Biofilm Forming Ability Of Salmonella Enteritidis In Vitro

Abstract

Salmonella enterica serotype Enteritidis is an important alimentary pathogen that recently gained special attention due to the ability of a large number of strains to form biofilms. Qualitative testing of biofilm forming ability was performed by observing the morphotype of the colonies on Congo Red agar and by conducting the pellicle test, while quantitative testing was carried out by Cristal violet assay on microtiter plates. A total of 14 isolates of S. Enteritidis were tested for biofilm forming ability, while Salmonella Enteritidis ATTC 13076 was used as the reference strain. Based on the morphotype of colonies cultivated on Congo Red agar at 25°C incubation temperature, among tested isolates three morphotypes were detected – red, dry and rough (rdar), brown, dry and rough (bdar) and smooth and white (saw). Half of the tested isolates demonstrated rdar morphotype. All isolates that showed a specific morphotype at this incubation temperature also formed the corresponding type of pellicle at the air-liquid interface. Additionally, comparing OD (optical density) values obtained by crystal violet test between groups of isolates that represent one of the three detected morphotypes (rdar, bdar and saw), statistically significant differences were detected. Based on OD values obtained by crystal violet test at both applied incubation temperatures, isolates were classified into three categories, regarding their ability to form biofilms: strong, moderate and weak biofilm producers. By comparing the amounts of the biofilms formed after 48h at 25°C and 37°C, statistically significant differences were noted (P<0.05). In this research we presented micrographs and a reconstruction of three-dimensional projections of biofilm developing phases of rdar morphotype isolates, which were obtained using confocal laser scanning microscopy.

Alternate abstract:

Salmonella enterica serotip Enteritidis je važan alimentarni patogen, a zbog sposobnosti formiranja biofilma koja je ustanovljena kod velikog broja različitih izolata, danas mu se pridaje sve veći značaj. Kvalitativna ispitivanja sposobnosti formiranja biofilma su izvedena određivanjem morfotipa kolonija na Kongo crvenom agaru i primenom pelikula testa, dok su kvantitativna ispitivanja izvedena primenom kristal violet testa na mikrotitar pločama. Testovi sposobnosti produkcije biofilma su izvedeni sa 14 izolata Salmonella Enteritidis, a kao referentni soj korišćen je Salmonella Enteritidis ATCC 13076.

Na osnovu 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 pokazivala rdar morfotip. Izolati koji su pokazali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.

Osim toga, upoređivanjem vrednosti apsorbanci izmerenih u kristal violet testu između grupa izolata koji reprezentuju jedan od tri detektovana morfotipa (rdar, bdar i saw) ustanovljeno je postojanje statistički značajnih razlika. Na temperaturi inkubiranja od 37°C detektovan je samo jedan morfotip (saw). Na osnovu vrednosti apsorbanci izmerenih primenom kristal violet testa na obe temperature inkubacije, izolati su na osnovu sposobnosti produkcije biofilma klasifikovani u tri kategorije - jaki, umereni i slabi biofilm. Pored toga su ustanovljene statistički značajne razlike u količini formiranog biofilma nakon 48h (P<0.05) u odnosu na temperature inkubiranja 25°C i 37°C. U istraživanju su prikazane mikrografije i rekonstrukcija trodimenzionalne strukture razvojnih faza biofilma izolata rdar morfotipa na površini stakla upotrebom konfokalne laserske skening mikroskopije.