Candida albicans is a normally harmless inhabitant of the oral cavity. However, unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is an opportunistic pathogen. The fungus’s behaviour correlates with its ability to grow in distinct morphogenic states, including budding yeast or blastospores, pseudo hyphae, and true hyphae ^{1– 3} . This morphogenic transition from yeast to hypha form is important for the pathogenesis of C. albicans, is dependent on how the fungus cell responds to the environmental cues ⁴ . As shown in the literature, cigarette smoke is one factor that can aid and accelerate this transformation ^{5– 7} , and there is a clear association between oral candidiasis and smoking habit ⁸ . In this regard, the presence of nicotine in cigarette smoke promotes C. albicans growth rate and adhesion, in which, a higher level of expression of genes related to adherence, such as ALS3 and HWP1 have been reported ⁹ .

Smoking is a social habit in many countries ¹⁰ , and it is an important public health problem, including in Indonesia ¹¹ . However, despite the extensive research exploring the deleterious role of a smoking habit on oral microorganisms, there is little information about the effect of non-conventional tobacco, i.e., clove cigarettes, on the virulence attributes of C. albicans. By searching the literature, we found that, like conventional cigarettes, the clove cigarette, commonly known as kretek, is the most popular ¹² in Indonesia. It is also a source of numerous toxicants, and they have a potential implication in the oral ecosystem ¹³ , which may also influence oral Candida pathogenicity ¹⁴ . The unique aspect of kretek is the dried clove buds it contains ¹⁵ which have never been identified in a conventional cigarette. The involvement of clove cigarette smoke condensate (CCSC) on the biofilm formation of C. albicans remains obscure. Given that cigarette smoke contains many toxicants ¹³ , it is important to explore the involvement of CCSC on the growth and morphogenesis of C. albicans, as this fungus is the most implicated oral pathogen in the clinical setting.

During the last several years, aptamers, either single strain DNA or RNA, and against different microorganism species, have become the focus of growing interest. In an earlier study, we reported an anti- Candida activity of an RNA-aptamer (Ca-apt1) against the fungus while growing as biofilm ¹⁶ . Here, we evaluated the aptamer's beneficial properties against C. albicans biofilm formation induced by cigarette smoke. Particularly, the aptamer's ability to restrict the transition of fungus phenotype from yeast to hyphal form.

Written informed consent was obtained from participants for use of their data.

The samples were collected, after informed consent was obtained from all the participants ¹⁷ , in accordance with the approved protocol of the Bioethics Committee of the Faculty of Dentistry, Universitas Indonesia (protocol number 020950818). The protocol conformed to the criteria of the Helsinki Declaration and the good clinical practical guidelines of the International Council on Harmonization.

The current study attempted to get further insight into clove cigarettes' effect on non-mammalian eukaryotes cells, taking the fungus C. albicans as a model system. Initially, as a means to determine that the restriction effect of C. albicans to switch its phenotype from yeast to hypha form was due to the presence of the precoating aptamer on yeast, we separated biofilm development into adhesion and growth phases. The result of the transcription assay during the initial stage of biofilm formation indicated that the aptamer involvement in reducing the expression of adhesion and hypha-associated gene ( ALS3 and HWP3) ²⁰ , and the transcription profile was in line with the increased concentration of the aptamer, but not on C. albicans strains. This result suggests that the aptamer (Ca-apt1) might bind to a common chemical structure on the fungal yeast cell, probably glucans and chitins ²¹ that form the basic cell wall scaffold ²² . We presume that this aptamer may interfere with the adherence mechanism where these molecules are involved ²² .

Our study is different from most in vitro studies, which use an antibody that recognizes cell-wall proteins, that are commonly related to the host immunological status ^{23, 24} . In this study, the precoating aptamer (Ca-apt1) does not directly recognize the cell wall proteins as an antibody would. In this way, we assumed that based on the ALS3/ HWP1 profiles explained above, the obvious virulence factors (morphology transformation from yeast to hypha) were more likely to be the main effect of precoating Ca-apt1. This assumption was supported by microscopic observation that at the attachment phase (90 min time point) the morphology of C. albicans consisted of both yeast cells and blastospore only. This result confirmed the transcription assay that the aptamer involvement in reducing adhesion and hypha-associated gene ( ALS3 and HWP3) ²⁰ was in line with the increased concentration, but not on C. albicans strains. Likewise, this qualitative data exhibited a significant potentiate of Ca-apt1 in modulating biofilm-associated genes, as well as C. albicans morphogenesis, at the early step of biofilm formation.

Although the pattern was similar, in general, the biofilm formation (90 min time point) treated with CCSC resulted in a higher viable cell than its NCSC counterpart, as we observed that after being treated for 48-h by CCSC or NCSC. Different cellular response of C. albicans was noted, both by analysing the gene ( ALS3 and HWP1) profiles and observing microscopically. At this maturation biofilm state, the induction of hypha-associated gene ( HWP1) was significantly different, but not for the gene's adhesion ( ALS3). We noted that the transcription level of HWP1 by either C. albicans strain was higher than ALS3 only when the preformed biofilm was treated with CCSC. This result indicates that unlike ALS3, the transcription level of HWP1 was dependent on the source of the condensate smoke used. The effect of condensate smoke on C. albicans cells viability in biofilm development does not relate to C. albicans strain. We assumed that different tobacco components, specifically the principal component alkaloid of tobacco (nicotine), between kretek and conventional cigarettes ^{25, 26} , may have a different effect on C. albicans when the fungus was growing in distinct environmental conditions that trigger the hyphal growth. Additionally, the HWP1 product (Hwp1) is a hypha-association protein commonly expressed on germ tube ^{27, 28} . Indeed, this result can be considered after 90 min, when germ tube was induced ²⁹ . The upregulation of HWP1 when the biofilm is maturing (48-h) is a survival pathway used by C. albicans to resist, or be tolerant against, the effect of chemical toxic-containing CCSC, by modulating the hypha form.

It has been reported that, like NCSC, CCSC comprises a high number of dangerous alkaloid chemical compounds, the most abundant of these being nicotine ²⁵ . A previous report also showed that the CCSC and NCSC used in this study deliver significant quantities of carbon monoxide (CO), tar and presumably other toxic components of tobacco smoke. Their smoke chemistry were found to be similar ³⁰ . The other chemical compound in the gas phase of cigarette smoke is nitrogen (N2) ³¹ . The nicotine in smoke yields from CCSC is equally delivered as by NCSC ³² . However, the nitrogen content in CCSC is lower than found in conventional cigarettes/NCSC ³⁰ . The most distinguished particle-phase, which is only found in CCSC, is eugenol ¹⁵ . Eugenol is a phenylpropanoid compound reported to have antimicrobial activity against planktonic cells of C. albicans and sessile cells within C. albicans biofilms ³³ .

In contrast to the eugenol, the presence of nicotine in cigarette smoke promotes a high level of adhesins leading to the increased adherence of C. albicans ³⁴ on a solid surface, as found in this study. Hence, it has the potential to increase biofilm formation by C. albicans. Considering that eugenol impairs the growth of C. albicans, we assume that under the experimental condition set in this study, C. albicans sensed eugenol in CCSC, and the presence of nitrogen helped it to grow. Since we grew the fungus in a hypha conditioning medium, the result obtained in this study indicates that by comparing with NCSC, the lower nitrogen in CCSC is the likely reason for the augmentation of C. albicans biofilm formation ^{9, 35} enhanced by the CCSC. As we evaluated microscopically, more hypha in 48-h biofilm was observed in response to the presence of CCSC than NCSC.

As shown in the literature, cigarette smoke exposure (CSC) has a potential adverse health effect on the oral ecosystem ³⁶ , and the growth rate of C. albicans increases when the fungus is grown in the presence of non-clove cigarette smoke condensate/NCSC ⁵ . Indeed, cigarette smoke is an important predisposing factor for oral candidiasis ³⁵ . Although kretek/clove cigarette smoke condensate (CCSC) and NCSC contains many toxicants, they have some different chemical constituents ^{30– 32} , which may lead to the differences in Candida cell behaviour that we observed in this study.

We further address whether the precoating aptamer could adversely affect the preformed biofilm formation's viability when treated with CCSC or NCSC. The experiment was done to confirm that, on the one hand, experimental C. albicans biofilm formation promoted by cigarette smoke (CCSC and NCSC) could be induced. On the other, that the precoating aptamer is needed for the success of this model. In this way, the discrepancy between CCSC and NCSC on C. albicans susceptibility was measured using a MTT reduction assay. This is a reliable test for an indirect method to quantify biofilm cell numbers ^{37, 38} . It has been demonstrated that when growing as a biofilm, the metabolic activity of C. albicans increases over time ³⁹ . However, another factor (the aptamer) was added in the current study. Here, we observed that the control group ( C. albicans of untreated condensate cigarette smoke/ CSC, without precoating aptamer (Ca-apt1) remained growing as a biofilm throughout the experimental time period set in this study. This result may suggest that C. albicans used the tobacco compound as a nutritional source, as aromatic hydrocarbons in cigarette smoke can be converted by the fungus ³⁵ .

Further, we found that after treating with NCSC for 48-h, the lowest concentration of tested aptamer (1ng/µL) was enough to reduce the cell viability > 50%. This indicates that the effectivity of NCSC as an accelerator of biofilm formation ⁵ was significantly suppressed. Hence, it affirms the anti-adhesion effect of the aptamer on biofilm development accelerated by conventional cigarette smoke ^{5, 7} . Surprisingly, aptamer-precoated C. albicans behaves differently upon exposure to growth medium-containing CCSC. We noted that at one ng/µL of the aptamer, significant cell growth changes were not observed compared to the control (uncoated aptamer). However, at 10 ng/µL, the aptamer could inhibit the biofilm cell growth until C. albicans reached a steady state at a 48-h time period, more and less at a similar rate to those cells exposed by NCSC. The result was supported by the light microscopy data, in which we observed that when the fungus was exposed with the minimal concentration (1 ng/µL) of Ca-apt1, the composition of 48-h-old biofilms consisted of pseudo hypha, almost similar to the control ( C. albicans without pre-coated aptamer). We also observed that when the concentration of the tested aptamer was increased by 10-fold, it prevented the cell's viability and successful germination of the adherence cells, resulting in scant biofilms, and more yeast cells were observed microscopically in treated groups (CCSC and NCSC exposure). For NCSC, we observed that regardless of the tested aptamer's concentration in precoating step, the NCSC-treated biofilms were predominantly in the blastospore or yeast form compared to the control. After 48-h exposure, many of these biofilm cells were unbudded and had an unelongated morphology. At the end of the experiment time, both C. albicans strains, which belonged to the treated groups ( C. albicans with precoating aptamer), showed a similar phenotype response to either CCSC or NCSC exposure. Our result contradicted a previous report that demonstrated that the clinical isolate showed less biofilm growth activity than the laboratory strain ⁴⁰ . We reasoned that the similar trend found in either C. albicans strain used in this study is probably because the clinical isolate and the ATCC strain used in this study have a similar karyotype as it has been previously reported ⁴¹ .

Regardless of the mechanism involved, this study demonstrated that both CCSC and NCSC have a potentiate to enhance biofilm formation by C. albicans. By exposing either cigarette smoke prepared from clove and non-clove cigarettes, C. albicans, either clinical isolate or the reference strain, shows a similar trend in its capacity to form biofilm. However, the pattern of biofilm formation was different. Besides, this study clearly indicates that the aptamer (Ca-apt1) had an impact on the degree of cell adhesion and its concentration affected the profile of biofilm formation at the maturity phase. However, the underlying mechanisms remain to be investigated at molecular level. The underlying mechanism is particularly important for pathogenic fungus, such as C. albicans, during exposure to tobacco smoke.