The diversity of coliphages and indigenous coliform strains (ICSs) simultaneously present in horse feces was investigated by culture-based and molecular methods. The richness of coliforms (as estimated by the Chao1 method) is about 1,000 individual ICSs distinguishable by genomic fingerprinting present in a single sample of feces. This unexpectedly high value indicates that some factor limits the competition of coliform bacteria in the horse gut microbial system. In contrast, the diversity of phages active against any selected ICS is generally limited to one to three viral genotypes present in the sample. The sensitivities of different ICSs to simultaneously present coliphages overlap only slightly; the phages isolated from the same sample on different ICSs are usually unrelated. As a result, the titers of phages in fecal extract as determined for different Escherichia coli strains and ICSs may differ by several orders of magnitude. Summarizing all the data, we propose that coliphage infection may provide a selection pressure that maintains the high level of coliform diversity, restricting the possibility of a few best competitors outgrowing other ICSs. We also observed high-magnitude temporal variations of coliphage titers as determined using an E. coli C600 test culture in the same animal during a 16-day period of monitoring. No correlation with total coliform count was observed. These results are in good agreement with our hypothesis.Bacteriophages exert a significant influence on natural microbial communities (2, 24, 33). They are responsible for 20 to 80% of bacterial mortality in freshwater and marine ecosystems (25, 33) and increase bacterial biodiversity (references 14, 15, 31, and 33 and references therein) due to preferential attack on the dominant species or strains and redistribution of the organic matter. The role of bacteriophages could be even more important in microbial systems where high densities of active bacteria are achieved. Among these systems are intestinal microbial populations of animals and humans where bacteria (17, 28) and bacteriophages (5,6,9,10,13,16,17) are present at high densities. The gut is the natural habitat for Escherichia coli and for coliphages, which are highly suitable for work in culture, making this system attractive as a model for phage ecology.For our study, we selected the horse as the macro host. The cellulolytic microbial community localized in the horse large intestine is very complex and includes bacteria, archaea, fungi, and protozoa (18). In contrast to rumen communities, the microbial biomass in the horse intestine is not subjected to digestion and is excreted with the feces. The conditions in the horse gut seem more stable than those in the intestines of many other species, as the time taken to digest grass is about 72 h (18), and the intervals between food intake and defecation are normally much shorter. A spatial complexity is present in the gut (9). The mucosal surface and the lumen contents are different ecological niches for bacteria. It has been show...

Summary Felix d’Herelle first demonstrated, about 90 years ago, the presence of bacteriophages in human and animal body microbiota. Our comprehension of the impact of naturally occurring bacteriophages on symbiotic bacteria, and of their role in general homeostasis of macro‐organism, nevertheless remains quite fragmentary. Analysis of data in various human‐ and animal body‐associated microbial systems on phage occurrence, diversity, host specificity and dynamics, as well as host occurrence, specificity and dynamics, suggests that mechanisms which stabilize phage–bacteria coexistence are not identical for either different species or different body sites. Regulation by phage infection instead probably depends on specific physical, chemical and biological conditions, e.g. pH, nutrient densities, host prevalence, relation to mucosa and other surfaces and presence of phage inhibiting substances. In some animal species intestinal bacteriophages thus appear to exert significant selective pressure over at least some resident bacterial populations, resulting in phages playing important roles in the self‐regulation of these microbial systems while at the same time contributing to maintenance of bacterial diversity (i.e. ‘killing the winner’). Emerging data additionally suggest that bacteriophage particles could play roles in regulating the immune reactions of the macro‐organism. Alternatively, for many systems links between phages and community characteristics have not been established.

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.

Lytic coliphage vB_EcoP_G7C and several other highly related isolates were obtained repeatedly from the samples of horse feces held in the same stable thus representing a component of the normal indigenous intestinal communities in this population of animals. The genome of G7C consists of 71,759 bp with terminal repeats of about 1160 bp, yielding approximately 73 kbp packed DNA size. Seventy-eight potential open reading frames, most of them unique to N4-like viruses, were identified and annotated. The overall layout of functional gene groups was close to that of the original N4 phage, with some important changes in late gene area including new tail fiber proteins containing hydrolytic domains. Structural proteome analysis confirmed all the predicted subunits of the viral particle. Unlike N4 itself, phage G7C did not exhibit a lysis-inhibited phenotype.

Bacteriophage 9g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family.

The biological functions of bacteriophage virions come down to the solution of three basic problems: to provide protection of viral nucleic acid from the factors of extracellular environment, to recognize a host suitable for phage replication, and to provide the delivery of nucleic acid through bacterial cell envelopes. This review considers the main regularities of phage-cell interaction at the initial stages of infection of tailed bacteriophages, from the reversible binding with receptors on the surface to the beginning of phage DNA entry. Data on the structure and functions of the phage adsorption apparatus, the main quantitative characteristics of the adsorption process, and the mechanisms of adaptation of phages and their hosts to each other effective at the stage of adsorption are presented.

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host’s O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages.

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.