was indicated by the presence of bacterial growth, and a bactericidal effect was indicated by the absence of growth in agar plates. Nano-copper had a bactericidal effect against P. gingivalis at a concentration of 0.8 μg/mL and a bacteriostatic effect against the bacterium at a concentration of 0.4 μg/mL [Figure 3]A and B. For A. actinomycetemcomitans, nano-copper had a bactericidal effect at a concentration of 3.12 μg/mL and a bacteriostatic effect at a 1.6 μg/mL concentration [Figure 3]C and D and [Table 1].
Figure 3: (A-D) MBC of nano-copper against P. gingivalis and A. actinomycetemcomitans

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Table 1: Minimum bactericidal concentration for P. gingivalis and A. actinomycetemcomitans

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  Discussion Top


Copper is less expensive than gold and silver, and it is readily miscible with polymers and relatively stable, both chemically and physically. NPs have been the focus of much attention due to their potential in biomedical and pharmaceutical applications. A limited number of studies have investigated their use in the field of dentistry.[15],[16] Thus, in the present study, we investigated the effect of nano-copper on the periodontal pathogens P. gingivalis and A. actinomycetemcomitans to determine its potential antimicrobial and anti-inflammatory usage in periodontitis patients.

In our study, P. gingivalis was sensitive to copper NPs up to 0.8 μg/mL dilution, and A. actinomycetemcomitans was sensitive to copper NPs up to 3.12 μg/mL. The optimum activity of copper NPs against both bacterial species was observed at a concentration of 3.12 μg/mL.

Toodehzaeim et al.[15] incorporated copper NPs into an adhesive composite (3M Unitek Transbond XT light cure adhesive paste) at concentrations of 0.01 and 0.5 mg/μL and 1 wt.% and observed no adverse effects on shear bond strength. Ahmed et al.[16] concluded that Cryptococcus neoformas, Bacillus subtilis, and Escherichia coli were highly sensitive to copper sulfate—copper oxide NPs (Cs—CuO NPs) at a concentration of 10,000 mg/L, whereas copper sulfate NPs (Cs NPs) at the same concentration (10,000 mg/L) was effective only against C. neoformas.

There are very few reports about antimicrobial activity of nano-copper against dental pathogens.[17],[18] The ability of copper to donate and accept electrons in a continuous process is responsible for its bactericidal action.[7] The generally accepted mechanism of inhibition of bacteria by copper NPs is rupture of the negatively charged bacterial cell wall, protein degeneration, and finally cell death due to the release of copper ions from the metallic NPs.[19]

Chatterjee et al.[20] reported that treatment of E. coli cells with copper NPs led to overproduction of reactive oxygen species in the bacterial cells, which increased lipid peroxidation, protein oxidation, and DNA degradation, finally culminating in cell death. Rakhmetova et al.[21] studied the wound healing properties of copper NPs and observed that the effectiveness of modified copper NPs in the form of an ointment differed, depending on their physicochemical parameters. Thus, based on the literature and the findings of the present study, copper NPs appear to have potential in the prevention and treatment of periodontitis. In-vivo studies are required to validate the results of this in-vitro study. This study had small sample size and hence the results cannot be generalized. Moreover, in-vivo studies are required to validate the results of this in-vitro study.


  Conclusion Top


The results of this study suggest that the most effective bactericidal concentrations of copper NPs against P. gingivalis and A. actinomycetemcomitans were 0.8 and 3.12 μg/mL, respectively. Copper NPs appear to have potential as an antimicrobial agent against P. gingivalis and A. actinomycetemcomitans. In patients with periodontal disease, the incorporation of copper NPs into allografts or local drug delivery systems has potential as a treatment option. Local applications of copper NPs have potential as a treatment for open wounds. It will be important for future studies to concentrate on specifically controlled experiments, to determine explicit details, and to shed light on how NPs kill microbes under dry conditions without releasing ions. Further studies are needed to evaluate the effects of these NPs on the physical properties of the restorative material and the clinical applicability of it into periodontitis patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There is no conflict of interest.



 
  References Top

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Piñón-Segundo E, Ganem-Quintanar A, Alonso-Pérez V, Quintanar-Guerrero D. Preparation and characterization of triclosan nanoparticles for periodontal treatment. Int J Pharm 2005;294:217-32.  Back to cited text no. 3
    
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Grass G, Rensing C, Solioz M. Metallic copper as an antimicrobial surface. Appl Environ Microbiol 2011;77:1541-7.  Back to cited text no. 7
    
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Borkow G, Gabbay J. Copper, an ancient remedy returning to fight microbial, fungal and viral infections. Curr Chem Biol 2009;3:272-8.  Back to cited text no. 8
    
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Usman MS, El Zowalaty ME, Shameli K, Zainuddin N, Salama M, Ibrahim NA. Synthesis, characterization, and antimicrobial properties of copper nanoparticles. Int J Nanomed 2013;8:4467-79.  Back to cited text no. 9
    
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Ruparelia JP, Chatterjee AK, Duttagupta SP, Mukherji S. Strain specificity in antimicrobial activity of silver and copper nanoparticles. Acta Biomater 2008;4:707-16.  Back to cited text no. 11
    
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Pramanik A, Laha D, Bhattacharya D, Pramanik P, Karmakar P. A novel study of antibacterial activity of copper iodide nanoparticle mediated by DNA and membrane damage. Colloids Surf B Biointerfaces 2012;96:50-5.  Back to cited text no. 12
    
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Borkow G, Gabbay J. Putting copper into action: Copper-impregnated products with potent biocidal activities. Fed Am Soc Exp Biol 2004;18:1728-30.  Back to cited text no. 13
    
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Krishnan R, Arumugam V, Vasaviah SK. The MIC and MBC of silver nanoparticles against Enterococcus faecalisA facultative anaerobe. J Nanomed Nanotechnol 2015;6:285.  Back to cited text no. 14
    
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Toodehzaeim MH, Zandi H, Meshkani H, Hosseinzadeh Firouzabadi A. The effect of CuO nanoparticles on antimicrobial effects and shear bond strength of orthodontic adhesives. J Dent (Shiraz) 2018;19:1-5.  Back to cited text no. 15
    
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Ahmed SB, Mohamed HI, Al-Subaie AM, Al-Ohali AI, Mahmoud NMR. Investigation of the antimicrobial activity and hematological pattern of nano-chitosan and its nano-copper composite. Sci Rep 2021;11:9540.  Back to cited text no. 16
    
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Ramyadevi J, Jeyasubramanian K, Marikani A, Rajakumar G, Rahuman AA. Synthesis and antimicrobial activity of copper nanoparticles. Mater Lett 2012;71:114-6.  Back to cited text no. 17
    
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Ren G, Hu D, Cheng EW, Vargas-Reus MA, Reip P, Allaker RP. Characterisation of copper oxide nanoparticles for antimicrobial applications. Int J Antimicrob Agents 2009;33:587-90.  Back to cited text no. 18
    
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Yu-sen EL, Vidic RD, Stout JE, McCartney CA, Victor LY. Inactivation of Mycobacterium avium by copper and silver ions. Water Res 1998;32:1997-2000.  Back to cited text no. 19
    
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Chatterjee AK, Chakraborty R, Basu T. Mechanism of antibacterial activity of copper nanoparticles. Nanotechnology 2014;25:135101.  Back to cited text no. 20
    
21.
Rakhmetova AA, Alekseeva TP, Bogoslovskaya OA, Leipunskii IO, Ol’khovskaya IP, Zhigach AN, Glushchenko NN. Wound-healing properties of copper nanoparticles as a function of physicochemical parameters. Nanotechnol Russia 2010;5:271-6.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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  [Table 1]



 

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