Osnovna mikrobiologija

Bridging the gap between the theoretical and the practical application of what students are learning is the first step towards understanding the central roles of microorganisms in nature and in our daily lives. Our goal for these instructions on laboratory exercises is to provide a bridge that helps our foreign students, who choose the Microbiology course (Erasmus exchange program), to integrate lectures with laboratory experience.Znanje študentov sloni na gradnji mostu med teoretično osnovo in praktičnimi vajami ter ponazarja prvi korak na poti do razumevanja poglavitne vloge mikroorganizmov v naravi in našem vsakdanjiku. Namen navodil za laboratorijske vaje pri predmetu Mikrobiologija v angleškem jeziku je omogočiti gradnjo tega mostu ter tako naše tuje študente, ki si v okviru programa Erasmus izberejo učni predmet Mikrobiologija, naučiti povezovanja teoretičnih osnov s praktičnimi prvinami

THE EFFECTS OF LACTOBACILLUS GENERA PROBIOTIC BACTERIA ON SGLT1 AND GLUT2 GENE EXPRESSION IN INTESTINAL EPITHELIAL FUNCTIONAL CELL MODELS

VPLIV PROBIOTIČNIH BAKTERIJ IZ RODU LACTOBACILLUS NA IZRAŽANJE GENOV SGLT1 IN GLUT2 V FUNKCIONALNIH CELIČNIH MODELIH ČREVESNEGA EPITELIJA SPLOŠNO: Ohranjanje zdravja je odvisno od sožitja med gostiteljevim organizmom in mikrobioto. Probiotične bakterije (probiotiki) so živi mikroorganizmi, ki ob zaužitju v zadostnih količinah vplivajo blagodejno na zdravje gostitelja. Že zgodnje raziskave so temeljile na proučevanju probiotičnih prehranskih lastnosti, ki ob zaužitju prispevajo tudi k strukturni spremembi črevesne mikrobiote in s tem med drugim vplivajo na stanje telesne presnove in njeno homeostazo. Ohranjanje homeostaze v telesu je tako ključnega pomena in se odraža na vseh nivojih, tudi na nivoju glukoze, ki predstavlja nujno gorivo za normalno delovanje celice. Kljub številnim kliničnim študijam, ki prikazujejo vplive različnih probiotikov na nivo glukoze in njeno homeostazo, je poznavanje točnega mehanizma njihovega delovanja še vedno skromno. CILJI: Cilji doktorske disertacije so bili ugotoviti, ali kateri izmed izbranih probiotičnih sevov, to so Lactobacillus plantarum PCS 20 (PCS 20), Lactobacillus plantarum PCS 26 (PCS 26), Lactobacillus rhamnosus GG ATCC 53103 (LGG) in Lactobacillus acidophilus ATCC 4356 (L. acidophilus), vpliva na izražanje genov SGLT1 in/ali GLUT2, kot tudi, ali kateri izmed izbranih probiotičnih sevov iz rodu Lactobacillus vpliva na spremenjeno količino prehoda glukoze skozi črevesne epitelijske celice (transepitelijski transport) v prašičjem in humanem funkcionalnem celičnem modelu. METODE: Črevesna celična linija nekarcinogenega izvora (CLAB) in humana črevesna celična linija karcinogenega izvora Caco2 (ATCC HTB-37) (Caco2) sta bili izpostavljeni probiotičnim sevom PCS 20, PCS 26, LGG in L. acidophilus. Učinek probiotičnih sevov je bil določen z merjenjem transkripcije in translacije genov SGLT1 in GLUT2 v celicah. Morebitna raznolikost med vplivi probiotičnih sevov je bila nadalje ocenjena z merjenjem transepitelijskega transporta glukoze po gojenju celic na mikroporoznih membranah. REZULTATI: Spremembe v transkripciji in izražanju genov SGLT1 in GLUT2 so bile različne glede na probiotični sev in/ali celično linijo. V celični liniji CLAB je dodatek LGG statistično značilno najbolj povečal transkripcijo gena SGLT1, medtem ko je dodatek PCS 26 statistično značilno najbolj zmanjšal transkripcijo gena GLUT2, kar se je odražalo tudi na proteinski ravni. V celični liniji Caco2 je vpliv vseh testiranih sevov pokazal tendenco k zmanjšanju transkripcije in izražanja gena GLUT2, pri čemer je sev L. acidophilus najučinkoviteje znižal raven proteina GLUT2. Statistično značilne spremembe na molekularni in proteinski ravni GLUT2 pri kombinacijah PCS 26/CLAB in L. acidophilus/Caco2 je spremljalo tudi značilno znižanje hitrosti transepitelijskega transporta glukoze. ZAKLJUČEK: Naša študija in vitro je izpostavila nekatere probiotične seve rodu Lactobacillus kot potencialne kandidate pri uravnavanju izražanja glukoznih prenašalcev in posledično pri uravnavanju transepitelijskega transporta glukoze v črevesnih epitelijskih celicah. Specifični probiotični sevi Lactobacillus lahko zmanjšajo transkripcijo in izražanje prenašalcev glukoze v črevesnih epitelijskih celicah ter s tem hitrost absorpcije glukoze iz hrane. Zmanjšana celokupna absorpcija glukoze iz črevesja kot posledica uporabe probiotičnega prehranskega dodatka bi lahko koristila v podpornem zdravljenju vseh patoloških stanj, povezanih s homeostazo glukoze. Nova dognanja bi tako prispevala k izboljšanju vodenja omenjenih patoloških stanj in tudi h kakovosti življenja pacientov.THEORETICAL BACKGROUND: The maintenance of a healthy status depends on the co - existence between the host organism and the microbiota. Probiotic bacteria (probiotics) are living microorganisms which, when consumed in sufficient quantities, have beneficial effects on the health of the host. Early studies have already focused on probiotic nutritional properties, which may also contribute to the structural changes in the intestinal microbiota, thereby affecting host metabolism and its homeostasis. Homeostasis maintenance in the body is therefore crucial and is reflected on all levels, including those of glucose, a simple sugar molecule which is essential fuel for a normal cell functioning. Despite numerous clinical studies that have shown effects of several probiotics on glucose and its homeostasis, knowledge of the exact function of their mechanism is still scarce. AIM: The aim of the doctoral dissertation was to determine if any of the selected probiotic strains Lactobacillus plantarum PCS 20 (PCS 20), Lactobacillus plantarum PCS 26 (PCS 26), Lactobacillus rhamnosus GG ATCC 53103 (LGG) and Lactobacillus acidophilus ATCC 4356 (L. acidophilus) has an effect on SGLT1 and / or GLUT2 gene expression and transcription. Moreover, our aim was to investigate the effects of selected Lactobacillus probiotic strains on glucose transepithelial transport in porcine and human polarized intestinal epithelial cell models. METHODS: Probiotic strains PCS 20, PCS 26, LGG and L. acidophilus were co-cultivated with non-carcinogenic porcine enterocytes (CLAB) and human epithelial colorectal adenocarcinoma cells ATCC HTB-37 (Caco2). The effect was determined by measuring the SGLT1 and GLUT2 gene transcription and protein expression levels upon exposure of cells to probiotics. Changes in glucose transepithelial transport were quantified in polarized cells growing on a microporous membrane support. RESULTS: Changes in transcription and expression of SGLT1 and GLUT2 were strain and cell line specific. In the CLAB cell line, LGG was statistically significantly the most potent SGLT1 up-regulator and PCS 26 the most potent down-regulator of GLUT2 transcription, which was also reflected on the protein level. In Caco2 cell line, all tested strains tended to downregulate the GLUT2 gene expression, while L. acidophilus most effectively reduced the GLUT2 protein level. The statistically significant effect of PCS 26 and L. acidophilus on GLUT2 molecular and protein level in CLAB and Caco2 cell lines, respectively, was also followed by a statistically significant decreased rate of glucose transepithelial transport. CONCLUSION: Our in vitro study has disclosed some probiotic strains of Lactobacillus genus as potential candidates in regulating the expression of glucose transporters and, consequently, controlling the transepithelial glucose transport in intestinal cells. Specific Lactobacillus probiotic strains may downregulate transcription and expression of glucose transporters in intestinal epithelial cells, thereby decreasing the rate of glucose absorption from food. Decreased glucose absorption could be beneficial as a support treatment of pathologies related to glucose homeostasis. New scientific findings would thus contribute to improve the management of these pathological conditions, as well as improve the quality of patients\u27 lives

Nanotechnology in food safety and quality assessment: potentiality of nanoparticles in diagnosis of foodborne pathogens

A rapid microbial detection in different biological and environmental material is a key of preventing several foodborne diseases. By implementing nanotechnology into food safety sector, a great step towards successful, reliable and sensible detection methods of foodborne pathogens has been achieved. Therefore, the aim of this review was to illustrate some of the principal functions of nanotechnology-based techniques, used for microbial detection in the last few years. Regarding consumer’s health, the review also discusses the question of safety, concerning human exposure to nanomaterials (NMs). Due to their different composition-unique properties, such as greater penetrability, reactivity and high surface to volume ratio, NMs have been coupled to several biomolecules and integrated in special system devices, resulting in improvement of sensitivity in transmitting biological signal informations in a shorter time. Among all the NMs, gold, magnetic and fluorescent nanoparticles (NPs) have been widely used, also in microbial diagnosis. Despite the success of linking nanotechnology to detection of foodborne pathogens, the exposure to various NMs could also be a matter of potential risk to human health, although conclusions still need to be definitely proven