Effect of enterocin AS-48 singly or in combination with biocides on planktonic and sessile B. cereus

Abstract

Enterocin AS-48 was tested singly or in combination with biocides on a cocktail of six Bacillus cereus strains in planktonic state and in biofilms formed on polystyrene microtiter plates. The biocides tested were benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, triclosan, chlorhexidine, polyhexamethylen guanidinium chloride and commercial sanitizers P3 oxonia and P3 topax 66. The numbers of survivors were determined after 60 min incubation with biocides or the biocide-bacteriocin combinations. Addition of enterocin AS-48 (25 mg/l) increased the inactivation of planktonic cells by the quaternary ammonium compounds, bisphenols and biguanines tested in a range of biocide concentrations from 0.25 to 2.5 g/l, and by 4 g/l polyguanine. Increased inactivation of the bacilli was also observed for the combination of enterocin AS-48 with 2.5% P3 oxonia, but not by P3 topax 66. In the sessile state, the bacilli were more resistant to biocides and also to the bacteriocin-biocide combinations. Hexadecylpyridium chloride was the most active biocide on biofilms in the single treatments. In the combined treatments with 50 mg/l bacteriocin, hexadecylpyridinium (2.5 g/l), polyhexamethylen guanidinium chloride (4 g/l) and P3 oxonia (2.5%) achieved complete inactivation of bacilli populations. P3 topax 66 showed the lowest performance among all treatments tested, either singly or in combination with bacteriocin. A cocktail of endospores was challenged with biocides and enterocin AS-48 for 60 min at temperatures of 22 °C, 40 °C, and 60 °C. Enterocin AS-48 did not significantly (p > 0.05) reduce viable counts or increase the lethal effect of biocides. However, treatments with 5 g/l benzalkonium chloride at 60 °C, 2.5 g/l hexadecypyridinium at 60 °C or P3 oxonia at 0.025% and 60 °C or at 0.25% at 22–60 °C achieved complete inactivation of bacterial endospores, both singly and in combination with bacteriocin. Significant reductions of viable counts (1–2 log cycles) were also obtained for some treatments with cetrimide, triclosan or polyhexamethylen guanidinium chloride, but not for chlorhexidine (up to 5 g/l) or P3 topax 66 (up to 1%). Polystyrene surfaces dosed with enterocin AS-48 (25 or 50 mg/l) remained free of detectable bacilli from 2 to 24 h after being inoculated with a cocktail of endospores, but stainless steel surfaces dosed with 50 mg/l bacteriocin did not prevent bacterial growth from endospores. Results from this study suggest that enterocin AS-48 could be applied as enhancer of biocide activity against planktonic and sessile B. cereus cells.