Clinical microbiologyPrebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile
Highlights
► Probiotic bifidobacteria were tested for stress tolerance, prebiotic degradation and AMA against CD. ► Bifidobacterium breve 46 and Bifidobacterium lactis 8:8 were robust to acid and porcine bile. ► B. breve 46, B. lactis 8:8 and Bifidobacterium longum 6:18 degraded GOS, IMOS and lactulose. ► These strains exhibited high AMA against CD when grown in medium with and without prebiotics. ► B breve 46 and B. lactis 8:8cells and their CFS inhibited growth of CD NAP1/027 and toxin production.
Introduction
Bifidobacteria is a major beneficial bacterial group in the healthy human gut microbiota, colonising the intestinal mucosa [1], [2]. They exhibit several health-promoting effects such as antimicrobial activity (AMA) against various enteric pathogens, immune-modulating properties decrease the serum cholesterol levels, reduce the incidence of colon cancer, and effective against gut disorders on a regular consumption as fermented food products [2], [3], [4], [5], [6]. They metabolize many complex carbohydrates that escape hydrolysis by digestive enzymes in the gut and reach the colon unabsorbed [2], [5], [7], [8]. Many non-digestible oligosaccharides (NDOs) in the gut act as prebiotics enhancing the growth of bifidobacteria, some lactic acid bacteria (LAB) and other beneficial gut microbes [9], [10], [11]. The two most studied prebiotic-NDOs are fructooligosaccharides (FOS) and galactooligosaccharides (GOS). Other prebiotic-NDOs such as xylooligosaccharides (XOS); isomaltooligosaccharides (IMOS), glucooligosaccharides, pectin oligosaccharides (POS) mannanooligosacharides (MOS), gentiooligosaccharides (GTO), chitooligosaccharides (CHOS), soy bean oligosaccharides (SOS) and polydextrose have also been evaluated but these substances, with high purity, are not commercially available [10], [11], [12].
To develop a synbiotic formulation, containing a single or a multi-strain probiotic and prebiotic mix, it is essential to screen probiotic strains based on a prebiotic index i.e. ability of strains to degrade several prebiotics and subsequently determine the prebiotic score i.e. the ability of each strain to degrade specific prebiotic-NDOs and explore how each prebiotic increase cell yields when grown in a defined medium. Some in vitro studies showed that a number of NDO's stimulate the growth of bifidobacteria and lactobacilli [13], [14], [15].
A long term antibiotic therapy may cause intestinal overgrowth of Clostridium difficile (CD), extended spectrum of β-lactamase producing Escherichia coli, methicillin-resistant Staphylococcus aureus, vancomycin resistant Enterococcus and other multiple resistant bacteria and Candida albicans [16]. The epidemic CD NAP1/027 strain could produce up to 16 times higher levels of toxin A and B and a binary toxin, than most hospital outbreak associated strains [17], [18]. Thus, there is an urgent need to develop alternative non-antibiotic based therapies such as probiotic and synbiotic dietary supplements, to stimulate and restore a healthy indigenous gut microbiota after various antimicrobial therapies [8], [9]. Several synbiotic supplements are reported to reduce inflammation, infection and promote immune modulation and alleviate other gastrointestinal disorders [8]. However, knowledge on the compatibility of strains in multi-strain synbiotic combinations, minimum effective dose to impart the desired health benefit without any side effects and the appropriate biomarkers in the in vivo trials are lacking, hence there is a need to address such issues. In the present study, potential probiotic properties of three enteric human bifidobacterial strains were analysed for (i) acid and bile tolerance, (ii) in vitro fermentation of selected prebiotic-NDOs i.e. FOS, GOS, IMOS, XOS, and lactulose to design optimal synbiotic combination and (iii) antimicrobial activity (AMA) against four clinical C. difficile strains including the virulent CD NAP1/027 strain. These properties were compared with three reference strains from two international type culture collections.
Section snippets
Chemicals
Proteinase K, pronase E, p-nitrophenol β-d-galactopyranoside, p-nitrophenol β-d-xylopyranoside and bromocresol purple were purchased from Sigma–Aldrich, St. Louis, Mo, USA. deMan Rogosa Sharp with l-cysteine-HCl (MRSC) agar was purchased from Oxoid Ltd, Basingstoke, UK. Native porcine bile (PB) was prepared as described previously [19]. Prebiotic oligosaccharides and polysaccharides studied are listed in Table 1.
Bacterial strains and culture conditions
All strains (Table 2) were maintained at −110 °C in Trypticase soy broth (TSB,
Acid and bile tolerance
The six bifidobacterial strains showed significant variations in the acid and bile tolerance tests (Fig. 1A). B. lactis strains showed the highest survival rate at pH 2.5 with no significant reduction (P > 0.05) compared with the other five strains. All strains were viable in MRSC broth at pH 2.5 after 30 min incubation (Fig. 1A). Four of the six tested strains retained 100% viability when grown in MRSC broth with 5% porcine bile for 4 h, except Bifidobacterium longum 6:18 and Bifidobacterium
Discussion
In the present study, acid and bile stress tolerance tests of bifidobacterial strains provided the first-level relevant strain selection criteria (Fig. 1). Two B. lactis strains were highly resistant to acid stress as reported by Masco et al., [24]. This acid tolerance could be due to an over expression of the F1Fo-ATPase as shown in the wild type and mutant strains for B. longum [25].
All strains except B. longum 6:18 showed ≥90% viability in the bile tolerance assay. The porcine bile simulate
Conclusions
B. breve 46 and B. lactis 8:8 strains were the robust as determined by acid and bile stress tolerance assays. Although B. longum 6:18 exhibited a high AMA against CD and a high prebiotic-NDO metabolizing ability, it did not survive bile stress, i.e. a less robust strain. These three in-house strains and three reference strains are highly active in degrading GOS, FOS, IMOS and lactulose. B. lactis 8:8 and B. longum 6:18 also metabolized XOS. All the bifidobacterial strains exhibited high AMA
Acknowledgements
Authors thank Ellen Ertmann at Friesland Campina for expert advice on the choice of GOS for this study. Authors also thank Prof A. Weintraub (Karolinska Institute, Stockholm, Sweden) for kindly providing the CD NAP1/027 strain, Dr. Torbjörn Norén (Clinical Microbiology Laboratory, University hospital, Örebro, Sweden) for performing PCR ribotyping of the CD strains and Sweet Town Biotech (Taiwan) for providing the XOS. This study was supported by a grant from the European community's seventh
References (38)
- et al.
Gut flora in health and disease
Lancet
(2003) - et al.
Progress in genomics, metabolism and biotechnology of bifidobacteria
Int J Food Microbiol
(2011) Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens
FEMS Microbiol Rev
(2004)- et al.
Probiotics in the gastrointestinal diseases of the elderly
J Nutr Health Aging
(2012) - et al.
Prebiotics for prevention of gut infections
Trends Food Sci Technol
(2012) Prebiotics: the concept revisited
J Nutr
(2007)- et al.
In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics
Anaerobe
(2010) - et al.
The role of toxin A and toxin B in the virulence of Clostridium difficile
Trends Microbiol
(2012) - et al.
Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe
Lancet
(2005) - et al.
Production of β-galactosidase by Bifidobacteria as influenced by various culture conditions
Int J Food Microbiol
(2005)
In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium
J Dairy Sci
Dietary fructooligosaccharides affect intestinal barrier function in healthy men
J Nutr
Effect of processing conditions on the prebiotic activity of commercial prebiotics
Int Dairy J
Inhibition of Clostridium difficile growth and adhesion to enterocytes by Bifidobacterium supernatants
Anaerobe
Surface protein from Lactobacillus kefir antagonizes in vitro cytotoxic effect of Clostridium difficile toxins
Anaerobe
In vitro investigations of the effect of probiotics and prebiotics on selected human intestinal pathogens
FEMS Microbiol Ecol
The in vitro inhibition of gram-negative pathogenic bacteria by bifidobacteria is caused by the production of organic acids
Int Dairy J
Lactic acid bacteria as probiotics
Curr Issues Intest Microbiol
The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome
Proc Nat Acad Sci U S A
Cited by (86)
Inulinase and fructooligosaccharide production from carob using Aspergillus niger A42 (ATCC 204447) under solid-state fermentation conditions
2023, International Journal of Biological MacromoleculesA synbiotic combination of Bifidobacterium longum Bif10 and Bifidobacterium breve Bif11, isomaltooligosaccharides and finger millet arabinoxylan prevents dextran sodium sulphate induced ulcerative colitis in mice
2023, International Journal of Biological MacromoleculesIron in infectious diseases friend or foe?: The role of gut microbiota
2023, Journal of Trace Elements in Medicine and Biology1.17 - Probiotics, Microbiome and the Concept of Cross-Feeding
2022, Comprehensive Gut Microbiota
- 1
K. K. K & P. A contributed equally to the experimental design, laboratory work and manuscript writing and are the first authors of this manuscript.