Overcome the Resistance of Pseudomonas aeruginosa
O1 Preclinical study of a host-directed therapy based on Metformin and bioactive liposomes for the control of multidrug resistant P. aeruginosa infection
Noemi Poerio1, Federica De Santis1, Alice Rossi2, Ana Henriquez1, Federica Di Sano1, Vincenzina Lucidi3, Alessandra Bragonzi2, Maurizio Fraziano1
1Dipartimento di Biologia, Università di Roma ‘’ Tor Vergata ‘’, Roma, Italy; 2Unità di Infezioni e Fibrosi Cistica, Istituto Scientifico San Raffaele, Milano, Italy; 3Unità Operativa Complessa Fibrosi Cistica, Dipartimento di Medicina Pediatrica, Ospedale Pediatrico Bambino Gesù, Roma, Italy.
Correspondence: Maurizio Fraziano (fraziano@bio.uniroma2.it)
Background
We have recently showed that apoptotic body like liposomes (ABL) loaded with phosphatidylinositol 5-phosphate (PI5P) significantly enhance bactericidal response in macrophages from Cystic Fibrosis (CF) patients against P. aeruginosa and in bronchoalveolar lavage cells from patients with pneumonia caused by different multidrug resistance (MDR) bacterial pathogens. Moreover, Metformin (Met) has been recently reported to augment airway surface hydration, in in vitro models of CF, and to enhance antimicrobial innate immune response and to reduce inflammation in in vivo models of tuberculosis.
Hypothesis and objectives
The main goal of this project was the development of a novel immunotherapeutic approach based on bioactive liposome in combination with Met aimed to enhance antimicrobial innate immune response, while simultaneously improve airway surface hydration and mitigate inflammation to control multidrug resistant infections in CF.
Methods
Macrophages generated by peripheral monocytes derived from healthy donors, treated or not with a pharmacological inhibitor of CFTR (INH172), and from CF patients were infected with a panel of MDR P. aeruginosa clinical isolates and stimulated with ABL/PI5P, alone or in combinations with Met. We have evaluated intracellular bacterial clearance and uptake, and phagosome maturation by CFU and fluorimetric assays, respectively. Finally, treatments were tested in in vivo murine model of MDR-RP73 P. aeruginosa acute infection, in terms of leukocytes recruitment and bactericidal action enhancement.
Results
We showed that treatment with ABL/PI5P and/or Met rescues impaired phagosome acidification in CFTR-pharmacologically inhibited macrophages and promotes intracellular bacterial killing in INH172- and CF primary macrophages infected with MDR P. aeruginosa 2113 strain, although only ABL/PI5P stimulation increase bacterial uptake. Finally, preliminary results in in vivo model of MDR-RP73 P. aeruginosa acute infection, show that treatment with ABL/PI5P or Met induces an overall decrease in leukocytes recruitment, associated to an increase of macrophage component and to a reduction of pulmonary bacterial load, although following ABL/PI5P treatment only.
Conclusions
Our results show that bioactive liposome and metformin-based strategy could represent a promising host-directed therapeutic option for the control of drug resistant bacterial infections and for the reduction of the inflammation-based pathology in CF.
Acknowledgment
This study was supported by Italian Cystic Fibrosis Research Foundation (FFC), Research project Number FFC#14/2017
O2 Exploiting the potential of gallium for the treatment of Pseudomonas aeruginosa pulmonary infection
Paolo Visca1, Francesco Peri2, Raffaella Sorrentino3
1Dipartimento di Scienze, Lab. Microbiologia Clinica e Virologia, Università Roma Tre, Roma, Italy; 2Dip. Di Biotecnologie e Bioscienze, Università Milano Bicocca, Milano, Italy; 3Dip. Di Farmacia, Università di Napoli Federico II , Napoli, Italy
Correspondence: Paolo Visca (paolo.visca@uniroma3.it)
Background and rationale
Morbidity and mortality in cystic fibrosis (CF) patients is attributable to infectious sequelae caused by different pathogens[1]. Antibiotic resistance in CF calls for the development of new antimicrobials. Ga(III) inhibits bacterial growth, acting as an iron mimetic, and is already used in medicine (Ganite®) for treatment of non-infectious disorders[2]. Bacteria erroneously incorporates Ga(III) instead of Fe(III) within essential molecules because unable to discriminate between these two ions, resulting inhibited[3].
Hypothesis and objectives
The main objective of this project was a comparative assessment of the antibacterial activity of different Ga(III) formulations on major CF pathogens, and the development of safe Ga(III)-based drugs that can specifically directed in the lung of CF patients, via inhalable formulations.
Essential methods
We capitalized upon expertise in organic synthesis, pharmaceutical chemistry and microbiology to: i) compare the antibacterial activity of different Ga(III) formulations on major CF pathogens; ii) generate new formulations for in vivo administration and determine their pharmacological characteristics; iii) investigate acute toxicity and the organ distribution of Ga(III), upon intra-tracheal and intravenous administration in rats.
Results
New Ga(III) testing methods have been developed for major CF pathogens. Two compounds showed potent broad-spectrum antibacterial properties. To overcome limitations of systemic administration, a novel inhalable Ga(III)-based dry powder has been developed. The new formulation showed high Ga(III) content and stability in the long-term, good antimicrobial proprieties and an excellent biodistribution in rats after intra-tracheal aerosol administration.
Spin-off for research & clinical purposes
In the worrying scenario of increasing antibiotic resistance in CF-associated bacterial pathogens, Ga(III)-derived compounds are good candidates for broad-spectrum antimicrobials, and hold great promise for the progression into drugs with potential clinical applicability in the short-medium perspective.
Acknowledgment
This study was supported by Italian Cystic Fibrosis Research Foundation (FFC), Research project Number FFC#18/2017
References
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1.
Folkesson A, Jelsbak L, Yang L, Johansen HK, Ciofu O, Høiby N, Molin S. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol. 2012; 10: 841-51.
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2.
Bonchi C., Imperi F., Minandri F., Visca, P., Frangipani E. Repurposing of gallium-based drugs for antibacterial therapy. Biofactors. 2014; 40; 303-312.
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3.
Kaneko Y., Thoendel M., Olakanmi O., Britigan B.E., Singh P.K. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J. Clin. Invest. 2007; 117; 877-888.
O3 Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model
Marco Cafora1, Gianluca Deflorian2, Francesca Forti3, Laura Ferrari2, Giorgio Binelli4, Federica Briani3, Daniela Ghisotti3, Anna S Pistocchi1
1Dipartimento di Biotecnologie Mediche e Medicina Traslazionale – Università degli Studi di Milano – LITA – via Fratelli Cervi 93 – 20090 Segrate (MI) – Italy; 2Istituto FIRC di Oncologia Molecolare – IFOM – Via Adamello 16 – 20139 Milano – Italy; 3Dipartimento di Bioscienze - Università degli Studi di Milano - Via Celoria 26 - 20133 Milano – Italy; 4Dipartimento di Biotecnologie e Scienze della Vita - Università degli Studi dell’Insubria - Via J.H. Dunant 3 – Varese- Italy.
Correspondence: Anna S Pistocchi (anna.pistocchi@unimi.it)
Background and Rationale
We have recently isolated virulent phages capable of infecting P. aeruginosa and used them to treat P. aeruginosa infections in mouse and Galleria mellonella larvae. The positive outcome obtained by phage therapy encouraged us to further investigate its use in a cystic fibrosis (CF) background. Indeed, P. aeruginosa infections are particularly serious in CF patients and, as a consequence, CF patients are subject to frequent antibiotic treatments to control the infections. The appearance and diffusion of multidrug resistant (MDR) isolates of P. aeruginosa is responsible for the increasingly unsuccessful use of antibiotics and alternative therapies are urgently needed. Phages, the natural enemies of bacteria, can be a possible solution as they infect only very specific bacterial hosts, they self-control their dose multiplying only when and where the target bacterial host strains are present, and are able to kill also MDR bacteria.
Hypothesis and Objectives
The scientific question addressed by this work is the validation of phage therapy against P. aeruginosa infection in a cystic fibrosis background (CF). We chose zebrafish (Danio rerio) as in vivo model. The zebrafish model has two main advantages: it lacks an adaptive immune response for the first 4-6 weeks of life representing an ideal model for studying innate immunity and it is a good model for CF as the CFTR channel is conserved between fish and mammals.
Essential Methods
We deregulated the CFTR function in zebrafish, obtaining CF embryos. We infected control (WT) and CF embryos with P. aeruginosa and we compared lethality, bacterial burden and inflammatory cytokines after infection followed by phage administration.
Results
We demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and immune response in WT and in CF embryos. We also show an improvement by combining the action of phages and antibiotics against P. aeruginosa infection in CF zebrafish embryos. In addition, we found that phage administration, in the absence of bacterial infection, relieves the constitutive inflammatory state of CF embryos.
Conclusions
Our data suggest promising therapeutic approaches to reduce antibiotic doses and time of administration, avoiding the development of MDR in a CF background. To our knowledge this is the first time that phage therapy is used to cure P. aeruginosa infections in an in vivo CF model.
Acknowledgment
This study was supported by Italian Cystic Fibrosis Research Foundation (FFC), Research project Number FFC#22/2017