P-013. Survival of Listeria monocytogenes Serotype 4b and Serotype 1/2a Strains in Mixed Culture Biofilms

Y. Pan1, F. Breidt2;
1North Carolina State Univ., Raleigh, NC, 2USDA/ARS, Raleigh, NC.

Most outbreaks of listeriosis are caused by the foodborne pathogen Listeria monocytogenes serotype 4b, however, serotype 1/2a is frequently isolated from food and food processing environments. The objective of this study was to determine whether a frequently isolated serotype 1/2a strain could preferentially survive in biofilms which are formed with a mixture of a serotype 1/2a strain and a serotype 4b strain in a simulated food processing (SFP) environment. Stainless steel coupons were used to form biofilms in a diluted growth medium for 7 days at 22.5oC. Following biofilm formation, the coupons were then subjected to a 24 hour cycle which was composed of three sequential steps including treatment by peroxides, dehydration, and incubation in the diluted growth medium. Pure and mixed culture biofilms were subjected to the SFP cycle continuously for 5 weeks. The cell viability was measured using real-time PCR with serotype specific primers. Replicate coupons were swabbed, and cells were treated with propidium monoazide prior to DNA isolation. As we have shown previously, this treatment prevented subsequent amplification of cell free DNA or dead cell DNA from biofilms. During biofilm formation with pure cultures, starting with equal cell numbers, the serotype 1/2a strain formed higher density biofilms (2.6 × 107 CFU/cm2) than the serotype 4b strain (3.25 ×106 CFU/cm2) (P < 0.05). Interestingly, there was no significant difference (P > 0.05) in cell density of each strain in biofilms formed using mixed cultures with the same initial cell populations. The results indicate that differences in biofilm formation with single strains may not reflect the cell densities achieved using mixed cultures. During the 5-week SFP cycle, neither strain survived preferentially in the mixed culture biofilms. There were no significant differences in the cell densities of each strain (P > 0.05), both strain densities decreased by 3 log cycles. The real-time PCR technique with propidium monoazide treatment may be useful for investigating the persistence of L. monocytogenes strains in mixed culture biofilms.