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.

• Klíčová slova
• Dosage forms, Engineered endolysin, Enzyme-based antibacterial, Gram-negative bacteria, Preclinical efficacy,
• MeSH
• antibakteriální látky farmakologie   aplikace a dávkování   MeSH
• endopeptidasy * farmakologie   aplikace a dávkování   MeSH
• gramnegativní bakteriální infekce * farmakoterapie   MeSH
• gramnegativní bakterie * účinky léků   MeSH
• krysa rodu Rattus MeSH
• myši inbrední BALB C * MeSH
• myši MeSH
• proteinové inženýrství metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• endolysin MeSH Prohlížeč
• endopeptidasy * MeSH

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts' exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

• MeSH
• antibakteriální látky * farmakologie   chemie   MeSH
• biologické pigmenty farmakologie   MeSH
• biologické přípravky farmakologie   chemie   MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky farmakologie   chemie   MeSH
• grampozitivní bakterie účinky léků   účinky záření   MeSH
• komplexní směsi farmakologie   chemie   MeSH
• mikrobiální testy citlivosti * MeSH
• Monascus * chemie   metabolismus   MeSH
• mycelium * chemie   účinky záření   účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• red yeast rice MeSH Prohlížeč

We studied the impact of a sublethal concentration of erythromycin on the fitness and proteome of a continuously cultivated population of Escherichia coli. The development of resistance to erythromycin in the population was followed over time by the gradient plate method and minimum inhibitory concentration (MIC) measurements. We measured the growth rate, standardized efficiency of synthesis of radiolabeled proteins, and translation accuracy of the system. The proteome changes were followed over time in two parallel experiments that differed in the presence or absence of erythromycin. A comparison of the proteomes at each time point (43, 68, and 103 h) revealed a group of unique proteins differing in expression. From all 35 proteins differing throughout the cultivation, only three were common to more than one time point. In the final population, a significant proportion of upregulated proteins was localized to the outer or inner cytoplasmic membranes or to the periplasmic space. In a population growing for more than 100 generations in the presence of antibiotic, erythromycin-resistant bacterial clones with improved fitness in comparison to early resistant culture predominated. This phenomenon was accompanied by distinct changes in protein expression during a stepwise, population-based development of erythromycin resistance.

• Klíčová slova
• Continuous cultivation system, Escherichia coli, erythromycin, fitness, proteome, resistance,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• časové faktory MeSH
• erythromycin farmakologie   MeSH
• Escherichia coli účinky léků   genetika   metabolismus   MeSH
• genetická zdatnost účinky léků   MeSH
• kultivační média MeSH
• mikrobiální testy citlivosti MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• erythromycin MeSH
• kultivační média MeSH
• proteiny z Escherichia coli MeSH
• proteom MeSH