Variants of smooth Salmonella enterica serovar Enteritidis that grow to higher cell density than the wild type are more virulent.

Salmonella enterica serovar Enteritidis that grows to a higher cell density (SE-HCD) than wild type while retaining O-chain lipopolysaccharide was isolated by transforming wild type serovar Enteritidis with the cell density sensor plasmid pSB402 and selecting for bioluminescence. A luminescent strain, SE-HCD, that emitted light in proportion with cell density and opacity through stationary phase was isolated. After a peak cell density of 1.5 x 10(11) CFU/ml was observed, luminescence decreased, although opacity continued to increase. Scanning electron microscopy revealed that changes in luminescence and opacity past peak cell density were associated with lysis of a swarming hyperflagellated coccobacillary cell type and emergence of a 10-to-30-fold-elongated rod cell type that lacked cell surface structures. Vigorous aeration was required to induce this dramatic cellular differentiation. The virulence of two isogenic variants with different patterns of light emission at an opacity of 0.2 after the culture was diluted 10-fold (1/10 OD) was assessed in animal models. Whereas SE-HCD1 killed 70% of 6-day-old chicks challenged subcutaneously, the same dose of SE-HCD2 did not kill any chicks. Conversely, subcutaneous challenge of hens with SE-HCD2 contaminated eggs five and seven times more often, respectively, than did SE-HCD1 or wild type serovar Enteritidis. Intravenous challenge with SE-HCD2 contaminated 22% of eggs versus 0. 5% with wild type, depressed egg production for 4 weeks, and caused clinical signs of Gallinarum Disease (Fowl Typhoid) in hens. SE-HCD2 produced no contaminated eggs following oral infection, whereas wild type contaminated 1.3% of eggs. Thus, SE-HCD2 is better at contaminating eggs than wild type, but only by parenteral challenge. These results suggest that it may be possible to separate luminescent serovar Enteritidis into groups that infect different age groups and organs and contaminate eggs.