The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.

A method based on nanosecond laser treatment was used to design superhydrophobic and superhydrophilic aluminum alloy substrates showing enhanced cytotoxic activity with respect to Escherichia coli K12 C600 strain. It was shown that the survival of cells adhered to the superhydrophobic substrates was significantly affected by the presence of organic contaminants, which are ubiquitous in hospital practice and the food industry. The peculiarities of the texture also played a notable role in antibactericidal activity. It was found that the superhydrophilic surfaces had much higher toxicity than the superhydrophobic ones, which was explained by the mechanisms of adhesion of cells to the surface. Scanning electron microscopy and tomographic reconstruction of the adhered cells were used to study the variation of cell morphology after attachment to surfaces with different wettability. It was shown that the cytotoxicity of superhydrophobic surfaces could be significantly enhanced by using the combined antimicrobial action of bacteriophages and the superhydrophobicity of the objects.

Fibrin glue has been used clinically for decades in a wide variety of surgical specialties and is now being investigated as a medium for local, prolonged drug delivery. Effective local delivery of antibacterial substances is important perioperatively in patients with implanted medical devices or postoperatively for deep wounds. However, prolonged local application of antibiotics is often not possible or simply inadequate. Biofilm formation and antibiotic resistance are also major obstacles to antibacterial therapy. In this paper we test the biocompatibility of bacteriophages incorporated within fibrin glue, track the release of bacteriophages from fibrin scaffolds, and measure the antibacterial activity of released bacteriophages. Fibrin glue polymerized in the presence of the PA5 bacteriophage released high titers of bacteriophages during 11 days of incubation in liquid medium. Released PA5 bacteriophages were effective in killing Pseudomonas aeruginosa PA01. Overall, our results show that fibrin glue can be used for sustained delivery of bacteriophages and this strategy holds promise for many antibacterial applications.

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Endolysin-based therapeutics are promising antibacterial agents and can successfully supplement the existing antibacterial drugs array. It is specifically important in the case of Gram-negative pathogens, e.g., ESKAPE group bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and are highly inclined to gain multiple antibiotic resistance. Despite numerous works devoted to the screening of new lytic enzymes and investigations of their biochemical properties, there are significant breaches in some aspects of their operating characteristics, including safety issues of endolysin use. Here, we provide a comprehensive study of the antimicrobial efficacy aspects of four Gram-negative bacteria-targeting endolysins LysAm24, LysAp22, LysECD7, and LysSi3, their in vitro and in vivo activity, and their biological safety. These endolysins possess a wide spectrum of action, are active against planktonic bacteria and bacterial biofilms, and are effective in wound and burn skin infection animal models. In terms of safety, these enzymes do not contribute to the development of short-term resistance, are not cytotoxic, and do not significantly affect the normal intestinal microflora in vivo. Our results provide a confident base for the development of effective and safe candidate dosage forms for the treatment of local and systemic infections caused by Gram-negative bacterial species.