Mikotoksinogenost kliničkih i okolišnih Aspergillus fumigatus i A. flavus sojeva

Clinical isolates of fifty strains of A. fumigatus and 30 strains of A. flavus from immmunocompromised patients from the hematological unit were analyzed for mycotoxin production and compared with the same number of environmental isolates (from soil, compost, and air). Only 9 (18%) strains of A. fumigatus produced gliotoxin in a mean concentration 2.22 mg mL-1 (range 0.5-5 mg mL-1). Aflatoxin B1 was detected in 7 (23%) isolates (range from 0.02 to 1.2 mg L-1) and aflatoxin G1 in one (3%) of clinical A. flavus isolates (0.12 mg L-1). In the group of environmental isolates, 11 (37%) were positive for aflatoxin B1 production (range from 0.02 to 1.2 mg L-1) and one for aflatoxin G1 (0.02 mg L-1). Bioautoantibiogram ("bioassay in situ") on TLC plates against Bacillus subtilis NCTC 8236 showed that only gliotoxin-producing strains have bactericidal activity of Rf values corresponding to gliotoxin. The secondary-metabolite profiles of clinical and environmental A. fumigatus and A. flavus isolates were homogenous, except for gliotoxin production, which was detected only in the group of clinical isolates of A. fumigatus (18%).Analizirana je mikotoksinogenost pedeset kliničkih sojeva vrste A.fumigatus i trideset sojeva vrste A. flavus izoliranih iz imunokompromitiranih ispitanika u hematološkom odjelu. Kao usporedba, izoliran je isti broj okolišnih sojeva iz zemlje, komposta i zraka. Utvrđeno je da samo 9 sojeva (18%) vrste A. fumigatus tvori gliotoksin sa srednjom vrijednošću 2,22 mg mL–1 (u rasponu od 0,5 do 5 mg mL–1). Tvorba aflatoksina B1 utvrđena je u 7 sojeva (23%) (raspon od 0,024 do 1,2 mg L–1) i u jednog kliničkog izolata (3%) A.flavus vrste (0,12 mg L–1). U skupini okolišnih sojeva utvrđena je tvorba aflatoksina B1u 11 sojeva (37%) (u rasponu od 0,024 do 1,2 mg L–1) i u jednog soja tvorba aflatoksina G1(0,024 mg L–1). Bioautoantibiogramom na tankoslojnoj kromatografskoj ploči s bakterijskom vrstom Bacillus subtilis NCTC 8236 utvrđeno je da samo gliotoksinogeni sojevi pokazuju baktericidnu aktivnost oko Rf vrijednosti koja odgovara gliotoksinu. Profili sekundarnih metabolita kliničkih i okološnih sojeva A. funmigatus i A. flavus vrsta su bili homogeni, osim za tvorbu gliotoksina koji je utvrđen samo u skupini kliničkih izolata A. fumigatus sojeva (18%)

Odvezivanje sidra lipopolisaharida: sustavi za strukturne modifikacije lipida A nude dijagnostičke i terapijske opcije za borbu protiv polimiksinske rezistencije

Polymyxin antibiotics are the last resort for treating patients in intensive care units infected with multiple-resistant Gram-negative bacteria. Due to their polycationic structure, their mode of action is based on an ionic interaction with the negatively charged lipid A portion of the lipopolysaccharide (LPS). The most prevalent polymyxin resistance mechanisms involve covalent modifications of lipid A: addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN). The modified structure of lipid A has a lower net negative charge, leading to the repulsion of polymyxins and bacterial resistance to membrane disruption. Genes encoding the enzymatic systems involved in these modifications can be transferred either through chromosomes or mobile genetic elements. Therefore, new approaches to resistance diagnostics have been developed. On another note, interfering with these enzymatic systems might offer new therapeutic targets for drug discovery. This literature review focuses on diagnostic approaches based on structural changes in lipid A and on the therapeutic potential of molecules interfering with these changes.Polimiksinski antibiotici zadnja su linija liječenja pacijenata u intenzivnim jedinicama liječenja koji su inficirani multiplorezistentnim gramnegativnim bakterijama. S obzirom na njihovu polikationsku strukturu, njihov mehanizam djelovanja temelji se na ionskoj interakciji s negativno nabijenim dijelom lipopolisaharida koji se naziva lipid A. Najčešći mehanizmi rezistencije na polimiksine uključuju kovalentne modifikacije lipida A: adiciju kationskog šećera 4-amino-L-arabinoze (L-Ara4N) i/ili fosfoetanoloamina (pEtN). Modificirana struktura lipida A sadrži niži neto negativni naboj, što prouzročuje odbojne sile između polimiksina i lipida A. To dovodi do bakterijske rezistencije na urušavanje integriteta stanične membrane. Geni koji kodiraju za enzimske sustave koji sudjeluju u navedenim modifikacijama mogu se prenositi kromosomima ili mobilnim genskim elementima. Stoga su i razvijeni novi pristupi dijagnostici rezistencije. Osim toga, u navedenim enzimskim sustavima postoje i moguće nove terapijske mete za razvoj lijekova. Ovaj pregledni rad usredotočuje se na dijagnostičke metode koje se temelje na strukturnim promjenama u lipidu A i na terapijskom potencijalu molekula koje interferiraju s navedenim strukturnim modifikacijama

Utjecaj medija i temperature na tvorbu gliotoksina u sojeva Aspergillus fumigatus

Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 °C resulted in higher gliotoxin production than at 25 °C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL-1 (in YES at 37 °C) and 1.07 mg mL-1 (in CZA at 25 °C). YES broth as a medium containing 4 % sucrose and 2 % of yeast extract is a very rich substrate for the production of gliotoxin in vitro.Gliotoksin je sekundarni metabolit iz skupine epipolitiodioksipiperazina s biološki aktivnim internim disulfidnim mostom u molekuli, koji tvore razne plijesni i gljivica Candida albicans. Plijesan Aspergillus fumigatus vodeći je uzročnik invazivnih aspergiloza i također može tvoriti gliotoksin. Pretpostavlja se da in situ tvorba gliotoksina utječe na patogenezu aspergiloze. U ovom radu ispitali smo in vitro tvorbu gliotoksina u devet sojeva A. fumigatus vrste, izoliranih iz imunokompromitiranih pacijenata. Praćen je utjecaj medija i temperature inkubacije na tvorbu gliotoksina. Gliotoksin je ekstrahiran iz biomase i koncentracija mu je utvrđena polukvantitativno tankoslojnom kromatografijom. Tvorba gliotoksina uočena je već nakon trodnevne inkubacije u koncentracijama 4,06 mg mL-1 (u bujonu s kvaščevim ekstraktom – YES na 37 °C) i 1,07 mg mL-1 (u sintetskom Czapek-Dox bujonu – CZA na 25 °C). Tvorba gliotoksina bila je veća u YES bujonu, za razliku od sintetskog CZA bujona. Viša temperatura inkubacije (37 °C) također utječe na jaču tvorbu gliotoksina nego niža temperatura (25 °C). YES bujon s dodatkom 4 % saharoze i 2 % kvaščeva ekstrakta vrlo je bogat supstrat za in vitro tvorbu gliotoksina, kao jednog od lipofilnih sekundardnih metabolita plijesni vrste A. fumigatus

Welcome to the emerging third era of antimicrobial therapy

Problem otpornosti mikroba na antibiotike može se sagledati kao globalni, kompleksan i sistemski problem s društvenim, kulturnim, socijalnim i političkim posljedicama. Nekoliko je strategija kako prevladati otpornost mikroba na antibiotike, no danas postoji potreba za globalnom strategijom u solidarnoj borbi protiv ove sporo-razvijajuće i progresivne nepogode. Budući da na sve uključene dionike u ovom dinamičnom procesu postoji javni pritisak, ujedno je taj pritisak usmjeren i na znanstvenu zajednicu. Odgovor je na povratak na specifičnu terapiju putem primjene terapijskih monoklonalnih protutijela, ali i primjene inhibitora mikrobne virulencije, bakteriofagnih lizina, antimikrobnih peptida, potencijatora učinka antibiotika, imunomodulatora i dr. Ti pristupi, od kojih je većina orjentirana mikrobno-specifično, uvode nas u treće doba antimikrobne terapije koje se polako rađa napuštajući dominaciju antibiotske molekularne paradigme.The problem of antimicrobial resistance (AMR) could be seen as a global, complex and systemic problem with social, cultural and political consequences. There are several strategies how to overcome AMR, but today we need a unique global and solidary strategy to combat this slowly emerging and progressive disaster. Because all stakeholders in this dynamic challenging process are involved, scientists are also under public pressure to resolve this problem. Nowadays, scientific approach to combat AMR is focused on the need for a specific therapy. This approach includes the use of therapeutic monoclonal antibodies, microbial virulence inhibitors, bacteriophage lysins, antimicrobial proteins, potentiators of current antibiotics, immunomodulators, etc. These approaches, most of which are microbe-specific oriented, introduce us to the third era of antimicrobial therapy, which slowly arises from the domination of molecular paradigmatic era of antibiotics

Onečišćenja u ljekovitom bilju i biljnim proizvodima

Medicinal plants have a long history of use in therapy throughout the world and still make an important part of traditional medicine. Thus, medicinal plants and herbal products must be safe for the patient (consumer). This review addresses biological contaminants (microbes and other organisms) and chemical contaminants (mycotoxins, toxic elements such as heavy metals, and pesticide residues) as major common contaminants of medicinal herbs and herbal products. To prevent and screen for contamination and ensure safety and conformity to quality standards, medicinal herbs and herbal products should be included in appropriate regulatory framework.Ljekovito bilje i biljni proizvodi već tisućljećima nalaze široku primjenu u različitim sustavima tradicionalnog liječenja. Stoga je njihova neškodljivost, ponajprije uvjetovana kakvoćom biljne sirovine, od izuzetne važnosti za javno zdravstvo. Od mogućih čimbenika koji utječu na kakvoću ljekovitog bilja i biljnih proizvoda ovaj pregledni rad osvrće se na najčešće prisutna biološka (mikroorganizmi) i kemijska onečišćenja (mikotoksini, toksični elementi poput teških metala te ostaci pesticida). S ciljem postizanja ujednačenih standarda kakvoće te osiguranja sigurnosti primjene biljnih proizvoda od vitalne su važnosti zakonski propisi koji moraju u odgovarajućim regulatornim okvirima obuhvatiti ovu skupinu proizvoda s naglaskom na sprječavanju i ispitivanju njihovih mogućih onečišćenja

Antimikotsko djelovanje tekućeg ekstrakta i eteričnog ulja iz plodova aniša (Pimpinella anisum L., Apiaceae)

Antifungal activities of fluid extract and essential oil obtained from anise fruits Pimpinella anisum L. (Apiaceae) were tested in vitro on clinical isolates of seven species of yeasts and four species of dermatophytes. Diffusion method with cylinders and the broth dilution method were used for antifungal activity testing. Anise fluid extract showed antimycotic activity against Candida albicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis and C. krusei with MIC values between 17 and 20% (V/V). No activity was noticed against C. glabrata, and anis fruits extracts showed growth promotion activity on Geotrichum spp. Anise fruits extract inhibited the growth of dermatophyte species (Trichophyton rubrum, T. mentagrophytes, Microsporum canis and M. gypseum) with MIC values between 1.5 and 9% (V/V). Anise essential oil showed strong antifungal activity against yeasts with MIC lower than 1.56% (V/V) and dermatophytes with MIC lower than 0.78% (V/V). Significant differences in antifungal activities were found between anise fluid extract and anise essential oil (p < 0.01). Anise essential oil exhibited stronger antifungal activities against yeasts and dermatophytes with MIC values between 0.10 and 1.56% (V/V), respectively.Ispitana je antimikotsko djelovanje tekućeg ekstrakta i eteričnog ulja iz plodova aniša (Pimpinella anisum L., Apiaceae) na kliničke izolate sedam vrsta kvascima sličnih gljivica i četiri vrste dermatofita. Za ispitivanje antifungalne aktivnosti upotrebljene su metode difuzije cilindrima i metode razrjeđivanja u tekućem hranilištu. Tekući ekstrakt aniša pokazuje antifungalnu aktivnost na vrste Candida albicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis i C. krusei s vrijednostima minimalne inhibitorne koncentracije (MIK) između 17 i 20% (V/V). Vrsta C. glabrata je rezistentna na djelovanje esktrakta, a uočeno je promotivno djelovanje ekstrakta na rast Geotrichum sp. Esktrakt aniša pokazuje inhibiciju rasta dermatofita (Trichophyton rubrum, T. mentagrophytes, Microsporum canis i M. gypseum) s MIK vrijednostima između 1,5 i 9,0% (V/V). Eterično ulje aniša pokazuje jaku antifungalnu aktivnost na kvascima slične gljivice s vrijednostima MIK-a nižima od 1,56% (V/V), a na dermatofite s vrijednostima MIK-a nižima od 0.78% (V/V). Utvrđena je značajna razlika (p < 0,01) između MIK-a tekućeg ekstrakta aniša i aniševog eteričnog ulja. Eterično ulje aniša pokazuje jaču antifungalnu aktivnost na kvascima slične gljivice i dermatofite s vrijednostima MIK-a između 0,10 i 1,56% (V/V)

Protective effects of olive oil phenolics oleuropein and hydroxytyrosol against hydrogen peroxide-induced DNA damage in human peripheral lymphocytes

This study investigates antioxidant capacity and protective effects of phenolic compounds oleuropein (OLP) and hydroxytyrosol (HT), present in olive oil and olive leaves, against H2O2-induced DNA damage in human peripheral lymphocytes. Antioxidant potency was determined using the measurement of radical-scavenging activity (ABTS∙+ assay), ferric reducing power (FRAP assay) and cupric reducing antioxidant capacity (CUPRAC assay). Both substances were found to be potent antioxidant agents due to their free radical-scavenging activities. Antigenotoxic effects of oleuropein and hydroxytyrosol against H2O2-induced damage in human lymphocytes were evaluated in vitro by alkaline comet assay. At tested concentrations (1, 5, 10 µmol L–1), oleuropein and hydroxytyrosol did not induce a significant increase of primary DNA damage in comparison with the negative control. Pretreatment of human lymphocytes with each of the substances for 120 min produced a dose-dependent reduction of primary DNA damage in the tested cell type. Hydroxytyrosol showed a better protective effect against H2O2-induced DNA breaks than oleuropein which could be associated with their free radical-scavenging efficacy

Tvorba verukulogena u kliničkih i iz zraka izoliranih sojeva vrste Aspergillus fumigatus Fres.

Among airborne aspergilli sampled in outdoor air of the Zagreb area (2002/2003), Aspergillus niger (v. Teigh.) and A. fumigatus (Fres.) were the most abundant species (20-30%), with low mean annual concentrations (0.21-1.04 CFU m-3). Higher concentrations of A. fumigatus were observed in autumn and winter (0.5-1.05 CFU m-3) than in spring and summer (0-0.4 CFU m-3). On the other hand, A. fumigatus was found to be the most frequent isolate from upper and/or lower respiratory tracts of imunocompromised patients in many studies. This species produces several mycotoxins including the tremorgenic mycotoxin verruculogen that can be found in spores and during myceliar growth. Verruculogen production ability was tested by biosynthesis on 30 airborne and 33 clinical isolates of A. fumigatus. In both groups, high percentage of verruculogen-producing strains was noticed (84% of airborne and 91% of clinical isolates). Verruculogen production was not significantly different in the groups of airborne isolates (0.34 ± 0.16 mg mL-1) and clinical isolates (0.26 ± 0.19 mg mL-1, p > 0.05).Uzorkovanje spora plijesni na području grada Zagreba obavljeno je tijekom godine 2002./2003. Pri tome su Aspergillus niger (v. Teigh.) i A. fumigatus (Fres.) bile najučestalije (20–30%) među aerogenim Aspergillus vrstama. Srednje godišnje koncentracije njihovih spora bile su izrazito niske (0,21–1,04 CFU m–3). Veće koncentracije spora A. fumigatus zabilježene su tijekom jeseni i zime (0,50–1,05 CFU m–3) u odnosu na koncentracije izmjerene tijekom proljeća i ljeta (0–0,40 CFU m–3). S druge strane, mnogobrojne studije ukazuju na visoku učestalost A. fumigatus izolata iz gornjih i donjih dišnih puteva imunokompromitiranih bolesnika. Ova plijesan tvori nekoliko različitih mikotoksina, uključujući i tremorgeni mikotoksin verukulogen koji se može naći u sporama i miceliju vrste A. fumigatus. Tvorba verukulogena ispitana je u 30 aerogenih i 33 klinička izolata A. fumigatus vrste. U obje skupine dokazana je visoka učestalost verukulogen-pozitivnih sojeva (84% aerogenih i 91% kliničkih izolata). Potencijal tvorbe verukulogena u aerogenih (0.34 ± 0.16 mg mL–1) i kliničkih sojeva (0.26 ± 0.19 mg mL–1) nije se značajnije razlikovao

Antimikrobni učinak eteričnog ulja borovice (Juniperus communis L., Cupressaceae)

Juniper essential oil (Juniperi aetheroleum) was obtained from the juniper berry, and the GC/MS analysis showed that the main compounds in the oil were alpha-pinene (29.17%) and betha-pinene (17.84%), sabinene (13.55%), limonene (5.52%), and mircene (0.33%). The juniper essential oil was evaluated for the antimicrobial activity against sixteen bacterial species, seven yeast-like fungi, three yeast and four dermatophyte strains. Juniper essential oil showed similar bactericidal activities against Gram-positive and Gram-negative bacterial species; with MIC values between 8 and 70% (V/V) as well as a strong fungicidal activity against yeasts, yeast-like fungi and dermatophytes, with the MIC values below 10%, V/V. The strongest fungicidal activity was recorded against Candida spp. (MIC from 0.78 to 2 %, V/V) and dermatophytes (from 0.39 to 2%, V/V).Eterično ulje borovice (Juniperi aetheroleum) dobiveno je iz plodova (bobica Juniperus communis L., Cupressaceae) GC/MS analizom dokazane su glavne sastavnice alpha-pinen (29,17%) i beta-pinen (17,84%), sabinen (13,55%), limonen (5,52%) i mircen (0,33%). Ispitan je antimikrobni učinak eteričnog ulja borovice na 16 bakterijskih vrsta, 7 kvasaca i kvascima sličnim gljivicama, te 4 vrste dermatofita. Eterično ulje borovice pokazuje slični baktericidan učinak na Gram-pozitivne i Gram-negativne bakterijske vrste s MIK vrijednostima između 8 i 70% (V/V). Ulje pokazuje snažni protugljivični učinak s MIK vrijednostima ispod 10% (V/V). Najjača protugljivična aktivnost uočena je na vrste roda Candida, s MIK vrijednostima između 0,78 i 2% (V/V) i na dermatofite s MIK vrijednostima između 0,39 i 2% (V/V)