Control of Pathogens in Biofilms

March 2014

Various bacteria, including foodborne pathogens, can form biofilms on stainless steel surfaces, potentially leading to contamination of foods. Enclosed in a matrix primarily consisting of polysaccharides, pathogens in biofilms are often more resistant to environmental stresses such as heat and chemical sanitizers than are their planktonic counterparts.

We did a study to (1) investigate biofilm formation by Listeria monocytogenes, Salmonella Typhimurium, and Shiga toxin-producing Escherichia coli (STEC) on the surface of stainless steel held at 100% relative humidity (RH) and 21°C for up to 72 h; and (2) evaluate the efficacy of heat and chemical sanitizer treatments on inactivation of pathogens in biofilms formed on stainless steel.

Multistrain mixtures (ca. 8 log CFU) of L. monocytogenes, S. Typhimurium, and STEC were separately deposited in an encircled area (Ø = 1.27 cm) on stainless steel coupons (type 304; 4 by 2.5 cm) and immersed in brain heart infusion broth (BHI) or tryptic soy broth (TSB) (100% RH) at 21°C for 72 h. At 24-h intervals, the marked area was washed five times with 0.1% peptone water and replenished with fresh broth.

Coupons were then either treated by heating (60, 80, and 100°C) for 10 min or with 0.1 ml of a chemical sanitizer for 10 min, or both. Test sanitizers and chemicals were a quaternary ammonium compound (QAC, 150 µg/ml), lactic acid (LA, 3%), sodium hypochlorite (SHC, 100 µg/ml), hydrogen peroxide (HP, 2%), levulinic acid (LVA, 3%), sodium dodecyl sulfate (SDS, 2%), and three different concentrations of LVA plus SDS (0.5% LVA+0.05% SDS, 1% LVA+0.1% SDS, and 2% LVA+0.5% SDS).

Biofilms on treated and untreated coupons were subsequently enriched by immersing coupons in BHI or TSB, and in aqueous filtrates from suspensions of cooked ground turkey or beef, cereal, and peanut butter. To prepare turkey, beef, and peanut butter filtrates, 80 g were homogenized with 20 ml of sterile water; a cereal homogenate was prepared by combining 10 g with 40 ml of water. Filtrates from filter-sterilized homogenates served as enrichment media. Cells attached to coupons immersed in TSB, BHI, and food filtrates for 24 h at 37ºC were dislodged using a bead vortex method and samples were spread on TSA and on MOX, XLD, or MAC agars to enumerate L. monocytogenes, S. Typhimurium, and STEC, respectively.

Populations of 8.6 to 9.2 log CFU of pathogen/coupon were recovered from biofilms after incubating in BHI and TSB for 72 h. Heating biofilms at 80°C resulted in significant (P < 0.05) log CFU/coupon reductions of 1.0, 1.4, and 1.7 for L. monocytogenes, S. Typhimurium, and STEC, respectively. Not surprisingly, inactivation was greater when the treatment temperature was increased from 60 to 80 to 100ºC.

The three pathogens were reduced by 0.3 to > 6.9 log CFU/coupon within 10 min when coupons were treated with QAC (150 µg/ml), SHC (100 µg/ml), HP (2%), LVA (3%), SDS (2%), or LVA+SDS (0.5% LVA+0.05% SDS and 1% LVA+0.1% SDS). Significantly higher numbers (P < 0.05) of cells in biofilms treated with LA (3%) were recovered on TSA than on selective media. When LA (3%) and 2% LVA+0.5% SDS were applied to biofilms following treatment at 80ºC, all three pathogens were reduced to undetectable levels, i.e., samples were negative by enrichment.

Results of this study indicate that treatment of L. monocytogenes, S. Typhimurium, and STEC biofilms with lactic acid and levulinic acid with SDS, followed by heating is effective in reducing populations by up to 6.9 log CFU/coupon. These treatments show promise for use as interventions to control biofilms in food processing facilities.