Generation and studies of BKRF4- deficient mutants of Epstein-Barr virus

Epstein-Barr virus (EBV) BKRF4 gene product is a tegument protein encoded by a gene with no sequence homology outside of the gamma subfamily of Herpesviridae. Its positional homologs are necessary for an efficient viral lytic program, in particular viral progeny egress and primary infection. To characterize BKRF4 in this regard, EBV recombinant viruses deficient for BKRF4 were developed using site-directed mutagenesis and a bacterial artificial chromosome (BAC)-based recombineering system. Stable human embryonic kidney (HEK) 293 cell lines containing these genomes were generated and the phenotypes of these mutants were analyzed following stimulation of the viral lytic cycle. During the lytic program, BKRF4-null cell lines showed decreased protein expression of various EBV lytic genes that were analyzed using immunostaining and flow cytometry. Reduced amounts of extracellular viral progeny were observed when quantified by real-time PCR and infectivity assays as compared to wild type. These findings suggest an active role of BKRF4 in EBV infection, possibly in viral egress

Multilocus Sequence Typing for Interpreting Blood Isolates of Staphylococcus epidermidis

Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is also a common contaminant of blood cultures and, as a result, there is frequently uncertainty as to its diagnostic significance when recovered in the clinical laboratory. One molecular strategy that might be of value in clarifying the interpretation of S. epidermidis identified in blood culture is multilocus sequence typing. Here, we examined 100 isolates of this species (50 blood isolates representing true bacteremia, 25 likely contaminant isolates, and 25 skin isolates) and the ability of sequence typing to differentiate them. Three machine learning algorithms (classification regression tree, support vector machine, and nearest neighbor) were employed. Genetic variability was substantial between isolates, with 44 sequence types found in 100 isolates. Sequence types 2 and 5 were most commonly identified. However, among the classification algorithms we employed, none were effective, with CART and SVM both yielding only 73% diagnostic accuracy and nearest neighbor analysis yielding only 53% accuracy. Our data mirror previous studies examining the presence or absence of pathogenic genes in that the overlap between truly significant organisms and contaminants appears to prevent the use of MLST in the clarification of blood cultures recovering S. epidermidis

Molar Mass, Entanglement, and Associations of the Biofilm Polysaccharide of Staphylococcus epidermidis

U okviru ovog završnog rada dat će se detaljan pregled vrsta motornih ulja i objasnit će se procesi koji uzrokuju njihovo starenje. Kad je nakon korištenja ulje klasificirano kao otpad, ono se može i mora zbrinuti na pravilan način koji je propisan zakonskim zahtjevima u Republici Hrvatskoj, a koji će također biti obuhvaćeni ovim radom. Bit će objašnjene i dvije osnovne mogućnosti oporabe otpadnih mazivih ulja: materijalna i termička. U poglavlju koje opisuje materijalnu oporabu otpadnog mazivog ulja razmatrat će se komercijalizirani procesi materijalne oporabe u industriji te će ovi procesi biti detaljnije objašnjeni. Kod termičke oporabe opisat će se područja primjene takvog načina oporabe otpadnih mazivih ulja te metode pomoću kojih se emisije štetnih tvari nastalih tijekom oporabe drže ispod graničnih vrijednosti emisija koje su propisane. U zaključnom djelu završnog rada bit će prikazani podaci o prikupljanju i oporabi otpadnih mazivih ulja prikupljeni za područje Republike Hrvatske, grada Zagreba i Zagrebačke županije. Dat će se detaljan pregled prikupljenih količina otpadnih mazivih ulja u vremenskom periodu od 2007. do 2013. godine po ovlaštenim sakupljačima te će se analizirati učinkovitost prikupljanja. Također će se iznijeti podaci o oporabljenim količinama otpadnih ulja po ovlaštenim oporabiteljima. Kako je u Republici Hrvatskoj najzastupljenija termička oporaba otpadnog ulja s primjenom u cementnoj industriji, opisat će se na primjeru tvrtke Našicecement d.o.o. prednosti korištenja otpadnog ulja u kombinaciji s konvencionalnim fosilnim gorivima u svrhu dobivanja potrebne toplinske energije u proizvodnom procesu za dobivanje klinkera. Usporednim dijagramima bit će prikazane vrijednosti emisije štetnih tvari izmjerenih od strane tvrtke i graničnih vrijednosti emisija propisanih Uredbom o graničnim vrijednostima emisija (NN 117/12)

Role of Environmental and Antibiotic Stress on Staphylococcus epidermidis Biofilm Microstructure

Cellular clustering and separation of Staphylococcus epidermidis surface adherent biofilms were found to depend significantly on both antibiotic and environmental stress present during growth under steady flow. Image analysis techniques common to colloidal science were applied to image volumes acquired with high-resolution confocal laser scanning microscopy to extract spatial positions of individual bacteria in volumes of size ∼30 × 30 × 15 μm³. The local number density, cluster distribution, and radial distribution function were determined at each condition by analyzing the statistics of the bacterial spatial positions. Environmental stressors of high osmotic pressure (776 mM NaCl) and sublethal antibiotic dose (1.9 μg/mL vancomycin) decreased the average bacterial local number density 10-fold. Device-associated bacterial biofilms are frequently exposed to these environmental and antibiotic stressors while undergoing flow in the bloodstream. Characteristic density phenotypes associated with low, medium, and high local number densities were identified in unstressed S. epidermidis biofilms, while stressed biofilms contained medium- and low-density phenotypes. All biofilms exhibited clustering at length scales commensurate with cell division (∼1.0 μm). However, density phenotypes differed in cellular connectivity at the scale of ∼6 μm. On this scale, nearly all cells in the high- and medium-density phenotypes were connected into a single cluster with a structure characteristic of a densely packed disordered fluid. However, in the low-density phenotype, the number of clusters was greater, equal to 4% of the total number of cells, and structures were fractal in nature with <i>d</i>_f =1.7 ± 0.1. The work advances the understanding of biofilm growth, informs the development of predictive models of transport and mechanical properties of biofilms, and provides a method for quantifying the kinetics of bacterial surface colonization as well as biofilm fracture and fragmentation