RESUMO
BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.
Assuntos
Endopeptidases , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas , Camundongos Endogâmicos BALB C , Animais , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Camundongos , Endopeptidases/farmacologia , Endopeptidases/administração & dosagem , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/administração & dosagem , Ratos , Masculino , Engenharia de Proteínas/métodosRESUMO
A method based on nanosecond laser processing was used to design superhydrophilic and superhydrophobic copper substrates. Three different protocols were used to analyze the evolution of the bactericidal activity of the copper substrates with different wettability. Scanning electron microscopy was used to study the variation of cell morphology after the attachment to superhydrophilic and superhydrophobic surfaces. The dispersions of Escherichia coli K12 C600 and Klebsiella pneumoniae 811 in Luria Bertani broth in contact with the superhydrophilic copper surface showed enhanced bacterial inactivation, associated with toxic action of both hierarchically textured copper surface and high content of Cu2+ ions in the dispersion medium. In contrast, the bacterial dispersions in contact with the superhydrophobic copper substrates demonstrated an increase in cell concentration with time until the development of corrosion processes. The resistance of bacterial cells to contact the copper substrates is discussed on the basis of surface forces, determining the primary adhesion and of the protective action of a superhydrophobic state of the surface against electrochemical and biological corrosion.
Assuntos
Ligas/farmacologia , Antibacterianos/farmacologia , Cobre/farmacologia , Corrosão , Condutividade Elétrica , Escherichia coli K12/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Klebsiella pneumoniae/efeitos dos fármacos , Propriedades de Superfície , MolhabilidadeRESUMO
In the present study, we investigate the biological properties and genomic organization of virulent bacteriophage AM24, which specifically infects multidrug-resistant clinical Acinetobacter baumannii strains with a K9 capsular polysaccharide structure. The phage was identified as a member of the family Myoviridae by transmission electron microscopy. The AM24 linear double-stranded DNA genome of 97,177 bp contains 167 open reading frames. Putative functions were assigned for products of 40 predicted genes, including proteins involved in nucleotide metabolism and DNA replication, packaging of DNA into the capsid, phage assembly and structural proteins, and bacterial cell lysis. The gene encoding the tailspike, which possesses depolymerase activity towards the corresponding capsular polysaccharides, is situated in the phage genome outside of the structural module, upstream of the genes responsible for packaging of DNA into the capsid. The data on characterization of depolymerase-carrying phage AM24 contributes to our knowledge of the diversity of viruses infecting different capsular types of A. baumannii.
Assuntos
Acinetobacter baumannii/virologia , Cápsulas Bacterianas/metabolismo , Genes Virais/genética , Myoviridae/classificação , Myoviridae/genética , Acinetobacter baumannii/efeitos dos fármacos , Cápsulas Bacterianas/classificação , DNA Viral/genética , Farmacorresistência Bacteriana Múltipla , Genoma Viral/genética , Microscopia Eletrônica de Transmissão , Myoviridae/isolamento & purificação , Fases de Leitura Aberta/genética , Análise de Sequência de DNARESUMO
Antibacterial therapy with phage-encoded endolysins or their modified derivatives with improved antibacterial, biochemical and pharmacokinetic properties is one of the most promising strategies that can supply existing antibacterial drugs array. Gram-negative bacteria-induced infections treatment is especially challenging because of rapidly spreading bacterial resistance. We have developed modified endolysin LysECD7-SMAP with a significant antibacterial activity and broad spectra of action against gram-negative bacteria. Endolysin was formulated in a bactericidal gel for topical application with pronounced effectivity in local animal infectious models. Here we present preclinical safety studies and pharmacokinetics of LysECD7-SMAP-based gel. We have detected LysECD7-SMAP in the skin and underlying muscle at therapeutic concentrations when the gel is applied topically to intact or injured skin. Moreover, the protein does not enter the bloodstream, and has no systemic bioavailability, assuming no systemic adverse effects. In studies of general toxicology, local tolerance, and immunotoxicology it was approved that LysECD7-SMAP gel local application results in the absence of toxic effects after single and multiple administration. Thus, LysECD7-SMAP-containing gel has appropriate pharmacokinetics and can be considered as safe that supports the initiation of the phase I clinical trials of novel antibacterial drug intending to treat acute wound infections caused by resistant gram-negative bacteria.
RESUMO
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.
RESUMO
The bactericidal activity of copper and copper alloys is well appreciated and was already exploited in medical practice in 19th century. However, despite of being an essential nutrient required by organisms to perform life functions, excess copper is extremely toxic and detrimental to health. Recent studies have shown that superhydrophobic surfaces have a significant antibacterial potential for reduction of nosocomial infections. At the same time, the prolonged contact with biological liquids may cause a degradation of the superhydrophobic copper surface and corrosion with increasing egress of toxic copper ions. These aspects are poorly studied so far. In this paper, we analyze the evolution of the properties of both the superhydrophobic copper surface and the suspension of Escherichia coli bacteria during their prolonged contact and study the impact of such contact on the bactericidal activity of the surface. It is shown that by controlling the corrosion resistance and the wettability of the superhydrophobic copper substrate, it becomes possible to sustain the bactericidal action of copper substrates for a long time, simultaneously avoiding the excessive corrosive degradation and release of copper ions in the environment.
Assuntos
Antibacterianos/farmacologia , Cobre/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Corrosão , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , MolhabilidadeRESUMO
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.
Assuntos
Antibacterianos , Endopeptidases/química , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , Proteínas Citotóxicas Formadoras de Poros , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , OvinosRESUMO
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.