Identification of Synbiotics Conducive to Probiotics Adherence to Intestinal Mucosa Using an In Vitro Caco-2 and HT29-MTX Cell Model

Abstract

The ability of bacteria to adhere to the intestinal mucosa is a critical property necessary for the long-term colonization of the intestinal tract. This ability can be highly sensitive to the presence of prebiotics. However, limited data are available in this respect for beneficial bacteria such as probiotics or resident gut microbiota. We previously demonstrated that the presence of prebiotics may decrease adherence in several pre- and probiotic combinations. Thus, characterizing the interactions between numerous combinations involving different classes of pre- and probiotics can be crucial in identifying new synbiotics. Accordingly, here, we extend our prior analyses to evaluate the adhesion of five lactobacilli, six bifidobacteria, and one probiotic Escherichia coli strains, as commercial probiotics or promising probiotic candidates, together with the cariogenic Bifidobacterium dentium strain. As an in vitro intestinal mucosa model, Caco-2 and mucin-secreting HT29-MTX cells were co-cultured at 9:1 in the presence or absence of prebiotics. Commercial inulin-type fructooligosaccharide prebiotics Orafti® GR, Orafti® P95, and galactooligosaccharide-based prebiotic formula Vivinal®, including purified human milk oligosaccharides (HMOs) were added into the cultivation media as the sole sugar source (2.5% each). Adherence was tested using microtiter plates and was evaluated as the percentage of fluorescently labeled bacteria present in the wells after three washes. Consistent prebiotics-mediated enhanced adherence was observed only for the commercial probiotic strain E. coli O83. For the remaining strains, the presence of HMO or prebiotics Orafti® P95 or Orafti® GR decreased adherence, reaching statistical significance (p < 0.05) for three of out of eight (HMO) or five of out of 11 strains tested, respectively. Conversely, Vivinal® enhanced adhesion in six out of the 12 strains tested, and notably, it significantly attenuated the adherence of the cariogenic Bifidobacterium dentium Culture Collection of Dairy Microorganisms (CCDM) 318. To our knowledge, this represents the first report on the influence of commercial prebiotics and HMOs on the adhesion of the cariogenic Bifidobacterium sp. Vivinal® seems to be a promising prebiotic to be used in the formulation of synbiotics, supporting the adhesion of a wide range of probiotics, especially the strains B. bifidum BBV and BBM and the probiotic Escherichia coli O83.

2′‐Fucosyllactose promotes Lactobacillus rhamnosus KLDS 8001 to repair LPS‐induced damage in Caco‐2 cells

“…reported that the addition of inulin‐based prebiotics enhanced the adhesion of L . rhamnosus CCDM to Caco‐2 cells, which was speculated to be associated with enhanced negative charge strength of the strain (Krausova et al., 2021). These suggest that 2′‐FL, as an adhesion promoter, might provide an intestinal development advantage for KLDS 8001.…”

A Review on In vitro Cell Culture Model for Bacterial Adhesion and Invasion: From Simple Monoculture to Co-Culture Human Intestinal Epithelium Model

“…The co-cultures previously proposed in literature for evaluating the mechanism of bacterial adhesion and invasion were obtained by using mucus secreting HT29 subclones which is HT29-MTX to generate a mixed population of enterocytes and mucus secreting cells (Caco-2/HT29-MTX) resembling as closely as possible the intestinal epithelium (Table 2). Bacterial adhesion is influenced by surface characteristics, which are influenced by the structure and composition of the cell wall [1,12]. Krausova et al [1] aimed to identify potentially effective synbiotics by analyzing the adherence of bacterial strains to a Caco-2/HT29-MTX coculture cell line model that mimicking the intestinal epithelium.…”

“…Bacterial adhesion is influenced by surface characteristics, which are influenced by the structure and composition of the cell wall [1,12]. Krausova et al [1] aimed to identify potentially effective synbiotics by analyzing the adherence of bacterial strains to a Caco-2/HT29-MTX coculture cell line model that mimicking the intestinal epithelium. They reported high adherence for all strains tested after integrating HT29-MTX cells for mucin synthesis in this model.…”

“…The lack of mucus in Caco-2, on the other hand, permits easy access to the cells, leading to an overestimation of their permeability [13]. Consequently, the presence of intestinal mucus may significantly affect adherence, as observed in studies with cell lines that secrete or do not secrete mucus [1].…”

“…The intestinal adhesion and invasion of the epithelium are important stages in initiating the bacterial pathogenesis, as the virulent effects of intestinal pathogens rely on their ability to colonize and invade the intestinal mucosa [1][2][3]. Hence, the entry of bacteria into epithelial cells is essential for its pathogenicity, intracellular replication, spread to other tissues, and cause intestinal disease [4][5][6][7][8].…”