Phage Titer Research Articles

Pharmacokinetics and Pharmacodynamics of a Novel Virulent Klebsiella Phage Kp_Pokalde_002 in a Mouse Model.

Phage therapy is one of the most promising alternatives to antibiotics as we face global antibiotic resistance crisis. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of phage therapy are largely unknown. In the present study, we aimed to evaluate the PK/PD of a locally isolated virulent novel øKp_Pokalde_002 (Podoviridae, C1 morphotype) that infects carbapenem-resistant Klebsiella pneumoniae (Kp56) using oral and intraperitoneal (IP) route in a mouse model. The result showed that the øKp_Pokalde_002 rapidly distributed into the systemic circulation within an hour via both oral and IP routes. A higher concentration of phage in plasma was found after 4 h (2.3 x 105 PFU/ml) and 8 h (7.3 x 104 PFU/ml) of administration through IP and oral route, respectively. The phage titer significantly decreased in the blood and other tissues, liver, kidneys, and spleen after 24 h and completely cleared after 72 h of administration. In the Kp56 infection model, the bacterial count significantly decreased in the blood and other organs by 4–7 log10 CFU/ml after 24 h of øKp_Pokalde_002 administration. Elimination half-life of øKp_Pokalde_002 was relatively shorter in the presence of host-bacteria Kp56 compared to phage only, suggesting rapid clearance of phage in the presence of susceptible host. Further, administration of the øKp_Pokalde_002 alone in healthy mice (via IP or oral) did not stimulate pro-inflammatory cytokines (TNF-α and IL-6). Also, treatment with øKp_Pokalde_002 resulted in a significant reduction of pro-inflammatory cytokines (TNF-α and IL-6) caused by bacterial infection, thereby reducing the tissue inflammation. In conclusion, the øKp_Pokalde_002 possess good PK/PD properties and can be considered as a potent therapeutic candidate for future phage therapy in carbapenem-resistant K. pneumoniae infections.

Occurrence and Accumulation of Human Enteric Viruses and Phages in Process Water from the Fresh Produce Industry.

The virological quality of process water (PW) used by the produce industry has received limited attention. As a first step to overcoming technical limitations in monitoring viruses in PW, the analytical performance of ultrafiltration was assessed to concentrate viral particles from 20 L of spiked PW. The selected method used for sample concentration of PW was carefully validated, thus enabling the accurate quantification and estimation of viral titers of human enteric viruses and phages. PW from the produce industry was collected periodically from the washing tanks of commercial facilities. The analysis of coliphages was performed by plaque assay, while the occurrence of enteric viruses and crAssphage was determined by molecular techniques. Significant differences in the physicochemical composition of PW, mostly due to the different nature of fresh produce types and differences in the sanitizer used in commercial operation, were observed. Accumulation of crAssphage and coliphages was observed in PW, but correlation with human enteric viruses was not possible due to the low prevalence of these pathogens in the PW analyzed. The obtained results showed that depending on the type of product washed, the product/water ratio and the residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly.

Evolution of the complex treatment of purulent diseases of the hand including laser irradiation (a review)

Effects of 1270 nm laser light irradiation at phage particles of virulent klebsiellosis bacteriophage were studied. The medical klebsiellosis bacteriophage, manufactured industrially, was taken as the study object. Klebsiella pneumonia N 296, sensitive to the selected phage, was used as a test-culture. An experimental device manufactured by LTD «New surgical technologies» was used as a source of light. Semiconductor diodes generate light with wavelength 1270 nm (1268–1272 nm) in the continuous mode. The number of viable phage particles in the initial solution of klebsiellosis bacteriophage was 5×108. Irradiation of the phage with 1270 nm laser light decreased the number of viable phage particles to 105. Results did not practically depend on the exposure time, i. e. phage titers were equally reduced when exposed to laser light for 5 min, 10 min and 15 min. Irradiation of Klebsiella bacteriophage with 1270 nm laser light reduced the number of viable phage particles by 3 log orders (initial titer was 108; after irradiation – 105 negative phage colonies). It is indicative of their damage. Mechanisms of phage particle damage should be the object of further research so as to defi ne if laser irradiation with the above mentioned wavelengths can be used in medical practice.

Bacteriophage Therapy for Difficult-to-Treat Infections: The Implementation of a Multidisciplinary Phage Task Force (The PHAGEFORCE Study Protocol).

In times where only a few novel antibiotics are to be expected, antimicrobial resistance remains an expanding global health threat. In case of chronic infections caused by therapy-resistant pathogens, physicians have limited therapeutic options, which are often associated with detrimental consequences for the patient. This has resulted in a renewed interest in alternative strategies, such as bacteriophage (phage) therapy. However, there are still important hurdles that currently impede the more widespread implementation of phage therapy in clinical practice. First, the limited number of good-quality case series and clinical trials have failed to show the optimal application protocol in terms of route of administration, frequency of administration, treatment duration and phage titer. Second, there is limited information on the systemic effects of phage therapy. Finally, in the past, phage therapy has been applied intuitively in terms of the selection of phages and their combination as parts of phage cocktails. This has led to an enormous heterogeneity in previously published studies, resulting in a lack of reliable safety and efficacy data for phage therapy. We hereby present a study protocol that addresses these scientific hurdles using a multidisciplinary approach, bringing together the experience of clinical, pharmaceutical and molecular microbiology experts.

Comparison of Delivery Methods in Phage Therapy against Flavobacterium columnare Infections in Rainbow Trout.

Viruses of bacteria, bacteriophages, specifically infect their bacterial hosts with minimal effects on the surrounding microbiota. They have the potential to be used in the prevention and treatment of bacterial infections, including in the field of food production. In aquaculture settings, disease-causing bacteria are often transmitted through the water body, providing several applications for phage-based targeting of pathogens, in the rearing environment, and in the fish. We tested delivery of phages by different methods (via baths, in phage-coated material, and via oral delivery in feed) to prevent and treat Flavobacterium columnare infections in rainbow trout fry using three phages (FCOV-S1, FCOV-F2, and FCL-2) and their hosts (FCO-S1, FCO-F2, and B185, respectively). Bath treatments given before bacterial infection and at the onset of the disease symptoms were the most efficient way to prevent F. columnare infections in rainbow trout, possibly due to the external nature of the disease. In a flow-through system, the presence of phage-coated plastic sheets delayed the onset of the disease. The oral administration of phages first increased disease progression, although total mortality was lower at the end of the experiment. When analysed for shelf-life, phage titers remained highest when maintained in bacterial culture media and in sterile lake water. Our results show that successful phage therapy treatment in the aquaculture setting requires optimisation of phage delivery methods in vivo.

Improving Phage-Biofilm In Vitro Experimentation.

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

In Vitro and In Vivo Gastrointestinal Survival of Non-Encapsulated and Microencapsulated Salmonella Bacteriophages: Implications for Bacteriophage Therapy in Poultry.

The therapeutic use of bacteriophages is recognized as a viable method to control Salmonella. Microencapsulation of phages in oral dosage forms may protect phages from inherent challenges of the gastrointestinal tract in chickens. Therefore, the main objective of this study was to assess the survival of Salmonella BP FGS011 (non-encapsulated and microencapsulated) through the gastrointestinal tract under in vitro as well as in vivo conditions after oral administration to 1-day-old chicks. To this end, the phage FGS011 was encapsulated in two different pH-responsive formulations with polymers Eudragit® L100, and Eudragit® S100 using the process of spray drying. Phages encapsulated in either of the two formulations were able to survive exposure to the proventriculus-gizzard in vitro conditions whereas free phages did not. Moreover, phages formulated in polymer Eudragit® S100 would be better suited to deliver phage to the caeca in chickens. In the in vivo assay, no statistically significant differences were observed in the phage concentrations across the gastrointestinal tract for either the free phage or the encapsulated phage given to chicks. This suggested that the pH of the proventriculus/gizzard in young chicks is not sufficiently acidic to cause differential phage titre reductions, thereby allowing free phage survival in vivo.

Broad host range bacteriophages found in rhizosphere soil of a healthy tomato plant in Bulgaria

The urgent need of research of new approaches to control bacterial disease on economical important crops, focuses our attention on bacteriophages as alternative biocontrol agents. Thus, the purpose of this paper is to present the isolation and initial characterization of three bacteriophages (SfXv124t/1, 2 and 3) isolated from rhizosphere soil of a healthy tomato plant in Bulgaria that are capable to lyse three phytopathogenic bacteria. The initial characterization includes determination of: their host range, plaque morphology, optimal storage temperature of pure phage lysates, their sensitivity to UV light, thermal inactivation, optimal multiplicity of infection (MOI) and virion morphology. The obtained results showed that one of the phage isolates was capable to lyse wild strains from three phytopathogenic bacterial species: Xanthomonas vesicatoria, Xanthomonas euvesicatoria and Xanthomonas gardneri, and the two remaining phages were active against X. vesicatoria and X. euvesicatoria. On X. vesicatoria lawn, the phages produced the same plaque types that differed only in their size. Storage at 4 °C for 26 days did not lead to decrease in phage titer as opposed to storage at 28 °C followed by decrease to varying degree for all three phages. The results obtained after exposure of the phage lysates to sunlight (UVA + B) and UVC light in separate experiments showed that UVC had a potent phagocidal effect as after 50 min of exposure there were no viable phages in the samples. UVA an UVB had lethal effect for two of the phage isolates and absolutely no lethal effect for the third one as after 50 min of exposure to sunlight there was no decrease in the initial phage titer. Phage isolates were tested for their thermal inactivation after incubation of pure phage lysates at three different temperatures: 55 °C, 75 °C and 95 °C for a period of 10 and 30 min. The most lethal temperature turned out to be 95 °C as after 10 min there were no viable phages in the samples. Phage isolate SfXv124t/1 was the most susceptible as its titer decreased by 1 lg after 10 min of incubation at 55 °C and by another 1 lg after 30 min. The most thermally resistant isolate was SfXv124t/3 as its titer remained stable after 30 min of incubation at 55 °C and decreased only by lg after incubation at 75 °C for 10 min. The optimal MOI for SfXv124t/3 was 0,01 (tested range 0,01–100) with maximal phage titer, reported at the 24th hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae.

English

Brucellosis, being a zoonotic disease, is difficult to control despite the availability of vaccines. Bovine brucellosis could be controlled effectively using brucellaphages. This study was conducted to isolate bacteriophages against Brucella abortus from cattle farms sewage/slurry samples (n=50). Isolation and propagation of brucellaphages was made through spot and plaque assay. Two samples (n=2) were found positive on the plates as circular, clear, and pinpoint plaques (0.3 to 0.5 mm) with 4.6×106 PFU/mL of brucellaphage titre against Brucella abortus RB51. One- step growth experiments revealed latent period of 120 min and burst size of 93 and 114 PFU for two isolated brucellaphages (ϕP1 and ϕP2) respectively. These phages were unable to lyse Staphylococcus aureus, Streptococcus spp., Salmonella spp., Escherichia coli, Bacillus subtilis and Pasteurella multocida. Isolated brucellaphages were stable up to 60°C and between 7 to 9 pH. No loss in phage titres were observed at 4°C but phage titres were reduced by one log at (-20°C) overnight. There was no effect of SM buffer, normal saline and EDTA on stability of brucellaphages and addition of divalent salts in medium showed significant increase in PFU. However, treatment with SDS and chloroform destroyed the phages in one hour exposure. The phages were found carrying double stranded DNA (~40 kb size) and two prominent protein bands of 45 kDa and 70 kDa. Biocontrol activity of brucellaphages showed average Brucella abortus count reduction to 4.5×103 from 5.0×108 CFU/ g of soil sample within 48 h when treated with maximum phage titre of 5.0×1011 PFU/ mL. Hence, this study revealed that brucellaphages are present in our environment and can potentially be consider for their cost-effective practical applications in inactivation of Brucella abortus after comprehensive experimental evaluations. © 2021 Friends Science Publishers

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A.hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24hr. Without the existence of the phage, the highest mortality rate was 68.3±2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33±2.9% or 16.67±2.9% at the phage dose of log 6.2±0.09 or log 4.2±0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Efficacy assessment of PEV2 phage on Galleria mellonella larvae infected with a Pseudomonas aeruginosa dog otitis isolate

Pseudomonas (P.) aeruginosa is the most frequently isolated Gram-negative bacteria in dog otitis. Antimicrobial resistance is particularly prevalent in P. aeruginosa and phage therapy represents a promising alternative therapeutic strategy. The aim of this study was to assess the efficacy of the PEV2 phage against a clinical P. aeruginosa isolate from a canine otitis using a Galleria (G.) mellonella larvae model. The genomic DNA of PAV237 P. aeruginosa isolate was sequenced and analysed. In a first main experiment, the efficacy of PEV2 phage against PAV237 was assessed at different multiplicities of infection (MOI) (50,000, 5000, 500, 50) by analyzing the larvae survival rate during 4 days. In a second experiment, the bacterial and phage titer evolutions were assessed depending on two MOIs (50,000, 5000). No significant survival increase was observed with PEV2 therapy in the infected larvae groups. The generated Kaplan-Meier curves showed that the rate of alive larvae was significantly higher in the non-infected larvae compared to the infected-treated ones irrespective of phage MOIs. An increase of the phage titer was observed at 24 and 48 h post-inoculation (HPI) with both MOIs and the P. aeruginosa titers were lower with MOI 50,000 and 5000 compared to the infectivity control at 24 and 48 HPI. Even if an ineffectiveness of the PEV2 phage was observed on the larvae survival, PEV2 is active against P. aeruginosa in this model and PEV2 replication is correlated with a lower bacterial proliferation in the phage treated larvae.

Evaluation of anhydrous processing and storage methods of the temperate bacteriophage ɸV10 for integration into foodborne pathogen detection methodologies.

Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.

Development of thermosensitive hydrogel wound dressing containing Acinetobacter baumannii phage against wound infections

With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, respectively. Excellent storage stability of the IME-AB2 phage in a ~18 wt% Poloxamer 407 (P407) hydrogel solution was first demonstrated with negligible titer loss (~0.5 log) in 24 months at 4 °C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37 °C demonstrated >5 log10 CFU/ml reduction against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% respectively). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% reduction in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections.

Bacteriophage liquid formulation: A potential green tool for the management of pomegranate bacterial blight

Pomegranate is one of the majorly grown fruit cash crops in India. For the past few decades, it has been observed suffering from bacterial blight disease, hampering the growers' economic benefits. The present study aimed to evaluate bacteriophage-based liquid formulation's potential in managing pomegranate bacterial blight disease caused by Pseudomonas sp. Isolated two phages (vB_Psp.S_PRɸL2 and vB_Psp.M_SSɸL8) against the etiology of pomegranate bacterial blight disease were used for the biocontrol study. Phage based different liquid formulations were developed and evaluated for six month shelf life period at 4 ° and 30 °C temperatures. Among studied liquid formulations for both the phages, the 0.5% whitening clay was found to maintain the most active phage titre. Foliar spray of 0.5% whitening clay liquid formulation of the phages PRɸL2 and SSɸL8 efficiently reduced the pomegranate bacterial blight disease compared to standard streptocycline. Moreover, the preventive application of phages based 0.5% whitening clay liquid formulation showed promising results in the pot experiment under greenhouse conditions.

An Assessment of the Viability of Lytic Phages and Their Potency against Multidrug Resistant Escherichia coli O177 Strains under Simulated Rumen Fermentation Conditions.

Preslaughter starvation and subacute ruminal acidosis in cattle are known to promote ruminal proliferation of atypical enteropathogenic Escherichia coli strains, thereby increasing the risk of meat and milk contamination. Using bacteriophages (henceforth called phages) to control these strains in the rumen is a potentially novel strategy. Therefore, this study evaluated the viability of phages and their efficacy in reducing E. coli O177 cells in a simulated ruminal fermentation system. Fourteen phage treatments were allocated to anaerobic serum bottles containing a grass hay substrate, buffered (pH 6.6–6.8) bovine rumen fluid, and E. coli O177 cells. The serum bottles were then incubated at 39 °C for 48 h. Phage titres quadratically increased with incubation time. Phage-induced reduction of E. coli O177 cell counts reached maximum values of 61.02–62.74% and 62.35–66.92% for single phages and phage cocktails, respectively. The highest E. coli O177 cell count reduction occurred in samples treated with vB_EcoM_366B (62.31%), vB_EcoM_3A1 (62.74%), vB_EcoMC3 (66.67%), vB_EcoMC4 (66.92%), and vB_EcoMC6 (66.42%) phages. In conclusion, lytic phages effectively reduced E. coli O177 cells under artificial rumen fermentation conditions, thus could be used as a biocontrol strategy in live cattle to reduce meat and milk contamination in abattoirs and milking parlours, respectively.

Isolation of Diarrheagenic Escherichia coli and their Specific Phages from Rabbits

This study designed to isolation virulence E. coli, specific phages and identify in rabbits biochemically, serologically, detect virulence genes using PCR and susceptibility to antimicrobial range. For propose fifty-fecal-swab samples collected from diarrheic and freshly-dead-young-rabbits were presented to bacteriological examination. Data showed twenty-seven samples (54%) gave positive results of Escherichia coli. Data in-vitro Pathogenicity-test using Congo-red(CR) binding assay showed eight E. coli isolates (29.63%) were Congo-red positive. Antimicrobial susceptibility to several antibiotics was studied. E. coli isolates showed different susceptibility degrees to antibiotics, and isolates 3,4 and 8 categorized as multidrug resistant isolates. Three selected E. coli isolates were serologically identified classified as O169, O125 and O158 serotypes for isolates 4,8,3 respectively. Conventional polymerase chain reaction for detection eaeA and Stx1 virulence genes revealed E. coli serotypes O158 expressed eaeA gene, but others, O169 and O125 don't expressed. While, none serotypes expressed Stx1 gene. Phages may be used effectively to control of pathogenic E. coli, colonizing farm rabbit's intestines. However, harsh acidic-conditions and digestive enzymes activities influence phage infectivity, and decrease efficiency in application-trails. Natural-defensive-barrier development was being suitable for oral administration to farm poultry presented acid-stability. Encapsulated pahge beads in gelatin chitosan -matrix showed partial phage titer reductions. Phage beads titers were constant for storage in water, but complete release achieved after 6hr in simulated intestinal solution at 37°C. Finding multidrug resistant enterohaemorrhagic, E. coli serotypes created severe health hazard for rabbits and contact human. Encapsulated phages beads are promising and cost-effective method for bacteriophage targeting intestinal bacteria of farm-rabbits.

Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication.

Cell-free systems allow interference with gene expression processes without requiring elaborate genetic engineering procedures. This makes it ideally suited for rapid prototyping of synthetic biological parts. Inspired by nature's strategies for the control of gene expression via short antisense RNA molecules, we here investigated the use of small DNA (sDNA) for translational inhibition in the context of cell-free protein expression. We designed sDNA molecules to be complementary to the ribosome binding site (RBS) and the downstream coding sequence of targeted mRNA molecules. Depending on sDNA concentration and the promoter used for transcription of the mRNA, this resulted in a reduction of gene expression of targeted genes by up to 50-fold. We applied the cell-free sDNA technique (CF-sDNA) to modulate cell-free gene expression from the native T7 phage genome by suppressing the production of the major capsid protein of the phage. This resulted in a reduced phage titer, but at the same time drastically improved cell-free replication of the phage genome, which we utilized to amplify the T7 genome by more than 15 000-fold in a droplet-based serial dilution experiment. Our simple antisense sDNA approach extends the possibilities to exert translational control in cell-free expression systems, which should prove useful for cell-free prototyping of native phage genomes and also cell-free phage manipulation.

Broad-spectrum nanoparticles against bacteriophage infections.

Viral infections caused by bacteriophages, i.e., viruses that kill bacteria are one of the most dangerous and common threats for bacteria-based bioreactors. More than 70% of biotechnology companies have admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, there are no effective methods to avoid them to date. Herein, we present a novel technology based on nanoparticles that irreversibly deactivates bacteriophages and is safe for bacteria. Our method allows for the unsupervised protection of bacterial processes in the biotechnology industry. Gold nanoparticles coated with a mixture of negatively charged 11-mercapto 1-undecanesulfonic acid (MUS) and hydrophobic 1-octanethiol (OT) ligands are effective at deactivating various types of Escherichia coli-selective phages: T1, T4, and T7. The nanoparticles can lower the titer of phages up to 2 and 5 logs in 6 and 24 h at 50 °C, respectively. A comparative analysis of nanoparticles with different ligand shells illustrates the importance of the combination of negatively charged and hydrophobic ligands that is the key to achieving a good inhibitory concentration (EC50 ≤ 1 μg mL−1) for all tested phages. We show that the nanoparticles are harmless for the commonly used bacteria in industry Escherichia coli and are effective under conditions simulating the environment of bioreactors.

Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells.

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

РАЗРАБОТКА СХЕМЫ ИНДИКАЦИИ БАКТЕРИЙ AEROMONAS SALMONICIDA С ИСПОЛЬЗОВАНИЕМ БАКТЕРИОФАГОВ

The causative agent of furunculosis Aeromonas salmonicida is widely spread around the world and usually affects many cold-water fish (trout), salmon, and less often fish from other families. Currently, there is no scheme for indicating the bacterium of the species A. salmonicida from objects of the external environment, using bacteriophages and accumulation media, which complicates the study of the distribution area of the mentioned microorganism and rapid indication of the pathogen. For this reason, the aim of the study was to develop a scheme for the indication of A. salmonicida bacteria using bacteriophages. To identify field strains of A. salmonicida, we examined 56 samples of water objects in Ulyanovsk and the Ulyanovsk region. Using the environment of accumulation of A. Sl.1 and the differential diagnostic environment A.Sl.2.7 A. salmonicida strains were isolated and typed from 56 samples. We have developed a scheme for the indication of A. salmonicida bacteria based on the modification of the phage titer increase reaction (RSF) using the accumulation medium A.Sl.1. This scheme allows detecting A. salmonicida in the amount of 103m.K./ml within 24 hours. The proposed scheme has the potential to be modified and applied to a whole range of microorganisms, sensitive and specific, allowing to detect bacteria in the substrate in a short time, in the presence of foreign microflora without isolation of pure cultures.