Antimicrobial effects of liquid anesthetic isoflurane on Candida albicans

Candida albicans is a dimorphic fungus that can grow in yeast morphology or hyphal form depending on the surrounding environment. This ubiquitous fungus is present in skin and mucus membranes as a potential pathogen that under opportunistic conditions causes a series of systemic and superficial infections known as candidiasis, moniliasis or simply candidiasis. There has been a steady increase in the prevalence of candidiasis that is expressed in more virulent forms of infection. Although candidiasis is commonly manifested as mucocutaneous disease, life-threatening systemic invasion by this fungus can occur in every part of the body. The severity of candidal infections is associated with its morphological shift such that the hyphal morphology of the fungus is most invasive. Of importance, aberrant multiplication of Candida yeast is also associated with the pathogenesis of certain mucosal diseases. In this study, we assessed the anti-candidal activity of the volatile anesthetic isoflurane in liquid form in comparison with the anti-fungal agent amphotericin B in an in vitro culture system. Exposure of C. albicans to isoflurane (0.3% volume/volume and above) inhibited multiplication of yeast as well as formation of hyphae. These data suggest development of potential topical application of isoflurane for controlling a series of cutaneous and genital infections associated with this fungus. Elucidiation of the mechanism by which isoflurane effects fungal growth could offer therapeutic potential for certain systemic fungal infections.     

CX3CR1 Is Dispensable for Control of Mucosal Candida albicans Infections in Mice and Humans

Candida albicans is part of the normal commensal microbiota of mucosal surfaces in a large percentage of the human population. However, perturbations of the host''s immune response or bacterial microbiota have been shown to predispose individuals to the development of opportunistic Candida infections. It was recently discovered that a defect in the chemokine receptor CX₃CR1 increases susceptibility of mice and humans to systemic candidiasis. However, whether CX₃CR1 confers protection against mucosal C. albicans infection has not been investigated. Using two different mouse models, we found that Cx3cr1 is dispensable for the induction of interleukin 17A (IL-17A), IL-22, and IL-23 in the tongue after infection, as well as for the clearance of mucosal candidiasis from the tongue or lower gastrointestinal (GI) tract colonization. Furthermore, the dysfunctional human CX₃CR1 allele CX₃CR1-M280 was not associated with development of recurrent vulvovaginal candidiasis (RVVC) in women. Taken together, these data indicate that CX₃CR1 is not essential for protection of the host against mucosal candidiasis, underscoring the dependence on different mammalian immune factors for control of mucosal versus systemic Candida infections.     

Adhesion of Candida albicans to Endothelial Cells under Physiological Conditions of Flow

Candida albicans is a commensal organism that under certain circumstances can become pathogenic. During systemic infection C. albicans is disseminated via the circulation to distant organs, where it causes multiple organ failure. Despite the severity of systemic C. albicans infection, little is known about the mechanisms involved in the adhesion of this organism to the endothelium lining blood vessels. Previous studies have used static assays to examine adhesion. However, these do not realistically model blood vessels, where circulating C. albicans cells must adhere to the endothelium under conditions of flow and shear stress. Furthermore, there is conflicting evidence concerning the role played by yeast, pseudohyphal, and hyphal forms of C. albicans in adhesion to endothelium. To test the hypothesis that there may be differences in the abilities of these three morphogenic forms of C. albicans to adhere to endothelium under conditions of flow, we developed an in vitro flow adhesion assay. We found that all three forms of C. albicans rapidly bound to confluent endothelial cells under conditions of flow. Maximum adhesion was found at low shear stress levels similar to that found in postcapillary venules. Moreover, yeast forms bound in significantly greater numbers than did pseudohyphal and hyphal forms, respectively, contrasting with previous findings from static assays. These findings are consistent with recent in vivo data suggesting that yeast forms may be capable of adhering to the endothelium and migrating into the tissues before undergoing morphogenic change to cause tissue damage.Candida albicans is a normal commensal organism of the oral cavity, gastrointestinal tract, and vagina. Usually the organism colonizes these mucosal surfaces without causing infection. However, under certain circumstances it is capable of becoming pathogenic. Systemic candidiasis is a serious infection with a high mortality rate and morbidity in those who survive (26). After infections caused by methicillin (meticillin)-resistant Staphylococcus aureus, it is probably the second most common cause of death from hospital-acquired infections and involves the hematogenous spread of C. albicans to multiple organs (11, 27). Mucocutaneous candidal infections seldom lead to systemic candidiasis. Even fungal colonization of the lungs and gastrointestinal tract does not always lead to systemic infection but may occur when surface integrity and host defenses are impaired. Systemic candidiasis appears to occur most often when direct access of fungi is facilitated by major gastrointestinal or cardiac surgery, organ transplantation, or the use of indwelling intravascular catheters and devices (13).During systemic dissemination, circulating C. albicans must first adhere to the endothelial cells lining blood vessels before transmigrating across the vessel wall and entering the tissues. Despite the morbidity caused by systemic candidiasis, little is known about the mechanisms involved in the adhesion of C. albicans to endothelial cells or their subsequent transmigration. Currently, there are two different theories as to how C. albicans adheres to the endothelium and enters the tissues (reviewed in reference 10). The first theory proposes that C. albicans must undergo morphogenic conversion from yeast to hyphal forms within the circulation to be able to invade the tissues (6, 19, 21). However, more-recent data indicate that morphogenic change may not be necessary, suggesting that yeast cells may be able to adhere to the endothelium and transmigrate into the tissues before undergoing morphogenic change (3, 4, 23, 24).Studies investigating candidal adhesion to the endothelium have mainly used static in vitro assays where C. albicans is left in prolonged contact with cultured monolayers of endothelial cells. This is very different from the fleeting interactions that C. albicans has with endothelial cells under conditions of shear stress and flow that occur in blood vessels in vivo. Numerous studies with leukocytes, tumor cells, and microorganisms have shown that static assays do not replicate the dynamic interactions that occur with endothelium under conditions of flow and are poor at elucidating the contribution of specific adhesion molecules (8, 14). However, to date no studies have investigated the adhesion of the three different morphogenic forms of C. albicans to endothelium under conditions of flow.To test the hypothesis that there may be differences in the abilities of yeast, pseudohyphal, and hyphal forms of C. albicans to adhere to the endothelium under conditions of flow, we developed an in vitro flow adhesion assay that mimics the conditions found within blood vessels. Here we show for the first time that adhesion of all three forms of C. albicans to the endothelium under conditions of flow is rapid. In addition, yeast forms adhere with greater numbers than do pseudohyphal or hyphal forms, suggesting that yeast forms may be the most predominant form adhering to endothelial cells during systemic infection.     

Dectin-1 Is Not Required for Controlling Candida albicans Colonization of the Gastrointestinal Tract

Candida albicans is normally found as a commensal microbe, commonly colonizing the gastrointestinal tract in humans. However, this fungus can also cause mucosal and systemic infections once immune function is compromised. Dectin-1 is an innate pattern recognition receptor essential for the control of fungal infections in both mice and humans; however, its role in the control of C. albicans colonization of the gastrointestinal tract has not been defined. Here, we demonstrate that in mice dectin-1 is essential for the control of gastrointestinal invasion during systemic infection, with dectin-1 deficiency associating with impaired fungal clearance and dysregulated cytokine production. Surprisingly, however, following oral infection, dectin-1 was not required for the control of mucosal colonization of the gastrointestinal tract, in terms of either fungal burdens or cytokine response. Thus, in mice, dectin-1 is essential for controlling systemic infection with C. albicans but appears to be redundant for the control of gastrointestinal colonization.     

Interleukin-17-Induced Protein Lipocalin 2 Is Dispensable for Immunity to Oral Candidiasis

Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4⁺ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2^−/− mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA^−/− or Act1^−/− mice). However, Lcn2^−/− mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis.     

Mutative expression in Candida albicans infection and cytokine signaling network in gene knockout mice

The interactions of Candida species with host cells are crucial for candidiasis. Recognition and adherence to host constituents are the keys to initial colonization of mucosal surfaces and the invasion of host cells. Resistance to mucosal candidiasis is mediated by cell-mediated immunity. Knowledge about host receptors on immune cells and the adhesins on the surface of C. albicans are more redundant, respectively, than about the ligands on the fungal surface and the structures on the host cells bound by adhesions. Silencing or disrupting specific genes both in the pathogen and the host are developing optimistically. The research on IL-12/23 p40 knockout (KO) mice is sound in providing preliminary array data about the principal pathways in oral C. albicans infection and clues about the molecular differences between wild-type (WT) and p40 KO mice of candidiasis in different sites, thus, hints that certain specific drug targets accessible to topical agents for the clinical treatment of oral candidiasis and relevant mucocutaneous precancerous lesions.     

The pH of the Host Niche Controls Gene Expression in and Virulence of Candida albicans

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.     

A Candida albicans Strain Expressing Mammalian Interleukin-17A Results in Early Control of Fungal Growth during Disseminated Infection

Candida albicans is normally a commensal fungus of the human mucosae and skin, but it causes life-threatening systemic infections in hospital settings in the face of predisposing conditions, such as indwelling catheters, abdominal surgery, or antibiotic use. Immunity to C. albicans involves various immune parameters, but the cytokine interleukin-17A (IL-17A) (also known as IL-17) has emerged as a centrally important mediator of immune defense against both mucosal and systemic candidiasis. Conversely, IL-17A has been suggested to enhance the virulence of C. albicans, indicating that it may exert detrimental effects on pathogenesis. In this study, we hypothesized that a C. albicans strain expressing IL-17A would exhibit reduced virulence in vivo. To that end, we created a Candida-optimized expression cassette encoding murine IL-17A, which was transformed into the DAY286 strain of C. albicans. Candida-derived IL-17A was indistinguishable from murine IL-17A in terms of biological activity and detection in standard enzyme-linked immunosorbent assays (ELISAs). Expression of IL-17A did not negatively impact the growth of these strains in vitro. Moreover, the IL-17A-expressing C. albicans strains showed significantly reduced pathogenicity in a systemic model of Candida infection, mainly evident during the early stages of disease. Collectively, these findings suggest that IL-17A mitigates the virulence of C. albicans.     

Morphogenesis Is Not Required for Candida albicans-Staphylococcus aureus Intra-Abdominal Infection-Mediated Dissemination and Lethal Sepsis

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An established experimental mouse model of Staphylococcus aureus-Candida albicans intra-abdominal infection results in ∼60% mortality within 48 h postinoculation, concomitant with amplified local inflammatory responses, while monomicrobial infections are avirulent. The purpose of this study was to characterize early local and systemic innate responses during coinfection and determine the role of C. albicans morphogenesis in lethality, a trait involved in virulence and physical interaction with S. aureus. Local and systemic proinflammatory cytokines were significantly elevated during coinfection at early time points (4 to 12 h) compared to those in monoinfection. In contrast, microbial burdens in the organs and peritoneal lavage fluid were similar between mono- and coinfected animals through 24 h, as was peritoneal neutrophil infiltration. After optimizing the model for 100% mortality within 48 h, using 3.5 × 10⁷ C. albicans (5× increase), coinfection with C. albicans yeast-locked or hypha-locked mutants showed similar mortality, dissemination, and local and systemic inflammation to the isogenic control. However, coinfection with the yeast-locked C. albicans mutant given intravenously (i.v.) and S. aureus given intraperitoneally (i.p.) failed to induce mortality. These results suggest a unique intra-abdominal interaction between the host and C. albicans-S. aureus that results in strong inflammatory responses, dissemination, and lethal sepsis, independent of C. albicans morphogenesis.     

Oral bacteria modulate invasion and induction of apoptosis in HEp-2 cells by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen, causing infections of the respiratory and other organ systems in susceptible hosts. P. aeruginosa infection is initiated by adhesion to and invasion of mucosal epithelial cells. The failure of host defenses to eliminate P. aeruginosa from mucosal surfaces results in P. aeruginosa proliferation, sometimes followed by overt infection and tissue destruction. There is growing evidence that associates poor oral health and respiratory infection. An in vitro model system for bacterial invasion of respiratory epithelial cells was used to investigate the influence of oral bacteria on P. aeruginosa epithelial cell invasion. Oral pathogens including Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter (Actinobacillus) actinomycetemcomitans increased invasion of P. aeruginosa into HEp-2 cells from one- to threefold. In contrast, non-pathogenic oral bacteria such as Actinomyces naeslundii and Streptococcus gordonii showed no significant influence on P. aeruginosa invasion. P. aeruginosa together with oral bacteria stimulated greater cytokine production from HEp-2 cells than did P. aeruginosa alone. P. aeruginosa in combination with periodontal pathogens also increased apoptosis of HEp-2 cells and induced elevated caspase-3 activity. These results suggest that oral bacteria, especially periodontal pathogens, may foster P. aeruginosa invasion into respiratory epithelial cells to enhance host cell cytokine release and apoptosis.     

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.     

Oral candidiasis: clinical features and control

Candidiasis is the most commonly encountered fungal infection, and oral candidiasis is often observed as a local opportunistic infection. Oral candidiasis is clinically divided into three types: acute forms, chronic forms, and Candida-associated lesions. Candida adhesion and multiplication are largely regulated by the local and systemic factors of the host. The local factors include impairment of the oral mucosal integrity, which is usually impaired by hyposalivation, anticancer drugs/radiation for head and neck cancers, denture wearing, a decrease in the oral bacterial population, and poor oral hygiene. Among Candida species, oral candidiasis is mostly caused by Candida albicans (C. albicans), C. glabrata, or C. tropicalis. Oral Candida induces a variety of symptoms, such as oral mucosal inflammation manifesting as an uncomfortable feeling, pain, erythema, erosion, taste abnormalities, and hyperplasia of the oral mucosa. Candida overgrowth in the oral cavity may disseminate to distant organs. Therefore, in order to avoid the sequelae of systemic candidiasis, oral candidiasis should be rapidly controlled. Oral candidiasis is usually treated by the local application of antifungal drugs. However, oral candidiasis occasionally escapes the control of such local treatment due to the development of multi-drug resistant Candida strains and species or due to the suppression of salivation or cellular immune activity. When drug-resistant strains are suspected as the pathogens and when the host is generally compromised, the oral administration of combinations of antifungal drugs, enhancement of cellular immune activity, and improvement of the nutritional condition are recommended.     

The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity

The host immune status is critical for preventing opportunistic infections with Candida albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of interleukin (IL)-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Altogether, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal–host interactions in vivo.     

Effects of Psychological Stress and Fluoxetine on Development of Oral Candidiasis in Rats

Psychological stress has been found to suppress cell-mediated immune responses that are important for limiting the proliferation of Candida albicans. Fluoxetine has been observed to reduce negative consequences of stress on the immune system in experimental and clinical models, but there are no data on its effects on oral candidiasis. We designed experiments to evaluate the effects of fluoxetine on the development of oral candidiasis in Sprague-Dawley rats exposed to a chronic auditory stressor. Animals were submitted to surgical hyposalivation in order to facilitate the establishment and persistence of C. albicans infection. Stress application and treatment with drugs (placebo or fluoxetine) were initiated 7 days before C. albicans inoculation and lasted until the end of the experiments, on day 15 postinoculation. Establishment of C. albicans infection was evaluated on days 2 and 15 after inoculation. Tissue injury was determined by the quantification of the number and type (normal or abnormal) of papillae on the dorsal tongue per microscopic field. A semiquantitative scale was devised to assess the degree of colonization of the epithelium by fungal hyphae. Our results showed that stress exacerbates C. albicans infection in the tongues of rats. Significant increases in Candida counts, the percentage of the tongue''s surface covered with clinical lesions, the percentage of abnormal papillae, and the colonization of the epithelium by hyphae were found in stressed rats compared to the nonstressed ones. Treatment with fluoxetine significantly reversed these adverse effects of stress. Besides the psychopharmacological properties of fluoxetine against stress, it has consequences for Candida infection.Candida albicans is an example of an opportunistic pathogen frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. The most common predisposing factors to oral candidiasis are immunosuppresive therapy, immunoincompetence, and immunodeficiencies, indicating that the host immune system provides a protective mechanism(s) against superficial invasion by Candida.Several lines of evidence indicate that cell-mediated immunity is important in limiting the proliferation of Candida; thus, this opportunistic human pathogen preferentially causes invasive and disseminated infections in patients with defective phagocytic defenses and serious mucocutaneous infections in patients with deficiencies in T-cell function. Phagocytes appear to protect the host from fungal colonization even in the absence of adaptive immune mechanisms, while as-yet-undefined T-cell-dependent factors seem necessary for the control of C. albicans on body surfaces (29).Previous research demonstrated adverse effects of stress on natural and specific immune responses (1, 4, 9, 18, 21) that might predispose the host to more severe Candida infections. On the other hand, treatment with fluoxetine, a nontricyclic antidepressant drug, was found to attenuate some effects of stress on the immune systems of rodents, such as T-cell depletion, the inhibition of the blastogenic (15) and cytotoxic activities of spleen cells (34), and defects in phagocytosis (17). Nevertheless, there are few data on the effects of this compound on the development of fungal infection. In order to further elucidate this relationship, we studied the effects of fluoxetine on the development of oral candidiasis in rats exposed to a repeated auditory stressor.     

Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans

The Gram-positive bacterium Enterococcus faecalis and the fungus Candida albicans are both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using a Caenorhabditis elegans model of polymicrobial infection, we discovered that E. faecalis and C. albicans negatively impact each other''s virulence. Much of the negative effect of E. faecalis on C. albicans was due to the inhibition of C. albicans hyphal morphogenesis, a developmental program crucial to C. albicans pathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence in E. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibit C. albicans filamentation in the context of an in vitro biofilm. Finally, a screen of an E. faecalis transposon mutant library identified other genes required for suppression of C. albicans hyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.     

Antivirulence activity and in vivo efficacy of a thiazole derivative against candidiasis

Candida albicans is a pathogen equipped with a variety of commensal and virulence traits that help it colonize the microbiota and invade host tissue during infection. In this study, we investigated the potential anticandidal activity of 3-[2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazino)]butan-1-ol (MT), a thiazolylhydrazone compound synthesized by our group, and identified it as a promising antifungal agent. The activity of MT was evaluated in vitro and in vivo against C. albicans as well as its ability to inhibit virulence factors. For this, the ability of MT to inhibit the adhesion of C. albicans to human buccal epithelial cells and biofilm formation and filamentation was tested. In addition, the potential in vivo activity of MT was evaluated in murine models of oral candidiasis.Our results confirmed the antifungal activity of MT, with a minimal inhibitory concentration range of 0.5–2 µg/mL. Indeed, MT treatment in vitro decreased the expression of C. albicans genes involved in biofilm formation and morphogenesis and encoding hydrolytic enzymes, which was also confirmed through phenotypic observations. In addition, MT promoted a decrease in the colony forming units recovered from the tongues of mice with oral candidiasis. In this work, we present a potent antivirulence compound that shows potential for candidiasis therapy, especially for topical use.     

Competitive Binding Inhibition Enzyme-Linked Immunosorbent Assay That Uses the Secreted Aspartyl Proteinase of Candida albicans as an Antigenic Marker for Diagnosis of Disseminated Candidiasis

The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10⁷ C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition ± standard error [SE] compared with preinfection control urine, 15.7% ± 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% ± 4.8% to 44.5% ± 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 × 10⁸ C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition ± SE by day 3 after infection, 32.9% ± 2.7% [P < 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.     

Novel Nystatin A<Subscript>1</Subscript> derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A₁, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A₁ had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A₁ was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A₁ derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A₁. Furthermore, compared with Nystatin A₁, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.