Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.Enterohemorrhagic Escherichia coli O157:H7 was first recognized as the probable cause of hemorrhagic colitis in humans in 1982 (50). Since then, this organism has emerged as a major cause of food-borne illness in countries around the world, including the United States (49), Europe (17, 18, 24, 36), Japan (39), and Australia (28). Outbreaks have been associated with a variety of food sources, including ground beef (50), green leafy vegetables (1, 41), and nonpasteurized milk (26), and environments such as municipal water and lakes (27, 55). Symptoms in infected humans range from mild diarrhea to severe, hemorrhagic colitis, with 5 to 10% of patients developing hemolytic uremic syndrome (HUS), making E. coli O157:H7 one of the leading causes of acute renal failure in children and the elderly (38).Early studies have shown that some strains of E. coli O157:H7 form biofilms on both abiotic and biotic surfaces outside the host (15, 51, 58). Biofilms are exopolymeric matrix-enclosed bacterial populations that are firmly adherent to each other and/or to surfaces (9). Biofilms have been shown to be responsible for protection from a variety of environmental stresses, such as acidification, high temperatures, and desiccation (52). Moreover, microbes in biofilms are highly resistant to other adverse conditions, such as sanitizers and household cleaners (44, 45) as well as antibiotics (23, 34). The ability of this pathogen to form biofilms on a wide range of food surfaces as well as food processing surfaces makes E. coli O157:H7 problematic in both the health and food industries (29).Biofilm formation is a dynamic and complex process and includes initial attachment of cells to the substratum, physiological changes within the organism, multiplication of the cells to form microcolonies, and eventually maturation of the biofilm (42). Because of this complexity, the process of biofilm formation and its regulation is poorly understood. Previous studies in E. coli O157:H7 have focused on individual genes and the specific genetic pathways that are responsible for biofilm formation (10, 51, 58, 60). In contrast, few studies have focused on studying genetic factors that control E. coli O157:H7 biofilm formation on a global scale, although studies of this type have been performed with other bacterial pathogens (43, 47, 59).The goal of this study was to gain additional insights into biofilm formation in E. coli O157:H7. A global mutational approach with a mini-Tn5 transposon was used to study the process of biofilm formation in strain EDL933, a strong biofilm-forming strain. This strain was first isolated during a multistate outbreak involving contaminated hamburgers (50). A library of >11,000 mutants was generated and screened for a biofilm-negative phenotype. Our results reinforced the fact that biofilm formation is a complex process involving a large number of genes and genetic pathways. This study discovered several pO157 genes that were not previously known to be linked to biofilm formation.