Topical application of antimicrobial peptides (AMPs) released from local carriers into the focus of bacterial infection appears to be promising therapeutic method for the cure or prevention of osteomyelitis. For the experiments carried out on the model of induced osteomyelitis we will use local carriers combined with AMPs invented in our laboratory. In another experimental model we will study the efficacy of AMPs in the prevention of bacterial biofilm formation on the surfaces of implants prepared by the combination of peptides and synthetic bone cement implanted into infected bone marrow. In preliminary experiments carried out using both models we have found out that AMPs are surprisingly more efficient than antibiotics. In the forthcoming experiments we want to find the optimal combination of the peptide and local carrier that shows antimicrobial effect, but low toxicity against bone cells. Then we will verify the efficacy and safety of topically applied AMPs in carrier for treatment of osteomyelitis or prevention of implant related-infections by experiments on laboratory animals.

Lokální použití antimikrobiálních peptidů (AMP) uvolňovaných z lokálních nosičů do ložiska infekce se nabízí jako velmi nadějná terapeutická metoda pro léčbu nebo prevenci osteomyelitidy způsobenou rezistentními patogeny. K experimentům prováděných na modelech indukované osteomyelitidy použijeme lokální nosiče kombinované s AMP vyvinutými v naší laboratoři. Na dalším experimentálním modelu budeme studovat účinek AMPs na prevenci tvorby bakteriálních biofilmů na površích implantátů připravených kombinací peptidů a syntetického kostního cementu a poté vložených do infikované kostní dřeně. Předběžnými experimenty provedenými na obou modelech jsme zjistili, že AMPs jsou překvapivě účinnější než antibiotika. Ve výzkumu budeme pokračovat tak, abychom získali optimální kombinaci peptidu s nosičem vykazující antimikrobiální účinek, ale s minimální toxicitou vůči kostním buňkám. Poté budeme ověřovat účinky a bezpečnost lokálně aplikovaných AMP v nosiči k léčbě osteomyelitidy nebo prevenci infekcí implantátů experimenty na laboratorních zvířatech.

• MeSH
• antimikrobiální peptidy terapeutické užití   MeSH
• bakteriální léková rezistence MeSH
• biofilmy MeSH
• infekce spojené s protézou prevence a kontrola   MeSH
• kosti a kostní tkáň MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• osteomyelitida farmakoterapie   MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• preventivní medicína
• infekční lékařství
• ortodoncie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Candida albicans has several virulence factors at its disposal, including yeast-hyphal transition associated with biofilm formation, phospholipases, proteases and hemolytic activity, all of which contribute to its pathogenesis. We used synthetic derivative LL-III/43 of antimicrobial peptide lasioglossin LL-III to enhance effect of azoles on attenuation of C. albicans virulence factors. LL-III/43 was able to inhibit initial adhesion or biofilm formation of C. albicans strains at 50 µM. Azoles, however, were ineffective at this concentration. Using fluorescently labeled LL-III/43, we observed that peptide covered C. albicans cells, partially penetrated through their membranes and then accumulated inside cells. LL-III/43 (25 µM) in combination with clotrimazole prevented biofilm formation already at 3.1 µM clotrimazole. Neither LL-III/43 nor azoles were able to significantly inhibit phospholipases, proteases, or hemolytic activity of C. albicans. LL-III/43 (25 µM) and clotrimazole (50 µM) in combination decreased production of these virulence factors, and it completely attenuated its hemolytic activity. Scanning electron microscopy showed that LL-III/43 (50 µM) prevented C. albicans biofilm formation on Ti-6Al-4 V alloy used in orthopedic surgeries and combination of LL-III/43 (25 µM) with clotrimazole (3.1 µM) prevented biofilm formation on urinary catheters. Therefore, mixture of LL-III/43 and clotrimazole is suitable candidate for future pharmaceutical research.

• MeSH
• antifungální látky farmakologie   MeSH
• azoly farmakologie   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• Candida albicans účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• faktory virulence MeSH
• fosfolipasy antagonisté a inhibitory   MeSH
• hemolýza účinky léků   MeSH
• hydrofobní a hydrofilní interakce účinky léků   MeSH
• kationické antimikrobiální peptidy chemická syntéza   farmakokinetika   MeSH
• lidé MeSH
• proteasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The α-defensins (AD) present in synovial fluid have been regarded as constituting the most accurate periprosthetic joint infection (PJI) biomarker. The methods most commonly used for estimating AD as a biomarker are the qualitative Synovasure® PJI tests, based on the technique of lateral flow, and quantitative enzyme-linked immunosorbent assay (ELISA). Here, we propose a novel test based on detecting α-defensins in synovial fluid by high-performance liquid chromatography (HPLC). Synovial fluid was collected from 157 patients diagnosed with PJI, infectious arthritis (IA), arthrosis, reactive arthritis, and rheumatoid arthritis. AD concentrations in the fluid were determined by HPLC, and these same samples were used for additional diagnostic analyses. The results were statistically processed to calculate cutoff concentrations for PJI and IA. HPLC testing showed a sensitivity of 94% and a specificity of 92% for diagnosis of PJI, and a sensitivity of 97% and a specificity of 87% for diagnosis of IA. Using HPLC, we detected in synovial fluid a combination of three α-defensins: human neutrophil peptides HNP1, HNP2, and HNP3. All measured AD concentration values shown in this work refer to the sum of these three individual concentrations. Our study shows that the HPLC method meets the conditions for measuring precise concentrations of the sum of AD and can be recommended as part of a diagnostic array for PJI and IA diagnostics. By this method, we have verified that higher levels of AD in synovial fluid can also be seen in rheumatoid illnesses, crystal arthropathies, and reactive arthritis.

• Publikační typ
• časopisecké články MeSH

PURPOSE: Antibiotic-loaded polymethylmethacrylate-based bone cement has been implemented in orthopaedics to cope with implant-related infections associated with the formation of bacterial biofilms. In the context of emerging bacterial resistance to current antibiotics, we examined the efficacy of short antimicrobial peptide-loaded bone cement in inhibiting bacterial adhesion and consequent biofilm formation on its surface. METHODOLOGY: The ability of α-helical antimicrobial peptides composed of 12 amino acid residues to prevent bacterial biofilm [methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli] formation on the surface of model implants made from polymethylmethacrylate-based bone cement was evaluated by colony-forming unit (c.f.u.) counting of bacteria released by sonication from the biofilms formed on their surfaces. The biofilms on model implant surfaces were also visualized by light microscopy after staining with tetrazolium dye (MTT) and by scanning electron microscopy. RESULTS: When incorporated in the implants, these peptides caused a mean reduction in the number of bacterial cells attached to implants' surfaces (by five orders of magnitude), and 88 % of these implants showed no bacterial adhesion after being exposed to growth media containing various bacteria. CONCLUSION: The results showed that the antibiofilm activity of these peptides was comparable to that of the antibiotics, but the peptides exhibited broader specificity than the antibiotics. Given the rapid development of antibiotic resistance, antimicrobial peptides show promise as a substitute for antibiotics for loading into bone cements.

• MeSH
• antibakteriální látky farmakologie   MeSH
• antiinfekční látky farmakologie   MeSH
• bakteriální adheze účinky léků   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• kostní cementy MeSH
• methicilin rezistentní Staphylococcus aureus účinky léků   růst a vývoj   MeSH
• mikrobiální testy citlivosti MeSH
• peptidy chemická syntéza   farmakologie   MeSH
• polymethylmethakrylát MeSH
• protézy a implantáty mikrobiologie   MeSH
• Pseudomonas aeruginosa účinky léků   růst a vývoj   MeSH
• Staphylococcus epidermidis účinky léků   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH

Candida albicans is the main causative agent of vulvovaginal candidiasis (VVC), a common mycosis in women, relapses of which are difficult to manage due to biofilm formation. This study aimed at developing novel non-toxic compounds active against Candida spp. biofilms. We synthesised analogues of natural antifungal peptides LL-III (LL-III/43) and HAL-2 (peptide VIII) originally isolated from bee venoms and elucidated their structures by nuclear magnetic resonance spectroscopy. The haemolytic, cytotoxic, antifungal and anti-biofilm activities of LL-III/43 and peptide VIII were then tested. LL-III/43 and VIII showed moderate cytotoxicity to HUVEC-2 cells and had comparable inhibitory activity against C. albicans and non-albicans spp. The lowest minimum inhibitory concentration (MIC90) of LL-III/43 was observed towards Candida tropicalis (0.8 µM). That was 8-fold lower than that of antimycotic amphotericin B. Both peptides can be used to inhibit Candida spp. bio film f ormation. Biofilm inhibitory concentrations (BIC50) ranged from 0.9 to 58.6 µM and biofilm eradication concentrations (BEC50) for almost all tested Candida spp. strains ranged from 12.8 to 200 µM. Als o pro ven were the peptides' abilities to reduce the area colonised by biofilms , inhibit hyphae formation and permeabilise cell membranes in biofil ms . LL-III/43 and VIII are promising candidates for further development as therapeutics against VVC.

• MeSH
• amfotericin B farmakologie   MeSH
• antifungální látky chemická syntéza   farmakologie   MeSH
• biofilmy účinky léků   MeSH
• Candida účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) účinky léků   MeSH
• hyfy účinky léků   MeSH
• kandidóza vulvovaginální mikrobiologie   MeSH
• kationické antimikrobiální peptidy chemická syntéza   farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• včelí jedy chemie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Joint replacement infections and osteomyelitis are among the most serious complications in orthopaedics and traumatology. The risk factors for these infections are often bacterial resistance to antimicrobials. One of the few solutions available to control bacterial resistance involves antimicrobials, which have a different mechanism of action from traditional antibiotics. Antimicrobial peptides (AMP) appear to be highly promising candidates in the treatment of resistant infections. We have identified several AMP in the venom of various wild bees and designed analogues that show potent antimicrobial activity and low toxicity against eukaryotic cells. The aim of the present study was to test the efficacy of one of those synthetic peptide analogues for the treatment of acute osteomyelitis invoked in laboratory rats. Femoral cavities of 20 laboratory Wistar rats were infected with Staphylococcus aureus. After 1 week, eight rats received an injectable calcium phosphate carrier alone, another eight rats were treated with a calcium phosphate mixed with AMP, and four rats were left without any further treatment. After another week, all rats were euthanized and radiographs were made of both the operated and healthy limbs. The animals with the carrier alone exhibited more severe acute osteomyelitis on radiographs in comparison to the recipients of the calcium phosphate carrier loaded AMP and untreated infected individuals. Based on the results of the above mentioned experiment, it was concluded that when injected directly into the site of femoral acute osteomyelitis, the calcium phosphate carrier mixed with AMP reduced osteomyelitis signs visible on radiographs.

• MeSH
• antibakteriální látky aplikace a dávkování   MeSH
• fosforečnany vápenaté farmakologie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• osteomyelitida farmakoterapie   mikrobiologie   MeSH
• peptidy aplikace a dávkování   MeSH
• potkani Wistar MeSH
• stafylokokové infekce farmakoterapie   mikrobiologie   MeSH
• Staphylococcus aureus účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Antimikrobiální peptidy jsou významnou složkou nespecifické imunity téměř všech forem života a významně se podílí na obraně jedince proti patogenním mikroorganismům. Během jejich dlouholetého studia byly popsány fyzikálně‑chemické vlastnosti molekul peptidů, klíčové pro jejich antimikrobiální účinek (kationicita, hydrofobicita, amfipaticita), a pro mnohé byly navrženy různé mechanismy jejich působení. Počáteční fází působení kationických peptidů je elektrostatická interakce se záporně nabitým povrchem bakteriální buňky. V přítomnosti bakteriální membrány nabývají molekuly peptidů své amfipatické sekundární struktury umožňující postranním řetězcům jejich hydrofobních aminokyselin zanoření dovnitř fosfolipidové dvojvrstvy membrány. To má za následek narušení struktury membrány a/nebo vznik pórů. Dochází k rozvratu biologických funkcí membrány, což v samotném důsledku vede k zániku celé buňky. Účinek je rychlý a natolik odlišný od účinku antibiotik používaných v současné klinické praxi, že se obecně předpokládá, že by se antimikrobiální peptidy v boji s infekčními chorobami jednou mohli stát léčivy nové generace. V tomto článku jsou shrnuty jednotlivé kroky výše zmíně‑ ného procesu a představeny některé modely vzniku membránových pórů. Dále jsou nastíněny způsoby, kterými se mikroorganismy proti působení antimikrobiálních peptidů brání.

Antimicrobial peptides are an important part of nonspecific immunity of almost all living organisms; they contribute significantly to their defence against pathogenic microorganisms. A range of physical and chemical properties important for the effect of these compounds (cationicity, hydrophobicity, amphipathicity) have been described during decades of their study, and various models of their mechanism of action have been proposed. First stage of the action is the electrostatic interaction of cationic peptides with negatively charged surface of the bacterial cell. Peptide molecules adopt their amphipathic secondary structure in the environment of bacterial membranes, which enables hydrophobic amino acid side chains to immerse inside the phospholipid bilayer of the membrane. It leads to membrane disruption, or alternatively to transmembrane pore formation. Such a loss of biological functions of bacterial membrane results in destruction of the entire cell. The effect is quick and completely different from the effect of commonly used antibiotics. Due to this effect, it is expec‑ ted that antimicrobial peptides could become the antimicrobials of new generation. In this article, the whole process as described above is discussed in detail and some membrane pore forming models are presented. In addition, modes of microbial resistance towards antimicrobial peptides are also summarised.

• MeSH
• bakteriální léková rezistence účinky léků   MeSH
• kationické antimikrobiální peptidy * farmakologie   MeSH

We examined the benefits of short linear α-helical antimicrobial peptides (AMPs) invented in our laboratory for treating bone infection and preventing microbial biofilm formation on model implants due to causative microorganisms of osteomyelitis. For this purpose, we introduced a model of induced osteomyelitis that utilizes human femur heads obtained from the hospital after their replacement with artificial prostheses. We found that the focus of the infection set up in the spongy part of this bone treated with AMP-loaded calcium phosphate cement was eradicated much more effectively than was the focus treated with antibiotics such as vancomycin or gentamicin loaded into the same cement. This contradicts the minimum inhibitory concentrations (MIC) values of AMPs and antibiotics against some bacterial strains obtained in standard in vitro assays. The formation of microbial biofilm on implants made from poly(methylmethacrylate)-based bone cement loaded with AMP was evaluated after the implants' removal from the infected bone sample. AMPs loaded in such model implants prevented microbial adhesion and subsequent formation of bacterial biofilm on their surface. Biofilms did form, on the other hand, on control implants made from the plain cement when these were implanted into the same infected bone sample. These results of the experiments performed in human bone tissue highlight the clinical potential of antimicrobial peptides for use in treating and preventing osteomyelitis caused by resistant pathogens.

• Publikační typ
• časopisecké články MeSH

HYL-20 (GILSSLWKKLKKIIAK-NH2) is an analogue of a natural antimicrobial peptide (AMP) previously isolated from the venom of wild bee. We examined its antimicrobial activity against three strains of Enterococcus faecalis while focusing on its susceptibility to proteolytic degradation by two known proteases-gelatinase (GelE) and serine protease (SprE)-which are secreted by these bacterial strains. We found that HYL-20 was primarily deamidated at its C-terminal which made the peptide susceptible to consecutive intramolecular cleavage by GelE. Further study utilising 1,10-phenanthroline, a specific GelE inhibitor and analogous peptide with D-Lys at its C-terminus (HYL-20k) revealed that the C-terminal deamidation of HYL-20 is attributed to not yet unidentified protease which also cleaves internal peptide bonds of AMPs. In contrast to published data, participation of SprE in the protective mechanism of E. faecalis against AMPs was not proved. The resistance of HYL-20k to C-terminal deamidation and subsequent intramolecular cleavage has resulted in increased antimicrobial activity against E. faecalis grown in planktonic and biofilm form when compared to HYL-20.

• MeSH
• antibakteriální látky chemická syntéza   metabolismus   farmakologie   MeSH
• bakteriální proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• Enterococcus faecalis účinky léků   enzymologie   růst a vývoj   ultrastruktura   MeSH
• fenantroliny farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• kationické antimikrobiální peptidy chemická syntéza   metabolismus   farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• plankton účinky léků   enzymologie   růst a vývoj   ultrastruktura   MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• serinové endopeptidasy chemie   metabolismus   MeSH
• substituce aminokyselin MeSH
• včely chemie   fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• želatinasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• infekce spojené s protézou prevence a kontrola   MeSH
• kationické antimikrobiální peptidy terapeutické užití   MeSH
• kostní cementy terapeutické užití   MeSH
• lidé MeSH
• nosiče léků MeSH
• osteomyelitida etiologie   prevence a kontrola   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH