Uticaj novosintetisanih derivata halkona na rast, produkciju biofilma i faktore virulencije multirezistentnih sojeva Acinetobacter baumannii
Influence of newly-synthesized chalcone derivatives on growth, biofilm production, and virulence factors expression of multiresistant Acinetobacter baumannii strains
Author
Ušjak, DušanMentor
Milenković, MarinaCommittee members
Božić, DraganaIvković, Branka
Stevanović, Magdalena
Dinić, Miroslav
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Show full item recordAbstract
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 izola...ti 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
g...enome 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.