Phage Preparations Research Articles

Control of Salmonella enterica spp. enterica in milk and raw milk cheese using commercial bacteriophage preparations.

Control of Salmonella enterica spp. enterica in milk and raw milk cheese using commercial bacteriophage preparations.

Preparation and pharmacokinetic evaluation of Staphylococcus phage COP-80B for treatment of periprosthetic joint infections in a mouse model

Preparation and pharmacokinetic evaluation of Staphylococcus phage COP-80B for treatment of periprosthetic joint infections in a mouse model

Characterization and purification of Pseudomonas aeruginosa phages for the treatment of canine infections

BackgroundPseudomonas aeruginosa is an opportunistic pathogen that causes infections in both human and veterinary medicine, presenting significant challenges in treatment because of biofilm production and its intrinsic resistance. This problem is exacerbated by the increase in acquired antimicrobial resistance. Bacteriophage (phage) therapy has emerged as a promising alternative for treating infection classically treated with antibiotics, offering a targeted approach to combat this infection. This study aimed to evaluate the therapeutic potential of 7 phages, focusing on their suitability for treating canine infections, as well as their purification and safety analysis for therapeutic use.ResultsTwo self-isolated phages and five provided phages were analysed. All tested phages reduced bacterial load in vitro; however, their efficacy varied across different concentrations. The host range analysis revealed a spectrum between 9.8 and 68.6% of canine clinical P. aeruginosa isolates. In our in vitro tests 3 out of 7 phages were able to significantly reduce the biofilm biomass, achieving reductions up to 93.38%. The sequence analysis did not discover known virulence factors and genes connected to antimicrobial resistance mechanisms. The self-isolated phages were classified as lysogenic, whereas the other phages had a lytic infection cycle. Through the purification of the phages, high-titre phage preparations (> 1011 PFU/ml) were generated with high stability for at least 1.5 years. The tested endotoxin units are below the regulatory limits.ConclusionInvestigating phages as alternative treatment option seems promising with lytic phages covering a broad host range and a genomic potential for biofilm degradation. These findings support the development of phage cocktails as a targeted alternative for treating canine P. aeruginosa infections, particularly in cases of antibiotic resistance, and highlight the importance of selecting well-characterized lytic phages for therapeutic efficacy and safety.

Reversible excision of the wzy locus in Salmonella Typhimurium may aid recovery following phage predation.

Bacteriophage (phage) are promising novel antimicrobials but a key challenge to their effective implementation is the rapid emergence of phage resistance. An improved understanding of phage-host interactions is therefore needed. The Anderson phage typing scheme differentiates closely related strains of Salmonella enterica serovar Typhimurium (S. Typhimurium) based on sensitivity to a panel of phage preparations. Switches in phage type are indicative of changes in phage sensitivity and inform on the dynamics of phage interaction with their host bacteria. We investigated the molecular basis of switches between the relatively phage sensitive S. Typhimurium DT8 and phage resistant DT30 strains that are present in the same phylogenetic clade. DT30 strains emerged from DT8 strains predominantly by deletion of a genomic region affecting the wzy locus encoding an O-antigen polymerase. The deletion site was flanked by two perfect direct repeats designated attL and attR. During broth culture in the presence of a typing phage that used O-antigen as primary receptor the Δwzy genotype increased in frequency compared with culture in the absence of phage and removal of attL prevented deletion of the wzy locus. Co-culture of S. Typhimurium DT8 with a strain lacking wzy resulted in reversion of the latter to wild type. We propose a model in which reversible deletion of the wzy locus enables recovery of S. Typhimurium DT8 following predation by phage that use O-antigen as their primary receptor. This was consistent with ancestral state reconstruction of DT8 and DT30 phylogeny that supported a model of reversible transition from DT8 to DT30 in natural populations.

Scalable purification of bacteriophages preparations.

Scalable purification of bacteriophages preparations.

The Susceptibility of Klebsiella Pneumoniae Strains Isolated from COVID-19 Patients to Commercially Available Bacteriophage Medications

Background. Superinfection caused by Klebsiella pneumoniae occupies a leading position in the structure of bacterial complications in COVID-19 patients. The intensive circulation of Klebsiella in specialised hospitals has contributed to the consolidation of the most clinically and epidemiologically important strains of this pathogen, in particular, representatives of hypervirulent and carbapenem-resistant clonal lines, which have not lost their relevance even in the post-pandemic period. The use of bacteriophages as therapeutic and anti-epidemic agents seems justified given the widespread use of multidrugresistant strains of K. pneumoniae.Aim of the study. To evaluate the susceptibility of K. pneumoniae strains associated with nosocomial infections in patients with COVID-19 to polyvalent bacteriophage medications.Materials and methods. The study included 96 non-repeating K. pneumoniae strains isolated from clinical material of patients admitted to a major hospital in St. Petersburg with severe and moderate forms of COVID-19 from May 2020 to January 2021. The susceptibility of clinical strains to bacteriophages was assessed using the spot test analysis. Commercially available bacteriophage preparations used for testing included the following: purified polyvalent pyobacteriophage, sextaphage, and purified polyvalent Klebsiella pyobacteriophage. In order to identify the probable mechanisms of resistance of hospital strains of K. pneumoniae, the nucleotide sequences of the genomes of 6 strains of this pathogen belonging to the dominant hospital genetic lines ST3, ST39, ST307, ST395, ST874 were studied.Results. Negative results of spot tests were observed in 32.29% (95% CI=23.8–42.2) of cases; in general, the proportion of patients eligible for treatment with phage therapy was 49% (95% CI=39.2–58.8). Loci of class 1 subtypes IV-A3 and I-E, potentially associated with resistance to CRISPR-Cas, were identified in the genome structure of the studied strains, as well as a number of prophage sequences potentially associated with resistance to bacteriophages.Conclusion. The study demonstrated low activity of polyvalent bacteriophage medications against K. pneumoniae strains causing nosocomial infections in patients with COVID-19. Increasing the diversity of bacteriophage strains active against epidemiologically relevant K. pneumoniae clones can expand the possibilities of phage therapy for Klebsiella infections. The rational use of medications containing these bacteriophages is possible within the paradigm of personalised phage therapy.

Modern Approaches to Bacteriophage Therapy of Infectious Diseases

The literature review includes works by domestic and foreign authors concerning the use of bacteriophages as an alternative method of therapy and prevention of the development of the inflammatory process in case of bacterial infections. The paper provides historical information about phage therapy. The properties of moderate and virulent bacteriophages, the main mechanisms of interaction of bacteriophage-based preparations with a bacterial cell are considered. Special attention is paid to the characteristic differences between the bacteriophage–bacterium interaction and the effects of antibiotics on microorganisms. The review contains information about the features of the surface binding of phages to microorganisms, anti-phage bacterial protection systems, as well as about the molecular and genetic antibacterial mechanisms of phage counteraction. The data on experimental and clinical studies of anti-phage cellular and humoral immunity and the effect of phages on cytokine production are presented. The literature review highlights the current state of the issue of the therapeutic and prophylactic significance of bacteriophages in relation to a wide range of bacterial infections. Special attention is paid to the literature concerning the study of information on the use of bacteriophages for the prevention and treatment of diseases caused by particularly dangerous microorganisms. The review contains information on the use of phage therapy as an alternative to antibiotics method to combat bacterial infection, both as an independent means of prevention and treatment, and in combination with antibiotic therapy. The data on the various mechanisms of synergy of bacteriophage preparations and antibiotics are presented. The review includes literature sources describing the occurrence of pathological reactions to the introduction of bacteriophages, as well as examples convincingly proving the effectiveness of an integrated approach strategy with the inclusion of phages in infectious disease control schemes, which should occupy a certain niche in the future.

Development of an Anti-Zearalenone Nanobody Phage Display Library and Preparation of Specific Nanobodies.

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 1011 plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody's future integration into rapid on-site detection methods for ZEN in both animal feed and human food products.

Clinical Application of Bacteriophage Therapy in Children.

Bacteriophages are highly specific in targeting single pathogens in the treatment of bacterial infections and thus offer a promising alternative to antibiotic therapies with the potential for essentially no impact to the child's own microbiome. Bacteriophages have been evaluated by investigators for several pathogens and tissue site infections in adults, but data in children are lacking. The application of this therapy in pediatrics provides an extraordinary opportunity to redesign our approach to directed, precision medicine antimicrobial therapy for infants and children for both acute infections and for chronic infections, particularly those caused by multidrug-resistant pathogens. In this therapeutic approach, a bacteriophage preparation would be selected exclusively for and limited to the patient's specific pathogen, thus minimizing the collateral damage to the child's microbiome, as often demonstrated with antibiotic therapy. In the following article, we describe these novel anti-infective biological agents, review the recent literature, discuss practical considerations and limitations, and share potential applications for both topical and systemic administration of bacteriophages, either as primary therapy or in combination with traditional antibiotics. Additionally, important areas of interest for pediatric research are discussed.

Whole-body Bacteriophage Distribution Characterized by a Physiologically based Pharmacokinetic Model.

In 2019 there were over 2.8 million cases of antibiotic-resistant bacterial infection in the US with gram negative organisms having up to a 6% rate of mortality. Bacteriophage (phage) therapy holds great promise to treat such infections. However, the biologic features which influence the pharmacokinetics (PK) of phage have been difficult to characterize due to a lack of standardized protocols of phage purification, tissue assay, and labeling. Here we present robust methods for ultrapure phage preparation as well as non-destructive highly stable attachment of radio-iodide to phage using the well described Sulfo-SHPP linker. We purified and radiolabeled the phage strains, PAML-31-1, OMKO1, and Luz24 lytic to drug-resistant Pseudomonas aeruginosa for biodistribution assay in normal young adult CD-1 mice injected via penile vein. Groups of 5 mice were euthanized and tissues/organs removed for weighing and scintillation well counting of I-125 activity at 30 min, 1h, 2h, 4h, 8h, and 24h. A physiologically based PK (PBPK) model was then constructed focusing on compartments describing blood, lung, muscle, bone, liver, stomach, spleen, small intestines, large intestines, and kidney. Model permeability coefficient (PS) was estimated across all organs as being 0.0227. Tissue partition coefficients (KP) were estimated for high perfusion organs (lung and kidney) as 0.000138, GI organs (liver, spleen, and stomach) as 0.627, and all other organs as 0.220. Elimination was governed by MPS-mediated elimination (TMPS,deg) and active secretion at epithelial barriers (CLActive), which were estimated as 0.00301 h and 0.0145 L/h/kg, respectively. Monte Caro simulations showed that the rapid elimination phage in humans is expected, resulting in phage blood concentrations being lower than 102 PFU/mL (limit of quantification by plaque assay) by 12 hours. As such, multi-dose regimens and continuous infusion regimens were the only strategies that allowed continuously detectible phage concentrations. Evaluation of different dose levels showed that at a maximum dose of 1012 PFU, phage concentrations are expected to be approximately 107 PFU/g. Our physiologically based PK model of phage represents the first rigorous pre-clinical assessment of phage PK utilizing contemporary pharmacometric approaches amenable to both pre-clinical and clinical study design.

Isolation and characterization of a broad-spectrum bacteriophage against multi-drug resistant Escherichia coli from waterfowl field.

Isolation and characterization of a broad-spectrum bacteriophage against multi-drug resistant Escherichia coli from waterfowl field.

Evaluation of host immune responses to Mycobacteriophage Fionnbharth by route of delivery

For much of the last decade, tuberculosis (TB) was the leading cause of mortality due to an infectious pathogen (Mycobacterium tuberculosis, M.tb). Approximately 1.3 million deaths in 2023 worldwide were attributed to TB disease. Focused intervention strategies to block transmission would significantly reduce the global health burden of TB. Mycobacteriophages (phages) are a sorely underutilized biologic therapy for the pathogen M.tb, and here we aimed to address outstanding questions about their utility for clinical applications. We aimed to determine the impact of repeated mucosal or intravenous (IV) delivery of representative anti-M.tb phage FionnbharthΔ45Δ47 (Fionnbharth) in a preclinical mouse model. In addition, we specifically sought to understand which route induced anti-phage antibodies, which may reduce the long-term impact of phage therapy. C57BL/6 mice were dosed weekly for 6 weeks by either route and serum and bronchoalveolar lavage fluid (BALf) were evaluated for anti-phage humoral responses by ELISA. We found that aerosol delivery disperses phage across all lung lobes where M.tb is also found after experimental infection by the same route. Repeated aerosol delivery was well tolerated and did not induce robust neutralizing humoral immunity. In contrast, Mice receiving IV phage developed increasing magnitude and neutralizing total IgG and IgA responses over time. To determine whether pre-treatment environmental exposure to Fionnbharth-like phages could induce antibody responses that are potentially neutralizing, ~ 500 human plasma samples from normal donors were evaluated by ELISA. We observed that 5% of samples had antibodies to Fionnbharth (with end point titers > 10− 3 dilution), although none were neutralizing. Furthermore, we found that highly-purified phage preparations did not activate mouse or human derived toll like receptor (TLR) 4 or TLR9 in HEKblue reporter assays. These data together support using Fionnbharth in anti-M.tb therapy phage cocktail strategies and that aerosol delivery should be prioritized for further efficacy testing.

Possibilities of rational application of bacteriophages in the therapy of infections caused by polyresistant strains of Klebsiella pneumoniae

The issue of combating antimicrobial resistance has been relevant for several decades, since the arsenal of effective antibiotics used in infectious diseases is significantly reduced, and the development of new antibacterial drugs is significantly difficult and expensive. At the same time, the possibility of widespread use of bacteriophages in hospital settings is being discussed.Goal. Evaluation of the sensitivity to bacteriophages of hospital strains of K.pneumoniae isolated from patients in the departments of surgery and intensive care and intensive care of the Structure Clinical Infectious Disease Hospital named after S.P.Botkin.Materials and methods. The activity of bacteriophage preparations “Sextafag” (series p158, p220, p242), “Klebsiell polyvalent bacteriophage” (series y04, y07, y10, y16), “Polyvalent Pyobacteriophage purified” (series y10, y291022) (Manufacturer: NPO Microgen JSC, Moscow) was determined by preparing suspensions of microorganisms, their sowing on nutrient media with subsequent application of bacteriophages, taking into account and interpreting the results.Results. The majority of polyresistant K.pneumoniae strains showed sensitivity to the presented bacteriophage preparations, which allows them to be considered as additional antibacterial agents for the treatment of these groups of patients.Conclusion. The presented results of the study showed that hospital polyand pan­resistant strains of K.pneumoniae are sensitive to preparations of monoand polyvalent bacteriophages, which can be considered as a potential alternative in conditions of antibiotic resistance. The study did not reveal a link between the antibiotic resistance profile of the culture and sensitivity to bacteriophages.

Evaluation of lytic activity of commercial bacteriophage preparations against Klebsiella pneumoniae associated bronchopneumonia of cattle

As the number of reports of antimicrobial resistance among various bacterial pathogens is increasing, there has been a revitalisation of research aimed at improving therapeutic measures associated with phage therapy. However, the fact that data on the effective use of phage-containing preparations are limited is of considerable concern, which, in turn, significantly hampers the active introduction of this alternative to antibiotics in the complex of anti-epizootic measures. In this regard, the aim of the study is to analyses the effectiveness of liquid commercial preparations of bacteriophages in assessing the lytic activity against Klebsiella pneumoniae isolated from calves with signs of bronchopneumonia, in vitro. Determination of phage particles was carried out by the conventional Gratia method (agar layers). The lytic activity of bacteriophages was evaluated by «spot-test» method. The analysis of our results demonstrates the possibility of potential application of commercial phage -containing preparations as part of the scheme of therapeutic measures in infectious bronchopneumonia of farm animals. Klebsiella pneumoniae strains isolated from nasopharyngeal mucus of calves showed sensitivity to bacteriophages included in commercial biopreparations used in the study. As the problem of growing antibiotic resistance among microorganisms is increasing, phage therapy becomes an attractive alternative.

Оценка эффективности экспериментального препарата бактериофага против сальмонеллеза свиней

The prevalence of Salmonella strains that cause invasive forms of infection and the growing rates of antibiotic resistance of individual serotypes of the pathogen raise a number of pressing challenges to the search for new antimicrobial agents for the prevention and treatment of salmonellosis. Creating an effective product that can protect piglets from salmonellosis during a period of high risk of infection during weaning is an urgent task. As a result of the conducted research, the preventive and curative effectiveness of the bacteriophage preparation on laboratory and target animals – piglets of weaning age was studied, and a rational scheme for its use was proposed.

Bacteriophage therapy in the era of bacterial resistance: future prospects

Antibiotic misuse escalates antibacterial resistance. Bacteriophages are being explored as a viable solution. They not only destroy pathogens but also promote bacterial community homeostasis. This manuscript outlines bacteriophage therapeutic strategies, including cocktail therapy, synergistic therapy, and artificial phage modification. Also addressed are the phage effects on bacterial efflux pumps and their role in quorum sensing. The delivery methods and precautions of bacteriophage preparations are also listed. Potential future research may involve further exploring uncultured phage mechanisms using machine learning from the expanding phage gene database; or probe specific phage physiological activities and mechanisms using multiomics techniques.

Bacteriophage therapy and prevention of infectious and inflammatory complications after endoscopic urologic surgery

Relevance. Given the growing antibiotic resistance of pathogens of urologic infection to antimicrobial drugs, one of the urgent problems is the search for alternative methods of antimicrobial prevention of infectious and inflammatory complications after surgical interventions, as well as an adequate therapy regimen during the postoperative period. Bacteriophage preparation is an alternative method for the treatment and prevention of infectious and inflammatory urological diseases.Objective. To determine an effective method for preoperative antimicrobial prevention of infectious and inflammatory complications in patients with kidney stones subjected to percutaneous nephrolithotripsy (PNLT).Methods. The study included 90 patients with coralloid or multiple large kidney stones who underwent PNLT. Before PNLT, all patients underwent bacteriological urinalysis in order to determine sensitivity not only to antibiotics and bacteriophage preparations. Urine sampling was performed for further microbiological cultural examination during renal pelvic puncture on the 3rd and 7th days after PNLT. Depending on the technique of perioperative prophylaxis, three groups of 30 were formed. Group 1 patients were intravenously injected with 1000 mg of ciprofloxacin throughout the operation and then intravenously dipped with 1000 mg once a day for 3–5 days. Group 2 received intramuscular cefotaxime + sulbactam (1.0+0.5) twice a day 2 h before surgery intramuscularly once. In group 3, patients received oral mycobacteriophage 40 ml polyvalent purified orally 1 h before surgery and 40 ml orally 3 times a day for 3–5 days after surgery.Results. In all three groups, the development of infectious complications in patients was assessed: acute pyelonephritis, systemic inflammatory reaction syndrome (SERS) or urosepsis. No serious infectious or inflammatory complications were observed in any group during the early postoperative period. The development of CVD after PNLT was noted on the 1st–3rd postoperative day (in 26.6 %, 20 and 20 % of patients of the 1st, 2nd and 3rd groups, respectively). However, on the 4th–7th day after PNLT, normal blood parameters (leukocytes, rod-shaped neutrophils), temperature, and general well-being were noted.Conclusion. The same effectiveness of different antimicrobial regimens for preventing infectious and inflammatory complications after PNLT was similar. Bacteriophage preparations are effective and can prevent infectious and inflammatory complications following PNLT. The development of CVD after PNLT on the 1st–3rd day after surgery cannot be correlated only with the antimicrobial drugs used and the method of their administration (intravenously, intramuscularly and orally). Most likely, the development of CVD is associated with operational injury.

Lifespan and bioactivity improvement of bacteriophages immobilized on carboxymethylcellulose - Cationic starch-coated paper for food packaging.

Lifespan and bioactivity improvement of bacteriophages immobilized on carboxymethylcellulose - Cationic starch-coated paper for food packaging.

In Vitro Susceptibility of Clinical and Carrier Strains of Staphylococcus aureus to STAFAL® Phage Preparation.

The treatment of infections caused by Staphylococcus aureus is currently complicated by the increasing number of strains resistant to antimicrobial agents. One promising way to solve this problem is phage therapy. Due to the lack of data on the effectiveness and safety of phage preparations, STAFAL® is the only registered phage preparation for the treatment of infectious diseases in the Slovak Republic and the entire European Union. The aim of this work was to determine the effectiveness of the STAFAL® phage preparation against S. aureus strains of different origins with variable sensitivity to antimicrobial substances and with different genetic backgrounds. For this purpose, 111 carrier strains, 35 clinical isolates from bloodstream infections, and 46 strains from skin and soft tissue infections were analysed. The effectiveness of STAFAL® was determined by the plaque forming method. STAFAL® was effective against 74.0% of the strains tested. Susceptibility to this phage preparation was significantly higher in strains resistant to methicillin (MRSA), erythromycin and clindamycin (p < 0.05). The high efficiency of the STAFAL® preparation was confirmed against spa types t003, t024 and t032, typical of the hospital environment. The in vitro results indicate high therapeutic potential of the STAFAL® antistaphylococcal phage preparation, especially against MRSA strains.

Phage Therapy in Traumatology: A Review on Perspectives for Treating Acute Wounds and Post-Surgical Complications

Full-scale hostilities in Ukraine led to an unprecedented number of victims with serious injuries, including gunshot wounds, broken bones, and mine-explosive injuries. Wound infections are one of the main causes of non-combat losses of personnel. A prerequisite for the development of a wound infection, among other things, is high microbial contamination of combat wounds. Major bacteria causing such infections are staphylococci (Staphylococcus aureus, S. epidermidis), streptococci (Streptococcus pyogenes, St. agalictiae), enterococci (Enterococcus faecalis), gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii), anaerobic bacteria (Clostridium perfringens, Bacterioides spp.), etc. Modern data indicate a change in the current species composition of causative agents of wound infections, an increase both in the polyresistance of the microbiota to antibacterial drugs and in the frequency of biofilm formation protecting pathogenic microorganisms from antimicrobial therapy and the patient’s immune response. Such purulent bacterial infections require new approaches to therapy. Taking into account the large number of combat injuries in Ukraine, the use of bacteriophages as specific agents for the biological control of pathogenic microbiota is of particular importance. In view of the nature of injuries, phage preparations intended for the treatment of purulent-surgical infections of wound and burn surfaces are considered the most promising and in demand today. This review summarizes modern data on the use of phage preparations in clinics and in orthopedics and traumatology in particular. Issues of the delivery of phages to the focus of infection, their effective dose, duration of therapy, and the possibility of combining it with antibiotic therapy are discussed. Information about phage therapy programs that have already been implemented in some countries is presented. The advantages and disadvantages of the use of bacteriophages for personalized therapy of severe patients are highlighted, and the prospects for further research are indicated.