Acta veterinaria 2007 Volume 57, Issue 5-6, Pages: 429-440
doi:10.2298/AVB0706429M
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Investigation of biofilm formation in vitro ability of Listeria monocytogenes strains isolated from animals

Milanov Dubravka, Ašanin Ružica, Mišić D., Vidić Branka, Ratajac R.

Listeria monocytogenes is the causative agent of listeriosis in humans and animals and an important food-born pathogen. Control of its presence in food processing plants, particularly on sites where food contamination is expected, is of paramount importance with respect to food safety and protection of human health. Numerous studies demonstrated that this organism can be isolated during several months, even several years, from diverse sites in food processing plants, which is due to its ability to adsorb onto inert surfaces and form a biofilm, alone or in coexistence with other bacterial species. In this study we investigated the ability of 16 animal isolates of L. monocytogenes to form a biofilm on polystyrene microtiter plates. The investigation was performed at three different temperatures 4oC, 25 oC and 37oC that are commonly suitable for growth of Listeria monocytogenes. The research was carried out using three different nutritive media: trypthon-soy broth with yeast extract (TSB-YE), brainheart infusion (BHI) and 1/20 diluted trypthon-soy broth with yeast extract (1/20 TSB-YE). In order to investigate the biofilm formation in vitro an inoculum was prepared from 24-hours-old cultures of isolated strains of L.monocytogenes. The density of the inoculum was 2-10x107 cfu/mL (OD600=0.093 ± 0.009 in TSB-YE). The microtiter plates were incubated at cited temperatures during 48h. Colonization rate of L.monocytogenes strains on polystyrene surface and biofilm formation were monitored using crystal violet stain added to the microtiter plates, as well as using light microscopy. The tested strains demonstrated a diverse ability of biofilm formation, depending on the incubation temperature and nutritive medium. In this paper we presented the results obtained for four strains of Listeria monocytogenes isolated from brain samples of sheep, designated as 785/05, 593/05, 748/05, 1915/04 and one strain isolated from aborted calve's fetus, designated as 021/04. The referent strain of L.monocytogenes, 1071 (4b), was used as a control. Strains of L. monocytogenes cultured in nutrient-rich media (e.g. TSB-YE) and at higher temperatures (37oC) exhibited a higher ability of biofilm formation. However, non of the studied strains could be classified as a good biofilm producer, disregard of the incubation temperature and medium used. The highest OD values were obtained at the incubation temperature of 37oC in TSB-YE (OD595=0.346 ± 0.055), when three strains were quantified as moderate and two as weak biofilm producers. The decrease of incubation temperature resulted in decreased OD values, thus four strains were classified as weak biofilm producers at 25oC (OD595=0.289 ± 0.083), and none of the investigated strains was assessed as biofilm producers at 4oC (OD595=0.124 ± 0.011) Light-microscopy examination, confirmed by quantitative values obtained by the crystal violet microtiter test, proved to be a simple and rapid screening method for the quantification of the ability of L.monocytogenes to form biofilms in varying test conditions.

Keywords: Listeria monocytogenes, biofilm, microtiter test, crystal violet stain

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