Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus

Abstract

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens that are associated with deterioration of lung function, e.g., in persons with cystic fibrosis (CF). There is evidence that co-infections with these pathogens cause airway inflammation and aggravate pathology in CF lungs. Intermicrobial competition of P. aeruginosa and A. fumigatus has been described, but it is unknown how anti-fungal therapy is affected. The anti-fungal azole voriconazole (VCZ), supernatants of P. aeruginosa laboratory isolates PA14 or PAO1, or clinical isolate Pa10 independently inhibited biofilm metabolism of A. fumigatus isolates 10AF and AF13073. When VCZ and supernatants were combined at their IC50s, synergistic effects on A. fumigatus were found. Synergistic effects were no longer observed when P. aeruginosa supernatants were prepared in the presence of iron, or when P. aeruginosa mutants were lacking the ability to produce pyoverdine and pyochelin. Combination of pure P. aeruginosa products pyoverdine, pyochelin, and pyocyanin with VCZ showed synergistic anti-fungal effects. Combining VCZ with P. aeruginosa supernatants also improved its MIC and MFC against planktonic A. fumigatus. In summary, in the case of P. aeruginosa–A. fumigatus co-infections, it appeared that the P. aeruginosa co-infection facilitated therapy of the Aspergillus; lower concentrations of VCZ might be sufficient to control fungal growth.

Synergy Between Pseudomonas aeruginosa Filtrates And Voriconazole Against Aspergillus fumigatus Biofilm Is Less for Mucoid Isolates From Persons With Cystic Fibrosis

“…In a previous study we determined the IC50 for VCZ against 10AF forming biofilm metabolism within a 2-fold dilution step of 125 nM ( Sass et al., 2021 ). We now combined filtrates of each clinical isolate at the dilution closest to its individual IC50 ( Table 2 ) with VCZ at its IC50.…”

“…To determine how potent interactions of isolates of each group (CF mucoid, CF non-mucoid, non-CF) were with VCZ, we further combined each isolate at the same high filtrate dilution (1:256), with VCZ at its IC50. This also enabled comparisons of the data from studying clinical isolates to those previously published with reference laboratory isolates ( Sass et al., 2021 ), as the latter study investigated filtrates at a 1:256 dilution. Figure 5 shows that in each group ( Figure 5A : mucoid CF, Figure 5B : non-mucoid CF, Figure 5C : non-CF) all isolates acted synergistically with VCZ when used at a 1:256 dilution.…”

“…As an extreme example of poor VCZ anti-fungal activity we used a clinical Aspergillus isolate resistant to VCZ concentrations. We studied concentrations that produced an IC50 in 10AF ( Figure 10A ), and in another wildtype laboratory reference strain, AF13073 ( Sass et al., 2021 ). The VCZ-resistant strain AF21-23 showed an IC50 of 4–8 µM, which is about 50× higher than the IC50 of a susceptible strain ( Figure 10A ).…”

“…Pseudomonas competes with Aspergillus for crucial resources, e.g., iron, via a variety of their products, such as phenazines, its major siderophore pyoverdine ( Sass et al., 2017 ), pyocyanin (5-N-methyl-1-hydroxyphenazine) ( Kerr et al., 1999 ; Briard et al., 2015 ; Sass et al., 2020 ), 1-hydroxyphenazine ( Kerr et al., 1999 ; Briard et al., 2015 ), phenazine-1-carboxamide ( Briard et al., 2015 ), phenazine-1-carboxylic acid ( Briard et al., 2015 ), and rhamnolipids ( Briard et al., 2017 ; Sass et al., 2021 ). Using laboratory reference isolates (PA14, PAO1) we recently showed that the Pseudomonas products pyoverdine, pyocyanin, and pyochelin interacted synergistically with the anti-fungal drug VCZ against A. fumigatus forming biofilm in vitro ( Sass et al., 2021 ). As correctly emphasized in a recent excellent review, “Pathogens are known to adapt to the host environment during chronic infections; therefore, testing reference strains alongside clinical isolates is extremely important in polymicrobial communication studies” ( Reece et al., 2021 ).…”

Metrics of Antifungal Effects of Ciprofloxacin on Aspergillus fumigatus Planktonic Growth and Biofilm Metabolism; Effects of Iron and Siderophores

“…Reagent interaction studies: Two-dimensional reagent interaction (“checkerboard”) studies were performed, as previously detailed [ 24 ], and the concentrations studied are delineated in the Results Section 3 .…”