Prenos gena za odpornost proti ampicilinu s konjugacijo in transdukcijo iz uropatogenih sevov bakterije Escherichia coli

Kapetanova, Zorica (author), Starčič Erjavec, Marjanca (mentor)

Bakterijska odpornost proti antibiotikom predstavlja globalni zdravstveni izziv. Velik delež bakterij ima namreč gene, katerih produkti omogočajo odpornost proti različnim antibiotikom. Bakterije takšne gene lahko pridobijo zaradi pridobljenih mutacij ali genskih prenosov med celicami. Cilj te magistrske naloge je bil preučiti možnost prenosa gena za odpornost proti antibiotiku ampicilinu iz izbranih sevov bakterije Escherichia coli (E. coli) zbirke uropatogenih sevov E. coli (zbirka DL) s konjugacijo in transdukcijo v laboratorijske seve E. coli. Zbirka DL vsebuje 110 uropatogenih sevov E. coli, za katere so že predhodno določili fenotipsko odpornost proti antibiotikom. Iz zbirke smo za magistrsko nalogo izbrali seve s fenotipsko odpornostjo proti ampicilinu (n = 16) in jih uporabili kot donorje v konjugaciji in transdukciji. Za izvedbo selekcije v konjugaciji in transdukciji smo uporabili štiri različne recipientske laboratorijske seve (J53, RU4406, RU4404 in HB101), ki so občutljivi za antibiotike, proti katerim so odporni izbrani donorski sevi. Konjugacija je bila izvedena na plošči LB, medtem ko smo za selekcijo transkonjugant uporabili selekcijska gojišča, ki so omogočala rast transkonjugant ob hkratni protiselekciji za donorske in recipientske celice. Gen za odpornost proti ampicilinu smo s konjugacijo uspeli prenesti iz sevov DL2, DL6, DL7, DL8, DL9, DL16, DL17, DL63, DL74. DL76. DL82 v recipientih sevov J53, RU4404, RU4406 in HB101. Transdukcijo smo izvajali na tri različne načine: (T1, T2 in P1). S transdukcijo nismo uspeli prenesti gen za odpornost proti ampicilinu iz nobenega od 16 donorskih sevov DL. Za potrditev, da pridobljene transkonjugante izvirajo iz recipientskih sevov in da niso spontane mutante donorskih sevov smo uporabili ERIC-PCR (ang. enterobacterial repetitive intergenic consensus). Poleg tega smo izvedli filogenetski kvadrupleks PCR, s katerim smo vse uporabljene seve, glede na štiri različne pomnožke PCR, uvrstili v filogenetsko skupino.

• Language: Slovenian
• Keywords: Escherichia coli, konjugacija, transdukcija, odpornost proti antibiotikom, ampicilin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [Z. Kapetanova]
• Year: 2024
• PID: 20.500.12556/RUL-163676
• UDC: 579.25:602.64
• COBISS.SI-ID: 210961923
• Publication date in RUL: 10.10.2024

Secondary language

• Language: English
• Title: Transfer of ampicillin resistance gene with conjugation and transduction from uropathogenic strains of Escherichia coli

Abstract

Bacterial resistance to antibiotics is a global health challenge. A large proportion of bacteria carry genes whose products confer resistance to various antibiotics. Bacteria can acquire such genes through acquired mutations or horizontal gene transfer between cells. The aim of this master thesis was to investigate the potential transfer of the gene for resistance to the antibiotic ampicillin from selected Escherichia coli (E. coli) strains from a collection of uropathogenic E. coli strains (DL collection) into laboratory E. coli strains by conjugation and transduction. The DL collection consists of 110 uropathogenic E. coli strains for which phenotypic antibiotic resistance has already been determined. For this master thesis strains from the collection that exhibited phenotypic resistance to ampicillin (n = 16) were selected and used as donors in conjugation and transduction experiments. Four different laboratory recipient strains (J53, RU4406, RU4404, and HB101) sensitive to the antibiotics to which the selected donor strains were resistant were used for selection in conjugation and transduction. Conjugation was performed on an LB plate, while selection media that allowed the growth of transconjugants with simultaneous counter-selection for donor and recipient cells were used for transconjugant selection. We successfully transferred the gene for ampicillin resistance by conjugation from strains DL2, DL6, DL7, DL8, DL9, DL16, DL17, DL63, DL74, DL76, and DL82 to recipient strains J53, RU4404, RU4406 and HB101. Transduction was performed using three different methods (T1, T2 and P1). We were unable to transfer the ampicillin resistance gene from any of the 16 DL donor strains by transduction. To confirm that transconjugants obtained originated from the recipient strains and were not spontaneous mutants of the donor strains, we used ERIC-PCR (enterobacterial repetitive intergenic consensus). Additionally, we performed phylogenetic quadruplex PCR to asign all the strains used into specific phylogenetic groups based on four different PCR amplicons.

• Keywords: Escherichia coli, conjugation, transduction, antibiotic resistance, ampicillin