Abstract
Established (48- and 72-h) Salmonella enteritidis biofilms grown in glass flow cells with or without artificial crevices (0.5-, 0.3-, and 0.15-mm widths) were subjected to a 10% trisodium phosphate (TSP) solution under different flow regimens (0.3, 0.6, 1.2, and 1.8 cm s-1). The abundance of biofilm remaining after TSP treatment, the biocidal efficacy of TSP, and the factors which contributed to bacterial survival were then evaluated by using confocal laser microscopy and a fluorescent viability probe. Biofilm age affected the amount of biofilm which remained following a 15-s exposure to TSP. After TSP treatment of 48-h biofilms, 29% of the original biofilm remained at the biofilm-liquid interface, whereas 75% of the biofilm remained at the base (the attachment surface). Following TSP treatment of 72-h biofilms, 27% of the biofilm material remained at the biofilm-liquid interface, 73% remained at the 5-micron depth, and 91% remained at the biofilm base. Results obtained using the BacLight viability probe indicated that TSP exposure killed all the cells in 48-h biofilms, whereas in the thicker 72-h biofilms, surviving bacteria (approximately 2% of the total) were found near the 5- and 0-micron depths. In the presence of artificially constructed crevices, an inverse relationship was shown to exist between bacterial survival (ranging from approximately 13 to 83% of total biofilm material) and crevice width. This relationship was further influenced by the velocity of TSP flow; high TSP flow velocities (1.8 cm s-1) resulted in the lowest number of surviving bacteria at the base of crevices (approximately 42% survival). Extended time courses demonstrated that after TSP stress was relieved, biofilms continued to grow within crevices but not in systems without crevices. It is suggested that advective TSP flux into crevices and through the biofilm matrix was enhanced under conditions of high flow. These results suggest that the inherent roughness of the substratum on which the biofilm was grown and the timing of TSP application are important factors controlling the efficacy of TSP treatment.