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Influence of essential oils on selected enterobacteria strains biofilms formed on the vegetable leaf surfaces

dc.contributor.advisorKocić-Tanackov, Sunčica
dc.contributor.otherTepić, Horecki, Aleksandra
dc.contributor.otherKocić-Tanackov, Sunčica
dc.contributor.otherČabarkapa, Ivana
dc.creatorVarga, Ana
dc.date.accessioned2022-09-06T08:07:10Z
dc.date.available2022-09-06T08:07:10Z
dc.date.issued2022-08-02
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/Disertacija165123016681955.pdf?controlNumber=(BISIS)120429&fileName=165123016681955.pdf&id=19845&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/record.jsf?recordId=120429&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/IzvestajKomisije165123017704566.pdf?controlNumber=(BISIS)120429&fileName=165123017704566.pdf&id=19846&source=NaRDuS&language=srsr
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/20579
dc.description.abstractPojava i učestalost epidemija izazvanih konzumiranjem hrane beleži stalni rast. Tome su doprinele promene u ishrani i težnja savremenih potrošača za konzumiranjem minimalno obrađene hrane koja u najvećoj meri obuhvata sveže voće i lisnato povrće. Kao jedan od čestih izvora kontaminacije navodi se zelena salata koja se pre konzumiranja podvrgava samo pranju. S obzirom na način gajenja, putevi kontaminacije ovog povrća patogenim bakterijama kao što su Escherichia coli - E. coli i Salmonella Typhimurium - S. Typhimurium su mnogobrojni. Dodatni problem predstavlja sposobnost ovih patogena da formiraju biofilmove u kojima su zaštićeni od nepovoljnih uslova spoljašnje sredine, a koje je nemoguće potpuno ukloniti pranjem vodom. Činjenica da mikroorganizmi u biofilm formaciji pokazuju znatno veću otpornost prema dejstvu antibiotika i raznih dezinficijenasa u odnosu na planktonski oblik, doprinela je izboru etarskih ulja (EU) kao prirodnih jedinjenja velikog i do sada nepotpuno iztraženog antimikrobnog i antibiofilm potencijala. Kulinarske i začinske biljke, bogate EU se od davnina koriste u kulinarstvu i farmaciji, te svako novo saznanje vezano za njihov antimikrobni potencijal može doprineti iznalaženju novog biološkog rešenja za eliminisanje patogena i njihovih biofilmova u hrani, posebno onoj koja se konzumira sveža, kao što je lisnato povrće. Imajući u vidu navedene činjenice, cilj rada je bio da se ispita antibiofilm potencijal EU prema biofilmovima E. coli i S. Typhimurium ATCC 14028 formiranim u in vitro uslovima i na površini lista zelene salate. S tim u vezi prvo je ispitivana sposobnost izolata E. coli različitog porekla da formiraju biofilmove in vitro uz prethodno određivanje morfotipa kolonija na Kongo crvenom agaru (KCA) na dve različite temperature, 25 oC i 37 oC. Zatim je izvršeno ispitivanje sposobnosti preživljavanja odabranog izolata E. coli (koji je prethodnim testiranjem svrstan u jake biofilm producere) i laboratorijskog tipskog soja S. Typhimurium u zemljištu pri konstantnim i promenljivim uslovima vlage i temperature tokom 120 dana inkubiranja. Ispitana je sposobnost adherencije i formiranje biofilma ova dva mikroorganizma na površini lista zelene salate na temperaturi od 25 oC i 37 oC. Na kraju je određen uticaj odabranih EU na adherenciju i formirane biofilmove E. coli i S. Typhimurium u in vitro uslovima i na listu zelene salate. Vizuelizacija biofilma formiranog na površini lista zelene salate izvedena je primenom skening elektronske mikroskopije (SEM). Određivanjem morfotipa kolonija na KCA na temperaturi inkubiranja od 25 oC utvrđeno je da su testirani izolati E. coli formirali kolonije morfotipa RDAR (eng. red, dry and rough), BDAR (eng. brown, dry and rough), RAS (eng. red and smooth) i SAW (eng. smooth and white), dok su na temperaturi inkubiranja od 37 oC formirali kolonije morfotipa, RDAR, BDAR i SAW. Laboratorijski tipski soj S. Typhimurium formirao je kolonije morfotipa RDAR na temperaturi inkubiranja od 25 oC, odnosno SAW na temperaturi inkubiranja od 37 oC. Ispitivanjem sposobnosti formiranja biofilma in vitro uz kvantifikaciju ukupne mase biofilma primenom kristal violet testa na obe temperature uočen je jak uticaj temperature u smislu većeg broja izolata koji su svrstani u jake biofilm producere na nižoj temperaturi inkubiranja. Uporednom analizom rezultata dobijenih za određivanje morfotipa i sposobnosti formiranja biofilma na obe temperature uočena je pozitivna korelacija između broja izolata E. coli koji su formirali RDAR morfotip i broja izolata koji su svrstani u jake biofilm producere. Ispitivanjem sposobnosti preživljavanja odabranog izolata E. coli i S. Typhimurium u zemljištu, nakon 120 dana inkubiranja, ustanovljeno je prisustvo vijabilnih ćelija oba mikroorganizma pri konstantnim i pri promenljivim uslovima vlažnosti i temperature. Stopa preživljavanja S. Typhimurium bila je veća i pri konstantnim i pri promenljivim uslovima temperature i vlažnosti i iznosila je 0,60% odnosno 0,27%. Stepen adherencije na površinu lista zelene salate tokom 3 h na temperaturi od 25 oC iznosio je 6,75 log cfu/cm2 lista za S. Typhimurium, odnosno 6,64 log cfu/cm2 lista za E. coli. Viša temperatura inkubiranja (37 oC) različito je uticala na stepen adherencije ispitivanih mikroorganizama na list zelene salate, generalno ga redukujući na nižu vrednost, pri čemu je kod S. Typhimurium redukcija iznosila 50%, a kod E. coli 10%. Primenom SEM, na mikrografijama manjeg uvećanja uočeno je formiranje ćelijskih agregata E. coli i S. Typhimurium na površini lista zelene salate, koji su sastavni deo biofilma dok je na mikrografijama većeg uvećanja uočena interkonekcija između ćelija i formirani biofilm. Drugi deo ispitivanja sproveden je u cilju određivanja uticaja EU začinskih biljaka bosiljka, mirođije, nane i vreska na inicijalnu ćelijsku adheziju i prethodno formirani biofilm. Pre ispitivanja antimikrobnog i antibiofilm potencijala, analiziran je hemijski sastav EU, koji je pokazao da sva četiri EU sadrže visok % monoterpena. Najzastupljenije identifikovano jedinjenje EU bosiljka bilo je linalool (69,2%), EU mirođije karvon (66,6%) i limonen (25,3%), EU nane mentol (30,8%) i menton (28,8%) i EU vreska p-cimen (33,8%) i karvakrol (32,9%). Najbolji antimikrobni potencijal prema bujonskim kulturama E. coli i S. Typhimurium pokazalo je EU mirođije, sledi EU bosiljka i vreska, dok je EU nane pokazalo najmanji antimikrobni potencijal, koji je vrednovan dobijenim vrednostima za minimalnu inhibitornu koncentraciju (eng. minimal inhibitory concentration, MIC) i minimalnu baktericidnu koncentraciju (eng. minimal bactericidal concentration, MBC). Etarska ulja pokazala su dozno-zavistan uticaj na inicijalnu ćelijsku adheziju i posledično na formiranje biofilma u in vitro uslovima, prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane (odnosi se na koncentracije EU od 2 MIC). Ispitivanjem uticaja EU na ukupnu masu biofilma in vitro dokazano je da EU u primenjenim koncentracijama od 0,5 MIC, MIC i 2 MIC ispoljavaju uticaj na redukciju ukupne mase formiranog biofilma na dozno-zavistan način u funkciji vremena, tako da je najveći % eradikacije biofilma E. coli i S. Typhimurium postignut nakon 60 minuta ekspozicije primenom EU koncentracije 2 MIC prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane. Uticaj EU na prethodno formirani biofilm E. coli i S. Typhimurium na površini lista zelene salate takođe je zavisio od koncentracije. Posmatrajući njihov uticaj sa aspekta broja vijabilnih ćelija biofilma, najefikasnije je bilo EU mirođije koncentracije 2 MIC, a najmanje efikasno EU nane koncentracije MIC. Najveći % redukcije ukupne mase biofilma i E. coli i S. Typhimurium postignut je primenom EU koncentracije 2 MIC prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane. Poređenjem rezultata redukcije ukupne mase biofilma E. coli i S. Typhimurium, formiranog u in vitro uslovima i na površini lista zelene salate, veći procenat redukcije zabeležen je kod biofilmova formiranih u in vitro uslovima, što se može objasniti hrapavom strukturom površine lista zelene salate koja je ograničila kontakt EU i biofilma ispitivanih mikroorganizama. Na osnovu izvedenih ispitivanja, u skladu sa ciljevima ove doktorske disertacije i postignutih rezultata, može se zaključiti da je EU mirođije pokazalo najbolji antimikrobni i antibiofilm potencijal u in vitro uslovima i na kuponima lista zelene salate prema oba ispitivana mikroorganizma. Sledi EU bosiljka, vreska i nane. Dobijeni rezultati upućuju da EU ovih biljaka na osnovu dokazanog antimikrobnog i antibiofilm potencijala koji poseduju mogu naći primenu kao sastojci salatnih preliva, mogu da se inkorporiraju u materijale za pakovanje ili u atmosferu pakovanja doprinoseći time zdravstvenoj bezbednosti zelene salate.sr
dc.description.abstractThe frequency of foodborne outbreaks is constantly increasing. This trend has been contributed to by changes in diet and the desire of modern consumers to consume minimally processed foods, which primarily include fresh fruits and leafy vegetables. Lettuce is one of the frequent sources of contamination, consumed after washing only. Due to the growing techniques, the contamination pathways of this vegetable with pathogenic bacteria, such as Escherichia coli - E. coli and Salmonella Typhimurium - S. Typhimurium, are numerous. An additional problem is the ability of these pathogens to form a biofilm. In biofilms, pathogens are protected from adverse environmental conditions and cannot be entirely removed by water washing. The fact that microorganisms in biofilm formation show significantly higher resistance to antibiotics and various disinfectants compared to planktonic form, contributed to the choice of essential oils (EOs). EOs as natural compounds of great and until now incompletely investigated antimicrobial and antibiofilm potential. Culinary and spice plants rich in EOs have long been used in cooking and pharmacy, and any new knowledge about their antimicrobial potential can contribute to finding a new biological solution to eliminate pathogens and their biofilms in food, especially those consumed fresh as leafy vegetables. Having in mind this fact, this study aimed to examine the effect EOs on the removal of biofilms of E. coli and S. Typhimurium ATCC 14028 formed in vitro and on lettuce leaves. In this regard, the ability of E. coli isolates of different origins to form biofilms in vitro was examined. Previous, the morphotype of the colonies on the Congo Red Agar (CRA) at two different temperatures, 25 °C and 37 °C, was determined, as well as the viability of the selected E. coli isolates, previously classified as a strong biofilm producer. Furthermore, the viability of the laboratory-type strain S. Typhimurium in soil under constant and variable humidity and temperature during 120 days of incubation was investigated, along with these two microorganisms’ ability to adhere and form a biofilm on the lettuce leaves surface at temperatures of 25 °C and 37 °C. Finally, the influence of selected EO on adherence and formed biofilms of E. coli and S. Typhimurium in vitro and on lettuce leaf was examined. Obtained biofilm visualization was performed using scanning electron microscopy (SEM). By determining the colony morphotypes on CRA at an incubation temperature of 25 °C, the tested isolates of E. coli formed colony morphotypes RDAR (red, dry and rough), BDAR (brown, dry and rough), RAS (red and smooth) and SAW (smooth and white), while at an incubation temperature of 37 °C they formed colony morphotypes, RDAR, BDAR and SAW. The laboratory-type strain S. Typhimurium formed the RDAR and SAW morphotype at incubation temperatures of 25 °C and 37 °C, respectively. Examination of the ability to form biofilms in vitro with quantification of total biofilm mass using crystal violet test at both temperatures showed a strong influence of temperature in terms of a higher number of isolates classified as strong biofilm producers at lower incubation temperature. A comparative analysis of the results obtained to determine morphotype and biofilm formation ability at both temperatures showed a positive correlation between the number of E. coli isolates that formed the RDAR morphotype and the number of isolates classified as strong biofilm producers. By examining the viability of the selected isolate of E. coli and S. Typhimurium in soil, after 120 days of incubation, the presence of viable cells of both microorganisms was detected under both constant and variable conditions of humidity and temperature. The survival rate of S. Typhimurium was higher under both constant and variable temperature and humidity conditions and was 0.60% and 0.27%, respectively. The adhesion degree to the surface of lettuce leaves for 3 h at a temperature of 25 °C was 6.75 log cfu/cm2 leaves for S. Typhimurium and 6.64 log cfu/cm2 leaves for E. coli. Higher incubation temperature (37 °C) differently affected the degree of adhesion of the tested microorganisms to a lettuce leaf, generally reducing it to a lower value, with a reduction of 50% in S. Typhimurium and 10% in E. coli. Using SEM, low magnification micrographs showed the formation of E. coli and S. Typhimurium cell aggregates on the surface of lettuce leaves, which are an integral part of the biofilm, while high magnification micrographs showed interconnection between cells and the formed biofilm. Therefore, the second part of the research within this doctoral dissertation aimed to examine the influence of EOs of spice plants of basil, dill, mint, and heather on the initial cell adhesion and preformed biofilm. Before testing the antimicrobial and antibiofilm potentials, the chemical composition of the EOs was analyzed, this showed that all four EOs contain a high share of monoterpenes. The major compounds of basil EO were linalool (69.2%), dill EO carvone (66.6%) and limonene (25.3%), mint EO menthol (30.8%) and menthone (28.8 %) and heather EO p- cymene (33.8%) and carvacrol (32.9%). The best antimicrobial potential against the broth cultures of E. coli and S. Typhimurium showed dill EO, followed by EOs of basil and heather, while mint EO showed the lowest antimicrobial potential. The antimicrobial potential was evaluated based on the obtained values for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Tested EOs showed a dose-dependent effect on initial cell adhesion and consequently on biofilm formation in vitro according to the following order: dill EO > basil EO > heather EO > mint EO (refers to EO concentrations of 2 MIC). By examining the influence of the EO on the total mass of biofilm in vitro, it was proved that EOs in applied concentrations of 0.5 MIC, MIC, and 2 MIC have an effect on reducing the total mass of preformed biofilm in a dose-dependent manner as a function of time. Eradication of preformed biofilms of E. coli and S. Typhimurium were achieved after 60 minutes of exposure to EOs in concentration of 2 MIC in the following order: EO dill > EO basil > EO heather > EO mint. The influence of the EO on the preformed biofilm of E. coli and S. Typhimurium on the surface of lettuce leaves showed also a dose-dependent manner as a function of time. Observing their influence regarding to the number of viable biofilm cells, the most effective was EO dill in a concentration of 2 MIC, and the least efficient was EO dill in a concentration of MIC. The highest % reduction of the total biofilm mass of both E. coli and S. Typhimurium was determined by applying the EO concentration of 2 MIC in the following order: EO dill > EO basil > EO heather > EO mint. By comparing the results of reducing the total mass of E.coli and S. Typhimurium biofilms formed in vitro and on the surface of lettuce leaves, a higher percent reduction was observed in biofilms formed in vitro. This result can be explained by the rough surface structure of lettuce leaves, which limited the contact between the EO and the biofilm of the tested microorganisms. Based on the performed tests following the goals of this doctoral dissertation and the achieved results, it can be concluded that dill EO showed the best antimicrobial and antibiofilm potential in vitro and on lettuce coupons against both tested microorganisms. It is followed by basil, heather and mint EOs. Obtained results indicated that investigated EOs next to be used as ingredients in salad dressings also can be incorporated into packaging materials as well as packaging atmosphere. Based on their proven antimicrobial and antibiofilm potential their application can contribute to food safety and lead to improving health safety in general.en
dc.languagesr (latin script)
dc.publisherУниверзитет у Новом Саду, Технолошки факултетsr
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Новом Садуsr
dc.subjectbiofilmovi, enteropatogene bakterije E. coli i S. Typhimurium, etarska ulja, antimikrobna i antibiofilm aktivnost etarskih ulja, lisnato povrćesr
dc.subjectbacterial biofilms, enteropathogenic bacteria E. coli and S. Typhimurium, essential oils, antimicrobial and antibiofilm activity of essential oils, leafy vegetablesen
dc.titleUticaj etarskih ulja na biofilmove odabranih sojeva enterobakterija formiranih na lisnoj površini povrćasr
dc.title.alternativeInfluence of essential oils on selected enterobacteria strains biofilms formed on the vegetable leaf surfacesen
dc.typedoctoralThesissr
dc.rights.licenseBY-NC-ND
dc.identifier.fulltext$DSPACE_URL/bitstream/id/145326/Disertacija_12540.pdf
dc.identifier.fulltext$DSPACE_URL/bitstream/id/145327/Izvestaj_komisije_12540.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_20579


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