Biofilm Formation by 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups.

is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of and produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. The major source of contamination of food with is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by ; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of , , , and and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.