Gray phenotype: enhanced fitness strategy for Candida dubliniensis

In this study Yue H. et al described for the first time the gray phenotype and tristable white-gray-opaque transitions in Candida dubliniensis. Here we discuss some intriguing aspects related to this virulence trait of Candida dubliniensis in comparison to Candida albicans and within the wider and complex phenotypic switch phenomenon so typical of the virulence program of these opportunistic pathogenic fungi. In particular, the relationship between the presence of gray phenotype and prevalence of Candida dubliniensis in the oral cavity of HIV-positive subjects is pointed out

New approaches in the development of a vaccine for mucosal candidiasis: progress and challenges

The commensal fungus Candida albicans causes mucosal candidiasis in the rapidly expanding number of immunocompromised patients. Mucosal candidiasis includes oropharyngeal, esophageal, gastrointestinal, and vaginal infections. Vulvovaginal candidiasis (VVC) and antimycotic-refractory recurrent VVC is a frequent problem in healthy childbearing women. Both these mucosal infections can affect the quality of life and finding new therapeutical and preventive approaches is a challenge. A vaccine against candidal infections would be a new important tool to prevent and/or cure mucosal candidiasis and would be of benefit to many patients. Several Candida antigens have been proposed as vaccine candidates including cell wall components and virulence factors. Here we discuss the recent progress and problems associated with vaccination against mucosal candidiasis

Effects of Cupral® on the formation and persistence of microbial biofilms in vitro

Introduction: endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are frequently found pathogenic microorganisms, such as Gram+, Gram- and opportunistic fungi, belonging to Candida spp, responsible for several endodontic pathologies. As clinical importance is the fact that biofilm is extremely resistant to common intra-canal irrigants, antimicrobial drugs and host immune defenses. The aim of this in vitro study was to evaluate the efficacy of Cupral® on planktonic forms of some pathogens, as well as to assess its ability to prevent and affect the formation/persistence of microbial biofilms. Materials and Methods: ATCC strains of S. aureus, P. aeruginosa and C. albicans were exposed to various concentrations of Cupral® (an antiseptic compound based on calcium and copper hydroxide, used in endodoncy) to investigate its antimicrobial efficacy. This activity has been evaluated in terms of microbial growth and cellular doubling time (optical density, colony forming units and doubling time assays), inhibition/persistence (crystal violet staining), viability of microbial cells embedded in the biofilms (live/dead stain) and pyoverdine production (fluorimetric assay). Finally, the morphology of Cupral®-treated biofilms was investigated by optical/confocal microscopy analysis. Results: the addition of Cupral® to microbial cultures, influences, in a significantly and dose-dependent manner, the doubling time and growth of microbial cultures. Cupral® antimicrobial activity was also assessed on biofilms formation and persistence with meaningful decreases of residual biomass (observed reductions of 47-94% for S. aureus, 28-95% for P. aeruginosa and 27-75 % for C. albicans). Cupral®-treated biofilms analyzed by optical and confocal microscopy revealed loss of typical sessile structure, with few scattered microbial cells and a reduced thickness. Finally, the addition of Cupral® reduced both the number of embedded alive cells in the biofilms and the levels of pyoverdine in the culture supernatants. Discussion and Conclusions: this pilot in vitro study provided the first evidences on Cupral® efficacy against microbial biofilms. The wide range of action (vs Gram+, Gram- and fungi) of Cupral® strongly suggests its use as compound in the prevention and treatment of main oral biofilm-associated infections

The Propionibacterium spp. extract reduces Candida albicans-induced damage to vaginal epithelial cells and increases mitochondrial response to Candida albicans infection in vitro

Introduction. Bacterial lysates are prepared by inactivated microorganisms and are extensively employed in clinical settings as immunomodulants and to improve mucosal immunity. However, despite their extensive clinical use, their effects on the host are only partially known. The Propionibacterium spp. extract (PE) is a bacterial lysate included as an active compound in a gel formulation used to treat the symptoms of vulvovaginal candidiasis. Here, we analyzed its possible beneficial effects in an in vitro model of vaginal epithelial cells infected with Candida. Materials and Methods. Initially, we analyzed the PE effects on C. albicans and C. parapsilosis growth by the microdilution method. We then assessed the capacity of PE to reduce C. albicans-induced damage of vaginal epithelial cells through the quantification of lactate-dehydrogenase released by damaged cells in the growth medium. Moreover, in order to test the capacity of the PE to modulate epithelial mitochondrial activity, we evaluated Reactive-Oxygen-Species (ROS) production by the infected epithelial cells, stimulated or not with PE. This was kinetically monitored through the analysis of emitted fluorescence, after addition of the MitoSOX Red probe. Results. Our results show that PE did not affect directly microbial growth. In addition, the epithelial cells stimulation with PE reduced C. albicans-induced cell damage. Moreover, the treatment with PE increased the epithelial cells mitochondrial activity in response to C. albicans infection in vitro. Discussion and Conclusions. Taken together, our results show that PE increases ROS production by epithelial cells in response to C. albicans infection. Therefore, our results suggest that the increased mitochondrial activity induced by PE, could protect epithelial cells against the damage induced by C. albicans infection

Compounds released from Lactobacillus (L.) acidophilus, L. plantarum, L. rhamnosus and L. reuteri inhibit Candida parapsilosis pathogenic potential after infection of vaginal epithelial cells in vitro.

INTRODUCTION. Lactobacillus spp. are the most represented microorganisms in the vaginal microbiota of healthy women, where they provide a shelter against infections from several pathogens, such as the yeasts belonging to the genus Candida. The latter are responsible for the vulvovaginal candidiasis (VVC), a condition affecting up to 75% of women during their child-bearing age at least once in their lifetime. Moreover, 5-8% of such women develop the recurrent form of the disease (RVVC), consisting of at least 5 VVC episodes per year. Notwithstanding C. albicans is the main responsible of VVC cases, in the last decades, the incidence of VVC cases by non-albicans Candida (NAC) species has become prevalent, especially in some geographical areas. C. parapsilosis, in particular, has been reported to be second species most commonly isolated from women affected by VVC. However, little is known on this species, and on its role in the pathogenesis of VVC. MATERIALS AND METHODS. Cell-free supernatants (CFS) were obtained following an overnight culture of 4 different Lactobacilli species (L. acidophilus, L. plantarum, L. rhamnosus, L. reuteri). Lactobacilli-released compounds, contained in CFS, were assessed for their effect on several virulence factors of C. parapsilosis (strain CLIB214), such as growth rate, capacity to form pseudohyphae, capacity to adhere to a vaginal epithelium in vitro (A-431 cells monolayer) and to induce cell damage. The latter was evaluated by measuring lactate dehydrogenase (LDH) release from A431 cells. RESULTS. C. parapsilosis growth inhibition by L. acidophilus, L. plantarum and L. reuteri CFS was 47%, 55% and 52% respectively, whereas L. rhamnosus CFS effect was weaker (33% inhibition growth). All the Lactobacilli significantly inhibited C. parapsilosis adhesion to vaginal epithelial cells: upon incubation with CFS, only 5-7% of fungal cells adhered to epithelial cells, after 90 minutes incubation; differently, the adhesion of the control reached 19%. Interestingly, no effect on pseudohyphae formation by any of the CSF was ever observed. Finally, the C. parapsilosis-induced damage on A-431 cells was significantly reduced by the addition of the CSF. DISCUSSION AND CONCLUSIONS. Our results show that the investigated species of Lactobacilli release compounds capable to impair several C. parapsilosis virulence factors, such as growth rate and adhesion to vaginal epithelial cells; interestingly, while not affecting fungal capacity to form pseudohyphae, such compounds significantly reduce Candida-mediated epithelial damage.. These data suggest that, in the context of vaginal microbiota, these Lactobacilli species may play an important role in counteracting the onset of mucosal Candida infections

In vitro analysis of epithelial tolerability and anti-Candida effect of a new lactic acid-based vaginal gel formulation

INTRODUCTION. Vulvovaginal candidiasis (VVC) is the most prevalent vaginal infection in adult women. It is mainly caused by Candida albicans, and it affects 75% of healthy women at least once during their reproductive age; 5-10% of such women have recurrent episodes (RVVC), with more of 4 episodes of acute VVC per year. Symptoms of VVC include itching, burning, swelling and redness of the vaginal mucosa with white vaginal discharge. The urinary system can also be affected, with pain and burning when urinating. This condition seriously damages the well-being and the life quality of the affected women. Since Candida is a commensal fungus of the vaginal mucosa of healthy women, the main question is how the fungus can switch from harmless component of the vaginal microbiota to virulent pathogen. In this work we analyzed the capacity of lactic acid-based vaginal gel formulation Respecta® Balance Gel (RBG) to counteract C. albicans virulence after epithelial cells infection in vitro. MATERIALS AND METHODS. For the establishment of the in vitro infection model, we used a monolayer of the A-431 vaginal epithelial cell line and two different strains of C. albicans (strain SC5314 and the bioluminescent strain gLUC59). Dose-dependent experiments were performed to test the epithelial tolerability to RBG (IHS srl, Biofarma Group) by monitoring lactate-dehydrogenase (LDH) release from damaged cells. The capacity of RGB to counteract Candida-induced epithelial damage were analysed by monitoring LDH release from cells. Fungal growth and adhesion capacity during vaginal epithelial cells infection in the presence of RGB were evaluated by quantify the Relative Luminescent Units (RLU) and CFU counts, respectively. RESULTS. Our results show that, at dilution 1:150, RGB is well tolerated by the vaginal epithelium and consequently we used this dose for the subsequent experiments. RBG was able to significantly reduce (by 65%) C. albicans-induced damage of vaginal epithelial cells. This effect was accompanied with the capacity of RGB to significantly reduce Candida adhesion to the epithelium (adhesion reduction by 34%). Intriguingly, no inhibition of fungal growth was observed after 24h of infection in the presence of RGB in our experimental conditions. DISCUSSION AND CONCLUSIONS. Our results show that RGB significantly reduce C. albicans-induced damage of vaginal epithelial cells. One of the mechanisms underlying this effect is the inhibition of C. albicans adhesion to the vaginal epithelial cells, which may prevent Candida from penetrating and damaging epithelial cells, hence counteract Candida virulence. Collectively our preliminary results suggest that RBG can strengthen the VVC therapy favoring the establishment of an ecosystem that prevent Candida virulence

Antibacterial Effects of MicroRepair®BIOMA-Based Toothpaste and Chewing Gum on Orthodontic Elastics Contaminated In Vitro with Saliva from Healthy Donors: A Pilot Study

Several new products with innovative formulations are being proposed to facilitate oral care. Here, we evaluated the effects of a commercially available product, a toothpaste and chewing gum named Biorepair Peribioma, on oral microorganisms of healthy subjects. Saliva from six volunteers was collected during 20 min of mastication of a traditional gum (gum A) and the Biorepair Peribioma gum (gum P). Orthodontic elastics (OE) were in vitro contaminated with salivary samples, both A and P, and subsequently exposed or not to a Biorepair Peribioma toothpaste-conditioned supernatant (Tp-SUP). The salivary samples were tested for initial microbial load; hence, the contaminated OE were assessed for microbial growth, adhesion, biofilm formation and persistence; moreover, species identification was assessed. We found that the salivary samples A and P had similar microbial load; upon contamination, microbial adhesion onto the OE was detected to a lower extent when using saliva P with respect to saliva A. Microbial growth and biofilm formation, assessed at 24 h, remained at lower levels in OE exposed to saliva P, compared to saliva A. This difference between salivary samples A and P was confirmed when measuring biofilm persistence (48 h), while it was lost in terms of microbial re-growth (48 h). The Tp-SUP treatment drastically affected microbial load at 24 h and strongly impaired biofilm formation/persistence, in OE exposed to both salivary samples A and P. Finally, such treatment resulted in consistent overgrowth of Lactobacilli, bacterial species originally present both in the Biorepair Peribioma toothpaste and gum. In conclusion, by an in vitro pilot study, we show that the Biorepair Peribioma toothpaste and gum deeply affect oral microorganisms’ behavior, drastically impairing their ability to contaminate and produce plaque onto orthodontic devices

EDTA and Taurolidine affect Pseudomonas aeruginosa virulence in vitro: impairment of secretory profile and biofilm production onto peritoneal dialysis catheters

Introduction: peritoneal catheter-associated biofilm infection is reported to be the main cause of refractory peritonitis in peritoneal dialysis patients. The application of antimicrobial lock therapy, based on results on central venous catheters, may be a promising option also for treatment of biofilm-harboring peritoneal catheters. In this study, we investigated the effects of two lock solutions, EDTA and Taurolidine, on an “in-vitro” model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Materials and Methods: silicon peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. After washing, serial concentrations of Taurolidine (0.5, 0.25 and 0.125 %) and EDTA (2.5, 0.75 and 0.25 %), either alone or in combination, were applied for 24 h, once or twice, onto the contaminated catheters and then P. aeruginosa viability/persistence was evaluated in real time up to 120 h, by a Fluoroskan reader. Moreover, on selected supernatants from biofilm treated or not with EDTA and/or Taurolidine, High-Performance Liquid Chromatography-Mass (HPLC) analysis was performed to measure phenazine and pyocianine production. Results: Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence onto the contaminated catheters. The lock solution treatment did not lead to the sterilization of the devices; yet, it resulted in a substantial destructuration of the peritoneal catheter-associated P. aeruginosa biofilm. Moreover, HPLC analysis showed that the treatment of biofilm-harboring catheters with EDTA and Taurolidine deeply affected the secretion of some key virulence-related molecules by P. aeruginosa, such as phenazines and pyocianines. Discussion and conclusions: EDTA and Taurolidine affect the formation and persistence of P. aeruginosa biofilm onto peritoneal catheters; moreover, also the secretion of P. aeruginosa virulence factors is profoundly compromised. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheter-related infections more susceptible to antibiotic treatment, thus avoiding/reducing the onset of the antibiotic resistance phenomena