In-situ inhibicija Staphylococcus aureus bakterijama mliječne kiseline iz dva tradicionalna slovenska sira proizvedena od sirovog mlijeka

Bacteriocin(s) producing lactic acid bacteria naturally present in traditional cheeses represent an inexhaustive pool of microbes with safeguarding potential. Some bacteriocins produced by cheese lactic acid bacteria were already described as successful anti-staphylococcal agents. The presence of genes for bacteriocins with potential anti-staphylococcal activity was also demonstrated in two Slovenian traditional raw milk cheeses, “Tolminc” and “Kraški ovčji sir”. Same bacteriocin genes were also detected in viable lactic acid bacteria consortia’s isolated from “bacteriocin positive cheeses” on Rogosa, M17 and CATC agar media. The aim of the research was to elucidate whether or not this particular cheese consortia, in which bacteriocin genes were detected, actually exhibit anti-staphylococcal activity in milk and/or cheese. For this purpose different cheese consortia were selected in relation to versatility of detected bacteriocin genes and used to perform challenge tests against Staphylococcus aureus in milk and cheese. In milk following the time/temperature regime of traditional cheese production all cheese consortia effectively inhibited growth of Staphylococcus aureus in the range of app. 2 to 3 log. In cheese the inhibition of staphylococci was less pronounced but still evident since inhibition of app. 1.5 log was detected. Sole inhibition by lactic acid production was ruled out while linking inhibition directly to bacteriocin production would take some additional work.Bakterije mliječne kiseline (BMK), prirodno prisutne u tradicionalnim sirevima, predstavljaju neograničen izvor mikroba sa zaštitnim svojstvima. Za neke bakteriocine, koje proizvode BMK iz sira, antistafilokokna svojstva već su poznata. Prisutnost gena za bakteriocine s antistafilokoknim svojstvima potvrđena je u dva tradicionalna slovenska sira iz sirovog mlijeka; to su sir “Tolminc” i “Kraški ovčji sir”. Prisutnost istih bakteriocina potvrđena je i kod bakterija mliječne kiseline, koji su izolirali iz tih sireva na agar podlogama Rogosa, M17 i CATC. Cilj je bio provjeriti da li izolirani sojevi bakterija mliječne kiseline, koji nose gene za bakteriocine, zaista pokazuju antistafilokokna svojstva u mlijeku i/ili siru. Na temelju prisutnih gena za bakteriocine ispitana je antimikrobna aktivnost združene kulture bakterija mliječne kiseline prema bakteriji Staphylococcus aureus u mlijeku i siru. U mlijeku kojeg smo podvrgnuli vremensko-temperaturnom režimu proizvodnje tradicionalnih sireva, združena kultura bakterija mliječne kiseline inhibirala je rast bakterije Staphylococcus aureus u rasponu od 2 do 3 logaritamske jedinice. U siru je inhibicija stafilokoka bila nešto manje izražena, ali još uvijek očita sa 1,5 logaritamskih jedinica slabijim rastom S. aureus nego u kontrolnom siru. Učinak inhibicije izazvane mliječnom kiselinom bio je isključen, ali bi bilo potrebno izvesti još dodatna ispitivanja, da se utvrdi izravno inhibicijsko djelovanje bakteriocina na stafilokoke

Kompetitivna prednost bakteriocinogenih sojeva konzorcija bakterija mliječne kiseline izoliranih iz sira proizvedenog od sirovog mlijeka

The presence of gene determinants for different bacteriocins has been already demonstrated in traditional Slovenian types of raw milk cheeses ‘Tolminc’ and ‘Kraški’. These genes were present also in the cultivable microbiota. In this research the aim was to establish how the presence of gene determinants for bacteriocins in microbial consortia is reflected in its antimicrobial activity. In addition, one of the goals was to determine whether the strains that carry gene determinants for bacteriocins have any competitive growth advantage in microbial population. Microbial consortium of ‘Tolminc’ cheese was propagated in milk and examined at the end of propagation its antimicrobial activity and the presence of gene determinants for bacteriocins. Comparison of the results obtained before and after propagation leaded to the conclusion that most of the strains possessing gene determinants for bacteriocins were unable to persist during propagation. The strains which did persist during propagation carried gene determinants for enterocins P, L50B and cytolysin. Antimicrobial activity of consortium before and after propagation was not substantially different and cannot be attributed to any of detected bacteriocins.Prisutnost genskih determinanti za stvaranje različitih vrsta bakteriocina već je ranijim istraživanjima utvrđena u slovenskim ovčjim sirevima “Tolminac” i “Kraški sir” koji se proizvode na tradicionalan način. Isti geni za izoliranu mikrobnu populaciju (konzorcij) također su potvrđeni i u kultiviranim uvjetima. U ovom radu pokušalo se utvrditi kako se prisutnost tih genskih determinanti u mikrobnom konzorciju odražava na njegovu antimikrobnu aktivnost. Osim toga, utvrđivao se i utjecaj bakterijskih sojeva koji nose gene za stvaranje bakteriocina na kompetitivnost rasta u mješovitoj populaciji. Mikrobni konzorcij izoliran iz sira “Tolminac” propagiran je u mlijeku 10 dana, nakon čega je utvrđena antimikrobna aktivnost kulture i prisutnost genskih determinanti za stvaranje bakteriocina. Usporedbom rezultata za sposobnost stvaranja bakteriocina prije i nakon precjepljivanja u mlijeku, utvrđen je gubitak te sposobnosti za većinu izoliranih sojeva. Sojevi koji su izdržali ponovljena precjepljivanja u mlijeku nosili su genske determinante za bakteriocine: enterocin P, enterocin L50B i citolizin. Antimikrobna aktivnost konzorcija prije i nakon precjepljivanja nije se značajno razlikovala te se ne može pripisati niti jednom od u ovom pokusu potvrđenih bakteriocina

In-situ inhibicija Staphylococcus aureus bakterijama mliječne kiseline iz dva tradicionalna slovenska sira proizvedena od sirovog mlijeka

Bacteriocin(s) producing lactic acid bacteria naturally present in traditional cheeses represent an inexhaustive pool of microbes with safeguarding potential. Some bacteriocins produced by cheese lactic acid bacteria were already described as successful anti-staphylococcal agents. The presence of genes for bacteriocins with potential anti-staphylococcal activity was also demonstrated in two Slovenian traditional raw milk cheeses, “Tolminc” and “Kraški ovčji sir”. Same bacteriocin genes were also detected in viable lactic acid bacteria consortia’s isolated from “bacteriocin positive cheeses” on Rogosa, M17 and CATC agar media. The aim of the research was to elucidate whether or not this particular cheese consortia, in which bacteriocin genes were detected, actually exhibit anti-staphylococcal activity in milk and/or cheese. For this purpose different cheese consortia were selected in relation to versatility of detected bacteriocin genes and used to perform challenge tests against Staphylococcus aureus in milk and cheese. In milk following the time/temperature regime of traditional cheese production all cheese consortia effectively inhibited growth of Staphylococcus aureus in the range of app. 2 to 3 log. In cheese the inhibition of staphylococci was less pronounced but still evident since inhibition of app. 1.5 log was detected. Sole inhibition by lactic acid production was ruled out while linking inhibition directly to bacteriocin production would take some additional work.Bakterije mliječne kiseline (BMK), prirodno prisutne u tradicionalnim sirevima, predstavljaju neograničen izvor mikroba sa zaštitnim svojstvima. Za neke bakteriocine, koje proizvode BMK iz sira, antistafilokokna svojstva već su poznata. Prisutnost gena za bakteriocine s antistafilokoknim svojstvima potvrđena je u dva tradicionalna slovenska sira iz sirovog mlijeka; to su sir “Tolminc” i “Kraški ovčji sir”. Prisutnost istih bakteriocina potvrđena je i kod bakterija mliječne kiseline, koji su izolirali iz tih sireva na agar podlogama Rogosa, M17 i CATC. Cilj je bio provjeriti da li izolirani sojevi bakterija mliječne kiseline, koji nose gene za bakteriocine, zaista pokazuju antistafilokokna svojstva u mlijeku i/ili siru. Na temelju prisutnih gena za bakteriocine ispitana je antimikrobna aktivnost združene kulture bakterija mliječne kiseline prema bakteriji Staphylococcus aureus u mlijeku i siru. U mlijeku kojeg smo podvrgnuli vremensko-temperaturnom režimu proizvodnje tradicionalnih sireva, združena kultura bakterija mliječne kiseline inhibirala je rast bakterije Staphylococcus aureus u rasponu od 2 do 3 logaritamske jedinice. U siru je inhibicija stafilokoka bila nešto manje izražena, ali još uvijek očita sa 1,5 logaritamskih jedinica slabijim rastom S. aureus nego u kontrolnom siru. Učinak inhibicije izazvane mliječnom kiselinom bio je isključen, ali bi bilo potrebno izvesti još dodatna ispitivanja, da se utvrdi izravno inhibicijsko djelovanje bakteriocina na stafilokoke

Bioraznolikost mikrobnih konzorcija izoliranih iz tradicionalnog svježeg ovčjeg sira Karakačanski skakutanac

The aim of this study was to assess the structure of indigenous microbial community associated with traditional fresh sheep cheese Karakačanski skakutanac and to preserve autochthonous microbial consortia. Eleven cheeses were sampled during production season (April-September) and subjected to microbiological analysis. Bacterial DNA was isolated by Maxwell®16 DNA system from 99 microbial consortia harvested from three culture media (M17, Rogosa, CATC) on the 1st, 2nd and 3rd day of the cheese shelf life. The extracted bulk DNA (n = 99) was used as a template for PCR-ARDRA and PCR-DGGE analysis. There were no dramatic shifts in the bacterial number and structure of the microbial consortia harvested on the 1st, 2nd or 3rd day of the cheese shelf life neither during period of sampling. Lactococcus lactis subsp. lactis reached the number of 107-108 CFU g-1, while Leuconostoc pseudomesenteroides, Enterococcus faecalis, and Lactobacillus versmoldensis were identified only at lower dilutions (10-2 - 10-3). This first polyphasic microbiological-molecular study of the Karakačanski skakutanac indicated the main LAB representatives associated with the cheese. Obtained autochthonous microbial consortia present a valuable pool of strains for further genetic and functional characterizations.Cilj ovog rada bio je analizirati strukturu mikrobnih konzorcija tradicionalnog svježeg ovčjeg sira Karakačanski skakutanac. Jedanaest sireva sakupljeno je tijekom sezone proizvodnje od travnja do rujna. Mikrobni konzorciji sakupljeni su sa 3 hranjive podloge (M17, Rogosa, CATC) od 11 sireva nakon prvog, drugog, i trećeg dana proizvodnje. Mikrobna DNA je izolirana iz 99 konzorcija, te korištena u PCR-ARDRA i PCR-DGGE analizi. Nije bilo promjene u strukturi mikrobnih konzorcija sakupljenih prvog, drugog i trećeg dana nakon proizvodnje, niti tijekom sezone. Utvrđena je dominantnost populacije Lactococcus lactis subsp. lactis (107-108 CFU g-1), dok su ostale vrste bakterija mliječne kiseline, Leuconostoc pseudomesenteroides, Enterococcus faecalis i Lactobacillus versmoldensis, identificirane samo na nižim razrjeđenjima (10-2-10-3). Ova prva mikrobiološko- molekularna analiza tradicionalnog sira Karakačanski skakutanac omogućila je uvid u strukturu njegove specifične mikrobne populacije. Sakupljeni mikrobni konzorciji predstavljaju značajan izvor sojeva za daljnju genetsku i funkcionalnu karakterizaciju

Karakterizacija bakteriocinogenih sojeva bakterija mliječne kiseline iz tradicionalnog slovenskog sira ‘Tolminc’

The purpose of this research was to examine traditional Slovenian ‘Tolminc’ cheese for the presence of lactic acid bacteria that produce several bacteriocins. The presence of gene determinants for different bacteriocins in this type of cheese and in the cultivable population of ‘Tolminc’ microbiota, have already been demonstrated, as well as its antimicrobial activity. Due to the difficulties in connecting the presence of gene determinants for bacteriocins with the observed antimicrobial activity it was decided to examine in this study the same features on the level of individual bacteriocinogenic strains. Like in previous results, enterococci and their bacteriocins prevailed in cheese microbial consortia. None of isolated strains inhibited growth of Staphylococcus aureus, while the other indicator strains were inhibited in a strain specific manner. Most of isolated strains carried gene determinants for cytolysin. On the basis of gene determinants for bacteriocins, antimicrobial activity, phenotyping by PhP (PhenePlateTM) system and PCR identification, some similarities found were among Enterococcus isolates.Cilj ovog rada bio je provjeriti prisutnost sojeva bakterija mliječne kiseline koji proizvode različite bakteriocine u tradicionalnom slovenskom siru ‘Tolminc’. Prisutnost genskih determinanti za pojedine bakteriocine u ovoj vrsti sira i izoliranoj populaciji mikrobiote sira ‘Tolminc’ već je bila prikazana, a također i njihova antimikrobna aktivnost. Zbog poteškoća pri povezivanju detektiranih genskih determinanti za bakteriocine i antimikrobne aktivnosti, odlučeno je u ovom radu analizirati ista svojstva i kod pojedinačnih bakteriocinogenih sojeva. Slično prethodnim istraživanjima, enterokoki i njihovi bakteriocini najbolje su bili zastupljeni. Nijedan od izoliranih sojeva nije inhibirao bakteriju Staphylococcus aureus, dok su ostale indikatorske mikroorganizme inhibirali različito. Većina sojeva nosila je genske determinante za bakteriocin citolizin. Na temelju genskih determinanti za bakteriocine, antimikrobne aktivnosti, fenotipizacije s PhP (PhenePlateTM) sistemom i identifikacije roda i vrste sojeva, mogu se naći neke sličnosti između Enterococcus sojeva

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival