ABSTRACTWe investigated the phylogeny of biofilm forming (BF) and nonbiofilm forming (NBF)Escherichia coli(n= 114) from a beef processing environment as well as genetic elements in their BF and persistence via a comparative genomic analysis. Phylogroup B1 made up the largest proportion of both the BF (73.8%) and NBF (50.9%) groups.E. colifrom all of the sources that were examined had mixed phylogroups, except for those that were recovered from equipment after cleaning, which were exclusively from phylogroup B1. Both the core genome and gene content trees showed a tree-wide spread of BF strains, with clusters, including both BF and NBF strains. Genome-wide association studies (GWAS) via Scoary or Pyseer did not find any genes or mutations that were overrepresented in the BF group. A retrospective analysis of phenotypes found a significant correlation (P< 0.05) between BF ability and curli production, cellulose synthesis, and/or mobility. However, the BF group also included strains that were negative for curli and cellulose and/or missing encoding genes for the two traits. All curli and cellulose encoding genes were present in most genomes, regardless of their BF status. The degree of motility was correlated with both curli and cellulose production, and 80 common genes were overrepresented in all three of the trait-positive groups. A PTS enzyme II, a subsidiary gluconate catabolism pathway, and an iron-dicitrate transport system were more abundant in the persistingE. coligroup. These findings suggest gene function redundancy inE. colifor biofilm formation as well as additional substrate utilization and iron acquisition in its persistence.IMPORTANCEThe persistence of potentially hazardous bacteria is a major challenge for meat processing environments, which are conducive for biofilm formation. Marker genes/phenotypes are commonly used to differentiate biofilm formingE. colistrains from their nonbiofilm forming counterparts. We took a comparative genomic analysis approach to analyzeE. colistrains that were from the same environment but were differentiated by their biofilm forming ability. A diversification of the genes involved in the biofilm formation ofE. coliwas observed. Even though there is a correlation on the population level between biofilm formation and the expression of curli and cellulose, uncertainties exist on the individual strain level. Novel substrate utilization and iron acquisition could contribute to the persistence ofE. coli. These findings not only advance our understanding of the ecology ofE. coliwith respect to its persistence but also show that a marker gene/phenotype driven approach for the biofilm control ofE. colimay not be prudent.