Cell-Free Spent Media Obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis Grown in Media Supplemented with 3′-Sialyllactose Modulate Virulence Gene Expression in Escherichia coli O157:H7 and Salmonella Typhimurium

Abstract

Complex oligosaccharides from human milk (HMO) possess an antimicrobial activity and can promote the growth of bifidobacteria such as Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulence effect on several pathogenic bacteria. Whey is rich in complex bovine milk oligosaccharides (BMO) structurally similar to HMO and B. crudilactis, a species of bovine origin, is able to metabolize some of those complex carbohydrates. This study focused on the ability of B. bifidum and B. crudilactis to grow in a culture medium supplemented in 3′-sialyllactose (3′SL) as the main source of carbon, a major BMO encountered in cow milk. Next, the effects of cell-free spent media (CFSM) were tested against virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Both strains were able to grow in presence of 3′SL, but B. crudilactis showed the best growth (7.92 ± 0.3 log cfu/ml) compared to B. bifidum (6.84 ± 0.9 log cfu/ml). Then, CFSM were tested for their effects on virulence gene expression by ler and hilA promoter activity of luminescent mutants of E. coli and S. Typhimurium, respectively, and on wild type strains of E. coli O157:H7 and S. Typhimurium using RT-qPCR. All CFSM resulted in significant under expression of the ler and hilA genes for the luminescent mutants and ler (ratios of −15.4 and −8.1 respectively) and qseA (ratios of −2.1 and −3.1) for the wild type strain of E. coli O157:H7. The 3′SL, a major BMO, combined with some bifidobacteria strains of bovine or human origin could therefore be an interesting synbiotic to maintain or restore the intestinal health of young children. These effects observed in vitro will be further investigated regarding the overall phenotype of pathogenic agents and the exact nature of the active molecules.

Bifidobacterium mongoliense genome seems particularly adapted to milk oligosaccharides digestion leading to production of antivirulent metabolites

“…The metabolites obtained from the fermentation of 3'SL by B. mongoliense could decrease pathogenicity of S. Typhimurium. This effect had already been observed with CFSM fermented by B. bifidum BBA1, where all the tested genes were under-expressed [42].…”

“…Furthermore, with CFSM obtained from medium fermented without any glucose, three genes were significantly down-regulated (fliC, luxS and stx1). In the study of Bondue et al [42], CFSM obtained from medium supplemented with 3'SL and fermented by B. bifidum BBA1 and B. crudilactis FR/62/B/3 presented a significant effect against E. coli O157:H7 ATCC 43890 virulence genes expression. Indeed, most of the virulence genes were down-regulated (ler, luxS, stxB2 and qseA), except the fliC gene, which tended to be up-regulated.…”

“…The media MRS2-G and MRS2-Wh are rich in carbohydrates, and fermentation products such as lactate and acetate are synthesised. To prevent an inhibition of pathogenic bacteria growth due to media acidification, all CFSM were neutralised before testing them with E. coli or S. Typhimurium as described previously [16,42].…”

“…Typhimurium [42] and these bioactive molecules could be related to some carbohydrate residues, if they are obtained from carbohydrate metabolism. β-galactosidase genes, necessary for 3'SL degradation, have been identified in the B. crudilactis LMG 23609 genome but do not seem sufficient to easily metabolise the BMO.…”

“…The applied method was fully described in the previous study of Bondue et al [42]. E. coli O157:H7 with a heating rate of 0.1℃/s.…”