Uvedba metode za ugotavljanje odpornosti bakterije Chlamydia trachomatis proti makrolidom

Marušić, Monika (author), Keše, Darja (mentor)

Chlamydia trachomatis je najpogostejša bakterijska povzročiteljica spolno prenosljivih okužb v svetovnem merilu. Večinoma okužbe potekajo asimptomatsko, ki v odsotnosti zdravljenja vodijo v resne zdravstvene zaplete. Za zdravljenje klamidijske okužbe se priporoča azitromicin ali doksiciklin. Oba antibiotika spadata v skupino bakteriostatičnih antibiotikov, ki z vezavo na 23S rRNK in 16S rRNK blokirajo sintezo beljakovin. Stopnje neuspešnega zdravljenja so dokaj visoke (8–23 %), najpogostejša vzroka sta ponovna okužba po prekinitvi zdravljenja ali razvoj heterotipske odpornosti. Odpornost proti makrolidom je lahko posledica mutacij v genu za 23S rRNK, ki ovirajo vezavo in interakcijo antibiotika s tarčnim mestom delovanja. Za ugotavljanje občutljivosti klamidije za antibiotike in določanje minimalne inhibitorne koncentracije (MIK) se najpogosteje uporablja metoda gojenja v celični kulturi ob prisotnosti antibiotika v različnih koncentracijah, vendar se test zaradi zahtevnosti izvedbe in manjše občutljivosti uporablja le izjemoma. Namen magistrske naloge je vzpostavitev učinkovite metode za preverjanje prisotnosti mutacij v domeni V 23S rRNK, ki posredujejo odpornost proti makrolidom. V raziskavo smo vključili 58 vzorcev DNK kliničnih izolatov z večkrat potrjeno okužbo s C. trachomatis, kljub antibiotični terapiji. 36 vzorcev DNK smo uspešno pomnožili z alelno specifičnimi oligonukleotidi in izvedli sekvenciranje. Na podlagi bioinformacijske analize na pozicijah A2057, A2058, A2059 in T2611 (štetje po E. coli) nismo našli mutacij, ki vodijo v razvoj odpornosti proti makrolidom. V skladu s pričakovanji smo uspešno uvedli molekularno metodo, ki nam omogoča dokazovanje mutacij, ki posredujejo odpornost proti makrolidom.

• Language: Slovenian
• Keywords: klamidije, Chlamydia trachomatis, spolno prenosljive okužbe, protimikrobna odpornost, makrolidi, 23S rRNK, mutacije
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [M. Marušić]
• Year: 2021
• PID: 20.500.12556/RUL-134071
• UDC: 579.61:616.98:579.882:577.2.08
• COBISS.SI-ID: 91211267
• Publication date in RUL: 23.12.2021

MARUŠIĆ, Monika, 2021, Uvedba metode za ugotavljanje odpornosti bakterije Chlamydia trachomatis proti makrolidom [online]. Master’s thesis. Ljubljana : M. Marušić. [Accessed 23 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=134071

Secondary language

• Language: English
• Title: Introduction of a method for detecting the resistance of Chlamydia trachomatis to macrolides

Abstract

Chlamydia trachomatis is the most common sexually transmitted bacterial infection worldwide. Often, infections occur asymptomatically, leading to serious health complications in the absence of treatment. The use of azithromycin and doxycycline is recommended for the treatment of sexually transmitted chlamydial infections. Both antibiotics belong to the group of bacteriostatic antibiotics that bind to 23S rRNA and 16S rRNA, blocking protein synthesis. Treatment failure rates are high (8-23 %), for which the most common cause is re-infection after discontinuation of treatment or the development of heterotypic resistance. The most commonly used test for antibiotic susceptibility and MIC determination is cultivation in cell culture in the presence of antibiotics in various concentrations. Resistance to macrolides may be due to mutations in the 23S rRNA gene that cause reduction in interaction of antibiotics with target site of action. The aim of this master's thesis is to establish an effective method for determination of mutations in the domain V of 23S rRNA that mediate resistance to macrolides, so we included 58 DNA samples of clinical isolates with confirmed C. trachomatis infection after antibiotic treatment. 36 DNA samples were successfully amplified with allele-specific oligonucleotides and sequencing was performed. Based on bioinformatics analysis at positions A2057, A2058, A2059 and T2611 (E. coli numbering), we did not find mutations that are important for the development of resistance to macrolides. Based on expectations, we have successfully established a molecular method to obtain nucleotide sequences, whereby the presence of important mutations that mediate resistance to macrolides can be determined based on alignments with reference sequences.

• Keywords: Chlamydia trachomatis, sexually transmitted diseases, antibiotic resistance, macrolides, mutation, 23S rRNK