Search

Odpornost proti antibiotikom v bakterijah postaja vse bolj resen problem za globalni zdravstveni sistem, saj postajajo številne prej ozdravljive okužbe manj ozdravljive ali celo neozdravljive. Svetovna zdravstvena organizacija (WHO) je bakterijsko odpornost proti antibiotikom uvrstila med tri največje grožnje za javno zdravstvo v 21. stoletju. Razvoj novih protimikrobnih učinkovin tako postaja ena glavnih prioritet, saj se lahko le na ta način izognemo tej težavi. Peptidoglikan je pomemben gradnik bakterijske celične stene, ki se sintetizira v treh različnih delih celice: v citoplazmi, celični membrani in zunaj celice. Prva stopnja reakcijske poti poteka v citoplazmi, kjer encim MurA katalizira pomemben korak. Večina trenutnih protibakterijskih zdravil zavira sintezo peptidoglikana v zadnjih stopnjah sinteze, ki potekajo izven celice. Kljub temu nekaj antibiotikov deluje znotraj celice, med njimi je fosfomicin, ki zavira aktivnost encima MurA v citoplazmi. Ta magistrska naloga se osredotoča na sintezo dveh derivatov glukozamina, ki temeljita na molekularnem modeliranju in posnemanju naravnega substrata. Za pravilno usmerjenost funkcionalnih skupin in sintezo reverzibilnih zaviralcev smo kot osnovno spojino uporabili N-acetilglukozamin, ki lahko prodira skozi celično steno in membrano v notranjost bakterij, kjer se nahaja encim MurA. Glavni cilj je bil razviti sintezno pot za pripravo spojin, v katerih so kisli fragmenti pripeti na hidroksilno skupino na mestu 6. Najprej smo zaščitili vse funkcionalne skupine N-acetilglukozamina, razen hidroksilne skupine na mestu 6. Spojino 11 smo sintetizirali z direktno uvedbo ocetne kisline na hidroksilno skupino, s pomočjo kloroocetne kisline. Pri sintezi spojine 9 pa smo sprva morali hidroksilno skupino aktivirati s tvorbo tozilata, preko katerega smo potem tvorili derivat z dimetil malonatom, ki smo ga na koncu hidrolizirali do karboksilne kisline. Z encimskim testom smo potrdili, da sta obe spojini zavirali encimsko aktivnost MurA, pri čemer je imela spojina 9 vrednost IC50 84 μM in spojina 11 vrednost IC50 14 µM. Antibiotic resistance in bacteria is becoming an increasingly serious problem for the global healthcare system, leading to a reduction in the effectiveness of previously curable infections and even making some infections incurable. The World Health Organization (WHO) has listed it as one of the biggest threats to public healthcare in the 21st century. To address this issue, significant research efforts are being invested into discovery of new antimicrobial drugs. Peptidoglycan is an important component of the bacterial cell wall, and its biosynthesis occurs in three regions: the cytoplasm, the cell membrane, and outside the cell. The MurA enzyme is present in the cytoplasm and initiates the pathway by catalyzing the first reaction. The majority of currently available antibiotics inhibit the peptidoglycan synthesis pathway, with their site of action in the later stages outside the cell. However, a few antibiotics act inside the cell, such as fosfomycin, which inhibits the activity of the MurA enzyme. This master’s thesis focused on synthesizing two glucosamine derivatives based on molecular modeling and mimicking of the natural substrate. N-acetylglucosamine was utilized to correctly align functional groups and develop reversible inhibitors capable of penetrating the cell wall and membrane, reaching the bacteria’s interior where the MurA enzyme is located. The primary purpose of the master’s thesis was to develop a synthetic route for preparing compounds that have acidic fragments attached to the hydroxyl group located at position 6. Firstly, all functional groups of N-acetylglucosamine, except for the hydroxyl group at position 6, were protected. Compound 11 was synthesized by directly introducing acetic acid onto the hydroxyl group using chloroacetic acid. For the synthesis of compound 9, the hydroxyl group was first activated by forming a tosylate, which was then utilized to form a derivative with dimethyl malonate. This derivative was finally hydrolyzed to yield carboxylic acid form. Enzyme testing confirmed that both compounds inhibited enzyme activity, with compound 9 having an IC50 value of 84 μM and compound 11 having an IC50 value of 14 µM.

Proteus anguinus ali človeška ribica sodi med dvoživke in velja za največjo obligatno jamsko žival ter za edinega jamskega vretenčarja v Evropi. Njen obstoj je zaradi onesnaževanja okolja kot posledice človeške dejavnosti in neposrednega vpliva na njen habitat ogrožen. Raziskave kože človeških ribic in kraške ter akvarijske vode so potrdile prisotnost za dvoživke potencialno nevarnih sevov bakterij iz rodu Aeromonas, povzročiteljev t.i. sindroma rdečih nog. Sevom rodu Aeromonas smo določili virulentni potencial in ocenili grožnjo, ki jo predstavljajo človeškim ribicam. Seve rodu Aeromonas smo do vrste natančno identificirali s filogenetsko analizo na podlagi konkateniranih nukleotidnih zaporedij hišnih genov dnaJ, dnaX, gyrB, recA in rpoD ter gena za 16S rRNA v programu MEGA-X. Na podlagi gena za 16S rRNA prepoznane izolate najbolj patogene vrste A. hydrophila smo identificirali kot A. media, potrdili pa smo prisotnost za ribe in druge vodne živali potencialno patogeni vrsti A. salmonicida in A. veronii. Analize so nakazale na večjo vrstno raznolikost aeromonasov v kožni mikrobioti zdravih človeški ribic napram bolnim ter potrdili dejstvo, da kožna mikrobiota promovira rast v okolju manj zastopanih bakterijskih vrst. Z metodo PCR smo ugotavljali prisotnosti genov za virulentne dejavnike act, alt, ast, aerA, ahpA, epr, hlyA in GCAT in s povprečno 2,13 geni na sev rodu Aeromonas potrdili nizek virulentni potencial. Kljub temu smo odkrili 2 potencialno citotoksična seva z genotipom act+/alt+/aerA+/hlyA+, 19 potencialno citotoksičnih sevov genotipa act+/alt+/aerA+ in 4 potencialno zelo virulentne seve genotipa alt+/aerA+/hlyA+. Kar 93 % sevov je bilo odpornih proti vsaj enemu testiranemu antibiotiku, proti gentamicinu (93,49 % sevov), imipenemu (49,11 % sevov), eritromicinu (34,91 % sevov), ceftriaksonu (34, 32 % sevov), aztreonamu (2,96 % sevov) in enrofloksacinu (2,96 % sevov), ki se uporabljajo v veterini ali humani medicini, kar je zaskrbljujoče. Seve z največjim povprečnim številom antibiotikov, proti katerim so bili odporni, smo izolirali iz lokacije, kjer je v preteklosti prišlo do onesnaženja s težkimi kovinami, kar se sklada s konceptom navskrižne odpornosti. Ugotovitve nakazujejo na verjetno onesnaženje kraških vod z odpadnimi vodami, živinorejo in človeškimi fekalijami ter posledično širjenje genskega materiala, ki vsebuje zapise za mehanizme odpornosti in virulence med bakterijami. The amphibian Proteus anguinus, also known as the olm, is considered the largest obligate cave animal and the only cave-dwelling vertebrate in Europe. Its existence is threatened by the direct impact on its habitat caused by environmental pollution due to human activities. Microbiome studies of its skin and aquatic enviroment led to the discovery of potentially dangerous and red-leg syndrome causing bacteria of the genus Aeromonas. We determined the virulence potential and assessed the threat Aeromonas spp. pose to the olm. We identified the isolated strains of Aeromonas spp. to species by phylogenetic analyzes based on the concatenated sequences of the housekeeping genes dnaJ, dnaX, gyrB, recA and rpoD and the 16S rRNA gene using the MEGA-X program. We were able to identify strains previously recognized as A. hydrophila as A. media and confirm the presence of the potential fish pathogen species A. salmonicida and A. veronii. The results indicated a greater diversity of Aeromonas species isolated from the skin microbiome of healthy olms compared to unhealthy animals, and confirmed the fact that the skin microbiome promotes the growth of the less represented Aeromonas species from the aquatic enviroment. Using the PCR method, we determined the presence of the virulence factor genes act, alt, ast, aerA, ahpA, epr, hlyA and GCAT and confirmed an average of 2.13 genes per Aeromonas spp. strain, indicating a low virulence potential of the isolated strains. However, we succeeded in detecting 2 potentially cytotoxic act+/alt+/aerA+/hlyA+ strains, 19 potentially cytotoxic act+/alt+/aerA+ strains, and 4 potentially highly virulent alt+/aerA+/hlyA+ strains. No less than 93 % of the strains were resistant to at least one of the antibiotics tested: Gentamicin (93.49 % of all strains), Imipenem (49.11 % of all strains), Erythromycin (34.91 % of all strains), Ceftriaxone (34.32 % of all strains), Aztreonam (2.96 % of all strains), and Enrofloxacin (2.96 % of all strains), which are used in veterinary or human medical practice, raising concern. The strains with the highest average number of antibiotics to which they were resistant were isolated from a site with a history of heavy metal contamination, consistent with the concept of cross-resistance in bacteria. The results of this study suggest that karst waters are likely contaminated by sewage, livestock and human faeces, leading to the spread of resistance and virulence mechanisms among environmental bacteria.

Namen magistrske naloge je bil v vzorcih surovega mleka iz slovenskih mlekomatov raziskati prisotnost različnih genov, ki kodirajo odpornost proti tetraciklinom, eritromicinu, kloramfenikolu in aminoglikozidnim antibiotikom. V nalogi smo analizirali 50 vzorcev mleka, pri katerih smo z metodo PCR ugotavljali prisotnost genov za odpornost proti tetraciklinom (tet(A), tet(B), tet(C), tet(M), tet(O), tet(W)), eritromicinu (erm(A), erm(B), erm(C)), aminoglikozidnim antibiotikom (aac(6')-aph(2''), ant(6)-la, aph(3')-llla) in kloramfenikolu (cat). Največkrat smo ugotovili prisotnost gena aac(6')-aph(2'') in sicer v 21 vzorcih surovega mleka, sledijo mu preostala aminoglikozidna gena, tetraciklinski in eritromicinski geni, gena za odpornost proti kloramfenikolu nismo zaznali. Ugotovili smo tudi, da se pri analizi z metodo PCR meje zaznavnosti za posamezen gen razlikujejo in so v območju od 2 x 103 do 1 x 108 KE/mL mleka. Surovo mleko iz slovenskih mlekomatov vsebuje različne gene za odpornost proti antibiotikom, vendar še zdaleč ne toliko, kot poročajo iz drugih držav Evrope in sveta. The aim of the study was to investigate the presence of various genes encoding resistance against tetracyclines, erythromycin, chloramphenicol and aminoglycoside antibiotics in 50 samples of raw milk from Slovenian vending machines. By using PCR method we analysed the presence of genes encoding resistance to tetracyclines (tet(A), tet(B), tet(C), tet(M), tet(O), tet(W)), erythromycin (erm(A), erm(B), erm(C)), aminoglycoside antibiotics (aac(6')-aph(2''), ant(6)-la, aph(3')-llla) and chloramphenicol (cat). The most commonly determined antibiotic resistance gene was aac(6')-aph(2''), presented in 21 samples of raw milk, followed by the remaining aminoglycoside genes, tetracyclines and erythromycin genes. However, the gene encoding resistance to chloramphenicol was not detected. In addition, we found that in PCR method, selected antibiotic resistance genes had different limits of detection and were in range between 2 x 103 to 1 x 108 CFU/mL of milk. In conclusion, raw milk from Slovenian milk vending machines contains various antibiotic resistance genes, although the prevalence is lower compared to reports from other countries in Europe and world.

Zaradi naraščajočega pojavljanja večkratno odpornih bakterij (VOB) se v zdravstvu povečuje potreba po novih strategijah za zdravljenja okužb s tovrstnimi povzročitelji. Fosfomicin je starejši antibiotik, ki spada v skupino fosfonskih antibiotikov. Zaradi nizke molekulske mase in nizke vezave na plazemske proteine v telesu dosega dobro distribucijo in visoko difuzibilnost v tkiva. Najvišje serumske koncentracije doseže po 2–2,5 ure. Intravensko apliciran fosfomicin se nespremenjen izloči z glomerulno filtracijo v ledvicah v 90 odstotkih. Fosfomicin v bakterijsko citoplazmo vstopa s pomočjo heksoza fosfatnega in glicerol-3-fosfatnega transportnega sistema ter deluje kot analog fosfoenolpiruvata in zavira encimsko katalizirano reakcijo prve stopnje sinteze peptidoglikana. Ima širok spekter delovanja, saj je med drugim učinkovit proti večkratno odpornim po Gramu negativnim in po Gramu pozitivnim bakterijam. V splošnem je varen antibiotik, ki je v Evropi in ZDA indiciran za zdravljenje nezapletenih okužb sečil, v Evropi pa se poleg tega lahko uporablja tudi za zdravljenje drugih okužb sečil ter za zdravljenje sepse in okužb mehkih tkiv. Najpomembnejši mehanizem rezistence je encimska inaktivacija. Referenčna metoda za določanje občutljivosti bakterij za fosfomicin po priporočilih EUCAST je agar dilucijska metoda, s pomočjo katere smo določili distribucijo minimalnih inhibitornih koncentracij (MIK) izolatov izbranih vrst enterobakterij (N 506) ter Acinetobacter baumannii (N 55). Med posameznimi vrstami enterobakterij so velike razlike v MIK50, MIK90 in okvirnih epidemioloških mejnih vrednosti. Najnižje vrednosti smo določili pri Escherichia coli in Citrobacter spp., pri ostalih pa so bile bistveno višje. Občutljivost izolatov E. coli za peroralni fosfomicin je bila visoka in je znašala 93,4 %. Za intravenski fosfomicin je bilo občutljivih 95,5 % izolatov E. coli, 66,2 % izolatov Klebsiella pneumoniae, 92,2 % izolatov K. oxytoca, 50,0 % izolatov Enterobacter cloacae kompleks in 97,1 % izolatov Citrobacter spp. Pomembno razliko v občutljivosti med ne-VOB in VOB izolati smo opažali samo pri K. pneumoniae (79,8 % in 39,6 %). Ob upoštevanju razmejitvenih vrednosti za enterobakterije bi bilo za intravenski fosfomicin občutljivih 1,8 % izolatov A. baumannii. Za določanje MIK se pogosto uporabljajo preprostejši komercialni gradient difuzijski testi. EUCAST jih zaradi nezanesljivih rezultatov odsvetuje za testiranje občutljivosti za fosfomicin. Med našo raziskavo smo analizirali kategorično ujemanje. Pri peroralnem fosfomicinu smo opažali veliko napako v 0,8 % in zelo veliko napako v 0,8 %, pri intravenskem pa 2,4 % zelo velikih napak, kar za diagnostični test ni več sprejemljivo. The increasing emergence of multidrug-resistant (MDR) bacteria has led to a growing need for new strategies to treat MDR bacterial infections. Fosfomycin is an older antibiotic that belongs to the group of phosphonic antibiotics. Due to its low molecular weight and low binding to plasma proteins in the body, it achieves good distribution and high tissue diffusibility. It reaches peak serum concentrations after 2-2.5 hours. Intravenously administered fosfomycin is excreted in the kidneys unchanged by glomerular filtration at a rate of 90%. Fosfomycin enters the bacterial cytoplasm through the hexose phosphate and glycerol-3-phosphate transport systems and functions as an analogue of phosphoenolpyruvate inhibiting the enzyme-catalysed first-stage reaction of peptidoglycan synthesis. It has a wide spectrum of activity as it is effective against several MDR Gram-negative and Gram-positive bacteria. It is an overall safe antibiotic indicated for treating uncomplicated urinary tract infection both in Europe and in the USA and may also be used in Europe for treating other urinary tract infections, sepsis and soft tissue infections. The most relevant resistance mechanism is enzyme inactivation. The reference method for determining the susceptibility of bacteria to fosfomycin according to EUCAST is the agar dilution method, which was performed to determine the distribution of minimum inhibitory concentrations (MIC) of selected Enterobacteriaceae isolates (N 506) and Acinetobacter baumannii (N 55). There are significant differences in MIC50, MIC90, and in tentative epidemiological cut-off values between individual Enterobacteriaceae species with the lowest values determined for Escherichia coli and Citrobacter spp., whereas the others were significantly higher. The susceptibility of E. coli isolates to oral fosfomycin was high and amounted to 93.4%. 95.5% of E. coli isolates, 66.2% of Klebsiella pneumoniae isolates, 92.2% of K. oxytoca isolates, 50.0% of Enterobacter cloacae complex isolates, and 97.1% of Citrobacter spp. isolates were susceptible to intravenous fosfomycin. A significant difference in susceptibility between non-MDR and MDR isolates was observed only for K. pneumoniae (79.8% and 39.6%). Considering the enterobacterial cut-off values, 1.8% of A. baumannii isolates would be susceptible to intravenous fosfomycin. Simpler commercial gradient diffusion tests are often used to determine MICs. EUCAST advises against them for fosfomycin susceptibility testing due to unreliable results. During our study, we analyzed categorical agreement and observed a major error rate of 0.8% and a very major error rate of 0.8% for oral fosfomycin, and a major error rate of 2.4% for intravenous fosfomycin which is no longer acceptable for a diagnostic test.

Proti antibiotikom odporne bakterije predstavljajo enega izmed glavnih zdravstvenih problemov 21. stoletja, še posebej bakterije z geni za encime ESBL in karbapenemaze. K razvoju in širjenju odpornosti proti antibiotikom vplivajo tako človeški, živalski kot okoljski rezervoarji. V Sloveniji je malo znanega o prisotnosti bakterij z zmanjšano občutljivostjo za antibiotike v izvenbolnišničnem okolju, še predvsem na odlagališčih. Namen naloge je bila osamitev bakterij, ki so fenotipsko odporne proti betalaktamom z razširjenim spektrom delovanja in karbapenemom, iz površinskih in podzemnih vod, povezanih z izbranim odlagališčem odpadkov ter genotipska opredelitev fenotipske odpornosti, ki temelji na izločanju encimov za betalaktamaze z razširjenim spektrom delovanja (ESBL) in karbapenemaz. Bakterije iz vzorcev podzemnih in površinskih vod smo osamili z uporabo 3 izbranih selektivno diferencialnih gojišč in jih identificirali z metodo MALDI TOF. Encime ESBL in karbapenemaze smo najprej potrdili s fenotipskimi testi (metoda kombiniranih diskov za potrditev ESBL in CIM testi), nato s tržno dostopnimi kiti določili njihove gene. Ugotovili smo, da so izolati iz površinskih vod odporni proti več antibiotikom, kot izolati iz podzemnih vod. S pomočjo molekularnih testov smo določili gene za odpornost proti betalaktamom pri dveh izolatih, ki smo ju izolirali iz površinskih vod. Pri prvem izolatu Klebsiella oxytoca (ZZV20-11045), izoliranem iz površinskih padavinskih vod, smo potrdili gen za encim CTXM-1 in gen za encim OXA-48. Pri drugem izolatu Pseudomonas aeruginosa (ZZV20-11064), ki smo ga izolirali iz površinskih izcednih vod pa smo potrdili prisotnost gena za encim iz skupine VIM. Iz površinskih voda smo osamili več proti betalaktamom in karbapenemom odpornih bakterij kot v podzemnih vodah. Pri obeh odpornih mikroorganizmih smo potrdili prisotnost genov za encime iz skupine ESBL in karbapenemaze (dokazali smo gene blaVIM, blaCTXM-1 and blaOXA-48). Antibiotic resistance is one of the major health problems of the 21st century. The development and spread of antibiotic resistance is influenced by human, animal and environmental reservoirs. Little is known in Slovenia about the presence of bacteria with reduced sensitivity to antibiotics in the non-hospital environment. Among the major resistant bacteria that pose a threat to human health are bacteria with genes for the enzymes ESBL and carbapenemase. The aim of the study was to isolate bacteria that are phenotypically resistant to betalactams with extended spectrum and carbapenem from surface and groundwater associated with the selected landfill and genotypic definition of phenotypic resistance based on the secretion of enzymes for betalactamase (ESBL) and carbapenemases. Bacteria from groundwater and surface water samples were isolated by three specific selective-differential media and identified using MALDI-TOF. ESBL enzymes and carbapenemases were first confirmed by phenotypic tests (combined disk method to confirm ESBL and CIM tests), then their genes were determined by commercially available kits. Surface water isolates were found to be resistant to more antibiotics than groundwater isolates. Molecular tests confirmed the presence of resistance genes in two isolates isolated from surface waters. In the case of Klebsiella oxytoca isolate (ZZV20-11045) isolated from surface rainwater, the gene for the enzyme CTXM-1 and the gene for the enzyme OXA-48 were confirmed. In the isolate of Pseudomonas aeruginosa (ZZV20-11064), which was isolated from surface leachate, the presence of the gene for the enzyme from the VIM group was confirmed. More betalactam and carbapenem resistant bacteria were isolated from surface waters than in groundwater. In both resistant microorganisms, the presence of genes for ESBL and carbapenemase enzymes were confirmed (genes blaVIM, blaCTXM-1 and blaOXA-48 were detected).

S. enterica je s hrano prenosljiva patogena bakterija, ki že izkazuje odpornost na medicinsko pomembne antibiotike. Odličen alternativni pristop h konvencionalni antibiotični terapiji je probiotična terapija. Med probiotike uvrščamo tudi bakterije iz rodu Bacillus, vendar so interakcije med bakterijo S. enterica in temi probiotiki v biofilmu slabo raziskane. Ni znano kako vplivajo interakcije med sevi ene vrste na interakcije z drugimi vrstami bakterij. Znano pa je, da bakterije B. subtilis lahko razlikujejo med filogenetsko visoko in manj sorodnimi sevi. V nalogi smo preučevali kako to sorodstveno razlikovanje med sevi B. subtilis vpliva na rast in prostorsko razporeditev S. enterica. Uporabili smo seve, označene z genom za odpornost proti antibiotiku (AmpR, SpR ali CmR) in s fluorescenčnim označevalcem (GFPmut3, mKate2 ali CFP), kar nam je omogočilo spremljanje sprememb rasti in razporeditve celic S. enterica. Testirali smo izrabljena gojišča različnih kombinacij sevov B. subtilis in interakcije s S. enterica ob sogojenju obeh vrst v tekočem in na trdnem gojišču. Ugotovili smo, da izrabljeno gojišče sevov B. subtilis, ne glede na tip mešanice sevov, ne zavira rasti S. enterica, temveč jo spodbuja, tako v tekočem kot tudi na trdnem gojišču. Sogojenje S. enterica z enim ali z mešanico sevov B. subtilis je negativno vplivalo na rast S. enterica, pri čemer v tekočem gojišču nismo zaznali značilnega vpliva sorodnosti. V kolonijah na trdnem gojišču so bile mešanice visoko sorodnih sevov B. subtilis nekoliko uspešnejše v zmanjševanju števila celic S. enterica, v primerjavi z manj sorodnimi mešanicami. Prostorska razporeditev S. enterica se tako v kolonijah kot v tekočem gojišču ni spreminjala v odvisnosti od sorodnosti in števila sevov B. subtilis. Ugotovili smo, da ima sorodnost med sevi B. subtilis značilen vpliv tudi na odpornost S. enterica na antibiotike. Bakterija S. enterica postane bolj občutljiva na tetraciklin in kotrimoksazol ter manj občutljiva na spektinomicin po inkubaciji z visoko sorodnimi sevi B. subtilis, v primerjavi z enovrstnim biofilmom. A food-borne pathogen Salmonella enterica is already showing resistance to antibiotics and new approaches are needed to combat this problem. An excellent alternative approach to the conventional antibiotic therapy is the probiotic therapy. Strains of the Bacillus genus are in use as probiotics, but the interactions between S. enterica probiotic strains in biofilms are poorly understood. Even less is known about how intraspecific bacterial interactions influence interactions with another bacterial species. B. subtilis has been recently recognized for its ability to discriminate between less and more related strains. In order to study how relatedness between B. subtilis strains affects the growth and spatial distribution of S. enterica, we used mixtures of highly related and less related B. subtilis strains and cocultured them with S. enterica. Each strain was labelled with an antibiotic resistance gene (AmpR, SpR or CmR) and a fluorescent marker (GFPmut3, mKate2 or CFP). We aimed to determine whether and how the growth and distribution of S. enterica was changing in co-culture with B. subtilis and how kin discrimination between strains of B. subtilis impacts S. enterica. In experiments with spent media of B. subtilis strain mixtures and their relatedness have not inhibited growth of S. enterica, on contrary, spent media contained components that promoted the growth of S. enterica in liquid and on solid media. On the other hand, co-cultivation with B. subtilis mixtures had a negative impact on S. enterica growth, with no correlation with the strain number or relatedness. In colonies on a solid medium, the mixtures of highly-related strains of B. subtilis seemed to be more effective in reducing number of S. enterica cells, however, the size of Salmonella's colony has not changed. We also found that the relatedness between B. subtilis strains had a significant impact on antibiotic resistance of S. enterica, which became more susceptible to tetracycline and cotrimoxazole and less susceptible to spectinomycin after incubation with highly-related B. subtilis strains.

Bakterije vrste C. jejuni so najpogostejše povzročiteljice kampilobakterioze ter gastroenteritisov pri človeku. Za svoje razmnoževanje in rast potrebujejo optimalne pogoje, zato so znani kot zelo zahtevni in občutljivi mikroorganizmi. Sposobnost tvorbe biofilmov jim omogoča večjo odpornost na protimikrobne učinkovine ter s tem lažje preživetje. Nadzor bakterij v odporni obliki biofilmov zahteva uporabo še večjih koncentracij antibiotikov. Problem naraščajoče odpornosti bakterij zato zahteva vpeljavo novih alternativnih strategij za nadzor nad odpornimi sevi in odpornimi oblikami biofilma. Ena od alternativnih strategij je uporaba protimikrobnih učinkovin naravnega izvora in sicer v njihovih sub-inhibitornih koncentracijah. V teh koncentracijah učinkovine ne vplivajo na rast, ampak na druge lastnosti bakterij in tako ne povzročajo razvoja odpornosti. Namen naloge je bil: (i) predstaviti različne učinkovine naravnega izvora in njihovo dokazano delovanje na inhibicijo tvorbe in redukcijo nastalega biofilma bakterij C. jejuni, (ii) opisati mehanizme delovanja na različnih celičnih nivojih C. jejuni ter (iii) primerjati tarčno delovanje antibiotikov in učinkovin naravnega izvora pri bakterijah C. jejuni. V zaključku naloge smo opisali modulacijo genov oz- proteinov, ključnih za nastanek biofilma ter vključene v mehanizme gibljivosti, adhezije in medcelične komunikacije, v celicah tretiranimi z učinkovinami naravnega izvora. Na ta način smo dokazali tarčne mehanizme delovanja protimikrobnih učinkovin naravnega izvora na C. jejuni. Bacteria of the species C. jejuni are the most common causative agents of campylobacteriosis and gastroenteritis in humans. They require optimal conditions for their replication and growth, which is why they are known to be very fastidious and sensitive microorganisms. The ability to form biofilms makes them more resistant to antimicrobial agents and thus more survivable. To control bacteria in the resistant form of biofilms require the use of even higher concentrations of antibiotics. Therefore increasing bacterial resistance requires the introduction of new alternative strategies to combat resistant strains and resistant forms of biofilms. One of these alternative strategies is the use of antimicrobial agents of natural origin at their subinhibitory concentrations. At these concentrations, the agents do not affect the growth but other properties of the bacteria and thus do not lead to development of resistance. The purpose of this thesis was: (i) to present different compounds of natural origin and to demonstrate their ability to inhibit biofilm formation or reduce the formed biofilm of C.jejuni, (ii) to describe the mechanisms of action at different cellular levels of C. jejuni, and (iii) to compare the target effect of antibiotics and compounds of natural origin on C. jejuni. At the end, we described the modulation of genes or proteins crucial for biofilm formation and their involvement in the mechanisms of motility, adhesion and intercellular communication, in cultures treated with natural antimicrobial agents. In this way, we have explained the target mechanisms of action of antimicrobial agents of natural origin in C. jejuni cells.