Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment

Abstract

The objective of this study was to evaluate the resistance of biofilms of Listeria monocytogenes to sanitizing agents under laboratory conditions simulating a food processing environment. Biofilms were initially formed on stainless steel and Teflon coupons using a five-strain mixture of L. monocytogenes. The coupons were then subjected to repeated 24-h daily cycles. Each cycle consisted of three sequential steps: (i) a brief (60 s) exposure of the coupons to a sanitizing agent (a mixture of peroxides) or saline as a control treatment, (ii) storage of the coupons in sterile plastic tubes without any nutrients or water for 15 h, (iii) and incubation of the coupons in diluted growth medium for 8 h. This regimen was repeated daily for up to 3 weeks and was designed to represent stresses encountered by bacteria in a food processing environment. The bacteria on the coupons were reduced in number during the first week of the simulated food processing (SFP) regimen, but then adapted to the stressful conditions and increased in number. Biofilms repeatedly exposed the peroxide sanitizer in the SFP regimen developed resistance to the peroxide sanitizer as well as other sanitizers (quaternary ammonium compounds and chlorine). Interestingly, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitizing agents. These data suggest that the resistance of the treated biofilms to sanitizing agents may be due to attributes of extracellular polymeric substances and is not an intrinsic attribute of the cells in the biofilm.

FIG. 1.

The 24-h cycle of the SFP regimen. The SFP regimen consisted of three sequential steps as indicated in the schematic: (i) sanitation by Matrixx (or saline treatment) for 60 s, followed by neutralization and rinsing; (ii) storage without water or nutrients for 15 h; and (iii) incubation with growth medium (TSB-YE/10) for 8 h, followed by two rinses with sterile water. The arrows indicate the sampling times before and after the sanitation step (step i).

FIG. 2.

Sanitizer treatments of L. monocytogenes biofilms. The viable cell counts from stainless steel (A) and on Teflon (B) coupons during the 3-week SFP system are shown. The dotted lines (open symbols) represent the control samples without sanitizer treatment. The solid lines (filled symbols) represent the Matrixx samples. The error bars indicate the standard deviation.

FIG. 3.

Resistance of L. monocytogenes biofilms to sanitizer treatments. The log reduction for biofilms on stainless steel (A-1, B-1, and C-1) and Teflon (A-2, B-2, and C-2) coupons during the 3-week SFP regimen is shown. Coupons were treated with peroxides (Matrixx) (A), a mixture of quaternary ammonium compounds (Multi-Quats) (B), and chlorine (C). The dotted lines represent data for the control group, and the solid lines represent data for the Matrixx-treated group. The error bars indicate the standard deviation.

FIG. 4.

Inactivation of detached cells by Matrixx. The log reduction of viable cells for the 24-h culture (FP 24 h) and the cells from the Matrixx-treated biofilms on stainless steel (SS) and Teflon (Tef) coupons from the SFP regimen is shown: 1wk-c, control without sanitizer treatment for 1 week; 1wk, treated with Matrixx for 1 week; 2wk-c, control without sanitizer treatment for 2 weeks; 2wk, treated with Matrixx for 2 weeks. Tef 48h represents the initial biofilm from the 48-h incubation prior to the start of the SFP regimen. (Only data for cells from Teflon coupons are shown.)

FIG. 5.

Photomicrographs of L. monocytogenes biofilms. The images shown were from stainless steel coupons only. (A) Control group without peroxide treatment. (B) Peroxide-treated group.