Simone Guglielmetti

The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept)

The probiotic approach represents a potentially effective and mild alternative strategy for the prevention and treatment of either inflammatory or allergic diseases. Several studies have shown that different bacterial strains can exert their probiotic abilities by influencing the host’s immune system, thereby modulating immune responses. However, the emerging concern regarding safety problems arising from the extensive use of live microbial cells is enhancing the interest in non-viable microorganisms or microbial cell extracts, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. The purpose of this review is to provide an overview of the scientific literature concerning studies in which dead microbial cells or crude microbial cell fractions have been used as health-promoting agents. Particular attention will be given to the modulation of host immune responses. Possible mechanisms determining the effect on the immune system will also be discussed. Finally, in the light of the FAO/WHO definition of probiotics, indicating that the word ‘probiotic’ should be restricted to products that contain live microorganisms, and considering the scientific evidence indicating that inactivated microbes can positively affect human health, we propose the new term ‘paraprobiotic’ to indicate the use of inactivated microbial cells or cell fractions to confer a health benefit to the consumer.

Six-Week Consumption of a Wild Blueberry Powder Drink Increases Bifidobacteria in the Human Gut

Wild blueberries are a rich source of polyphenols and other compounds that are highly metabolized by the intestinal microbiota and may, at the same time, affect the intestinal environment itself. A repeated-measure, crossover dietary intervention on human volunteers was designed to study the effect of six week consumption of a wild blueberry ( Vaccinium angustifolium ) drink, versus a placebo drink, in modulating the intestinal microbiota. Relative to total eubacteria, Bifidobacterium spp. significantly increased following blueberry treatment (P ≤ 0.05), while Lactobacillus acidophilus increased after both treatments (P ≤ 0.05). No significant differences were observed for Bacteroides spp., Prevotella spp., Enterococcus spp., and Clostridium coccoides . Bifidobacteria, which have been largely proposed to be of benefit for the host, appeared to be selectively favored suggesting an important role for the polyphenols and fiber present in wild blueberries. Results obtained suggest that regular consumption of a wild blueberry drink can positively modulate the composition of the intestinal microbiota.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium–host interactions

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Implication of an Outer Surface Lipoprotein in Adhesion of Bifidobacterium bifidum to Caco-2 Cells

ABSTRACT We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.

Oral Bacteria as Potential Probiotics for the Pharyngeal Mucosa

ABSTRACT The research described here was aimed at the selection of oral bacteria that displayed properties compatible with their potential use as probiotics for the pharyngeal mucosa. We included in the study 56 bacteria newly isolated from the pharynges of healthy donors, which were identified at the intraspecies level and characterized in vitro for their probiotic potential. The experiments led us to select two potential probiotic bacterial strains (Streptococcus salivarius RS1 and ST3) and to compare them with the prototype oral probiotic S. salivarius strain K12. All three strains efficiently bound to FaDu human epithelial pharyngeal cells and thereby antagonized Streptococcus pyogenes adhesion and growth. All were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. Immunological in vitro testing on a FaDu layer revealed different responses to RS1, ST3, and K12. RS1 and ST3 modulated NF-κB activation and biased proinflammatory cytokines at baseline and after interleukin-1β (IL-1β) induction. In conclusion, we suggest that the selected commensal streptococci represent potential pharyngeal probiotic candidates. They could display a good degree of adaptation to the host and possess potential immunomodulatory and anti-inflammatory properties.

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.

Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product

ABSTRACT Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties.

Modulation of Fecal Clostridiales Bacteria and Butyrate by Probiotic Intervention with Lactobacillus paracasei DG Varies among Healthy Adults

BACKGROUND The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ∼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.

Study of the adhesion of Bifidobacterium bifidum MIMBb75 to human intestinal cell lines.

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B.bifidum to only restricted distal sites of the gut.

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity.

ABSTRACT The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the hosts defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.