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Gutierrez et al. Microbiome Res Rep 2023;2:36 https://dx.doi.org/10.20517/mrr.2023.37 Page 9 of 23

signaling compounds [18,95,96] . Furthermore, the presence of Bifidobacterium within the mucus layer influences
the spatial organization and composition of the gut microbiota, thereby impacting the overall microbial
ecosystem. In several studies, the ability to bind to the mucus layer allowed Bifidobacterium species to create
a niche and exclude pathogens [54,56,57,62,64,92,97] . One study found that a probiotic containing Bifidobacterium
could inhibit pathogenic colonization of Escherichia coli, and this protective effect was dependent on MUC2
[98]
expression by Caco-2 cells . This data suggests that mucus adhesion is critical for excluding pathogens. In
addition to excluding pathogens, Bifidobacterium species likely have synergistic interactions with other
commensal microbes in the mucus layer. Bifidobacterium has been shown to cross-fed commensal
Eubacterium rectale , E. hallii [100,101] , and Faecalibacterium prausnitzii . In each of these scenarios,
[99]
[102]
Bifidobacterium-commensal co-cultures generated elevated levels of butyrate, a beneficial short-chain fatty
acid, compared to the mono-cultures. The literature clearly indicates that Bifidobacterium species readily
bind to mucus, and this mucus adhesion likely sets the stage for a range of beneficial effects on both the host
and the gut microbial community.

MUCUS DEGRADATION BY BIFIDOBACTERIUM SPECIES
In addition to serving as a binding site for bacteria, mucus can act as a nutrient source. The mucin protein is
heavily O-glycosylated and has multiple structures of repeating α- and β-linked N-acetyl-galactosamine
(GalNAc), N-acetyl-glucosamine (GlcNAc), and galactose (Gal) residues, terminated with α-linked fucose
(Fuc), and sialic acid (Neu5Ac) residues . Mucus-degrading bacteria harbor specific glycosyl hydrolases
[103]
(GHs) that enzymatically degrade mucin glycans [3,4,103-106] . After cleavage, the released glycan oligosaccharides
can feed the bacteria or other microbes in the vicinity [3,107] . In order to degrade mucin glycans, intestinal
bacteria must possess GH33 sialidases (also known as neuraminidases), which cleave terminal sialic acid
residues. For efficient glycan cleavage, bacteria can also generate GH29 or GH95 to remove fucose residues.
Once the terminal sugars are removed, the underlying GalNAc, GlcNAc, and galactose residues can be
removed. Bacteria can have GH101 or GH129 to remove GalNAc, GH84, GH85, GH89, or GH20 to remove
GlcNAc, or GH2, GH35, GH42, and GH98 to remove galactose residues. Some bacteria also encode for
GH16, endo-acting O-glycanases that remove larger glycan structures. A recent genome analysis confirmed
that B. bifidum harbored the largest repertoire of mucus-degrading GHs among the Bifidobacterium
[103]
species . All B. bifidum genomes had GH33, GH29, GH95, GH20, GH2, GH42, GH101, GH129, GH89,
and GH84 , suggesting that this species was capable of cleaving sialic acid, fucose, GalNAc, GlcNAc, and
[103]
galactose from mucus glycans. B. breve, B. longum, and B. scardovii were also found to possess multiple
mucus-associated GHs. This finding is consistent with other genome studies and in vitro studies, which
report that B. bifidum, B. longum, and B. breve can degrade mucus [19,100,103,108-112] . In contrast, B. adolescentis,
B. angulatum, B. animalis, B. dentium, B. pseudolongum, and B. thermophilum possessed few mucus-
associated GHs . In vitro work confirmed that B. dentium and B. angulatum were unable to grow on pig
[103]
colonic mucus as the sole carbon source . Separate studies have also found that B. animalis subsp. lactis
[103]
and B. pseudolongum do not degrade mucus [100,113-115] . These studies suggest that, unlike mucus adhesion,
mucus degradation is not conserved in Bifidobacterium species .
[103]
Mucin degradation is considered to be a normal process of intestinal mucus turn-over and begins within
[116]
the first few months of life [117,118] . Infants are commonly colonized with mucin-degrading B. bifidum,
B. longum subsp. infantis, and B. breve [28-30,118] , as well as Akkermansia muciniphila and Bacteroides
species . Interestingly, breast-fed babies that are dominated by Bifidobacterium species exhibit a delay in
[116]
the mucin degradation profile as compared with babies fed with formula milk . Consistent with this
[118]
notion, Karav et al. found that supplementation of B. longum subsp. infantis EVC001 to healthy breast-fed
infants significantly reduced the proportion of free colonic mucin-derived O-glycans in the total glycan pool
to 1.87% compared to 37.68% in the control infants who did not receive supplemented B. longum . The
[119]