Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus
Abstract
:1. Introduction
2. Results
2.1. Determination of IC50s for P. aeruginosa Strain PA14 Supernatants or VCZ against A. fumigatus 10AF Biofilm Formation
2.2. PA14 Supernatant Supported VCZ Anti-Fungal Activity against 10AF
2.3. Synergistic Interaction of VCZ with P. aeruginosa Supernatants Was Independent of the P. aeruginosa or A. fumigatus Strain Used
2.4. Iron Interfered with P. aeruginosa/VCZ Synergy
2.5. Pyoverdine Contributed to P. aeruginosa/VCZ Synergy
2.6. Pyochelin Contributed to P. aeruginosa/VCZ Synergy
2.7. Pyocyanin Contributed to P. aeruginosa/VCZ Synergy
2.8. PA14 Supernatant Supported Anti-Fungal Activity of VCZ against Planktonic A. fumigatus Growth
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Strains and Isolates
4.3. P. aeruginosa Planktonic Supernatant Production and Dilution
4.4. Assay for Measurement of Aspergillus Forming Biofilm Metabolism
4.5. Minimal Inhibitory Concentration (MIC), Minimal Fungicidal Concentration (MFC), Fractional Inhibitory, and Fungicidal Concentration Indexes (FICi and FFCi)
4.6. BLISS Independence Model for Analysis of Drug Combination Effects
- -
- Observed > Predicted: Synergy
- -
- Observed = Predicted: Independent (5% range of Ypab)
- -
- Observed < Predicted: Antagonism
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Riordan, J.R.; Rommens, J.M.; Kerem, B.; Alon, N.; Rozmahel, R.; Grzelczak, Z.; Zielenski, J.; Lok, S.; Plavsic, N.; Chou, J.L.; et al. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 1989, 245, 1066–1073. [Google Scholar] [CrossRef]
- Collins, F.; Jurivich, D.; Sistonen, L.; Kroes, R.; Morimoto, R. Cystic fibrosis: Molecular biology and therapeutic implications. Science 1992, 256, 774–779. [Google Scholar] [CrossRef]
- Rowe, S.M.; Miller, S.; Sorscher, E.J. Cystic fibrosis. N. Engl. J. Med. 2005, 352, 1992–2001. [Google Scholar] [CrossRef]
- King, J.; Brunel, S.F.; Warris, A. Aspergillus infections in cystic fibrosis. J. Infect. 2016, 72, S50–S55. [Google Scholar] [CrossRef]
- O’Brien, S.; Fothergill, J.L. The role of multispecies social interactions in shaping Pseudomonas aeruginosa pathogenicity in the cystic fibrosis lung. FEMS Microbiol. Lett. 2017, 364, fnx128. [Google Scholar] [CrossRef]
- Gentzsch, M.; Mall, M.A. Ion Channel Modulators in Cystic Fibrosis. Chest 2018, 154, 383–393. [Google Scholar] [CrossRef]
- Baxter, C.G.; Dunn, G.; Jones, A.M.; Webb, K.; Gore, R.; Richardson, M.D.; Denning, D.W. Novel immunologic classification of aspergillosis in adult cystic fibrosis. J. Allergy Clin. Immunol. 2013, 132, 560–566. [Google Scholar] [CrossRef] [PubMed]
- Moss, R.B. Fungi in Cystic Fibrosis and Non–Cystic Fibrosis Bronchiectasis. Semin. Respir. Crit. Care Med. 2015, 36, 207–216. [Google Scholar] [CrossRef]
- Agarwal, R.; Chakrabarti, A.; Shah, A.; Gupta, D.; Meis, J.F.; Guleria, R.; Moss, R.; Denning, D.W.; ABPA complicating asthma ISHAM working group. Allergic bronchopulmonary aspergillosis: Review of literature and proposal of new diagnostic and classification criteria. Clin. Exp. Allergy 2013, 43, 850–873. [Google Scholar] [CrossRef]
- Chishimba, L.; Niven, R.M.; Cooley, J.; Denning, D.W. Voriconazole and Posaconazole Improve Asthma Severity in Allergic Bronchopulmonary Aspergillosis and Severe Asthma with Fungal Sensitization. J. Asthma 2012, 49, 423–433. [Google Scholar] [CrossRef]
- Hogan, C.; Denning, D.W. Allergic Bronchopulmonary Aspergillosis and Related Allergic Syndromes. Semin. Respir. Crit. Care Med. 2011, 32, 682–692. [Google Scholar] [CrossRef]
- Perisson, C.; Destruys, L.; Grenet, D.; Bassinet, L.; Derelle, J.; Sermet-Gaudelus, I.; Thumerelle, C.; Prevotat, A.; Rosner, V.; Clement, A.; et al. Omalizumab treatment for allergic bronchopulmonary aspergillosis in young patients with cystic fibrosis. Respir. Med. 2017, 133, 12–15. [Google Scholar] [CrossRef] [Green Version]
- Amin, R.; Dupuis, A.; Aaron, S.D.; Ratjen, F. The Effect of Chronic Infection with Aspergillus fumigatus on Lung Function and Hospitalization in Patients with Cystic Fibrosis. Chest 2010, 137, 171–176. [Google Scholar] [CrossRef] [PubMed]
- Reece, E.; Segurado, R.; Jackson, A.; McClean, S.; Renwick, J.; Greally, P. Co-colonisation with Aspergillus fumigatus and Pseudomonas aeruginosa is associated with poorer health in cystic fibrosis patients: An Irish registry analysis. BMC Pulm. Med. 2017, 17, 70. [Google Scholar] [CrossRef] [Green Version]
- Briard, B.; Bomme, P.; Lechner, B.E.; Mislin, G.L.A.; Lair, V.; Prévost, M.-C.; Latgé, J.-P.; Haas, H.; Beauvais, A. Pseudomonas aeruginosa manipulates redox and iron homeostasis of its microbiota partner Aspergillus fumigatus via phenazines. Sci. Rep. 2015, 5, 8220. [Google Scholar] [CrossRef]
- Briard, B.; Rasoldier, V.; Bomme, P.; Elaouad, N.; Guerreiro, C.; Chassagne, P.; Muszkieta, L.; Latgé, J.-P.; Mulard, L.; Beauvais, A. Dirhamnolipids secreted from Pseudomonas aeruginosa modify antifungal susceptibility of Aspergillus fumigatus by inhibiting β1,3 glucan synthase activity. ISME J. 2017, 11, 1578–1591. [Google Scholar] [CrossRef]
- Nazik, H.; Choudhary, V.; Stevens, D.A. Verapamil Inhibits Aspergillus Biofilm, but Antagonizes Voriconazole. J. Fungi (Basel) 2017, 3, 50. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Elliot, R.P. Some properties of pyoverdine, the water-soluble fluorescent pigment of the pseudomonads. Appl. Microbiol. 1958, 6, 241–246. [Google Scholar] [CrossRef]
- Chatterjee, P.; Sass, G.; Swietnicki, W.; Stevens, D.A. Review of Potential Pseudomonas Weaponry, Relevant to the Pseudomonas–Aspergillus Interplay, for the Mycology Community. J. Fungi (Basel) 2020, 6, 81. [Google Scholar] [CrossRef]
- Stites, S.W.; Walters, B.; O’Brien-Ladner, A.R.; Bailey, K.; Wesselius, L.J. Increased Iron and Ferritin Content of Sputum from Patients with Cystic Fibrosis or Chronic Bronchitis. Chest 1998, 114, 814–819. [Google Scholar] [CrossRef]
- Wang, J.; Lory, S.; Ramphal, R.; Jin, S. Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients. Mol. Microbiol. 1996, 22, 1005–1012. [Google Scholar] [CrossRef] [PubMed]
- Nazik, H.; Sass, G.; Ansari, S.R.; Ertekin, R.; Haas, H.; Déziel, E.; Stevens, D.A. Novel intermicrobial molecular interaction: Pseudomonas aeruginosa Quinolone Signal (PQS) modulates Aspergillus fumigatus response to iron. Microbiology 2020, 166, 44–55. [Google Scholar] [CrossRef]
- Sass, G.; Ansari, S.R.; Dietl, A.-M.; Déziel, E.; Haas, H.; Stevens, D.A. Intermicrobial interaction: Aspergillus fumigatus siderophores protect against competition by Pseudomonas aeruginosa. PLoS ONE 2019, 14, e0216085. [Google Scholar] [CrossRef] [Green Version]
- Bargon, J.; Dauletbaev, N.; Köhler, B.; Wolf, M.; Posselt, H.-G.; Wagner, T.O.F. Prophylactic antibiotic therapy is associated with an increased prevalence of Aspergillus colonization in adult cystic fibrosis patients. Respir. Med. 1999, 93, 835–838. [Google Scholar] [CrossRef] [Green Version]
- Denning, D.W.; Park, S.; Lass-Florl, C.; Fraczek, M.G.; Kirwan, M.; Gore, R.; Smith, J.; Bueid, A.; Moore, C.B.; Bowyer, P.; et al. High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease. Clin. Infect. Dis. 2011, 52, 1123–1129. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Morio, F.; Aubin, G.G.; Danner-Boucher, I.; Haloun, A.; Sacchetto, E.; Garcia-Hermoso, D.; Bretagne, S.; Miegeville, M.; Le Pape, P. High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis. J. Antimicrob. Chemother. 2012, 67, 1870–1873. [Google Scholar] [CrossRef] [Green Version]
- Mortensen, K.L.; Jensen, R.H.; Johansen, H.K.; Skov, M.; Pressler, T.; Howard, S.J.; Leatherbarrow, H.; Mellado, E.; Arendrup, M.C. Aspergillus Species and Other Molds in Respiratory Samples from Patients with Cystic Fibrosis: A Laboratory-Based Study with Focus on Aspergillus fumigatus Azole Resistance. J. Clin. Microbiol. 2011, 49, 2243–2251. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Burgel, P.-R.; Baixench, M.-T.; Amsellem, M.; Audureau, E.; Chapron, J.; Kanaan, R.; Honoré, I.; Dupouy-Camet, J.; Dusser, D.; Klaassen, C.H.; et al. High Prevalence of Azole-Resistant Aspergillus fumigatus in Adults with Cystic Fibrosis Exposed to Itraconazole. Antimicrob. Agents Chemother. 2012, 56, 869–874. [Google Scholar] [CrossRef] [Green Version]
- Stevens, D.A.; Moss, R.B.; Hernandez, C.; Clemons, K.V.; Martinez, M. Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center. Antimicrob. Agents Chemother. 2016, 60, 2180–2184. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Denning, D.W.; Clemons, K.V.; Hanson, L.H.; Stevens, D.A.; Morrison, D.C.; Silverstein, R.; Bright, S.W.; Chen, T.-Y.; Flebbe, L.M.; Lei, M.-G. Restriction Endonuclease Analysis of Total Cellular DNA of Aspergillus fumigatus Isolates of Geographically and Epidemiologically Diverse Origin. J. Infect. Dis. 1990, 162, 1151–1158. [Google Scholar] [CrossRef] [PubMed]
- Denning, D.W.; Stevens, D.A. Efficacy of cilofungin alone and in combination with amphotericin B in a murine model of disseminated aspergillosis. Antimicrob. Agents Chemother. 1991, 35, 1329–1333. [Google Scholar] [CrossRef] [Green Version]
- O’Toole, G.A.; Kolter, R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 1998, 30, 295–304. [Google Scholar] [CrossRef] [PubMed]
- Lee, D.G.; Urbach, J.M.; Wu, G.; Liberati, N.T.; Feinbaum, R.L.; Miyata, S.; Diggins, L.T.; He, J.; Saucier, M.; Déziel, E.; et al. Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol. 2006, 7, R90. [Google Scholar] [CrossRef] [Green Version]
- Fischer, S.; Klockgether, J.; Losada, P.M.; Chouvarine, P.; Cramer, N.; Davenport, C.F.; Dethlefsen, S.; Dorda, M.; Goesmann, A.; Hilker, R.; et al. Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14. Environ. Microbiol. Rep. 2016, 8, 227–234. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Stover, C.K.; Pham, X.Q.; Erwin, A.L.; Mizoguchi, S.D.; Warrener, P.; Hickey, M.J.; Brinkman, F.S.L.; Hufnagle, W.O.; Kowalik, D.J.; Lagrou, M.; et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000, 406, 959–964. [Google Scholar] [CrossRef] [PubMed]
- Ferreira, J.A.G.; Penner, J.C.; Moss, R.B.; Haagensen, J.A.J.; Clemons, K.V.; Spormann, A.M.; Nazik, H.; Cohen, K.; Banaei, N.; Carolino, E.; et al. Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium. PLoS ONE 2015, 10, e0134692. [Google Scholar] [CrossRef] [Green Version]
- Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.; Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988, 48, 4827–4833. [Google Scholar] [PubMed]
- Eliopoulos, G.M.; Eliopoulos, C.T. Antibiotic combinations: Should they be tested? Clin. Microbiol. Rev. 1988, 1, 139–156. [Google Scholar] [CrossRef]
- Zhao, W.; Sachsenmeier, K.; Zhang, L.; Sult, E.; Hollingsworth, R.E.; Yang, H. A New Bliss Independence Model to Analyze Drug Combination Data. J. Biomol. Screen. 2014, 19, 817–821. [Google Scholar] [CrossRef] [PubMed] [Green Version]
VCZ 0.250 | VCZ 0.125 | VCZ 0.063 | |
---|---|---|---|
Pa sup 1:256 | I | S | I |
Pa sup 1:512 | S | S | S |
Pa sup 1:1024 | S | S | S |
A | PA14 Sup (10AF) | PA14 Sup (AF13073) | PAO1 Sup (10AF) |
---|---|---|---|
Pa sup 1:256 | S | I | I |
Pa sup 1:512 | S | S | S |
Pa sup 1:1024 | S | S | S |
VCZ 0.125 | VCZ 0.125 | VCZ 0.125 | |
B | Pa10 Sup (10AF) | ||
Pa sup 1:256 | I | ||
Pa sup 1:512 | I | ||
Pa sup 1:1024 | S | ||
VCZ 0.125 |
PA14 Sup (In RPMI with Iron) | PA14ΔpvdD/ΔpchE | |
---|---|---|
Pa sup 1:256 | A | I |
Pa sup 1:512 | A | A |
Pa sup 1:1024 | A | I |
VCZ 0.125 | VCZ 0.125 |
MIC | MFC | FICi | FFCi | |
---|---|---|---|---|
PA14 supernatant | 1:32 | No MFC | - | - |
VCZ | 0.5 µM | 16 µM | - | - |
Combination | 1:32 + 0.125 µM | 1:256 + 8 µM | 0.28 | 0.5 |
Organism | Isolate | Description | ATCC | References |
---|---|---|---|---|
Aspergillus fumigatus | 10AF | Virulent patient isolate | 90,240 | [30,31] |
Aspergillus fumigatus | AF13073 | 13,073 | ||
Pseudomonas aeruginosa | PA14 | Parental strain of all PA14 mutants studied | [32,33,34] | |
Pseudomonas aeruginosa | PAO1 | 15,692 | [35] | |
Pseudomonas aeruginosa | Pa10 | Reference non-mucoid CF isolate | [36] | |
Pseudomonas aeruginosa | PA14ΔpvdD/ΔpchE | Pyoverdine-pyochelin double siderophore mutant |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Sass, G.; Shrestha, P.; Stevens, D.A. Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus. Pathogens 2021, 10, 519. https://doi.org/10.3390/pathogens10050519
Sass G, Shrestha P, Stevens DA. Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus. Pathogens. 2021; 10(5):519. https://doi.org/10.3390/pathogens10050519
Chicago/Turabian StyleSass, Gabriele, Pallabi Shrestha, and David A. Stevens. 2021. "Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus" Pathogens 10, no. 5: 519. https://doi.org/10.3390/pathogens10050519
APA StyleSass, G., Shrestha, P., & Stevens, D. A. (2021). Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus. Pathogens, 10(5), 519. https://doi.org/10.3390/pathogens10050519