Folia Microbiol (2015) 60:357–363 DOI 10.1007/s12223-014-0367-6

Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens Ait Meddour Amel & Bendali Farida & Sadoun Djamila

Received: 4 March 2014 / Accepted: 20 November 2014 / Published online: 3 December 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014

Abstract It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P0.06; moderately adherent (moderate biofilm producer), 0.24≥A>0.12; and strongly adherent (strong biofilm producer), A>0.24. Following this classification, En. durans was a moderate biofilm producer at 25 °C (A=0.14) and a weak biofilm producer at 30 and 37 °C (A=0.09). The absorbance registered for S. aureus and E. coli at 25 °C were 0.26 and 0.45, respectively, which testify that they were strong biofilm producers at this temperature. However, at 30 and 37 °C, these two strains (Fig. 1) showed a moderate potential (0.24≥A>0.12). On the contrary, P. aeruginosa (0.12≥A> 0.06) and L. innocua (A0.12). Statistical analysis showed significant differences (P0.05) was noted between the values recorded at 30 and 37 °C. These results led us to conclude on the high impact of growth temperature (25 vs. 30 °C and 25 vs. 37 °C) on the adherence ability and the disparity among the strains. Adherence to stainless steel coupons En. durans showed an adherence level of ≈104 CFU/mL after 3-h incubation and a maximum level of ≈106 CFU/mL was obtained after 18 h (Fig. 2). Similarly, the target strains adhered to the steel after 3 h with levels ranging from 103 CFU/ mL (S. aureus) to ≈105 CFU/mL (E. coli). These levels reached high values after 18 h ranging from 104 CFU/mL (P. aeruginosa) to ≈108 CFU/mL (E. coli). No significant differences (P>0.05) were recorded on the adhesion ability of all the strains at the three temperatures tested (25, 30 and 37 °C). Anti-adherence potential of En. durans

Results

Using En. durans cells

Adherence to polystyrene microtitre plates

After 3-h incubation, a similar adhesion level of the target strains on stainless steel coupons was observed in pure and mixed cultures at the three temperatures tested. The reduction of the target strain adhesion levels was observed within 18-h incubation in the presence of En. durans compared to the controls. A reduction level of ≈1 log CFU/mL for L. innocua and of ≈2 log CFU/mL for the other strains was registered (Fig. 2). No significant differences (P>0.05) were recorded when incubating at 25, 30 and 37 °C.

A quantitative evaluation of the biofilm formation by En. durans and the four target strains (E. coli, S. aureus, P. aeruginosa and L. innocua) was performed in 96-well microplates. Absorbance values at 630 nm after staining with crystal violet and subsequently to culture at different temperatures (25, 30 and 37 °C) were measured. These values were considered an index of bacterial adhesion to the surface and consequently their ability to form biofilms. The results (Fig. 1) revealed that the strains were able to adhere and consequently to form biofilms under the tested conditions. The strains were classified into four categories following the classification of Stepanovic et al. (2000) using the absorbance (A) of the sterile broth as a control (Ac): non-adherent (non-biofilm producer),

Using En. durans CFS En. durans CFS (pH 4.6) showed the ability to influence the adhesion of the target strains. Significant differences (P

Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of...
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