Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Towards Standardization of Phage Susceptibility Testing: The Israeli Phage Therapy Center "Clinical Phage Microbiology"-A Pipeline Proposal.

Using phages as salvage therapy for nonhealing infections is gaining recognition as a viable solution for patients with such infections. The escalating issue of antibiotic resistance further emphasizes the significance of using phages in treating bacterial infections, encompassing compassionate-use scenarios and clinical trials. Given the high specificity of phages, selecting the suitable phage(s) targeting the causative bacteria becomes critical for achieving treatment success. However, in contrast to conventional antibiotics, where susceptibility-testing procedures were well established for phage therapy, there is a lack of standard frameworks for matching phages from a panel to target bacterial strains and assessing their interactions with antibiotics or other agents. This review discusses and compares published methods for clinical phage microbiology, also known as phage susceptibility testing, and proposes guidelines for establishing a standard pipeline based on our findings over the past 5 years of phage therapy at the Israeli Phage Therapy Center.

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program.

Complete Genome Sequence of Pseudomonas aeruginosa Bacteriophage PASA16, Used in Multiple Phage Therapy Treatments Globally.

PASA16 is a Pseudomonas aeruginosa phage isolated from a soil sample and used to treat several patients suffering from persistent infections in various countries. PASA16's genome was sequenced, analyzed, and deposited in GenBank.

Expanding clinical phage microbiology: simulating phage inhalation for respiratory tract infections.

Phage therapy is a promising antibacterial strategy for resistant respiratory tract infections. Phage inhalation may serve this goal; however, it requires a careful assessment of their delivery by this approach. Here we present an in vitro model to evaluate phage inhalation. Eight phages, most of which target pathogens common in cystic fibrosis, were aerosolised by jet nebuliser and administered to a real-scale computed tomography-derived 3D airways model with a breathing simulator. Viable phage loads reaching the output of the nebuliser and the tracheal level of the model were determined and compared to the loaded amount. Phage inhalation resulted in a diverse range of titre reduction, primarily associated with the nebulisation process. No correlation was found between phage delivery to the phage physical or genomic dimensions. These findings highlight the need for tailored simulations of phage delivery, ideally by a patient-specific model in addition to proper phage matching, to increase the potential of phage therapy success.

Bacterial lysis, autophagy and innate immune responses during adjunctive phage therapy in a child.

Adjunctive phage therapy was used in an attempt to avoid catastrophic outcomes from extensive chronic Pseudomonas aeruginosa osteoarticular infection in a 7-year-old child. Monitoring of phage and bacterial kinetics allowed real-time phage dose adjustment, and along with markers of the human host response, indicated a significant therapeutic effect within two weeks of starting adjunctive phage therapy. These findings strongly suggested the release of bacterial cells or cell fragments into the bloodstream from deep bony infection sites early in treatment. This was associated with transient fever and local pain and with evidence of marked upregulation of innate immunity genes in the host transcriptome. Adaptive immune responses appeared to develop after a week of therapy and some immunomodulatory elements were also observed to be upregulated.

Isolation and Characterization of Streptococcus mutans Phage as a Possible Treatment Agent for Caries.

Streptococcus mutans is a key bacterium in dental caries, one of the most prevalent chronic infectious diseases. Conventional treatment fails to specifically target the pathogenic bacteria, while tending to eradicate commensal bacteria. Thus, caries remains one of the most common and challenging diseases. Phage therapy, which involves the use of bacterial viruses as anti-bacterial agents, has been gaining interest worldwide. Nevertheless, to date, only a few phages have been isolated against S. mutans. In this study, we describe the isolation and characterization of a new S. mutans phage, termed SMHBZ8, from hundreds of human saliva samples that were collected, filtered, and screened. The SMHBZ8 genome was sequenced and analyzed, visualized by TEM, and its antibacterial properties were evaluated in various states. In addition, we tested the lytic efficacy of SMHBZ8 against S. mutans in a human cariogenic dentin model. The isolation and characterization of SMHBZ8 may be the first step towards developing a potential phage therapy for dental caries.

Complete Genome Sequences of Two Enterococcus faecalis Bacteriophages, EFGrKN and EFGrNG, Targeted to Phage Therapy.

EFGrKN and EFGrNG are new Enterococcus faecalis phages that were isolated from sewage samples as part of the Israeli Phage Bank (IPB). The complete genomes were sequenced, analyzed, and deposited in GenBank. According to their lytic activity in vitro, it seems that these phages have a potential to be used in future phage therapy treatments.

Targeting Biofilm of MDR Providencia stuartii by Phages Using a Catheter Model.

Providencia spp. are emerging pathogens mainly in nosocomial infections. Providencia stuartii in particular is involved in urinary tract infections and contributes significantly to the high incidence of biofilm-formation in catheterized patients. Furthermore, recent reports suggested a role for multiple drug resistant (MDR) P. stuartii in hospital-associated outbreaks which leads to excessive complications resulting in challenging treatments. Phage therapy is currently one of the most promising solutions to combat antibiotic-resistant infections. However, the number of available phages targeting Providencia spp. is extremely limited, restricting the use of phage therapy in such cases. In the present study, we describe the isolation and characterization of 17 lytic and temperate bacteriophages targeting clinical isolates of Providencia spp. as part of the Israeli Phage Bank (IPB). These phages, isolated from sewage samples, were evaluated for host range activity and effectively eradicated 95% of the tested bacterial strains isolated from different geographic locations and displaying a wide range of antibiotic resistance. Their lytic activity is demonstrated on agar plates, planktonic cultures, and biofilm formed in a catheter model. The results suggest that these bacteriophages can potentially be used for treatment of antibiotic-resistant Providencia spp. infections in general and of urinary tract infections in particular.

Clinical Phage Microbiology: a suggested framework and recommendations for the in-vitro matching steps of phage therapy.

Phage therapy is a promising solution for bacterial infections that are not eradicated by conventional antibiotics. A crucial element of this approach is appropriate matching of bacteriophages and antibiotics to the bacterial target according to the clinical setting. However, there is currently little consistency in the protocols used for the laboratory evaluation of bacteriophages intended for antibacterial treatment. In this Personal View, we suggest a framework aimed to match appropriate bacteriophage-based treatments in clinical microbiology laboratories. This framework, which we have termed Clinical Phage Microbiology, is based on the current research on phage treatments. In addition, we discuss special cases that might require additional relevant evaluation, including bacteriophage interactions with the host immune response, biofilm-associated infections, and polymicrobial infections. The Clinical Phage Microbiology pipeline could serve as the basis for future standardisation of laboratory protocols for personalised phage therapy.

The Israeli Phage Bank (IPB).

A key element in phage therapy is the establishment of large phage collections, termed herein "banks", where many well-characterized phages, ready to be used in the clinic, are stored. These phage banks serve for both research and clinical purposes. Phage banks are also a key element in clinical phage microbiology, the prior treatment matching of phages and antibiotics to specific bacterial targets. A worldwide network of phage banks can promote a phage-based solution for any isolated bacteria. Herein, we describe the Israeli Phage Bank (IPB) established in the Hebrew University, Jerusalem, which currently has over 300 phages matching 16 bacteria, mainly pathogens. The phage bank is constantly isolating new phages and developing methods for phage isolation and characterization. The information on the phages and bacteria stored in the bank is available online.

Isolation and Characterization of Live Yeast Cells from Ancient Clay Vessels.

Ancient fermented food has been studied mainly based on residue analysis and recipes and reconstruction attempts were performed using modern domesticated yeast. Furthermore, microorganisms which participated in fermentation were studied using ancient-DNA techniques. In a recent paper, we presented a novel approach based on the hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. Here we present a pipeline for isolation of yeast from clay vessels uncovered in archeological sites and transferred to the microbiology lab where they can be isolated and characterized. This method opens new avenues for experimental archeology and enables attempts to recreate ancient food and beverages using the original microorganisms.

Successful Treatment of Antibiotic-resistant, Poly-microbial Bone Infection With Bacteriophages and Antibiotics Combination.

A patient with a trauma-related left tibial infection associated with extensively drug-resistant Acinetobacter baumannii and multidrug-resistant Klebsiella pneumoniae was treated with bacteriophages and antibiotics. There was rapid tissue healing and positive culture eradication. As a result, the patient's leg did not have to be amputated and he is undergoing rehabilitation.