Molecular epidemiology of two international sprout-borne Salmonella outbreaks.

Sprout-borne Salmonella outbreaks in Finland have increased during the last 10 years. The latest two were caused by Salmonella enterica serovar Bovismorbificans (antigenic structure 6,8:r:1,5) in 1994 and S. enterica serovar Stanley (4,5, 12:d:1,2) in 1995. In this study, the restriction fragment length polymorphism of genomic DNA after pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance profiles of the outbreak and nonoutbreak strains were compared. In each separate outbreak, the PFGE patterns of the outbreak strains (40 strains of S. enterica serovar Bovismorbificans and 28 strains of S. enterica serovar Stanley) after digestion of genomic DNA with restriction enzyme XbaI were indistinguishable from each other but differed clearly from those of the nonoutbreak strains (26 strains of S. enterica serovar Bovismorbificans and 40 strains of S. enterica serovar Stanley). The restriction enzyme XhoI did not differentiate the outbreak and nonoutbreak strains. The S. enterica serovar Stanley strains associated with the outbreak also had a unique antimicrobial resistance pattern, whereas all S. enterica serovar Bovismorbificans strains, both outbreak and nonoutbreak strains, were sensitive to all antimicrobial agents tested. Thus, the molecular typing confirmed that the S. enterica serovar Bovismorbificans outbreak isolates from humans and sprout salad were identical and strongly supported the epidemiological finding that S. enterica serovar Stanley outbreak isolates also originated from contaminated alfalfa seeds. It also confirmed that the sources of similar outbreaks in Sweden in 1994 caused by S. enterica serovar Bovismorbificans and in the United States in 1995 caused by S. enterica serovar Stanley and the source of the Finnish outbreaks were common.

Conformation of Escherichia coli outer membrane protein OmpA produced in Bacillus subtilis: influence of lipopolysaccharide.

The conformation of the outer membrane protein OmpA of Escherichia coli produced in Bacillus subtilis and solubilized in Sarkosyl was studied by measuring its ability to bind OmpA-specific phage K3 and to inhibit F-mediated conjugation. The partially purified protein was inactive in both of these assays. Refolding of the protein in the presence of lipopolysaccharide resulted in preparations with full phage-binding and conjugation-inhibiting capacity, indicating the formation of surface-exposed loops of OmpA of native conformation. The finding is of importance for the potential use of outer membrane proteins of Gram-negative bacteria as vaccines.

Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: fusions with secretable peptides.

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step.

When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.

High level production of Escherichia coli outer membrane proteins OmpA and OmpF intracellularly in Bacillus subtilis.

A high yield of Escherichia coli outer membrane proteins OmpA (about 200 mg/l) and OmpF (about 100 mg/l) was obtained in Bacillus subtilis when produced intracellularly. The yield was more than 100-fold higher than the yield of these proteins by a similar vector containing the complete signal sequence of alpha-amylase of B. amyloliquefaciens. Both proteins isolated after breakage of the B. subtilis cells by low-speed centrifugation were about 70% pure and could be solubilized by Sarkosyl, SDS and guanidine hydrochloride.

A strong antibody response to the periplasmic C-terminal domain of the OmpA protein of Escherichia coli is produced by immunization with purified OmpA or with whole E. coli or Salmonella typhimurium bacteria.

We produced in Bacillus subtilis the complete, as well as the N-terminal two-thirds, OmpA protein of Escherichia coli (called here Bac-OmpA and Bac-OmpA-dN, respectively). These Bac-OmpA proteins were used to examine the immunological properties of different parts of OmpA, free of lipopolysaccharide and other components of the outer membrane. The full-length Bac-OmpA was indistinguishable from the authentic protein isolated from E. coli (Coli-OmpA) both as immunogen and as antigen in enzyme immunoassay (EIA). The N-terminal Bac-OmpA-dN was a poor immunogen which gave rise to significantly lower titers of anti-OmpA antibody than did the full-length OmpA preparations. When used as an antigen in EIA, the Bac-OmpA-dN detected anti-OmpA antibody in serum samples from animals immunized with the full-length OmpA much less efficiently than did either Bac-OmpA or Coli-OmpA. The periplasmic C-terminal domain therefore appears to be an immunodominant epitope of the purified OmpA protein. Also, when rabbits and mice were immunized with intact, live or dead E. coli, the antibody response detected by EIA with the full-length protein, Bac-OmpA, was much stronger than that detected with the N-terminal two-thirds, Bac-OmpA-dN. Similar results were obtained with the OmpA of Salmonella typhimurium. Because the ompA gene of enterobacteria is highly conserved, the Bac-OmpA might be useful as a group-specific EIA antigen to diagnose diseases caused by members of the family Enterobacteriaceae.