Evaluation of Antimicrobial Effect of Air-Polishing Treatments and Their Influence on Human Dental Pulp Stem Cells Seeded on Titanium Disks.

Dental implants are one of the most frequently used treatment options for tooth replacement, and titanium is the metal of choice due to its demonstrated superiority in resisting corrosion, lack of allergic reactions and mechanical strength. Surface roughness of titanium implants favors the osseointegration process; nevertheless, its topography may provide a suitable substrate for bacterial biofilm deposition, causing peri-implantitis and leading to implant failure. Subgingival prophylaxis treatments with cleansing powders aimed to remove the bacterial accumulation are under investigation. Two different air-polishing powders-glycine and tagatose-were assayed for their cleaning and antimicrobial potential against a Pseudomonas biofilm and for their effects on human dental pulp stem cells (hDPSCs), seeded on sandblasted titanium disks. Immunofluorescence analyses were carried out to evaluate cell adhesion, proliferation, stemness and osteogenic differentiation. The results demonstrate that both the powders have a great in vitro cleaning potential in the early period and do not show any negative effects during hDPSCs osteogenic differentiation process, suggesting their suitability for enhancing the biocompatibility of titanium implants. Our data suggest that the evaluated cleansing systems reduce microbial contamination and allow us to propose tagatose as an adequate alternative to the gold standard glycine for the air-polishing prophylaxis treatment.

Evaluation of Biological Response of STRO-1/c-Kit Enriched Human Dental Pulp Stem Cells to Titanium Surfaces Treated with Two Different Cleaning Systems.

Peri-implantitis-an infection caused by bacterial deposition of biofilm-is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems-Ni-Ti Brushes (Brush) and Air-Polishing with 40 µm bicarbonate powder (Bic40)-might alter the physical/chemical features of two different titanium surfaces-machined (MCH) and Ca++ nanostructured (NCA)-and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.

Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation. RESULTS: We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm. CONCLUSIONS: Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

Impact of Candida albicans hyphal wall protein 1 (HWP1) genotype on biofilm production and fungal susceptibility to microglial cells.

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.

The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae.

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Contribution of different pneumococcal virulence factors to experimental meningitis in mice.

BACKGROUND: Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM. METHODS: Mice were challenged by the intracranial route with the serotype 4 TIGR4 strain (wt) and three isogenic mutants devoid of PspA, PspC, and the capsule. Survival, bacterial counts, and brain histology were carried out. To study the interaction between S. pneumoniae mutants and microglia, phagocytosis and survival experiments were performed using the BV2 mouse microglial cell line. RESULTS: Virulence of the PspC mutant was comparable to that of TIGR4. In contrast, survival of animals challenged with the PspA mutant was significantly increased compared with the wt, and the mutant was also impaired at replicating in the brain and blood of infected mice. Brain histology indicated that all strains, except for the unencapsulated mutant, caused PM. Analysis of inflammation and damage in the brain of mice infected with TIGR4 or its unencapsulated mutant demonstrated that the rough strain was unable to induce inflammation and neuronal injury, even at high challenge doses. Results with BV2 cells showed no differences in phagocytic uptake between wt and mutants. In survival assays, however, the PspA mutant showed significantly reduced survival in microglia compared with the wt. CONCLUSIONS: PspA contributed to PM pathogenesis possibly by interacting with microglia at early infection stages, while PspC had limited importance in the disease. The rough mutant did not cause brain inflammation, neuronal damage or mouse death, strengthening the key role of the capsule in PM.

Novel Options to Counteract Oral Biofilm Formation: In Vitro Evidence.

Biofilm production on biotic and abiotic surfaces is crucial in the pathogenesis of most infections, particularly those occurring in the oral cavity. Its prevention and/or control may greatly facilitate the management of patients with oral diseases. Here, the antibiofilm activity of a biomimetic hydroxyapatite and a natural compound, MicroRepair (MicroR) and pomegranate (PomeGr), respectively, was assessed. By luminescence/fluorescence-based assays, Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were tested for biofilm production in the presence of MicroR and/or PomeGr. We found that both MicroR and PomeGr could affected biofilm production; however, the efficacy of the two, given alone or in combination, varied according to the microbial agent considered. These data open to clinical studies aimed at defining the most efficacious protocols to counteract oral biofilm-associated infections.

Attenuation of Pseudomonas aeruginosa Virulence by Pomegranate Peel Extract.

Pseudomonas aeruginosa is an opportunistic pathogen often responsible for biofilm-associated infections. The high adhesion of bacterial cells onto biotic/abiotic surfaces is followed by production of an extracellular polysaccharidic matrix and formation of a sessile community (the biofilm) by the release of specific quorum-sensing molecules, named autoinducers (AI). When the concentrations of AI reach a threshold level, they induce the expression of many virulence genes, including those involved in biofilm formation, motility, pyoverdine and pyocyanin release. P. aeruginosa embedded into biofilm becomes resistant to both conventional drugs and the host's immune response. Accordingly, biofilm-associated infections are a major clinical problem underlining the need for new antimicrobial therapies. In this study, we evaluated the effects of pomegranate peel extract (PomeGr) in vitro on P. aeruginosa growth and biofilm formation; moreover, the release of four AI was assessed. The phenolic profile of PomeGr, exposed or not to bacteria, was determined by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. We found that bacterial growth, biofilm production and AI release were impaired upon PomeGr treatment. In addition, the PomeGr phenolic content was also markedly hampered following incubation with bacterial cells. In particular, punicalagin, punicalin, pedunculagin, granatin, di-(HHDP-galloyl-hexoside) pentoside and their isomers were highly consumed. Overall, these results provide novel insights on the ability of PomeGr to attenuate P. aeruginosa virulence; moreover, the AI impairment and the observed consumption of specific phenolic compounds may offer new tools in designing innovative therapeutic approaches against bacterial infections.

Pomegranate Extract Affects Fungal Biofilm Production: Consumption of Phenolic Compounds and Alteration of Fungal Autoinducers Release.

Candida albicans expresses numerous virulence factors that contribute to pathogenesis, including its dimorphic transition and even biofilm formation, through the release of specific quorum sensing molecules, such as the autoinducers (AI) tyrosol and farnesol. In particular, once organized as biofilm, Candida cells can elude conventional antifungal therapies and the host's immune defenses as well. Accordingly, biofilm-associated infections become a major clinical challenge underlining the need of innovative antimicrobial approaches. The aim of this in vitro study was to assess the effects of pomegranate peel extract (PomeGr) on C. albicans growth and biofilm formation; in addition, the release of tyrosol and farnesol was investigated. The phenolic profile of PomeGr was assessed by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis before and after exposure to C. albicans. Here, we showed that fungal growth, biofilm formation and AI release were altered by PomeGr treatment. Moreover, the phenolic content of PomeGr was substantially hampered upon exposure to fungal cells; particularly pedunculagin, punicalin, punicalagin, granatin, di-(HHDP-galloyl-hexoside)-pentoside and their isomers as well as ellagic acid-hexoside appeared highly consumed, suggesting their role as bioactive molecules against Candida. Overall, these new insights on the anti-Candida properties of PomeGr and its potential mechanisms of action may represent a relevant step in the design of novel therapeutic approaches against fungal infections.

Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo-in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

EDTA and Taurolidine Affect Pseudomonas aeruginosa Virulence In Vitro-Impairment of Secretory Profile and Biofilm Production onto Peritoneal Dialysis Catheters.

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 for treatment of biofilm-harboring peritoneal catheters. This study investigated the effects of two lock solutions, EDTA and taurolidine, on an in vitro model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Silicone peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. Then, serial dilutions of taurolidine and/or EDTA were applied (for 24 h) once or twice onto the contaminated catheters, and P. aeruginosa viability/persistence were evaluated in real time up to 120 h using a Fluoroskan reader. On selected supernatants, high-performance liquid chromatography mass spectrometry (HPLC-MS) analysis was performed to measure the production of autoinducers (AI), phenazines, and pyocyianines. Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence on the contaminated catheters. The treatment did not lead to the sterilization of the devices, yet it resulted in a substantial destructuration of the catheter-associated P. aeruginosa biofilm. HPLC-MS analysis showed that the treatment of biofilm-harboring catheters with taurolidine and EDTA also affected the secretory activity of the pathogen. EDTA and taurolidine affect P. aeruginosa biofilm produced on peritoneal catheters and profoundly compromise the microbial secretory profile. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheter-related infections more susceptible to antibiotic treatment. IMPORTANCE An in vitro model allows studies on the mechanisms by which the lock solutions exert their antimicrobial effects on catheter-associated biofilm, thus providing a better understanding of the management of devise-associated infections.

Copper-Calcium Hydroxide and Permanent Electrophoretic Current for Treatment of Apical Periodontitis.

Endodontic failure has been and continues to be a problem for endodontics-specialists. Complicated anatomy, numerous foramens, and accessory canals are an environment for microorganisms to infect the teeth. The purpose of the present work was to evaluate the regeneration of copper-calcium hydroxide (Cupral)-endodontically treated teeth diagnosed with apical periodontitis using an electrophoresis technique. In total, 132 patients, aging from 19 to 65 years old, underwent endodontic treatment mono- and multi-radicular teeth, with complicated canals from January 2019 to June 2020. The patients were divided into two groups: (i) the control group-which included 54 patients (n = 62 teeth) receiving endodontic paste (Calcipast + 1) and, as final filling, the AH-PlusTM cement-and (ii) the Cupral group, which included 78 patients (n = 80 teeth) receiving Cupral paste plus the electrophoretic current and, as final filling, the Atacamit-alkaline cement. The clinical cases were periodically observed along an 18-month follow-up period via radiography. Data were expressed as focal size of the lesions (mean ± standard error (SEM) of all the radiographic outcomes) observed in each group at each interval point. Statistical analysis was performed using the Student's t-test that allowed us to compare the control and Cupral groups; the statistical significance was set at p < 0.05 and p < 0.01, where the latter was highly significant. Before treatments, the focal sizes were 4.8 mm and 4.95 mm for control and Cupral-treated groups, respectively. After 6 months, the mean focal sizes were 3.9 mm and 2.14 mm for the control and Cupral groups, respectively. After 12 months, in the control group, the mean focal size was measured at 2.8 mm, while, in Cupral group, the lesion size decreased down to 0.31 mm and a highly dynamic regeneration of the destructive focal-bone occurred. After 18 months, the lesions were further significantly reduced in the control group (mean values of 2.62 mm), while they were barely detectable in the Cupral group (0.2 mm). In conclusion, we provide initial evidence that the Cupral-electrophoresis methodology is effective in treating destructive periodontitis of teeth with problematic canals up to 18 months, thus allowing teeth preservation.

Candida metapsilosis as the least virulent member of the 'C. parapsilosis' complex.

Results of recent molecular studies have provided evidence of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. While there are initial data pertaining to the virulence of these Candida species with respect to reconstituted epidermal and oral epithelial tissues, there have been no studies, as of yet, on their interaction with immune cells. Employing an in vitro infection model using microglial cells, we investigated the pathogenetic potential of different isolates of each of these three species. We show that C. metapsilosis isolates are more susceptible to microglia-mediated antifungal activity, as compared with those of C. parapsilosis and C. orthopsilosis. Interestingly, C. metapsilosis isolates are also phagocytosed to a lower extent, but the yeast-containing phagosomes exhibit the highest degree of acidification in comparison with the phagosomes containing C. parapsilosis or C. orthopsilosis. Furthermore, when assessing microglia secretory response to infection, comparable high levels of MIP-1α and little or no TNF-α production are observed with all of these Candida species. Finally, unlike C. metapsilosis infected cells, microglial cells infected with C. parapsilosis and C. orthopsilosis release high and time-dependent levels of lactate dehydrogenase (LDH). Overall, these findings point to C. metapsilosis as the least virulent member of the 'C. parapsilosis' complex.

The ß-Lactamase Inhibitor Boronic Acid Derivative SM23 as a New Anti-Pseudomonas aeruginosa Biofilm.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great capacity to form biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), a cell-to-cell communication mechanism used by many bacteria. Selective inhibition of QS-controlled pathogenicity without affecting bacterial growth may represent a novel promising strategy to overcome the well-known and widespread drug resistance of P. aeruginosa. In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as ß-lactamase inhibitor, on biofilm formation and virulence factors production by P. aeruginosa. Our results indicated that SM23: (1) inhibited biofilm development and production of several virulence factors, such as pyoverdine, elastase, and pyocyanin, without affecting bacterial growth; (2) decreased the levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules, in line with a dampened lasR/lasI system; (3) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; and (4) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model closely mimicking clinical settings. Taken together, our results indicate that, besides inhibiting ß-lactamase, SM23 can also act as powerful inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

Propolis Affects Pseudomonas aeruginosa Growth, Biofilm Formation, eDNA Release and Phenazine Production: Potential Involvement of Polyphenols.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for a wide range of clinical conditions, from mild infections to life-threatening nosocomial biofilm-associated diseases, which are particularly severe in susceptible individuals. The aim of this in vitro study was to assess the effects of an Albanian propolis on several virulence-related factors of P. aeruginosa, such as growth ability, biofilm formation, extracellular DNA (eDNA) release and phenazine production. To this end, propolis was processed using three different solvents and the extracted polyphenolic compounds were identified by means of high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. As assessed by a bioluminescence-based assay, among the three propolis extracts, the ethanol (EtOH) extract was the most effective in inhibiting both microbial growth and biofilm formation, followed by propylene glycol (PG) and polyethylene glycol 400 (PEG 400) propolis extracts. Furthermore, Pseudomonas exposure to propolis EtOH extract caused a decrease in eDNA release and phenazine production. Finally, caffeic acid phenethyl ester (CAPE) and quercetin decreased upon propolis EtOH extract exposure to bacteria. Overall, our data add new insights on the anti-microbial properties of a natural compound, such as propolis against P. aeruginosa. The potential implications of these findings will be discussed.

Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation.

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1Delta mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1-overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans-microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.

Efficacy of a Copper-Calcium-Hydroxide Solution in Reducing Microbial Plaque on Orthodontic Clear Aligners: A Case Report.

The aim of this study was to investigate the ability of a copper-calcium-hydroxide-based compound to remove microbial plaque naturally produced onto orthodontic clear aligners. A commercially available dental paste, named Cupral, based on copper-calcium-hydroxide, was used. A healthy volunteer (female, 32 years old), undergoing orthodontic treatment with thermoplastic clear aligners was enrolled. By conventional/confocal microscopy and colony-forming unit (CFU) assay, 2-week used aligners were examined for microbial plaque, prior and following exposure to Cupral. Confocal microscopy revealed abundant plaque irregularly distributed onto the aligner surface. Following Cupral treatment, a drastic decrease occurred in plaque thickness and matrix presence. As assessed by the CFU assay, total microbial load approached 109 CFUs/aligner, with slight differences in aerobiosis and anaerobiosis culture conditions; six macroscopically different types of colonies were detected and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Following Cupral treatment, microbial load dropped to undetectable levels, irrespectively of the conditions considered. Exposure of clear aligners to Cupral results in the elimination of contaminating microorganisms; the antimicrobial activity is retained up to 1.25% concentration. Overall, our data describe a novel use of Cupral, a copper-calcium-hydroxide-based compound, in daily hygiene practices with promising results.

Antimicrobial and antibiofilm efficacy of a copper/calcium hydroxide-based endodontic paste against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans.

Endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are found pathogens, like bacteria and opportunistic fungi responsible for various endodontic pathologies. As clinical importance is the fact, that biofilm is extremely resistant to common intracanal irrigants, antimicrobial drugs and host immune responses. The aim of this study was to evaluate the in vitro efficacy of a Cu/CaOH2-based endodontic paste, against bacteria and fungi, such as Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. We found that such compound significantly reduced microbial replication time and cell growth. Moreover, biofilm formation and persistence were also affected; treated biofilms showed both a reduced number of cells and levels of released pyoverdine. This study provides the first evidence on effectiveness of this endodontic compound against microbial biofilms. Given its wide range of action, its use in prevention and treatment of the main oral biofilm-associated infections will be discussed.

Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization.

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.