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

Abstract

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 fibronektin i kolagen, i aktivnost sistema međućelijske komunikacije (Quorum- Sensing, QS). Klinički izolati A. baumannii gotovo uniformno bili su rezistentni na karbapeneme, a čak skoro 19% izolata bilo je rezistentno na kolistin, pripadajući tako ekstenzivno rezistentnom ili panrezistentnom fenotipu. Izolati su pokazali sposobnost kontaminacije antiseptika i produkcije velikih količina biofilma. Nutritivni sastav hranljivih medijuma značajno je uticao na nivo produkcije biofilma, dok se visok nivo produkcije održao pri širokom opsegu različitih temperatura inkubacije i u prisustvu subinhibitornih koncentracija antibiotika. Sintetisani halkoni ispoljili su umerenu antimikrobnu aktivnost, pri čemu su metoksi-supstituisani derivati u proseku najjače inhibirali rast. Takođe, zabeleženo je nekoliko sinergističkih interakcija halkona sa meropenemom, a inhibicija efluksnih pumpi predložena je kao potencijalni mehanizam. Halkoni su pokazali sposobnost značajne inhibicije motiliteta i produkcije biofilma, a metoksi-supstituisani derivat (o- OCH3) ispoljio je značajnu antivirulentnu aktivnost posredstvom nishodne regulacije ekspresije ompA, bap i abaI gena i inhibicije adhezije na komponente ECM. Na osnovu ovih rezultata, o- OCH3 halkon je identifikovan kao potentni antivirulentni agens protiv A. baumannii.

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 clinical isolates expressed extensive drug-resistant or pan-drug resistant phenotypes, being nearly uniformly resistant to carbapenems. Almost 19% of isolates were resistant to colistin as well. The isolates proved to be able to contaminate the antiseptic solutions and to produce large quantities of biofilms. Nutritional composition of growth media significantly affected the level of biofilm production. In contrast, wide range of different incubation temperatures and the presence of antibiotics at subinhibitory concentrations had little effect, and the bacteria managed to maintain high level of biofilm production. Moderate antimicrobial activity was displayed by synthesized chalcones, among which methoxy-substituted derivatives achieved greatest growth inhibition in average. Also, synergistic activity of chalcones and meropenem was present in several cases, for which efflux pump inhibition was proposed as the potential mechanism. The chalcones significantly inhibited motility and biofilm production, whereas methoxy-substituted derivative (o- OCH3) also displayed significant antivirulence activity, by downregulating the ompA, bap, and abaI gene expression and by inhibiting fibronectin- and collagen-mediated adhesion. It can be concluded that o-OCH3 has been identified as a potent antivirulence agent against A. baumannii.