Effects of culture conditions on the biofilm formation of Cronobacter sakazakii strains and distribution of genes involved in biofilm formation

Abstract

Biofilm of Cronobacter sakazakii on the surfaces of equipment and processing environments is the important source of persistent contamination in food samples. Based on 828 tests (23 × 4 × 3 × 3), the biofilm-forming abilities of 23 C. sakazakii isolates were assessed under different pH values, temperatures, and culture time. Biofilm formed by C. sakazakii was evaluated in tryptone soy broth in 96-well plates using crystal violet staining and its quantification was counted using the optical density measurements (OD570 nm). Among the evaluated conditions, C. sakazakii formed highest amount of biofilm at pH 5.0 (9 strains, 39.1%), at 28 °C (23 strains, 100%) and for 48 h (14 strains, 60.9%). In addition, the biofilm under pH7-24 h-28 °C, pH5-24 h-28 °C, and pH7-48 h-28 °C was detected by confocal laser scanning microscopy for detailed analysis of biofilm. Finally, PCR methods were newly developed for detection of the genes involved in biofilm formation. The bcsC, bcsG, flgJ, bcsA, fliD, and flhE were present in 100%, 100%, 91.3%, 95.7%, 100%, and 34.8% of tested strains respectively. Results indicated that the properties of polysaccharides in biofilm formation of C. sakazakii were affected by microbial environments and the biofilm-forming ability was strain-specific. The findings presented here provide useful information for development of efficient procedures against biofilm formation.