MASTER THESIS

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U NIVERSITY OF L JUBLJANA

F ACULTY OF CHEMISTRY AND CHEMICAL TECHNOLOGY

MASTER THESIS

Nives Ražnjević

Ljubljana, 2021

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UNIVERSITY OF LJUBLJANA

FACULTY OF CHEMISTRY AND CHEMICAL TECHNOLOGY

SECOND CYCLE MASTER'S PROGRAMME (BIOCHEMISTRY)

ANTIMICROBIAL ACTIVITY OF SELECTED

HETEROCYCLIC ORGANIC COMPOUNDS FROM THE FCCT COMPOUND LIBRARY

MASTER THESIS

Nives Ražnjević

MENTOR: Assoc. Prof. Dr. Marko Novinec

Ljubljana, 2021

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Statement of Authorship

of the master thesis

I, Nives Ražnjević, hereby declare that I am the sole author of the master thesisunder the title “Antimicrobial activity of selected heterocyclic organic compounds from the FCCT compound library”.

With my signature, I assure that:

● This master thesis is entirely the result of my own research work under the mentorship of the Assoc. Prof. Marko Novinec;

● I made sure to state and cite the meanings and work of other people according to the instructions;

● I am aware that plagiarism, where the other people’s ideas and work are presented as mine, is a criminal offense;

● I took care of grammatical and design correctness of the master thesis;

● The electronic form of the master thesis is identical to the printed form of the master thesis.

_________________________ _____________________

(Place, Date) (Signature)

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Spremni list

Magistrsko delo je zaključek Magistrskega študijskega programa 2. stopnje Biokemija.

Delo je bilo opravljeno na Fakulteti za kemijo in kemijsko tehnologijo UL.

Senat UL FKKT je za mentorja imenoval izr. prof. dr. Marka Novinca.

Recenzenta: izr. prof. dr. Marko Dolinar, doc. dr. Vera Župunski

Komisija za oceno in zagovor magistrskega dela

Predsednica komisije: doc. dr. Vera Župunski, Katedra za biokemijo, UL FKKT

Član: izr. prof. dr. Marko Dolinar, Katedra za biokemijo, UL FKKT

Član: izr. prof. dr. Marko Novinec, Katedra za biokemijo, UL FKKT

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Acknowledgment

First of all, I would like to thank Assoc. Prof. Dr. Marko Novinec for his guidance, advice, support, and all the knowledge and skills he passed on to me.

Then Assoc. Prof. Dr. Marko Dolinar for an overview, advice, and his final touches on this work.

To Tjaša Goričan and Luka Ciber, for their openness to questions and all help and clarifications.

None of this would have been possible without great emotional and financial support.

I thank the associations "Študentski Tolar" and "Ocean Znanja in the Republic of Croatia" for their financial contribution and material security.

To Jelka, for providing me the opportunity to enroll at the college in the first place and to embark on this adventure.

To Barbara, for support when it was most difficult, and for allowing me to devote myself fully to my studies and the learning process.

To Tihomir, who believed in me and encouraged me when I thought I would not make it.

To Ema, my faithful buddy, who went through every part of this journey with me and with whom I sisterly shared everything.

And finally, to all my friends, family, and partner Jure.

Thank you from the bottom of my heart.

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List of abbreviations and symbols

AMPA – 2-amino-2-methylpropanoic acid AHL – acyl-homoserine lactone

AK – amikacin AM – ampicillin

AMC – amoxicillin / clavulanic acid CAZ – ceftazidime

CC – clindamycin

CFU – colony-forming unit CIP – ciprofloxacin

CRO – ceftriaxone CXM – cefuroxime

DMSO – dimethyl sulfoxide EA – ethyl acetate

ER – erythromycin

EUCAST – European Committee for Antimicrobial Susceptibility Testing FEP – cefepime

FID – free induction decay FOX – cefoxitin

G – gentamycin

HNA – hydroxynaphthalene acid IMP – imipenem

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LEV – levofloxacin LPS – lipopolysaccharide LZ – linezolid

MEM – meropenem

MIC – minimum inhibitory concentration

MRSA – methicillin resistant Staphylococcus aureus NCI – National Cancer Institute

NMR – nuclear magnetic resonance NOR – norfloxacin

OD – optical density PE – petroleum ether RA – rifampicin RG – receiver gain

SHV-1 – sulfhydryl variable-1

SXT – trimethoprim / sulfamethoxazole TEM-1 – temoniera-1

TZP – piperacillin / tazobactam

UL FCCT – the University of Ljubljana, Faculty of Chemistry and Chemical Technology VA – vancomycin

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Antimicrobial activity of selected heterocyclic organic compounds from the FCCT compound library

Abstract

The development of antibiotic-resistant strains of bacteria poses a real threat and burden to the healthcare system. In this master thesis, 100 different compounds available in the compound library of the Chair of Biochemistry and the Chair of Organic Chemistry UL FCCT were tested for bacterial growth inhibition on four bacterial species - Staphylococcus aureus, Bacillus thuringiensis, Escherichia coli, and Pseudomonas aeruginosa. Growth inhibition was monitored spectrophotometrically. All compounds were tested at a concentration of 100 µg/mL. Inhibition was confirmed by testing using a modified Kirby-Bauer disk diffusion method with the disks impregnated with 20 µg of the respective compound. The presence of the inhibition zone was considered as confirmation of inhibitory activity. After the inhibitory activity was confirmed, the minimum inhibitory concentration was determined. Among the tested compounds, 9 compounds showed antimicrobial activity, out of which two were further characterized.

2-[(4-chloro-1-hydroxynaphthalene-2-carbonyl)amino]-2-methylpropanoic acid (compound 1) showed selective antimicrobial effect only on S. aureus with a minimum inhibitory concentration of 125 µg/mL, while piperidyn-2-yl-{2-(trifluoromethyl)-6-[4- (trifluoromethyl) phenyl] pyridin-4-yl}methanol (compound 2) inhibited the growth of all strains tested. The minimum inhibitory concentration of compound 2 for S. aureus and B. thuringiensis was 88 µg/mL, for E. coli 175 µg/mL, and for P. aeruginosa 316 µg/mL.

Growth dynamics was monitored for compound 2 and compared with non-treated cells over 6 hours. During the observed period, there was no increase in bacterial concentration in the presence of the test compound. Compound 1 proved to be interesting due to its selective inhibitory activity. The analogs of the reactants for the compound 1 synthesis were readily available at the Chair of Organic Chemistry FCCT and were used to synthesize an analog of compound 1, which was designated as compound 8. After synthesis, the structure and purity of the synthesized analog were verified. NMR spectroscopy showed that 287 mg of the pure compound was synthesized and was visible as a yellow powder. Then, the inhibitory activity of compound 8 was investigated. It was found to slightly inhibit the growth of all tested strains; however, the inhibition was less than 50% compared to the negative control. In parallel, we also tested the reactants used in the synthesis of compound 8, i.e. 2-hydroxy-1-naphthoic acid (HNA) and 2-amino-2- methylpropanoic acid methyl ester. Interestingly, HNA showed significant inhibitory activity (> 90%) on S. aureus and E. coli. Together, these results open novel possibilities in the discovery of antibacterials.

Keywords: Bacterial Infections, Drug Resistance, Anti-Bacterial Compounds, Chemical Synthesis

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Protimikrobno delovanje nekaterih organskih spojin iz knjižnice spojin FKKT Razširjeni povzetek

Razvoj na antibiotike odpornih sevov bakterij predstavlja resno grožnjo in breme za zdravstveni sistem. Od odkritja penicilina so bile razvite številne protimikrobne spojine.

Ker pa so bakterije živi organizmi, imajo sposobnost prilagajanja in tako razvijejo odpornost. Čeprav gre za globalni svetovni problem, je razvoj novih protimikrobnih spojin precej redek. V zadnjih letih so predmet raziskav zlasti novi pristopi pri zdravljenju okužb, ki jih povzročajo odporni sevi, kot je terapija s fagi. Ta pristop lahko prinese dobre rezultate, vendar je za razvoj in sprejetje takšne terapije v praksi potrebnega kar nekaj časa. Trenutno uporabljene antibiotike razdelimo v 9 strukturnih skupin (beta laktamski antibiotiki, cefalosporini, aminoglikozidi, makrolidi, tetraciklini, sulfonamidi, fluorokinoloni, linkozamidi in ostale strukturne skupine), novi antibiotiki pa v glavnem temeljijo na modifikaciji doslej znanih struktur. Antibiotik kanamicin, uporabljen kot negativna kontrola v delu, pripada skupini aminoglikozidov.

Bakterije se na različne načine zaščitijo pred protimikrobnimi snovmi. Najbolj znani načini delovanja bakterijske odpornosti so sprememba tarče antibiotika, proizvodnja encimov, ki spreminjajo ali razgrajujejo antibiotik, zmanjšanje prepustnosti antibiotikov v celico zaradi strukturnih sprememb porina in aktivno izločanje antibiotikov iz bakterijskih celic. Nekatere bakterije so naravno odporne na določene spojine, če nimajo ustreznih vezavnih mest, kar je primarna bakterijska odpornost. Vendar so mutacije med bakterijami pogoste in lahko povzročijo novo odpornost. Ko govorimo o odpornosti, je pomembno omeniti nastanek biofilma. Nastanek biofilma je naravna lastnost večine bakterijskih vrst in je najpogostejša oblika, v kateri jih najdemo v okolju. Biofilm je kompleksna struktura, sestavljena iz živih in odmrlih bakterij, ujetih v zunajcelični matriks, sestavljen iz različnih polimernih molekul, ki so presnovni produkti bakterij znotraj biofilma. Primeri takšnih polimernih molekul so eksopolisaharidne snovi, beljakovine in nukleinske kisline, sproščene iz odmrlih bakterij. Te strukture bakterijam zagotavljajo mehansko zaščito, poleg tega pa bakterije v biofilmu komunicirajo z uporabo signalnih molekul. Zaradi zgoraj naštetega, pravila klasične mikrobiologije pogosto niso uporabna za biofilm. Koncentracije, potrebne za zaviranje rasti bakterij v biofilmu, pa so pogosto bistveno višje od tistih, ki veljajo za planktonske bakterijske celice.

V delu smo preizkusili protimikrobne lastnosti sto različnih spojin, ki so bile na voljo v knjižnicah spojin Katedre za biokemijo in Katedre za organsko kemijo Fakultete za kemijo in kemijsko tehnologijo Univerze v Ljubljani. Spojine so bile testirane na dveh Gram-pozitivnih vrstah (Staphylococcus aureus in Bacillus thuringiensis) ter dveh Gram- negativnih vrstah (Escherichia coli in Pseudomonas aeruginosa). Vse seve smo dobili od izr. prof. dr. Mateja Butale z Biotehniške fakultete Univerze v Ljubljani.

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Navedene vrste, razen B. thuringiensis, so čez zgodovino razvili odporne seve in so zato zanimiv predmet raziskave. Poleg tega so tako divji kot tudi mutirani odporni sevi pogosti izolati v klinični praksi. Ker se število razpoložljivih učinkovitih antibiotikov sčasoma in z razvojem mutacij zmanjšuje, je treba razviti nova protimikrobna sredstva in nove pristope k zdravljenju okužb, ki jih povzročajo odporni sevi.

Tako je med odpornimi Gram-pozitivnimi znan Staphylococcus aureus, odporen na meticilin (MRSA), medtem ko se pri Gram-negativnih bakterijah pojavljajo sevi ESBL (beta laktamaza razširjenega spektra, angl. extended spectrum beta-lactamase), odporni na novejše generacije cefalosporinskih antibiotikov. Sevi ESBL so, med ostalim, prisotni pri vrstah Escherichia coli in Pseudomonas aeruginosa.

Namen te raziskave je bil preveriti, ali imajo izbrane organske spojine protimikrobne lastnosti proti izbranim sevom bakterij. S spojinami, ki so delovale protimikrobno, smo določili rastne krivulje in minimalne inhibitorne koncentracije. To delo predstavlja začetek sistematičnega raziskovanja protimikrobnega delovanja sintetiziranih organskih spojin iz omenjenih knjižnic na rast bakterij in bi moralo zagotoviti podlago za prihodnje raziskave. Naši hipotezi sta bili, da (1) v knjižnici obstajajo spojine s protimikrobnim učinkom, in (2) da se njihov učinek na testirane bakterijske vrste razlikuje.

Zaviranje rasti smo spremljali z gojenjem bakterij do eksponentne faze rasti, kar pomeni 16 do 18 ur oz. preko noči, v prisotnosti oz. odsotnosti testiranih spojin. Bistre raztopine po inkubaciji so bile pokazatelj možnega zaviralnega učinka testiranih spojin. Rezultate smo potrdili še spektrofotometrično z merjenjem absorbance pri 600 nm. Vse spojine smo testirali pri koncentraciji 100 μg/mL. Pozitivno kontrolo so predstavljale bakterije gojene v tekočem LB gojišču brez dodane spojine, kot negativno kontrolo smo uporabili dodatek antibiotika kanamicina, ki zavira rast vseh preučevanih bakterij. Kot slepi vzorec smo uporabili samo LB gojišče ter vrednosti absorbance odšteli od absorbanc vzorcev s testnimi spojinami. V primerih obarvanih spojin je slepi vzorec predstavljalo gojišče LB z dodano spojino. Spojine, ki se kazale zaviranje bakterijske rasti, smo dodatno testirali s prilagojeno Kirby-Bauerjevo difuzijsko metodo, kjer smo na trdno gojišče z nacepljenimi bakterijami položili sterilni celulozni disk, nanj odpipetirali 10 μL koncentrirane preučevane spojine (20 mg/mL) in po 24-urni inkubaciji preverili prisotnost in velikost cone inhibicije, ki je potrdila zaviralni učinek. V primeru potrditve zaviralnega učinka smo naredili razredčitve antibiotikov in določili najmanjšo inhibitorno koncentracijo (MIC). Med preizkušenimi spojinami je devet spojin kazalo protimikrobno delovanje, od katerih smo dve podrobneje okarakterizirali, in sicer 2-[(4-kloro-1-hidroksinaftalen-2- karbonil) amino] -2-metilpropanojska kislina (spojina 1) in piperidin-2-il-{2- (trifluorometil)-6-[4- (trifluorometil) fenil] piridin-4-il} metanol tudi znana kot enpirolin (spojina 2). V prisotnosti spojine 2 smo izmerili krivulje rasti, tako da smo spremljali rast bakterij v časovnem obdobju 6 ur, kjer so bakterije prišle v osrednji del ekponencialne fazi rasti (za vse izbrane vrste je vrednost A600 kontrole iznaša med 0,6 in

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0,8). Pri dodatku spojine 2 in pri negativni kontroli rast nobene od preučevanih bakterij ni bila opazna oz. vrednost A600 je bila blizu nič.

Čeprav smo pri spojinah 4 (1-hidroksi-2,2,2-trikloroetil)urea) in 5 (N,N- dimetiletilenamin-4-metilenamin piperidin) opazili zaviralni učinek na rast bakterij, se je izkazalo, da imajo same spojine visoke vrednosti absorbance slepih vzorcev. Po Kirby- Bauerjevi difuzijski metodi preskušanja občutljivosti bakterij na ti dve spojini območja inhibicije v nobenem primeru nismo opazili, zato sklepamo, da spojini nimata dejanskega zaviralnega učinka. Spojina 6 (N²-cikloheksil-N¹-(6-metoksi-8-kinolinil)-1,2- butandiamin) je selektivno zavirala rast S. aureus. Pri preizkušanju občutljivosti z difuzijsko metodo smo izmerili območje inhibicije 12 mm. Spojina 7 (derivat pirazola, sintetiziran na Katedri za organsko kemijo) je selektivno zavirala rast P. aeruginosa, njeno protimikrobno delovanje pa je treba še potrditi z dodatnimi testi in podrobneje preučiti.

Spojina 1 je pokazala selektivni protimikrobni učinek le na S. aureus z najmanjšo inhibitorno koncentracijo 125 µg/mL, medtem ko je spojina 2 zavirala rast vseh testiranih sevov. Najnižje inhibitorne koncentracije spojine 2 za S. aureus in B. thuringiensis so bile 88 µg/mL, za E. coli 175 µg/mL in za P. aeruginosa 316 µg/mL. Za spojino 2 smo pripravili rastne krivulje v njeni prisotnosti in odsotnosti in v obdobju 6 ur spremljali rast bakterij. V opazovanem obdobju ni bilo povečanja števila bakterij v prisotnosti spojine.

Spojina 1 se je izkazala zanimivo, zaradi selektivnega zaviralnega učinka. Zaradi majhne količine razpoložljive spojine ni bilo mogoče izvesti podrobnejših raziskav. Zato smo morali za nadaljnje eksperimente sami sintetizirati zadostno količino te spojine. Sinteza spojine 1 je razmeroma preprosta in poteka iz po dveh reaktantov (4-kloro-1- hidroksinaftalenkarboksilna kislina in 2-metilpropanojska kislina) in pomožnih snovi (karbodiimid, hidroksibenzotriazol hidrat in N-metilmorfolin). Ker reaktanti za sintezo spojine 1 niso bili na voljo na Katedri za organsko kemijo, smo se odločili za sintezo njenega analoga (metilni ester [(2-hidroksinaftalen-1-karbonil) amino]-2- metilpropanojske kisline, spojina 8), ki se od spojine 1 razlikuje po položaju hidroksilne skupine in odsotnosti atoma klora, vezanih na naftalenski obroč, ter po dodatku metilnega estra, vezanega na aminokislinski del molekule. Novo sintetizirano spojino smo očistili na koloni s silika-gelom, čistost frakcij pa smo preverili s tankoplastno kromatografijo.

Čiste frakcije smo združili in jih uparili. Po sintezi smo pregledali strukturo in čistost sintetiziranega analoga z NMR spektroskopijo. ¹H-NMR spekter je pokazal, da mso sintetizirali 287 mg čiste spojine z videzom rumenega prahu.

Nato smo preučili inhibitorni učinek spojine 8 in ugotovili, da sicer nekoliko zavira rast vseh testiranih sevov, vendar je bilo zaviranje v vseh primerih manjše od 50%. Vzporedno s smo testirali tudi reaktanta, uporabljena pri sintezi, in sicer, 2-hidroksi-1-naftojsko

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kislino (HNA, spojina 9) in metilni ester 2-amino-2-metilpropanojske kisline. Spojina 9 je pokazala zaviralni učinek (> 90 % zaviranje rasti) na S. aureus in E. coli. Spojina 1 in spojina 9 imata enako ogrodje, kar kaže, da je za zaviranje ključen tisti del, ki je obema molekulama skupen. Razlog za neučinkovitost spojine 8 v primerjavi s spojinama 1 in 9 bi lahko bila večja velikost molekule na račun metilestrske skupine v aminokislinskem delu molekule. Spojina 9 je sestavni del naftalenskega dela spojine 8. Iz tega sklepamo, da je ključ za selektivno zaviranje rasti S. aureus bodisi hidroksilni del molekule spojine 9, ki se v spojini 8 izgubi zaradi tvorbe amidne vezi z 2-amino-2-metilpropanojsko kislino ali zaradi dodatka metilnega estra, ki je lahko med reakcijo s ciljnimi bakterijskimi strukturami sterična ovira. Zaradi podobnosti z antibiotikom rifampicinom domnevamo, da bi bil mehanizem delovanja spojin 1 in 9 na S. aureus lahko podoben, torej da se veže na β podenoto bakterijske RNA-polimeraze. Vendar pa so potrebne nadaljnje raziskave, da bi ugotovili natančno povezavo med strukturo in aktivnostjo ter določili mehanizem delovanja.

Rezultati te magistrske naloge tako predstavljajo izhodišče za obsežnejše biokemijske in molekularnobiološke raziskave učinkov in mehanizmov doslej neraziskanih spojin iz knjižnice spojin FKKT na zaviranje rasti bakterij.

Ključne besede: bakterijske okužbe, odpornost na zdravila, protibakterijske spojine, kemična sinteza

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Nives Ražnjević, University of Ljubljana, Master’s Degree Thesis

1. Introduction

A growing problem in today's world is the increasing number of microorganisms that are developing resistance to antimicrobial agents. In the clinical setting, this poses a threat to the healthcare system due to higher morbidity and mortality rates as well as higher economic costs. The development of resistance implies that even diseases caused by bacterial infection and considered relatively harmless could have a fatal outcome due to the lack of effective antibiotics (1). Fortunately, the development of science and technology makes it possible to deal with the development of bacterial resistance step by step and find a possible solution.

As is already known, the first antibiotic, penicillin, was discovered in 1928 by the Scottish bacteriologist Alexander Fleming. The discovery of penicillin was fortuitous, but also serendipitous, as Fleming noticed in the moldy petri dish that the mold killed certain bacteria he had cultured. The fungus was isolated and its activity on bacterial growth was studied. It is now known that penicillin prevents the development of bacterial cell walls.

After the discovery of penicillin, extensive research began on substances with antibiotic activity and the development of bacterial resistance. Although today more than 100 antibiotics are known, we can divide them into 9 most commonly known categories - β- lactam antibiotics, cephalosporins, macrolides, fluoroquinolones, sulfonamides, tetracyclines, glycopeptides, lincosamides, and other antibiotics (2).

The Chair of Biochemistry at UL FCCT has a library of compounds that have been tested for their activity as protease inhibitors. To date, most of the compounds have been tested against enzymes belonging to the cathepsin group, and numerous inhibitory and activating effects have been observed (3,4). Many of these compounds have been synthesized with the aim of finding a chemotherapeutic effect. The effect on cathepsins has been studied for several years, as certain groups of cathepsins have been associated with the development of numerous pathological processes in the body.

Testing of the compound library was extended to the study of antimicrobial activity on selected bacteria, which is the subject of this work. The library was enriched with the compounds synthesized at the Chair of Organic Chemistry FCCT. Two Gram-positive and two Gram-negative microorganisms were selected for this study. These microorganisms are Staphylococcus aureus RN1442, Bacillus thuringiensis, Escherichia coli MG 1655, and Pseudomonas aeruginosa. Microorganisms were obtained by the courtesy of Assoc. Prof. Matej Butala from the Biotechnical Faculty, University of Ljubljana.

The selected species are suitable for the study of antimicrobial activity because all except Bacillus thuringiensis have a strain that has already developed resistance to a number of antibiotics that have been effective against them throughout history (5–7). Examples of such resistant strains include methicillin resistant Staphylococcus aureus (MRSA) and

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extended spectrum β-lactamase (ESBL) Escherichia coli (5,6). A particular and growing problem with nosocomial infections is Pseudomonas aeruginosa, which is otherwise a ubiquitous microorganism. However, contact with the hospital environment has led to the development of specialized strains that are resistant to most antibiotics currently in use (7). P. aeruginosa causes opportunistic infections, which means that it infects people with already impaired immunity, and such patients are being found in the hospital environment, where the resistant strain of Pseudomonas is usually present (2). Therefore, it is not difficult to conclude that such a combination can present dangerous and possibly fatal outcome for the patient. Bacillus thuringiensis was chosen since it is a Gram-positive bacterium and offers the possibility for concluding the antimicrobial selectivity of a compound to bacteria depending on the structure of their cell wall.

Gram-positive and Gram-negative bacteria differ in the composition of their cell walls.

Therefore, they stain and are seen differently under the microscope as either purple or red when stained by the Gram staining technique. The Gram stain is the best known and most widely used stain in microbiology which consists of four components added in the order given - crystal violet, Lugol's solution, decolorizing agent, and safranin (red dye). The cell wall of Gram-positive bacteria is largely composed of peptidoglycans that form a thick layer (Figure 1A). Gram-positive bacteria acquire a primary purple color incorporated into the network-like structure of the peptidoglycans, which is not lost by treatment with the decolorizing agent and is seen as purple on the slide. In contrast, Gram- negative bacteria have a thin peptidoglycan layer within the cell wall (Figure 1B) that does not retain the primary staining, crystal violet.

Figure 1. The difference between Gram-positive and Gram-negative bacteria cell wall structure. A) Gram- positive bacteria cell wall structure. B) Gram-negative bacteria cell wall structure.¹

1 The cell wall structure was drawn using app.biorender.com tool, according to (2)

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Upon decolorization, the primary, purple color disappears while the secondary, red dye, safranin, remains bound and the bacteria appear stained red on the slide. Due to differences in the cell wall structure, the process of entry of the compound into the bacterial cell is different. Therefore, it is necessary to examine at least two members of each group of bacteria to determine whether the compound under investigation has selective antimicrobial activity against Gram-positive or Gram-negative bacteria (2).

1.1. Antibiotic mechanisms of action

The main characteristic of antibiotics is their toxicity to bacteria, while at the same time being weakly toxic or not toxic at all to the human body. They are classified into four main categories according to their mechanism of action and further according to their molecular structure. The four main mechanisms of action are (2):

- the influence on bacterial cell wall synthesis;

- the influence on protein synthesis;

- the influence on the function of the cytoplasmic membrane;

- the influence on nucleic acid synthesis.

Table 1 shows which mechanism of action is a property of each structural group of antibiotics (2).

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Table 1. Mechanism of action as a property of each structural group of antibiotics.

Antibiotic mechanism of action Antibiotic structural group

Inhibition of bacterial cell wall synthesis β-lactam antibiotics (penicillins, cephalosporins, monobactams,

carbapenems), glycopeptide antibiotics, bacitracin

Inhibition of protein synthesis Aminoglycosides, tetracyclines, chloramphenicol, macrolides, lincosamides, streptogramins, oxazolidinones

Inhibition of the function of the cytoplasmic membrane

Polymyxins

Inhibition of nucleic acid synthesis Quinolones, rifamycins, metronidazole, sulfonamides, trimethoprim,

nitrofurantoin

To better understand the relationship between the structure of the molecule and its function, the mechanism of action of some of the most commonly used antibiotics is explained in more detail.

Disruption of cell wall synthesis can occur either by binding to bacterial regulatory enzymes responsible for peptidoglycan synthesis within the cell wall layer (e.g.

carboxypeptidases, transpeptidases, transglycosylases) or by interaction with molecules required for the synthesis of bacterial cell wall components. Peptidoglycan is formed of pentaglycans. Pentaglycans contain D-alanine termini, which is a binding target for vancomycin. By binding to these amino acid termini, vancomycin disrupts peptidoglycan synthesis and consequently cell wall synthesis.

The inhibition of protein synthesis may be a consequence of the binding of the drug to the ribosomal subunits. For example, gentamicin binds irreversibly to the 30S ribosomal subunit, resulting in premature release of the ribosome from the bacterial mRNA, whereas erythromycin binds reversibly to the 50S ribosomal subunit and the 23S rRNA, resulting in blockage of polypeptide elongation.

On the other hand, polymyxins interact with phospholipids and polysaccharides in the bacterial cell membrane, increasing its permeability. An example of antimicrobial activity by inhibiting nucleic acid synthesis is ciprofloxacin, which binds to the bacterial

Table SEQ Table \* ARABIC 1. The distribution of structural groups of antibiotics according to their mechanism of action

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topoisomerase II, thereby interfering with DNA replication, recombination, and repair.

Rifampin impairs transcription of bacterial RNA by binding to the β-subunit of DNA- dependent RNA polymerase (2).

The use of antibiotics is common in the selection of resistant mutants. Bacterial resistance can be acquired by spontaneous mutations in the bacterial genome or by horizontal transmission via plasmids (2).

The action of antibiotics can be broadly divided into a bacteriostatic action and a bactericidal action. Bacteriostatic action prevents the growth and multiplication of bacteria, while bactericidal antibiotics kill bacteria. Some antibiotics can have a bactericidal effect on all bacteria. Meanwhile, certain antibiotics may have a bacteriostatic effect on some bacteria and a bactericidal effect on some other members of certain bacterial genera (2).

1.2. The types of bacterial resistance to antimicrobial substances

During their evolution, bacteria have developed different types of antibiotic resistance.

To date, the following mechanisms of antibiotic resistance are known: alteration of the antibiotic target site, production of enzymes that modify or degrade the antibiotic, reduction of antibiotic permeability by structural modification of porin, and use of cellular efflux to actively excrete antibiotics from the cell. Active efflux of antimicrobial agents contributes to lowering the efficacy of the drug. It is the most common cause of resistance to tetracyclines. Considering aminoglycosides, efflux is rarely observed and occurs only in Gram-negative bacteria (2).

Some bacteria are naturally resistant to certain compounds if they do not have suitable binding sites, which is called primary bacterial resistance. However, mutations are common and can lead to new resistance. Resistance genes can also be transferred by horizontal transfer, which in this case is called secondary resistance (8). Thus, bacterial resistance can be either innate or acquired. Some bacterial species are resistant to certain types of antimicrobial agents because of their cell wall composition or other structural specifics within the bacterial cell. On the other hand, it is possible for resistance to antimicrobial agents to transfer either vertically or horizontally (2).

Considering resistance mechanisms, it is necessary to mention biofilms. A biofilm is a structure consisting of an extracellular matrix produced by bacteria of bacterial cells it contains. It provides protection from negative environmental factors. Bacteria trapped in the biofilm structure usually exhibit significantly higher resistance to antimicrobial compounds. Microorganisms in the biofilm are attached to the substrate surface as well as to each other and are immersed in the extracellular matrix they produce. After the

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formation of a mature biofilm, about 80% of the biomass is composed of a slimy extracellular matrix, while bacteria make up only about 20%. Planktonic cells, in the context of biofilms, are bacteria that are not part of the biofilm but move freely as individuals (9). Treatment of infections caused by bacteria forming the biofilm usually requires prolonged therapy with high doses of antibiotics (10).

When it comes to orthopedic prosthetic infections, biomaterials such as endoprostheses or artificial heart valves often present a challenge in treatment because the bacteria within the biofilm are protected and often more resistant than planktonic bacterial cells. Most of the research on antimicrobial agents is based on planktonic bacterial cells. The activity on the biofilm is then extrapolated from the data. However, the bacteria in biofilm differ from planktonic bacteria in terms of metabolism, growth rate and gene expression.

Therefore, new approaches are needed to study the properties and susceptibility of biofilms to antibiotics (9). Communication within the biofilm occurs according to the principle of quorum sensing. The release of autoinducer molecules signals the biofilm inhabitants that their numbers are sufficient to activate virulence genes, which differ among bacterial genera (8). For example, for E. coli the presence in the genome of fimH, pap, sfa and afa genes shows the strong ability for biofilm creation (11). Meanwhile, genes responsible for biofilm formation in Pseudomonas are ppyR, pslA, pelA, algU, algL, algD, exoA and fliC (12). The studies have shown that sub-MIC doses of antimicrobial agents can promote biofilm formation (13,14).

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1.3. Characteristic features and resistance development in studied bacterial species

Species from the four bacterial genera, Staphylococcus, Bacillus, Escherichia, and Pseudomonas, were selected for this study. Staphylococcus aureus and Bacillus thuringiensis are Gram-positive bacteria while Escherichia coli and Pseudomonas aeruginosa are representatives of Gram-negative bacteria. This chapter provides a brief introduction to the characteristics and an overview of the typical resistances of each species studied.

1.3.1. Staphylococcus aureus

Looking at the micromorphology, members of the species S. aureus are Gram-positive cocci that form clusters in a microscopic preparation (Figure 2A) which was the reason for the name of the genus (Greek staphylos = cluster, Greek coccus = berry, round fruit).

When grown on agar plates, wild types usually appear as golden-cream colonies from which this species takes its name (lat. aureus = golden). The characteristic growth color and micromorphology can be seen in Figure 2 (B and C). The species possess the enzyme catalase which is used in classical microbiology as a diagnostic tool to distinguish it from streptococci, which are also Gram-positive, round bacteria.

Figure 2. Staphylococcus aureus. A) Microscopic slide preparation, magnification: 1000×. B) Bacterial culture with visible separate colonies grown on the solid LB medium in Petry dish. C) Bacterial culture with visible separate colonies grown on the blood agar medium in Petry dish.²

S. aureus can be distinguished from most other species within the genus Staphylococcus by expression of the enzyme coagulase. In vitro assays for catalase and coagulase in the diagnosis of infections help identify the bacterial genus and/or species (15). S. aureus is normally part of the nasopharyngeal microbiota. However, by invading the lower respiratory tract it can cause numerous complications, especially its methicillin-resistant strains (MRSA). S. aureus often causes problems and infections of the skin and wounds

2 Source: obtained by the courtesy of the Department of Microbiology and Parasitology, Faculty of Medicine, University of Rijeka.

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as well as complications in patients with orthopedic implants. With the increasing number of resistant strains, this could become a growing problem in healthcare (16).

Resistance development in S. aureus started with the resistance to penicillin due to the production of the enzyme penicillinase. Penicillinases are the subset of the most abundant, SHV-1 and TEM -1 class A β-lactamases. β-Lactamases are enzymes that inactivate β- lactam antibiotics (2). The problem of developed resistance was solved by the discovery of the synergy of β-lactam antibiotics and clavulanic acid. This combination of the two bioactive molecules was effective against the mutant S. aureus strain but resulted in a mutation in PBP (penicillin-binding protein), the target molecule for binding of the antibiotic to the bacterial membrane. The mutated version of the protein was named PBP2A. This mutation led to the development of another resistance. A new strain of S.

aureus evolved that was highly resistant to β-lactam antibiotics and thus to methicillin, hence the name methicillin-resistant Staphylococcus aureus (MRSA). The genes encoding the PBP protein are called mec genes, and confirmation of their presence is the gold standard for MRSA diagnosis (17).

In addition to β-lactam antibiotics, S. aureus can develop resistance to aminoglycosides, tetracyclines and streptogramin A. Another type of resistance found in S. aureus is resistance to macrolide-lincosamide-streptogramin B. The genes responsible for each resistance are found in Table 2 (18).

Table 2. Genes conferring resistance to antibiotics in S. aureus (18)

Antibiotic or antibiotic group Genes encoding resistance β-lactam antibiotics mecA

Aminoglycosides aacA-aphD

Tetracyclines tetK, tetM

Macrolide-lincosamide-streptogramin B erm(A), erm(C)

Streptogramin A vat(A), vat(B), vat(C)

Special attention should be paid to the development of biofilm. Biofilm development in staphylococci is accompanied by an increase in cyclic peptide concentration. Biofilm formation begins when a sufficient number of bacteria is reached within the colony.

Bacteria use the quorum sensing system to detect how many members are present in the colony, and in Staphylococcus, the quorum sensing system is encoded on the agr operon.

Staphylococcus secretes small amounts of the autoinduction protein (AIP). When the quorum is reached, i.e., the concentration of AIP increases, it binds to the membrane molecule AgrC, which promotes the transcription of two RNAs, RNA II and RNA III,

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from the agr operon. RNA II encodes 4 proteins - AgrA, AgrB, AgrC and AgrD. AgrB is a membrane protein whose function is to convert AgrD into AIP, which then leaves the bacterial cell, binds to another AgrC protein, and continues the cycle according to the principle of positive feedback (2,19).

1.3.2. Bacillus thuringiensis

Bacillus thuringiensis is a sporogenic Gram-positive rod. Microscopic preparation is visible in Figure 3A. When cultured, it grows in characteristically large white mat colonies and requires no special growth conditions. Figure 3 (B and C) shows the macromorphology of the colonies when grown on LB and blood agar medium.

Figure 3. Bacillus thuringiensis. A) Microscopic slide preparation, magnification: 1000×. B) Bacterial culture with visible separate colonies on the solid LB medium in a Petry dish. C) Bacterial culture with the visible separate colonies on the blood agar medium.³

Bacillus thuringiensis primarily infects plants. Its parasporal crystals contain the Bt toxin which has insecticidal activity. Thus, it can be used to protect plants against insects and parasites. The mechanism of action of the toxin is interaction with specific receptors in the larval gut once the insect larvae have ingested the toxin. A pore forms in the intestinal epithelium which allows the leakage of intestinal contents resulting in the deaths of the insect. In the last 40 years, it has been used in agriculture either as an insecticidal spray or as a product of genetically modified crops (20).

Initially, it was assumed that B. thuringiensis does not cause infections in humans and was selected for this study only to have another representative of the Gram-positive microorganisms, in addition to S. aureus, to conclude. Namely, experiments performed on two Gram-positive and two Gram-negative strains could suggest whether an agent has a selective activity for specific members of the group. However, data from European

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Committee for Antimicrobial Susceptibility Testing (EUCAST) show 8 clinical cases of human infections caused by B. thuringiensis from January to June 2021 (21).

1.3.3. Escherichia coli

Escherichia coli is a Gram-negative fermentative non-sporogenic rod (Figure 4A) of the family Enterobacteriaceae. It is the most common member of the genus Escherichia. In terms of its metabolism and structure, it is described as a facultative anaerobic bacterial species. Most members of the Enterobacteriaceae family look macromorphologically similar when cultured on non-selective media. They usually form shiny, slimy, greyish colonies and E. coli is no exception. When clinical specimens are examined, culture must be subcultured on specific selective media to be properly identified. Figure 4 (B and C) shows the macromorphology of E. coli.

Figure 4. Escherichia coli. A) Microscopic slide preparation, magnification: 1000×. B) Bacterial culture with visible separate colonies on the solid LB medium in a Petry dish. C) Bacterial culture with the visible separate colonies on the blood agar medium.⁴

E. coli is mesophilic and requires no special additives during cultivation. Escherichia are classified into three main groups based on their clinical and genetic criteria - commensal strains that lack specialized virulence factors, intestinal pathogenic strains, and extraintestinal pathogenic strains (22). Pathogenic strains have the potential to cause various infections such as gastrointestinal infections, urinary tract infections (UTIs), and sepsis and meningitis. In certain subgroups (pathovars) of intestinal pathogens, E. coli can cause severe diarrhea. These subgroups of E. coli are enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteroaggregative (EAEC) and enterotoxic E. coli (ETEC).

The greatest threat to the healthcare system comes from enterohemorrhagic E. coli. It is defined by serogroup O157:H7 and is responsible for the hemorrhagic uremic syndrome

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(HUS) and outbreaks of bloody diarrhea. The reservoir for the disease is usually cattle.

Therefore, most outbreaks are associated with the consumption of contaminated beef (23).

A serious problem of our time is the development of antibiotic resistance among E. coli strains, especially ESBL strains (8). ESBL stands for extended spectrum β-lactamase, which are also known as cephalosporinases. The term ESBL refers to the enzymes that can inactivate new generation β-lactam antibiotics, such as cephalosporins.

Cephalosporins are usually effective against strains that are resistant to penicillins (2).

However, the development of ESBLs is another challenge in the fight against resistant pathogenic bacteria.

A recent study has shown that infection with certain strains of E. coli, along with other microorganisms, plays a role in the development of irritable bowel diseases (IBD) such as ulcerative colitis and Crohn's disease. However, a prerequisite for the development of IBD is a genetic predisposition. Diet has been shown to play a key role, as saturated fats and sugary foods alter the composition of the intestinal mucus and allow pathogenic groups of E. coli to invade the tissues. The use of antibiotic therapy in combination with probiotics and corticosteroids has been shown to be beneficial in prolonging the duration of remission in IBD patients (24). However, as the increase in antibiotic-resistant strains has become of increasing concern over time, it is important to prescribe antibiotic therapy judiciously and to understand how resistance to different bacterial strains develops.

When it comes to E. coli, antibiotic resistance is usually mentioned in the context of urinary tract infections, as this microorganism has been shown to be the leading cause of them. Antibiotics that are predominantly used in different countries are β-lactams, trimethoprim, nitrofurantoins and quinolones. Nonetheless, widespread use and misuse have led to an increase in antimicrobial resistance and there is a need to search for suitable substitutes. Since any antimicrobial therapy can have side effects, various vaccines have been investigated to prevent infections with resistant E. coli strains (25).

1.3.4. Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous microorganism that is becoming a major cause of hospital-acquired infections because of its ability to develop resistance to antimicrobial agents. It is a non-fermentative, obligate aerobic microorganism visible under the microscope as Gram-negative rods (Figure 5A) usually arranged in pairs (2). It has a distinctive odour reminiscent of ripe grapes (26). When cultivated in general media, it appears greenish yellowish on account of the pigments it produces - pyocyanin and pyoverdine (Figure 5B). Its characteristic feature is metallic reflection when cultivated on certain media (Figure 5C).

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Figure 5. Pseudomonas aeruginosa. A) Microscopic slide preparation, magnification: 1000×. B) Bacterial culture with visible separate colonies on the solid LB medium in a Petry dish. C) Bacterial culture with the visible separate colonies on the blood agar medium.⁵

Certain strains can produce red-brownish pigment named pyorubin (2). The production of the pigments is shown in Figure 6.

Figure 6. The production of Pseudomonas aeruginosa's pigments when cultivated on the nutrient agar plate (26). A) Pyoverdine. B) Pyocyanine. C) Pyorubine.

Virulence of P. aeruginosa is mainly due to exotoxin A, two elastases (LasA and LasB) and phospholipase C. Exotoxin A acts on eukaryotic cells by blocking peptide chain elongation, thereby impairing protein synthesis. LasA is a serine protease, while LasB is a zinc metalloprotease. Both act synergistically in degrading elastin, resulting in damage to a tissue composed primarily of elastin, such as lung parenchyma. Phospholipase C facilitates tissue destruction during P. aeruginosa infection as well. It is a hemolysin capable of degrading lecithin and lipids. The pigments pyocyanin and pyoverdin also contribute to virulence. Pyocyanin stimulates the release of interleukin 8, which contributes to the attraction of neutrophil leukocytes. It also catalyzes the production of toxic forms of oxygen - superoxide and hydrogen peroxide. Meanwhile, pyoverdine acts as a siderophore and therefore binds iron from the organism for its metabolism (2).

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Infections caused by P. aeruginosa are primarily opportunistic, that is, they mainly affect immunocompromised individuals (8). The problem encountered in such infections are highly resistant strains, which are a typical feature of P. aeruginosa. This microorganism has one of the largest genomes among known bacterial genomes, indicating a high degree of adaptability when it comes to the threats to which the species is exposed. Some strains are resistant to most antibiotics used today, which can make therapy frustrating and exhausting, as it usually affects already debilitated patients with weakened immunity (2).

Resistance in Pseudomonas is an intrinsic property due to the rapid efflux of antibiotics.

Most non fermentative bacteria are thought to have highly efficient efflux pumps that allow the excretion of hydrophobic and amphipathic molecules (27). However, it can also acquire resistance through horizontal transfer (conjugation) of plasmids and triggering expression of the ampC gene, which inactivates most β-lactam antibiotics. Up to this point, specific antibiotics such as anti-Pseudomonas penicillins, 3rd and 4th generation cephalosporins, carbapenems, monobactams, and in the case of multidrug-resistant strains, more toxic antibiotics such as polymyxins are used (8). Resistance among Pseudomonas spp. is no exception when it comes to biofilm formation, which in this genus is triggered by the production of a sufficient concentration of acyl-homoserine lactone (AHL) (2).

The unique feature of P. aeruginosa is the low permeability of its outer membrane, which prevents small molecules such as penicillin from diffusing into the bacterial cytosol. Most Gram-negative bacteria have a thick outer layer membrane, but P. aeruginosa has an outer layer that is 12 to 100 times less permeable than E. coli (27).

A theory suggests the presence of a "self-promoted ingestion" system in the Pseudomonas genus. It is a system that regulates the uptake of gentamicin, tobramycin and colistin.

Polycationic molecules interact with binding sites for divalent cations located on the surface of LPS, resulting in membrane destabilization and thus increased permeability.

All non-fermentative bacteria, except Burkholderia cepacia, are thought to possess this system of antibiotic uptake (2).

Three proteins - MexA, MexB and OprM are the main contributors to intrinsic resistance in P. aeruginosa. Knock-out of the genes encoding these proteins resulted in bacterial strains that exhibited 4- to 10-fold higher susceptibility to chloramphenicol, tetracycline, quinolones, and β-lactams (except imipenem) (27). Every non-fermentative bacterium has an inducible gene for cephalosporinase encoded on the chromosome which is activated by the sub-MIC doses of β-lactams (25).

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2. Research aim and hypotheses

This thesis aimed to test whether selected organic compounds from the UL FCCT compound library have antimicrobial properties against the selected bacterial strains.

The results of the screening should provide a starting point for further research into the mechanisms of action on the particular strains.

Growth curves and minimum inhibitory concentrations should be determined for the compounds showing antimicrobial activity.

This work is at the beginning of the exploration of the antimicrobial activity of synthesized organic compounds on bacterial growth and should provide initial directions for future research.

Our hypotheses were:

(1) that the library of organic compounds from FCCT contains a compound(s) with antimicrobial activity, and

(2) the compounds with the antimicrobial properties will have different influence on each of the treated bacterial species.

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3. Materials and methods 3.1. Materials

The materials used for this study are 4 strains of bacteria, 100 compounds from the UL FCCT compound library, and other chemicals and equipment needed to perform the study.

3.1.1. Bacterial species and strains

Microorganisms examined in this study are:

- Staphylococcus aureus RN1442 - Bacillus thuringiensis

- Escherichia coli MG1655 - Pseudomonas aeruginosa

Microorganisms were obtained by the courtesy of Assoc. Prof. Matej Butala from the Biotechnical Faculty, University of Ljubljana and presented in more detail in the introductory chapter.

3.1.2. The compound library

The compound library consists of two parts - one is the official library of UL FCCT (located at the Chair of Organic Chemistry) and the other is the library of compounds of the Chair of Biochemistry. One hundred compounds (in total) from both libraries were tested.

Compounds that showed antimicrobial activity are shown in Table 3

.

Most of the compounds are heterocyclic organic compounds that differ by the number and position of heterocyclic rings and bonded substituents.

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Table 3. The list of compounds showing antimicrobial activity.

Compound Structure IUPAC name ZINC number Source

1. 2-[(4-chloro-1-

hydroxynaphthalene-2- carbonyl)amino]-2- methylpropanoic acid

ZINC01560636 U.S. NCI/DTP Open Chemical Repository (code NSC270063)

2. Piperidyn-2-yl-{2-

(trifluoromethyl)-6-[4- (trifluoromethyl) phenyl] pyridin-4- yl}methanol

ZINC13143009 U.S. NCI/DTP Open Chemical Repository (code NSC305798)

3. The Chair of Organic

Chemistry FCCT

4. (1-hydroxy-2,2,2-

trichloroethyl) urea

ZINC08603305

NSC13182

5. N,N-

dimethylethyleneamine- 4-methyleneamine piperidyn

ZINC19843238 UkrOrg Synthesis

6. N²-cyclohexyl-N¹-(6-

methoxy-8-quinolinyl)- 1,2-butanediamine

ZINC01591915

NSC13616

7. The Chair of Organic

Chemistry FCCT

8. [(2-

hydroxynaphthalene-1- carbonyl) amino] -2- methylpropanoic acid methyl ester

ZINC436753116 The Chair of Organic Chemistry and The

Chair of

Biochemistry FCCT

9. 2-hydroxy-1-naphthoic

acid

ZINC155999 TCI, USA

Compound 3 is a pyrazole derivative that has been newly synthesized and has not yet been published in a scientific paper. The general structure of the compounds is shown in

.

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Analogs of this structure differ from each other by the number of 4 different substituents.

Compound 7 is a 1-substituted derivative of 5-[2-(acylamino)ethyl]-1H-pyrazole-4- carboxamides. Kralj et al. developed the seven-step synthesis of the listed compounds at the Chair of Organic Chemistry, FCCT in 2009 (28).

3.1.3. Other chemicals

In addition to the compounds used from the compound library, Table 4 contains all other chemicals used in this work.

Table 4. The list of the other used chemicals

Chemical Manufacturer

1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

Merck, Germany 2-amino-2-methylpropanoic acid methyl

ester

Fluorochem, United Kingdom

Dimethylformamide Merck, Germany

Dimethyl sulfoxide Merck, Germany

Ethanol ECP, Slovenia

Ethyl acetate Carlo-Erba, France

Glycerol Merck, Germany

Hydroxy benzotriazole hydrate Fluka, Switzerland

Kanamycin Merck, Germany

LB medium Merck, Germany

Muller-Hinton solid medium Merck, Germany

N-methyl morpholine Merck, Germany

Petroleum ether VWR, France

Silica gel Merck, Germany

Sodium hydrogen sulfate (synthesized from H2SO4)

Riedel-de Haen, Germany

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3.1.4. Devices and equipment

Table 5 provides information about the devices and tools used in this work.

Table 5. The list of the used equipment

Equipment Model Manufacturer

Centrifuge miniSpin Eppendorf, Germany

Magnetic mixer FB15045 Bio-Rad, USA

Microcentrifuge tubes Eppendorf, Germany

Microtiter plates (96 well)

Corning NMR spectrometer Bruker Avance III 500

MHz NMR Spectrometer

Bruker, USA

Ph-meter Seveneasy Mettler Toledo, USA

Pipettes Eppendorf Research

Plus (0,5-10 μL)

Eppendorf, Germany Eppendorf Research

Plus (10-100 μL)

Eppendorf, Germany Eppendorf Research

Plus (100-1000 μL)

Eppendorf, Germany

Pipette tips Sarstedt, Germany

Plate reader Tecan Sunrise Tecan, Switzerland Rotary evaporator Rotavapor Heidolph Heidolph, Germany

Shaker Rotomix Ty.50800 ThermoFisher Scientific, USA

Shaking incubator Sanyo, Japan

Scales WLC2/A2 Radwag, Poland

XA/60/220/X Radwag, Poland Spectrophotometer UV/VIS Cary 50 Varian, USA

Thermoblocks Bio TDB-100 Biosan, Lithuania

Thermomixer 5436 Eppendorf, Germany Ultrasonic

homogenizer

Hielscher, Germany

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3.2. Methods

3.2.1. The preparation of bacterial suspensions

To determine the antimicrobial activity of the selected compounds on the selected bacterial strains, the most suitable growth conditions must be determined for each bacterial strain. There are several ways to prepare a bacterial suspension for further experiments. In this thesis, the method used to prepare the bacterial suspension is the overnight culture growth method. The culture is incubated overnight followed by the measurement of A600. Strain viability is determined by the correlation between A600 and CFU per milliliter after the strains are plated on the agar plates overnight (14).

The preliminary experiments are carried out in triplicate. Three to five morphologically identical colonies (from the solid medium) are selected, transferred to the broth medium and incubated overnight. After 16 to 20 h of incubation, the A600 is measured (Cary Varian spectrophotometer) and diluted until it is below an absorbance of 1.0, since a loss of linearity occurs when the absorbance is above 1.0. The suspension is diluted in a series of 10-fold dilutions to 1:10^-7. Following the procedure, 100 µL of the final three suspensions (from 10^-4 to 10^-7) are inoculated on the agar plate and incubated overnight.

After incubation, the colonies on the plate are counted and the CFU per milliliter is given with.

𝑁 =𝐶 × 10 10^−𝐷

Equation 1. Calculation of colony-forming units per milliliter. N is the number of CFU/mL, C is the number of colonies per plate and D is the number of the 1:10 dilution.

The calculated number is averaged over the triplicate experiments, providing reliable data on the correlation between overnight culture and A600. This allows determination between CFU and dilution factor for further research applications. After determining the correlation, the fresh suspensions can be incubated and used for further experiments.

Following incubation, the A600 is measured, and the sample is diluted until the absorbance is below 1.0. The factor previously calculated to obtain a solution containing approximately 1×10⁸ CFU/mL is used for dilution (14).

3.2.2. The preparation of permanent bacterial cultures

For the preparation of permanent bacterial cultures, 8.5 mL of LB medium inoculated with the respective strain and cultured overnight was mixed with 15 mL of glycerol to achieve a 15% glycerol dilution. The 1 mL aliquots were transferred to 10 microcentrifuge tubes and frozen at -18°C. These suspensions were used for further experiments.

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3.2.3. Screening of the compound library and the inhibitory assays in microtiter plates

Screening in microtiter plates allows the identification of compounds that inhibit bacterial growth. The bacterial division is an exponential process that can be followed by measuring the optical density of the suspension. Growth inhibition was detected spectrophotometrically measuring the optical density of the suspension at 600 nm. The rate of division is determined by the generation time of a particular bacterial strain, which is its specific innocuous property. It is the time it takes for one cell to divide into two.

Cells with a shorter generation time require a shorter period for cultivation, while cells with a longer generation time require a longer period for cultivation. The growth period during cultivation in a closed batch system forms a typical bacterial growth curve characterized by four phases, namely lag phase, exponential phase, stationary phase and bacterial death. Most experiments can be performed when cell division enters the exponential phase. The generation time of the selected bacterial strains is usually similar and ranges from 20 to 30 minutes (2).

Thanks to this fact, simultaneous cultivation of all four selected bacterial strains is facilitated as it takes almost the same time to reach the peak of the exponential phase. All strains are mesophilic, which means that they can be cultivated at 37°C.

The principle of determining growth inhibition is as follows: bacteria are cultivated simultaneously in three suspensions based on the LB medium - one without additives (negative control), one with antibiotic (positive control) and one with the added tested organic compound (100 µg/mL). The duration of the cultivation is 16 to 20 hours. This procedure can be performed in test tubes and measured individually on the Cary Varian spectrophotometer after cultivation. However, the use of microtiter plates facilitates the whole procedure as the test is performed in smaller volumes, and the reading of the results can be done with the plate reader (Tecan Sunrise). The standard procedure is based on the method of Rufian-Henares & Morales (27) and was modified accordingly to suit the conditions for this study.

Previously prepared permanent cultures were brought to the log phase of their growth and stored at -80°C. Prior to the assay, an aliquot of each bacterial species was thawed on ice and once again brought to the log phase. The volume of 2 µL of the bacterial suspension containing approximately 10⁶ CFU/mL was inoculated into the LB medium. The corresponding volume of liquid LB medium was added according to the volume of the selected ligand to reach a total volume of 200 µL in a 96-well microtiter plate.

The selected compounds were arranged in the columns while the bacterial strains were arranged in rows. This arrangement creates a matrix. Plain LB medium was added to a well to serve as a blank to test the sterility of the medium. LB with the organic compound without added bacteria was also added to the microtiter plate as some of the compounds precipitated or stained the sample. This served as an individual blank sample for all the

MASTER THESIS

U NIVERSITY OF L JUBLJANA

F ACULTY OF CHEMISTRY AND CHEMICAL TECHNOLOGY

MASTER THESIS

Nives Ražnjević

Ljubljana, 2021

UNIVERSITY OF LJUBLJANA

FACULTY OF CHEMISTRY AND CHEMICAL TECHNOLOGY

SECOND CYCLE MASTER'S PROGRAMME (BIOCHEMISTRY)

ANTIMICROBIAL ACTIVITY OF SELECTED

HETEROCYCLIC ORGANIC COMPOUNDS FROM THE FCCT COMPOUND LIBRARY

MASTER THESIS

Nives Ražnjević

MENTOR: Assoc. Prof. Dr. Marko Novinec

Ljubljana, 2021

Statement of Authorship

of the master thesis

Spremni list

Acknowledgment

Table of Contents

List of abbreviations and symbols

1. Introduction

1.1. Antibiotic mechanisms of action

1.2. The types of bacterial resistance to antimicrobial substances

1.3. Characteristic features and resistance development in studied bacterial species

2. Research aim and hypotheses

3. Materials and methods 3.1. Materials

.

.

3.2. Methods