A comparative study on the antimicrobial activity of irreversible hydrocolloid mixed with silver nanoparticles and chlorhexidine.

BACKGROUND: Impressions taken from patients have the potential of cross-transmission of infection among dentistry personnel. The present study aimed to compare the antimicrobial activity of chlorhexidine (CHX) and silver nanoparticles (AgNPs) combined with irreversible hydrocolloid.
MATERIALS AND METHODS: This experimental study examined the in vitro antimicrobial effects of irreversible hydrocolloid mixed with silver nanoparticles and chlorhexidine using four groups, namely CHX (0.2%) solution and mouthwash mixed with irreversible hydrocolloid Groups 1 and 2), AgNPs (0.1 and 0.2%) (Groups 3 and 4), and specimens mixed with distilled water as a control group (Group 5) on bacterial strains, namely, Staphylococcu saureu s,Enterococcu sfaecali s,Pseudomona saeruginos a,Escherichi acoli, andStaphylococcu sepidermidi s through disc diffusion method. There were three replications per bacterial species. As data were not normally distributed, Kruskal-Wallis test was used at a significance level of 0.05.
RESULTS: No antimicrobial activity was observed in the control groups. In S. aureus, CHX mouthwash had the highest antimicrobial activity, and AgNPs 0.1% and 0.2% groups had lower antimicrobial activity, and there was a significant difference between the two concentrations of AgNPs (P < 0.05). InE.faecali s, the effects of CHX compounds and AgNPs 0.2% were similar to each other and were higher than the effect of AgNPs 0.1% (P < 0.05). In E. coli, CHX compounds exhibited the highest efficacy relative to other materials (P < 0.05), and the AgNPs had no effect. In P. aeruginosa, AgNPs showed the highest growth inhibition zone, which had a significant difference compared to other materials (P ≤ 0.01), whereas the CHX compounds were not effective. InS.epidermidi s, the effect of CHX compounds was similar to one another and was higher than the effect of AgNPs (P ≤ 0.01).
CONCLUSION: According to our observations, the antimicrobial activity of AgNPs at 0.1 and 0.2% against five tested bacterial strains was similar to those of pure CHX 0.2% solution and CHX 0.2% mouthwash. Copyright:

INTRODUCTION

Dental impression materials may carry pathogenic microorganisms because of direct contact with the blood, saliva, and bacterial plaque of patients, which can transmit infectious diseases to dentistry and/or laboratory personnel.[123] Irreversible hydrocolloid is usually used to record primary impressions in dentistry.[4] While impression recording, the surface tissue and hydrophilic nature of the irreversible hydrocolloid allow maintaining maximum microbial pathogens not only on the surface but also inside the material.[5] Research has shown that a large number of impressions have been so far sent to laboratories without passing through any kind of disinfection process.[678] It has been shown that a combination of disinfectants with irreversible hydrocolloid powder provides an additional disinfection method with no adverse effects in dimensional stability and surface accuracy of the impression.[9] Chlorhexidine (CHX) and silver nanoparticles (AgNPs) are two disinfectants that have been demonstrated to be effective in combination with irreversible hydrocolloid while maintaining physical properties of the impression.[101112] The main aim of the current study is to determine the selective material with the most effective antimicrobial activity, when mixed with irreversible hydrocolloid. The null hypothesis of the study was that there is no difference of antimicrobial effect between CHX and AgNPs.

MATERIALS AND METHODS

In this experimental study the irreversible hydrocolloid impression powder (Lascod Kromopan type A, Florence, Italy) was mixed in a ratio of 20 mL liquid with 9 g of the powder according to the manufacturer's instruction. AgNPs (US Research, USA, No. 4-22-7440) were used at concentrations of 0.2% and 0.1% with a size range of 5–8 nm. CHX was used in the following two forms:

CHX 2% solution (Stalowa Wola,Cerkamed, Poland) diluted to a 0.2 solution

Alcohol-free CHX 0.2% mouthwash (Hexadiene, Donyaye Behdasht pharmaceutical Company, Tehran, Iran).

In this study, the antimicrobial activity of five substances in combination with irreversible hydrocolloids was studied, which included the following groups:

Irreversible hydrocolloid mixed with CHX 0.2% solution (CHXs)

Irreversible hydrocolloid mixed with CHX 0.2% mouthwash (CHXm)

Irreversible hydrocolloid mixed with AgNPs 0.1% (AgN1)

Irreversible hydrocolloid mixed with AgNPs 0.2% (AGN2)

Irreversible hydrocolloid mixed with sterile distilled water only as a control group (C).

Preparation of discs

The irreversible hydrocolloid impression powder used for the specimens had already been weighed and found to be of equal weights. Nine grams of irreversible hydrocolloid powder was put in a plastic bowl and 20 ml of the study solution was added to the bowl to prepare the discs of each study group. The mixture was stirred by a user for 45 s using an alginate spatula. Thereafter, the materials were filled in an impression cast with 15-mm internal diameter and 19-mm height placed on a glass slap. The impression cast was then compressed from above by a smooth glass slap to remove extra materials, which was left to set the material, followed by cutting discs of 4 mm thick and 15 mm in diameter by a sterile blade [Figure 1]. Three discs were obtained from each of the five study groups per test bacterium (i.e., a total of 15 discs were obtained from each of the five groups).

Figure 1

Schematic view of disc preparation.

Evaluation of antimicrobial effect

The antimicrobial activity was measured by disc diffusion method in Mueller–Hinton agar medium (Merck, Darmstadt, Germany). The standard bacterial strains, namely, Staphylococcus aureus (ATCC 20739), Staphylococcus epidermidis (ATCC 14990), Enterococcus faecalis (PTCC 1237), Pseudomonas aeruginosa (Pao1), and Escherichia coli (ATCC 35218), were obtained as lyophilized ampoule from the Iranian Research Organization for Science and Technology. The lyophilized ampoules were broken by observing aseptic standards and microbiological methods to prepare a suspension containing the target microorganism, which was cultured and incubated to grow. McFarland 0.5 standard was used to have similar bacterial concentrations. Afterward, a very little amount of precultured bacteria was collected using a sterile swab, mixed in sterile saline, and compared with the opacity of 0.5 McFarland standard. More saline was added in case of a higher opacity, and if it was more transparent, a little amount of bacteria was added to reach the opacity of 0.5 McFarland. Similar dilutions of bacterial strains were prepared and inoculated in the media. Each individual microbe was cultured in three separate plates to ensure the data. After that, the test discs were placed in the peripheral of the plates containing microbe-infected Mueller–Hinton agar medium. The control samples were placed at the central part of the plates.

The plates containing microorganisms and the discs were incubated at 37°C for 24 h. The inhibition zone diameter for individual microorganisms was measured with a ruler in millimeter by a user. The diameter of the shortest path was considered in the examination.

Data were assigned appropriate codes and were analyzed by SPSS statistics 24 software (I. B. M. Company, New York, U. S. A.). Mean values with standard deviations were reported for continuous data in descriptive statistics. As data were not normally distributed, Kruskal–Wallis test was used at a significance level of 0.05.

RESULTS

Following incubation, growth inhibition zone (GIZ) was obtained for assessing the antimicrobial activity of each bacterial strain [Figure 2]. Table 1 summarizes the GIZs observed in the tested samples. All control groups of the bacterial strains exhibited no antimicrobial activity. In S. aureus, the greatest and lowest GIZs were recorded in CHX 0.2% mouthwash and AgNPs 0.1% groups, respectively. Both CHX mouthwash and CHX solution displayed an uppermost effectiveness with a statistically significant difference (P < 0.05). A lower antimicrobial activity was detected with AgNPs 0.1% and 0.2% than those of the other two groups, with a' statistically significant difference between the two concentrations (P < 0.05).

Figure 2

Growth inhibition zone for individual bacterial strains: (1)Escherichia coli, (2)Staphylococcus aureus, (3)Staphylococcus epidermidis,(4)Pseudomonas aeruginosa, and (5)Enterococcus faecalis under the effects of 0.2% chlorhexidine solution (G1), chlorhexidine mouthwash (G2), 0.1% silver nanoparticles (G3), 0.2% silver nanoparticles (G4), and control group (c).

Table 1

Mean±standard deviation diameter of inhibition zone (mm) for each bacterial species and group

Species	CHX 0.2% solution	CHX 0.2% mouthwash	AgNPs 0.1%	AgNPs 0.2%	Control
Staphylococcus aureus	24.33±0.57	27.66±1.52	17.00±0.00	18.33±0.57	0
Enterococcus faecalis	19.00±0.00	19.00±0.00	17.00±0.00	18.33±0.57	0
Escherichia coli	16.00±0.00	16.00±0.00	0	0	0
Pseudomonas aeruginosa	0	0	17.33±0.57	18.00±1.00	0
Staphylococcus epidermidis	25.33±0.57	27.33±1.15	17.33±0.57	17.00±0.57	0

CHX: Chlorhexidine; AgNPs: Silver nanoparticles

CHX compounds showed the largest GIZs in the case of E. faecalis, upon which both CHX solution and mouthwash presented a fully similar effect (P = 1) followed by AgNPs 0.2% with maximum effectiveness. No statistically significant differences were found between these three groups (P > 0.1). However, all the three groups were statistically significantly different from AgNPs 0.1% group with lowermost effectiveness (P < 0.05).

In E. coli, GIZs were of the highest size as a result of CHX compounds with the greatest effectiveness compared to the other substances (P < 0.05), while their effects were quite similar to one another (P = 1). Both concentrations of AgNPs showed no antibacterial property against this bacterial strain.

AgNPs represented the largest GIZ in P. aeruginosa, which had statistically significant differences with the other substances (P ≤ 0.01), but both concentrations of AgNPs were not statistically significantly different (P = 1). CHX compounds exerted no antimicrobial impacts on this bacterial strain.

CHX mouthwash led to the greatest GIZ size in S. epidermidis. With almost similar effects (P > 0.05), both CHX mouthwash and solution were more effective than AgNPs (P ≤ 0.01). Moreover, AgNPs at concentrations of 0.1% and 0.2% were not statistically significantly different (P > 0.1). Overall, the antibacterial activities of CHX solution and mouthwash on the five tested microorganisms were not statistically significantly different from those of AgNPs at concentrations of 0.1% and 0.2% (P > 0.1).

DISCUSSION

Based on the study results, our null hypothesis was that there is no difference of antimicrobial effect between CHX and AgNPs. Our results indicated that all the tested materials showed antibacterial effects on S. aureus, E. faecalis, and S. epidermidis. However, CHX compounds and AgNPs revealed no antibacterial impacts on P. aeruginosa and E. coli, respectively. Even so, when all GIZs were examined among the five experimental groups, no significant differences were observed among CHX 0.2% mouthwash, CHX 0.2% solution, and both AgNP 0.1% and 0.2% groups. Nonetheless, all the tested agents were significantly different from those of the control group. CHX mouthwash exhibited the highest antibacterial effect on S. aureus, even higher than its pure solution, with AgNPs 0.1% and 0.2% showing the least influences. The different influences of CHX mouthwash and solution on S. aureus can be attributed to the ingredients of CHX mouthwash used herein, which consist of glycerin, methyl paraben, paraben profile, and C.116035 flavor. Because paraben is an antimicrobial preservative, it could have sensitized most of the S. aureus strains to CHX mouthwash compared to pure CHX solution.[13]

CHX compounds and AgNPs 0.2% displayed the highest and lowest antibacterial activities against E. faecalis, respectively.

The uppermost antibacterial activity toward S. epidermidis was recorded in both CHX mouthwash and solution, whereas AgNPs 0.1% and 0.2% showed the lowermost impacts.

As noted above, CHX compounds presented no antibacterial activities against P. aeruginosa, whereas AgNPs at concentrations of 0.1% and 0.2% similarly affected this strain. In line with our results, Wang et al.[10] reported a high resistance of P. aeruginosa toward CHX treatment, although they used relatively lower concentrations than those applied in this study. Such a difference in the response of P. aeruginosa to CHX can be attributed to different strains of this bacterium, some of which are more CHX resistant. The present research, however, demonstrated a high antibacterial resistance of P. aeruginosa to CHX compared to the other bacterial strains examined. CHX binds to the bacterial cellular membrane, thereby causing intracellular matrix leakage, inhibition of respiratory system, and cytoplasmic coagulation in bacteria. Gram-negative bacteria are more resistant to CHX than Gram-positive ones; in particular, P. aeruginosa that has acquired an improved natural resistance to this agent owing to its outer membrane. In addition, the presence of flow pumps (that expel any potentially damaging agent out of the cell) can account for more resistance of this strain toward disinfectants. P. aeruginosa can generally resist CHX even at high concentrations.[14]

Contrary to the former strain, AgNPs showed no antibacterial activity against E. coli. This strain was highly affected by the antibacterial activities of both CHX solution and mouthwash while showing almost similar effects. The antibacterial effect of AgNPs on E. coli has been reported in some studies.[111516] The discrepant reports are attributable to differences in the size, concentration, and shape of the used nanoparticles.[1718] Furthermore, E. coli is a Gram-negative species with a robust polysaccharide cellular wall, which is more resistant to AgNP infiltration into the cell by taking a longer exposure time. Durán et al. have also presented evidence that mutant strains of this bacterium are six times more resistant to AgNPs because of lacking OMPG and OMPF prions, and that E. coli has a rapid evolutionary ability to resist AgNPs.[19] The differential mechanism in various strains of this bacterium can explain the different observations reported on E. coli.

As mentioned, E. coli is also Gram negative similar to P. aeruginosa; hence, it is expected that AgNPs and CHX yield similar outcomes on these two bacteria. Inconsistent results, however, can be explained by the unique characteristics of individual bacterial species.

Results of studies by Ginjupalli et al. and Penden Wangchuk et al. suggest a high antibacterial activity of AgNPs.[1112]

Kollu et al. reported a minimum inhibitory concentration of 0.02–0.05 for CHX and found that 0.01% CHX solution was sufficient to inhibit the growth of most microbes present in the irreversible hydrocolloid (before impression forming).[20] Casemiro et al. found a better antimicrobial effect of 0.02% CHX than the other used agents,[21] which is in agreement with a high antimicrobial activity of 0.02% CHX observed herein.

Kangarlou Haghighi et al. compared the antimicrobial activity of AgNPs-containing solution with those of sodium hypochlorite and CHX against E. faecalis, Candida albicans, E. coli, and P. aeruginosa. Their results suggested a rather similar antimicrobial effectiveness of AgNPs and CHX,[15] corroborating our observations. Jafari et al., on the other hand, noticed that AgNPs at 1000 ppm were more effective on all the tested microorganisms compared to other agents used (CHX 2%).[16] The inconsistent results can be ascribed to differently sized NPs and various bacterial strains used in the two studies.

This study is the first one to examine the antimicrobial activities of AgNPs and CHX compounds with similar concentrations in combination with irreversible hydrocolloid impression material. The two materials showed comparatively identical antimicrobial activities at similar concentrations, with no difference between AgNP concentrations at 0.1% and 0.2%. CHX mouthwash is widely available at a lower price than the other materials tested. Based on the research by Wang et al. and Amalan et al., which unveiled the satisfactory physical properties of irreversible hydrocolloid in combination with CHX,[1022] our research findings, which showed the strong antimicrobial effect of this substance, and the low cost and availability of this substance as a mouthwash, it is recommended to conduct clinical trials to clinically analyze irreversible hydrocolloid in combination with this substance. Besides, contrary to CHX, both AgNP powder and solution are available. Hence, alginate-producing factories can integrate this material in irreversible hydrocolloid ingredients to induce its antimicrobial properties. Nonetheless, further investigations should scrutinize the effects of size, concentration, and shape of AgNPs as well as its long-term interaction on the physical properties of impression material.

Despite what mentioned above, the present study suffers from some limitations as follows:

It was not possible to determine the antimicrobial activities of samples against harmful and communicable pathogens (e.g., tuberculosis and hepatitis) likely present in the mouth ambiance.

Another study limitation was its in vitro nature, the results of which cannot be generalized to in vivo conditions. However, the current results may be considered as a base for in vivo examinations.

CONCLUSION

Overall, the antibacterial activities of 0.2% CHX compounds against the five tested microorganisms were similar to those of AgNPs at concentrations of 0.1% and 0.2%. CHX mouthwash and pure solution acted similarly in relation to one another.

Financial support and sponsorship

Nil

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial, in this article.