Ekspresija i primarna struktura gena ompK35 i ompK36 u kliničkih izolata Klebsiella pneumoniae (ST437) neosjetljivih na ertapenem

master's thesis

• Title (english): Expression and primary structure of ompK35 and ompK36 genes of Klebsiella pneumoniae (ST437) clinical isolates non-susceptible to ertapenem
• Author: Mia Pejković
• Mentor: Arjana Tambić-Andrašević (mentor)
• Mentor: Jasna Hrenović (sumentor)
• Committee member: Jasna Hrenović (predsjednik povjerenstva)
• Committee member: Renata Šoštarić (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Karbapenemi su antibiotici zadnje linije obrane u liječenju infekcija izazvanih višestruko otpornim enterobakterijama otpornim na cefalosporine proširenog spektra, među kojima značajnu ulogu ima Klebsiella pneumoniae. Otpornost na karbapeneme postao je globalni zdravstveni problem. Proizvodnja karbapenemaza je najefikasniji mehanizam otpornosti na karbapeneme, no mehanizmi otpornosti na ovu klasu antibiotika, poglavito ertapenem, uključuju i hidrolizu ESBL enzimima u uvjetima smanjene koncentracije antibiotika u bakterijskoj stanici, proizašlih iz promjena u porinskim kanalima. OmpK35 i OmpK36 su hidrofilni porinski kanali K. pneumoniae koji omogućavaju unos nutrijenata i antibiotika u bakteriju. U ovom radu analizirane su promjene unutar gena ompK35 i ompK36 u kliničkih izolata K. pneumoniae ST437 smanjene osjetljivosti na ertapenem prikupljenih tijekom 2011./2012. godine u zagrebačkim bolnicama. Mehanizmi otpornosti su utvrđeni korištenjem metode lančane reakcije polimerazom, elektroforezom u agaroznom gelu i sekvenciranjem. Otpornost na ertapenem se utvrdila mjerenjem minimalnih inhibitornih koncentracija (MIC, mg/L) metodom mikrodilucije u kation-prilagođenom Muller-Hinton bujonu. Svi izolati su pokazali visoku stopu otpornosti na ertapenem i imali su značajno sniženu ekspresiju OmpK35 porinskog kanala (p<0.001). 47.8% izolata je imalo inserciju GD unutar L3 omče koja je povezana s pojačanom otpornosti na ertapenem. Svi izolati su imali promjene unutar gena ompK36 (insercije, delecije, supstitucije).

Abstract (english)

Carbapenems are last line antibiotics that are used as a treatment of infections caused by Enterobacteriaceae non-susceptibile to extended spectrum cephalosporins, with Klebsiella pneumoniae as one of the most significant members of this family. Carbapenem resistance is major global health problem. Production of carbapenemases is the most efficient mechanism of carbapenem resistance, although mechanisams involved in carbapenem resistance involve ESBL enzymes that are able to hydrolyze this class of antibiotics in the presence of decreased concentration of antibiotics as a consequence of alternations of porins. Klebsiella pneumoniae hydrophilic porins OmpK35 and OmpK36 play role in intake of nutrients and antibiotics in bacterial cell. In this thesis structural alternations were analysed in ompK35 and ompK36 genes in clinical isolates of K. pneumoniae ST437. Isolates were collected during 2011/2012 in hospitals in Zagreb. Resistance mechanisms were determined by using polymerase chain reaction, agarose gel-electrophoresis and sequencing. Minimal inhibitory concentrations (MIC, mg/L) were measured by microdilution method in cation-adjusted Mueller-Hinton broth. All isolates showed high level of ertapenem resistance and significatly lower level of expression of gene ompK35 (p<0.001). 47.8% isolates had GD insertions in loop L3, previously shown to affect high level ertapenem resistance. Clinical isolates had alterations in ompK36 gene (insertions, deletions, substitutions).

• Keywords: Klebsiella pneumoniae; beta-laktami; ertapenem; MDR; otpornost; ompK35 i ompK36
• Keywords (english): Klebsiella pneumoniae; beta-lactams; ertapenem; MDR; resistance; ompK35 and ompK36
• Language: croatian
• URN:NBN: urn:nbn:hr:217:304802
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-02-23 08:11:36