Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumen

Abstract

Salmonella enterica consists of over 2,000 serovars that are major causes of morbidity and mortality associated with contaminated food. Despite similarities among serovars of Salmonella enterica, many demonstrate unique host specificities, epidemiological characteristics, and clinical manifestations. One of the unique epidemiological characteristics of the serovar Enteritidis is that it is the only bacterium routinely transmitted to humans through intact chicken eggs. Therefore, Salmonella enterica serovar Enteritidis must be able to persist inside chicken eggs to be transmitted to humans, and its survival in egg is important for its transmission to the human population. The ability of Salmonella enterica serovar Enteritidis to survive in and transmit through eggs may have contributed to its drastically increased prevalence in the 1980s and 1990s. In the present study, using transposon-mediated mutagenesis, we have identified genes important for the association of Salmonella enterica serovar Enteritidis with chicken eggs. Our results indicate that genes involved in cell wall structural and functional integrity, and nucleic acid and amino acid metabolism are important for Salmonella enterica serovar Enteritidis to persist in egg albumen. Two regions unique to Salmonella enterica serovar Enteritidis were also identified, one of which enhanced the survival of a Salmonella enterica serovar Typhimurium isolate in egg albumen. The implication of our results to the serovar specificity of Salmonella enterica is also explored in the present study.

FIG. 1.

Survival of clinical isolates of Salmonella enterica serovar Enteritidis (SE), Salmonella enterica serovar Typhimurium (ST), and animal and laboratory strains of E. coli after incubation with egg albumen for 24 h. Each datum point represents an independent bacterial strain listed in Table 1. The percentage of survival represents the ratio of surviving bacteria after 24 h of incubation to the input bacteria. At least three experiments were performed, and results from a representative experiment performed in triplicate are shown.

FIG. 2.

Transposon used for the construction of the Salmonella enterica serovar Enteritidis transposon mutant library. The structure of the transposon encoded by the plasmid pMOD3-Kan is shown. The 19-bp mosaic end sequence for transposition flanks the transposon. R6Kγ ori was used to rescue the adjacent chromosomal sequence into a plasmid for insertion site identification. The kanamycin resistance cassette (Kan^r) was cloned into the construct to provide a selection marker. The PshAI sites were used to release the transposon sequence from the vector.

FIG. 3.

3 Salmonella enterica serovar Enteritidis unique DNA regions surrounding the transposon insertion sites in ES16 and ES47 mutants. (A) ORFs near the Tn insertion site in ES16; (B) ORFs near the Tn insertion site in ES47. The insertion site in each mutant is marked by an arrowhead.

FIG. 4.

Survival of Salmonella enterica serovar Typhimurium isolate ST3665 derivatives after incubation with egg albumen for 24 h. Salmonella enterica serovar Typhimurium ST3665, ST3665 transformed with plasmid vector pRB3-273C (ST3665[RB3-273C]), ST3665 transformed with plasmid containing SEN4287 (ST3665[RB3-SEN4287]), and Salmonella enterica serovar Enteritidis SE2472 were incubated with egg albumen at 37°C. The percentage of survival represents the ratio of surviving bacteria after 24 h of incubation to the input bacteria. The results from three independent experiments are shown. Error bars indicate standard deviation.