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http://hdl.handle.net/11607/5381
Title: | RESTORATION OF VANCOMYCIN SENSITIVITY USING ANTISENSE RNA AND INVESTIGATION OF ITS EFFICACY IN VANCOMYCIN-RESISTANT ENTEROCOCCI IN INFECTED MICE |
Other Titles: | ANTİSENS RNA KULLANARAK VANKOMİSİN HASSASİYETİNİN RESTORASYONU VE VANKOMİSİN DİRENÇLİ ENTEROKOK İLE ENFEKTE FARELERDE ETKİNLİĞİNİN ARAŞTIRILMASI |
Authors: | Bozdoğan, Bülent Yalçın Benli, Melis Aydın Adnan Menderes Üniversitesi, Sağlık Bilimleri Enstitüsü, Moleküler Biyoteknoloji Anabilim Dalı |
Keywords: | antisense RNA (asRNA), gene silencing, vanA, vancomycin, vancomycin-resistant enterococci (VRE) |
Issue Date: | 2025 |
Publisher: | Aydın Adnan Menderes Üniversitesi, Sağlık Bilimleri Enstitüsü |
Abstract: | Purpose: This study aimed to restore susceptibility in vancomycin-resistant Enterococcus strains using plasmid-encoded antisense RNA (asRNA) of the vanA/vanB genes, and to examine their in vivo efficacy in an experimental mouse model. Materials and Methods: The vanA and vanB genes were amplified by PCR from clinical E. faecium VRE50 and E. faecalis V583, respectively, then reverse cloned into pUC19 plasmid to generate antisense RNAs via restriction enzyme sites. These constructed plasmids with reversly oriented genes were transformed into E. coli DH10B. Then, asRNAs, for vanA and vanB were extracted and their abilities to restore vancomycin susceptibility were tested in vitro in the presence of vancomycin. The vancomycin MIC was done with or without asRNA and during 24-hour OD595 measurements were taken over 24 hours to generate growth curves. For in vivo analysis, liposome encapsulated and free asRNAs were administered intraperitoneally to VRE-infected CD-1 mice with vancomycin, and treatment outcomes were assessed by measuring CFU to calculate bacterial loads in peritoneal fluid. Additionally, murine sepsis scores of the mice were recorded and determined daily. Results: Acording to in vitro studies, while antibiotic susceptibility tests showed a remarkable reduction in vancomycin MIC values, decreasing from >256 µg/mL in the wild-type strain to 32 µg/mL in expressing vanA-antisense RNA by fusion plasmid in clinical VRE50, vanB-asRNA reduced in vancomycin MIC value from 64 µg/mL (MIC value of E.faecalis V583) to 16 µg/mL. Growth kinetics analyses revealed extended lag phases and reduced growth rates in antisense-expressing VRE strains, supporting the functional repression of resistance gene expression. In the murine sepsis model, mice treated with the combination of intraperitoneal antisense RNA and vancomycin exhibited significantly lower bacterial burdens in peritoneal fluid and improved clinical scores compared to control groups. While liposomal formulations were tested for in vivo delivery, their therapeutic efficacy appeared limited, possibly due to insufficient uptake or delayed release. Collectively, these results indicate that antisense RNA constructs can effectively impair van gene function both in vitro and in vivo, restoring antibiotic susceptibility and attenuating infection severity. Conclusion: This study demonstrates that plasmid-derived antisense RNAs targeting the vanA and vanB genes can effectively resensitize vancomycin-resistant Enterococcus strains both in vitro and in vivo. As a reversible and highly specific gene-silencing approach that does not require genome editing, antisense RNA technology emerges as a promising new strategy in the fight against antimicrobial resistance. Beyond its application to VRE, this method holds potential for targeting resistance genes in a wide range of multidrug-resistant pathogens. |
URI: | http://hdl.handle.net/11607/5381 |
Appears in Collections: | Doktora |
Files in This Item:
File | Description | Size | Format | |
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Melis Yalçın Benli TEZ 20,08,25.pdf | Doktora tezi | 1.97 MB | Adobe PDF | View/Open Request a copy |
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