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)

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

Interactions of Bacteriophages with Animal and Human Organisms—Safety Issues in the Light of Phage Therapy

Bacteriophages are viruses infecting bacterial cells. Since there is a lack of specific receptors for bacteriophages on eukaryotic cells, these viruses were for a long time considered to be neutral to animals and humans. However, studies of recent years provided clear evidence that bacteriophages can interact with eukaryotic cells, significantly influencing the functions of tissues, organs, and systems of mammals, including humans. In this review article, we summarize and discuss recent discoveries in the field of interactions of phages with animal and human organisms. Possibilities of penetration of bacteriophages into eukaryotic cells, tissues, and organs are discussed, and evidence of the effects of phages on functions of the immune system, respiratory system, central nervous system, gastrointestinal system, urinary tract, and reproductive system are presented and discussed. Modulations of cancer cells by bacteriophages are indicated. Direct and indirect effects of virulent and temperate phages are discussed. We conclude that interactions of bacteriophages with animal and human organisms are robust, and they must be taken under consideration when using these viruses in medicine, especially in phage therapy, and in biotechnological application

Mucosal adjuvant activity of IL-2 presenting spores of bacillus subtilis in a murine model of Helicobacter pylori vaccination.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization

Western blotting analysis of expression of the <i>cot-linker-IL-2</i> fusion gene.

<p>Western blotting analysis with anti-CotB (Panel A) or anti-IL-2 (Panel B) antibodies. Spore coat proteins from spores of the 168 (lane 1) or BKH122 (fusion CotB-GGGEAAAKGGG-IL-2) (lane 2) strain. Each lane of panel A and B was loaded with 20 µg of total proteins. Arrows point to fusion proteins.</p

IFN-γ response of sensitized mouse splenocytes to UreB as assessed by the ELISpot.

<p>The splenocytes were isolated from naïve mice (open bars), mice orally immunized with 168 spores (dotted open bars), BKH108 (CotC-UreB, vegetative expression of UreB) (bars with horizontal hatching), BKH122 (CotB-linker-IL-2) (bars with vertical hatching) or 1∶1 mixture of BKH108 and BKH122 (dotted closed bars). Cells were treated with purified UreB protein for 72 h and then the IFN-γ cells were enumerated by ELISpot procedure. Error bars represent standard deviation. * p-value <0.05, ** p-value <0.005. I – day 22, II – day 43, III – day 61.</p

Densitometric analysis.

<p>Densitometric analysis.</p

Localisation of fusion proteins as assessed by immunofluorescence microscopy.

<p>Purified, free spores of wild type strain 168, BKH108 (CotC-UreB), BKH122 (CotB- GGGEAAAKGGG -IL-2) were visualised by phase contrast (PC) and immunofluorescence (IF) microscopy. The spores were incubated with mouse anti-UreB or anti-IL-2 antibodies, followed by anti-mouse IgG-Cy3 conjugates. The same exposure time was used for all IF images.</p