Studying Bacteriophage Efficacy Using a Zebrafish Model.

The rise of bacteria resistant to the antibiotics currently in use (multiple drug-resistant, MDR) is a serious problem for patients affected by infections. This situation is even more worrying in the case of chronic bacterial infections, such as those caused by Pseudomonas aeruginosa (Pa), in patients with cystic fibrosis (CF). As an alternative to antibiotic treatments, the use of bacteriophages (phages) to fight bacterial infections has gained increasing interest in the last few years. Phages are viruses that specifically infect and multiply within the bacteria without infecting eukaryotic cells. It is well assumed that phage therapy has a high bacterial specificity, which, unlike antibiotics, should limit the damage to the endogenous microbiome. In addition, phages can kill antibiotic-resistant bacteria and perform self-amplification at the site of the infection.The protocol detailed in this chapter describes how the antimicrobial effect of phages can be studied in vivo in the zebrafish (Danio rerio) model infected with Pa. The same procedure can be applied to test the effectiveness of several different phages killing other bacterial species and for the rapid preclinical testing of phages to be used as personalized medicine.

Identification and impact on Pseudomonas aeruginosa virulence of mutations conferring resistance to a phage cocktail for phage therapy.

IMPORTANCE: In this work, we identified the putative receptors of 16 Pseudomonas phages and evaluated how resistance to phages recognizing different bacterial receptors may affect the virulence. Our findings are relevant for the implementation of phage therapy of Pseudomonas aeruginosa infections, which are difficult to treat with antibiotics. Overall, our results highlight the need to modify natural phages to enlarge the repertoire of receptors exploited by therapeutic phages and suggest that phages using the PAO1-type T4P as receptor may have limited value for the therapy of the cystic fibrosis infection.

Restoring airway epithelial homeostasis in Cystic Fibrosis.

Cystic fibrosis (CF), the most common life-threatening genetic disorder in Caucasians, is caused by recessive mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene encoding a chloride ion channel. Aberrant function of CFTR involves mucus- and sweat-producing epithelia affecting multiple organs, including airways and lungs. This condition facilitates the colonization of fungi, bacteria, or viruses. Recurrent antibiotic administration is commonly used to treat pathogen infections leading to the insurgence of resistant bacteria and to a chronic inflammatory state that jeopardizes airway epithelium repair. The phenotype of patients carrying CFTR mutations does not always present a strict correlation with their genotype, suggesting that the disease may occur because of multiple additive effects. Among them, the frequent microbiota dysbiosis observed in patients affected by CF, might be one cause of the discrepancy observed in their genotype-phenotype correlation. Interestingly, the abnormal polarity of the CF airway epithelium has been observed also under non-infectious and non-inflammatory conditions, suggesting that CFTR dysfunction "per se" perturbs epithelial homeostasis. New pathogen- or host-directed strategies are thus needed to counteract bacterial infections and restore epithelial homeostasis in individuals with CF. In this review, we summarized alternative cutting-edge approaches to high-efficiency modulator therapy that might be promising for these patients.

Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models.

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr-loss-of-function (cftr-LOF) zebrafish embryos infected with Pseudomonas aeruginosa (Pa) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 "eater", improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del-CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF.

Phages as immunomodulators and their promising use as anti-inflammatory agents in a cftr loss-of-function zebrafish model.

Cystic Fibrosis (CF), one of the most frequent hereditary diseases due to mutations in the CFTR gene, causes mortality in humans mainly due to infection in the respiratory system. However, besides the massive inflammatory response triggered by chronic bacterial infections, a constitutive pro-inflammatory state associated with the most common CFTR mutations has been reported in paediatric cases before the onset of bacterial colonization. In previous works we isolated and characterized a mix of virulent bacteriophages (phage cocktail) able to efficiently counteract Pseudomonas aeruginosa infection in a zebrafish model with cftr loss-of-function (LOF), but also showing anti-inflammatory effects in zebrafish embryos not infected by bacteria. On these premises, in this work we demonstrated the anti-inflammatory role of the phage cocktail both in the wild-type (WT) and hyper-inflamed cftr LOF zebrafish embryos in terms of reduction of pro-inflammatory markers. We also dissect that only the virion proteinaceous components, but not the phage DNA, are responsible for the immune-modulatory effect and that this action is elicited through the activation of the Toll-like Receptor (TLR) pathway. In the cftr LOF zebrafish embryos, we demonstrated that phages injection significantly reduces neutrophil migration following acute inflammatory induction. The elucidation of the molecular interaction between phages and the cells of vertebrate immune system might open new possibility in their manipulation for therapeutic benefits especially in diseases such as cystic fibrosis, characterized by chronic infection and inflammation.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos.

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Assessment of innate immune response activation following the injection of extracellular vesicles isolated from human cell cultures in zebrafish embryos.

The release of extracellular vesicles (EVs) is a common feature of cells but the specific functional role of this secretion still remains poorly understood. EVs carry on their surface and in their lumen several molecules that act as signals, making EVs abundant and effective messengers for cell-to-cell communications. For instance, EVs released from cancer cells can modulate tumor invasiveness, and EVs produced in autoinflammatory diseases can improperly activate the immune system. We recently described an effect of EVs released from colorectal cancer cells in the immune-modulation of cytokine expression in zebrafish. Here, we detail a simple methodological approach to purify EVs from human cell media and to inject them in the zebrafish embryo circulation to follow in vivo the response of the innate immune system to EVs injection.

Extracellular Vesicles Released by Colorectal Cancer Cell Lines Modulate Innate Immune Response in Zebrafish Model: The Possible Role of Human Endogenous Retroviruses.

Extracellular vesicles (EVs) are important components of the metastatic niche and are crucial in infiltration, metastasis, and immune tolerance processes during tumorigenesis. We hypothesized that human endogenous retroviruses (HERV) positive EVs derived from tumor cellsmay have a role in modulating the innate immune response. The study was conducted in two different colorectal cancer cell lines, representing different stages of cancer development: Caco-2, derived from a non-metastatic colorectal adenocarcinoma, and SK-CO-1, derived from metastatic colorectal adenocarcinoma (ascites). Both cell lines were treated with decitabine to induce global hypomethylation and to reactivate HERV expression. EVs were quantified by nanoparticle tracking analysis, and HERV-positive EV concentrations were measured by flow cytometry. The effect of EVs isolated from both untreated and decitabine-treated cells on the innate immune response was evaluated by injecting them in zebrafish embryos and then assessing Interleukin 1ß (IL1-ß), Interleukin 10 (IL-10), and the myeloperoxidase (mpx) expression levels by real-time qPCR. Interestingly, HERV-K positive EVs concentrations were significantly associated with a reduced expression of IL1-ß and mpx, supporting our hypothesis that HERV-positive EVs may act as immunomodulators in tumor progression. The obtained results open new perspectives about the modulation of the immune response in cancer therapy.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Design of a Broad-Range Bacteriophage Cocktail That Reduces Pseudomonas aeruginosa Biofilms and Treats Acute Infections in Two Animal Models.

The alarming diffusion of multidrug-resistant (MDR) bacterial strains requires investigations on nonantibiotic therapies. Among such therapies, the use of bacteriophages (phages) as antimicrobial agents, namely, phage therapy, is a promising treatment strategy supported by the findings of recent successful compassionate treatments in Europe and the United States. In this work, we combined host range and genomic information to design a 6-phage cocktail killing several clinical strains of Pseudomonas aeruginosa, including those collected from Italian cystic fibrosis (CF) patients, and analyzed the cocktail performance. We demonstrated that the cocktail composed of four novel phages (PYO2, DEV, E215 and E217) and two previously characterized phages (PAK_P1 and PAK_P4) was able to lyse P. aeruginosa both in planktonic liquid cultures and in biofilms. In addition, we showed that the phage cocktail could cure acute respiratory infection in mice and treat bacteremia in wax moth (Galleria mellonella) larvae. Furthermore, administration of the cocktail to larvae prior to bacterial infection provided prophylaxis. In this regard, the efficiency of the phage cocktail was found to be unaffected by the MDR or mucoid phenotype of the pseudomonal strain. The cocktail was found to be superior to the individual phages in destroying biofilms and providing a faster treatment in mice. We also found the Galleria larva model to be cost-effective for testing the susceptibility of clinical strains to phages, suggesting that it could be implemented in the frame of developing personalized phage therapies.