*2.1. Enzymatic Activity*

Several studies have established an association between hydrolytic enzymes activity and an increase in the pathogenic ability of *Candida* sp. [55,56].

It has been demonstrated that, due to higher blood glucose concentration, diabetic *Candida* sp. isolates present significantly higher hemolytic and esterase enzymatic activity, which may contribute to increased enzyme activity among diabetic patients [57–60]. The same authors also hypothesized that these species are more pathogenic under abnormal conditions such as DM [61,62]. Secreted aspartyl proteinases (SAP) capable of degrading numerous substrates that constitute host proteins in the oral cavity have also been studied. These enzymes are thought to help *Candida* sp. to acquire essential nitrogen for growth, to attach to and penetrate oral mucosa, or both [63,64]. They can also

cause amplified vascular permeability, leading to inflammatory reactions [65] and clinical symptoms, which may disturb the humoral host defense [66]. Similarly, phospholipase (PL) targets the membrane phospholipids and digests these components, initiating cell lysis and facilitating the penetration of the infecting fungi [67]. This enzyme induces the accumulation of inflammatory cells and plasma proteins, releasing several inflammatory mediators in vivo [67].

Very recently, it was revealed that *C. albicans* hyphae induce both epithelial damage and innate immunity through the secretion of a cytolytic peptide toxin called candidalysin [68,69]. This enzyme is encoded by the hypha-associated *ECE1* gene and is the first peptide toxin to be identified in any human fungal pathogen. Candidalysin induces calcium ion influx and lactate dehydrogenase (LDH) release in oral epithelial cells, which are features of cell damage and membrane destabilization. Importantly, the study also reported that *C. albicans* mutants where the entire *ECE1* gene or the candidalysin-encoding region had been deleted have full invasive potential in vitro but are incapable of inducing tissue damage or cytokine release and are highly weakened in a mouse model of oropharyngeal candidiasis and a zebrafish swim bladder mucosal model [68].

## *2.2. Biofilm Formation*

Biofilms are communities of microorganisms embedded in an extracellular matrix [70,71], which confer substantial resistance to antifungal therapy and increased host immune responses [72,73]. These communities can be formed in both biotic (e.g., mouth mucosae) or abiotic (e.g., catheters) surfaces [74,75]. In fact, candidemia are the most prevalent invasive mycoses worldwide with mortality rates close to 40% [76]. *Candida* sp. are often recognized as the origin of candidemia, urinary tract infections, and hospital pneumonia. In practically all of these cases, the infections are related with the use of a medical device and biofilm formation on its surface [20]. The most frequently colonized medical device is the central venous catheter used for administration of fluids, nutrients, and medicines [77]. The contamination of the catheter or the infusion fluid can arise from the skin of the patient, the hands of health professionals [77], or by migration into the catheter from a pre-existing lesion. Less commonly, if *Candida* sp. that colonize the gastrointestinal tract as a commensal start to develop a pathogenic behavior, they are able to infiltrate the intestinal mucosa and diffuse through the bloodstream. Consequently, circulating yeas<sup>t</sup> may colonize the catheter endogenously. This is more common in cancer patients, as chemotherapy leads to damage to the intestinal mucosa [78]. In the other patients, infected catheters are the most significant source of bloodstream infections, followed by widespread invasive candidiasis. The catheter removal is recommended in patients with disseminated *Candida* sp. infection to enable disinfection of the blood and to increase prognosis [79,80].

Biofilm development of *Candida* sp. (Figure 3) can be explained in four chronological steps: adherence-initial phase in which the yeas<sup>t</sup> in suspension and planktonic cells adhere to the surface (first 1–3 h); intermediate phase-development of biofilm (11–14 h); maturation phase-the polymeric matrix (PEM) completely penetrates all layers of the cells adhered to the surface in a three-dimensional structure (20–48 h); dispersion-the most superficial cells leave the biofilm and colonize areas surrounding the surface (after 24 h) [81]. Hence, a mature biofilm comprises of a dense network of cells in the form of yeasts, hyphae, or pseudohyphae (or not, depending on the *Candida* sp.) involved by PEM and with water channels between the cells. These help in the diffusion of nutrients from the environment through the biomass to the lower layers and also allow the removal of waste [81,82]. Formed using in vivo models, *Candida* sp. biofilms seem to follow the same sequence of in vitro formation [83]. Nonetheless, the maturation step happens more quickly, and the thickness is increased. The final architecture of the biofilm is variable and depends, in part, on the *Candida* sp. involved, the growing conditions, and the substrate on which it is formed [81,84].

High levels of glucose are thought to serve as the carbohydrate energy source necessitated by *Candida* sp. for the biofilm formation and are probably required to produce the polysaccharide matrix [85], which is secreted by sessile cells, providing protection against environmental challenges [86]. Biofilm formation has been shown to be dependent on the *Candida* sp. and its

clinical origin. Biofilms are refractory to antifungal drugs and more difficult to treat than infections with planktonic cells [44]. Moreover, it has been verified that *Candida* sp. isolated from patients with DM have a higher pathogenic potential for biofilm-forming [87]. The communities are extremely common on medical devices.

**Figure 3.** Development of a *Candida* sp. biofilm in a surface.
