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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.

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.

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.

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.