Behavior of Listeria monocytogenes in a multi-species biofilm with Enterococcus faecalis and Enterococcus faecium and control through sanitation procedures

• L. monocytogenes favored biofilm formation in multi-species cultures at 25 °C.
• Negative effect was observed for L. monocytogenes biofilm in mixed cultures at 39 °C.
• Peracetic acid eliminated the multi-species biofilms under all tested conditions.
• The biguanide sanitizer was unable to eliminate any of the biofilms.

The formation of mono-species biofilm (Listeria monocytogenes) and multi-species biofilms (Enterococcus faecium, Enterococcus faecalis, and L. monocytogenes) was evaluated. In addition, the effectiveness of sanitation procedures for the control of the multi-species biofilm also was evaluated. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39 °C) and contact times (0, 1, 2, 4, 6 and 8 days). In all tests, at 7 °C, the microbial counts were below 0.4 log CFU/cm2 and not characteristic of biofilms. In mono-species biofilm, the counts of L. monocytogenes after 8 days of contact were 4.1 and 2.8 log CFU/cm2 at 25 and 39 °C, respectively. In the multi-species biofilms, Enterococcus spp. were present at counts of 8 log CFU/cm2 at 25 and 39 °C after 8 days of contact. However, the L. monocytogenes in multi-species biofilms was significantly affected by the presence of Enterococcus spp. and by temperature. At 25 °C, the growth of L. monocytogenes biofilms was favored in multi-species cultures, with counts above 6 log CFU/cm2 after 8 days of contact. In contrast, at 39 °C, a negative effect was observed for L. monocytogenes biofilm growth in mixed cultures, with a significant reduction in counts over time and values below 0.4 log CFU/cm2 starting at day 4. Anionic tensioactive cleaning complemented with another procedure (acid cleaning, disinfection or acid cleaning + disinfection) eliminated the multi-species biofilms under all conditions tested (counts of all micro-organisms < 0.4 log CFU/cm2). Peracetic acid was the most effective disinfectant, eliminating the multi-species biofilms under all tested conditions (counts of the all microorganisms < 0.4 log CFU/cm2). In contrast, biguanide was the least effective disinfectant, failing to eliminate biofilms under all the test conditions.