Uticaj Etarskih Ulja na Biofilmove Odabranih Sojeva Enterobakterija Formiranih na Lisnoj Površini Povrća

Abstract

Pojava 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.

Alternate abstract:

The 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.