From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense

Abstract

The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. Bifidobacterium mongoliense, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.

Keywords: bifidobacteria; dairy food microorganisms; microbe–host interactions; microbial genomics; microbiome; microbiota.

Fig 1

B. mongoliense pangenome, core genome, and phylogenetic tree. Panel (a) illustrates the pangenome size based on the sequential insertion of the 12 B. mongoliense genomes. Panel (b) shows the core-genome size via the sequential addition of the genomes, whereas panel (c) reports the number of TUGs per each B. mongoliense genome. Panel (d) represents the phylogenetic tree of the B. mongoliense species (left) and a color scheme incorporating the ANI values between each analyzed strain (right).

Fig 2

Carbohydrate-active enzymes of B. mongoliense and other bifidobacterial species harboring fermented foods. The graphical representation shows a map of GHs, GTs, and PL, with the percentage within each genome representing the enzyme’s corresponding gene frequency in the chromosome sequences of the species analyzed. The final column displays the cumulative prevalence of GH, GT, and PL in families with all strains.

Fig 3

Growth abilities of the B. mongoliense strains on different carbon sources. Growth performances of the B. mongoliense strains were measured by assessing optical density (OD) values after 96 h of incubation. For OD values between 0 and 0.3, the strain was assumed to be unable to grow in the sugar, whereas for OD values between 0.3 and 1, the strain was determined to be able to sustain growth on the particular carbohydrate. LNT, lacto-N-tetraose; FOS, fructooligosaccharides.

Fig 4

B. mongoliense BMONG18 growth observed through co-cultivation with other bifidobacterial species. The four panels show the cell number quantification of B. mongoliense BMONG18, B. bifidum PRL2010, B. longum PRL2022, and B. adolescentis PRL2023 strains in mono- and bi-association on the MRS medium supplemented with HMOs, starch, and mucin, indicated above each panel by qPCR. Results of the qPCR experiments are expressed by bars indicating fold change (FC) of cell number quantification after 24 h of growth on MRS medium compared with the respective inoculum (T0). The y-axis indicates the FC of genome copy number per milliliter of bacterial culture, whereas the x-axis displays the name of the strains involved in mono- and bi-associations. The dotted bars represent the amount of each bifidobacterial strain present at T0.

Fig 5

Transcriptional modulation of B. mongoliense BMONG18 genes when grown under three different broth conditions. Panels (a–b) summarize the number of genes per functional category. The image reports transcriptional modulation of genes, expressed as the average of the normalized count reads obtained from each independent biological triplicate.

Fig 6

Adhesion of B. mongoliense cells to HT29-MTX cell monolayers and mucin. Panel (a) depicts the quantification of the adhesion performances of different B. mongoliense strains to HT29-MTX cell monolayers, expressed as the adhesion index. The vertical bars indicate standard deviations, and the asterisks indicate Kruskal-Wallis test P values. Panel (b) shows light microscopic images of HT29-MTX monolayer cells as observed with Giemsa staining of B. mongoliense strains. The bifidobacterial strains shown in each image are (i) B. mongoliense 2256B, (ii) B. mongoliense BMONG18, (iii) B. mongoliense 2258B, (iv) B. mongoliense DSM 21395, (v) B. bifidum PRL2010, and (vi) B. animalis subsp. lactis Bb-12. Panel (c) represents the percent adhesion values of B. mongoliense strains to mucin. The percentage of relative adhesion for each strain is determined by calculating the CFU values before and after the adhesion assay. The error bars represent the standard deviation of all assays, and the asterisks indicate Kruskal-Wallis test P values < 0.05.