The defect in the metabolism of erythritol of the Brucella abortus B19 vaccine strain is unrelated with its attenuated virulence in mice.

The role of the defect in erythritol catabolism in the attenuated virulence of Brucella abortus B19 vaccine strain in mice was investigated by means of five different strains: (i) the erythritol sensitive B19 vaccine strain; (ii) a natural erythritol tolerant (NET) mutant obtained spontaneously from B19; (iii) an erythritol resistant derivative from B19 (FJS19) obtained by gene replacement of the deleted ery region; (iv) the erythritol resistant B. abortus 2308 reference virulent strain; and (v) an erythritol sensitive mutant (227 strain) obtained from strain 2308 by transposon insertion in the chromosomal ery region. Besides virulence for mice, erythritol oxidation as well as other phenotypic markers were tested in all the strains. The 2308 and FJS19 strains grew in the presence of erythritol and oxidized the sugar, whereas the B19 and 227 strains did not. The NET strain grew in presence of erythritol but was unable to oxidize it. The B19 vaccine strain and its two erythritol resistant derivatives, NET and FJS19, showed similar residual virulence and splenic time courses in mice. Moreover, the virulent strain 2308 and its erythritol sensitive derivative (227 strain) exhibited similar levels of splenic infection. Altogether, these results demonstrate that the genetic region implicated in erythritol catabolism is not related to the low virulence exhibited by B19 in mice.

Analysis and genetic manipulation of Shigella virulence determinants for vaccine development.

Shigellosis is a major public health problem in developing countries. Current epidemics of Shigella dysenteriae serotype 1 strains are particularly serious and are characterized by high mortality rates. A high proportion of the isolates are resistant to many of the antibiotics currently in use in these countries, a feature which seriously compromises clinical treatment of the infections. Efficacious vaccines are thus urgently needed. Basic studies on Shigella virulence factors, infections in laboratory models, and host responses has led to the development of several strategies for the production of vaccines. All of these are live oral vaccines involving bacteria capable of at least limited survival in the animal intestine and of carrying selected antigens to the mucosal immune system. One type of vaccine involves non-pathogenic shigellae, attenuated either by introduction of a requirement for aromatic amino acids (aroD) or by loss of the large plasmid that specifies bacterial invasion of the mucosal epithelium. S. dysenteriae 1 strains under development as vaccines need to be engineered to eliminate high level Shiga toxin production, and a rapid and effective method to achieve this was recently elaborated. The second type of vaccine is represented by hybrid strains consisting of a carrier organism, such as an attenuated Salmonella or an Escherichia coli K-12 strain carrying the Shigella invasion plasmid, and the selected foreign antigen that it produces, in all cases so far the Shigella O antigen polysaccharide.

Biological activities specified by antibiotic resistance plasmids.

Bacteria can display resistance to a wide spectrum of noxious agents and environmental conditions, and these properties are often mediated by genes located on extrachromosomal DNA elements called plasmids. Replication, vertical and horizontal transmission and evolution of these elements are discussed, and examples of the genes responsible for the resistance phenotypes are given. Selective forces that drive the evolution of new combinations of bacterial properties of particular importance in clinical situations are analysed.

Purification of alpha-hemolysin from an overproducing E. coli strain.

The genetic determinant of the alpha-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions. alpha-Hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified alpha-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the alpha-hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.