Browsing by Author "Özkütük A.A."

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    Pyrazinamide monoresistance in clinical isolates
    (2013) Kalir N.; Özkütük A.A.; Esen N.; Özkütük N.
    Aim: To determine pyrazinamide (PZA) monoresistance in clinical isolates of Mycobacterium tuberculosis complex (MTBC) isolated in hospitals in and around İzmir. Materials and methods: The study was performed between 2004 and 2009 with 150 streptomycin, isoniazid, rifampicin, and ethambutol susceptible MTBC clinical strains isolated in the university hospitals of İzmir and Manisa. The PZA susceptibility of the isolates was determined by the BACTEC 460 TB test. Results: The results indicated that resistance to PZA was present in 5 (3.3%) of the 150 MTBC isolates susceptible to all primary drugs except PZA. Conclusion: It is not essential to perform PZA susceptibility tests routinely along with other primary drugs due to the low PZA monoresistance level in our region. © TÜBİTAK.
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    Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis
    (Brieflands, 2024) Arslan N.; Demiray-Gurbuz E.; Ozkutuk N.; Esen N.; Özkütük A.A.
    Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods:Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis. © 2024, Arslan et al.