<p>Poznavanje i razumevanje adhezivne sposobnosti i formiranja biofilma patogenih bakterija, kao i njihovog odnosa prema faktorima koji mogu stimulisati ili inhibirati razvoj biofilma, je od fundamentalnog značaja za iznalaženje mera za njihovu efikasnu prevenciju i eliminaciju.<br />Imajući u vidu navedenu činjenicu kao i da je Salmonella enterica serotip Enteritidis epidemiološki najfrekventniji serotip cilj ovog istraživanja je bio da se ispita: sposobnost različitih sojeva Salmonella Enteritidis izolovanih iz kliničkog materijala, hrane za životinje i odabranog referentnog soja da formiraju biofilm, adherentnost na površine od stakla i nerđajućeg čelika, sposobnost preživljavanja odabranih biofilm produkujućih sojeva kao i mogućnost primene konfokalne laserske i skening elektronske mikroskopije u vizuelizaciji trodimenzionalne strukture biofilma.<br />Određivanjem morfotipa kolonija na Kongo red agaru na temperaturi inkubiranja od 25°C među testiranim izolatima detektovana su tri morfotipa RDAR (red, dry and rough), BDAR (brown dry and rough) i SAW (smooth and white). Polovina testiranih izolata je eksprimirala RDAR morfotip. Izolati koji su eksprimirali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.<br />Uporednom analizom rezultata primenjenih skrining testova za utvrđivanje sposobnosti formiranja biofilma pri temperaturi inkubiranja od 25°C ustanovljena je korelacija između pojave određenog morfotipa na Kongo crvenom agaru i sposobnosti formiranja biofilma u kristal violet i pelikula testu. Međutim, sa povećanjem temperature inkubiranja na 37oC, ova korelacija nije ustanovljena, sa izuzetkom izolata SE8.<br />Svi testirani izolati su pokazali sposobnost adherencije na površinu stakla i nerđajućeg čelika, ali u različitoj meri. Na sposobnost adherencije povoljniji uticaj je imala temperatura inkubiranja od 25°C (p<0,05), sa izuzetkom izolata SE3 (p>0,05). Između stepena adherencije izolata na površine stakla i nerđajućeg čelika nisu ustanovljene statistički značajne razlike (p>0,05).<br />Praćenjem stope preživljavanja tokom 28 dana u uslovima isušivanja evidentirana je znatno veća stopa preživljavanja ćelija izolata RDAR morfotipa u odnosu na stopu preživljavanja ćelija BDAR morfotipa (p<0,05). Praćenjem stope preživljavanja tokom 90 dana u uslovima povremene dostupnosti hranljivih materija, zabeležena je veća stopa preživljavanja u odnosu na stopu preživljavanja u uslovima isušivanja. U uslovima povremene dostupnosti hranljivih materija nakon devedeset dana ispitivanja kod obe grupe izolata procenat vijabilnih ćelija je iznosio više od 50%.<br />Primenjenim mikroskopskim tehnikama (CLSM i SEM) omogućena je detaljna vizualizacija formiranih biofilmova. Na modelu izolata SERDAR morfotipa, ustanovljeno je da se formiranje biofilma pod primenjenim eksperimentalnim uslovima, odvija u tri faze: 1) inicijalna adhezija za površinu i formiranje manjih ćelijskih agregata (24h); 2) formiranje većih ćelijskih agregata uz produkciju EPS (48h); 3) sazrevanje biofilma uz značajnu produkciju EPS što omogućava formiranje stabilne trodimenzionalne strukture biofilma (96h).<br />Nasuprot karakteristikama koje bakterije pokazuju tokom rasta u medijumima koji obiluju hranljivim materijama, bakterije u biofilmovima pokazuju drugačije osobine u pogledu ekspresije gena i karakteristika rasta. Zahvaljujući ovim razlikama, bakterije u biofilmovima pokazuju povećanu rezistenciju na antibiotike i dezinficijense, zbog čega se konstantno razvijaju nove kontrolne strategije u cilju iznalaženja potencijalnih bioloških rešenja koja pored različitih enzima, faga, antimikrobnih jedinjenja proizvedenih od strane mikroorganizama uključuju i antimkrobna jedinjenja biljnog porekla kao što su biljni ekstrakti, etarska ulja i različiti začini. Stoga, je u okviru drugog segmenta ovog istraživanja ispitivan hemijski sastav etarskih ulja, antimikrobni efekat etarskih ulja (O. heracleoticum , O. vulgare , Th. vulgaris i Th. serpyllum) i pojedinačnih komponenti etarskog ulja (karvakrola i timola) na bujonske kulture testiranih sojeva Salmonella Enteritidis kao i uticaj odabranih koncentracija etarskih ulja na inicijalnu adheziju i već formirani biofilm odabranih sojeva Salmonella Enteritidis.<br />Etarska ulja je karakterisao visok zbirni udeo glavnih fenolnih komponenti karvakrola i timola: O. heracleoticum (71,6%), O. vulgare (63,6%), Th. vulgaris (59,77%), Th. serpyllum (40,04%). Etarska ulja su ispoljila antimikrobni efekat sledećim redosledom: O. heracleoticum >O. vugare =Th. vulgaris >Th. serpyllum. Antimikrobni efekat etarskih ulja je bio direktno srazmeran zbiru fenolnih komponenti (karvakrola i timola) u etarskom ulju. U odgovoru na tretman etarskim uljima između izolata S. Enteritidis nisu ustanovljene razlike.<br />Etarska ulja, karvakrol i timol su pokazali inhibitorni efekat na inicijalnu adheziju i posledično na formiranje biofilma testiranih izolata S. Enteritidis na dozno zavisan način. Upoređivanjem uticaja etarskih ulja na inhibiciju inicijalne adhezije i metabolitičke aktivnosti ćelija između izolata RDAR i BDAR morfotipa ustanovljene razlike nisu bile statistički značajne (p>0,05).<br />Ispitivanjem uticaja etarskih ulja, karvakrola i timola na ukupnu biomasu biofilma i metabolitičku aktivnost ćelija dokazano je da etarska ulja u primenjenim koncentracijama ispoljavaju uticaj na redukciju ukupne biomase formiranog biofilma i metaboličke aktivnosti bakterijskih ćelija na dozno zavisan način u funkciji vremena. Znatno veća efikasnost primenjenih tretmana je pokazana u slučaju njihove primene na biofilmove formirane od strane izolata BDAR morfotipa (p<0,05).</p> / <p><span style="font-size:10px;">Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination.<br />Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure.<br />Determination of colony morphotype on Congo red agar at incubation temperature of 25°C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface.<br />Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25ºC. In the case of assays conducted at 37°C, this correlation was not established, except for the isolate SE8.<br />All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25ºC had more favorable effect on the adherence, with the exception of isolate SE3 (p>0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p>0.05).<br />Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was</span> <span style="font-size:10px;">obtained for RDAR than BDAR morphotype isolates (p<0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates.<br />Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h).<br />Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices.<br />Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates.<br />Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum > O. vugare = Th. vulgaris > Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference.<br />Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p>0.05).<br />Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity</span> <span style="font-size:10px;">of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p<0.05).</span></p>
| Identifer | oai:union.ndltd.org:uns.ac.rs/oai:CRISUNS:(BISIS)94814 |
| Date | 18 June 2015 |
| Creators | Čabarkapa Ivana |
| Contributors | Škrinjar Marija, Tomović Vladimir, Lević Jovanka, Milanov Dubravka |
| Publisher | Univerzitet u Novom Sadu, Tehnološki fakultet Novi Sad, University of Novi Sad, Faculty of Technology at Novi Sad |
| Source Sets | University of Novi Sad |
| Language | Serbian |
| Detected Language | Unknown |
| Type | PhD thesis |
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