Učinak fosfomicina in vitro u kombinaciji s drugim antibioticima prema kliničkim izolatima i biofilmu bakterije Pseudomonas aeruginosa

doctoral thesis

• Title (english): In vitro effect of fosfomycin combined with other antibiotics on clinical isolates and biofilm of Pseudomonas aeruginosa
• Author: Mia Slade Vitković
• Mentor: Branka Bedenić (mentor)
• Mentor: Gordana Maravić Vlahoviček (komentor)
• Committee member: Maja Šegvić Klarić (predsjednik povjerenstva)
• Committee member: Ivana Perković (član povjerenstva)
• Committee member: Amarela Lukić-Grlić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2024-12-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Pseudomonas aeruginosa (PA) jedan je od vodećih uzročnika bolničkih infekcija. Uzrokuje zabrinutost s obzirom na stalni porast otpornosti na antibiotike i mogućnost stvaranja biofilma. Stari antibiotik fosfomicin sve se češće koristi u terapiji sistemnih infekcija uzrokovanih otpornim bakterijama. Bolja učinkovitost može se postići kombiniranim davanjem antibiotika. Cilj istraživanja bio je odrediti in vitro sinergiju fosfomicina s 8 antibiotika koji se koriste u liječenju PA infekcija na izolatima te bakterije s različitim determinantama rezistencije. Cilj je također bio ispitati postantibiotički učinak fosfomicina samog i u kombinaciji s istim antibioticima i utvrditi učinak kombinacija vršnih koncentracija antibiotika na inhibiciju i razaranje biofilma koji stvaraju otporni izolati te bakterije. Testiranje osjetljivosti na antibiotike provedeno je metodom disk difuzije i određivanjem minimalnih inhibitornih koncentracija. Prisustvo β-laktamaza utvrđeno je fenotipskim testovima i lančanom reakcijom polimeraze. Metodom ukriženih trakica i „time-kill“ metodom ispitana je sinergija. Postantibiotički učinak je određen metodom brojanja poraslih kolonija po uklanjanju antibiotika kojemu su bakterije bile izložene. Stvaranje biofilma, inhibicija formiranja i disrupcija biofilma ispitani su statičkim testom u mikrotitarskim pločicama s kristal violetom. Otpornost na fosfomicin registrirana je kod značajnog dijela kliničkih izolata P. aeruginosa (52,9 %). Najveća stopa sinergije određena metodom ukriženih trakica postignuta je u kombinacijama fosfomicina s ceftazidimom (21,6 %) i gentamicinom (17,7 %), a „time-kill“ metodom s kolistinom i gentamicinom. Iako su sve kombinacije s antibioticima produljile postantibiotički učinak, najbolji rezultat postignut je u kombinaciji s kolistinom. Negativne vrijednosti PAE nisu zabilježene niti s jednom kombinacijom. Prema rezultatima ispitivanja biofilma u mikrotitarskim pločicama svi ispitani sojevi bili su proizvođači biofilma. Predominantno je registriran učinak kombinirane terapije na inhibiciju stvaranja biofilma. Kombinirana terapija bila je učinkovitija u sprječavanju nastanka biofilma od monoterapije, čak i kod izolata otpornih na pojedinačno primijenjene antibiotike. Najviše stope inhibicije stvaranja biofilma zabilježene su u kombinacijama s kolistinom, cefepimom i ceftazidimom. Kombinacije s ispitivanim β-laktamskim antibioticima i kolistinom imale su snažniji učinak na inhibiciju formiranja biofilma od inhibitora sinteze proteina. Rezultati dobiveni u ovoj studiji mogli bi biti klinički značajni u pogledu terapije različito otpornog P. aeruginosa, ali i profilakse stvaranja biofilma kod takvih izolata.

Abstract (english)

Pseudomonas aeruginosa (PA) is one of the leading causes of hospital infections. It is a cause for concern given the rapid antibiotic resistance development and the ability of biofilm formation. Fosfomycin has recently reemerged as an interesting treatment option for antimicrobial-resistant infections, and combination therapy is an important treatment alternative. The aim of this research was to determine in vitro synergy of fosfomycin with 8 antibiotics used for treatment of PA infections on variously resistant PA isolates. The aim was also to examine the postantibiotic effect of fosfomycin alone and in combination with those antibiotics and to determine inhibition and disruption of PA biofilm in the presence of peak plasma concentrations of those antibiotics alone and in combination with fosfomycin. Antibiotic susceptibility was determined using the disk diffusion method and the microdilution method. The presence of β-lactamases was determined using phenotypic tests and polymerase chain reaction. Synergy was determined using the gradient diffusion strip cross method (GDSC) and the "time-kill" method. The post-antibiotic effect was determined by the viable colony counting method after antibiotic removal. Biofilm formation, inhibition of biofilm formation and disruption were determined using the crystal violet microtiter plate assay. Resistance to fosfomycin was registered in a significant part of clinical isolates of P. aeruginosa (52.9 %). The highest synergy rate determined using the GDSC method was observed for combinations with ceftazidime (21.6 %) and gentamicin (17.7 %) and using the "time-kill" method with colistin and gentamicin. Although all antibiotic combinations prolonged the postantibiotic effect, the best result was achieved in combination with colistin. Negative PAE values were not recorded. All tested isolates were biofilm producers. The effect of combination therapy was predominantly registered in prevention of biofilm formation. Combination therapy was more effective in preventing biofilm formation than monotherapy, even in isolates resistant to individually administered antibiotics. The highest inhibition rates of biofilm formation were recorded in combinations with colistin, cefepime and ceftazidime. Combinations with β-lactam antibiotics and colistin had a stronger effect on the inhibition of biofilm formation than protein synthesis inhibitors. The results of this study could be clinically valuable not only regarding treatment of variously resistant P. aeruginosa isolates, but also regaring biofilm formation profilaxis of those isolates.

• Keywords: Pseudomonas aeruginosa; višestruko otporna bakterija; prošireno otporna bakterija; fosfomicin; sinergija antibiotika; „time-kill“ metoda; metoda ukriženih trakica; postantibiotički učinak; biofilm; inhibicija stvaranja biofilma; razaranje biofilma
• Keywords (english): Pseudomonas aeruginosa; multidrug-resistant; extensively drug-resistant; fosfomycin; antibiotic synergy; "time-kill" method; cross strip method; post-antibiotic effect; biofilm; inhibition of biofilm formation; biofilm destruction
• Language: croatian
• URN:NBN: urn:nbn:hr:163:775411
• Promotion: 2025
• Study programme: Title: Pharmceutical-biochemical sciences - doctoral study - university study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području biomedicine i zdravstva (doktor/doktorica znanosti u području biomedicine i zdravstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2025-03-28 13:45:22