*2.3. Hydrophobicity*

In *Candida* sp., the adhesion phenomenon is mediated by agglutinin-like (Als) sequence proteins [88], which are glycosylphosphatidylinositol (GPI)-linked to β-1-6 glucans in the fungal cell wall. Als-dependent cellular adhesion is connected with increases in cell surface hydrophobicity (CSH) [89]. The CSH of *Candida* sp. enhances virulence by promoting adhesion to host tissues [89,90]. *C. albicans* Als3p (hypha-associated) is a major epithelial adhesin that is strongly upregulated during epithelial infection in vitro [90], and the disruption of the *ALS3* gene reduces epithelial adhesion in vitro. Likewise, decreasing the expression of the *ALS2* gene also reduces adhesion [91,92]. On the other side, deletion of the ALS5, ALS6, or ALS7 genes increased adhesion [93], demonstrating that the Als proteins can have opposing roles in fungal attachment to surfaces. Putative homologues of Als proteins have also been identified in NCAC [94]. In *C. glabrata*, for example, epithelial adhesins (Epa) have a comparable structure to the Als proteins [95,96].

Together with adhesion ability, hydrophobicity is a virulent factor that is gene-regulated and usually positively correlated with biofilm metabolic activity [97,98] since hydrophobic interactions seem to be crucial in promoting tissue invasion by the mycelial phase of *Candida* sp. It is presumed that *Candida* sp. can grow under anaerobic conditions, although in these conditions fermentation is the dominant pathway for ATP production [99]. The results of Sardi et al. [100] indicated that 51.97% of diabetic patients' isolates were highly hydrophobic under anaerobic conditions when compared to 21.90% under the aerobic atmosphere [100]. It is recognized that the germ tubes are able to adhere to fibronectin, fibrinogen, and complement via cell surface receptors [101], helping the attachment of filament yeasts to extracellular matrix components (ECM) [102] and producing impairment of phagocytosis, consequently increasing the resistance to blood clearance and the virulence of *Candida* sp. [103].

#### **3. Candidiasis and DM**

## *3.1. Oral Diseases*

## 3.1.1. Oral Candidiasis

The prevalence of oral candidiasis is increasing, as it is one of the most common fungal infections [104]. Oral candidiasis can be diagnosed by the differential patterns of mucosal changes like erythematous, pseudomembranous, and curd-like plaques (biofilms) [105,106]. Higher *Candida* sp. colonization rates were reported in patients with DM type 1 when compared to DM type 2 patients (84% vs. 68%, respectively), while the percentage in nondiabetic subjects was around 27% [18]. The studies also describe how this colonization does not always lead to infection. Nonetheless, it is a prelude to infection when host immunity is compromised and the risk of a disseminated infection is high [107]. Such infections continue to be a major healthcare challenge [108].

The risk factors for oral candidal infection are complex, but it is known that tongue lesions, tobacco smoking, denture wearing, and immunosuppression (e.g., diabetes mellitus) clearly influence oral *Candida* sp. carriage and the upsurge of oral candidiasis [30,109–114]. The causes influencing the higher incidence of oral candidiasis in diabetic patients are presented in Table 1.

Higher expressions in enzymatic activity and the biofilm forming capacity of *Candida* sp. are two of the most important features in oral candidiasis. In a study by Sanitá et al. [115], the virulence of 148 clinical isolates of *C. albicans* from oral candidiasis was characterized by measuring the expression of PL and SAP in healthy subjects (HS), diabetics with oral candidiasis (DOC), and non-diabetics with oral candidiasis (NDOC). For PL, *C. albicans* from NDOC and DOC had the highest enzymatic activity (76.6%); for SAP, *C. albicans* from NDOC exhibited lower enzymatic activity (48.9%). Similar results have been reported in the past [59,87], with percentages greater than 90% for both PL and SAP activity among clinical isolates of *C. albicans* [26,116,117] found. Arlsan and colleagues found 12 different genotypes and compared the virulence factors of several *Candida* sp. isolated from the oral cavities of 142 healthy and diabetic individuals, with and without caries. Although the most isolated species was also *C. albicans*, there were statistical differences in terms of isolated *Candida* frequency between healthy subjects and diabetic patients. DM showed no effect on the activities of virulence factors (biofilm production, proteinase, and phospholipase activity) of *Candida* sp. Yet, different genotypes of *C. albicans* exhibited different virulence activities [118]. Other authors showed that the activity of SAPs suggestively rises in denture wearers with signs of candidiasis compared to denture wearers with a normal palatal mucosa [119]. The inconsistency of results of these reports can be explained by the large variation of intra-and interspecies of *Candida* and deviations in the methodology in most reports [24,26,59,87,116,117,119–123]. A longitudinal study by Sanchés-Vargas et al. [124] quantified biofilms in oral clinical isolates of *Candida* sp. from adults with local and systemic predisposing factors for candidiasis. Between the isolates, authors found *C. albicans*, *C. tropicalis*, *C. glabrata*, *C. krusei*, *C. lusitaniae*, *C. kefyr*, *C. guilliermondii*, and *C. pulcherrima* from the oral mucosa of totally and partially edentulous patients (62.3%) and the oral mucosa of diabetics (37.7%). On average, the oral isolates of *C. glabrata* were considered strong biofilm producers, whereas *C. albicans* (the most common species) and *C. tropicalis* were moderate producers. This might be because *C. glabrata* has been shown to have a higher aggressiveness, producing a grea<sup>t</sup> quantity of biofilm matrices ye<sup>t</sup> also increasing chitin concentrations in the cell walls [125–127].

Additional important features are the oral pH and the glycemic control. A study performed by Samaranayake et al. [128] demonstrated that pyruvates and acetates are the major ionic species, generating a quick decrease in pH with *Candida* sp., as found in batch cultures of mixed saliva supplemented with glucose [128].

Other authors indicated that yeasts have a superior ability to adhere to epithelia and denture acrylic surfaces at a low pH of approximately 2–4.14 [31,129]. Balan and colleagues stated that during hyperglycemic episodes, the environmental alteration in the oral cavity increased salivary glucose and acid production, which favored the transition of *Candida* sp. from commensal to pathogen [31]. In another report, while comparing diabetics and non-diabetics, Pallavan et al. [130] verified that 70% of the healthy individuals had lower colonization and 43.3% of the diabetic patients had severe colonization by *Candida* sp., which was also observed in other studies [14,18,21,131]. Prediabetes is a condition where there is an elevation of plasma glucose above the normal range but below that of clinical diabetes [132]. Javed and their colleagues isolated oral *Candida* sp. from 100% of patients with prediabetes and from 65.7% of the controls, observing that the carriage of *C. albicans* was greater among patients with prediabetes (48.7%) than with controls (25.7%) [133]. They also observed that the colonization with *Candida* sp. reduced the salivary flow rate and was independent of glycemic status in patients with prediabetes [132].

In fact, while some studies found a direct significant association between glycosylated hemoglobin and oral *Candida* colonization [134,135], other authors found no relationship between glycosylated hemoglobin and high *Candida*-burden in patients with DM [14,18,136,137]. Furthermore, studies indicate that the concentration of glucose present in the gingival crevicular fluid is related to the blood glucose level [138]. The quality of glycemic control can also partially explain the presence of a significant relationship between subgingival plaque candidal colonization and higher concentrations of glucose [138].

Although most of the scientific community believes that diabetes is a risk factor associated with oral yeas<sup>t</sup> infections, in a recent paper, Costa and colleagues [139] reported that the presence of yeasts in the oral cavity of patients with type 1 DM (60% of total) was not affected by diabetes, metabolic control, duration of the disease, salivary flow rate, or saliva buffer capacity by age, sex, place of residence, number of daily meals, consumption of sweets, or frequency of tooth brushing. *Candida albicans* was the most prevalent yeas<sup>t</sup> species, but a higher number of yeas<sup>t</sup> species was isolated in nondiabetics [139]. The fact that this study was developed exclusively in children may be related to this conclusion.


**Table1.** Physiopathologyandetiologyrelated to theoccurrenceoforalcandidiasisindiabetics.

#### 3.1.2. Antifungal Treatment of Oral Candidiasis

Importantly, several reports have stated the importance of the evaluation of the susceptibility of the oral isolates to the antifungal drugs in order to choose proper therapy in diabetic patients to control the fungal diseases. Aitken-Saavedra et al. [160] revealed that 66% of the yeasts isolated in diabetic patients were *C. albicans*, followed by *C. glabrata* (20.7%). In patients with decompensated type 2 DM, higher levels of salivary acidification and a greater diversity and quantity of yeasts of the genus *Candida* were observed. When nystatin was administered in these patients, higher inhibition was observed at a lower pH [160]. The study presented by Lydia Rajakumari and Saravana Kumari [161] showed a lower glycemic control leads to a higher candidal colonization in diabetic patients. The predominant species was *C. albicans*, but among denture wearers, *C. glabrata* was predominant. More importantly, ketoconazole, fluconazole, and itraconazole were effective against the isolated *Candida* sp. [161].

Similarly, Premkumar et al. [162] stated that, although *C. albicans* was the most predominantly isolated species, *C. dubliniensis*, *C. tropicalis*, and *C. parapsilosis* were also observed. The authors showed variable resistance toward amphotericin B, and fluconazole was observed in clinical isolates from diabetics but not from healthy patients. Again, a positive correlation was observed between glycemic control and candidal colonization [162]. In 2011, Bremenkamp et al. [163] found no significant differences in antifungal susceptibility to the tested agents between *Candida* sp. isolates from diabetic and non-diabetic subjects, which was consistent with the study by Manfredi et al. [164]. Furthermore, a high prevalence of *C. dubliniensis* in diabetic patients was found, which may sugges<sup>t</sup> a potential misdiagnosis of its morphologically-related species, *C. albicans*. Other authors found the same two species in DM patients [1,16,57,135]. In ye<sup>t</sup> another study, Sanitá and their colleagues [165] investigated the susceptibility of 198 oral clinical isolates of *Candida* sp. against caspofungin, amphotericin B, itraconazole, and fluconazole. Their findings confirmed the resistant profile of *C. glabrata* isolates against azole antifungals—especially itraconazole—in individuals with diabetes and denture stomatitis. The clinical sources of the isolates were shown to have no effect on the mininum inhibitory concentration (MIC) values obtained for all antifungals tested, which was in accordance with previous reports [26,119]. Additionally, *Candida* sp. isolates with higher rates of resistance to flucytosine, ketoconazole, miconazole, and econazole were confirmed in patients with diabetes when compared to healthy controls [163,166]. The increase in the environment glucose concentration may trigger the expression of several genes responsible for several carbohydrate cell wall and biofilm matrices components, consequently leading to resistant strains. This has been demonstrated before in gene and drug studies [126]. The variability in the susceptibility results may be related to the different antifungal drugs tested in those works.

Using a different approach, Mantri et al. [149] evaluated *Candida* sp. colonization in dentures with a silicone soft liner in diabetic and non-diabetic patients, assessing the antifungal efficacy of chlorhexidine gluconate [149]. The results showed normal oral flora in diabetics and non-diabetics and no difference between groups. They also showed a significant reduction of the colonization after cleaning the dentures with 4% chlorhexidine gluconate. This suggests that this drug has a good antiseptic effect on *Candida* sp. by killing it and preventing new adhesion. In 2015, Indira et al. [167] conducted a study that compared the common opportunistic infections (OIs) between 37 people living with HIV with DM (PLHIV-DM) and 37 people living with HIV without DM (PLHIV). Both of the groups were treated with anti-retroviral therapy (ART) [167]. The most common Ois included oral candidiasis (49% of PLHIV-DM and 35% of PLHIV) and *C. krusei* was the most common *Candida* sp. isolated (50%). No significant difference in the profile of Ois was found between PLHIV with and without DM.

## 3.1.3. Periodontal Diseases

Periodontal diseases of fungal origin are relatively unusual in healthy individuals but arise more often in immunocompromised people or in cases when normal oral flora has been distressed (e.g., the use of broad-spectrum antibiotics) [168,169]. Diabetes is a stated risk factor for periodontitis, which is the sixth-leading complication of diabetes [170,171]. Alterations in host response, collagen metabolism, vascularity, gingival crevicular fluid, heredity patterns, and immunosuppressive treatment (drugs, dosage, and treatment duration) are known factors that promote periodontitis in diabetes [172,173]. The etiology and pathogenesis of periodontitis is still imprecise, but it is recognized that *Candida* sp. is part of the oral and subgingival microbiota of individuals who suffer from severe periodontal inflammation [174]. The virulence factors of *Candida* sp. simplify the colonization and the proliferation in the periodontal pockets by co-aggregating with bacteria in dental biofilms and adhering to epithelial cells [30], which are essential in the microbial colonization, thereby contributing to the evolution of oral diseases [175,176]. As much as 20% of patients with chronic periodontitis have been shown to have periodontal pockets that are colonized by several species of *Candida* sp., predominantly *C. albicans* [177,178], but *C. dubliniensis* [174], *C. glabrata*, and *C. tropicalis* have been reported too [176,179]. Furthermore, *C. albicans* strains isolated from subgingival sites of diabetic and periodontal patients showed high PL in cases of chronic periodontitis. Environmental oxygen concentration demonstrated influence on the virulence factors [100,180,181]. Sardi et al. [100], in a study using PCR experiments, demonstrated that the quantities of several *Candida* sp. were higher in diabetic patients with a chronic periodontal disease than in patients without diabetes. *C. albicans*, *C. dubliniensis*, *C. tropicalis*, and *C. glabrata* were detected in 57%, 75%, 16%, and 5% of the periodontal pockets, respectively. In non-diabetic patients, *C. albicans* and *C. dubliniensis* were present in 20% and 14%, respectively. Periodontal inflammation has been described to be worse in prediabetics when compared to healthy controls [182–185], assuming that the oxidative stress induced by chronic hyperglycemia with a reduced unstimulated whole salivary flow rate (UWSFR) in these patients may contribute to deteriorating periodontal status [133]. Thus, it has been suggested that glycemic control enhances healing and reduces periodontal inflammation in patients with DM and prediabetes [134,182,185–191]. As a result, some authors believe that it may reduce oral *Candida* sp. carriage in patients with prediabetes [133]. The HbA1c concentration is an important diagnostic tool for monitoring long-term glycemic control [192]. Also, in these cases, higher *Candida* sp. infection levels have also been associated with low diabetic control (HbA1c > 9), occurring less frequently in subjects with well-controlled blood sugar levels (HbA1c < 6).

#### 3.1.4. Denture Prosthetics and Candidiasis

Since the oral cavity is highly populated with several polymicrobial communities, each one occupies very precise niches that diverge in both anatomical location and as well as nutrient availability [193]. A consequence of strong commensal bacteria/yeast colonization is the inhibition of pathogenic microorganism colonization through resistance. The vital function of commensal yeas<sup>t</sup> and bacteria and the harmful effects of commensal depletion through the use of broad spectrum antibiotics [194,195] are well recognized. Recent studies disclosed that commensal microorganisms not only protect the host by niche occupation, but also interact with host tissue, promoting the development of proper tissue structure and function [196,197].

Dentures represent a protective reservoir for oral microorganisms, mostly in biofilm form, favoring yeas<sup>t</sup> proliferation, improving their infective potential, and protecting fungal cells against several medications [198–202]. Elderly edentulous denture wearers, patients with debilitating diseases, and users of acrylic prosthetics have a significant risk of virulent oral yeas<sup>t</sup> infections [124]. Furthermore, elderly patients with diabetes have a 4.4 times higher estimated risk of developing oral candidiasis when compared with individuals without this disease. No statistically significant relation was determined between xerostomia, the use of prosthesis, and oral candidiasis [154], as suggested by some studies [203,204]. Sanitá et al. [199] studied the prevalence of *Candida* sp. in diabetics and non-diabetics

with and without denture stomatitis (DS) and found that *C. albicans* was the predominant species isolated in the three groups (81.9%). They also detected *C. tropicalis* (15.71%) and *C. glabrata* (15.24%), as found in previous studies [10,14,15,21,24,57,136,144,173,205–210]. Interestingly, and contrary to other reports, the authors did not detect *C. dubliniensis* among any *Candida* sp. isolates. Even though these results confirm previous findings [10,14,16,21,24,26,136,144,173,207,209,210], this species has been isolated in both diabetic [12,57,206] and non-diabetic [208] patients. This discrepancy among studies may be related to problems with identification techniques, since *C. dubliniensis* and *C. albicans* have similar phenotypic characteristics. The same authors also found that the prevalence of *C. tropicalis* significantly increased, showing the highest degree of inflammation in DS, as observed in previous studies [12,16,24,136,144,173,208–210].

#### 3.1.5. Co-Occurrence of Dental Plaque, Periodontitis, and Gingivitis

The existence of numerous different oral diseases in a single patient is frequent in diabetics. Hammad et al. [29] studied the relationship between the tongue and subgingival plaque *Candida* sp. colonization, as well as its relationship with the quality of glycemic control in type 2 diabetics with periodontitis. The results showed that *Candida* sp. colonized 59% and 48.7% of the patients' tongues and subgingival plaque, respectively. In this cross-sectional study, the authors concluded that poorly controlled type 2 diabetics and female patients with periodontitis showed a higher prevalence of subgingival plaque *Candida* sp. colonization than men, regardless of oral hygiene, tobacco smoking, age, or duration of DM [29]. The authors could not correlate oral candidal colonization and the amount of dental plaque, a patient's gingival status, or oral hygiene, as in other studies [211], but noticed *Candida* sp. present in the dental plaque in the form of biofilm. Remarkably, and compared with the control group, they found that *C. albicans* cells isolated from the subgingival plaque of patients with periodontitis adhered more to epithelial cells [212], suggesting that the oxygen concentration in the periodontal pockets affects the virulence of *C. albicans* [213]. A study that evaluated the effect of *Candida* sp. and general disease- or treatment-related factors on plaque-related gingivitis severity in children and adolescents with Nephrotic syndrome (NS, a clinical condition with a proteinuria level exceeding the body's compensating abilities) and diabetes concluded that poor hygiene control was the main cause of gingivitis. Olczak-Kowalczyk and colleagues [172] showed in their work that *Candida* sp. often occurred in healthy patients, but oral candidiasis was found only in the NS and diabetes groups (9.37% and 11.43%). Their work also showed that gingivitis occurred more frequently in patients with NS/diabetes. Moreover, gingivitis severity was most likely correlated to age, lipid disorders, and an increase in body mass and *Candida* sp. In uncompensated diabetes and in those patients using immunosuppressive treatment, it was assumed that NS would increase the plaque-related gingivitis.

#### 3.1.6. Esophagitis and Oropharyngeal Candidiasis

*Candida* sp. esophagitis and oropharyngeal are also oral complications found to moderately affect DM patients. Recently, Takahashi et al. [214] determined long-term trends in *Candida* sp. esophagitis (CE) prevalence and associated risk factors for patients with or without HIV infections. A risk analysis revealed that, among other factors, DM is associated with CE. Also, Mojazi and their colleagues [215] identified risk factors for oropharyngeal *Candida* sp. colonization in critically ill patients, with the results confirming DM as a risk factor [215]. Likewise, Owotade and colleagues [216] investigated the role of anti-retroviral (ARV) therapy and other factors related to oral candidiasis. Results demonstrated that 59.4% of the individuals were colonized by yeasts. *C. albicans* was the most common species (71%) and *C. dubliniensis* was the most frequent non-*Candida albicans Candida* species (NCAC). The probabilities of colonization were five times greater in patients with diabetes [216], confirming previous findings [16].

Oral and esophageal candidiasis sometimes leads to mucosal hyperplasia and progresses to carcinoma. There are many reports of the antibacterial effects of probiotics, but consensus about their antifungal effect has not been reached. In order to find alternative therapies, Terayama et al. [217]

investigated whether probiotic (yogurt) containing *Lactobacillus gasseri* OLL2716 (LG21 yogurt) could prevent proliferative and inflammatory changes caused by *C. albicans* in a mucosal candidiasis animal model. Diabetes was induced in eight-week-old WBN/Kob rats by the intravenous administration of alloxan. The results suggested that probiotic (yogurt) containing *L. gasseri* OLL2716 can suppress squamous hyperplastic change and inflammation associated with *C. albicans* infection in the forestomach [217].
