Clarifying the molecular mechanisms by which bacteria acquire virulence traits is important toward understanding the bacterial virulence system. In the present study, we utilized a bacterial evolution method in a silkworm-infection model and revealed that deletion of the opgGH operon encoding synthases for osmoregulated periplasmic glucan (OPG) increased the virulence of non-pathogenic laboratory strain of Escherichia coli against silkworms. The opgGH knockout mutant exhibited resistance to the host antimicrobial peptides and antibiotics. Compared with the parent strain, the opgGH knockout mutant produced greater amounts of colanic acid, which is involved in E. coli resistance to antibiotics. RNA sequence analysis revealed that the opgGH knockout altered the expression of various genes, including the evgS/evgA two-component system that functions in antibiotic resistance. In both a colanic acid-negative background and evgS-null background, the opgGH knockout increased E. coli resistance to antibiotics and increased the silkworm killing activity of E. coli In the null background of the envZ/ompR two-component system, which genetically interacts with opgGH, the opgGH knockout increased the antibiotic resistance and the virulence in silkworms. These findings suggest that the absence of OPG confers antimicrobial resistance and virulence of E. coli in a colanic acid-, evgS/evgA-, and envZ/ompR- independent manner.IMPORTANCEThe gene mutation types that increase bacterial virulence of Escherichia coli remain unclear, in part due to the limited number of methods available for isolating bacterial mutants with increased virulence. We utilized a bacterial evolution method in the silkworm infection model, in which silkworms were infected with mutagenized bacteria and highly virulent bacterial mutants were isolated from dead silkworms. We revealed that knockout of OPG synthases increases E. coli virulence against silkworms. The OPG-knockout mutants were resistant to host antimicrobial peptides as well as antibiotics. Our findings not only suggest a novel mechanism for virulence acquisition in E. coli, but also support the usefulness of utilizing the bacterial experimental evolution method in the silkworm infection model.