Effectiveness of the two forms of Drugs and Disinfected Solutions on -Oral Microorganism Adhered to Denture Base Materials

Aims : Microbial adherence to different denture base materials causes denture stomatitis. This study sought to assess the effectiveness of certain antibacterial, antifungal drugs and disinfected solutions. Materials and methods : the drugs were applied in two forms: gel and mouth rinse against various types of candida and bacterial strains that adhered to the two types of denture base materials the acrylic-chrome cobalt and the heat cure acrylic resin denture base materials. The microbial strains used in this study were isolated, by using two different agar mediums Sabouraud's dextrose agar was used for Cryptococcus neoformans, Candida famata, and Candida albicans while blood agar includes Staphylococcus aureus, Corynebacterium diphtheriae, Pseudomonas aeruginosa, lactobacillus acidophilus, Actinomyces Spp., Enterococcus Spp., Streptococcus spp., and E. coli. The susceptibility of candida and bacterial strains against the three types of drugs was determined using nystatin (Nys) in the form of 100000IU /ml suspension and 15gm ointment, and metronidazole (metro) in the form of 250mg tablet diluted in 100000IU/ml of distilled water and 15g 0.75% gel also the chlorhexidine (CHX) 0.20% in the form of both mouthwash and gel used disk diffusion method. The level of inhibition zones was determined in the median and interquartile range due to the small sample size of strain (n=5). The effectiveness of different drugs was examined in the Kruskal-Wallis test. Nonparametric Comparisons for All Pairs. Results : The study showed remarkable outcomes related to the effectiveness of the two forms of the three different drugs against fungi and the bacterial strains associated with the fungi in our samples. Conclusions : The finding specifies that the gel forms are the most effective.


‫ااألأل‬ ‫اااار‬ ‫لألديصقألدير‬ ‫لألمع‬ ‫ااأل‬ ‫ةر‬ ‫ان‬ ‫ااألدي‬ ‫اليألديلة‬ ‫ا‬ ‫ال‬ ‫لألدين‬ ‫علأل‬ ‫الألديرة‬ ‫ااألةديل‬ ‫ااألدوية‬ ‫الية‬ ‫فع‬ ‫بليراديألدوسلسةاأليةرقألدوسالن‬
among the elderly population (1).Since its introduction in 1937, Polymethyl methacrylate (PMMA) has emerged as the predominant material in the fabrication of removable full dentures.This material offers multiple benefits, such as appealing esthetic qualities, minimal water absorption and solubility, sufficient strength, low toxicity, ease of repair, and a straightforward molding characteristics (2) Microbial adhesion, the initial phase in biofilm formation, occurs during mastication.This microbial biofilm might be home to opportunistic pathogens including Candida species, which have been linked to precancerous lesions, as well as grampositive Streptococci, rods, and gramnegative bacteria (3) .Denture stomatitis is one of the most prevalent oral illnesses among people who use dental prostheses.
Denture stomatitis, a prevalent oral illness among dental prosthesis users, is characterized by inflammation of the mucosa under the prosthesis.The multifactorial etiology of denture stomatitis encompasses bacterial plaque accumulation, resin porosity, trauma from ill-fitting prostheses, and inadequate oral hygiene, all of which predispose the mucosa beneath the prosthesis to infection (4) .Approximately 65% of upper full denture wearers are impacted by this lesion.Predominantly located in the palatine mucosa, it is usually asymptomatic, yet may present with symptoms like discomfort, halitosis, itching, and burning sensations (5) .Post-delivery of removable dentures, patient education, maintenance of cleanliness, and regular monitoring are essential to ensure the proper functioning and to prevent complications in the supporting tissues (6) .However, the preferred therapy for denture stomatitis consists mostly of hygiene instructions for washing the prosthesis by manual technique, which is frequently and generally adopted by patients, administration of a topical antifungal, and verification of the need for a new prosthesis (5).
The direct application of medication, either as an ointment on the lesion or on the sterilized prosthesis, enhances medicine contact time with the lesions.This results in more effective treatment and faster healing furthermore it has been demonstrated that exposure of oral microorganisms to disinfectants and drug solutions in the form of mouth rinse or Immersion of dentures in disinfectants and drug solution suppresses the adhesion ability of oral microbial to buccal epithelial cells and adhesion to the prosthesis (7) .
While the direct application of medication as ointment or mouth rinse has been described for a long time, the appropriate therapy has yet to be determined, due to the high rate of relapse.hours at 37°C, based on preliminary trials that determined optimal growth times for the microorganisms of interest (8) then identification for candida and bacterial species was done by observing the morphology and color of the colonies, then staining and microscopically identifying them finally on the Vitek 2 microbial ID/AST (9), The isolated single colonies of fungi and bacteria were cultivated using spread plate method on separate agar plates and incubated for 24 hours at 37°C to get pure isolated colonies (10 ) .
As   agars ten for each one of the eight bacterial strains (12) .

Statistical Analysis
The level of inhibition zones was determined in the median and interquartile range due to the small sample size of strain

Susceptibility of Fungi to the three different types of Drugs
The study showed remarkable outcomes related to the effectiveness of the two forms of the three different drugs against fungi, as shown in Table 1 and Figure 2 the highest median of inhibition zones in the gels form of the three medications than in solutions form.with (M=1mm).The comparison between them is illustrated in Table 1 ) Candida famata showed high sensitivity The pairwise comparisons among all the different drugs toward each fungus are illustrated in Table 1 )   among denture wearers (13) .It is critical to practice regular oral hygiene, including a combination of mechanical and chemical methods as the most appropriate choice in terms of denture cleaning (5) .The most significant aspect in the prevention and treatment of denture stomatitis is the quality of prosthesis cleaning, which requires frequent inspection in a dental clinic for the evaluation of the prosthetic and the buccal cavity as a preventive measure to avoid and regulate the triggering variable, in addition to prosthesis hygiene guidelines, topical antifungal and antibacterial treatments should be used (14) .As topical medications are favored over systemic medicines owing to the Nys.Suspension Nys.Ointment CHX.mouthwash CHX.Gel Metro.Table Metro.Gel renal and hepatotoxicity linked to the latter, as well as their efficacy against oral candidiasis 15 .

Nystatin Drug (ointment& suspension) against Candida and Bacterial strains
According to the Nys medication, the ointment form showed the highest effect on the three candida species than the suspension form as Nys is an antimicrobial agent with both fungicidal and fungistatic properties, In addition to its superior efficacy on the three species as compared to metro gel, CHX gels form a close second, this finding advocated the results of Sartawi et al. (16) et al., 2021 and Shaikh et al. (17) they were summarized that nystatin is the standard topical treatment for oral candidiasis, with the total inhibition of both the binding and colonization of C.
albicans.In addition to that the finding of this article agreed with Czajka et al. 18 , 2023 which reported that the topical application of Nys to that showed by study of Rai et al. (19) , 2022 and Labban et al. (20) they reported that using nystatin is still the mainstay for the treatment of oral candidiasis, because of its increased efficacy, can be the treatment regimen of choice for denture stomatitis.Although Nys suspension demonstrated effectiveness toward the three species of candida but slightly less effect than the ointment this agrees with the Lyu et al. (21) , 2016 study which showed also that the efficacy of Nys suspension in treating denture stomatitis was inferior to that of miconazole also Gonoudi et al. 22 study showed the greater satisfaction with the use of garlic rather than nystatin in denture stomatitis.
While the two forms of nystatin administration had the least effect on the oral bacteria that were paired with the candida strains, the ointment kind of nystatin had a higher effect than the mouth wash type since most of those bacteria shown resistance agonist Nys.This finding was acceptable because nystatin has no antibacterial properties which is close to Baldino et al. 23 study that reported Nys alone displayed lower CFU/mL for

Streptococcus mutans than
Chlorhexidine/nystatin formulation.On the other side, the effectiveness of the drug on some oral bacterial strains disagreed with the lack of studies dealing with the same oral bacterial strains found with candida in the swab of wounds rather than in denture stomatitis as in Lotan et al. (24) in its case series of 5 patients with nosocomial.was that the recent study took the biofilm from denture stomatitis patients not from wound.
From this article's finding and all other studies, the comparison related to bacterial species was difficult because almost all of the articles deal with the candida species as they are prevalent among all microbial with denture stomatitis biofilm but as summarized the illustrates that Nys of its two forms showed a high effectiveness toward candida species and a lower toward various kinds of bacterial species besides that, the ointment form occupies a superior effect than the suspension form.

Bacterial strains
Chlorhexidine digluconate is an antiseptic agent with a broad antibacterial spectrum, which acts against Gram-positive and Gramnegative bacteria and some fungi, its formulations have been widely used to chemically control dental biofilm (25) .This process.Cobalt-Chromium (Co-Cr) alloys, known for their applications in orthopedics and dentistry, are primarily classified as base-metal alloys.Co-Cr alloys are extensively used in dentistry for the fabrication of metallic frameworks for removable partial dentures and, more recently, for the fabrication of porcelainfused-to-metal restorations and implant frameworks.The rising global interest in using Co-Cr alloys for dental applications can be attributed to their inexpensive cost and acceptable physicomechanical Figure1.

Figure 1 :
Figure 1: Inhibition zones for the antimicrobial gel and solution forms Disk diffusion method for testing the susceptibility of bacterial strains to the drugs and disinfectant solution For the determination of susceptibility of bacterial strains to the three types of the drugs in form of both solution and gel by

(
n=5).The effectiveness of different drugs on inhibition zones of the fungi and bacteria found in dentures was examined in the Kruskal Wallis test.Nonparametric Comparisons for All Pairs Using the Dunn Method for Joint Ranking was used for pairwise comparisons.The statistical calculations were performed using JMP Pro 17.1.0(JMP ® , Version 17.1.0.SAS Institute Inc., Cary, NC, 1989-2023).

Figure 2 :
Figure 2: Susceptibility of Fungi to the two forms of three different types of Drugs to both Nys ointment and CHX gel, with median (M) inhibition zones of 22mm and 20mm, respectively.Whereas the strain showed approximately the same response toward Metro solution and CHX solution with (M=16mm), (M=16mm) respectively.The lowest sensitivity was shown toward Nys solution and Metro gel with median (M) inhibition zone of (10mm) and (8mm) in order Candida albicans exhibited remarkable sensitivity toward the gel and solution forms of Nys with (M=21mm), (M=16mm) in sequence, while the sensitivity of this strain slightly decreased toward CHX gel with Median inhibition zones of (16mm) and prominent decreased toward CHX solution with (M=8mm), whereas the two forms of Metro drug displayed nearly the same effect toward candida albicans of (M=12mm), (M=11mm) for both Metro gel and Metro solution respectively Bacterial strains to the three different types of Drugs Tables 2 and Figure 3 summarize our findings, indicating that, in general, the gel form of the three medications demonstrated greater efficacy against the bacterial strains associated with the fungi in our samples, compared to the solution forms.However, some bacterial strains exhibited resistance towards both forms Staphylococcus aureus showed the highest sensitivity toward Metro solution with a median (M) inhibition zone of (24mm) then showed lesser sensitivity to CHX gel with (M=20mm).The sensitivity of the Staphylococcus aureus decreased toward CHX solution and Nys ointment with (M=10mm) and (M=9mm) in sequent, while this bacteria showed resistance toward the effect of Metro gel and Nys solution with (M=1mm) and (M=0) respectively Corynebacterium diphtheria displayed the greatest sensitivity to CHX gel, with a median inhibition zone of (19mm), followed by Metro solution, with a median of (14mm).While its sensitivity decreased toward the remaining forms of the three drugs, toward CHX solution with (M=9mm), and (M=7mm) toward Metro gel, Nys solution, and Nys ointment Pseudomonas aeruginosa showed high sensitivity to CHX gel, with a median of (22mm), and slightly less sensitivity to Metro gel, with a median of (16mm).Pseudomonas aeruginosa responsiveness decreased to (15mm) M toward CHX solution and (8mm) M toward Nys ointment.

Figure 3 :
Figure 3: Susceptibility of Bacterial strains to the two forms of the three different types of Drugs ointment form was the treatment of oral yeast infection associated with Cryptococcus neoformans and Candida famata,.The uncommon presence of nystatin-resistant fungi and its affordable cost are the other advantages of this drug compared to other antifungals, Nys suspension also illustrated higher effectiveness on the three strains of candida than CHX and metro solution, as the means been shown in the descriptive analysis of about (15, 10.2,15.8)for Cryptococcus, this finding near study found that the two forms of CHX have the same as the two forms of the Nys in their powerful effect in inhibiting the ability of candida species to grow, in addition, the use of CHX in the form of gel exhibited more antibiofilm activity than mouthwash type as CHX causes several cellular alterations, such as the fragmentation of the cell wall and the degeneration of the cytoplasm, resulting in the fungicidal effect of the drug these finding matched Sajjan, et al.26 reviews and Scheibler et al.27 studies concluded that application of CHX gel to the fitting surface of maxillary denture reduced inflammation and significantly reduced fungal activity also rinsing with CHX together with soaking the denture overnight the same solution eliminated candida albicans on the denture surface which approximately near to the outcome of the gel form toward the three candida strain in this article.The finding of the recent study disagrees with in Akay et al.28 study which concluded that CHX disinfectant solutions weren't meant to be used as the first line of defense against biofilm since their efficiency was less than that of sodium hypochlorite solutions when they evaluated the effectiveness of the most used of antimicrobial agent.Furthermore, CHX in its two forms shows the highest effect among bacterial species except with Lactobacillus acidophilus and Streptococcus spp.So, research conducted by Sajjan, et al.(26) reviews showed a nearly similar outcome that CHX in the form of mouthwash and gel is effective in reducing the level of microorganisms, it was found to be significantly effective in reducingStreptococcus, Actinomyces, and other bacterial species related to peri-implantitis.Also, Baldino et al.23 study matched the current study which concluded that the combination of the drugs did not produce significantly different results compared to chlorhexidine alone.Considering its broad spectrum of action, chlorhexidine alone was already expected to be efficacious against S. mutans and C. albicans biofilm.It is worth noting the slight differences in results of the current research with the other research mentioned, as their sampling from oral candidiasis in addition to denture stomatitis while this study from only denture stomatitis.In summary, CHX not only an excellent antiplaque agent but it also possesses very good antimicrobial properties.Its broad antimicrobial spectrum can be considered a boon for overall oral health.A wealth of research supports its use in various forms in treating denture stomatitis.Metronidazole drug (gel & mouthwash solution) against Candida and Bacterial strainsIn dental practice, metronidazole is the next most widely given antibiotic after amoxicillin.Metronidazole is very effective in treating infections that involve anaerobic or microaerophilic microorganisms because of the mode of action of metronidazole that it passes through the cell target's membrane by passive diffusion, and its nitro group is converted to nitro radicals by ferredoxin or flavodoxin the redox capability of their electron transport components, which are liable for nitro group reduction and the generation of toxic metabolites29,30 .In this study in contrast to Nys and CHX, the use of the metro in the form of mouth rinse showed a moderate effect in reducing both candida albicans and candida famata while cryptococcus neoformans appear it's resistant against it.Also, the outcome of this article advocated the Sivakumar et al. 31 studies on the use of metro in Pediatric Dentistry which stated that metro is the drug of choice in strictly anaerobic infections and it is a concentration-dependent, not timedependent, antibiotic, also study recommended that these finding might have better results if used double or triple concentration.On the other hand, the bacterial strains associated with denture stomatitis of this article demonstrated resistance only toward Pseudomonas aeruginosa and Streptococcus species.While the drug demonstrated a high effect on all other bacteria, particularly the solution forms the gel form showed less effect and several bacteria showed resistance as Actinomyces species and Enterococcus species.This outcome appeared to match Wolff et al. 32 studies in oro-facial infection which reported that metronidazole should not be used to treat actinomycotic infections without added antimicrobial agents since metronidazole is not active against the pathogenic actinomycetes, also, this finding are close to Cooper et al. 11 review article , deal with treating dentoalveolar infections in a general dentistry practice context, which conclusion is that metronidazole, either alone or in combination, did not produce better clinical outcomes in terms of clearance or symptomatic alleviation when compared to other antibiotics.In summary, because of the poor use of Metro in research related to denture stomatitis, despite the acceptable result in this study for effectiveness against candida albicans the drug

Table 1 :
Effectiveness of different drugs on inhibition zones of the fungi adhered to denture base materials Nystatin 100000IU/ml oral-suspension > Chlor-hexidine 0.20% mouth-wash (p=0.0342)Nystatin 15g Ointment > Metro-nidazole 15g 0.75% Gel (p=0.0460)Kruskal Wallis test was performed for statistical analyses.Nonparametric Comparisons For All Pairs Using the Dunn Method For Joint Ranking was used for pairwise comparisons.

Table 2 :
Effectiveness of different drugs on inhibition zones of the bacteria adhered to denture base materials