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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.

Odpornost proti antibiotikom med anaerobnimi bakterijami je močno narasla v zadnjih tridesetih letih. Najobsežnejša slovenska raziskava o protimikrobni odpornosti anaerobnih bakterij iz leta 2015 je pokazala, da je odpornost proti amoksicilinu s klavulansko kislino pri najpogosteje izolirani skupini B. fragilis znašala 8 %, odpornost proti imipenemu pa 1 %. Podatkov o občutljivosti za ostale betalaktamske antibiotike v kombinaciji z zaviralci betalaktamaz ni, saj se v rutinski diagnostiki pri nas testiranje zanje ne izvaja. Zato smo se odločili, da v našem magistrskem delu z gradient difuzijskim testom preverimo občutljivost izbranih anaerobnih bakterij, iz rodu Bacteroides in Parabacteroides, za amoksicilin s klavulansko kislino, piperacilin s tazobaktamom in ceftolozan s tazobaktamom. Skupno smo testirali 120 prospektivno zbranih izolatov (104 izolati rodu Bacteroides, 16 izolatov rodu Parabacteroides), ki smo jih izolirali iz kužnin in 87 izbranih izolatov iz zbirke IMI z znano odpornostjo proti amoksicilinu s klavulansko kislino (77 izolatov rodu Bacteroides, 10 izolatov rodu Parabacteroides). Odpornost prospektivno zbranih izolatov rodu Bacteroides za amoksicilin s klavulansko kislino je bila 17,3 %, med izolati rodu Parabacteroides je bilo odpornih 31,3 % testiranih izolatov. Odpornost izolatov rodu Bacteroides proti piperacilinu s tazobaktamom je znašala 11,5 %, za izolate rodu Parabacteroides pa kar 43,8 %. Odstotek odpornosti pri izbranih izolatih z znano odpornostjo proti amoksicilinu s klavulansko kislino je bil še višji in je za izolate rodu Bacteroides znašal 58,4 %, za izolate rodu Parabacteroides pa 50 %. Ker o ceftolozanu s tazobaktamom še ni smernic za interpretacijo mejnih vrednosti, smo določili distribucijo vrednosti MIK. Za prospektivno zbrane izolate je bila vrednost MIK50 16 μg/mL, MIK90 pa 256 μg/mL. Vsi izbrani izolati rodu Parabacteroides z znano odpornostjo proti amoksicilinu s klavulansko kislino so imeli vrednost MIK 256 μg/mL, medtem ko je bil razpon vrednosti za izolate rodu Bacteroides od 1,5 do 256 μg/mL. Antibiotic resistance among anaerobic bacteria has risen sharply in the last thirty years. The most extensive Slovenian research about antimicrobial resistance of anaerobic bacteria from 2015 showed that resistance to amoxicillin/clavulanic acid of the most frequently isolated B. fragilis group was 8%, and the resistance to imipenem was 1%. There is no data about susceptibility for the other beta-lactam antibiotics in combination with beta-lactamase inhibitors as testing for them is not performed in our routine diagnostics. For this reason, we decided to test the susceptibility of selected anaerobic bacteria from the genus Bacteroides and Parabacteroides to amoxicillin/clavulanic acid, piperacilin/tazobactam, and ceftolozan/tazobactam with a gradient diffusion method. We tested a total of 120 prospectively collected clinically significant anaerobic bacteria (104 isolates of the genus Bacteroides, 16 isolates of the genus Parabacteroides) and 87 selected isolates from the IMI collection with known resistance to amoxicillin/clavulanic acid (77 isolates of the genus Bacteroides, 10 isolates of the genus Parabacteroides). The resistance of prospectively collected Bacteroides isolates to amoxicillin/clavulanic acid was 17.3%, and 31.3% for the Parabacteroides isolates. The resistance of isolates of the genus Bacteroides to piperacillin/tazobactam was 11.5%, and among isolates of the genus Parabacteroides resistance was as high as 43.8%. The percentage of resistance of the selected isolates with known resistance to amoxicillin with clavulanic acid was even higher: 58.4% for Bacteroides isolates and 50% for isolates of the genus Parabacteroides. As there are no guidelines for interpretive breakpoints for ceftolozane/ tazobactam, we only determined MIC distribution. For prospectively collected isolates, the MIC50 value was 16 μg/mL, and MIC90 was 256 μg/mL. All selected isolates of the genus Parabacteroides with known resistance to amoxicillin/clavulanic acid had a MIC value of 256 μg/mL, while the value range for isolates of the genus Bacteroides ranged from 1.5 to 256 μg/mL.

Razširjena uporaba antibiotikov je povzročila, da se njihov obseg v okolju dnevno globalno povečuje. Antibiotiki se v telesu ne presnovijo v celoti. Večina antibiotikov se v okolje vnese preko živali, saj se v veterini uporabljajo kot zdravstvena preventiva, za zdravljenje bakterijskih okužb, z namenom pospeševanja rasti in/ali prirasta živali, preostanek pa v okolje vnesemo ljudje, zaradi zdravljenja bakterijskih okužb. Vnos antibiotikov v okolje neposredno najbolj vpliva na vodne organizme, predvsem pa je vse bolj skrb vzbujajoč pojav odpornih bakterij oz. superbakterij. Te bakterije so odporne proti antibiotikom in posledično nanje antibiotiki ne delujejo več, zato je vse več še pred kratkim preprosto ozdravljivih bakterijskih okužb težje ozdravljivih oz. neozdravljivih. Odstranjevanje antibiotikov je torej nujno. Zaradi vstopa večine antibiotikov v vodno okolje, se osredotočamo predvsem na odstranjevanje iz odpadnih vod. Konvencionalne komunalne čistilne naprave antibiotikov niso sposobne odstraniti in posledično lahko ti preprosto vstopijo v površinske vode, podtalnico in tudi pitno vodo. Za odstranjevanje antibiotikov iz odpadnih vod se vse bolj uporabljajo napredni oksidacijski procesi. Namen magistrske naloge je bil primerjava različnih naprednih oksidacijskih procesov za čiščenje odpadnih vod, ki vsebujejo antibiotike in preveritev, ali so ti postopki primerni za odstranjevanje antibiotikov iz vodnega okolja. Preučevali smo naslednje tehnike oz. postopke: direktna ozonacija, ozonacija pri povišanem pH (pH=9,5), ozonacija pri povišanem pH (pH=9,5) ob dodatku H2O2, ozonacija, katalizirana z dodatkom Fe2+, ozonacija, katalizirana z dodatkom Fe2+ in UV svetlobe, ozonacija s Fentonovo oksidacijo in ozonacija s foto-Fentonovo oksidacijo. Učinkovitost tehnik smo ovrednotili s spremljanjem celotnega organskega ogljika (TOC) in kemijske potrebe po kisiku (KPK). Ugotovili smo, da pri vseh postopkih proces poteče dvostopenjsko z bolj učinkovito prvo stopnjo procesa. Direktna ozonacija se je izkazala za najmanj učinkovito od vseh preučevanih postopkov, tako glede na TOC, kot KPK rezultate. Učinkovitost ozonacije se je povečala ob povišanem pH, ob dodatku UV, H2O2 in/ali katalizatorjev. Najučinkovitejša je bila ozonacija s foto-Fentonovo oksidacijo, s katero smo dosegli med 83 in 96 % stopnjo oksidacije prisotnih antibiotikov, ter med 65 in 84 % stopnjo mineralizacije, torej popolno razgradnjo antibiotikov v anorganske komponente, CO2 in H2O. S tem smo potrdili, da so napredni oksidacijski procesi primerni postopki za odstranjevanje antibiotikov iz odpadnih vod. The widespread use of antibiotics has been causing increase of their extent in the environment. Antibiotics are not completely metabolised in the body. Most antibiotics come into the environment through animals, as they are used in the veterinary medicine for health prevention, for the treatment of bacterial infections, with the purpose to promote growth and/or animal increment. The rest come to the environment because of human consumption, due to the treatment of bacterial infections. Antibiotics into the environment directly affect the aquatic organisms, and above all, the increasing concern arouses phenomenon of resistant bacteria or superbacteria. These bacteria are resistant to antibiotics and, consequently, they are not affected by them, so nowadays more and more bacterial infections are more difficult to be cured or are even incurable. Therefore, the removal of antibiotics is necessary. Due to the entry of most antibiotics in the aquatic environment, their removal from wastewater is the most important. Conventional municipal wastewater treatment plants are not capable of removing antibiotics and thus they can easily enter surface water, groundwater, and even drinking water. The main focus nowadays is the addition of the tertiary cleaning using advanced oxidation processes. The purpose of this thesis was to compare the various advanced oxidation processes for wastewater treatment containing antibiotics and verification if these procedures are suitable for the removal of antibiotics from the aquatic environment. We studied the following experimental techniques/procedures: direct ozonation, ozonation at elevated pH (pH = 9.5), ozonation at elevated pH (pH = 9.5) at the addition of H2O2, catalysed ozonation by addition of Fe2+, catalysed ozonation by addition of Fe2+ and UV light, ozonation with Fenton oxidation and ozonation with photo-Fenton oxidation. The methods were evaluated according to the total organic carbon (TOC) and chemical oxygen demand (COD). It can be concluded that studied procedures occur in two phases with first stage of process to be more effective. Direct ozonation was the least effective in terms of TOC, as well as COD. The efficiency of the ozonation increased at elevated pH, with the addition of UV, H2O2 and/or catalysts. The most effective procedure was ozonation with photo-Fenton oxidation, where we achieved between 83 and 96% oxidation of the present antibiotics and between 65 and 84% mineralization, hence the complete decomposition of antibiotics into inorganic components, CO2, and H2O. Our results confirmed that advanced oxidation processes are appropriate for removing antibiotics from wastewater.

Močeril je, zaradi svoje edinstvene biologije, najbolj prepoznavna žival v dinarskem podzemlju. Sodi med najbolj ogrožene živali v Evropi, ogrožajo jo predvsem različni patogeni in spremembe v njenem naravnem okolju, ki so predvsem posledica onesnaževanja. Cilj magistrskega dela je bil raziskati sestavo bakterijske kožne mikrobiote močerila in njegovega vodnega okolja. S kože 32 osebkov podvrste Proteus anguinus anguinus in dveh osebkov podvrste Proteus anguinus parkelj ter 18 vzorcev vode različnih lokacij smo izolirali 2196 aerobnih kemoorganoheterotrofnih bakterij. Ugotovili smo, da sta sestavi kožne in vodne mikrobiote precej podobni, sestava pa je izredno pestra. Največ izolatov pripada rodovom Pseudomonas, Aeromonas, Acinetobacter, Flavobacterium in Bacillus, ki so ubikvitarni, izolirali smo jih iz brisov in vode, zato sklepamo, da večina kožne mikrobiote močerila izvira iz njegovega vodnega okolja. Izolati rodov Brevundimonas, Janthinobacter, Serratia in Stenotrophomonas, pa bi po literaturi lahko bili sestavni del zaščitne mikrobiote dvoživk in tako imeli podobno funkcijo tudi pri močerilu. Primerjava mikrobiote kože med prostoživečimi osebki in osebki v ujetništvu je pokazala, da je diverziteta izolatov večja pri živalih v ujetništvu. Primerjava med zdravimi in bolnimi osebki pa je pokazala, da je diverziteta bakterij večja pri zdravih osebkih. Preverili smo tudi odpornost 112 od 2196 izolatov proti ampicilinu, cefotaksimu, imipenemu, tetraciklinu, eritromicinu, kanamicinu, enrofloksacinu in kloramfenikolu. Največ testiranih sevov je bilo odpornih proti cefotaksimu (47,5 %), nižji delež pa proti tetraciklinu (3,3 %). Enrofloksacin, ki se uporablja v veterinarski medicini, je učinkoval proti večini testiranih izolatov. Z metodami strojnega učenja smo ugotovili, da ni podobnosti med vzorci glede lokacije, sezono vzorčenja ter tipom vzorca. Statistično značilno razliko med skupinami vzorcev smo dokazali samo med vzorci iz lokacij Jelševnik in jamski laboratorij Tular. Magistrska naloga je bila opravljena v okviru projekta 'Porajajoči mikrobni patogeni človeške ribice (Proteus anguinus), endemne troglobiontske dvoživke'. Due to its unique biology, the blind cave salamander (olm) is the most recognizable animal in the Dinaric underground. It is one of the most endangered animals in Europe, threatened mainly by various pathogens and changes in its natural environment, mainly by pollution. The aim of the master thesis was to investigate the composition of the bacterial skin microbiota of the olm and its aquatic environment. The 2196 aerobic chemoorganoheterotrophic bacteria were isolated from the skin of 32 specimens of the subspecies Proteus anguinus anguinus and two specimens of the subspecies Proteus anguinus parkelj, as well as 18 water samples from different locations. We found that the composition of microbiota is highly diverse and when comparing the cutaneous and aqueous microbiota they are quite similar. Most isolates belong to the genera Pseudomonas, Aeromonas, Acinetobacter, Flavobacterium and Bacillus, which are ubiquitous, we isolated them from swabs and water, so we conclude that most of the skin microbiota originates from their aquatic environment. Isolates of the genera Brevundimonas, Janthinobacter, Serratia and Stenotrophomonas could be an integral part of the protective microbiota of amphibians according to the literature and thus have a similar function in the salamander. The comparison of skin microbiota between wild and captive specimens showed that the diversity of isolates is higher in captive animals. Comparison of skin microbiota between healthy and diseased specimens showed that bacterial diversity was greater in healthy specimens. We also tested the resistance of 112 of the 2196 isolates to ampicillin, cefotaxime, imipenem, tetracycline, erythromycin, kanamycin, enrofloxacin and chloramphenicol. Most strains tested were resistant to cefotaxime (47.5%) and a smaller proportion to tetracycline (3.3%). Enrofloxacin, used in veterinary medicine, was effective against most of the isolates tested. Using machine learning methods, we found that there was no similarity between samples in terms of location, sampling season, and sample type. A statistically significant difference between sample groups was only found between samples from different sites, namely between Jelševnik and the Tular cave laboratory sites. The master thesis was completed as part of the project 'Emerging microbial threats to endemic troglobiotic amphibian Proteus anguinus'.

V magistrskem delu smo testirali občutljivost stafilokokov, osamljenih iz kozjega mleka, za veterinarsko najpogosteje uporabljane antibiotike, ki so penicilin, cefalotin, oksacilin, cefoksitin, enrofloksacin, fusidinska kislina, eritromicin, klindamicin, gentamicin, nitrofurantoin, tetraciklin in trimetoprim/sulfametoksazol. V analizo smo vključili 32 sevov stafilokokov, osamljenih iz mleka, prirejenega pri ekološkem načinu reje koz in 40 sevov stafilokokov, osamljenih iz mleka, prirejenega pri konvencionalnem načinu reje koz. Med stafilokoki so prevladovali koagulaza negativni stafilokoki (44 sevov), medtem ko je bilo 28 stafilokokov koagulaza pozitivnih in pripadnikov vrste Staphylococcus aureus. Testiranje občutljivosti za antibiotike je potekalo z metodo razredčevanja v mikrotitrski ploščici. Vsi stafilokoki so bili občutljivi za cefalotin, gentamicin in nitrofurantoin, stafilokoki iz mleka ekološke reje pa poleg teh še za fusidinsko kislino. Najbolj občutljiv stafilokok je bil sev vrste S. warneri. 80 % vseh stafilokokov je bilo odpornih proti trimetoprim/sulfametoksazolu, 20 % pa proti tetraciklinu in penicilinu. Sevi vrste S. aureus so bili najbolj odporni proti trimetoprim/sulfametoksazolu, proti penicilinu pa 7 % sevov, medtem ko so bili vsi sevi S. aureus občutljivi za enrofloksacin, fusidinsko kislino, cefalotin, nitrofurantoin in gentamicin. Med koagulaza negativnimi stafilokoki je bilo več kot 80 % sevov odpornih proti trimetoprim/sulfametoksazolu in 20 % proti penicilinu. Način reje koz (ekološki, konvencionalni) ni bistveno vplival na profil odpornosti proti antibiotikom pri stafilokokih, osamljenih iz kozjega mleka. In the master's thesis we tested antibiotic susceptibility of staphylococci, isolated from goat's milk, to the veterinary most used antibiotics (penicillin, cephalothin, cefoxitin, enrofloxacin, fusidic acid, erythromycin, clindamycin, gentamicin, nitrofurantoin, tetracycline and thrimethoprim/sulfamethoxazole). We analysed 32 staphylococci from milk of organic and 40 strains from milk of conventional goat farming. In general, coagulase negative staphylococci (44 strains) dominated over coagulase positive staphylococci (28 strains, S. aureus). Antibiotic susceptibility testing was performed using microdilution method. All staphylococci were sensitive to cephalothin, gentamicin and nitrofurantoin, while staphylococci from organic farming were additionaly sensitive to fusidic acid. Among staphylococci, the most sensitive was one strain of S. warneri species. 80 % of staphylococci were resistant to trimethoprim/sulfamethoxazole, and 20 % to tetracycline and penicillin. Strains of S. aureus were most resistant to trimethoprim/sulfamethoxazole and 7 % to penicillin, while all S. aureus strains were sensitive to enrofloxacin, fusidic acid, cephalothin, nitrofurantoin and gentamicin. More than 80 % coagulase negative staphylococci were resistant to trimethoprim/sulfamethoxazole and 20 % to penicillin. Type of farming (conventional or organic) did not significantly affected resistance of tested staphylococci, isolated from goat's milk.

Posvečena voda igra pomembno vlogo v različnih religijah. Prvič so jo prepoznali kot vir potencialno patogenih mikroorganizmov v Angliji leta 1992. Povezali so jo z okužbami ljudi, ki so jih povzročili Pseudomonas aeruginosa, Acinetobacter baumanii, Escherichia coli in druge enterobakterije. Posvečena voda se v cerkvah nahaja v kropilnikih, ki so običajno ob vhodu v cerkev ter rezervoarjih, ki se nahajajo nekje v cerkvi. Ko verniki vstopijo v cerkev in ko jo zapustijo, pomočijo svoje prste v kropilnik ter s tem vnesejo kožno mikrobioto v posvečeno vodo, nato pa s palcem naredijo križec na čelo, usta in na prsni koš. Iz rezervoarja si lahko verniki posvečeno vodo nalijejo v posodo in jo odnesejo domov. V procesu priprave posvečene vode Katoliška cerkev priporoča dodatek neregulirane količine blagoslovljene soli. Da bi bolje razumeli vpliv dodajanja soli na bakterijsko združbo posvečene vode in potencialno tveganje, ki ga ta voda predstavlja kot vir okužbe s patogenimi mikroorganizmi, smo preučili sestavo aerobne kultivabilne bakterijske združbe v posvečeni vodi iz kropilnikov in rezervoarjev izbranih katoliških cerkva v Ljubljani in okolici. Velik del izolatov so predstavljali predstavniki rodov, ki so tipični za človeško kožno mikrobioto (Acinetobacter, Staphylococcus) in pa vodovodno vodo (Pseudomonas). Izbrane izolate smo testirali na odpornost proti antibiotikom. V preiskovanih cerkvah soli ne dodajajo ali je dodatek premajhen za preprečevanje mikrobne kontaminacije posvečene vode. Potrebno bi bilo uvesti ustrezne higienske ukrepe, kot sta redna menjava vode in temeljito čiščenje kropilnikov. Holy water plays an important role in different religions. It was first recognized as a source of potentially pathogenic microorganisms in England in 1992. It has been associated with human infections caused by Pseudomonas aeruginosa, Acinetobacter baumanii, Escherichia coli and other enterobacteria. Holy water is kept in churches in the fonts, which are usually located at the entrance to the church and in reservoirs located somewhere in the church. When believers enter and leave the church, they wet their fingers in the font introducing a skin microbiota into the holy water and with their thumb they make a cross on their forehead, mouth and chest. Believers can pour holy water into a container and take it home. In order to better understand the impact of salt addition on the bacterial community and the potential risk posed by this water as a source of infection with pathogenic microorganisms, we examined the composition of cultivable aerobic bacterial community in holy water from the fonts and reservoirs of selected Catholic churches in and around Ljubljana. Large part of the isolates were genera typical of human skin microbiota (Acinetobacter, Staphylococcus) and tap water (Pseudomonas). Selected isolates were also tested for antibiotic resistance. In the studied churches, salt in not added or the addition of salt is too small to prevent the microbial contamination of holy water. Appropriate hygiene measures such as regular water exchange and thorough cleaning of fonts should be introduced.

Namen magistrske naloge je bil raziskati odpornost proti antibiotikom eritromicinu, tetraciklinu in kanamicinu med laktobacili, izoliranimi iz sirov. V raziskavi smo uporabili 34 vzorcev sirov, dostopnih na slovenskem tržišču, iz katerih smo osamili 176 izolatov. Z analizo RAPD-PCR smo izolate pregledali in med izolati z istim vzorcem pomnožkov izbrali po enega za nadaljnjo analizo. 106 izolatov z različnimi vzorci pomnožkov smo najprej identificirali na nivoju vrste in ugotovili, da je največ sevov pripadalo vrstama Lactobacillus (L.) plantarum (26) in L. paracasei (22), 18 sevov vrsti Pediococcus pentosaceus in 1 sev vrsti Bifidobacterium animalis subsp. lactis. Fenotipsko odpornost smo ugotavljali z metodo razredčevanja v mikrotitrski ploščici. Ker so pri 70 sevih dobljene vrednosti minimalne inhibitorne koncentracije presegle vrednosti, predpisane s strani EFSA, smo z metodo PCR pri njih preverili še morebitno prisotnost genov z zapisi za odpornost proti antibiotikom: tet(A), tet(B), tet(C), tet(M), tet(O), tet(W), ermA, ermB, ermC, ermT, aph(3')-llla, aac(6')-aph(2'') in ant(6)-la. Vseh 70 odpornih sevov je izkazalo fenotipsko odpornost proti kanamicinu, proti eritromicinu pa je bilo odpornih 5 sevov iz vrst L. parabuchneri, L. capillatus, L. plantarum, P. pentosaceus in L. paracasei, vendar pri nobenem sevu nismo potrdili prisotnosti preiskovanih genov. Odpornost proti tetraciklinu je izkazalo 9 sevov, prisotnost genov tet(A) in tet(M) smo potrdili le pri sevu vrste L. capillatus. Ugotovili smo, da laktobacili iz sirov ne predstavljajo resnejšega tveganja za horizontalen prenos genov, vendar je vseeno potrebno preverjanje odpornosti proti antibiotikom pri mlečnokislinskih bakterijah. The aim of the study was to investigate antibiotic resistance of erythromycin, tetracycline and kanamycin among lactobacilli isolated from cheese. We used 34 samples of cheese available on the Slovenian market, from which we isolated 176 isolates. Isolates were analysed by using RAPD-PCR and among isolates with the same RAPD-PCR band pattern, one was selected for further analysis. 106 isolates with different band pattern were first identified at species level and it was revealed that most strains belonged to the species of Lactobacilus plantarum (26) and L. paracasei (22), 18 strains belonged to Pediococcus pentosaceus species and 1 strain to Bifidobacterium animalis subsp. lactis species. Phenotypic resistance was determined by the microdilution method. 70 strains exerted MIC values above the cut-off values proposed by EFSA. Therefore, PCR was carried out to search for antibiotic resistance genes as follows: tet(A), tet(B), tet(C), tet(M), tet(O) , tet(W), ermA, ermB, ermC, ermT, aph(3')-llla, aac(6')-aph(2'') in ant(6)-la. Among resistant strains, all 70 strains revealed phenotypic resistance to kanamycin, while 5 strains, belonging to L. parabuchneri, L. capillatus, L. plantarum, P. pentosaceus and L. paracasei were resistant to erythromycin, but none of the strains carried the investigated antibiotic resistance genes. Resistance to tetracycline showed 9 strains but the presence of genes tet(A) and tet(M) was found only in the strain of L. capillatus. We can conclude that there is no serious risk of horizontal gene transfer between investigated lactobacilli, but there is still necessity to test LAB for antibiotic resistance.

Številne študije so potrdile ugoden vpliv uporabe probiotikov na gostitelja, tako pri ljudeh kot pri živalih. Uporaba probiotikov po vsem svetu narašča. V zadnjem času pa so znanstveniki začeli opozarjati na to, da tudi ti namerno zaužiti mikroorganizmi lahko predstavljajo potencialni vir genov za odpornost proti antibiotikom, ki se lahko prenašajo med bakterijami različnih vrst. Evropska agencija za varnost hrane EFSA (angl. European Food Safety Authority) v okviru koncepta QPS (angl. Qualified Presumption of Safety) skrbi, da se v živilih in prehranskih dopolnilih uporabljajo le tiste vrste bakterij, ki so varne, poleg tega pa je med kriteriji varnosti omenjena tudi odsotnost prenosljivih genov za odpornost proti antibiotikom. To je mogoče zagotoviti le z ugotavljanjem prisotnosti z odpornostjo proti antibiotikom povezanih genov v celotnih genomih posameznih sevov. Danes je postalo dostopno ugotavljanje nukleotidnega zaporedja celotnih bakterijskih genomov, v preteklosti pa so se posluževali zgolj fenotipskega testiranja. Razen intrinzične oziroma naravne odpornosti so pri mnogih sevih bakterij iz rodov Lactobacillus in Bifidobacterium, iz katerih izhajajo tudi številni probiotiki, odkrili tudi pridobljene gene za odpornost, med katerimi prednjačijo tisti za odpornost proti eritromicinu, tetraciklinom in aminoglikozidom. Prenos nekaterih takih genov, na primer prenos gena za odpornost proti vankomicinu, med enterokoki in laktobacili, so opazili tudi in vitro in in vivo v miših. Prenosa genov za odpornost proti antibiotikov iz mlečnokislinskih bakterij ali bifidobakterij v patogene in vivo še niso dokazali, potencialne nevarnosti pa ne moremo izključiti. Zato je zelo pomembna redna kontrola probiotičnih bakterij v izdelkih na trgu, ki zajema tudi ugotavljanje fenotipske in genotipske odpornosti proti antibiotikom. A number of studies have confirmed the beneficial effect of the use of probiotics on the host, both in humans and in animals. The use of probiotics worldwide is rising. Lately, scientists have begun to warn that even deliberately ingested microorganisms can present a potential source of antibiotic resistance genes that can be transmitted among bacteria of various species. The European Food Safety Authority (EFSA), within the framework of the Qualified Presumption of Safety (QPS) concept, ensures that only those species of bacteria that are safe are used in foods and dietary supplements, and beside this, also the absence of transferable antibiotic resistance genes is mentioned. This can be assured only by determination of the presence of the genes associated with antibiotic resistance in the entire genomes of individual strains. Nowadays, the detection of the nucleotide sequences of total bacterial genomes has become available, and in the past phenotypic testing was only performed. Apart from intrinsic or natural resistance, the acquired resistance genes were found in many strains of bacteria from Lactobacillus and Bifidobacterium genera, among which those for erythromycin, tetracycline and aminoglycoside resistances predominate. The transfer of some antibiotic resistance genes, for example the transfer of the vancomycin resistance gene between enterococci and lactobacilli, have also been observed in vitro and in vivo in mice. The transmission of antibiotic resistance genes from lactic acid bacteria or bifidobacteria to pathogens in vivo has not been demonstrated yet, and potential risks can not be ruled out. For this reason regular control of probiotic bacteria on the market is very important, which also includes the identification of phenotypic and genotypic resistance to antibiotics.

Zaradi neustrezne in nekritične uporabe protimikrobnih zdravil (PZ) pri živalih, ki vstopajo v prehransko verigo človeka, število odpornih bakterijskih sevov, osamljenih iz živali, kakor tudi živil, narašča. Kampilobakterioza, ki jo povzroča Campylobacter jejuni je ena izmed najpogosteje prijavljenih gastrointestinalnih bolezni pri ljudeh, večja prevalenca odpornih bakterijskih sevov pa povečuje tveganje za neučinkovito zdravljenje bolezni. Zato je pomembno na ta problem opozarjati vse deležnike živilske proizvodno-oskrbovalne verige in tudi zdravnike, da se redno izvajajo testi bakterijske odpornosti na najpomembnejša protimikrobna zdravila (PZ). V eksperimentalnem delu smo določali odpornost 8 sevov C. jejuni iz proizvodnega (klavniškega) in 8 sevov iz naravnega (površinskih voda) okolja proti šestim PZ (eritromicinu, ciprofloksacinu, tetraciklinu, gentamicinu, nalidiksni kislini in streptomicinu) na komercialno pripravljenih mikrotitrskih ploščicah SensititreTM EUCAMP2. Tako klavniški (KS) kot vodni (VS) sevi so pokazali odpornost proti istim vrstam PZ – ciprofloksacinu (CIP), tetraciklinu (TET) in nalidiksni kislini (NAL). Delež odpornih sevov proti posameznim PZ je bil višji pri klavniških (CIP 87,5 %, TET 50 %, NAL 50 %) kot vodnih (CIP 25 %, TET 12,5 %, NAL 25 %) sevih. Zasledili smo tudi večkratno odpornost, ki je bila pri klavniških sevih 87,5 % pri vodnih pa 25 %, in sicer med CIP in TET (KS: 37,5 %), ter CIP in NAL (KS: 50 %, VS: 25 %). Prišli smo do sklepa, da je vzrok večje odpornosti KS večja izpostavljenost PZ, vzrok odpornosti VS pa je verjetno prenos odpornih sevov iz klavnice v njeno okolico. Due to inadequate and uncritical use of antimicrobial drugs in animals, entering the human food chain, the number of resistant bacterial strains, isolated from humans as well as from the food, is increasing. Campylobacteriosis caused by Campylobacter jejuni is one of the most frequently reported gastrointestinal diseases in humans. The appearance of resistant bacterial strains increases the risk of ineffective treatment of the disease. Therefore, it is important to alert all stakeholders in the food production and supply chain, as well as doctors, to regularly carry out bacterial resistance tests on the most important antibiotics. In our experimental work, we tested the resistance of 8 strains of C. jejuni from the production (slaughterhouse) and 8 strains from the natural (surface water) environment against six antimicrobial drugs (erythromycin, ciprofloxacin, tetracycline, gentamicin, streptomycin nalidixic acids) on commercially prepared microtiter plates SensititreTM EUCAMP2. Both slaughtering (SS) and water (WS) strains have shown resistance to the same groups of antibiotics - ciprofloxacin (CIP), tetracycline (TET) and nalidixic acid (NAL). The proportion of resistant strains against individual AM was higher in slaughterhouse (CIP 87,5 %, TET 50%, NAL 50%) than water (CIP 25 %, TET 12,5 %, NAL 25 %) strains. Multidrug resistance was also present, which is in the case of slaughterhouse strains 87,5 % and 25 % in water strains, namely between CIP and TET (SS: 37,5 %) and CIP and NAL (SS: 50 %, WS: 25 %). We have come to the conclusion that the cause for the higher resistance levels of SS might be a higher exposure to antibiotics, and the cause of resistant WS is the transfer of resistant strains from the slaughterhouse to its surroundings.

Od sredine dvajsetega stoletja bakterijske infekcije pri ljudeh in živalih zdravimo z antibiotiki. Njihova uporaba je bistveno izboljšala kakovost in dolžino življenja. Zvečana uporaba antibiotikov pa je tudi povzročila razvoj številnih bakterijskih sevov odpornih proti antibiotikom. Vedno več je »super« bakterij, ki so odporne proti večini ali vsem v kliniki uporabljenim antibiotikom. Posledično je Svetovna zdravstvena organizacija razglasila odpornost proti antibiotikom za eno izmed treh največjih groženj človeštvu. Porast odpornosti proti antibiotikom je prinesla težnjo po razvoju učinkovin z novim načinom protibakterijskega delovanja, razvoju novih pristopov, s katerimi bi obvladovali super bakterije. Med alternative antibiotikom uvrščamo antimikrobne peptide, cepiva, probiotike, protitelesa, bakteriofage, molekule, ki stimulirajo imunski sistem gostitelja in še nekatere druge pristope. V teku so klinična testiranja alternativnih protibakterijskih učinkovin, vendar do sedaj večini kandidatov (z izjemo nekaterih probiotikov in protiteles) še ni uspelo preiti na trg. V svojem delu bom predstavila osrčje problema, mehanizme razvoja odpornosti proti antibiotikom pri bakterijah, in trenutno najbolj obetajoče alternative antibiotikom. From the middle of the 20th century antibiotics are used to treat bacterial infection in humans and animals, which led to significant improvement of the quality of our lives. Together with the elevated use of antibiotics, bacteria developed resistance to the most established antibiotics. Thus, the so called »superbacteria« emerged, which carry resistance genes against most or all antibiotics currently used in clinics. As a consequence, the World health organization announced the resistance to antibiotics as one of the biggest threat to the humankind. Thus, novel antimicrobials and ways to treat bacterial infections are urgently needed. Few alternatives are being studied and put into clinical trials for instance: antimicrobial peptides, vaccines, probiotics, antibodies, bacteriophages, molecules stimulating the host immune system and many others. Several alternative novel antibacterials are currently being tested in clinical trials, however so far just few alternatives to antibiotics are commercially available. In the diploma thesis I present the mechanisms that lead to antibiotic resistance development basic among bacteria and the most promising alternatives to antibiotics.

Raziskovali smo antibiotično odpornost mlečnokislinskih bakterij in bifidobakterij izoliranih iz črevesne sluznice. Zanimali sta nas morebiten pojav fenotipske in genotipske odpornosti. Po homogenizaciji črevesnih bioptov smo izolirali mlečnokislinske bakterije, bifidobakterije smo že imeli izolirane in smo jih vzeli iz mikrobiološke zbirke. Izolate smo z metodo RAPD pregledali za vrstno pestrost in za nadaljnjo analizo izbrali tiste z unikatnimi vzorci pomnožkov. Po izolaciji DNA in namnožitvi 16S rDNA, smo identifikacijo izvedli glede na rezultate sekvenciranja pomnožkov. Sledilo je preverjanje fenotipske odpornosti z metodo razredčevanja v mikrotitrski ploščici. Glede na mejne vrednosti, ki jih je postavila EFSA, je previsoke MIC izkazalo 14 sevov od skupno 104, devet proti eritromicinu, štirje proti tetraciklinu in eden proti kanamicinu. Sev B. longum 34-7 v svoji dednini nosi zapis za gen erm(B), sevu B. adolescentis IM677 smo potrdili prisotnost gena tet(O) in sevu B. longum 34-8 pa prisotnost gena tet(W). Pri nobenem izmed testiranih sevov nismo potrdili prisotnosti genov za odpornost proti kanamicinu. Ker odpornostnih genov nismo našli pri vseh sevih, ki so izkazali fenotipsko odpornost je ta najverjetneje posledica mutacij, ali pa genov, ki jih ne poznamo oz. jih nismo vključili v raziskavo. The aim of the study was to search for antibiotic (erythromycin, tetracycline and/or kanamycin) resistance among lactic acid bacteria (LAB) and bifidobacteria isolated from children's intestinal mucosa. We were searching for signs of phenotypic or genotypic antibiotic resistance. First we homogenized intestinal biopsy samples and isolated LAB, bifidoacteria strains had already been isolated. LAB isolates were screened with RAPD to see how related they are, for further analysis we chose the ones with uniqe M13 bands. After DNA isolation and 16S rDNA amplification isolates were sent for sequencing to determine their genus or species. Further on, we tested the LAB and bifidobacterial isolates for phenotypic resistance with microdilution method. At microdilution test 14 out of 104 strains showed resistance against antibiotics, nine strains against erythromycin, four strains against tetracycline and one strain against kanamycin. Those 14 strains with high MIC were later on screened with PCR to see if they contain any of resistance genes. Out of 14 strains that showed phenotypic resistance we discovered that one strain of B. longum (34-7) carries gene for erythromycin resistance erm(B) in his DNA, one strain of B. adolescetnis (IM677) carries gene for tetracycline resistance tet(O), strain B. longum (34-8) carries gene tet(W). None of the strains tested carried genes for resistance against kanamycine.

Namen magistrske naloge je bil raziskati, ali mlečnokislinske bakterije, predvsem laktobacili, in bifidobakterije iz humanega mleka, kolostruma in zrelega mleka, vsebujejo gene za odpornost proti tetraciklinu in eritromicinu, kar predstavlja določeno tveganje za prenos odpornosti na patogene bakterije v dojenčkovem črevesju. Odpornost proti kanamicinu smo priključili iz razloga, ker je bila v predhodnih raziskavah teh sevov nepričakovano pogosta. Tako smo raziskali, ali so v njihovem genomu prisotni pridobljeni geni z zapisi za odpornost proti izbranim antibiotikom. En vzorec kolostruma in 38 vzorcev mleka smo nacepili na gojišči Rogosa in WCA-mup ter osamili 243 izolatov. Rezultati sekvenciranja izolirane DNA so pokazali, da smo s teh gojišč osamili bakterije iz rodov Lactobacillus, Propionibacterium, Streptococcus, Bifidobacterium in Actinomyces. Tem izolatom smo priključili še 56 predhodno izoliranih bakterijskih sevov iz rodov Lactobacillus in Bifidobacterium. Fenotipsko odpornost smo ugotavljali s pomočjo mikrodilucije. Pri sevih, ki so izkazovali fenotipsko odpornost proti določenemu antibiotiku, smo z uporabo PCR metode preverjali prisotnost najbolj znanih genov, povezanih z odpornostjo proti temu antibiotiku. Fenotipsko odpornost proti eritromicinu smo zaznali pri 3 sevih, proti tetraciklinu pri 10 sevih in proti kanamicinu pri 52 sevih, medtem ko prisotnosti genov erm(A), erm(B), erm(C), erm(T), tet(A), tet(B), tet(C), tet(M), tet(O), tet(W), aac(6’)-aph(2’’), ant(6)-Ia in aph(3’)-IIIa nismo potrdili pri nobenem od teh sevov. The aim of this master's thesis was to investigate whether lactic acid bacteria, especially lactobacilli, and bifidobacteria from human milk, colostrum and mature milk, contain tetracycline and erythromycin resistance genes, which presents a certain risk of spread of antibiotic resistance to pathogenic bacteria in the infant's gut. Kanamycin was subsequently added to the study for a reason that in previous studies the resistance of lactobacili to this antibiotic was unexpectedly frequent. Thus, we investigated whether the genes associated with the resistance to selected antibiotics are present in the genome of lactic acid bacteria and bifidobacteria. One sample of colostrum and 38 samples of milk were plated on Rogosa and WCA-mup medium. 243 bacterial strains were isolated. The results of the sequencing of isolated DNA showed that bacteria belonged to the genera Lactobacillus, Propionibacterium, Streptococcus, Bifidobacterium and Actinomyces. We also included 56 pre-isolated bacterial strains from the genera Lactobacillus and Bifidobacterium. Phenotypic resistance was determined by microdilution. In phenotypic resistant strains we investigated the presence of the most well-known genes associated with resistance to a particular antibiotic with PCR. Phenotypic resistance to erythromycin was detected in 3 strains, tetracycline in 10 strains, and kanamycin in 52 strains, while the presence of investigated genes erm(A), erm(B), erm(C), erm(T), tet(A), tet(B), tet(C), tet(M), tet(O), tet(W), aac(6')-aph(2''), ant(6)-Ia and aph(3')-IIIa has not been confirmed in any of the strains.