Susceptibility of germ-free pigs to challenge with protease mutants of Salmonella enterica serovar Typhimurium.

Salmonella protease mutants, clpP and especially htrA, are candidate live oral vaccines in humans. A functional and mature immune system is, however, required to cope with them in mice. Here, we test the cytokine response of highly susceptible germ-free pigs to infection with Salmonella Typhimurium clpP and htrA mutants. Cytokine levels (IL-4, IL-10, IL-18 and IFN-gamma) were measured by ELISA in plasma and washes from the terminal small bowel 24h after oral challenge. Unlike the infection with the wild type strain, no IFN-gamma response and low IL-18 intestinal levels were found in pigs infected with the protease mutants. Despite this and regardless of partially reduced ability of htrA and clpP mutants to invade and multiply in a 3D4 porcine macrophage-like cell line, both the mutants were as virulent as was the wild type LT2 strain and caused fatal septicaemia in germ-free pigs. IFN-gamma and IL-18 response therefore did not correlate with the virulence of Salmonella Typhimurium. Our results indicate that htrA and clpP attenuations should be used with caution in populations in which an increased number of immunocompromised individuals can be expected.

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system.

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.

Salmonella enterica subsp. enterica serovar Enteritidis harbours ColE1, ColE2, and rolling-circle-like replicating plasmids.

Using DNA hybridization, at least three distinct groups of low molecular mass plasmids were identified in Salmonella enterica subsp. enterica serovar Enteritidis. After sequencing representative plasmids from each group, we concluded that they belonged to ColE1, ColE2, and rolling-circle-like replicating plasmids. Plasmid pK (4245 bp) is a representative of widely distributed ColE1 plasmids. Plasmid pP (4301 bp) is homologous to ColE2 plasmids and was present predominantly in single-stranded DNA form. The smallest plasmids pJ (2096 bp) and pB (1983 bp) were classified as rolling-circle-like replicating plasmids. Both encoded only a single protein essential for their own replication, and they must have existed in an unusual molecular structure, as (i) they were capable of hybridization without denaturation, (ii) their DNA could be linearized with S1 nuclease, and (iii) even after such treatment, the ability to hybridize without denaturation did not disappear.