Dry-Resistance of Salmonella enterica subsp. enterica Serovar Enteritidis is Regulated by Both SEp22, a Novel Pathogenicity-Related Factor of Salmonella, and Nutrients

Abstract

Environmental isolates of Salmonella enterica serover Enteritidis (S. Enteritidis) clones were grown to the logarithmic phase, washed and re-suspended in saline or Luria-Bertani (LB) medium, and then 10-µL aliquots of the suspensions were dried overnight at room temperature. The dried bacteria were mixed with 1 mL of ice-cold PBS, suspended and examined for colony-forming activity. All of the pathogenic clones with high levels of SEp22, identical to Salmonella Dps, maintained good viability if suspended in LB medium prior to drying. However, none of the nonvirulent strains, exhibiting low levels of SEp22, survived. Similar results were obtained with sep22-knocked out mutants, suggesting that SEp22 is important for the acquisition of dry-resistance. Nutritional factors, such as LB medium, cabbage extracts, and egg yolk but not egg white, were shown to be necessary for the acquisition of dry-resistance, because none of the clones remained viable irrespective of SEp22 expression if suspended in saline. Scanning electron micrograms also supported the importance of nutrition, showing re-growth of the bacteria after drying in LB but not in saline. These results suggest the importance of both SEp22 expression and nutrients for the acquisition of dryresistance by S. Enteritidis.

Lactoferrin Inhibits the Acquisition of Dry-Resistance of <i>Salmonella</i> spp.

“…We found that all of them, except egg white, elevated Salmonella survival rates under dry conditions after they were added to the bacterial suspension in saline before the drying protocol (25). In the present study, lactoferrin and apolactoferrin did not elevate Salmonella dry-resistance in saline, rather they inhibited it when they were added to 20% LB medium in saline.…”

“…2, SECl#15-1, a pathogenic strain of environmental isolates of S. Enteritidis, survived in considerable numbers in the presence of LB at concentrations of more than 10%, but it did not survive in saline alone, showing nutrition-dependence in the acquisition of dry-resistance as suggested in a previous paper (25). Therefore, we used 20% LB in saline for suspending Salmonella before drying throughout this study.…”

“…In an earlier report on the mechanisms underlying acquisition of dry-resistance by S. Enteritidis (25), we showed that SEp22, which is a novel pathogenicityrelated factor of S. Enteritidis (26), and is identical to Salmonella Dps (10) (Amano et al, manuscript in preparation), plays a critical role in the establishment of resistance to dry-stress. Because Dps is an iron-binding protein like ferritin (1), and reported to be involved in the protection of DNA from stresses such as H 2 O 2 (30), heat and chemicals (16,31), it is interesting to study the roles of SEp22, Salmonella Dps, in the protection of Salmonella from dry-stress.…”

“…In this study, we used a new assay method which we had established, to measure/estimate Salmonella dryresistance at room temperature with an automatic drykeeper, as described in a previous paper (25). This method is easy and enables us to test many samples in filter-capped tubes (Fig.…”

“…The method for assaying dry-resistance was reported in a previous paper (25). In brief, 10 l of the washed bacterial suspension were mixed on ice with various amounts of lactoferrin, apolactoferrin and/or LB in saline in a final volume of 1 ml, and 10 l of each bacterial mixture containing about 1×10 6 cfu was placed at the bottom of a 50-ml sterile centrifugation tube equipped with a sterile 0.45-m pore-filtered cap (FRP, Germany, Fig.…”