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ABSTRACT Acetylcholine modulates the virulence of Candida albicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans. The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections. IMPORTANCE Candida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Candida albicans and C. dubliniensis are related yeasts that differ in the expression of virulence-associated proteins involved in adherence and biofilm development. CR3-RP (complement receptor 3-related protein) is one of the surface antigens expressed by Candida species. The main objective of this research was to elucidate the effect of the polyclonal anti-CR3-RP antibody (Ab) on adherence and the biofilm formed by C. albicans SC5314 and C. dubliniensis CBS 7987 and two clinical isolates in vitro, ex vivo and in vivo. A comparison of species, and of treated vs. non-treated with the anti-CR3-RP Ab showed a reduction in adherence (22%-41%) that was dependent on the time point of evaluation (60, 90 or 120 min), but did not prove to be species-dependent. Confocal microscopy revealed a decreased thickness in biofilms formed by both species after pre-treatment with the anti-CR3-RP Ab. This observation was confirmed ex vivo by immunohistochemistry analysis of biofilms formed on mouse tongues. Moreover, anti-CR3-RP Ab administration, 1 h post-infection, has been shown to promote larval survival compared to the control group in a Galleria mellonella infection model. Our data suggest a potential activity of the anti-CR3-RP Ab relevant to immunotherapy or vaccine development against biofilm-associated Candida infections. [ABSTRACT FROM AUTHOR]

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy. [ABSTRACT FROM AUTHOR]

Aim: The aim of our study was to investigate whether biofilm production by Candida albicans clinical isolates could be a hallmark of virulence in vivo. Materials & methods: Twenty clinical isolates of C. albicans were examined via histological studies on larvae infected with various fungal doses (from 103 to 105 CFU/larva) of biofilm producer and nonproducer strains. Results: The poor prognostic role of infection due to a biofilm-producing isolate was confirmed by the Wald test (hazard ratio: 2.63; 95% CI: 2.03-3.41). Histological examinations at 24 h showed a strong innate immune response, with evidence of melanization for both infection groups. However, at 48 h, we found huge differences in filamentation and tissue invasion capability between biofilm nonproducing and producing isolates, the latter being highly organized into biofilm and invading the larval intestinal tract. Invasion corroborated survival data. Conclusion: The histological results demonstrate that the production of biofilm could enhance the invasiveness of C. albicans. [ABSTRACT FROM AUTHOR]

Aim: We investigated the pathogenic role of biofilm and the therapeutic efficacy of anidulafungin in experimental infections of the wax moth Galleria mellonella by Candida albicans clinical strains. Materials & methods: On the basis of the in vitro propensity to form biofilm, five biofilm-producer (BP) and four nonproducer (NP) C. albicans clinical strains were used in this study. For each strain, we assessed the virulence by infecting G. mellonella larvae and observing survival. Anidulafungin was administered 2 h after yeast inoculum at 0.6 μg, according to the therapeutic dose recommended for humans. Results: Biofilm-forming ability highly influenced the larva-killing rate. A significant (p < 0.0001) survival decrease was observed in the BP group, with 80% of the infected larvae dying within 72 h. NP isolates did not reach the same killing rate, even at the end of experiments (216 h). Larval survival was enhanced (p < 0.0001) by anidulafungin administration in both groups. Survival rate at 72 h was similar in both groups (BP 78.5% and NP 87.5%); whereas there were still differences at the end of the experiments, with a higher survival in the NP group (75 vs 48%). Conclusion: Our data confirm the pathogenic role of biofilm in C. albicans infections. Its importance was further enhanced by a lack of contribution from extracellular enzymes, detected in both NP and BP strains. In addition, we demonstrated anidulafungin efficacy in treating biofilm-related invasive candidiasis. [ABSTRACT FROM AUTHOR]

Abstract: The activity of voriconazole was tested in vitro against 1996 clinical yeast isolates collected in 20 Italian microbiology laboratories. Voriconazole susceptibility testing was carried out with the broth microdilution (NCCLS M27-A2), Etest and disk diffusion methods. The minimum inhibitory concentrations at which 90% of the isolates were inhibited (MIC90) obtained with the NCCLS method were 0.03mg/L for Candida albicans, 0.5mg/L for Candida non-albicans and 0.25mg/L for other genera; those obtained with Etesting were, respectively, 0.032mg/L, 0.125mg/L and 0.125mg/L. With the disk diffusion method, the majority of isolates (92.3%) showed inhibition zone diameters between 21mm and 40mm. Using a tentative MIC cut-off of 1mg/L as indicative of in vitro susceptibility, 98.1% of the isolates tested in our study would be classified as susceptible, and only 28 (1.4%) of the isolates, with MICs higher than 2mg/L, would be classified as resistant to the drug. Our findings confirm the broad-spectrum in vitro activity of voriconazole against yeasts, including Candida species that are generally less susceptible to other azoles. [Copyright &y& Elsevier]

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance. [ABSTRACT FROM AUTHOR]

Anti-Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin. Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin-sensitive Candida albicans cells. The course of candidosis was studied in comparison with mice non-immunized and immunized with an isotype-matched unrelated MoAb subdivided into control groups. Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin-like anti-Id antibodies. MoAb KT4 affinity chromatography purified mouse anti-Id antibodies were capable of killing in vitro the yeast cells of the Cimdidu albicans strain used for the experimental infection. This is the first report of antimicrobial protection that exploits the role of anti-idiotypic antibodies presumably acting in vivo as antibiotics (idiotypic vaccination). [ABSTRACT FROM AUTHOR]

A young Down's syndrome patient developed tricuspid valve endocarditis several years after undergoing surgical closure of a congenital ventricular septal defect. Fungal etiology was established by PCR amplification of the Candida albicans ERG11 gene. Although antifungal therapy was administered, surgical replacement of the infected valve was required to eliminate the infection. [ABSTRACT FROM AUTHOR]

Abstract: The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences. [Copyright &y& Elsevier]

Highlights: [•] Herpes Simplex Virus-1 and Coxsackievirus-B5 can be embedded in Candida albicans biofilm. [•] Viruses encompassed in biofilm retain infectivity and can be released. [•] HSV-1 infected cells remain embedded in biofilm and release infectious virus. [•] Candida albicans biofilm on patients’ catheters might be a source of virus infections. [•] Virus interaction with biofilm reduces sensitivity to disinfectants but no to antibodies. [Copyright &y& Elsevier]