Hasan Nazik

Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

ABSTRACT Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl3 alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.

Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Visualization of Aspergillus fumigatus biofilms with Scanning Electron Microscopy and Variable Pressure-Scanning Electron Microscopy: A comparison of processing techniques

Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components. Conventional and Variable Pressure Scanning Electron Microscopy were applied to analyze the structure of biofilms attached to plastic and formed at an air-liquid interface.

Effect of acute predation with bacteriophage on intermicrobial aggression by Pseudomonas aeruginosa

In persons with structural lung disease, particularly those with cystic fibrosis (CF), chronic airway infections cause progressive loss of lung function. CF airways can be colonized by a variety of microorganisms; the most frequently encountered bacterial and fungal pathogens are Pseudomonas aeruginosa and Aspergillus fumigatus, respectively. Co-infection with P. aeruginosa and A. fumigatus often results in a more rapid loss of lung function, indicating that interactions between these pathogens affect infection pathogenesis. There has been renewed interest in the use of viruses (bacteriophage, mycoviruses) as alternatives to antibiotics to treat these infections. In previous work, we found that filamentous Pf bacteriophage produced by P. aeruginosa directly inhibited the metabolic activity of A. fumigatus by binding to and sequestering iron. In the current study, we further examined how filamentous Pf bacteriophage affected interactions between P. aeruginosa and A. fumigatus. Here, we report that the antifungal properties of supernatants collected from P. aeruginosa cultures infected with Pf bacteriophage were substantially less inhibitory towards A. fumigatus biofilms. In particular, we found that acute infection of P. aeruginosa by Pf bacteriophage inhibited the production of the virulence factor pyoverdine. Our results raise the possibility that the reduced production of antimicrobials by P. aeruginosa infected by Pf bacteriophage may promote conditions in CF airways that allow co-infection with A. fumigatus to occur, exacerbating disease severity. Our results also highlight the importance of considering how the use of bacteriophage as therapeutic agents could affect the behavior and composition of polymicrobial communities colonizing sites of chronic infection.

Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas

Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.

Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation

Increased ferric iron in allografts triggers a switch in the growth of the mold Aspergillus fumigatus from a colonizing to an invasive phenotype. Irons in the fire Although transplantation is a lifesaving therapy, patients receiving new organs are at serious risk for invasive, potentially fatal infections. Aspergillus fumigatus is a particularly common and troublesome fungal pathogen, but its ability to invade transplant tissues is poorly understood. To evaluate this property, Hsu and colleagues infected transplants in mice. Bleeding, caused by damage to small vessels in grafted airways, led to increased tissue iron, a known growth factor for Aspergillus. Increased tissue iron is a newly identified risk factor for transplant damage by microorganisms. Therapies in development that block iron and protect blood vessels may extend the life of organ recipients. Invasive pulmonary disease due to the mold Aspergillus fumigatus can be life-threatening in lung transplant recipients, but the risk factors remain poorly understood. To study this process, we used a tracheal allograft mouse model that recapitulates large airway changes observed in patients undergoing lung transplantation. We report that microhemorrhage-related iron content may be a major determinant of A. fumigatus invasion and, consequently, its virulence. Invasive growth was increased during progressive alloimmune-mediated graft rejection associated with high concentrations of ferric iron in the graft. The role of iron in A. fumigatus invasive growth was further confirmed by showing that this invasive phenotype was increased in tracheal transplants from donor mice lacking the hemochromatosis gene (Hfe−/−). The invasive phenotype was also increased in mouse syngrafts treated with topical iron solution and in allograft recipients receiving deferoxamine, a chelator that increases iron bioavailability to the mold. The invasive growth of the iron-intolerant A. fumigatus double-knockout mutant (ΔsreA/ΔcccA) was lower than that of the wild-type mold. Alloimmune-mediated microvascular damage and iron overload did not appear to impair the host’s immune response. In human lung transplant recipients, positive staining for iron in lung transplant tissue was more commonly seen in endobronchial biopsy sections from transplanted airways than in biopsies from the patients’ own airways. Collectively, these data identify iron as a major determinant of A. fumigatus invasive growth and a potential target to treat or prevent A. fumigatus infections in lung transplant patients.

Pseudomonas phage inhibition of Candida albicans

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Verapamil Inhibits Aspergillus Biofilm, but Antagonizes Voriconazole

The paucity of effective antifungals against Aspergillus and increasing resistance, the recognition of the importance of Aspergillus biofilm in several clinical settings, and reports of verapamil—a calcium channel blocker—efficacy against Candida biofilm and hyphal growth, and synergy with an azole antifungal in vitro, led to a study of verapamil ± voriconazole against Aspergillus. Broth macrodilution methodology was utilized for MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) determination. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to verapamil, verapamil plus voriconazole, or voriconazole alone. For biofilm formation, we found less inhibition from the combinations than with either drug alone, or less inhibition from the combination than that of the more potent drug alone. For preformed biofilm, we found no significant change in activity comparing voriconazole alone compared to added verapamil, and no significant alteration of activity of the more potent voriconazole, at any concentration in the range tested, by addition of a concentration of verapamil that is inhibitory alone. In full checkerboard assays with planktonic fungus, there was no indication of any effect of one drug on the other (indifference). Although verapamil was similarly inactive against planktonic Aspergillus, as with Candida, verapamil was indeed active against Aspergillus biofilm. However, indifference and antagonism was found with voriconazole.