Animal Models to Translate Phage Therapy to Human Medicine

Phagotherapy, the use of bacteriophages to fight bacterial infections as an alternative to antibiotic treatments, has become of increasing interest in the last years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections that constitute a serious issue for public health. Phage therapy is gaining favor due to its success in agriculture and veterinary treatments and its extensive utilization for human therapeutic protocols in the Eastern world. In the last decades, some clinical trials and compassionate treatments have also been performed in the Western world, indicating that phage therapy is getting closer to its introduction in standard therapy protocols. However, several questions concerning the use of phages in human therapeutic treatments are still present and need to be addressed. In this review, we illustrate the state of art of phage therapy and examine the role of animal models to translate these treatments to humans

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

Communication and visiting policies in Italian intensive care units during the first COVID-19 pandemic wave and lockdown: a nationwide survey

Background: During the first coronavirus disease 2019 (COVID-19) pandemic wave, an unprecedented number of patients with respiratory failure due to a new, highly contagious virus needed hospitalization and intensive care unit (ICU) admission. The aim of the present study was to describe the communication and visiting policies of Italian intensive care units (ICUs) during the first COVID-19 pandemic wave and national lockdown and compare these data with prepandemic conditions. Methods: A national web-based survey was conducted among 290 Italian hospitals. Each ICU (active between February 24 and May 31, 2020) was encouraged to complete an individual questionnaire inquiring the hospital/ICU structure/organization, communication/visiting habits and the role of clinical psychology prior to, and during the first COVID-19 pandemic wave. Results: Two hundred and nine ICUs from 154 hospitals (53% of the contacted hospitals) completed the survey (202 adult and 7 pediatric ICUs). Among adult ICUs, 60% were dedicated to COVID-19 patients, 21% were dedicated to patients without COVID-19 and 19% were dedicated to both categories (Mixed). A total of 11,102 adult patients were admitted to the participating ICUs during the study period and only approximately 6% of patients received at least one visit. Communication with family members was guaranteed daily through an increased use of electronic devices and was preferentially addressed to the same family member. Compared to the prepandemic period, clinical psychologists supported physicians more often regarding communication with family members. Fewer patients received at least one visit from family members in COVID and mixed-ICUs than in non-COVID ICUs, l (0 [0–6]%, 0 [0–4]% and 11 [2–25]%, respectively, p < 0.001). Habits of pediatric ICUs were less affected by the pandemic. Conclusions: Visiting policies of Italian ICUs dedicated to adult patients were markedly altered during the first COVID-19 wave. Remote communication was widely adopted as a surrogate for family meetings. New strategies to favor a family-centered approach during the current and future pandemics are warranted

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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

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

In vivo validation of phage therapy against Pseudomonas aeruginosa infections using zebrafish as a new model for cystic fibrosis

To investigate the pathophysiology of cystic fibrosis (CF) several animal models have been developed including mouse, pig, and ferret; however, none of them perfectly recapitulates all human patient symptoms. On the contrary, zebrafish (Danio rerio) recently emerged as a powerful genetic model system to better understand CF onset and to develop new pharmacological treatments. Indeed, zebrafish embryos present only innate immune system and the zebrafish cftr gene is highly conserved with the human orthologue. cftr-loss-of-function zebrafish embryos mimic CF human defects in response to infection of P. aeruginosa, presenting a dampened respiratory burst response, a reduced neutrophil migration and defects in endocrine organs function. In our previous work, we demonstrated that P. aeruginosa infection in mice and Galleria mellonella larvae could be cured by administration of phages, the natural enemies of bacteria. Phage therapy, used for decades in Eastern Europe, is gathering interest as a therapeutic alternative or a complementary treatment to antibiotics. The goal of this project is to in vivo validate the efficacy of phage therapy against P. aeruginosa infections using the CF-zebrafish animal model. Both wild-type and cftr-loss-of-function zebrafish embryos, were infected with P. aeruginosa by microinjection, followed by phage administration. The therapeutic effects of phages was evaluated, following embryo mortality, bacterial burden, neutrophil migration and immune response. In addition, we plan to combine the phage treatment with antibiotics to verify if combination of the two treatments has a positive outcome against P. aeruginosa infections

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

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 (CK phi), already validated as a PAO1 "eater", improves the antimicrobial effects of single treatments, and it is effective also against CK phi-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CK phi 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