A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Novel multiplex-PCR test for Escherichia coli detection.

Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods. IMPORTANCE: Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Hypoxia modulates human mast cell adhesion to hyaluronic acid.

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

Examining the Effects of an Anti-Salmonella Bacteriophage Preparation, BAFASAL®, on Ex-Vivo Human Gut Microbiome Composition and Function Using a Multi-Omics Approach.

Salmonella infections (salmonellosis) pose serious health risks to humans, usually via food-chain contamination. This foodborne pathogen causes major food losses and human illnesses, with significant economic impacts. Overuse of antibiotics in the food industry has led to multidrug-resistant strains of bacteria, and governments are now restricting their use, leading the food industry to search for alternatives to secure food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics, as they are natural components of the ecosystem. However, when specifically used in the industry, they can also make their way into humans through our food chain or exposure, as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented to animal feeds. To our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in-vitro assay called RapidAIM to investigate the effect of a bacteriophage mixture, BAFASAL®, used in poultry farming on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex-vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment.

Targeting SIRT2 Sensitizes Melanoma Cells to Cisplatin via an EGFR-Dependent Mechanism.

Melanoma cells are resistant to most anticancer chemotherapeutics. Despite poor response rates and short-term efficacy, chemotherapy remains the main approach to treating this cancer. The underlying mechanisms of the intrinsic chemoresistance of melanoma remain unclear, but elucidating these mechanisms is important to improve the efficacy of chemotherapy regimens. Increasing evidence suggests that sirtuin 2 (SIRT2) plays a key role in the response of melanoma cells to chemotherapeutics; thus, in the present study, we evaluated the impact of shRNA-mediated and pharmacological inhibition of SIRT2 on the sensitivity of melanoma cells to cisplatin, which is used in several regimens to treat melanoma patients. We found that cells with SIRT2 inhibition revealed increased sensitivity to cisplatin and exhibited increased accumulation of γ-H2AX and reduced EGFR-AKT-RAF-ERK1/2 (epidermal growth factor receptor-protein B kinase-RAF kinase-extracellular signal-regulated kinase 1/2) pathway signaling compared to control cells. Thus, our results show that sirtuin 2 inhibition increased the in vitro efficacy of cisplatin against melanoma cells.

Recombinant immunotoxin targeting GPC3 is cytotoxic to H446 small cell lung cancer cells.

Glypican-3 (GPC3) is a cell membrane glycoprotein that regulates cell growth and proliferation. Aberrant expression or distribution of GPC3 underlies developmental abnormalities and the development of solid tumours. The strongest evidence for the participation of GPC3 in carcinogenesis stems from studies on hepatocellular carcinoma and lung squamous cell carcinoma. To the best of our knowledge, the role of the GPC3 protein and its potential therapeutic application have never been studied in small cell lung carcinoma (SCLC), despite the known involvement of associated pathways and the high mortality caused by this disease. Therefore, the aim of the present study was to examine GPC3 targeting for SCLC immunotherapy. An immunotoxin carrying an anti-GPC3 antibody (hGC33) and Pseudomonas aeruginosa exotoxin A 38 (PE38) was generated. This hGC33-PE38 protein was overexpressed in E. coli and purified. ADP-ribosylation activity was tested in vitro against eukaryotic translation elongation factor 2. Cell internalisation ability was confirmed by confocal microscopy. Cytotoxicity was analysed by treating liver cancer (HepG2, SNU-398 and SNU-449) and lung cancer (NCI-H510A, NCI-H446, A549 and SK-MES1) cell lines with hGC33-PE38 and estimating viable cells number. A BrdU assay was employed to verify anti-proliferative activity of hGC33-PE38 on treated cells. Fluorescence-activated cell sorting was used for the detection of cell membrane-bound GPC3. The hGC33-PE38 immunotoxin displayed enzymatic activity comparable to native PE38. The protein was efficiently internalised by GPC3-positive cells. Moreover, hGC33-PE38 was cytotoxic to HepG2 cells but had no effect on known GPC3-negative cell lines. The H446 cells were sensitive to hGC33-PE38 (IC50, 70.6±4.6 ng/ml), whereas H510A cells were resistant. Cell surface-bound GPC3 was abundant on the membranes of H446 cells, but absent on H510A. Altogether, the present findings suggested that GPC3 could be considered as a potential therapeutic target for SCLC immunotherapy.

Nucleocapsid and Spike Proteins of the Coronavirus SARS-CoV-2 Induce IL6 in Monocytes and Macrophages-Potential Implications for Cytokine Storm Syndrome.

The pandemic of the new coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has led to the deaths of more than 1.5 million people worldwide. SARS-CoV-2 causes COVID-19, which exhibits wide variation in the course of disease in different people, ranging from asymptomatic and mild courses to very severe courses that can result in respiratory failure and death. Despite the rapid progression of knowledge, we still do not know how individual cells of the immune system interact with the virus or its components, or how immune homeostasis becomes disrupted, leading to the rapid deterioration of a patient's condition. In the present work, we show that SARS-CoV-2 proteins induce the expression and secretion of IL-6 by human monocytes and macrophages, the first line cells of antiviral immune responses. IL-6 may play a negative role in the course of COVID-19 by inhibiting Th1-dependent immunity and stimulating Th17 lymphocytes, thus leading to an increased probability of a cytokine storm.

The Art of Mast Cell Adhesion.

Cell adhesion is one of the basic phenomena occurring in a living organism, affecting many other processes such as proliferation, differentiation, migration, or cell viability. Mast cells (MCs) are important elements involved in defending the host against various pathogens and regulating inflammatory processes. Due to numerous mediators, they are contributing to the modulation of many basic cellular processes in a variety of cells, including the expression and functioning of different adhesive molecules. They also express themselves many adhesive proteins, including ICAM-1, ICAM-3, VCAM-1, integrins, L-selectin, E-cadherin, and N-cadherin. These molecules enable MCs to interact with other cells and components of the extracellular matrix (ECM), creating structures such as adherens junctions and focal adhesion sites, and triggering a signaling cascade. A thorough understanding of these cellular mechanisms can create a better understanding of MC biology and reveal new goals for MC targeted therapy. This review will focus on the current knowledge of adhesion mechanisms with the involvement of MCs. It also provides insight into the influence of MCs or MC-derived mediators on the adhesion molecule expression in different cells.

SARS-CoV-2 Proteins Induce IFNG in Th1 Lymphocytes Generated from CD4+ Cells from Healthy, Unexposed Polish Donors.

The outbreak of the SARS-CoV-2 virus in December 2019 has caused the deaths of several hundred thousand people worldwide. Currently, the pathogenesis of COVID-19 is poorly understood. During the course of COVID-19 infection, many patients experience deterioration, which might be associated with systemic inflammation and cytokine storm syndrome; however, other patients have mild symptoms or are asymptomatic. There are some suggestions that impaired cellular immunity through a reduction in Th1 response and IFNG (interferon gamma) expression, as well as cross-reactivity with common cold coronaviruses, might be involved in the differential COVID-19 course. Here, we show that CD4+ cells isolated from unexposed healthy donors that were differentiated towards the Th1 lineage in the presence of SARS-CoV-2 proteins exhibited induction of IFNG. Interestingly, the same cells induced to differentiate towards a Th17 lineage did not exhibit changes in IFNG expression or Th17-related cytokines. This suggests the cellular response to SARS-CoV-2 viral proteins is primarily associated with Th1 lymphocytes and may be dependent on past infections with common cold coronaviruses or vaccinations that induce unspecific cellular responses, e.g., BCG (Bacillus Calmette-Guérin). Thus, our results might explain the high variability in the course of COVID-19 among populations of different countries.

Identification of Novel Molecular Markers of Human Th17 Cells.

Th17 cells are important players in host defense against pathogens such as Staphylococcus aureus, Candida albicans, and Bacillus anthracis. Th17 cell-mediated inflammation, under certain conditions in which balance in the immune system is disrupted, is the underlying pathogenic mechanism of certain autoimmune disorders, e.g., rheumatoid arthritis, Graves' disease, multiple sclerosis, and psoriasis. In the present study, using transcriptomic profiling, we selected genes and analyzed the expression of these genes to find potential novel markers of Th17 lymphocytes. We found that APOD (apolipoprotein D); C1QL1 (complement component 1, Q subcomponent-like protein 1); and CTSL (cathepsin L) are expressed at significantly higher mRNA and protein levels in Th17 cells than in the Th1, Th2, and Treg subtypes. Interestingly, these genes and the proteins they encode are well associated with the function of Th17 cells, as these cells produce inflammation, which is linked with atherosclerosis and angiogenesis. Furthermore, we found that high expression of these genes in Th17 cells is associated with the acetylation of H2BK12 within their promoters. Thus, our results provide new information regarding this cell type. Based on these results, we also hope to better identify pathological conditions of clinical significance caused by Th17 cells.

Comprehensive Evaluation of the Safety and Efficacy of BAFASAL® Bacteriophage Preparation for the Reduction of Salmonella in the Food Chain.

Bacteriophages are bacterial predators, which are garnering much interest nowadays vis-à-vis the global phenomenon of antimicrobial resistance. Bacteriophage preparations seem to be an alternative to antibiotics, which can be used at all levels of the food production chain. Their safety and efficacy, however, are of public concern. In this study, a detailed evaluation of BAFASAL® preparation was performed. BAFASAL® is a bacteriophage cocktail that reduces Salmonella in poultry farming. In vivo acute and sub-chronic toxicity studies on rats and tolerance study on targeted animals (chicken broiler) conducted according to GLP and OECD guidelines did not reveal any signs of toxicity, which could be associated with BAFASAL® administration. In addition, no evidences of genotoxicity were observed. The tolerance study with 100-times concentrated dose also did not show any statistically significant differences in the assessed parameters. The in vitro crop assay, mimicking normal feed storage and feed application conditions showed that BAFASAL® reduced the number of Salmonella bacteria in experimentally contaminated feed. Moreover, reductions were observed for all examined forms (liquid, powder, spray). Furthermore, the in vivo efficacy study showed that treatment with BAFASAL® significantly decreased Salmonella content in caeca of birds infected with Salmonella Enteritidis. Detailed examination of BAFASAL® in terms of safety and efficacy, adds to the body of evidence that bacteriophages are harmless to animals and effective in the struggle against bacteria.

Growing Trend of Fighting Infections in Aquaculture Environment-Opportunities and Challenges of Phage Therapy.

Phage therapy, a promising alternative to antimicrobial treatment of bacterial diseases, is getting more and more popular, especially due to the rising awareness of antibiotic resistance and restrictions in antibiotics' use. During recent years, we observed a growing trend of bacteriophages' application in aquaculture, which in each year reports high losses due to bacterial diseases. This review provides an update of the status of bacteriophage therapy for the treatment and prevention of infections in the aquatic environment. As it is still mostly in the scientific stage, there are a few constraints that may prevent effective therapy. Therefore, specific characteristics of bacteriophages, that can act in favor or against their successful use in treatment, were described. We underlined aspects that need to be considered: specificity of phages, bacterial resistance, safety, immune response of the host organism, formulation, administration and stability of phage preparations as well as bacteriophages' influence on the environment. The biggest challenge to overcome is finding the right balance between the desired and problematic characteristics of bacteriophages. Finally, regulatory approval challenges may be encountered by bacteriophage manufacturers. Even though there are still some technical constraints connected with the global use of bacteriophage therapy, it was concluded that it can be successfully applied in aquaculture.

Hypoxia regulates human mast cell adhesion to fibronectin via the PI3K/AKT signaling pathway.

A decrease in oxygen concentration is a hallmark of inflammatory reactions resulting from infection or homeostasis disorders. Mast cells interact with extracellular matrix and other cells by adhesion receptors. We investigated the effect of hypoxia on integrin-mediated mast cell adhesion to fibronectin. We found that it was mediated by the α5/ß1 receptor and that hypoxia significantly upregulated this process. Hypoxia-mediated increases in mast cell adhesion occurred without increased surface expression of integrins, suggesting regulation by inside-out integrin signaling. Hypoxia also mediated an increase in phosphorylation of Akt, and PI3'kinase inhibitors abolished hypoxia-mediated mast cell adhesion. Hypoxia upregulates the function of integrin receptors by PI3' kinase-dependent signaling. This process might be important for the location of mast cells at inflammatory sites.

Cardiac glycosides with target at direct and indirect interactions with nuclear receptors.

Cardiac glycosides are compounds isolated from plants and animals and have been known since ancient times. These compounds inhibit the activity of the sodium potassium pump in eukaryotic cells. Cardiac glycosides were used as drugs in heart ailments to increase myocardial contraction force and, at the same time, to lower frequency of this contraction. An increasing number of studies have indicated that the biological effects of these compounds are not limited to inhibition of sodium-potassium pump activity. Furthermore, an increasing number of data have shown that they are synthesized in tissues of mammals, where they may act as a new class of steroid hormones or other hormones by mimicry to modulate various signaling pathways and influence whole organisms. Thus, we discuss the interactions of cardiac glycosides with the nuclear receptor superfamily of transcription factors activated by low-weight molecular ligands (including hormones) that regulate many functions of cells and organisms. Cardiac glycosides of endogenous and exogenous origin by interacting with nuclear receptors can affect the processes regulated by these transcription factors, including hormonal management, immune system, body defense, and carcinogenesis. They can also be treated as initial structures for combinatorial chemistry to produce new compounds (including drugs) with the desired properties.

Anti-Salmonella Potential of New Lactobacillus Strains with the Application in the Poultry Industry.

Probiotics are considered an alternative to antibiotics in the prevention and treatment of Salmonella diseases in poultry. However, to use probiotics as proposed above, it is necessary to evaluate their properties in detail and to select the most effective bacterial strains in the application targeted. In this study, probiotic properties of new Lactobacillus sp. strains were investigated and their antimicrobial activity against 125 environmental strains of Salmonella sp. was determined using the agar slab method. Furthermore, their survival in the presence of bile salts and at low pH, antibiotics susceptibility, aggregation and coaggregation ability, adherence to polystyrene and Caco-2 cells, and cytotoxicity were investigated. Each strain tested showed antagonistic activity against at least 96% of the environmental Salmonella sp. strains and thus representing a highly epidemiologically differentiated collection of poultry isolates. In addition, the probiotic properties of new Lactobacillus strains are promising. Therefore, all strains examined showed a high potential for use in poultry against salmonellosis.Probiotics are considered an alternative to antibiotics in the prevention and treatment of Salmonella diseases in poultry. However, to use probiotics as proposed above, it is necessary to evaluate their properties in detail and to select the most effective bacterial strains in the application targeted. In this study, probiotic properties of new Lactobacillus sp. strains were investigated and their antimicrobial activity against 125 environmental strains of Salmonella sp. was determined using the agar slab method. Furthermore, their survival in the presence of bile salts and at low pH, antibiotics susceptibility, aggregation and coaggregation ability, adherence to polystyrene and Caco-2 cells, and cytotoxicity were investigated. Each strain tested showed antagonistic activity against at least 96% of the environmental Salmonella sp. strains and thus representing a highly epidemiologically differentiated collection of poultry isolates. In addition, the probiotic properties of new Lactobacillus strains are promising. Therefore, all strains examined showed a high potential for use in poultry against salmonellosis.

The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT.

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib.

Malignant melanoma is the most aggressive skin cancer and can only be cured if detected early. Unfortunately, later stages of the disease do not guarantee success due to the rapid rate of melanoma cell metastasis and their high resistance to applied therapies. The search for new molecular targets and targeted therapy may represent the future in the development of effective methods for combating this cancer. SIRT2 is a promising target; thus, we downregulated SIRT2 expression in melanoma cells in vertical growth and metastatic phases and demonstrated that sirtuin acts as regulator of the basic functions of melanoma cells. A detailed transcriptomic analysis showed that SIRT2 regulates the expression of multiple genes encoding the tyrosine kinase pathways that are molecular targets of dasatinib. Indeed, cells with low SIRT2 expression were more susceptible to dasatinib, as demonstrated by multiple techniques, e.g., neutral red uptake, 3/7 caspase activity, colony formation assay, and in vitro scratch assay. Furthermore, these cells showed an altered phosphorylation profile for proteins playing roles in the response to dasatinib. Thus, our research indicates new, previously unknown SIRT2 functions in the regulation of gene expression, which is of key clinical significance.

Bacteriophage-based cocktail modulates selected immunological parameters and post-challenge survival of rainbow trout (Oncorhynchus mykiss).

Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR® bacteriophage-based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A. hydrophila and P. fluorescens limited the mortality of rainbow trout.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture.

BACKGROUND: Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the 'novel mode of action' concept desired for all new antibacterial agents. METHODS: The bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny. RESULTS: In this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations. CONCLUSIONS: From 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.