Type 1 and type p fimbriae-adhesins isolated from novel e. coli strains, process for their preparation and uses thereof

A process for the preparation of the fimbriae-adhesins, the process comprising:

• (a) seeding E. coli bacterial strains CECT 4484 or CECT 4485, or both, in a liquid culture medium at about 37° C. until cultures are obtained at the beginning of the stationary phase (108-109 cfu/ml);
• (b) centrifuging the cultures and washing the obtained sediment;
• (c) resuspending the sediments washed in a physiological saline solution with a neutral pH and cold homogenizing same in a shear homogenizer for 2 to 10 minutes, at a speed of 10,000 to 25,000 rpm;
• (d) centrifuging the homogenates thus obtained at a speed of 25,000 to 45,000×g for 20-45 minutes, discarding the sediment containing the bacteria and retrieving the supernatant liquid;
• (e) subjecting the supernatant liquid containing the fimbriae to several cold saline precipitations for a time period between 2 and 15 hours, retrieving the precipitate by centrifugation;
• (f) reconstituting the precipitate thus obtained in an aqueous physiological solution of a neutral pH and dialyzing the obtained solution against distilled water;
• (g) treating the dialyzed, solution containing the fimbriae with sodium deoxycholate 5% at about 80° C.;
• (h) subjecting the product resulting from the previous step to chromatography in a gel filtration column containing Sephacryl S-200 and subjecting the chromatographed product located in the exclusion volume to another chromatography in a column containing Sepharose 4B, and collecting the inclusion volume to obtain the fimbriae.

A process for the preparation of the fimbriae-adhesins, the process comprising:

• (a) seeding E. coli bacterial strains CECT 4484 or CECT 4485, or both, in a liquid culture medium at about 37° C. until cultures are obtained at the beginning of the stationary phase (108-109 cfu/ml);
• (b) centrifuging the cultures and washing the obtained sediment;
• (c) resuspending the sediments washed in a physiological saline solution with a neutral pH and cold homogenizing same in a shear homogenizer for 2 to 10 minutes, at a speed of 10,000 to 25,000 rpm;
• (d) centrifuging the homogenates thus obtained at a speed of 10,000×g for 20 minutes, discarding the sediment containing the bacteria and retrieving the supernatant liquid;
• (e) subjecting the supernatant liquid containing the fimbriae to several cold saline precipitations for a time period between 2 and 15 hours, retrieving the precipitate by centrifugation;
• (f) reconstituting the precipitate thus obtained in an aqueous physiological solution of a neutral pH and dialyzing the obtained solution against distilled water;
• (g) treating the dialyzed solution containing the fimbriae with sodium deoxycholate 5% at about 80° C.;
• (h) subjecting the product resulting from the previous step to chromatography in a gel filtration column containing Sephacryl S-200 and subjecting the chromatographed product located in the exclusion volume to another chromatography in a column containing Sepharose 4B, and collecting the inclusion volume to obtain the fimbriae.

A method for the production of anti-fimbriae-adhesion antibodies for the diagnosis and typification of uropathogenic E. coli expressing type P fimbriae characteristic of CECT 4484 or type 1 fimbriae characteristic of CECT 4485, comprising;

• immunizing a mammal with a member selected from the group consisting of Type P fimbriae isolated from E. coli CECT 4484, Type I fimbriae isolated from E. coli CECT 4485, and mixtures thereof;
• collecting serum from said mammal;
• and
• isolating anti-fimbriae adhesion antibodies from said serum.