Preparation of live vaccines

A method of generating a stable attenuated bacterial strain carrying three to seven attenuating metabolic drift (MD) mutations, wherein said method comprises the steps of:

• (a) obtaining a starting natural bacterial strain and growing said strain in the presence of a first antibiotic to obtain a mini colony of the strain bearing the first attenuating MD mutation;
• (b) isolating colonies of the strain obtained in step (a) that are dependent on the first antibiotic, wherein the colonies have a mini colony size of ≤10% as compared to colonies of a corresponding starting natural bacterial strain;
• (c) growing a colony of the strain obtained in step (b) in the absence of the first antibiotic and isolating attenuated colonies characterized by a colony size of ≥50% as compared to colonies of the corresponding starting natural bacterial strain;
• (d) growing a colony of the strain obtained in step (c) in a culture medium supplemented with a second antibiotic;
• (e) isolating and serially passaging colonies of the strain obtained in step (d) to identify colonies stably being of greater than 25% in size as compared to colonies of the corresponding starting natural bacterial strain;
• and
• (f) isolating colonies of the strain obtained in step (e) having said size of greater than 25% as the stable attenuated bacterial strain,
• wherein the first antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, and nalidixic acid;
• wherein the second antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin;
• to provide the stable attenuated bacterial strain carrying said three to seven attenuating MD mutations
• wherein the bacterial strain is selected from the group consisting of Aeromonas species (sp.), Bacillus cereus, Bordetella sp., Campylobacter species (sp.), Escherichia coli, Klebsiella sp., Listeria sp., Ornithobacterium rhinotracheale, Pseudomonas sp., Riemerella sp., Pasteurella/Avibacterium sp., Salmonella sp., Shigella sp., S taphylococcus aureus, and Yersinia sp.;
• wherein a colony of the attenuated bacterial strain having the mini colony size of ≤10% as compared to a colony of the corresponding starting natural bacterial strain defines a mini colony;
• wherein the colony size is determined by the diameter of the colony;
• and
• wherein the stable attenuated bacterial strain carrying said three to seven attenuating MD mutations is not over-attenuated.