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ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 23-29
 

An in-vitro evaluation of antibacterial effect of Amalgomer CR and Fuji VII against bacteria causing severe early childhood caries


Department of Pedodontics and Preventive Dentistry, Maratha Mandal's Nathajirao G. Halgekar Institution of Dental Sciences and Research Centre, Belgaum, Karnataka, India

Date of Web Publication2-Feb-2016

Correspondence Address:
Praveenkumar S Mandroli
Department of Pedodontics and Preventive Dentistry, Maratha Mandal's Nathajirao G. Halgekar Institution of Dental Sciences and Research Centre, Bauxite Road, Belgaum - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.175506

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   Abstract 

Objective: To evaluate the antibacterial action of Amalgomer CR and Fuji VII against bacteria causing S-early childhood caries. Materials and Methods: Antibacterial activity of Amalgomer CR and Fuji VII was assessed using the agar diffusion test in triplicate. The powder and liquid of each test material was mixed and inserted in the punched wells (6 mm × 2 mm). A composition of 0.2% chlorhexidine digluconate acted as control. The agar plates were incubated at 37°C for 24 h for Streptococcus mutans, S. salivarius, S. parasanguinis and Actinomyces viscosus, whereas Lactobacillus casei was incubated for 48 h. Sizes of the inhibition zones were calculated by subtracting the diameter of the specimen (6 mm) from the average of the three measurements of the halo. For each test material against each bacteria, 9 measurements were made (3 measurements × 3 times). Kruskal-Wallis test was done to compare the zones of inhibition of test materials against individual bacteria. Pair-wise comparison was done by Mann-Whitney U-test. Results: Amalgomer CR had the most antibacterial against S. mutans (31.0 mm), followed by A. viscosus (21.87 mm), S. salivarius (13.87 mm), S. parasanguinis (10.80 mm), and L. casei (9.69 mm). Fuji VII had the most antibacterial action against S. salivarius (10.65 mm), followed by A. viscosus (9.10 mm). However, it did not inhibit the growth of S. mutans (0 mm), S. parasanguinis (0 mm), and L. casei (0 mm). Conclusion: Amalgomer CR and Fuji VII showed wide variation in antibacterial action against all test organisms.


Keywords: Amalgomer CR, dental caries, Fuji VII


How to cite this article:
Bariker RH, Mandroli PS. An in-vitro evaluation of antibacterial effect of Amalgomer CR and Fuji VII against bacteria causing severe early childhood caries. J Indian Soc Pedod Prev Dent 2016;34:23-9

How to cite this URL:
Bariker RH, Mandroli PS. An in-vitro evaluation of antibacterial effect of Amalgomer CR and Fuji VII against bacteria causing severe early childhood caries. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Oct 15];34:23-9. Available from: http://www.jisppd.com/text.asp?2016/34/1/23/175506



   Introduction Top


Dental caries (decay) is an international public health challenge, especially among young children. Early childhood caries (ECC) is a serious public health problem in both developing and industrialized countries. Complete excavation of the caries is not possible in all these cases. Hence, the antibacterial property of the material used for restoring the tooth plays a crucial role. ECC can begin early in life, progresses rapidly in those who are at high risk, and often goes untreated. Its consequences can affect the immediate and long-term quality of life of the child's family and can have significant social and economic consequences beyond the immediate family as well. [1] ECC is clinically defined as "The presence of one or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a preschool-age child between birth and 71 months of age." [2] ECC is a particularly virulent type of dental caries characterized by overwhelming infectious challenge and is associated with unusual dietary practices. [3] In advanced forms of this disease, severe ECC can destroy the primary dentition and is the major reason for hospital visits for young children and also disproportionately affects disadvantaged ethnic and socioeconomic groups and can affect over 50% of the children in these groups. [4] Culture studies have demonstrated a strong association of Streptococcus mutans with severe ECC and also reported significant associations with S. salivarius, S. parasanguinis, Lactobacillus casei, and Actinomyces viscosus with severe ECC using cloning analysis and checkerboard analysis. [4],[5],[6],[7]

Contemporary restorative dentistry continues to evolve with the development of new materials, techniques, and concepts. Recently, a new ceramic-reinforced glass ionomer (Amalgomer CR) has been introduced to the dental profession. This tooth-colored product is proposed by the manufacturer to combine the high strength of a metallic restorative, esthetics, and other advantages of glass ionomers. Fuji VII is the world's first conventional autocuring glass ionomer material that contains no resin yet, is capable of accelerated setting by light curing. This command set feature gives the users a choice of setting reaction. With high fluoride release and antibacterial properties, it offers greater protection to surrounding tooth surfaces. The high fluoride release and strontium release contribute to its antibacterial effect. Glass ionomer cements (GICs) fluoride-releasing property has been documented in the literature. [8],[9],[10] After being released from GIC, the fluoride ions take part in the de- and re-mineralization phenomena, and may act directly on the carious process. [11],[12] There are divergences regarding the antimicrobial activity of restorative materials since researches have been found to show both positive and unsatisfactory outcomes. [13]

Studies previously conducted have verified bacterial growth inhibition promoted by both conventional and modified GIC. In spite of that, it is important to consider that many of those studies have found differences with regard to GIC antibacterial effects according to the strain assayed. [14] In the literature, very few studies have been reported on the antibacterial action of Amalgomer CR and Fuji VII. Hence, with this in background, the present in-vitro study evaluated the antibacterial action of two restorative materials, namely, Amalgomer CR and Fuji VII on bacteria causing severe ECC.

Objectives

  1. To evaluate antibacterial action of Amalgomer CR against bacteria causing severe ECC, namely, S. mutans, S. salivarius, S. parasanguinis, L. casei, and A. viscosus.
  2. To evaluate antibacterial action of Fuji VII against bacteria causing severe ECC namely, S. mutans, S. salivarius, S. parasanguinis, L. casei, and A. viscosus.
  3. To compare antibacterial action of Amalgomer CR and Fuji VII against bacteria causing severe ECC, namely, S. mutans, S. salivarius, S. parasanguinis, L. casei, and A. viscosus.



   Materials and Methods Top


This in-vitro study was conducted in the Department of Pedodontics and Preventive Dentistry and Department of Microbiology, at Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belgaum - 590 010, Karnataka, India.

The following test materials were evaluated.



The standard specimens of the below-mentioned strains of bacteria causing severe ECC were used.



The study was carried out in the following steps.

Preparation of culture media

Standard strains obtained were inoculated in the Brain Heart Infusion (BHI) broth for 48 h at 37°C according to physiologic requirements of each bacteria. Lactobacillus casei and A. viscosus were incubated anaerobically in anaerobic jar and S. mutans, S. salivarius, and S. parasanguinis were incubated microaerophically in CO 2 jar. The resultant bacterial growth was checked for turbidity and inoculated in selective media, namely, rugosa agar for L. casei and blood agar for A. viscosus and Streptococcus species. Then, they were checked for colony characteristics and the inoculums were prepared by comparing the McFarland's turbidity standards, i.e., 10 5 . Exactly 10 μl of this inoculums suspension will be pipetted out into a sterile BHI agar plate and spread with a sterile cotton swab to get a lawn culture of the bacteria. Similar lawn cultures were done for other bacteria.

Preparation of wells

In each sterilized petri dish (20 × 100 mm), wells measuring 6 mm in diameter and 2 mm in depth were made in each plate using a custom-made suction device. Three such wells were made in each petri dish. Nine petri dishes for each bacteria and a total of 45 petri dishes were used for five bacteria, as the study was done in triplicate.

Preparation of samples

The test materials were mixed according to the powder liquid ratio of each material as given by the manufacturer with sterile plastic spatula and inserted into the punched wells measuring 6 mm in diameter and 2 mm in depth. A uniform surface was accomplished using a small flat-ended dental instrument such as dental spatula. The materials were allowed to set in accordance with the manufacturer's recommendation and were handled under aseptic conditions. Also, one microliter of μl of aqueous 0.2% chlorhexidine digluconate (Indeco Remedies Ltd., Mumbai, India) was applied on sterile filter paper discs, also 6 mm in diameter, which acted as control.

Incubation of samples

Lactobacillus casei and A. viscosus were be incubated anaerobically in anaerobic jar and S. mutans, S. salivarius and S. parasanguinis were kept for 2 h at room temperature for diffusion of the material and then incubated microaerophically in CO 2 jar. The BHI agar plates were incubated at 37°C and 24 h for Streptococcus species and A. viscosus and 48 h for L. casei.

Measurement of inhibition zones

Zones of growth inhibition around were measured in millimeters with a digital caliper at three different points. Sizes of the inhibition zones were calculated by Kerby-Bauer disk diffusion method. [15] For each test material against each bacteria, 9 measurements were made (3 measurements × 3 times). The mean value was considered for statistical analysis.

Statistical analysis

Results were tabulated and subjected to statistical analysis at α = 0.05. Kruskal-Wallis test was done to compare the zones of inhibition of test materials against individual bacteria. Pair-wise comparison was done by Mann-Whitney U-test. SPSS version 20.0 (Armonk, NY: IBM Corp) was used for analysis.


   Results Top


The results were presented in tables and graphs and were subjected to statistical analysis. Kruskal-Wallis test was done to compare the zones of inhibition of test materials against individual bacteria. Pair-wise comparison of three materials was done by Mann-Whitney U-test. Confidence interval was set at 95% (α = 0.05). SPSS version 20.0 was used for analysis.

The mean and standard deviation of zone of inhibition of test materials against each organism are shown in [Table 1] and Graph 1[Additional file 1], Graph 2[Additional file 2] and Graph 3[Additional file 3], respectively.
Table 1: Mean and standard deviation of zone of inhibition of test materials and control against all organisms

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Streptococcus salivarius

Comparison of test materials (Amalgomer CR and Fuji VII) with control group (chlorhexidine digluconate) with respect to size of inhibition zones against S. salivarius was done by Kruskal-Wallis ANOVA test. The mean values for Amalgomer CR, Fuji VII, and chlorhexidine digluconate were 13.87, 10.65, and 15.66 mm, respectively. The size of zones was statistically significant (P < 0.0001) [Figure 1].
Figure 1: Zone of inhibition observed against Streptococcus salivarius

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Pair-wise comparison done using Mann-Whitney U-test between the test materials and the control group proved highly significant with P = 0.0009 for Amalgomer CR and Fuji VII, P = 0.0015 for Amalgomer CR and CHX, and P = 0.0003 for Fuji VII and CHX.

Actinomyces viscosus

Comparison of test materials (Amalgomer CR and Fuji VII) with control group (chlorhexidine digluconate) with respect to size of inhibition zones against A. viscosus was done by Kruskal-Wallis ANOVA test. The mean values for Amalgomer CR, Fuji VII, and chlorhexidine digluconate were 21.87, 9.10, and 22.42 mm, respectively. The sizes of the zones were statistically significant (P < 0.0001) [Figure 2].
Figure 2: Zone of inhibition observed against Actinomyces viscosus

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Pair-wise comparison done using Mann-Whitney U-test between the test materials and the control group proved significant with P = 0.0003 for Amalgomer CR and Fuji VII and P = 0.0003 for Fuji VII and CHX, whereas no statistically significant difference was seen with Amalgomer CR and CHX with P = 0.4529.

Streptococcus mutans

Comparison of test materials (Amalgomer CR and Fuji VII) with control group (chlorhexidine digluconate) with respect to size of inhibition zones against S. mutans was done. The mean values for Amalgomer CR, Fuji VII, and chlorhexidine digluconate was 31.30, 0, and 17.78 mm, respectively. The sizes of the zones were statistically significant (P < 0.0003) [Figure 3].
Figure 3: Zone of inhibition observed against Streptococcus mutans

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Pair-wise comparison done using Mann-Whitney U-test between the test materials and the control group proved highly significant with P = 0.0003 for Amalgomer CR and CHX. Fuji VII showed no zone of inhibition and hence was not included in the statistical analysis.

Lactobacillus casei

Comparison of test materials (Amalgomer CR and Fuji VII) with control group (chlorhexidine digluconate) with respect to size of inhibition zones against L. casei was done. The mean values for Amalgomer CR, Fuji VII, and chlorhexidine digluconate were 9.69, 0, and 20.57 mm, respectively. The sizes of the zones were statistically significant (P < 0.0003) [Figure 4].
Figure 4: Zone of inhibition observed against Lactobacillus casei

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Pair-wise comparison done using Mann-Whitney U-test between the test materials and the control group proved highly significant with P = 0.0003 for Amalgomer CR and CHX. Fuji VII showed no zone of inhibition and hence was not included in the statistical analysis.

Streptococcus parasanguinis

Comparison of test materials (Amalgomer CR and Fuji VII) with control group (chlorhexidine digluconate) with respect to size of inhibition zones against S. parasanguinis was done. The mean values for Amalgomer CR, Fuji VII, and chlorhexidine digluconate were 10.80, 0, and 17.16 mm, respectively. The sizes of the zones were statistically significant (P < 0.0003) [Figure 5].
Figure 5: Zone of inhibition observed against Streptococcus parasanguinis

Click here to view


Pair-wise comparison done using Mann-Whitney U-test between the test materials and the control group proved highly significant with P = 0.0003 for Amalgomer and CHX. Fuji VII showed no zone of inhibition and hence was not included in the statistical analysis.


   Discussion Top


Acidogenic bacteria play the main role in the development of dental caries. Treatment procedures using various methods of excavating carious lesions do not always eliminate all the microorganisms from the carious focus. Therefore, antibacterial action is a desired feature of materials used for dental fillings. [16]

The results of the study indicated that Amalgomer CR showed antibacterial action against all bacteria causing severe ECC. Among the bacteria, Amalgomer CR showed highest antibacterial action against S. mutans (31.30 mm) followed by A. viscosus (21.87 mm), S. salivarius (18.87 mm), S. parasanguinis (10.80 mm) and least antibacterial action against L. casei (9.69 mm). Fuji VII, on the other hand, showed highest antibacterial action against S. salivarius (10.65 mm) followed by A. viscosus (9.10 mm). However, it did not inhibit the growth of S. mutans, S. parasanguinis, and L. casei. On comparison, Amalgomer CR had better antibacterial action against bacteria causing severe ECC.

According to its setting mechanism, Amalgomer CR is a conventional acid-base reaction GIC. The product includes a particulate ceramic component with the intention of increasing its strength, supposedly without sacrificing appearance (although it is opaque white) or other general characteristics of GIC. It has been shown elsewhere that zirconia is the major if not the only (crystalline) component of the additive of this product, similar to that of some laboratory studies. Zirconia is known to be an excellent material for strengthening and toughening in certain composite contexts because of its peculiar character of a phase transformation from tetragonal to monoclinic under stress. This transformation produces a 4% change of volume which generates a local compressive stress, which then offsets crack-opening tension and so inhibits crack propagation, increasing the incorporating material's fracture resistance. [17] The inhibitory action of Amalgomer CR can be attributed to the coarse nature of ceramic particles contributing to fluoride release. This is supported by DeSchepper et al., [18] who observed that the coarse silver alloy particles in Argion (a metal-reinforced glass ionomer), which are not bound to the cement-matrix, result in an increase in the microporosity of the cement, thus increasing the effective surface area available for elution of fluoride. Fluoride forms a major constituent of many GICs but it has no role in the cement-forming process as predicted and suggested by Crisp and Wilson. [19] The other components such as calcium and aluminum that are involved in cement-matrix formation and play a basic role in hardening and strengthening the ionomer salt hydrogel as postulated by Crisp, Lewis, and Wilson. [20]

In Fuji VII, light is captured by pink chroma of the material. An advantage of Fuji VII is that visible light accelerates the curing process, minimizing the risk of salivary contamination. Another major advantage of GIC materials is fluoride release by the sealant. The fluoride released by Fuji VII, 2 weeks after application was 9 times greater than that released by fluoride containing resin sealant, and it had a more pronounced effect in promoting remineralization of early artificial caries. [20],[21] Release of fluoride over the long-term could enhance the cariostatic ability of the surrounding enamel, and increase the acid resistance of the demineralized enamel in the fissures. [22] Reduced action of Fuji VII in this study is intriguing. This can be attributed to the specific strain of bacteria. Also there is a possibility of influence of methodology used.

Chlorhexidine is a clinically important antiseptic, disinfectant, and preservative. It is a potent membrane-active agent against bacteria and inhibits outgrowth, but not germination, of bacterial spores, although it is not sporicidal. It is widely used as gold standard in antibacterial activity assessment, shows its best performance of antimicrobial effectiveness in the agar diffusion test. [23]

According to Palenik et al. [24] and Barkhordar et al., [25] GICs have a degree of antibacterial activity on some strains of S. mutans, Streptococcus sanguis, A. viscosus, S. salivarius, Streptococcus mitis, and L. casei, but most studies used only one or two strains of each bacteria group. They co-related the inhibition activity to the low pH of GIC after setting and/or fluoride release from these dental materials. Since S. mutans is associated with pit and fissure caries and S. sobrinus with smooth surface dental caries, and strains of these genetic groups have different cariogenicity in animal models, it is important to include several strains of bacterial groups to make conclusions on inhibitory activity.

Loyola-Rodriguez et al. [26] conducted a study to identify the factors involved in the antibacterial activity of GIC on S. mutans. The study states that all GICs investigated possess some antibacterial effect when freshly mixed, against at least one of the indicator strains used, but they may not be effective against the whole genetic group. These results suggest that low pH after setting is not associated with GIC inhibition activity. On the other hand, fluoride release was associated directly with the antimicrobial activity of GIC when the pH was adjusted to 7.1-7.3 using 50 mM of Tris-HCl buffer (pH 8.0). Several investigators have demonstrated that fluoride has both direct and indirect effect on the bacterial cell wall of mutans streptococci, producing inhibition of acid production, and electrolyte metabolism in vitro.

It supports the in-vitro study conducted by Bahadure et al., which demonstrates Amalgomer CR to have significantly higher fluoride-releasing capacity. [27] Zone of inhibition tests do not necessarily indicate that microorganisms have been killed by an antimicrobial product - just that they have been prevented from growing. Microbial growth agars themselves may interfere with the function of some antimicrobial agents. Further studies and clinical research is required to confirm its antibacterial nature because of its high fluoride release.


   Conclusion Top


Within the limitations of the present study, the test materials were found to have antibacterial action in inhibiting bacteria associated with S-ECC. Thus, the following conclusions were drawn.

  1. Amalgomer CR had the most antibacterial against S. mutans (31.0 mm), followed by A. viscosus (21.87 mm), S. salivarius (13.87 mm), S. parasanguinis (10.80 mm), and L. casei (9.69 mm).
  2. Fuji VII had the most antibacterial action against S. salivarius (10.65 mm), followed by A. viscosus (9.10 mm). However, it did not inhibit the growth of S. mutans, S. parasanguinis, and L. casei.
  3. On comparison of both the test materials, Amalgomer CR was found to have most antibacterial action in inhibiting all the bacteria causing S-ECC. The mean zones of inhibition produced by three materials against the bacteria were statistically significant and were as follows: S. mutans, S. parasanguinis and L. casei (P < 0.0003), A. viscosus, and S. salivarius (P < 0.0001).


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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