Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms, and here we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic, although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure/function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era.

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (> 55.0 %). The Nterminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥ 5.5 mN m -1 ), inducing increased rigidity (Cs -1 ↑), thermodynamic instability (ΔGmix < 0 → ΔGmix > 0) and high levels of lysis (> 50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m -1 ) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m -1 ) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure / function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin. Highlights• E2EM-lin shows specificity and potent efficacy towards Gram-positive bacteria • PG-driven membranolysis promotes E2EM-lin action against Gram-positive bacteria • PE-driven membranolysis promotes E2EM-lin action against Gram-negative bacteria • CL-mediated mechanisms contribute to E2EM-lin action against both bacterial types

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with −  < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

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Relevance. Pasteurellosis is a group of zoonotic infectious diseases caused by Pasteurella multocida. According to the antigenic composition, P. multocida is divided into 5 serogroups (A, B, D, F and E). Pathogenic and virulent properties of various serogroups and serotypes of the pathogen in different animal species vary widely and are a marker for determining their role in the development of the disease. Typing of P. multocida strains by capsule groups is an important condition for a comprehensive assessment of the epizootic situation, including for solving the issue of specific disease prevention.Methods. 82 strains of P. multocida from the collection of FGBI «VGNKI», isolated in different years from various animals, were used in the work. Phenotypic typing of pasteurella strains by capsule groups according to Carter was carried out in a test for the detection of hyaluronic acid in a pasteurella capsule and by the type of reaction in a tripaflavin sample. Strains giving a positive reaction with staphylococcus hyaluronidase wereassigned to capsule group A. If the test culture did not belong to group A, but was positive when examined in a tripaflavin sample, it was assigned to capsule group D.Results. Discrepancies between the results of phenotypic typing of pasteurella strains by capsule groups and by PCR were established. The results of typing did not coincide between PCR and Carter typing for capsule group A with respect to 25 strains, for capsule group D with respect to 5 strains and for capsule group B with respect to 5 strains. The number of untyped or doubtful phenotypic properties of pasteurella strains was 29.73%, untyped by PCR 2.46%. Hyaluronidase and acriflavin tests, unlike the PCR-method, do not provide an opportunity for typing pasteurella groups E and F.

The results of the metagenomic analysis revealed a high phylogenetic and taxonomic diversity of the microbial community of both luminal and mucosal microflora of the bird intestinal microbiome. Analysis of the taxonomic structure of the compared microbiomes showed both similarities and differences in the taxonomic diversity and relative abundance (%) of bacteria of the compared microbiomes at the level of order, class, genus.At the type level, 24 taxonomic categories were identified in the luminal and mucosal intestinal microbiome of birds, at the class level 35, at the order level 75, at the family level 168, at the genus level 350.The main share of the microbial community of the luminal and mucosal microbiome was made up of microorganisms belonging to the Firmicutes, Proteobacteria, Actinobacteria Cyanobacteria, and Bacteroidetes phyla. Comparison of the biological diversity of the colonic and mucosal microbiomes at the phylum level showed that the relative value of Proteobacteria in relation to Firmicutes in the mucosal microbiome was 3.25 times higher, and the ratio of Firmicutes to Bacteroidetes in the colonic microbiome was 3.98 times higher. than in mucosal.At the genus level, differences in the composition of microbiomes were manifested by a greater relative share (in decreasing order) in the luminal microbiome of representatives of the genus Lactobacillus, Candidatus Arthromitus, Romboutsia, Gallibacterium, Campylobacter, Enterococcus, and representatives of the genus Acinetobacter, Staphylococcus, Bacillus and Bradyrhizobium in the mucosal microbiome. An in-depth study of the intestinal microbiota using cultivation techniques and high-tech methods for identifying microorganisms, taking into account the results of metagenomic analysis, can significantly expand the understanding of the species diversity of microbiomes and the functions of representatives of various taxa in maintaining bird health, as well as to form a census of representatives of normal bird microflora.

Relevance. A significant increase in microbiome-associated diseases, closely related to violations of the bacterial diversity and functions of the normal intestinal microbiota, dictates the need to develop and implement measures for the long-term preservation of individual representatives of the normal microbiota in order to create new strategies for modifying the composition of microbiomes.Methods. The influence of the technology of deep freezing and storage of intestinal isolates of lactic acid bacteria of 2 taxonomic groups isolated from poultry in the conditions of the Low-temperature automated storage of biological samples of the Departmental Collection of useful microorganisms for Agricultural purposes of the Russian Agricultural Academy (VKSM) on the MRS culture medium using 10 and 20% glycerin or 10 and 20% sucrose as cryopreservants was studied. The suspensions of the isolates were frozen at - 150 °C for 18 hours and then placed in an automated cryopreservation at -80 °C. Control of samples for safetyResults. The technology of cryofreezing of lactic acid bacteria on MRS-broth using 10 and 20% glycerin or 10 and 20% sucrose as cryopreservants allows preserving the viability, physiological and  biochemical properties of intestinal isolates of lactic acid bacteria when stored for 18 months. All the protective media used (MRS-broth with glycerin 10 and 20%, MRS -broth with sucrose 10 and 20%) showed comparable results in the preservation of viability and acid-forming activity of Lactobacillus fermentum-2, Pediococcus pentosaceus 6p-3, Pediococcus pentosaceus 28p-1 isolates. Then the storage of Pediococcus pentosaceus isolate (28p-1) in a given parameter on a protective medium with 10 and 20% sucrose led to a decrease in the activity of acid formation.

Relevance. The use of antibiotics to treat infections of the reproductive tract in cows can cause bacterial resistance to antibiotics and endanger human health. Potentially, preparations from representatives of the lactic acid vaginal microflora (probiotics) can be used as a biological control to reduce the use of antibiotics in animal husbandry.However, the specific composition of the vaginal lactic acid microflora of healthy dry cows and its probiotic properties remain poorly documented.Methods. The species identification of catalase-negative gram-positive isolates of bacteria forming colonies on MRS agar isolated from utero-cervical mucus of healthy lactating cows was carried out in accordance with standard procedures using a culturedependent approach, biochemical tests and tests for sugar fermentation using API50 CHL strips (bioMеrieux, France). For testing for antagonistic activity by the method of delayed antagonism against test cultures of staphylococci and Escherichia, 14 isolates were selected in accordance with typing assigned to 5 species of the genus Lactobacillus.Results. The results obtained indicate that the utero-vaginal biocenosis of healthy lactating cows is represented by microorganisms of the Lactobacillaceae family, including lactic acid enterococci and bacteria of the Leuconostocaceae family. The presence of representatives of the genera Aerococcus, Peptostreptococcus and Gardnerella vaginalis was found in the composition of the vaginal biocenosis in cows without signs of pathology of the birth canal. According to the results obtained, the vaginal population of lactic acid bacteria of the genus Lactobacillus has low or medium antagonistic activity against test cultures of indicatory strains of staphylococci and Escherichia.