Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

Antibiotic Susceptibility of Cutibacterium acnes Strains Isolated from Israeli Acne Patients.

Antibiotic-resistant Cutibacterium acnes has been reported worldwide, but data from Israeli patients with acne is currently lacking. This study evaluated the antibiotic susceptibility of C. acnes, isolated from 50 Israeli patients with acne to commonly prescribed antibiotics, using the Epsilometer test (E-test). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis, 16S rRNA sequencing and single locus sequence typing (SLST) molecular typing were used to identify and characterize C. acnes. Among 36 strains isolated, phylotype IA1 was most common. Resistance to at least one antibiotic was found in 30.6% of tested strains. Resistance rates were highest for erythromycin (25.0%), followed by doxycycline (19.4%), clindamycin (16.7%), minocycline (11.1%) and tetracycline (8.3%). Significant correlation was found between resistance to multiple antibiotics, with 5.6% of isolates resistant to all antibiotics tested. When reviewing resistances rate worldwide antibiotic resistance was found to be prevalent in Israel. Measures to limit the emergence of antibiotic-resistant strains of Cutibacterium acnes should be taken and alternative treatments should be sought.

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.

Targeting Enterococcus faecalis biofilms with phage therapy.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

Intravesical administration of green tea extract attenuates the inflammatory response of bacterial cystitis--a rat model.

OBJECTIVE: To explore the effect of intravesical instillation of green tea extract (GTE) on a rat model of bacterial cystitis. MATERIALS AND METHODS: In vitro bactericidal properties of GTE were analysed by adding GTE to a suspension of uropathogenic E. coli (UPEC), streaking on MacConkey agar, and incubating overnight. In vivo effects of intravesical instillation of GTE on bacterial cystitis was analysed using a rat model of bacterial cystitis. In all, 42 female Sabra rats weighing 200-260 g were divided into five groups. Parameters measured were bladder weight (percentage of the total rat weight), dipstick urine analysis and histopathological changes in the bladder. Histological changes evaluated were degree of oedema, mixed inflammatory infiltration, urothelial epithelial invasion by neutrophils and reactive atypia. RESULTS: No in vitro bactericidal activity was detected for GTE. Intravesical instillation of GTE did not cause damage to the rat bladders. Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis in this rat model. CONCLUSIONS: Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis and is a novel approach to the treatment of bacterial cystitis. High concentrations of intravesical GTE did not cause histologically evident damage to the rat bladder. The results of this study are preliminary and further studies will be needed to explore the feasibility of using this approach in humans.

Successful phage-antibiotic therapy of P. aeruginosa implant-associated infection in a Siamese cat.

Antibiotic-resistant pathogens are a growing global issue, leading to untreatable infectious diseases in both humans and animals. Personalized bacteriophage (phage) therapy, the use of specific anti-bacterial viruses, is currently a leading approach to combat antibiotic-resistant infections. The implementation of phage therapy has primarily been focused on humans, almost neglecting the impact of such infections on the health and welfare of companion animals. Pets also have the potential to spread resistant infections to their owners or the veterinary staff through zoonotic transmission. Here, we showcase personalized phage-antibiotic treatment of a cat with a multidrug-resistant Pseudomonas aeruginosa implant-associated infection post-arthrodesis surgery. The treatment encompassed a tailored combination of an anti-P. aeruginosa phage and ceftazidime, precisely matched to the pathogen. The phage was topically applied to the surgical wound while the antibiotic was administered intramuscularly. After two treatment courses spanning 7 and 3 weeks, the surgical wound, which had previously remained open for five months, fully closed. To the best of our knowledge, this is the first case of personalized phage therapy application in felines, which provides further evidence of the effectiveness of this approach. The successful outcome paves the way for personalized phage-antibiotic treatments against persistent infections therapy in veterinary practice.

Protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections.

Phage therapy has re-emerged as a promising treatment for non-resolving infections. Given the lack of approved phage treatments, there is a need to establish a compassionate use pipeline. Here, we present a protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections. We describe steps for consultation and request implementation, evaluating and comparing different aspects of phage activity, and phage production. We then detail procedures for multidisciplinary meetings, ethics approvals, phage therapy, and follow-up. For complete details on the use and execution of this protocol, please refer to Onallah et al.1,2.

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

Oxygen deprivation affects the antimicrobial action of LL-37 as determined by microplate real-time kinetic measurements under anaerobic conditions.

Some agents, including Escherichia coli and group A Streptococcus pyogenes cause infections in oxygen depleted sites. LL-37 is a human host defence peptide shown previously to play an important role in controlling infections caused by these bacteria. However, the effect of oxygen levels on the antimicrobial activity of LL-37 remains obscure. In order to test the effect of oxygen (or lack thereof) on LL-37's activity against E. coli and S. pyogenes, a method for adapting commonly used microtiter plates for real-time growth-kinetic (and growth-inhibition) measurements under anaerobic conditions was developed. Using the proposed method, anaerobic conditions were attained in the microplate within 30 min and were maintained for at least five days. Anaerobiosis was further confirmed by comparing the growth of two anaerobic oral species (Porphyromonas gingivalis and Fusobacterium nucleatum) in anaerobic compartments of microtiter plates versus aerobic ones. Both species grew only in the anaerobic compartments of the plates as indicated by the growth curves generated. The sensitivities of E. coli and S. pyogenes to LL-37 were tested under anaerobic conditions and compared to those in aerobic ones. The oxygen facultative E. coli grew to a higher density under aerobic conditions and its sensitivity to LL-37 was increased under anaerobiosis. The microaerophilic pathogen S. pyogenes grew faster and to a higher density under anaerobic conditions and was much more resistant to LL-37 under oxygen deprivation. Our results suggest that resistance to antimicrobial agents of microbes infecting anaerobic-microaerophilic sites should be tested under oxygen-restricted conditions.

Topical phage therapy in a mouse model of Cutibacterium acnes-induced acne-like lesions.

Acne vulgaris is a common neutrophil-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) is known to play a key role. For decades, antibiotics have been widely employed to treat acne vulgaris, inevitably resulting in increased bacterial antibiotic resistance. Phage therapy is a promising strategy to combat the growing challenge of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria. Herein, we explore the feasibility of phage therapy against C. acnes. Eight novel phages, isolated in our laboratory, and commonly used antibiotics eradicate 100% of clinically isolated C. acnes strains. Topical phage therapy in a C. acnes-induced acne-like lesions mouse model affords significantly superior clinical and histological scores. Moreover, the decrease in inflammatory response was reflected by the reduced expression of chemokine CXCL2, neutrophil infiltration, and other inflammatory cytokines when compared with the infected-untreated group. Overall, these findings indicate the potential of phage therapy for acne vulgaris as an additional tool to conventional antibiotics.

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.

Phage Therapy in Israel, Past, Present, and Future.

The fascinating scientific history of phage therapy has been documented in numerous publications. In this study, however, we focus on an angle of the story that hitherto has remained relatively neglected, namely, phage therapy treatments, and the protagonists that conducted these in Mandatory-Palestine and subsequently the state of Israel, as part of a global trend. We complete the story by describing efforts in the new era of phage therapy in present-day Israel.

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.

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.

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.

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.

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.

Bacteriophage Rescue Therapy of a Vancomycin-Resistant Enterococcus faecium Infection in a One-Year-Old Child following a Third Liver Transplantation.

Phage therapy is an experimental therapeutic approach used to target multidrug-resistant bacterial infections. A lack of reliable data with regard to its efficacy and regulatory hurdles hinders a broad application. Here we report, for the first time, a case of vancomycin-resistant Enterococcus faecium abdominal infection in a one-year-old, critically ill, and three times liver transplanted girl, which was successfully treated with intravenous injections (twice per day for 20 days) of a magistral preparation containing two Enterococcus phages. This correlated with a reduction in baseline C-reactive protein (CRP), successful weaning from mechanical ventilation and without associated clinical adverse events. Prior to clinical use, phage genome was sequenced to confirm the absence of genetic determinants conferring lysogeny, virulence or antibiotic resistance, and thus their safety. Using a phage neutralization assay, no neutralizing anti-phage antibodies in the patient's serum could be detected. Vancomycin-susceptible E. faecium isolates were identified in close relation to phage therapy and, by using whole-genome sequencing, it was demonstrated that vancomycin-susceptible E. faecium emerged from vancomycin-resistant progenitors. Covering a one year follow up, we provide further evidence for the feasibility of bacteriophage therapy that can serve as a basis for urgently needed controlled clinical trials.

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.