Phages and phage-borne enzymes as new antibacterial agents.

BACKGROUND: Persistent and resistant infections caused by bacteria are increasing in numbers and pose a treatment challenge to the medical community and public health. However, solutions with new agents that will enable effective treatment are lacking or delayed by complex development and authorizations. Bacteriophages are known as a possible solution for invasive infections for decades but were seldom used in the Western world. OBJECTIVES: To provide an overview of the current status and emerging use of bacteriophage therapy and phage-based products, as well as touch on the socioeconomic and regulatory issues surrounding their development. SOURCES: Peer-reviewed articles and authors' first-hand experience. CONTENT: Although phage therapy is making a comeback since its early discovery, there are many hurdles to its current use. The lack of appropriate standardized bacterial susceptibility testing; lack of a simple business model and authorization for the need of many phages to treat a single species infection; and the lack of knowledge on predictable outcome measures are just a few examples. In this review, we explore the possible routes for phage use, either based on local specialty centres or by industry; the current status of phage therapy, which is mainly based on single-centre or single-bacterial cohorts, and emerging clinical trials; local country-level frameworks for phage utilization even without full authorization; and the use of phage-derived products as alternatives to antibiotics. We also explore what may be the current indications based on the possible availability of phages. IMPLICATIONS: Although phages are emerging as a potential treatment for non-resolving and life-threatening infections, the models for their use and production still need to be defined by the medical community, regulatory bodies, and industry. Bacteriophages may have a great potential for infection treatment but many aspects still need to be defined before their routine use in the clinic.

Indwelling catheter vs intermittent catheterization: is there a difference in UTI susceptibility?

BACKGROUND: Patients with neurogenic lower urinary tract dysfunction (NLUTD) often rely on some type of catheterization for bladder emptying. Intermittent catheterization (IC) is considered the gold standard and is preferred over continuous catheterization, since it is considered to cause fewer urinary tract infections (UTIs) than indwelling catheterization. The main objective of our study was to describe UTI prevalence (at visit) and incidence (within the last 12 months) and urine culture characteristics between patients using an indwelling catheter versus (vs) those performing IC. METHODS: In this cross-sectional study, we prospectively evaluated from 02/2020 to 01/2021 patients with NLUTD undergoing urine cultures for prophylactic reasons or due to UTI symptoms. At visit, all patients underwent a standardized interview on current UTI symptoms as well as UTI history and antibiotic consumption within the past year. Patients using an indwelling catheter (n = 206) or IC (n = 299) were included in the analysis. The main outcome was between-group differences regarding UTI characteristics. RESULTS: Patients using an indwelling catheter were older (indwelling catheter vs IC: median 66 (Q1-Q3: 55-77) vs 55 (42-67) years of age) and showed a higher Charlson comorbidity index (indwelling catheter vs IC: median 4 (Q1-Q3: 2-6) vs 2 (1-4) (both p < 0·001). A total of 40 patients from both groups were diagnosed with a UTI at visit (indwelling catheters vs IC: 8% (16/206) vs 8% (24/299); p = 0·782), and the number of UTIs within the past 12 months was not significantly different between groups. Overall, Escherichia coli (21%), Enterococcus faecalis (17%), and Klebsiella spp. (12%) were the most frequently detected bacteria. CONCLUSIONS: In this cohort of patients with NLUTD, we did not find relevant differences in UTI frequency between groups. These results suggest that UTI-related concerns should not be given undue emphasis when counseling patients for catheter-related bladder emptying methods.

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine.

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

Management of uncomplicated urinary tract infection in the post-antibiotic era: select non-antibiotic approaches.

BACKGROUND: Given the high frequency of patients presenting with urinary tract infections (UTIs) and the ensuing high degree of antibiotic prescription, UTI is a critical point of intervention for non-antibiotic treatments to curb the further development of antimicrobial resistance and provide risk-appropriate care for patients. OBJECTIVES: To highlight several select non-antibiotic therapies for the treatment of uncomplicated UTI and relevant indications (prevention and complicated UTI) from recent literature. SOURCES: PubMed, Google Scholar, and clinicaltrials.gov were searched for clinical trials published in the English language corresponding to non-antibiotic treatments for UTI. CONTENT: The focus of this narrative review centres on a limited number of non-antibiotic therapies for the treatment of UTI based on (a) herbal extracts or (b) antibacterial strategies (e.g. bacteriophage therapy and D-mannose). The experience of treatment with non-steroidal anti-inflammatory drugs is also used to fuel discussion on the risk of developing pyelonephritis without antibiotics-compared with the projected harms of continuing their widespread use. IMPLICATIONS: Non-antibiotic treatment strategies for UTI have shown varying results in clinical trials, and the current evidence does not yet indicate a clear, better alternative to antibiotics. However, the collective experience with non-antibiotic treatments suggests that there is a need to weigh the actual benefits/risks of unfettered, non-culture-confirmed antibiotic use in uncomplicated UTI. Given the different mechanisms of action of proposed alternatives, more in-depth knowledge on microbiological and pathophysiological factors influencing UTI susceptibility and prognostic indicators are highly needed to stratify patients most likely to benefit. The feasibility of alternatives in clinical practice should also be considered.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Antibiotic Exposure Leads to Reduced Phage Susceptibility in Vancomycin Intermediate Staphylococcus aureus (VISA).

In the time of antimicrobial resistance, phage therapy is frequently suggested as a possible solution for such difficult-to-treat infections. Vancomycin-intermediate Staphylococcus aureus (VISA) remains a relatively rare yet increasing occurrence in the clinic for which phage therapy may be an option. However, the data presented herein suggest a potential cross-resistance mechanism to phage following vancomycin exposure in VISA strains. When comparing genetically similar strains differing in their susceptibility to vancomycin, those with intermediate levels of vancomycin resistance displayed decreased sensitivity to phage in solid and liquid assays. Serial passaging with vancomycin induced both reduced vancomycin susceptibility and phage sensitivity. As a consequence, the process of phage infection was shown to be interrupted after DNA ejection from adsorbed phage but prior to phage DNA replication, as demonstrated through adsorption assays, lysostaphin sensitivity assays, electron microscopy, and quantitative PCR (qPCR). At a time when phage products are being used for experimental treatments and tested in clinical trials, it is important to understand possible interference between mechanisms underlying antibiotic and phage resistance in order to design effective therapeutic regimens.

Short and oral antimicrobial therapy for diabetic foot infection: a narrative review of current knowledge.

Diabetic foot infection is a frequent complication in long-standing diabetes mellitus. For antimicrobial therapy of this infection, both the optimal duration and the route of administration are often based more on expert opinion than on published evidence. We reviewed the scientific literature, specifically seeking prospective trials, and aimed at addressing two clinical issues: (1) shortening the currently recommended antibiotic duration and (2) using oral (rather than parenteral) therapy, especially after the patient has undergone debridement and revascularization. We also reviewed some older key articles that are critical to our understanding of the treatment of these infections, particularly with respect to diabetic foot osteomyelitis. Our conclusion is that the maximum duration of antibiotic therapy for osteomyelitis should be no more than to 4-6 weeks and might even be shorter in selected cases. In the future, in addition to conducting randomized trials and propagating national and international guidance, we should also explore innovative strategies, such as intraosseous antibiotic agents and bacteriophages.

Bacteriophages: what role may they play in life after spinal cord injury?

Bacterial infections are the leading cause of death in people with a spinal cord injury (SCI). Bacteriophages (phages) are viruses that solely infect and kill bacteria. The idea of using phages to treat bacterial infections, i.e., phage therapy, is very promising and potentially allows a more specific and personalized treatment of bacterial infections than antibiotics. While multi-drug resistant infections affect individuals from the general population, alternative therapeutic options are especially warranted in high-risk populations, such as individuals with SCI. However, more clinical data must be collected before phage therapy can be implemented in clinical practice, with numerous possible, subsequent applications.

ITN-VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics.

Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Sklodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.

Intravesical bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomised, placebo-controlled, double-blind clinical trial.

BACKGROUND: Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. In the context of increasing antibiotic resistance, finding alternative treatments for UTIs is a top priority. We aimed to determine whether intravesical bacteriophage therapy with a commercial bacteriophage cocktail is effective in treating UTI. METHODS: We did a randomised, placebo-controlled, clinical trial, at the Alexander Tsulukidze National Centre of Urology, Tbilisi, Georgia. Men older than 18 years of age, who were scheduled for transurethral resection of the prostate (TURP), with complicated UTI or recurrent uncomplicated UTI but no signs of systemic infection, were allocated by block randomisation in a 1:1:1 ratio to receive intravesical Pyo bacteriophage (Pyophage; 20 mL) or intravesical placebo solution (20 mL) in a double-blind manner twice daily for 7 days, or systemically applied antibiotics (according to sensitivities) as an open-label standard-of-care comparator. Urine culture was taken via urinary catheter at the end of treatment (ie, day 7) or at withdrawal from the trial. The primary outcome was microbiological treatment response after 7 days of treatment, measured by urine culture; secondary outcomes included clinical and safety parameters during the treatment period. Analyses were done in a modified intention-to-treat population of patients having received at least one dose of the allocated treatment regimen. This trial is registered with ClinicalTrials.gov, NCT03140085. FINDINGS: Between June 2, 2017, and Dec 14, 2018, 474 patients were screened for eligibility and 113 (24%) patients were randomly assigned to treatment (37 to Pyophage, 38 to placebo, and 38 to antibiotic treatment). 97 patients (28 Pyophage, 32 placebo, 37 antibiotics) received at least one dose of their allocated treatment and were included in the primary analysis. Treatment success rates did not differ between groups. Normalisation of urine culture was achieved in five (18%) of 28 patients in the Pyophage group compared with nine (28%) of 32 patients in the placebo group (odds ratio [OR] 1·60 [95% CI 0·45-5·71]; p=0·47) and 13 (35%) of 37 patients in the antibiotic group (2·66 [0·79-8·82]; p=0·11). Adverse events occurred in six (21%) of 28 patients in the Pyophage group compared with 13 (41%) of 32 patients in the placebo group (OR 0·36 [95% CI 0·11-1·17]; p=0·089) and 11 (30%) of 37 patients in the antibiotic group (0·66 [0·21-2·07]; p=0·47). INTERPRETATION: Intravesical bacteriophage therapy was non-inferior to standard-of-care antibiotic treatment, but was not superior to placebo bladder irrigation, in terms of efficacy or safety in treating UTIs in patients undergoing TURP. Moreover, the bacteriophage safety profile seems to be favourable. Although bacteriophages are not yet a recognised or approved treatment option for UTIs, this trial provides new insight to optimise the design of further large-scale clinical studies to define the role of bacteriophages in UTI treatment. FUNDING: Swiss Continence Foundation, the Swiss National Science Foundation, and the Swiss Agency for Development and Cooperation. TRANSLATIONS: For the Georgian and German translations of the abstract see Supplementary Materials section.

A systematic review of phage therapy applied to bone and joint infections: an analysis of success rates, treatment modalities and safety.

Bone and joint infections are difficult to treat, and increasing antibiotic resistance has only made them more challenging. This has led to renewed interest in phage therapy (PT). The aim of this systematic review was to determine success rate, current treatment modalities and safety of PT in bone and joint infections.A systematic search of PubMed, EMBASE and Cochrane databases as well as the journal PHAGE for literature published between January 2000 and April 2021 was conducted according to PRISMA guidelines to identify all human studies assessing bacteriophages as therapy for bone and joint infections. All study designs and patient populations were eligible. The review's primary outcome was success rate.Twenty records describing a total of 51 patients and 52 treatment episodes were included. No randomized controlled studies were identified. The overall success rate was 71% (n = 37/52). Topical administration alone was the most frequent administration route (85%, n = 44/52). Antibiotics were administered concomitantly with PT in the majority of treatments (79%, n = 41/52), and surgery was performed for 87% (n = 45/52) of treatment episodes. Four minor adverse events related to PT were reported, representing 8% (n = 4/52) of treatment episodes.PT for bone and joint infections has not been evaluated in any randomized controlled clinical study, and current administration modalities are highly variable between case reports and case series. While publications included here show potential benefit and few adverse effects, clinical trials are warranted to assess the efficacy of PT for bone and joint infections and determine optimal treatment modalities. Cite this article: EFORT Open Rev 2021;6:1148-1156. DOI: 10.1302/2058-5241.6.210073.

Effect of C-Reactive Protein-Guided Antibiotic Treatment Duration, 7-Day Treatment, or 14-Day Treatment on 30-Day Clinical Failure Rate in Patients With Uncomplicated Gram-Negative Bacteremia: A Randomized Clinical Trial.

Importance: Antibiotic overuse drives antibiotic resistance. Gram-negative bacteremia is a common infection that results in substantial antibiotic use. Objective: To compare the clinical effectiveness of C-reactive protein (CRP)-guided, 7-day, and 14-day antibiotic durations 30, 60, and 90 days after treatment initiation. Design, Setting, and Participants: Multicenter, noninferiority, point-of-care randomized clinical trial including adults hospitalized with gram-negative bacteremia conducted in 3 Swiss tertiary care hospitals between April 2017 and May 2019, with follow-up until August 2019. Patients and physicians were blinded between randomization and antibiotic discontinuation. Adults (aged ≥18 years) were eligible for randomization on day 5 (±1 d) of microbiologically efficacious therapy for fermenting, gram-negative bacteria in blood culture(s) if they were afebrile for 24 hours without evidence for complicated infection (eg, abscess) or severe immunosuppression. Intervention: Randomization in a 1:1:1 ratio to an individualized CRP-guided antibiotic treatment duration (discontinuation once CRP declined by 75% from peak; n = 170), fixed 7-day treatment duration (n = 169), or fixed 14-day treatment duration (n = 165). Main Outcomes and Measures: The primary outcome was the clinical failure rate at day 30, defined as the presence of at least 1 of the following, with a non-inferiority margin of 10%: recurrent bacteremia, local suppurative complication, distant complication (growth of the same organism causing the initial bacteremia), restarting gram-negative-directed antibiotic therapy due to clinical worsening suspected to be due to the initial organism, or death due to any cause. Secondary outcomes included the clinical failure rate on day 90 of follow-up. Results: Among 504 patients randomized (median [interquartile range] age, 79 [68-86] years; 306 of 503 [61%] were women), 493 (98%) completed 30-day follow-up and 448 (89%) completed 90-day follow-up. Median antibiotic duration in the CRP group was 7 (interquartile range, 6-10; range, 5-28) days; 34 of the 164 patients (21%) who completed the 30-day follow-up had protocol violations related to treatment assignment. The primary outcome occurred in 4 of 164 (2.4%) patients in the CRP group, 11 of 166 (6.6%) in the 7-day group, and 9 of 163 (5.5%) in the 14-day group (difference in CRP vs 14-day group, -3.1% [1-sided 97.5% CI, -∞ to 1.1]; P < .001; difference in 7-day vs 14-day group, 1.1% [1-sided 97.5% CI, -∞ to 6.3]; P < .001). By day 90, clinical failure occurred in 10 of 143 patients (7.0%) in the CRP group, 16 of 151 (10.6%) in the 7-day group, and 16 of 153 (10.5%) in the 14-day group. Conclusions and Relevance: Among adults with uncomplicated gram-negative bacteremia, 30-day rates of clinical failure for CRP-guided antibiotic treatment duration and fixed 7-day treatment were noninferior to fixed 14-day treatment. However, interpretation is limited by the large noninferiority margin compared with the low observed event rate, as well as low adherence and wide range of treatment durations in the CRP-guided group. Trial Registration: ClinicalTrials.gov Identifier: NCT03101072.

Efficient decellularization of equine tendon with preserved biomechanical properties and cytocompatibility for human tendon surgery indications.

Chronic and acute tendon injuries are frequent afflictions, for which treatment is often long and unsatisfactory. When facing extended injuries, matrices and scaffolds with sufficient biomechanical properties are required for surgical repair and could additionally serve as supports for cellular therapies to improve healing. In this study, protocols of either commonly used detergents only (SDS 1%, Triton 1%, TBP 1%, and Tween-20 1%) or a combination of freeze/thaw (F/T) cycles with decellularization agents (NaCl 1M, ddH2 O) were evaluated for the decellularization of horse equine superficial digital flexor tendon (SDFT) for hand flexor or extensor tendon reconstruction. Decellularization efficiency was assessed microscopically by histological staining (HE, DAPI) and DNA quantification. Macroscopical structure and biomechanical integrity of the tendon matrices were further assessed by gross observation, histological staining (SR), and mechanical testing (ultimate strain and stress, Young's modulus, energy to failure) for select protocols. Decellularization with hypertonic NaCl 1M in association with F/T cycles produced the most robust tendon matrices, which were nontoxic after 10 days for subsequent recellularization with human fetal progenitor tendon cells (hFPTs). This standardized protocol uses a less aggressive decellularization agent than current practice, which allows subsequent reseeding with allogenic cells, therefore making them very suitable and bioengineered tendon matrices for human tendon reconstruction in the clinic.

Current State of Compassionate Phage Therapy.

There is a current unmet medical need for the treatment of antibiotic-resistant infections, and in the absence of approved alternatives, some clinicians are turning to empirical ones, such as phage therapy, for compassionate treatment. Phage therapy is ideal for compassionate use due to its long-standing historical use and publications, apparent lack of adverse effects, and solid support by fundamental research. Increased media coverage and peer-reviewed articles have given rise to a more widespread familiarity with its therapeutic potential. However, compassionate phage therapy (cPT) remains limited to a small number of experimental treatment centers or associated with individual physicians and researchers. It is possible, with the creation of guidelines and a greater central coordination, that cPT could reach more of those in need, starting by increasing the availability of phages. Subsequent steps, particularly production and purification, are difficult to scale, and treatment paradigms stand highly variable between cases, or are frequently not reported. This article serves both to synopsize cPT publications to date and to discuss currently available phage sources for cPT. As the antibiotic resistance crisis continues to grow and the future of phage therapy clinical trials remains undetermined, cPT represents a possibility for bridging the gap between current treatment failures and future approved alternatives. Streamlining the process of cPT will help to ensure high quality, therapeutically-beneficial, and safe treatment.

Metagenome analysis of Russian and Georgian Pyophage cocktails and a placebo-controlled safety trial of single phage versus phage cocktail in healthy Staphylococcus aureus carriers.

Bacteriophage therapy is a commonly used treatment for Staphylococcus aureus infections in countries of the former Soviet Union, using both single phages and phage cocktails. The scarce data available on Eastern phage cocktails prompted an investigation into commercially-available Pyophage cocktails from two different manufacturers used to treat skin and wound infections. Comparison of the metagenomic composition of two Pyophage products from Georgia and Russia revealed substantial differences in phage-types targeting Escherichia, Enterococcus, Salmonella, Pseudomonas aeruginosa and Proteus, therefore indicating multiple strategies for composing phage cocktails against these bacterial pathogens. Closely-related Kayvirus-like Myoviruses were, however, a shared component against S. aureus within all products, except for the inclusion of a secondary S. aureus Podovirus in one Microgen cocktail. Metagenomic analysis also revealed the presence of several probable prophage sequences but detected no genetic safety risks in terms of virulence factors or antibiotic resistance genes. The safety of broad-spectrum cocktails was tested by comparing the effects of nasal and oral exposure to Eliava Pyophage, a monospecies counterpart and placebo in healthy human carriers of S. aureus. The lack of adverse effects in any treatment groups supports the clinical safety of S. aureus phages administered as a single phage or as phage cocktail.

Silk Route to the Acceptance and Re-Implementation of Bacteriophage Therapy-Part II.

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.

Clinical Indications and Compassionate Use of Phage Therapy: Personal Experience and Literature Review with a Focus on Osteoarticular Infections.

The history of phage therapy started with its first clinical application in 1919 and continues its development to this day. Phages continue to lack any market approval in Western medicine as a recognized drug, but are increasingly used as an experimental therapy for the compassionate treatment of patients experiencing antibiotic failure. The few formal experimental phage clinical trials that have been completed to date have produced inconclusive results on the efficacy of phage therapy, which contradicts the many successful treatment outcomes observed in historical accounts and recent individual case reports. It would therefore be wise to identify why such a discordance exists between trials and compassionate use in order to better develop future phage treatment and clinical applications. The multitude of observations reported over the years in the literature constitutes an invaluable experience, and we add to this by presenting a number of cases of patients treated compassionately with phages throughout the past decade with a focus on osteoarticular infections. Additionally, an abundance of scientific literature into phage-related areas is transforming our knowledge base, creating a greater understanding that should be applied for future clinical applications. Due to the increasing number of treatment failures anticipatedfrom the perspective of a possible post-antibiotic era, we believe that the introduction of bacteriophages into the therapeutic arsenal seems a scientifically sound and eminently practicable consideration today as a substitute or adjuvant to antibiotic therapy.