Influence of newly-synthesized chalcone derivatives on growth, biofilm production, and virulence factors expression of multiresistant Acinetobacter baumannii strains

Acinetobacter baumannii je nozokomijalni, multirezistentni patogen, koga karakterišesposobnost perzistencije na neživim površinama i mogućnost veoma brzog sticanja rezistencije naantibiotike. Danas su u svetu rasprostranjeni izrazito rezistentni sojevi protiv kojih u mnogimzdravstvenim ustanovama ne postoji efikasna terapija, a pronalazak alternativnih terapijskihpristupa je od izuzetne važnosti. Halkoni su jedinjenja sa potvrđenim antimikrobnim svojstvima ipokazanim različitim antivirulentnim aktivnostima. Ciljevi istraživanja ovog rada bili suodređivanje profila rezistencije, ispitivanje mogućnosti kontaminacije antiseptika i ispitivanjeprodukcije biofilma identifikovanih kliničkih izolata A. baumannii, kao i sinteza derivatahidroksihalkona i ispitivanje njihovih antimikrobnih i antivirulentnih aktivnosti protiv ovih izolata.Osetljivost izolata na antibiotike ispitana je kombinacijom difuzionih, dilucionih iautomatizovanih metoda, a identifikovani kolistin-rezistentni izolati dodatno su podvrgnutisekvenciranju celog genoma (WGS) i genetički su okarakterisani. Takođe, mehanizmi rezistencijena kolistin ispitani su primenom komparativne analize genoma i Real-Time kvantitativne lančanereakcije polimeraze (RT-qPCR). Time-kill test je primenjen za ispitivanje perzistencije uantisepticima, a nivo produkcije biofilma ispitan je pod različitim uslovima kultivacije in vitrostatičkom 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 aktivnostiodabranih halkona procenjene su posredstvom uticaja na produkciju biofilma (monomikrobnog ipolimikrobnog), vijabilnost biofilmskih ćelija, ekspresiju motiliteta, gensku ekspresiju faktoravirulencije (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 čakskoro 19% izolata bilo je rezistentno na kolistin, pripadajući tako ekstenzivno rezistentnom ilipanrezistentnom fenotipu. Izolati su pokazali sposobnost kontaminacije antiseptika i produkcijevelikih količina biofilma. Nutritivni sastav hranljivih medijuma značajno je uticao na nivoprodukcije biofilma, dok se visok nivo produkcije održao pri širokom opsegu različitih temperaturainkubacije i u prisustvu subinhibitornih koncentracija antibiotika. Sintetisani halkoni ispoljili suumerenu antimikrobnu aktivnost, pri čemu su metoksi-supstituisani derivati u proseku najjačeinhibirali rast. Takođe, zabeleženo je nekoliko sinergističkih interakcija halkona sa meropenemom,a inhibicija efluksnih pumpi predložena je kao potencijalni mehanizam. Halkoni su pokazalisposobnost značajne inhibicije motiliteta i produkcije biofilma, a metoksi-supstituisani derivat (o-OCH3) ispoljio je značajnu antivirulentnu aktivnost posredstvom nishodne regulacije ekspresijeompA, 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 abioticsurfaces and to rapidly acquire antibiotic resistance. Nowadays, highly resistant strains are widelydisseminated throughout the world, and the discovery of alternative therapeutic strategies is of utterimportance. Chalcones are compounds whose antimicrobial properties are well-known and forwhich different antivirulence activities have been demonstrated. The aims of this research were todetermine resistance profiles, to evaluate the possibility of antiseptic contamination, and to analyzethe biofilm production of identified A. baumannii clinical isolates, as well as to synthesizehydroxychalcone derivatives and to investigate their antimicrobial and antivirulence activitiesagainst these isolates.Antibiotic susceptibility of the isolates was tested by combination of diffusion, dilution, andautomated methods, and additionally, identified colistin-resistant isolates were subjected to wholegenome sequencing (WGS) and were genetically characterized. Also, colistin resistancemechanisms were explored by using comparative genome analysis and Real-Time quantitativepolymerase chain reaction (RT-qPCR). Time-kill test was used for the measurement of bacterialsurvival in antiseptics, whereas the level of biofilm production under different cultivationconditions was quantified by in vitro static method using safranin stain. Hydroxychalconederivatives 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 onthe 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 resistantphenotypes, being nearly uniformly resistant to carbapenems. Almost 19% of isolates were resistantto colistin as well. The isolates proved to be able to contaminate the antiseptic solutions and toproduce large quantities of biofilms. Nutritional composition of growth media significantly affectedthe level of biofilm production. In contrast, wide range of different incubation temperatures and thepresence of antibiotics at subinhibitory concentrations had little effect, and the bacteria managed tomaintain high level of biofilm production. Moderate antimicrobial activity was displayed bysynthesized chalcones, among which methoxy-substituted derivatives achieved greatest growthinhibition in average. Also, synergistic activity of chalcones and meropenem was present in severalcases, for which efflux pump inhibition was proposed as the potential mechanism. The chalconessignificantly inhibited motility and biofilm production, whereas methoxy-substituted derivative (o-OCH3) also displayed significant antivirulence activity, by downregulating the ompA, bap, and abaIgene expression and by inhibiting fibronectin- and collagen-mediated adhesion. It can be concludedthat o-OCH3 has been identified as a potent antivirulence agent against A. baumannii