Marine Ascomycetes Extract Antifungal Susceptibility against Candida spp. Isolates from Oral Candidiasis HIV/AIDS Patient: An In Vitro Study

Abstract Objective  The etiology of oral candidiasis (OC) was Candida albicans , C. krusei , C. dubliniensis , C. tropicalis that are frequently found in human immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS) patients. Marine ascomycetes (MA) have been widely reported as an important producer of various antibiotic compounds. However, there is limited study of antifungal compounds from MA against Candida species. The aim of this study was to investigate the antifungal susceptibility of MA against Candida spp. isolates from OC HIV/AIDS patient. Materials and Methods   Trichoderma sp . is a sponge-associated fungus collected from Karimunjawa National Park, Central Java, Indonesia. The validation of C. albicans, C. krusei, C. dubliniensis, C. tropicalis . was done by ChromAgar. This study was true experimental with post-test only control group design; the sample was four replications for each group. Nystatin administration (K +), the golden standard antifungal drug, was used. The minimum fungicidal concentration (MFC), minimum inhibitory concentration (MIC), and diffusion zone methods were done. Analysis of variance difference test, and post-hoc Tukey's honest significant different were done to analyze the significant different between groups ( p ≤ 0.05). Results  The MFC and MIC of MA against C. albicans, C. krusei, C. dubliniensis , and C. tropicalis were found at 12.5%. In addition, the greatest diffusion zone of MA against C. albicans, C. krusei, C. dubliniensis , and C. tropicalis was found at 12.5%. There is no appreciable difference in antifungal activity between K + and 12.5% of MA extract ( p ≥ 0.05). Conclusion  Concentration of 12.5% MA extract has antifungal susceptibility against Candida spp . isolates from OC HIV/AIDS patient.


Introduction
The most prevalent oral symptom of the human immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS) with low count of cluster differentiation 4 (CD4) in adults or children is oral candidiasis (OC), which can affect the tongue and other oral mucosal locations. 1,2As the viral disease progresses, it has been observed that nearly all HIV-infected individuals have a Candida colonization and that up to 90 to 95% of them acquire clinical lesions.An earlier study performed in Dr. Soetomo, Surabaya, East Java, Indonesia, discovered that 68 patients out of the 88 adult HIV/AIDS patients exhibited clinical features of OC. 1 Additionally, out of a total of 28 HIV-positive pediatric patients, OC was discovered in 16 (57.14%) of them in an earlier study. 2everal OC manifestations, including pseudomembranous candidiasis, acute and chronic erythematous candidiasis, and chronic hyperplastic candidiasis, can occur in HIV/AIDS patients. 3Linear gingival erythema and oral hairy leukoplakia are two conditions that resemble the clinical sign of OC. 4,5 Immunosuppressive condition in HIV/AIDS patient may lead to decrease in oral mucosal immunity that gives the chance of normal flora in the oral cavity to become opportunistic.Dental caries and periodontal disease also can frequently be found in HIV/AIDS patient. 6,7Candida species are opportunistic infections that affect people with compromised immune systems and immunosuppression. 1,2 fungal overgrowth on the tissues and, occasionally, the appearance of a white flake on the mucosal areas describe this illness. 3The most common cause of OC is Candida albicans.][10] However, prior previous studies identified a number of other species, including C. glabrata, C. dubliniensis, C. krusei, and C. tropicalis, as the culprits of candidiasis at Dr. Soetomo General Hospital, Surabaya, East Java, Indonesia.These fungal infection causes a severe systemic illness called candidemia. 1,2urthermore, candidiasis will have a higher negative impact for AIDS patients.4][15] Additionally, in order to defend themselves, Candida spp.create biofilm that increases their resistance to antifungal drug. 16,17ccording to several researches, infections including coronavirus disease-2019 and multidrug resistant (MDR)-tuberculosis can coinfect people with Candida spp.This coinfection will increase the duration of the medicine and raise the mortality rate. 18,19Additionally, recent research has shown that xerostomia radiation makes patients with head and neck cancer more susceptible to OC infection. 20It is necessary to investigate for novel antifungal compounds due to the decreasing efficacy of medications and their negative effects. 21The inability of current antifungal medications to treat OC in HIV/AIDS patients as a result of drug resistance has sparked basic research to develop novel antifungal medicines and HIV/AIDS medicine. 3,22e sponge-associated microorganisms are renowned for being among the top producers of antibacterial chemicals.Moreover, numerous antibiotics including averantin, nidurufin, citrinin, Chlorohydroaspyrones A and B are isolated from sponge-associated fungi, especially from phylum ascomycetes. 23Therefore, marine ascomycetes (MA) from a specific niche were carried out to gather possible bioactive substances.][26][27][28] Indonesia, as an archipelagic and maritime country, is home to massive MA that exist as a companion microbe.A filter-feeding creature called a marine sponge deposits a lot of MA that produces antibacterial chemicals. 29However, this work did not employ the Candida spp.isolates from OC HIV/AIDS patient or extract the pure lead compounds, despite the fact that previous study showed that various sponge-associated fungus from Indonesia shown anti-C.albicans activity. 30Additionally, Aspergillus sp.LS78 linked with marine sponges effectively yielded a new aspericacids A with antifungal efficacy against non-MDR C. albicans. 31As a result, it may indicated that sponge-associated MA may possessed excellent potential as a new source of novel antifungal drugs against Candida spp. to treat OC in HIV/AIDS patients.Furthermore, the aim of this study is to investigate the antifungal susceptibility of MA against C. albicans, C. dubliniensis, C. krusei, and C. tropicalis isolates from OC HIV/AIDS patients.

Ethical Clearance, Study Design, and Sample Selection
At the Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Candida spp. was isolated from OC patients after receiving ethical approval and appointment number 681/ HRECC.FODM/IX/2022.In this cross-sectional study, patients who had been tested for HIV/AIDS positive by means of polymerase chain reaction examination and low CD4 about less than 200 cells/mm 3 examined by means of flowcytometry were recruited.The patient's parents or guardians provided written informed consent and informed to consent form prior to the oral evaluation.Without knowing the patient's immunologic condition or whether they had undergone highly active antiretroviral therapy or not, trained oral medicine professionals performed an oral examination on each Indonesian patient.
The patient was examined while seated in a dental chair, with the aid of disposable dental mirrors, sterile gauze pads, and tongue blades, all under good illumination.According to European Commission-Clearinghouse clinical diagnostic criteria, OC was identified by its clinical characteristics as (1) pseudomembranous type, (2) erythematous type, and (3) angular cheilitis.An oral swab sample was used in a microbiological test to pinpoint the specific Candida spp. that was infecting the patient.HIV/AIDS patients who had OC at the time of the evaluation had oral swabs taken.These swabs were brought to the lab for culture and immediately submerged in Sabouraud dextrose liquid medium.Colony morphology on ChromAgar and microscopic morphology on slide culture were used to identify the species of Candida. 1,2etabolite Production and Extraction Trichoderma sp.(code:KJMT.FP 3.2) was isolated from an unidentified marine sponge from Karimunjawa National Park, Central Java, Indonesia.The seed culture was prepared in V-22 (1%, glucose 0.5%, NZ-case [Sigma-Aldrich, Co., LLC.] 0.3%, yeast extract [Kyokuto Pharmaceutical Industrial, Co., Ltd.] 0.2%, Tryptone [Difco Laboratories] 0.5%, K 2 HPO 4 0.1%, MgSO 4 •7H 2 O 0.05%, and CaCO 3 0.3%, pH 7.0) broth for 3 days at 200 rpm and 30°C.For production culture, in total 1% (v/v) of the seed culture was transferred into A16 broth media (0.2% glucose, 1.5% Pharma media, 0.3% CaCO 3 , 1% Diaion HP-20, pH 7.0.)for 7 days (200 rpm, 30°C).Afterward 1-butanol was added for extraction with ratio 1:1, then evaporated at 35 °C. 32

Determination of MIC and MFC of the Lead Compounds
On Inorganic Salt Starch Agar number 4 agar and Ascomycetes Isolation Agar all sponge-associated MA will be grown for 7 days in order to be used in this screening.All pure lead compounds will be dissolved in dimethyl sulfoxide (DMSO) to reach concentration of 100, 75, 50, 25, 12.5, and 6.25%.The minimum inhibition concentration (MIC) assay will be conducted using microwell-dilution method in 96-well plate.Each well will be filled by 195 µL of Sabouraud dextrose broth (SDB) with the C. albicans, C. krusei, C. dubliniensis, and C. tropicalis (0.5 McFarland) and 5 µL of the diluted pure lead compound solution.Nystatin will be used as a positive control, DMSO 5 µL/plate as a negative control, and SDB without any additional substances as a blank.The plate will be incubated for 24 hours at 32 °C.After 24 hours of incubation, in total 10 µL of Resazurin will be added into each well to give blue/purple color then incubated for 2 hours.The color changes from blue/purple to bright yellow indicates the bacterial growth inhibition.The lowest concentration that able to inhibit the bacterial growth will be determined as the MIC.Further, in total 10 µL of SDB from concentration that able to inhibit the bacterial growth will be inoculated onto SDA and incubated for 24 h at 32 °C.The lowest concentration with no bacterial growth on Sabouraud dextrose agar (SDA) will be determined as MFC value. 33,34reening of Antifungal Activity against Candida spp.
To find the probable isolate with antifungal activity, the agar plug technique will be used.MA with concentration of 25 and 12.5% was used for diffusion zone method.C. albicans, C. krusei, C. dubliniensis, and C. tropicalis shall be maintained on SDA for 24 hours before to the test.The pathogen shall be completely inoculated on SDA on the implementation day after being diluted in physiological salt solution to a turbidity of 0.5 McFarland.After that, the MA will be cut into circles and placed on the SDA that has received the vaccination. 33fter being incubated for 24 hours at 32 °C, the MA's presence in a clear zone denotes their antifungal activity.

Statistical Analysis
The study data were then all compiled and evaluated both descriptively and inferentially.A bar chart showing the mean and standard deviation of the data is displayed.The statistical package for social science (SPSS) version 20.0 for Windows was used to analyze the data.This version included the normality and homogeneity test (p > 0.05), analysis of variance difference test, and post-hoc Tukey's honest significant different (HSD) with a different significance value of p < 0.05 (IBM corporation, Illinois, Chicago, United States).

Results
From HIV/AIDS patient with low CD4 þ with OC, the isolate of Candida spp. was obtained.ChromAgar was used to characterize and identify the species of Candida spp.HIV/AIDS patient isolates revealed that there are four Candida spp.such as C. albicans (light green color); C. dubliniensis (green to yellow color); C. krusei (pink to crème color); and C. tropicalis (dark green color ►Fig.1).
In this study, MA extract was found to have MIC and MFC and to be able to stop the development of the Candida species  The nystatin treatment produced the highest levels of MIC and MFC of C. albicans, which were followed by MA extract concentrations of 100, 75, 50, 25, and 12.5% with significant differences (p ¼ 0.0001; p < 0.05; ►Fig.2A-D).Nystatin treatment, followed by MA extracts of 25 and 12.5%, produced the widest zone of C. albicans suppression (►Fig.2E).On the C. albicans inhibitory zone, there was no discernible difference between the treatment groups of Kþ and 12.5% of MA extract (►Fig.2F).
The MA extract of 100, 75, 50, 25, and 12.5% was shown to have significant differences in MIC and MFC of C. krusei (p ¼ 0.0001; p < 0.05; ►Fig.4A-D) when compared to nystatin treatment.Following nystatin treatment, MA extracts of 25 and 12.5% were found to have the largest zones of C. krusei inhibition (►Fig.4E).On the C. krusei inhibitory zone, there was no statistically significant difference between Kþ and 12.5% of MA extract as treatment groups (►Fig.4F).With a significant difference (p ¼ 0.0001; p < 0.05; ►Fig.5A-D), the highest MIC and MFC of C. dubliniensis were discovered in the nystatin treatment.►Fig.5E shows that nystatin treatment, followed by MA extracts of 25 and 12.5%, produced the widest zone of inhibition of C. dubliniensis.The inhibition zone of C. dubliniensis showed no significant change between the treatment groups of Kþ and 12.5% of MA extract (►Fig.5F).

Discussion
Human medicine has greatly benefited from the discovery and development of MA secondary metabolites.Even though there are hundreds of antibiotics and antifungals on the market right now, there is still a need and opportunity for the development of novel antimicrobials.Thus, there is a growing need for new antibiotics or alternative strategies to address antibiotic or antifungal resistance. 35ne of the major concerns to medicine today is the emergence of antibiotic-resistant bacterial infections, particularly those brought on by Enterococcus faecium,   Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. 36The antibiotics produced by MA that have been isolated from marine sponges have been shown to be effective against a variety of MDR bacteria. 37f the aim is to identify clinical or veterinary medications, cytotoxicity to the patient should be ruled out when dealing with microbial crude extracts.From crude extracts generated from diverse species, methodologies have been established for the screening, isolation, identification, and characterization of important bioactive chemicals and secondary metabolites. 38MA crude extracts also showed comparable findings, with the latter showing potential properties of therapies for antibiofilm development and quorum quenching.Therefore, the development of new antibiotics to treat MDR infectious diseases can come from MA that originated from Indonesia. 39,40A might be a source of antibiofilm agents of pathogens and other undesirable bacteria, when cellular development is not specifically controlled, other from by the limitation of quorum sensing.Since the selection pressure is "softer" with such antibiofilm agents, the evolution of resistance to anti-microbial products would be less likely.MA can be employed to prevent the development of biofilms and quorum sensing while avoiding the development of bacteria-evolved resistance, an issue frequently brought on by the use of antibiotics. 32,41MA have been used in medicine as sources of secondary metabolites that have antibacterial, antifungal, anthelmintic, and anticancer properties. 29The use of MA as probiotics has also been studied recently, while the majority of the study in this area is focused on applications in aquaculture and other aquatic settings. 42The capabilities of additional MA may be investigated in this field, even if it is uncertain whether MA should be included in probiotics given to people.MA is expected to be as common in applied research as they are in the world's seas as more is learned about their special characteristics. 43mong the worst dangers to human health across the world are infectious fungus infections.Fungal infections cause over 2 million fatalities annually worldwide, and the death toll is rising due to an increase in immunocompromised populations at risk.The introduction of infections that are resistant to first-line antifungal medications worsens the results. 43,44The evolutionary history of fungus and mammals has impeded the development of novel antifungals, restricting treatment choices to medications due to low efficacy and/or harsh side effects.Like other antimicrobial leads, the majority of antifungal agents-including two of the three known antifungal classes-come from sources that are natural products. 45,46his study investigated the antifungal susceptibility of MA against C. albicans, C. dubliniensis, C. krusei, and C. tropicalis isolates from OC HIV/AIDS patients.This study revealed that MA isolated from Karimunjawa National Park, Central Java, Indonesia has MFC and MIC against C. albicans, C. krusei, C. dubliniensis, C. tropicalis isolated from OC of HIV/AIDS patient at 12.5%.In addition, MA has greatest inhibition zone against C. albicans, C. krusei, C. dubliniensis, C. tropicalis was found at 12.5%.There is no appreciable difference in antifungal activity between nystatin and 12.5% of MA extract.There are several side effects of nystatin oral drop administration for OC treatment in HIV/AIDS patient such as nausea, vomiting, stomachache, headache, and diarrhea. 21In addition, previous study also reports that there is Mangrove leaves ethanol extract (Aegiceras corniculatum) that had no antifungal efficacy against C. albicans isolated from HIV/AIDS with no significant difference in inhibitory zone (p > 0.05). 47ue to their effectiveness against MRSA and unique mode of action as antibacterials, MA will undoubtedly be the focus of intensive study.This is in addition to the rapidly expanding interest in natural products, particularly those originating from marine organisms. 37,48n many Southeast Asian countries, medicinal plants are commonly used for antifungal activities.Among others, three plant species, that is, S. aromaticum, C. citratus, C. xanthorrhiza, are widely used for treating candidiasis in Indonesia, Malaysia, and Thailand. 49One method for enhancing MA anti-Candida properties is by extracting their organic solvent, which releases associated bioactive chemicals. 50,51p to our knowledge, there have not been any previous reports on MIC and MFC values of MA isolated from Karimunjawa National Park, Central Java, Indonesia.To further improve MA extracts as the active ingredients in anti-Candida medications and dietary supplements, more research into the chemical components of the extracts as well as cytotoxicity tests on test subjects and mammalian cells is required.

Conclusion
The OC of an HIV/AIDS patient was successfully isolated by our investigation from C. albicans, C. krusei, C. tropicalis, and C. dubliniensis.Nystatin antifungal resistance was found in C. albicans that was isolated from an HIV/AIDS patient's OC.Concentration of 12.5% MA extract has antifungal susceptibility against Candida Spp isolates from OC HIV/AIDS patients.Following the experimentation, numerous recommendations for future research have been made, including the need for enrichment and modified medium to minimize the isolation time and get more isolates of Candida spp. to expedite the investigation, a straightforward yet precise antifungal is required.

Fig. 1
Fig. 1 ChromAgar was used to characterize and identify the species of Candida spp.Human immunodeficiency virus/acquired immunodeficiency syndrome patient isolates revealed that there are four candida species such as C. albicans (light green color); C. dubliniensis (green to yellow color); C. krusei (pink to cream color) and C. tropicalis (dark green color).
that are involved in OC in HIV/AIDS patients with low CD4þ levels: C. albicans, C. dubliniensis, C. krusei, and C. tropicalis.

Fig. 2
Fig. 2 Candida albicans is the target of MA extract's antifungal action.(A) The MA extract's significant antifungal activity was seen in C. albicans' MIC values of 12.5, 25, 50, 75, and 100% following administration.(B) Following ingestion of MA extract, the MFC of C. albicans showed 12.5, 25, 50, 75, and 100% strong antifungal activity.After administration of MA extract, the inhibitory zone employing disk diffusion analysis on C. albicans revealed no appreciable difference in antifungal activity between Kþ and 12.5% of MA extract.MA, marine actinomycetes; MFC, minimum fungal concentration; MIC, minimum inhibitory concentration.

Fig. 3
Fig. 3 Candida tropicalis is the target of MA extract's antifungal action.(A) The MA extract's significant antifungal activity was seen in C. tropicalis' MIC values of 12.5, 25, 50, 75, and 100% following administration.(B) Following ingestion of MA extract, the MFC of C. tropicalis showed 12.5, 25, 50, 75, and 100% strong antifungal activity.After administration of MA extract, the inhibitory zone employing disk diffusion analysis on C. tropicalis revealed no appreciable difference in antifungal activity between Kþ and 12.5% of MA extract.MA, marine actinomycetes; MFC, minimum fungal concentration; MIC, minimum inhibitory concentration.

Fig. 4
Fig. 4 Candida krusei is the target of MA extract's antifungal action.(A) The MA extract's significant antifungal activity was seen in C. krusei's MIC values of 12.5, 25, 50, 75, and 100% following administration.(B) Following ingestion of MA extract, the MFC of C. krusei showed 12.5, 25, 50, 75, and 100% strong antifungal activity.After administration of MA extract, the inhibitory zone employing disk diffusion analysis on C. krusei revealed no appreciable difference in antifungal activity between Kþ and 12.5% of MA extract.MA, marine actinomycetes; MFC, minimum fungal concentration; MIC, minimum inhibitory concentration.

Fig. 5
Fig. 5 Candida dubliniensis is the target of MA extract's antifungal action.(A) The MA extract's significant antifungal activity was seen in C. dubliniensis' MIC values of 12.5, 25, 50, 75, and 100% following administration.(B) Following ingestion of MA extract, the MFC of C. dubliniensis showed 12.5, 25, 50, 75, and 100% strong antifungal activity.After administration of MA extract, the inhibitory zone employing disk diffusion analysis on C. dubliniensis revealed no appreciable difference in antifungal activity between Kþ and 12.5% of MA extract.MA, marine actinomycetes; MFC, minimum fungal concentration; MIC, minimum inhibitory concentration.