The in vitro inhibition of Gram-negative pathogenic bacteria by bifidobacteria is caused by the production of organic acids

Several Bifidobacterium strains, including commercial probiotics, were screened for the production of antibacterial substances active towards Gram-negative pathogenic bacteria. Agar spot assays, microtitre plate assays, and time-kill studies were performed. Bifidobacteria were found to exert strong inhibitory activity towards Gram-negative indicator bacteria, namely Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli C1845. The inhibitory mechanism was investigated and was shown to be dependent on the lowering of the pH of the medium and the production of organic acids, in particular acetic acid and lactic acid. The production of other antibacterial substances was examined by concentrating the cell-free culture supernatants by ammonium sulphate precipitation and chloroform/methanol extraction. The extracts of cultures of seven Bifidobacterium strains inhibited the growth of Gram-positive indicator bacteria, indicating that they produced a bacteriocin. The extract of a culture of Bifidobacterium longum BB536 was active against E. coli as well. However, the contribution of such inhibitory substances to the antibacterial activity of bifidobacteria towards Gram-negative pathogenic bacteria was found to be negligible.