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    Screening Brucella spp. in bovine raw milk by real-time quantitative PCR and conventional methods in a pilot region of vaccination, Edirne, Turkey
    (Elsevier Science Inc, 2016) Kaynak-Onurdag, F.; Okten, S.; Sen, B.
    Brucellosis is a worldwide zoonotic disease transmitted to humans by consumption of contaminated milk and milk products. Brucellosis is endemic in Turkey, and Edirne has a high Brucella prevalence. Brucellosis is prevented by live-attenuated vaccines for animals and the vaccination program has been in place since 1984 in Turkey. Thrace is the pilot region for this vaccination program. The gold standard diagnostic technique for brucellosis is still the isolation of suspicious bacterial colonies followed by bacteriological identification, but it is very time consuming and laborious. In many studies, Brucella has been investigated by PCR techniques. However, PCR-based methods cannot differentiate between the vaccine strain and the virulent strain; thus, the vaccine strain may interfere with the virulent strain and causes false-positive reactions. To monitor brucellosis control programs effectively, it is important to distinguish vaccine and field strains of Brucella spp. In this study, raw milk samples were collected from 99 cows at 12 different barns in 5 villages of Edirne (Turkey). Bacteriological analyses and real-time quantitative (q)PCR experiments were applied to all samples. The DNA was isolated using Biospeedy DNA-Tricky Purification Kit (Bioeksen, Istanbul, Turkey). For all reactions, Roche Light Cycler Nano (Roche Diagnostics, Mannheim,.Germany) instrument and Biospeedy EvaGreen qPCR Pre-Mix (Bioeksen) were used.. The data were analyzed,using Roche Light-Cycler NanoSoftware 1.0. For samples that were negative by bacteriological analyses and positive by qPCR, we developed a novel qPCR-based method to differentiate the virulent B. abortus strains and B. abortus S19 vaccine strain. We designed qPCR primers targeting the outer membrane protein of B. abortus. The qPCR products were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). In total, 2.02% of the samples were Brucella positive, by both bacteriological method and the novel qPCR method. We concluded that, to obtain true-positive results in Brucella spp. screening studies for milk, differentiating the virulent and vaccine strain should not be disregarded.
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    Synthesis and investigation of binding interactions of 1,4-benzoxazine derivatives on topoisomerase IV in Acinetobacter baumannii
    (Taylor & Francis Ltd, 2017) Yilmaz, S.; Yalcin, I.; Okten, S.; Onurdag, F. K.; Aki-Yalcin, E.
    Acinetobacter baumannii has emerged as an important pathogen for nosocomial infections having high morbidity and mortality. This pathogen is notorious for antimicrobial resistance to many common antimicrobial agents including fluoroquinolones, which have both intrinsic and acquired resistance mechanisms. Fluoroquinolones targeting the bacterial topoisomerase II (DNA gyrase and Topo IV) show potent broad-spectrum antibacterial activity by the stabilization of the covalent enzyme-DNA complex. However, their efficacy is now being threatened by an increasing prevalence of resistance. Fluoroquinolones cause stepwise mutations in DNA gyrase and Topo IV, having alterations of their binding sites. Furthermore, the water-Mg+2 bridge, which provides enzyme-fluoroquinolone interactions, has a significant role in resistance. In this study, 13 compounds were synthesized as 1,4-benzoxazine derivatives which act as bacterial topoisomerase II inhibitors and their antibacterial activities were determined against multi-drug resistant Acinetobacter strains which have ciprofloxacin (CIP) resistant and GyrA mutation. Afterwards we performed docking studies with Topo IV (pdb:2XKK) of these compounds to comprehend their binding properties in Discovery Studio 3.5. The results of this study show significant conclusions to elucidate the resistance mechanism and lead to the design of new antibacterial agents as bacterial topoisomerase II inhibitors.