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

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

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

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 β-lactamase Inhibitor Boronic Acid SM23 as a new anti-Pseudomonas aeruginosa Biofilm Compound

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation can enhance bacterial resistance to antimicrobial agents due to a decreased penetration of the antibiotic and a reduced rate of bacterial cells in biofilm. Selective inhibition of biofilm formation may thus represent a novel promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa. METHODS: In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and production of virulence factors, using the P. aeruginosa bioluminescent strain P1242. RESULTS: Our results indicated that SM23: a) inhibited the development of biofilm and the production of the virulence factors pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of P. aeruginosa QS-related Autoinducers molecules 3-oxo-C12-HSL and C4-HSL by dampened lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model, closely mimicking the clinical settings. CONCLUSION: Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as potent inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa 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 severenosocomial and community-acquired infections. Although enterococcal meningitis is rare,mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of thisinfection remain poorly understood, even though the ability of E. faecalis to avoid or survivephagocytic attack in vivo may be very important during the infection process. We previouslyshowed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis wasimplicated in oxidative stress responses and, interestingly, in the survival within mouse peritonealmacrophages using an in vivo\u2013in vitro infection model. In the present study, we investigated therole of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. Byusing an in vitro infection model, murine microglial cells were challenged in parallel with the wildtypestrain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosedby microglia efficiently and to a similar extent, the DsodA mutant was found to be significantlymore susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protectionassay. In addition, a significantly higher percentage of acidic DsodA-containing phagosomes wasfound and these also underwent enhanced maturation as determined by the expression ofendolysosomal markers. In conclusion, these results show that the MnSOD of E. faecaliscontributes 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. faecalispathogenesis

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 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

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