Analiza plazmida pES6 seva Escherichia coli VF2017

magistrsko delo

Ana Kugovnik (Author), Cene Gostinčar (Reviewer), Jerneja Ambrožič (Mentor)

Abstract

Sevi Escherichia coli imajo pogosto genske zapise za encime betalaktamaze z razširjenim spektrom delovanja (ESBL) na konjugativnih plazmidih. V raziskavi smo analizirali konjugativni plazmid pES6, povezan z odpornostjo proti cefotaksimu (blaSHV-12) in nizkim koncentracijam fluorokinolonov (qnrS1), iz ESBL-pozitivnih sevov E. coli, vzorčenih v perutninskih rejah v Sloveniji. Ugotovljene frekvence konjugativnega prenosa odpornosti proti cefotaksimu so bile pri poskusih konjugacij z donorskim sevom VF2017-A5 (A5) od 10-7 do 10-5, z donorskim sevom VF2017-A6 (A6) pa od 10-5 do 10-3. S sekvenciranjem plazmidne DNA (pDNA) izbranih transkonjugant (TK) smo ugotovili, da so poleg plazmida pES6, ki je enak kot v literaturi opisani plazmid pEC-243, v navedenih donorskih sevih prisotni še dodatni plazmidi pV5, pV6 in pK6. Pri TK iz donorja A5 je prisoten domnevni plazmid pV5 z zapisom za kolicin B in kolicin M, pri TK iz donorja A6 pa domnevni plazmid pV6 z zapisom za kolicin Ia in mikrocin V ter kriptični plazmid pK6. Njihovo prisotnost smo pri TK potrdili tudi s PCR s plazmidno-specifičnimi začetnimi oligonukleotidi, plazmida pV5 in pV6 pa tudi fenotipsko na podlagi kolicinske aktivnosti. S primerjavo rezultatov, dobljenih z bioinformacijsko analizo nukleotidnih zaporedij plazmidov sevov A5 in A6 s podatki, dobljenih z analizo izolirane nerazrezane in razrezane pDNA po gelski elektroforezi, smo ugotovili, da z elektroforezo pDNA, zgolj vizualno, ne zaznamo in/ali težko opredelimo vse plazmide v izolirani pDNA posameznega seva.

Keywords

Escherichia coli;ESBL;konjugativni plazmidi;IncX3;frekvenca konjugacije;NGS;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2022
Typology:	2.09 - Master's Thesis
Organization:	UL BF - Biotechnical Faculty
Publisher:	[A. Kugovnik]
UDC:	577.2
COBISS:	120876291
Views:	27
Downloads:	12
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Analysis of plasmid pES6 from Escherichia coli strain VF2017
Secondary abstract:	Escherichia coli strains have extended-spectrum beta-lactamases (ESBL) genes that are frequently encoded on conjugative plasmids. In our study, we analyzed the conjugative plasmid pES6, isolated from ESBL-positive E. coli strains from selected poultry farms in Slovenia, that confer resistance to low concentrations of fluoroquinolones (qnrS1) and cefotaxime (blaSHV-12). We analyzed the frequency of conjugative transfer of resistance to cefotaxime from source donors VF2017-A5 (A5) and VF2017-A6 (A6). The frequency of A5 conjugation experiments varied from 10-7 to 10-5, and the frequency of A6 conjugation experiments from 10-5 to 10-3. By sequencing the plasmid DNA (pDNA) of selected transconjugants (TK), we found that in addition to plasmid pES6, which is identical to plasmid pEC-243 described in the literature, additional plasmids pV5, pV6, and pK6 are present. Supposed plasmid pV5, present in TK derived from the A5 donor, encodes the colicins B and M. Supposed plasmid pV6, encoding the colicin Ia and microcin V and cryptic plasmid pK6 are present in TK derived from the A6 donor. Their presence in TK was confirmed by PCR with plasmid-specific primers. For plasmids pV5 and pV6, their presence was also confirmed phenotipically by colicin activity. Comparison of the results of bioinformatic analysis with data obtained from analysis of isolated, uncut and cut pDNA after gel electrophoresis, led to the conclusion that pDNA electrophoresis methods are not sufficient to visually detect all plasmids in the isolated pDNA of a single strain.
Secondary keywords:	conjugative plasmids;conjugation frequency;master thesis;Molekularna biologija;Univerzitetna in visokošolska dela;
Type (COBISS):	Master's thesis/paper
Study programme:	0
Thesis comment:	Univ. v Ljubljani, Biotehniška fak., Oddelek za biologijo
Pages:	1 spletni vir (1 datoteka PDF (XIII, 75 str., [9] str. pril.))
ID:	16411046