Preparation of live vaccines

A stable attenuated bacterial strain comprising five to seven attenuating metabolic drift (MD) mutations;

• wherein a first attenuating MD mutation is marked by dependence of growth on a first antibiotic and the strain bearing the first attenuating MD mutation grows as a mini colony in the presence of the first antibiotic;
• wherein a second attenuating MD mutation is marked by reversion to independence of growth in the absence of the first antibiotic;
• and
• wherein a third attenuating MD mutation is marked by dependence of growth on a second antibiotic and the strain bearing the third attenuating MD mutation grows as a mini colony in the presence of the second antibiotic;
• wherein a fourth attenuating MD mutation is marked by reversion to a third antibiotic;
• wherein the bacterial strain with independence of growth in the absence of the first antibiotic and the second antibiotic grows in a colony having a reduced size at least 50% as compared to a colony of the corresponding starting natural bacterial strain;
• wherein the bacterial strain resistant to the third antibiotic grows in a colony of reduced size but greater than 25% in size as compared to the corresponding starting natural bacterial strain;
• wherein said growth of bacterial strain resistant to the third antibiotic in a colony of said reduced size is a stable phenotype of the bacterial strain carrying the five to seven attenuating 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, Pasteurella/Avibacterium sp., Pseudomonas sp., Riemerella sp., Salmonella sp., Shigella sp., Staphylococcus aureus, and Yersinia sp.;
• 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;
• wherein the third antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin;
• wherein a colony of the attenuated bacterial strain having a 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 is not over-attenuated.