High Incidence of an Emerging Opportunistic Pathogen <em>Candida parapsilosis</em> in Water-Related Domestic Environments

Candidiasis is one of the common fungal opportunistic infections, usually associated with diverse Candida species. Candida albicans, C. glabrata complex, C. parapsilosis complex, C. tropicalis and C. auris are often identified in affected patients. Candida parapsilosis sensu stricto is an emerging cause of hospital-acquired Candida infections, predominantly in Southern Europe, South America and Asia. Home environment is a less known source of infection despite frequent isolation of C. parapsilosis from kitchen surfaces and household appliances such as dishwashers, washing machines and refrigerators. C. parapsilosis is one of the first colonisers of novel dishwashers and a member of stable fungal communities on rubber seals worldwide in concentrations up to 102 CFU/cm2. It colonises also drawers for detergents in washing machines and drainage channels in refrigerators. Tap water and groundwater act as vector for entrance of C. parapsilosis in the indoor environments. Within C. parapsilosis, four clinically relevant phenotypes can be distinguished. Experimental data on the prevalence of C. parapsilosis isolates phenotypes, obtained from indoor environments, will be presented. Smooth phenotype prevails in dishwashers and washing machines, while crepe and crater dominate in water. In conclusion, the ability to colonise diverse environments and accordingly switch phenotypes defines C. parapsilosis as a versatile, domestic environment-related opportunistic pathogen

Synergistic interactions in microbial biofilms facilitate the establishment of opportunistic pathogenic fungi in household dishwashers

Biofilms formed on rubber seals in dishwashers harbor diverse microbiota. In this study, we focussed on the microbial composition of bacteria and fungi, isolated from a defined area of one square centimeter of rubber from four domestic dishwashers and assessed their abilities to in vitro multispecies biofilm formation. A total of 80 isolates (64 bacterial and 16 fungal) were analyzed. Multiple combinations of bacterial isolates from each dishwasher were screened for synergistic interactions. 32 out of 140 tested (23%) four-species bacterial combinations displayed consistent synergism leading to an overall increase in biomass, in all experimental trails. Bacterial isolates from two of the four dishwashers generated a high number of synergistically interacting four-species consortia. Network based correlation analyses also showed higher co-occurrence patterns observed between bacterial members in the same two dishwasher samples, indicating cooperative effects. Furthermore, two synergistic fourspecies bacterial consortia were tested for their abilities to incorporate an opportunistic fungal pathogen, Exophiala dermatitidis and their establishment as biofilms on sterile ethylene propylene diene monomer M-class (EPDM) rubber and polypropylene (PP) surfaces. When the bacterial consortia included E. dermatitidis, the overall cell numbers of both bacteria and fungi increased and a substantial increase in biofilm biomass was observed. These results indicate a novel phenomenon of cross kingdom synergy in biofilm formation and these observations could have potential implications for human health

Microbial diversity and putative opportunistic pathogens in dishwasher biofilm communities

Extreme habitats are not only limited to natural environments, but also exist in manmade systems, for instance, household appliances such as dishwashers. Limiting factors, such as high temperatures, high and low pHs, high NaCl concentrations, presence of detergents, and shear force from water during washing cycles, define microbial survival in this extreme system. Fungal and bacterial diversity in biofilms isolated from rubber seals of 24 different household dishwashers was investigated using next-generation sequencing. Bacterial genera such as Pseudomonas, Escherichia, and Acinetobacter, known to include opportunistic pathogens, were represented in most samples. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus, and Rhodotorula, also known to include opportunistic pathogenic representatives. This study showed how specific conditions of the dishwashers impact the abundance of microbial groups and investigated the interkingdom and intrakingdom interactions that shape these biofilms. The age, usage frequency, and hardness of incoming tap water of dishwashers had significant impact on bacterial and fungal community compositions. Representatives of Candida spp. were found at the highest prevalence (100%) in all dishwashers and are assumed to be one of the first colonizers in recently purchased dishwashers. Pairwise correlations in tested microbiomes showed that certain bacterial groups cooccur, as did the fungal groups. In mixed bacterial-fungal biofilms, early adhesion, contact, and interactions were vital in the process of biofilm formation, where mixed complexes of bacteria and fungi could provide a preliminary biogenic structure for the establishment of these biofilms. IMPORTANCE Worldwide demand for household appliances, such as dishwashers and washing machines, is increasing, as is the number of immunocompromised individuals. The harsh conditions in household dishwashers should prevent the growth of most microorganisms. However, our research shows that persisting polyextremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions and supported by the biofilm mode of growth. The significance of our research is in identifying the microbial composition of biofilms formed on dishwasher rubber seals, how diverse abiotic conditions affect microbiota, and which key microbial members were represented in early colonization and contamination of dishwashers, as these appliances can present a source of domestic cross-contamination that leads to broader medical impacts

The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi

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Selection of opportunistic microorganisms in the extreme environment of dishwashers

Vodovodna voda, priključena na gospodinjske naprave, predvsem pomivalne stroje, deluje kot vektor zlasti vnosa gliv in tudi bakterij v pomivalni stroj. Glive in predvsem bakterije lahko vstopajo v pomivalni stroj tudi preko prebivalcev in hišnih ljubljenčkov ter ostankov hrane na umazani posodi. Znotraj pomivalnega stroja se zaradi skrajnostnih dejavnikov, kot so visoka temperatura, oksidativni stres, povišane koncentracije soli (NaCl), mehanskih strižnih sil vode, velikega organskega bremena in občasne dehidracije, mikroorganizmi povežejo v preživetvene strukture – biofilme. Ti biofilmi so sestavljeni iz različnih vrst bakterij in gliv. Mikobiota tesnila je globalno konsistentna in jo sestavlja 6 vrst oportuno patogenih gliv E. dermatitidis, E. phaeomuriformis, Candida parapsilosis sensu stricto, Fusarium dimerum species complex in kompleks Saprochaete/Magnusiomyces. Bakterijsko floro tesnil večinoma sestavljajo rodovi Bacillus, Gordonia, Exiguobacterium, Acinetobacter, Stenotrophomonas in Micrococcus. Presenetljivo so na tesnilu pomivalnih strojev prisotne tudi oportuno patogene vrste bakterij, kot so E. coli, P. aeruginosa, S. maltophilia in E. faecium. Začetni naseljevalci tesnil pomivalnih strojev so kvasovke rodu Candida, ki nudijo biotsko površino za nadaljnjo vzpostavitev bakterijsko-glivnih biofilmov. Med bakterijami, izoliranimi iz pomivalnih strojev, smo opazili obsežne sinergije. Vrsti A. junii in P. aeruginosa sta bili najboljši proizvajalki biofilmov v pomivalnih strojih in sta pomembno prispevali k sinergiji v biofilmih in k skupnemu povečanju biomase. Ugotovili smo, da lahko mešani bakterijski biofilmi vključujejo oportunistično patogeno črno kvasovko E. dermatitidis in ji olajšajo naselitev na gumijastih tesnilih pomivalnih strojev in drugih površinah (npr. polipropilen). Mikrobiom tesnil in celotnega pomivalnega stroja se z leti in frekvenco uporabe spreminja. Predvsem na glive bistveno vpliva trdota vodovodne vode. Glive se lahko iz pomivalnega stroja razširjajo v zunanje okolje preko odpadne vodovodne vode in aerosola, ki se sprošča v okolje po končanem ciklu pranja. Pomivalni stroji vplivajo na naselitev kuhinjskih površin z glivami, predvsem na pojavnost črnih kvasovk. Le-teh je občutno manj v kuhinjah brez pomivalnega stroja, kjer prevladuje oportuno patogena bela kvasovka C. parapsilosis.Due to extreme environmental factors prevailing inside dishwashers, such as high temperature, oxidative stress, elevated salt concentrations (NaCl), mechanical shear forces, high organic loads and occasional dehydration, microorganisms colonizing dishwashers merge into survival structures - biofilms. They consist of mixed assemblages of various bacteria and fungi. The mycobiota of the rubber seals was globally consistent and it comprised 6 species of opportunistic pathogens such as E. dermatitidis, E. phaeomuriformis, Candida parapsilosis, Fusarium dimerum and Saprochaete/Magnusiomyces complex. Besides rubber seals, fungi populated also doors, walls, drains, sprinkler, side nozzle, rack for cutlery, rinse-air dispenser and detergent dispenser. Bacterial flora of dishwasher rubber seals was comprised of the genera Bacillus, Gordonia, Exiguobacterium, Acinetobacter, Stenotrophomonas and Micrococcus. Surprisingly, bacteriome of rubber seals could include also opportunistic pathogens, such as E. coli, P. aeruginosa, S. maltophilia and E. faecium. The initial settlers of the dishwasher seals were yeasts of the genus Candida, that offer a biotic surface for further establishment of microbes. Extensive synergies were observed among bacteria isolated from dishwashers. The species A. junii and P. aeruginosa were the best producers of biofilms in dishwashers and significantly contributed to the observed synergy in biofilms and to the total increase of biomass. Mixed bacterial biofilms included also the opportunistic pathogenic black yeast E. dermatitidis, enabling an easier settlement of rubber seals of dishwashers and other artificial surfaces (eg polypropylene). Changes of microbiome over time depended on the years of operation and frequency of use, while water hardness essentially influenced the fungal community. Tap water, connected to the household appliances, especially dishwashers, was the main entry point of fungi and to a lesser extent for bacteria. Additional sources of fungal and especially bacterial contamination were inhabitants, pets and food leftovers on dirty dishes. Fungi could be released from the dishwasher to the external environment at the end of the washing cycle via waste water and aerosols. Colonization of kitchen surfaces with fungi was influenced by the presence of dishwashers, resulting in higher incidence of black yeasts, especially E. dermatitidis, that were considerably less frequent in kitchens without dishwashers, where opportunistic pathogenic white yeast C. parapsilosis prevailed

COMPARATIVE LEGAL VIEW OF MAIN CHARACTERISTICS OF PATENT PROTECTION AND TRADE SECRET PROTECTION IN SLOVENIA, CROATIA AND AUSTRIA

Magistrska naloga v teoretičnem delu predstavlja primerjalno-pravni prikaz poglavitnih značilnosti zaščite patenta in varstva poslovne skrivnosti v Sloveniji, Avstriji in na Hrvaškem. Končna splošna ugotovitev predelane literature in virov je, da je na obeh področjih obravnavanih držav več podobnosti kot razlik. Obenem naloga predstavi tudi razmerje med področjema prava intelektualne lastnine / prava industrijske lastnine in konkurenčnega prava / prava nelojalne konkurence. Pravni teoretiki so različnega mnenja, vendar na splošno povezava med obema področjema obstaja. Sledi obravnava dileme, v kateri se ukvarjamo z vprašanjem, kateri mehanizem zaščite je boljši, patent ali poslovna skrivnost. Poudariti je potrebno, da ni mogoče določiti, kateri institut je primerneje uporabiti, saj je za vsak konkreten primer lahko boljši oziroma učinkovitejši drug mehanizem zaščite. Glede na situacijo mora vsak izumitelj pretehtati pozitivne in negativne strani zaščite in se nato odločiti za primernejšo zaščito oziroma varstvo. Na podlagi predelane teorije sledi praktično delo v obliki anketePatent ali poslovna skrivnost v slovenskih podjetjih. Z anketo želimo pridobiti čim več različnih podatkov, tako o inovativnosti podjetij, številu patentov, razlogov za odločitev za en ali drug mehanizem zaščite, ovirah in pozitivnih lastnostih patenta ali poslovne skrivnosti. Zanimali so nas tudi podatki o internih aktih, določbah v pogodbi o zaposlitvi in pogodbah o ne razkrivanju informacij, ki določajo varovanje poslovnih skrivnosti itd. Kljub slabi odzivnosti slovenskih podjetij vseeno lahko ugotovimo, da so rezultati primerljivi z že opravljeno, obsežnejšo raziskavo področja intelektualne lastnine v Sloveniji.In its theoretical part, this master’s thesis presents the comparative law’s overview of the main features of patent protection and protection of business secrets in Slovenia, Austria and Croatia. The final general conclusion of the analysed literature and sources is that there are more similarities than differences in both fields in all of the countries included in the research. The thesis also presents the ratio between the fields of intellectual property law / industrial property law and competition law / unfair competition law. Legal theoreticians provide us with several differing opinions, but, in general, there exists a connection between the two fields. What follows is the analysis of the dilemma of which mechanism of protection is better - a patent or business secret. It has to be pointed out that it is impossible to determine which one is more appropriate to use, as a different protection mechanism can be better or more effective for each individual case. Considering the situation, each inventor must weigh the positive and negative sides of protection and then decide on the more suitable protection or security. What follows is the practical work in the form of a questionnaire, which is based on the analysed theorypatent or business secret in Slovenian companies. The questionnaire aims to acquire as much data as possible on the companies’ innovativeness, number of patents, reasons for choosing one or the other mechanism, obstacles and positive aspects of a patent or business secret, as well as internal acts, employment contract terms and agreements on confidentiality, which stipulate the protection of business secrets, etc. In spite of the companies’ limited feedback, it was still possible to establish that the results are comparable to those of an earlier, wider research dealing with intellectual property in Slovenia

QAC resistance genes in ESBL-producing E. coli isolated from patients with lower respiratory tract infections in the Central Slovenia region—a 21-year survey

Biocidal products prevent the spread of pathogenic microorganisms, including extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), which is one of the most alarming health problems worldwide. Quaternary ammonium compounds (QACs) are surface-active agents that interact with the cytoplasmic membrane and are widely used in hospitals and food processing environments. A collection of 577 ESBL-EC, isolated from lower respiratory tract (LRT) samples, was screened for QAC resistance genes oqxAoqxBqacE∆1qacEqacF/H/IqacGsugE (p)emrEmdfAsugE (c)ydgEydgFand for class 1, 2, and 3 integrons. The prevalence of chromosome-encoded genes ranged from 77 to 100%, while the prevalence of QAC resistance genes encoded on mobile genetic elements (MGEs) was relatively low (0–0.9%), with the exception of qacE∆1 (54.6%). PCR screening detected the presence of class 1 integrons in 36.3% (n = 210) of isolates, which were positively correlated with qacE∆1. More correlations between QAC resistance genes, integrons, sequence type group ST131, and β-lactamase genes were presented. The results of our study confirm the presence of QAC resistance genes and also class 1 integrons commonly found in multidrug-resistant clinical isolates and highlight the potential role of QAC resistance genes in the selection of ESBL-producing E. coli in hospitals

Art education and environmental sustainability

Monografija je v slovenskem prostoru eden prvih poskusov celovitejše znanstvene obravnave odnosa med skrbjo za ohranjanje človekovega naravnega okolja zanamcem in likovno umetnostjo. V teoriji je predstavljen odnos med okoljsko trajnostjo na eni in likovno umetnostjo ter edukacijo in poučevanjem umetnosti na drugi strani. Empirične raziskave so izvedene na populaciji predšolskih in šolskih otrok, predšolskih pedagogov in univerzitetnih študentov in raziskujejo implementacije likovnih dejavnosti na predšolskem, osnovnošolskem in srednješolskem nivoju. Ugotovili smo, da je okoljska trajnost dandanes večplastno povezana s sodobno likovno umetnostjo in poučevanjem umetnosti. Ekološke vsebine tvorijo pomemben del vsebin v vrtcih in šolah. Vsebine univerzitetnih pedagoških študijskih programov bi naj vsebovali več tematik, povezanih z okoljsko trajnostjo, kar bi povečalo interes in zavedanje študentov o pomembnosti tega aktualnega problema.The monograph is one of the first attempts in Slovenia at a comprehensive scientific discussion of the relation between the care for preserving human being’s natural environment and art education. The theoretical section aims to juxtapose and examine the relations between environmental sustainability, on the one hand, and visual art, general education and art education, on the other. The empirical surveys cover preschool and school-age children, preschool teachers and university students. The studies showcase the implementation of artistic activities during the preschool period, in primary schools and in secondary school practices. We established that the environmental sustainability of today shows multifaceted connections with contemporary art and education. Ecological content forms an important part of the content in kindergartens and schools. The content of art education study programmes at universities should include more topics connected with sustainable development, which would increase students’ interest in and awareness of the importance of this current problem

Opportunistic Water-Borne Human Pathogenic Filamentous Fungi Unreported from Food

Clean drinking water and sanitation are fundamental human rights recognized by the United Nations (UN) General Assembly and the Human Rights Council in 2010 (Resolution 64/292). In modern societies, water is not related only to drinking, it is also widely used for personal and home hygiene, and leisure. Ongoing human population and subsequent environmental stressors challenge the current standards on safe drinking and recreational water, requiring regular updating. Also, a changing Earth and its increasingly frequent extreme weather events and climatic changes underpin the necessity to adjust regulation to a risk-based approach. Although fungi were never introduced to water quality regulations, the incidence of fungal infections worldwide is growing, and changes in antimicrobial resistance patterns are taking place. The presence of fungi in different types of water has been thoroughly investigated during the past 30 years only in Europe, and more than 400 different species were reported from ground-, surface-, and tap-water. The most frequently reported fungi, however, were not waterborne, but are frequently related to soil, air, and food. This review focuses on waterborne filamentous fungi, unreported from food, that offer a pathogenic potential