Described is a method for the generation of a live vaccine containing stable bacteria carrying at least three attenuating mutations and a vaccine containing bacteria obtained by said method. Particularly, said method comprises (i) growing a bacterial strain in the presence of a first antibiotic and isolating mini colonies that are antibiotic dependent, thereby introducing the first attenuating mutation, (ii) growing the mutant clone in the absence of the first antibiotic and isolating attenuated revertants characterised by a colony size which is >；50% of the colony size of the wild strain, and (iii) growing the obtained mutant clone in the presence of a second antibiotic wherein clones are isolated with a reduced colony size no less than 25% of the size of the wild strain. This method provides stably attenuated bacterial strains while avoiding over-attenuation.
