Uticaj novosintetisanih derivata halkona na rast, produkciju biofilma i faktore virulencije multirezistentnih sojeva Acinetobacter baumannii

Acinetobacter baumannii je nozokomijalni, multirezistentni patogen, koga karakteriše sposobnost perzistencije na neživim površinama i mogućnost veoma brzog sticanja rezistencije na antibiotike. Danas su u svetu rasprostranjeni izrazito rezistentni sojevi protiv kojih u mnogim zdravstvenim ustanovama ne postoji efikasna terapija, a pronalazak alternativnih terapijskih pristupa je od izuzetne važnosti. Halkoni su jedinjenja sa potvrđenim antimikrobnim svojstvima i pokazanim različitim antivirulentnim aktivnostima. Ciljevi istraživanja ovog rada bili su određivanje profila rezistencije, ispitivanje mogućnosti kontaminacije antiseptika i ispitivanje produkcije biofilma identifikovanih kliničkih izolata A. baumannii, kao i sinteza derivata hidroksihalkona i ispitivanje njihovih antimikrobnih i antivirulentnih aktivnosti protiv ovih izolata. Osetljivost izolata na antibiotike ispitana je kombinacijom difuzionih, dilucionih i automatizovanih metoda, a identifikovani kolistin-rezistentni izolati dodatno su podvrgnuti sekvenciranju celog genoma (WGS) i genetički su okarakterisani. Takođe, mehanizmi rezistencije na kolistin ispitani su primenom komparativne analize genoma i Real-Time kvantitativne lančane reakcije polimeraze (RT-qPCR). Time-kill test je primenjen za ispitivanje perzistencije u antisepticima, a nivo produkcije biofilma ispitan je pod različitim uslovima kultivacije in vitro statičkom metodom uz bojenje safraninom. Derivati hidroksihalkona sintetisani su pomoću Claisen- Schmidt kondenzacije i njihove antimikrobne aktivnosti, samih i u kombinaciji sa antibioticima, ispitane su bujon-mikrodilucionom, Time-kill i Checkerboard analizom. Antivirulentne aktivnosti odabranih halkona procenjene su posredstvom uticaja na produkciju biofilma (monomikrobnog i polimikrobnog), vijabilnost biofilmskih ćelija, ekspresiju motiliteta, gensku ekspresiju faktora virulencije (OmpA, Bap i AbaI), adheziju A. baumannii na komponente ekstracelularnog matriksa (ECM), kao što su fibrone
Acinetobacter baumannii is a nosocomial, multiresistant pathogen, able to persist on abiotic surfaces and to rapidly acquire antibiotic resistance. Nowadays, highly resistant strains are widely disseminated throughout the world, and the discovery of alternative therapeutic strategies is of utter importance. Chalcones are compounds whose antimicrobial properties are well-known and for which different antivirulence activities have been demonstrated. The aims of this research were to determine resistance profiles, to evaluate the possibility of antiseptic contamination, and to analyze the biofilm production of identified A. baumannii clinical isolates, as well as to synthesize hydroxychalcone derivatives and to investigate their antimicrobial and antivirulence activities against these isolates. Antibiotic susceptibility of the isolates was tested by combination of diffusion, dilution, and automated methods, and additionally, identified colistin-resistant isolates were subjected to whole genome sequencing (WGS) and were genetically characterized. Also, colistin resistance mechanisms were explored by using comparative genome analysis and Real-Time quantitative polymerase chain reaction (RT-qPCR). Time-kill test was used for the measurement of bacterial survival in antiseptics, whereas the level of biofilm production under different cultivation conditions was quantified by in vitro static method using safranin stain. Hydroxychalcone derivatives were synthesized by Claisen-Schmidt condensation, and their antimicrobial activities, alone and in combination with antibiotics, were investigated using broth-microdilution, Time-kill, and Checkerboard analyses. Antivirulence activities of selected chalcones were evaluated based on the impact on biofilm production (monomicrobial and polymicrobial), biofilm cell viability, motility, virulence factors (OmpA, Bap, and AbaI) gene expression, fibronectin- and collagen- mediated adhesion, and quorum-sensing (QS) activity. A. baumannii cl