Herpes Simplex Virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals and UVA1 laser treatment

Abstract Background: Recently, we published data suggesting a mutualistic relationship between HSV-1 and Candida. albicans; in particular: (a) HSV-1 infected macrophages are inhibited in their anti-Candida effector function and (b) Candida biofilm protects HSV-1 from inactivation. The present in vitro study is aimed at testing the effects of Candida biofilm on HSV-1 sensitivity to pharmacological and physical stress, such as antiviral drugs (acyclovir and foscarnet) and laser UVA1 irradiation. We also investigated whether fungus growth pattern, either sessile or planktonic, influences HSV-1 sensitivity to antivirals. Methods: Mature Candida biofilms were exposed to HSV-1 and then irradiated with laser light (UVA1, 355 \u3bb). In another set of experiments, mature Candida biofilm were co-cultured with HSV-1 infected VERO cells in the presence of different concentrations of acyclovir or foscarnet. In both protocols, controls unexposed to laser or drugs were included. The viral yield of treated and untreated samples was evaluated by end-point titration. To evaluate whether this protective effect might occur in relation with a different growth pattern, HSV-1 infected cells were co-cultured with either sessile or planktonic forms of Candida and then assessed for susceptibility to antiviral drugs. Results: UVA1 irradiation caused a 2 Log reduction of virus yield in the control cultures whereas the reduction was only 1 Log with Candida biofilm, regardless to the laser dose applied to the experimental samples (50 or 100 J/cm2). The presence of biofilm increased the IC90 from 18.4\u201325.6 J/cm2. Acyclovir caused a 2.3 Log reduction of virus yield in the control cultures whereas with Candida biofilm the reduction was only 0.5 Log; foscarnet determined a reduction of 1.4 Log in the controls and 0.2 Log in biofilm cultures. Consequently, the ICs50 for acyclovir and foscarnet increased by 4- and 12-folds, respectively, compared to controls. When HSV-1 was exposed to either sessile or planktonic fungal cells, the antiviral treatments caused approximately the same weak reduction of virus yield. Conclusions: These data demonstrate that: (1) HSV-1 encompassed in Candida biofilm is protected from inactivation by physical (laser) and pharmacological (acyclovir or foscarnet) treatments; (2) the drug antiviral activity is reduced at a similar extent for both sessile or planktonic Candida

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

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections

Effects of different mouthwashes on biofilm formation by oral streptococci

Introduction. Oral microbiota is an extremely complex and dynamic system including bacteria, archea and fungi. Different locations of the oral cavity host a large variety of microbiota mostly organized as biofilm, characterized by spatial and temporal differences in their distribution. Several bacteria belonging to the genus Streptococcus act as early colonizers, binding to the adhesive pellicle and providing a substrate for the attachment of late colonizers bacteria. The latter, in turn, may be responsible of severe pathologies, such as carious lesions, gingivitis and periodontal lesions. Therefore, we evaluated the effects of commercial mouthwashes (MoWs) on biofilm (BF) formation/persistence of early colonizers Streptococci. Materials and methods. Fourteen isolates belonging to 5 different species of oral streptococci (S. salivarius, S. mitis/oralis, S. sanguinis, S. parasanguinis, S. vestibularis) and 3 isolates of Enterococcus faecalis were obtained from pharyngeal swabs and employed for the present study. All the bacteria were incubated for 1 minute with 6 commercial MoWs, 4 with (MoWs 1, 2, 3 and 7) and 2 without (MoWs 4 and 5) chlorhexidine digluconate (CHX), which is known to exert antibacterial and antifungal activity. Control groups of each strain were treated for 1 minute with PBS and used as negative controls. After treatment with MoWs or PBS, bacteria were seeded in 96-well plates and allowed to form BF for up to 48 hours. BF formation was assessed by crystal violet assay. The capacity to form BF was expressed as the optical density (OD) percentage of each MoW-treated strain, as compared to the OD of the PBS-treated strain, which was considered as 100%. Statistical analyses were carried out by one-way ANOVA test with Bonferroni post-hoc test. Results. CHX-containing MoWs were capable to inhibit BF formation in most of the cases. In detail, CHX-containing MoWs significantly reduced BF formation by all the S. salivarius and E. faecalis isolates, while only several S. parasanguinis, S. mitis/oralis, S. sanguinis and S. vestibularis isolates were affected. One of the 2 CHX-free MoWs was capable to significantly inhibit BF formation by S. salivarius and S. vestibularis, whereas the remaining CHX-free MoW did not prove to be effective in impairing BF formation by any of the bacterial isolates assessed. Discussion and conclusions. Similarly to Candida albicans, BF formation by oral streptococci is affected by MoWs, provided that they include CHX in their formulation. Since the streptococci used in the present study act as early colonizers in the multispecies microbial BF of the dental surface, special attention should be used when choosing MoWs for prevention and/or treatment of oral pathologies of microbial origin

Effects of different mouthwashes on Candida albicans adhesion, susceptibility to phagocytic cells and capacity to elicit pro-inflammatory cytokine response

Introduction. Oral candidiasis is a frequent opportunistic fungal infection, occurring especially in susceptible individuals. This pathology, mainly associated with Candida albicans species, may be prevented by a good oral hygiene, including the daily use of toothbrush and mouthwashes (MoWs). Among several virulence factors, C. albicans has the ability to adhere to epithelial surfaces, to avoid phagocytosis and/or intracellular killing and to elicit proinflammatory cytokines production. We have previously demonstrated that both C. albicans hyphal development and biofilm formation/persistence are affected by MoWs, provided that they contain chlorhexidine digluconate. Therefore, in this study we aim to expand our knowledge on MoWs effects by investigating the behaviour of MoWs-treated C. albicans, in terms of adhesion to both abiotic and biotic surfaces, susceptibility to phagocytosis and capacity to elicit pro-inflammatory immune responses. Materials and methods. C. albicans SC5314 and 6 commercial MoWs have been employed: 4 with and 2 without chlorhexidine digluconate (CHX), a component known to have antibacterial and antifungal activity. Adhesion was assessed by a bioluminescent strain of C. albicans SC5314; MoWs-treated and PBS-treated fungal cells were incubated in 96-well plates containing or not a monolayer of TR-146 oral epithelial cell line; after 60 min, plates were washed and the residual bioluminescent signal recorded. Susceptibility to phagocytosis was assessed by exposing MoWs-treated and PBS-treated C. albicans to phagocytic cell line BV2 (effector:target=1:2). Following 24 hours incubation of TR-146 cells with MoWs-treated and PBS-treated C. albicans, cytokine levels in supernatants were measured. Results. Adhesion of MoWs-treated C. albicans to abiotic surfaces was significantly lower than PBS-treated Candida. Adhesion of MoWs-treated C. albicans to TR-146 cells was significantly lower than PBS-treated Candida, in all but MoW 4. No differences could be highlighted in terms of susceptibility to phagocytosis (percent phagocytic cells and phagocytosis index) between MoWs-treated and PBS-treated Candida. On the contrary, significantly higher acidic phagolysosomes percentages were recorded from Candida treated with 4 out of 6 MoWs, with respect to PBS-treated fungi. Finally, Candida pretreatment with 4 out of 6 MoWs and 5 out of 6 MoWs impaired the production of IL-1alpha and IL-1beta respectively. Discussion and conclusions. C. albicans adhesion, susceptibility to phagocytosis and capacity to elicit pro-inflammatory cytokine response are affected by MoWs, especially those containing CHX. Thus, special attention should be used when choosing MoWs whether prevention and/or treatment of Candida-associated oral pathologies was intended

Anti-Candida albicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans

Background: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. Aims: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. Methods: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. Results: CAGTA ≥50 μg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80 μg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90 min. Conclusions: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections

Candida albicans survival, growth and biofilm formation are differently affected by mouthwashes: an in vitro study

Candida albicans is the most common cause of oral mycoses. The aim of the present study was to investigate in vitro the susceptibility of C. albicans to mouthwashes, in terms of growth, survival and biofilm formation.Candida albicans, laboratory strain SC5314, and 7 commercial mouthwashes were employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F sodium fluoride; 3 with fluorine-containing molecules. None of the mouthwashes contained ethanol in their formulations. The anti-Candida effects of the mouthwashes were assessed by disk diffusion, crystal violet and XTT assays. By using five protocols combining different dilutions and contact times the mouthwashes were tested against:1) C. albicans growth;2) biofilm formation;3) survival of fungal cells in early, developing and mature Candida biofilm.Chlorhexidine digluconate-containing mouthwashes consistently exhibited the highest anti-Candida activity, irrespective of the protocols employed. Fungal growth, biofilm formation and survival of Candida cells within biofilm were impaired, the effects strictly depending on both the dilution employed and the time of contact.These in vitro studies provide evidence that mouthwashes exert anti-Candida activity against both planktonic and biofilm fungal structures, but to a different extent depending on their composition. This suggests special caution in the choice of mouthwashes for oral hygiene, whether aimed at prevention or treatment of oral candidiasis

In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity.

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule