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Early failure of silicone voice prostheses resulting from fungal colonization and biofilm formation poses a major concern in modern ear nose throat surgery. Therefore, developing new infection prevention techniques to prolong those implants’ survivorship is crucial. We designed an in vitro laboratory study to include nanomaterial-enhanced polymer coating with a plasma spraying technique against Candida albicans growth to address this issue. The anti-biofilm effects of high- and low-dose Al2O3 nanowire and TiO2 nanoparticle coatings were studied either alone or in conjunction with each other using checkerboard testing. It was demonstrated that both nanomaterials were capable of preventing fungal biofilm formation regardless of the anti-fungal agent concentration (median absorbance for high-dose Al2O3-enhanced polymer coating was 0.176 [IQR = 0.207] versus control absorbance of 0.805 [IQR = 0.381], p = 0.003 [98% biofilm reduction]; median absorbance for high-dose TiO2-enhanced polymer coating was 0.186 [IQR = 0.024] versus control absorbance of 0.766 [IQR = 0.458], p < 0.001 [93% biofilm reduction]). Furthermore, synergy was revealed when the Bliss model was applied. According to the findings of this work, it seems that simultaneous consideration of Al2O3 and TiO2 could further increase the existing antibiofilm potential of these nanomaterials and decrease the likelihood of localized toxicity.

Background Candida auris is a newly described multidrug-resistant fungal pathogen associated with biofilm formation and severe infections with high mortality. Objectives To study the activities of fluconazole, itraconazole, posaconazole, voriconazole, deoxycholate and liposomal amphotericin B, anidulafungin, caspofungin and micafungin against C auris biofilms and planktonic cells. Materials/methods C auris strains originating from 5 clades (South Asian, East Asian, African, South American and Iranian) were tested for biofilm production by safranin staining of the extracellular matrix polysaccharide structure as well as biofilm (BF) and planktonic (PLK) antifungal susceptibility to nine antifungal agents using the XTT reduction assay. Results Candida auris isolates produced mature BF as compared to non-C auris control (Candida albicans and Candida parapsilosis) strains. Four C auris isolates exhibited relatively high MIC's for fluconazole (32-128 mg/L for PLK MIC and 128-1024 mg/L for BF MIC) as compared to the Iranian strain that had PLK and BF MIC's 0.5 and 16, respectively. Itraconazole, posaconazole and voriconazole had relatively low PLK MICs but high BF MICs. A similar pattern was observed with echinocandins; relatively low PLK MIC (0.06-4 mg/L) but quite high BF MICs (4-2048 mg/L). While all isolates exhibited relatively low PLK MICs (0.06-4 mg/L) for both amphotericin B formulations, liposomal amphotericin B showed higher MICs compared to deoxycholate amphotericin B against C auris BF. Conclusion Triazoles, echinocandins and liposomal amphotericin B appear to have less activity against C auris biofilms than deoxycholate amphotericin B. Our in vitro model provides evidence for intrinsic C auris biofilm resistance to antifungal agents.

Εισαγωγή: Στη φύση, οι μικροοργανισμοί ζουν κυρίως σε σύνθετες λειτουργικές κοινότητες, οι οποίες αποτελούνται από ένα ή περισσότερα είδη μικροβίων. Παραδείγματα τέτοιων μικτών κοινοτήτων είναι εκείνες που σχηματίζονται από μικροοργανισμούς του είδους Candida spp. και Pseudomonas spp. Τα βιοϋμένια(ΒΥ) σχετίζονται με τη χρήση επεμβατικών ιατρικών εργαλείων. Σκοπός: Η διερεύνηση της δράσης 5 αντιβακτηριακών παραγόντων (αμικασίνη(ΑΜΚ), σιπροφλοξασίνη(CIP), κεφταζιδίμη(CEF), κολιμυκίνη(CST) και κλαριθρομυκίνη(CLAR) τόσο έναντι ΠΛΚ όσο και έναντι ΒΥ από PA και η μελέτη της δράσης των ανθρώπινων πολυμορφοπυρήνων (PMN) έναντι της PA. Επίσης, μελετήθηκε ο συνδυασμός αντιμικροβιακών παραγόντων έναντι μικτών βιοϋμενίων(MBY) από C.albicans(CA) και PA. Μελετήθηκε η συνδυαστική δράση της ΑΜΚ με τη βορικοναζόλη(VRC) ή τη ανιδουλαφουγκίνη(ΑΝID) και η φαγοκυτταρική ικανότητα των PMN έναντι MBY. Τέλος, μελετήθηκε η έκφραση των γονιδίων BGL2, SUN41 και ECE1 σε στέλεχος CA σε συνδυασμό με την δράση αντιμυκητιακών φαρμάκων.Υλικό-Μέθοδος: Για τα ΒΥ, αρχικά χρησιμοποιήθηκαν 106 μικροοργανισμοί/mL, ενώ για τα MBY που σχετίζονταν και με CA και με PA, αρχικά χρησιμοποιήθηκαν 106 μικροοργανισμοί/ml, σε RPMI-1640, μέσα σε πλάκες κυτταροκαλλιεργειών 96-πηγαδιών στους 37°C,48h,5%CO2, με συνεχή ανακίνηση. Οι συγκεντρώσεις που χρησιμοποιήθηκαν σε όλα τα πειράματα κυμάνθηκαν για όλα τα αντιβακτηριακά από 4-512mg/L και για όλα τα αντιμυκητιακά από 0,06-512 mg/L(n=8). Η δράση των αντιμικροβιακών φαρμάκων εκτιμήθηκε με ΧΤΤ. Τα PMNs χρησιμοποιήθηκαν σε αναλογία 1:1 και 5:1 για την Candida και σε αναλογία 1:20 και 1:10 για την Pseudomonas και συνδυάστηκαν με C1, C2, και C3 mg/L (n=4). Τρία ανεξάρτητα πειράματα με PCR πραγματοποιήθηκαν σε κάθε περίπτωση. Μεταβολή της έκφρασης κατά 2-φορές θεωρήθηκε σημαντική. Αποτελέσματα: Τα πρώτα δύο στελέχη ήταν το ένα ανθεκτικό(ΑΜΚr) και το άλλο ευαίσθητο(ΑΜΚs) στην αμικασίνη. Το ΑΜΚr εμφάνισε μεγαλύτερη ανθεκτικότητα στην αμικασίνη σε σχέση με το ΑΜΚs τόσο στα PLK όσο και στα ΒΥ. Παρόμοια συμπεράσματα μπόρεσαν να εξαχθούν και για τα άλλα δύο ζευγάρια στελεχών (CEFr vs CEFs και CΙΡr vs CΙΡs). Στη ταυτόχρονη προσθήκη, όταν προστέθηκε στην CA, CAS(0,008-0,25mg/L) και PSC(32-128mg/L) παρατηρήθηκαν συνεργικές αλληλεπιδράσεις. Επίσης, όλοι οι συνδυασμοί που μελετήθηκαν, CAS/PSC ή VRC/AND με PSC ή VRC, παρουσίασαν αδιάφορα αποτελέσματα στα ΒΥ. Σημαντική καταστροφή παρατηρήθηκε επίσης όταν συνδυάστηκε AND με VRC. Μετά την επώαση των διαφόρων μορφών των ΒΥ με VRC(128 και 512 mg/L) ή ANID(0,25 και 1 mg/L) για 12-24 h, το συνολικό RNA που απομονώθηκε από τα ΒΥ αναλύθηκε με RT-PCR. Η ANID προκάλεσε στατιστικά σημαντική υπερέκφραση των γονιδίων SUN41(3.7-9.3) και BGL2(2.2-2.8) στα ενδιάμεσα ΒΥ, ενώ η VRC προκάλεσε υπερέκφραση των αντίστοιχων γονιδίων στα ώριμα ΒΥ (SUN41:2.3, BGL2:2.1). Η έκφραση των γονιδίων μειώθηκε από την δράση της VRC στα ενδιάμεσα ΒΥ. Και τα δύο αντιμυκητιακά προκάλεσαν υποέκφραση του γονιδίου ECE1 στα ενδιάμεσα ΒΥ.Συμπεράσματα: Ακόμα και σε μεγάλες συγκεντρώσεις αντιψευδομοναδικών παραγόντων δεν κατέστη δυνατή η πλήρης εκρίζωση στελεχών των ΒΥ. Επίσης, πρέπει να τονίσουμε ότι τα ΒΥ όλων των στελεχών έχουν σαφώς υψηλότερες MIC σε σχέση με τα PLK. Τα αποτελέσματα των συνδυαστικών μελετών έδειξαν ότι όλοι οι συνδυασμοί της ΑΜΚ+ΡΜΝs είχαν συνεργικά ή προσθετικά αποτελέσματα. Επίσης, όλοι οι συνδυασμοί της ΑΜΚ+PMNs οδήγησε σε συνεργικά ή προσθετικά αποτελέσματα της PA. Η CEF ακολουθεί σε αποτελεσματικότητα ενώ η CIP επιδρά ανταγωνιστικά όταν συνδυάζεται με ΡΜΝ.

Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2 H -tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa . Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.

Yeasts and filamentous fungi both exist as single cells and hyphal forms, two morphologies used by most fungal organisms to create a complex multilayered biofilm structure. In this chapter we describe the most widely used assays for the determination of biofilm production and assessment of susceptibility of biofilms to antifungal agents or host phagocytes as various methods, the most frequent of which are staining, confocal laser scanning microscopy, quantification of extracellular DNA and protein associated with extracellular matrix and XTT metabolic reduction assay. Pathway-focused biofilm gene expression profiling is assessed by real-time reverse transcriptase polymerase chain reaction.

Yeasts and filamentous fungi both exist as single cells and hyphal forms, two morphologies used by most fungal organisms to create a complex multilayered biofilm structure. In this chapter we describe the most widely used assays for the determination of biofilm production and assessment of susceptibility of biofilms to antifungal agents or host phagocytes as various methods, the most frequent of which are staining, confocal laser scanning microscopy, quantification of extracellular DNA and protein associated with extracellular matrix and XTT metabolic reduction assay. Pathway-focused biofilm gene expression profiling is assessed by real-time reverse transcriptase polymerase chain reaction.

Biofilm formation complicates the treatment of various infections caused by Candida species. We investigated the effects of simultaneous or sequential combinations of two triazoles, voriconazole (VRC) and posaconazole (PSC), with two echinocandins, anidulafungin (AND) and caspofungin (CAS), against Candida albicans and Candida parapsilosis biofilms in comparison to their planktonic counterparts. Antifungal activity was assessed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. Antifungal-agent interactions were analyzed by the Bliss independence model in the simultaneous-treatment studies and by analysis of variance (ANOVA) in the sequential-treatment studies. Against C. albicans planktonic cells, the simultaneous combination of PSC (32 to 128 mg/liter) and CAS (0.008 to 0.25 mg/liter) was synergistic; the combinations of PSC (128 to 1,024 mg/liter) with AND (0.03 to 0.5 mg/liter) and VRC (32 to 512 mg/liter) with AND (0.008 to 0.03 mg/liter) were antagonistic. Against C. parapsilosis planktonic cells, the interaction between VRC (32 to 1,024 mg/liter) and CAS (1 to 16 mg/liter) was antagonistic. All simultaneous antifungal combinations demonstrated indifferent interactions against biofilms of both Candida species. Damage to biofilms of both species increased ( P < 0.01) in the presence of subinhibitory concentrations of echinocandins (0.008 to 0.064 mg/liter), followed by the addition of PSC (512 mg/liter for C. albicans and 64 to 512 mg/liter for C. parapsilosis ) or VRC (256 to 512 mg/liter for C. albicans and 512 mg/liter for C. parapsilosis ). Triazole-echinocandin combinations do not appear to produce antagonistic effects against Candida sp. biofilms, while various significant interactions occur with their planktonic counterparts.

Facial nerve oedema and anatomical predisposition to compression within the fallopian tube seem to be the only generally accepted facts in the pathophysiology of Bell's palsy. Several infectious causes have been suggested as possible triggers of this oedema. Most of the suggested pathogens have been associated with facial nerve lesions during latent infections, reinfections or endogenous reactivations. The aim of this study was to investigate the seroprevalence of three such pathogens Toxoplasma gondii, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in a population of patients with facial nerve palsy. Fifty-six patients with Bell's palsy were included in the study. A group of 25 individuals with similar age and gender distribution was used as control. Seropositivity for T. gondii, EBV viral capsid antigen (VCA) and CMV-specific IgM and IgG antibodies was investigated 2-5 days after the onset of the palsy. Comparisons for both IgM and IgG antibodies against T. gondii attributed significantly higher seroprevalence in the patients' group than in the control group (p = 0.024 and 0.013, respectively). The respective examinations for EBV and CMV attributed no significant results. The roles of EBV and CMV in the pathogenesis of Bell's palsy were not confirmed by this study. However, a significantly higher seroprevalence of IgM- and IgG-specific T. gondii antibodies was detected in patients with Bell's palsy when compared to healthy controls. The possibility that facial nerve palsy might be a late complication of acquired toxoplasmosis may need to be addressed in further studies.

Objectives: To investigate the activities of two newer triazoles and two echinocandins combined with human phagocytes against Candida parapsilosis biofilms. Methods: An in vitro model of C. parapsilosis biofilms was used. Biofilms were grown on silicone elastomer discs in 96-well plates at 37°C for 72 h. Biofilms or planktonic cells were incubated with voriconazole, posaconazole, caspofungin or anidulafungin, at clinically relevant concentrations, and human phagocytes (neutrophils or monocytes) alone or in combination with each of the antifungal agents for a further 22 h. Fungal damage induced by antifungal agents and/or phagocytes was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)2H-tetrazolium-5-carboxanilide] metabolic assay. Results: Each of the antifungal agents alone and in combination with human phagocytes induced less damage against C. parapsilosis biofilms compared with planktonic cells. No antagonistic interactions between antifungal agents and phagocytes were found. Furthermore, anidulafungin, but not caspofungin, and neutrophils exerted additive activity against C. porapsilosis biofilms. Conclusions: Besides a lack of antagonistic interactions between newer antifungal agents and phagocytes, anidulafungin exerts additive immunopharmacological activity against C. parapsilosis biofilms.

The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms.

Objectives: PA is implicated in an estimated 10–20% of all hospital-acquired infections and immunocompromised patients are susceptible to those infections. Cystic fibrosis (CF) airway bacteria are rarely eradicated by antibiotics and host immune defences due to their ability to form biofilms[for full text, please go to the a.m. URL], 18th Symposium on Infections in the Immunocompromised Host

Objectives: Candida albicans (CA) have frequently been associated with the formation of biofilms (BF) on biological and inert surfaces, such as intravascular catheters. Voriconazole (VRC) and anidulafungin (ANID) are the antifungal agents used to treat the resulting infections. BFs resistance to these[for full text, please go to the a.m. URL], 18th Symposium on Infections in the Immunocompromised Host

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.

Biofilm formation is an important component of vascular catheter infections caused by Candida albicans. Little is known about the interactions between human phagocytes, antifungal agents, and Candida biofilms.The interactions between C. albicans biofilms and human phagocytes alone and in combination with anidulafungin or voriconazole were investigated and compared with their corresponding planktonic counterparts by means of an in vitro biofilm model with clinical intravascular and green fluorescent protein (GFP)-expressing strains. Phagocyte-mediated and antifungal agent-mediated damages were determined by 2,3-bis[ 2- methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide assay, and structural effects were visualized by confocal microscopy. Oxidative burst was evaluated by flow cytometric measurement of dihydrorhodamine 123 oxidation, and cytokine release was measured by enzyme-linked immunosorbent assay.Phagocytes alone and in combination with antifungal agents induced less damage against biofilms compared with planktonic cells. However, additive effects occurred between phagocytes and anidulafungin against Candida biofilms. Confocal microscopy demonstrated the absence of phagocytosis within biofilms but marked destruction caused by anidulafungin and phagocytes. Anidulafungin but not voriconazole elicited tumor necrosis factor alpha release from phagocytes compared with that from untreated biofilms.C. albicans within biofilms are more resistant to phagocytic host defenses but are susceptible to additive effects between phagocytes and an echinocandin.

Background: The incidence of candidaemia is steadily increasing in n is considered a virulence factor responsible for catheter-related candidaemia. Objective: To investigate the capacity of bloodstream Candida isolates from NICU neonates to form BF and the degree of BF production. Methods: 5x10 5 planktonic (PL) cells/mL were grown in YNB medium with 2% glucose at 37°C for 24h. For BF formation, 10 6 cells/mL were grown on silicone disks placed at the bottom of 96-well plates in RPMI-1640 under constant shaking at 37°C for 48-72 h. BF production was then evaluated by XTT metabolic assay, safranin staining and light microscopy (LM). Documented BF producers (CA-M61 and CP/PA71) were used as positive controls (metabolic activity by XTT assay: 100%). Isolates that a) showed XTT conversion ≥80% of positive controls, b) stained with safranin and c) produced a microscopically visible dense fungal network were considered high BF producers. XTT conversion of