Phage Preparations Research Articles

Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Evaluation of the efficacy of inhalation application of bacteriophage preparations in bronchopneumonia associated with Klebsiella pneumoniae

Against the background of increasing antibiotic resistance among pathogens of bovine bronchopneumonia, interest in phagotherapy as a therapeutic tool is revived. Inhaled phagotherapy in turn has the potential to change the treatment regimen for bacterial respiratory infections, including those caused by antibiotic-resistant bacteria. Inhaled drug delivery tools allow the delivery of bacteriophages directly to the lesion. An important advantage, especially when it comes to bacteriophages, is the ability to use smaller doses of drugs than would be required if another route of administration were chosen. The article presents the results of evaluating the efficacy of the inhalation method of administration of commercial bacteriophage preparations in a mouse model. Mice were divided into five groups (1,2-animals exposed to <I>Klebsiella pneumonia</I> infection and undergoing bacteriophage inhalation (n=6), 3-animals exposed to infection and not undergoing therapy (n=3), 4- control group of animals not exposed to infection (n=3)). Inhalations were performed using a compressor four-mode Neb-Aid inhaler (Flaem Nuova, Italy). Rapidflyam 2 nebuliser was used in mode I (particle size 0.8-2.0 μm). The results demonstrate the effectiveness of the developed therapy scheme, which allows further use of bactriophage-containing preparations in infectious diseases of animals.

Commercial bacteriophage preparations for the control of Listeria monocytogenes and Shiga toxin-producing Escherichia coli in raw and pasteurized milk

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts ∼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation.

Specific aspects of bacteriophage regulation: international practices and future developments

INTRODUCTION. The increasing prevalence of multidrug-resistant strains of pathogens determines the need for fundamentally new antibacterial agents, including bacteriophage preparations. The consistent implementation of phage therapy is hindered by the lack of generally accepted standardised regulatory documents governing the legal and methodological aspects of the production and preclinical and clinical studies of bacteriophage preparations.AIM. This study aimed to analyse the international experience with the production and lifecycle management of bacteriophage preparations, as well as the main regulatory requirements for the control of their quality, safety, and efficacy.DISCUSSION. It is difficult to develop virulent bacteriophage preparations in accordance with the existing requirements for other medicinal products because of the biological characteristics of bacteriophages, the wide variety of bacteriophage strains, and the potential for rapid changes both in the bacteriophage population and in the pathogen population. Therefore, it is reasonable to develop streamlined marketing authorisation routes for phage therapies and methods for the assessment of their safety and efficacy. As part of these efforts, it is necessary to assess the adverse events specific to this group of medicinal products, such as the risks of lysogeny, resistance to bacteriophages, and antibiotic resistance gene transfer between bacterial strains. The pharmaceutical development of bacteriophage preparations can be based on several approaches. Many countries worldwide, including the United States, are implementing the concept of Quality by Design, considering approaches based on the Biological Master File, and conducting Expanded Access programmes. The Active Substance Master File procedure allows the submission of a separate document package covering only part of the registration dossier for regulatory approval. Expanded Access programmes provide individual patients with access to innovative medicinal products without approved treatment protocols. In the Russian Federation, the commercial production of bacteriophage medicinal products is conducted in accordance with the quality standards specified in the State Pharmacopoeia of the Russian Federation.CONCLUSIONS. There are fundamental differences in the approaches to phage therapy and its regulation around the world and in the Russian Federation. It is reasonable to supplement the current national guidelines for the safety and efficacy evaluation of bacteriophage preparations, in particular, to specify the requirements for conducting preclinical studies.

Efficacy of Bacteriophages in Wound Healing: An Updated Review.

We have attempted to summarize the efficacy of bacteriophage therapy, highlighting the recent advances and phage delivery methods in different clinical trials and animal studies for wound-associated infections. Bacteriophage therapy is the lyse of bacteria by bacteriophages at the site of invasion. As bacteria become more resistant to antibiotics, discovering an alternative is more important than ever, and bacteriophage therapy has yielded promising outcomes. A clear knowledge of the bacteriophage, microbiota, and human host and their interaction is necessary to implement bacteriophage treatment on a large scale. Much technological advancement and regulatory guidelines increased the credibility of phage therapy (PT). Still, the challenges include the development of efficient bacteriophage screening methods, phage therapy strategies for biofilms, and the quality and safety of phage preparations. However, much consideration is to be taken in designing a novel therapeutic approach for antibiotic-resistant infections by using phages, phage lytic proteins, bioengineered phages, or antibiotics in combination.

The Hydrophobic Stabilization of Pseudomonas aeruginosa Bacteriophage F8 and the Influence of Modified Bacteriophage Preparation on Biofilm Degradation

The bacteriophage F8 belongs to the Myoviridae group of phages and is a pathogen of Pseudomonas aeruginosa. Since Pseudomonas aeruginosa is a multidrug-resistant opportunistic bacterium and can cause serious challenges for health services, studying the potential use of phages against them is a promising approach. Pseudomonas aeruginosa can be found on medical devices because bacteria can attach to surfaces and develop biofilms, which are difficult to eradicate because of their high resistance to environmental conditions and antimicrobial therapeutics. Phage therapy is becoming promising as an alternative for the treatment of antibiotic-resistant infections, but there is still a lack of standardized protocols approved by health organizations for possible use in the clinic. In our research, we focused on the potential use of 1-octanol, which was previously used by our team to develop a method for phage purification from bacterial lysate. 1-octanol is a fatty alcohol that is mostly used in the cosmetics industry, and its advantage is that it is approved by the FDA as a food additive. In this paper, we studied the protective properties of the addition of 1-octanol for storing phage liquid preparations. We demonstrated the stabilization effect of 1-octanol addition on F8 bacteriophage preparation during storage under various conditions. Interestingly, more effective biofilm reduction was observed after treatment with the purified bacteriophage and with 1-octanol addition compared to crude lysate.

Pharmacokinetics and pharmacodynamics of bacteriophage therapy: a review with a focus on multidrug-resistant Gram-negative bacterial infections.

SUMMARYDespite the early recognition of their therapeutic potential and the current escalation of multidrug-resistant (MDR) pathogens, the adoption of bacteriophages into mainstream clinical practice is hindered by unfamiliarity with their basic pharmacokinetic (PK) and pharmacodynamic (PD) properties, among others. Given the self-replicative nature of bacteriophages in the presence of host bacteria, the adsorption rate, and the clearance by the host's immunity, their PK/PD characteristics cannot be estimated by conventional approaches, and thus, the introduction of new considerations is required. Furthermore, the multitude of different bacteriophage types, preparations, and treatment schedules impedes drawing general conclusions on their in vivo PK/PD features. Additionally, the drawback of acquired bacteriophage resistance of MDR pathogens with clinical and environmental implications should be taken into consideration. Here, we provide an overview of the current state of the field of PK and PD of bacteriophage therapy with a focus on its application against MDR Gram-negative infections, highlighting the potential knowledge gaps and the challenges in translation from the bench to the bedside. After reviewing the in vitro PKs and PDs of bacteriophages against the four major MDR Gram-negative pathogens, Klebsiella pneumoniae, Acinetobacter baumannii complex, Pseudomonas aeruginosa, and Escherichia coli, specific data on in vivo PKs (tissue distribution, route of administration, and basic PK parameters in animals and humans) and PDs (survival and reduction of bacterial burden in relation to the route of administration, timing of therapy, dosing regimens, and resistance) are summarized. Currently available data merit close scrutiny, and optimization of bacteriophage therapy in the context of a better understanding of the underlying PK/PD principles is urgent to improve its therapeutic effect and to minimize the occurrence of bacteriophage resistance.

Safety and Tolerability of ShigActive™, a Shigella spp. Targeting Bacteriophage Preparation, in a Phase 1 Randomized, Double-Blind, Controlled Clinical Trial.

Bacterial diseases of the gastrointestinal (GI) tract continue to be a major worldwide cause of human morbidity and mortality. Among various enteric pathogens, Shigella spp. are some of the most common and deadly bacterial pathogens. They are responsible for ~125 million worldwide cases of shigellosis, and ~14,000 deaths annually, the majority in children under the age of 5 and occurring in developing countries. Preventing and treating shigellosis with conventional drugs (e.g., vaccines and antibiotics) has proven to be very difficult. Here, we assessed the safety and tolerability of ShigActive™, a lytic bacteriophage preparation targeting Shigella spp., in a randomized, placebo-controlled, double-blind Phase 1 clinical trial. Ten participants randomized 4:1 received ShigActive™ or placebo co-administered with sodium bicarbonate orally three times daily for 7 days. Solicited and unsolicited adverse events (AEs) were observed for 29 days. Fifty percent of the subjects receiving ShigActive™ reported mild GI-related symptoms, while one participant experienced moderate fatigue. No serious or medically attended AEs occurred through day 90. Additionally, no significant differences in GI-associated inflammatory mediators or fecal microbiome changes were observed between placebo- and ShigActive™-treated subjects, or from a participants' baseline value. The results of this first-in-human (FIH) randomized, controlled Phase 1 trial of ShigActive™ demonstrate that it is safe and well tolerated when orally administered with no significant differences compared to placebo controls.

Directed evolution of bacteriophages: impacts of prolific prophage.

Directed evolution is a common strategy for evolving phages to expand host range, often targeting pathogenic strains of bacteria. In this study we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophage are active players in directed evolution and can contribute to observation of host-range expansion. Since prophage are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving experimental design of future directed evolution studies.

Neutralizing antibodies after nebulized phage therapy in cystic fibrosis patients

Cystic fibrosis (CF) patients are prone to recurrent multi-drug-resistant (MDR) bacterial lung infections. Under this scenario, phage therapy has been proposed as a promising tool. However, the limited number of reported cases hampers the understanding of clinical outcomes. Anti-phage immune responses have often been overlooked and only described following invasive routes of administration. Three monophage treatments against Staphylococcus aureus and/or Pseudomonas aeruginosa lung infections were conducted in cystic fibrosis patients. In-house phage preparations were nebulized over 10days with standard-of-care antibiotics. Clinical indicators, bacterial counts, phage and antibiotic susceptibility, phage detection, and immune responses were monitored. Bacterial load was reduced by 3-6 log in two of the treatments. No adverse events were described. Phages remained in sputum up to 33days after completion of the treatment. In all cases, phage-neutralizing antibodies were detected in serum from 10 to 42days post treatment, with this being the first report of anti-phage antibodies after nebulized therapy. Nebulized phage therapy reduced bacterial load, improving quality of life even without bacterial eradication. The emergence of antibodies emphasizes the importance of long-term monitoring to better understandclinical outcomes. These findings encourage the use of personalized monophage therapies in contrast to ready-to-use cocktails, which might induce undesirable antibody generation. This study was supported by the Spanish Ministry of Science, Innovation and Universities; Generalitat Valenciana; and a crowdfunding in collaboration with the Spanish Cystic Fibrosis Foundation.

Targeted inactivation of multidrug-resistant Alcaligenes faecalis in pig farm WWTPs by mixed bacteriophages to diminish the risk of pathogenicity and antibiotic resistance dissemination

Wastewater treatment plants (WWTPs) at pig farms are significant environmental reservoirs of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In particular, contamination by multidrug-resistant Alcaligenes faecalis (MDR-AF) poses an increasingly severe threat to human health. However, no safe and effective method is currently available to hinder its dissemination in the environment. In this study, two hitherto unreported bacteriophages were screened. In addition, qPCR experiments demonstrated that applying these bacteriophage preparations to wastewater significantly eradicates MDR-AF and reduces ARGs by 0.7–2.5 orders of magnitude (P < 0.05), thereby substantially diminishing the risk of antibiotic resistance transmission. Furthermore, various characterizations, bacteriophage genomic analysis, and microbial community analysis indicated that these bacteriophage preparations possess safety, specificity, and high resilience, rendering them an efficient and eco-friendly biological control measure.

Phage therapy in prosthetic joint infections caused by Staphylococcus aureus - A literature review.

Prosthetic joint infections (PJIs) are dreaded arthroplasty complications often caused by Staphylococcus aureus. Due to methicillin-resistant S. aureus (MRSA) strains or biofilm formation, successful treatment remains difficult. Currently, two-stage revision surgery constitutes the gold standard therapy of PJIs, sometimes replaced or supplemented by debridement, antibiotics, and implant retention (DAIR). Given the dire consequences of therapeutic failure, bacteriophage therapy might be another treatment option. Here we provide a comprehensive literature review addressing theefficacy of phages applied against S.aureus as causative agent of PJIs. The included 17 publications had in common that the applied phages proved to be effective against various S. aureus isolates including MRSA even in biofilms. Experiments with mice, rats, rabbits, and moth larvaeconfirmed favorable features of phage preparations in PJI treatment in vivo; including its synergistic with antibiotics. Case reports of PJI patients unanimously described the bacterial eradication following, alongside other measures, intravenous and intra-articular phage administration. Generally, no major side effects occurred, but in some cases elevated liver transaminases were observed. To conclude, our review compiled promising evidence suggesting the safety and suitability of phage therapy as an adjuvant to DAIR in S. aureus PJIs, and thus, underscores the significance of further research.

Use of the Naturally Occurring Bacteriophage Grouping Model for the Design of Potent Therapeutic Cocktails.

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

Study of the sensitivity of methicillin-resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt; to antibiotics and bacteriophage preparations

Objective. To evaluate the sensitivity of methicillin-resistant Staphylococcus aureus strains to antibiotics and therapeutic and prophylactic preparations of bacteriophages. Materials and methods. The antibiotic sensitivity of strains of methicillin-resistant S.aureus was studied by the disco-diffusion method, the determination of the minimum inhibitory concentrations of vancomycin and linezolid by broth microdilution method, and ceftaroline by E-tests. The sensitivity of methicillin-resistant S.aureus to bacteriophage preparations was studied. Results. It has been established that linezolid and vancomycin are quite active drugs against methicillin-resistant S.aureus (MRSA), however, resistance to them begins to develop. MRSA resistance to fluoroquinolones and cefoxitin was the highest of all antibiotics tested; the results obtained with cefoxitin can be interpreted for other cephalosporins (except ceftaroline) and all penicillins. Only 67.9% of MRSA strains retained sensitivity to ceftaroline, but ceftaroline-resistant strains had low levels of minimal inhibitory concentration. The preparations of the bacteriophages “Sextaphage” and “Piobacteriophage Phagio” showed their lytic activity only in relation to 35.8% and 18.9% of MRSA isolates, respectively. Conclusion. Monitoring the development of antibiotic resistance in methicillin-resistant S.aureus is an important part of the strategy of rational antibiotic therapy for staphylococcal infections. Only taking into account the preliminary step-by-step determination of the phage sensitivity of the causative agent of the disease, bacteriophage preparations can be considered as a completely possible alternative to antibiotics for the treatment of purulent inflammatory diseases caused by MRSA.

THE USE OF A POLYVALENT KLEBSIELLA BACTERIOPHAGE IN INFECTION AFTER RECONSTRUCTIVE PLASTIC SURGERY ON THE SPINE IN A CHILD (A CLINICAL CASE)

The incidence of infection after reconstructive plastic surgery on the spine (in particular, spondylodesis) in children remains at a fairly high level and ranges from 0.4% to 8.7%. These complications are often caused by polyresistant bacterial pathogens; this accentuates the need for new effective therapeutic approaches. Bacteriophage preparations in combination with antibiotics can optimize existing strategies for the treatment of infectious diseases, since they are able to selectively destroy bacteria, including biofilm-forming ones. The aim of the study was to draw the attention of practitioners to modern problems of treating polyresistant infections on the example of a case of purulent complication after spinal surgery in a child. Materials and methods. On the basis of the FSBI "Federal Center for Traumatology, Orthopedics and Endoprosthetics" under the Health Ministry of the Russian Federation (the town of Cheboksary) a retrospective analysis of a clinical case of treating early infection after reconstructive plastic surgery on the spine caused by a polyresistant strain of Klebsiella pneumoniae was conducted. Research results. Patient P., 2.2 years old, with congenital pathology of the musculoskeletal system, who repeatedly received medical care in various medical organizations, underwent surgical spine stabilization. In the early postoperative period, an infectious complication developed caused by a polyresistant strain of Klebsiella pneumoniae (a producer of extended-spectrum beta-lactamases and carbapenemases). Along with surgical debridement of the postoperative wound, a combined etiotropic antibacterial therapy was performed (12 weeks). However, complete healing of the postoperative wound was achieved in combination with the administration of a polyvalent klebsiella bacteriophage (10 weeks). Conclusions. This clinical case demonstrates that the use of a polyvalent klebsiella bacteriophage can increase the effectiveness of antibacterial pharmacotherapy in surgical treatment with preservation of metal structures.

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.

A thermosensitive hydrogel formulation of phage and colistin combination for the management of multidrug-resistant Acinetobacter baumannii wound infections.

Chronic skin wounds are often associated with multidrug-resistant bacteria, impeding the healing process. Bacteriophage (phage) therapy has been revitalized as a promising strategy to counter the growing concerns of antibiotic resistance. However, phage monotherapy also faces several application drawbacks, such as a narrow host spectrum, the advent of resistant phenotypes and poor stability of phage preparations. Phage-antibiotic synergistic (PAS) combination therapy has recently been suggested as a possible approach to overcome these shortcomings. In the present study, we employed a model PAS combination containing a vB_AbaM-IME-AB2 phage and colistin to develop stable wound dressings of PAS to mitigate infections associated with Acinetobacter baumannii. A set of thermosensitive hydrogels were synthesized with varying amounts of Pluronic® F-127 (PF-127 at 15, 17.5 and 20 w/w%) modified with/without 3 w/w% hydroxypropyl methylcellulose (HPMC). Most hydrogel formulations had a gelation temperature around skin temperature, suitable for topical application. The solidified gels were capable of releasing the encapsulated phage and colistin in a sustained manner to kill bacteria. The highest bactericidal effect was achieved with the formulation containing 17.5% PF-127 and 3% HPMC (F5), which effectively killed bacteria in both planktonic (by 5.66 log) and biofilm (by 3 log) states and inhibited bacterial regrowth. Good storage stability of F5 was also noted with negligible activity loss after 9 months of storage at 4 °C. The ex vivo antibacterial efficacy of the F5 hydrogel formulation was also investigated in a pork skin wound infection model, where it significantly reduced the bacterial burden by 4.65 log. These positive outcomes warrant its further development as a topical PAS-wound dressing.

Etiologic features of nosocomial sinusitis

To evaluate the qualitative and quantitative composition of the causative agents of nosocomial sinusitis and their sensitivity to antibacterial chemotherapy drugs in modern conditions. During the period 2022-2023, we selected 45 patients from 6 ICU based on a multidisciplinary hospital and 11 patients from the ICU based on an infectious profile hospital, who were diagnosed with nosocomial sinusitis based on anamnesis, clinical picture, and computed tomography results. The qualitative and quantitative composition of microorganisms in the discharge obtained during therapeutic and diagnostic puncture of the maxillary sinuses in all patients was studied. According to the results it was revealed that in the multidisciplinary hospital the following microorganisms were the most frequently detected in patients with nosocomial sinusitis: Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus haemolyticus, Enterococcus faecium, Enterococcus faecalis, Ochrobactrum anthropi. In patients with nosocomial sinusitis in the infectious diseases hospital the most frequently isolated were Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Proteus mirabilis. Fungal microorganisms were isolated much less frequently than bacterial microorganisms in both hospitals and were represented only by Candida albicans. In the multiprofile hospital the isolated strains of bacterial microorganisms were characterized by multidrug resistance in 89% of cases, and in the infectious diseases hospital - in 91% of cases. In the treatment of patients with nosocomial sinusitis, it is necessary to adjust empirical antibiotic therapy based on individual results of a bacteriological study. In case of ineffectiveness of antibacterial therapy, it is possible to use personalized biotherapy with virulent bacteriophage preparations in addition to prescribed antibacterial chemotherapy drugs, which makes it possible to enhance the antimicrobial effect of the therapy due to the additive action of drugs of two groups.

Возможности улучшения терапевтической активности препаратов бактериофагов.

Introduction. Growth of urological infection caused by antibiotic-resistant pathogens require the search for alternative antimicrobial treatments. Bacteriophage (phage) preparations are currently the only viable alternative to antibiotics. The therapeutic effect of bacteriophages is significantly different from antibiotics, making them active against multidrug-resistant bacteria. Bacteriophage therapy - creation of viral infection in bacteria. However, the effectiveness of treatment depends primarily on the quality of commercial bacteriophage preparations. The collections of bacteriophages is constantly updated in industrial production but in order to increase the lytic activity of bacteriophage preparations to the uropathogens of our clinic, it is necessary to constantly determine the sensitivity of the main pathogens of infection to commercial bacteriophage preparations and regularly update (adapting) commercial phage preparations. Purpose. Improve the lytic activity of commercial bacteriophage preparations; adapt them to the pathogens of a particular hospital. Materials and methods. From 2017 to the present, at the Research Institute of Urology and Interventional Radiology named after N.A. Lopatkin, a branch of the National Medical Research Center of Radiology of the Ministry of Health of Russia (hereinafter referred to as the Research Institute of Urology), regular work has been carried out to update (adapt) commercial bacteriophage preparations to the uropathogens. The most problematic multidrug-resistant microorganisms -pathogens of urological infection- were send for phage production. Wastewater from the clinical building of the Institute of Urology was also send to phage production. Results. Analysis of data on antibiotic- and phage resistance of uropathogens for 2022 indicates multi-resistance of microorganisms in our clinic. Antibiotic resistance phenotypes of pathogens in the urine of outpatients and inpatients have been defined as a whole: extended-spectrum beta-lactamases (ESBLs): 5.3-1.6%, respectively (E.coli-38%; Klebsiella spp. 62%; Proteus mir. 38,5%; P. aeruginosa -61%) methicillin-resistant Staphylococcus aureus (MRSA) 9.9-8.4%; carbapenem-resistant enterobacteria (CRE) 1.7-4.5% (E.coli-1.9%; Klebsiella spp. 29%); Proteus mir. 5%; P. aeruginosa -47.7%) and bacteriophage resistance of uropathogens 4-16.3% . After adaptation of bacteriophage preparations, an increase in their lytic activity to specific microorganisms was obtained by an average of 14% (from 78% to 92%), and an increase in the lytic activity of Sextafag, a polyvalent piobacteriophage containing phagolysate filtrates of Staphylococcus spp., Streptococcus spp., Proteus (P. vulgaris, P. mirabilis), Pseudomonas aeruginosa, Klebsiella pneumonia, E. coli by 16% (48% to 64%) Conclusions. To improve the lytic activity of bacteriophage preparations and improve the effectiveness of urological infection treatment, it is necessary to constantly update the composition of the cocktail of commercial bacteriophage preparations with phages from hospital wastewater and adapt them to freshly isolated microorganisms from urological patients.