Prophage isolation and characterization of carbapenem resistantKlebsiella pneumoniae isolates

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious global public health threat due to its intrinsic resistance mechanisms and ability to acquire additional resistance traits. In this thesis study, 20 CRKP isolates obtained from various clinical specimens and preserved in the culture collection of Başkent University Faculty of Medicine, Department of Medical Microbiology, were evaluated for the presence of prophages. Classical induction methods including UV irradiation, mitomycin C, levofloxacin, and ciprofloxacin were applied; however, no plaque formation was observed using the double-layer technique. Although MTT staining revealed plaque-like structures, sufficient amounts of phages could not be isolated. Therefore, four isolates with different sequence types were selected for whole genome sequencing, leading to the identification of 19 prophage regions. Among them, 14 were classified as "intact," 2 as "questionable," and 3 as "incomplete," with the latter excluded from further analysis. Re-induction of these isolates using previously isolated lytic phages confirmed the activation of 13 prophages through in silico analyses, indicating that some prophages, although undetectable by traditional methods, may still be inducible at the molecular level. Whole genome analysis revealed no direct antibiotic resistance genes in the prophages; however, genes associated with virulence, stress response, regulation, and defense were identified. Additionally, some prophages harbored anti-CRISPR proteins, tRNA genes, and mobile genetic elements related to horizontal gene transfer. While these elements do not directly encode resistance, they may contribute to bacterial adaptation under environmental stress and antibiotic pressure, thereby indirectly supporting resistance development. Pangenome analyses showed that the prophages were genetically diverse and primarily carried shell genes located in the accessory genome. Overall, the findings suggest that prophages integrated into CRKP genomes are molecularly active mobile genetic elements that may evade classical detection methods, yet significantly contribute to the evolutionary success and adaptability of their bacterial hosts.