Sex-dependent differences in behavioral and immunological responses to antibiotic and bacteriophage administration in mice.

Introduction: The problem of antibiotic resistance is a global one, involving many industries and entailing huge financial outlays. Therefore, the search for alternative methods to combat drug-resistant bacteria has a priority status. Great potential is seen in bacteriophages which have the natural ability to kill bacterial cells. Bacteriophages also have several advantages over antibiotics. Firstly, they are considered ecologically safe (harmless to humans, plants and animals). Secondly, bacteriophages preparations are readily producible and easy to apply. However, before bacteriophages can be authorized for medical and veterinary use, they must be accurately characterized in vitro and in vivo to determinate safety. Methods: Therefore, the aim of this study was to verify for the first time the behavioral and immunological responses of both male and female mice (C57BL/6J) to bacteriophage cocktail, composed of two bacteriophages, and to two commonly used antibiotics, enrofloxacin and tetracycline. Animal behavior, the percentage of lymphocyte populations and subpopulations, cytokine concentrations, blood hematological parameters, gastrointestinal microbiome analysis and the size of internal organs, were evaluated. Results: Unexpectedly, we observed a sex-dependent, negative effect of antibiotic therapy, which not only involved the functioning of the immune system, but could also significantly impaired the activity of the central nervous system, as manifested by disruption of the behavioral pattern, especially exacerbated in females. In contrast to antibiotics, complex behavioral and immunological analyses confirmed the lack of adverse effects during the bacteriophage cocktail administration. Discussion: The mechanism of the differences between males and females in appearance of adverse effects, related to the behavioral and immune functions, in the response to antibiotic treatment remains to be elucidated. One might imagine that differences in hormones and/or different permeability of the blood-brain barrier can be important factors, however, extensive studies are required to find the real reason(s).

The Influence of Bacteriophages on the Metabolic Condition of Human Fibroblasts in Light of the Safety of Phage Therapy in Staphylococcal Skin Infections.

Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages-vB_SauM-A, vB_SauM-C and vB_SauM-D-high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic.

Activity of Phage-Lactoferrin Mixture against Multi Drug Resistant Staphylococcus aureus Biofilms.

Biofilms are complex bacterial structures composed of bacterial cells embedded in extracellular polymeric substances (EPS) consisting of polysaccharides, proteins and lipids. As a result, biofilms are difficult to eradicate using both mechanical methods, i.e., scraping, and chemical methods such as disinfectants or antibiotics. Bacteriophages are shown to be able to act as anti-biofilm agents, with the ability to penetrate through the matrix and reach the bacterial cells. However, they also seem to have their limitations. After several hours of treatment with phages, the biofilm tends to grow back and phage-resistant bacteria emerge. Therefore, it is now recommended to use a mixture of phages and other antibacterial agents in order to increase treatment efficiency. In our work we have paired staphylococcal phages with lactoferrin, a protein with proven anti-biofilm proprieties. By analyzing the biofilm biomass and metabolic activity, we have observed that the addition of lactoferrin to phage lysate accelerated the anti-biofilm effect of phages and also prevented biofilm re-growth. Therefore, this combination might have a potential use in biofilm eradication procedures in medical settings.

Determination of Effects and Mechanisms of Action of Bacterial Amyloids on Antibiotic Resistance.

Bacterial functional amyloids, apart from their many other functions, can influence the resistance of bacteria to antibiotics and other antibacterial agents. Mechanisms of modulation of susceptibility of bacterial cells to antimicrobials can be either indirect or direct. The former mechanisms are exemplified by the contribution of functional amyloids to biofilm formation, which may effectively prevent the penetration of various compounds into bacterial cells. The direct mechanisms include the effects of bacterial proteins revealing amyloid-like structures, like the C-terminal region of the Escherichia coli Hfq protein, on the expression of genes involved in antibiotic resistance. Therefore, in this paper, we describe methods by which effects and mechanisms of action of bacterial amyloids on antibiotic resistance can be studied. Assessment of formation of biofilms, determination of the efficiency of antibiotic resistance in solid and liquid media, and determination of the effects on gene expression at levels of mRNA abundance and stability and protein abundance are described.

Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens.

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed.

Efficacy and safety of phage therapy against Salmonella enterica serovars Typhimurium and Enteritidis estimated by using a battery of in vitro tests and the Galleria mellonella animal model.

In light of spreading antibiotic resistance among pathogenic bacteria, the development of novel approaches to combat such microorganisms is crucial. Salmonella enterica is pathogenic to humans, however, it can also infect poultry, being a potential foodborne pathogen when poultry-derived food is contaminated by this bacterium. Phage therapy is one of the alternative ways to treat Salmonella-infected animals while the establishment of this method and its introduction to a general practice requires detailed studies on safety and efficacy. Here, we present the results of such studies with two previously isolated and characterized bacteriophages, vB_SenM2 and vB_Sen-TO17, and four strains of S. enterica belonging to two serovars, Typhimurium and Enteritidis. We demonstrated effective reduction of bacterial cell number and cell culture density when using each phage alone, and in combination (as a cocktail). These phages were also effective in reducing bacterial biofilm. The efficacy of this in vitro phage therapy was compared to the action of known antibiotics, as was the efficiency of appearance of bacteria resistant to both these types of antibacterial agents. Safety of the use of bacteriophages was demonstrated using the LAL chromogenic test and the chicken fibroblast viability assay. Finally, the efficacy of phage therapy was assessed with the in vivo model of S. enterica-infected Galleria mellonella larvae, showing a significant improvement in the survival of the animals. In conclusion, we demonstrated high efficacy and acceptable safety profiles of phage therapy against S. enterica strains using vB_SenM-2 and vB_Sen-TO17 phages (both alone and in a cocktail). These results open a possibility for a trial with the use of poultry and these phages which might potentially allow to introduce of this method for practical use in poultry farming.

Into the Unknown: Microbial Communities in Caves, Their Role, and Potential Use.

Caves have been an item of amateur and professional exploration for many years. Research on the karst caves has revealed great diversity of bacteria, algae, and fungi living on stone walls and speleothems, in mud puddles or sediments. They have become the source of interest for various research groups including geologists, chemists, ecologists, or microbiologists. The adaptations of cave-dwelling organisms applied to their survival are complex and some of their properties show potential to be used in various areas of human life. Secondary metabolites produced by cave's bacteria show strong antimicrobial, anti-inflammatory, or anticancer properties. Furthermore, bacteria that can induce mineral precipitation could be used in the construction industry and for neutralization of radioisotopes. In this review we focus on bacteria and algae present in cave ecosystems, their role in shaping such specific environment, and their biotechnological and medical potential.

Antibacterial, Antifungal and Anticancer Activities of Compounds Produced by Newly Isolated Streptomyces Strains from the Szczelina Chocholowska Cave (Tatra Mountains, Poland).

Resistance of bacteria, fungi and cancer cells to antibiotics and other drugs is recognized as one of the major problems in current medicine. Therefore, a search for new biologically active compounds able to either kill pathogenic cells or inhibit their growth is mandatory. Hard-to-reach habitats appear to be unexplored sources of microorganisms producing previously unknown antibiotics and other molecules revealing potentially therapeutic properties. Caves belong to such habitats, and Actinobacteria are a predominant group of microorganisms occurring there. This group of bacteria are known for production of many antibiotics and other bioactive compounds. Interestingly, it was demonstrated previously that infection with bacteriophages might enhance production of antibiotics by them. Here, we describe a series of newly isolated strains of Actinobacteria that were found in caves from the Tatra Mountains (Poland). Phage induction tests indicated that some of them may bear active prophages able to produce virions upon treatment with mitomycin C or UV irradiation. Among all the examined bacteria, two newly isolated Streptomyces sp. strains were further characterized to demonstrate their ability to inhibit the growth of pathogenic bacteria (strains of Staphylococcus aureus, Salmonella enterica, Enterococcus sp., Escherichia coli, and Pseudomonas aeruginosa) and fungi (different species and strains from the genus Candida). Moreover, extracts from these Streptomyces strains reduced viability of the breast-cancer cell line T47D. Chemical analyses of these extracts indicated the presence of isomers of dichloranthrabenzoxocinone and 4,10- or 10,12-dichloro-3-O-methylanthrabenzoxocinone, which are putative antimicrobial compounds. Moreover, various previously unknown (unclassified) molecules were also detected using liquid chromatography-mass spectrometry, suggesting that tested Streptomyces strains may synthesize a battery of bioactive compounds with antibacterial, antifungal, and anticancer activities. These results indicate that further studies on the newly isolated Actinobacteria might be a promising approach to develop novel antibacterial, antifungal, and/or anticancer drugs.

Bacteriophage-encoded enzymes destroying bacterial cell membranes and walls, and their potential use as antimicrobial agents.

Appearance of pathogenic bacteria resistant to most, if not all, known antibiotics is currently one of the most significant medical problems. Therefore, development of novel antibacterial therapies is crucial for efficient treatment of bacterial infections in the near future. One possible option is to employ enzymes, encoded by bacteriophages, which cause destruction of bacterial cell membranes and walls. Bacteriophages use such enzymes to destroy bacterial host cells at the final stage of their lytic development, in order to ensure effective liberation of progeny virions. Nevertheless, to use such bacteriophage-encoded proteins in medicine and/or biotechnology, it is crucial to understand details of their biological functions and biochemical properties. Therefore, in this review article, we will present and discuss our current knowledge on the processes of bacteriophage-mediated bacterial cell lysis, with special emphasis on enzymes involved in them. Regulation of timing of the lysis is also discussed. Finally, possibilities of the practical use of these enzymes as antibacterial agents will be underlined and perspectives of this aspect will be presented.

Bacteriophages vB_Sen-TO17 and vB_Sen-E22, Newly Isolated Viruses from Chicken Feces, Specific for Several Salmonella enterica Strains.

Two newly discovered bacteriophages, isolated from chicken feces and infecting Salmonella enterica strains, are described in this report. These phages have been named vB_Sen-TO17 and vB_Sen-E22, and we present their molecular and functional characterization. Both studied viruses are able to infect several S. enterica strains and develop lytically, but their specific host ranges differ significantly. Electron microscopic analyses of virions have been performed, and full genome sequences were determined and characterized, along with molecular phylogenetic studies. Genomes of vB_Sen-TO17 (ds DNA of 41,658 bp) and vB_Sen-E22 (dsDNA of 108,987 bp) are devoid of homologs of any known or putative gene coding for toxins or any other proteins potentially deleterious for eukaryotic cells. Both phages adsorbed efficiently (>95% adsorbed virions) within 10 min at 42 °C (resembling chicken body temperature) on cells of most tested host strains. Kinetics of lytic development of vB_Sen-TO17 and vB_Sen-E22, determined in one-step growth experiments, indicated that development is complete within 30-40 min at 42 °C, whereas burst sizes vary from 9 to 79 progeny phages per cell for vB_Sen-TO17 and from 18 to 64 for vB_Sen-E22, depending on the host strain. Virions of both phages were relatively stable (from several percent to almost 100% survivability) under various conditions, including acidic and alkaline pH values (from 3 to 12), temperatures from -80 °C to 60 °C, 70% ethanol, chloroform, and 10% DMSO. These characteristics of vB_Sen-TO17 and vB_Sen-E22 indicate that these phages might be considered in further studies on phage therapy, particularly in attempts to eliminate S. enterica from chicken intestine.

Characteristics of a Series of Three Bacteriophages Infecting Salmonella enterica Strains.

Molecular and functional characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, infecting various Salmonella enterica serovars and strains is presented. All these phages were able to develop lytically while not forming prophages. Moreover, they were able to survive at pH 3. The phages revealed different host ranges within serovars and strains of S. enterica, different adsorption rates on host cells, and different lytic growth kinetics at various temperatures (in the range of 25 to 42 °C). They efficiently reduced the number of cells in the bacterial biofilm and decreased the biofilm mass. Whole genome sequences of these phages have been determined and analyzed, including their phylogenetic relationships. In conclusion, we have demonstrated detailed characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, which reveal favorable features in light of their potential use in phage therapy of humans and animals, as well as for food protection purposes.

Characterization of a bacteriophage, vB_Eco4M-7, that effectively infects many Escherichia coli O157 strains.

The characterization of a recently isolated bacteriophage, vB_Eco4M-7, which effectively infects many, though not all, Escherichia coli O157 strains, is presented. The genome of this phage comprises double-stranded DNA, 68,084 bp in length, with a GC content of 46.2%. It contains 96 putative open reading frames (ORFs). Among them, the putative functions of only 35 ORFs were predicted (36.5%), whereas 61 ORFs (63.5%) were classified as hypothetical proteins. The genome of phage vB_Eco4M-7 does not contain genes coding for integrase, recombinase, repressors or excisionase, which are the main markers of temperate viruses. Therefore, we conclude that phage vB_Eco4M-7 should be considered a lytic virus. This was confirmed by monitoring phage lytic development by a one-step growth experiment. Moreover, the phage forms relatively small uniform plaques (1 mm diameter) with no properties of lysogenization. Electron microscopic analyses indicated that vB_Eco4M-7 belongs to the Myoviridae family. Based on mass spectrometric analyses, including the fragmentation pattern of unique peptides, 33 phage vB_Eco4M-7 proteins were assigned to annotated open reading frames. Importantly, genome analysis suggested that this E. coli phage is free of toxins and other virulence factors. In addition, a similar, previously reported but uncharacterized bacteriophage, ECML-117, was also investigated, and this phage exhibited properties similar to vB_Eco4M-7. Our results indicate that both studied phages are potential candidates for phage therapy and/or food protection against Shiga toxin-producing E. coli, as the majority of these strains belong to the O157 serotype.

Characterization of the Three New Kayviruses and Their Lytic Activity Against Multidrug-Resistant Staphylococcus aureus.

The development of antimicrobial resistance has become a global concern. One approach to overcome the problem of drug resistance is the application of bacteriophages. This study aimed at characterizing three phages isolated from sewage, which show lytic activity against clinical isolates of multidrug-resistant Staphylococcus aureus. Morphology, genetics and biological properties, including host range, adsorption rate, latent time, phage burst size and lysis profiles, were studied in all three phages. As analyzed by transmission electron microscopy (TEM), phages vB_SauM-A, vB_SauM-C, vB_SauM-D have a myovirion morphology. One of the tested phages, vB_SauM-A, has relatively rapid adsorption (86% in 17.5 min), short latent period (25 min) and extremely large burst size (~500 plaque-forming units (PFU) per infected cell). The genomic analysis revealed that vB_SauM-A, vB_SauM-C, vB_SauM-D possess large genomes (vB_SauM-A 139,031 bp, vB_SauM-C 140,086 bp, vB_SauM-D 139,088 bp) with low G+C content (~30.4%) and are very closely related to the phage K (95-97% similarity). The isolated bacteriophages demonstrate broad host range against MDR S. aureus strains, high lytic activity corresponding to strictly virulent life cycle, suggesting their potential to treat S. aureus infections.