Bifidobacteria—Insight into clinical outcomes and mechanisms of its probiotic action

Accepted Manuscript Title: Review article: Bifidobacteria- Insight into clinical outcomes and mechanisms of its probiotic action Author: Amrita Sarkar Santanu Mandal PII: DOI: Reference:

S0944-5013(16)30432-3 http://dx.doi.org/doi:10.1016/j.micres.2016.07.001 MICRES 25919

To appear in: Received date: Revised date: Accepted date:

23-2-2016 12-6-2016 7-7-2016

Please cite this article as: Sarkar Amrita, Mandal Santanu.Review article: Bifidobacteria- Insight into clinical outcomes and mechanisms of its probiotic action.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review article: Bifidobacteria- Insight into clinical outcomes and mechanisms of its probiotic action Amrita Sarkar†,* Santanu Mandal† †

School of Chemistry and Manchester Institute of Biotechnology. The University of Manchester, 131 Princess

Street, Manchester, M1 7DN, UK. *

Corresponding author: Amrita Sarkar; Email: [email protected]

Graphical Abstract

Abstract The invasion of pathogens causes a disruption of the gut homeostasis. Innate immune responses and those triggered by endogenous microbiota form the first line of defence in our body. Pathogens often successfully overcome the resistances offered, calling for therapeutic intervention. Conventional strategy involving

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antibiotics might eradicate pathogens, but often leave the gut uncolonised and susceptible to recurrences. Probiotic supplements are useful alternatives. Bifidobacterium is one of widely studied probiotic genus, effective in restoring gut homeostasis. Mechanisms of probiotic action of bifidobacteria are several, often with strain-specificity. Analysis of streamlined literature reports reveal that although most studies report the probiotic aspect of bifidobacteria, sporadic documented contradictory results exist, challenging its therapeutic application and prompting studies to unambiguously establish the strain-associated probiotic activity and negate adverse effects prior to its clinical administration. Multi-strain/combinatorial therapy possibly relies on a combination of underlying operating mechanisms, each contributing towards enhanced probiotic efficacy, understanding which could help in developing customised formulations against targeted pathogens. Bifidogenic activity is also mediated by surface-associated structural components such as exopolysaccharides, lipoteichoic acids along with metabolites and bifidocins. This highlights scope for developing advanced structural therapeutic strategy which might be pivotal in replacing intact cell probiotics therapy. Keywords: gut microbiome; pathogen protection; clinical application; adverse effects; immunomodulatory effect; combinatorial therapy

1. Introduction Increasing population of antibiotic resistant virulent pathogens has triggered the search of alternate means of pathogen combat. Photopharmacotherapy and probiotics in combination with prebiotics has become routes of major diversion from conventional use of antibiotics (Velema et al. 2014; Wang et al. 2013). They not only serve as green channels in reducing built-up of active antimicrobial elements in the environment but also lower drug resistivity in deadly pathogens. Since the acceptance by United Nations FAO and WHO in 2001 (Castellazzi et al. 2013), probiotics have found extensive applications. Healthy gut bacteria in humans are acquired during birth, which confer health promoting effects to the host. This neonatal microbial population gradually alters with age serving as a biomarker for health. Reduction in intestinal probiotic community often poses serious risks of pathogenic invasions and colonisation of gut epithelium, intake of probiotic supplements can be particularly useful. Numerous bifidobacterial strains have found to be useful in treating different clinical conditions. Therefore it is likely that different strains operate through a combination of more than one pathway instead of a single mechanistic route. The present communication aims to provide the current perspective of the concepts evolving about the mechanisms of probiotic action of bifidobacteria in humans rather than a comprehensive

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literature summary. Understanding mechanisms could be helpful in identification of suitable probiotic strains for prevention and customised therapeutic treatment of infections with known pathogenesis and prognosis. Interestingly these apparently benign and mostly probiotic microorganisms have been reported to have adverse effects on human health. Although infrequent, but these reports emphasise on the need for in-depth study prior to developing probiotic formulation for clinical administration.

2. Clinical prospect of bifidobacteria Bifidobacterial flora in neonatal gut is established within the first few days after delivery and constitutes upto almost 80% of the microfloral composition during infancy (Turroni et al. 2012). The gut flora established at birth and weaning, gradually alters with age, dietary, seasonal and geographical variations along with intake of probiotics along with other endogeneous and exogeneous factors (Balamurugan et al. 2008; Lagier et al. 2012; Davenport 2014; Mitsuoka 1990). Clinical study shows that usage of antibiotics on neonates not only causes a delay in developing a normal bifidobacterial intestinal flora, but also increases risk of colonisation by pathogenic bacteria such as Kleibsella, Enterobacteria, Citrobacter, Pseudomonas etc (Hussey et al. 2011). The delayed and disturbed bifidobacterial colonisation during infancy increases the risks of childhood diseases and could also influence the health of the host in future (Marra et al. 2006; Alm et al 2008; Bailey et al 2014). 2.1. Beneficial effects. Breakdown of probiotic barrier against pathogens often leads to gastrointestinal problems such as gastroenteritis, enterocolitis, irritable bowel syndrome (IBS), diarrhoea and food allergies/intolerances (Bermudez-Brito et al. 2012; Guglielmetti et al. 2011; Groschwitz and Hogan 2009). Bifidobacteria shows efficacy in alleviating related symptoms of constipation, abdominal pain, flatulence, bloating by rebalancing the gut flora and preventing abnormal bacterial fermentation of food residues (O’Mahony et al. 2005). Specific strains of B. infantis and B. lactis along with Streptococcus thermophiles, L. acidophilus and L. rhamnosus show efficacy in alleviating symptoms of acute gastroenteritis in infants (Erdogan et

al.

2012;

Vandenplas

and

Hert

2011).

Diarrhoea

often

follows

antibiotic

therapy

or

chemotherapy/radiotherapy in cancer patients (Fox et al. 2015; Demers 2014). Administration of B. lactis BB12, B94, B. longum BB-536 and specific strains of B. Bifidum in combination with L. rhamnosus GG, L. acidophilus La-5, LAC-361 is reported to reduce episodes and symptoms of diarrhoea in infants and adults (Demers et al. 2014; Fox et al. 2015; İşlek et al. 2014; Dinleyici et al. 2013; El-Soud et al. 2015). The increasing trend of IBS among people (~15-20%) raises a major concern about the prevention and treatment strategies. Clinical study shows that administration of B. bifidum MIMBb75 strain as nutritional supplement could

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substantially relieve patients of IBS symptoms and promote a healthy life compared to those on placebo (Guglielmetti et al 2011). B. bifidum KCTC12199BP, B. lactis KCTC 11904BP, W51, B. longum KCTC12200BP are some of the other strains exhibiting efficacy in IBS therapy (Rossi et al. 2015; Yoon et al. 2014). While B. animalis DN-173010 is particularly found to be helpful in improving faecal colonic transit time in people with long transit times (Picard et al. 2005). In vitro studies using human intestinal HT-29 cell lines also revealed the anti-inflammatory effects of B. animalis subsp. lactis PBS075 (Presti et al. 2015). Similar studies using human colonic microbiota model reveal that B. longum subsp. infantis NCIMB 702205 strain has the ability to reduce the gut-derived lipopolysaccharide which is related to chronic inflammatory and metabolic diseases (Rodes et al. 2013). Specific strains of B. breve and B. lactis along with L. casei have been reported to improve intestinal motility along with lowering of nosocomial sepsis and mortality in neonates and infants diagnosed with necrotising enterocolitis (Braga et al. 2011; Dilli et al. 2015). Bifidobacteria is also reported to exhibit probiotic effects against Crohn’s disease, ulcerative colitis and pouchitis (Shen et al. 2014; Tamaki et al. 2015; Saez-Lara et al. 2015). Other health benefits include antagonistic effect on colorectal cancer and treatment of Helicobacter pylori infections (Khodadad et al. 2013; Chitapanarux et al. 2015; Chong 2014; Yoon et al. 2014; Lee et al. 2008; Bindels et al. 2012). Particularly interesting study recently showed B. bifidum PRL2010 assisting in cholesterol assimilation through upregulation of genes encoding putative transporters and reductases (Zanotti et al. 2015). While double blinded, randomised, placebo controlled trial involving Japanese obese adults showed Bifidobacterium animalis ssp. lactis GCL2505 assisted reduction of visceral fat (Takahashi et al. 2016). Bifidobacteria not only improves lactose utilisation but also helps in stabilisation of gut mucosal barrier (Kailasapathy and Chin 2000). Atopic dermatitis/eczema is a chronic and pruritic skin disorder affecting infants and adults of all age group (Eichenfield et al. 2014; Wollenberg e al. 2016). Although its cause is not clearly understood, may arise due to immunological disorder related to IgE mediated reactions. The first line of treatment includes hydrational and anti-inflammatory topical medications (Wollenberg et al. 2016; Ring et al. 2012) with continuous search for alternative nonpharmacologic therapies. The first role of probiotic bifidobacteria in prevention and therapeutic intervention in such atopic diseases is well established, with B. breve LMG 23729, B. longum BB536, B. lactis Bb-12 being some of the strains with reported efficacy (Enomoto et al. 2014; Meneghin et al. 2012), while B. lactis NCC 2818 is reported to alleviate symptoms of seasonal allergic rhinitis (Singh et al. 2013). Such allergies are possibly related to limited Th1/Th2 levels and delayed development of immune tolerances due to lower exposure to extrinsic allergens such as pollens during infancy. The probiotic microorganism mitigates

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immune parameters and lowers Th2 cytokine levels (Singh et al. 2013). Similarly double blinded, placebo controlled and randomised clinical trial with B. bifidum G9-1 and B. longum MM-2 in combination with Lactobacillus gasseri KS-13 is reported to improve quality of life in individuals suffering from seasonal allergies (Dennis et al. 2016). Urogenital infection (including bacterial vaginosis, urinary tract infections, fungal and yeast vaginitis) affects women globally. Development of antibiotic resistance and limited usage during pregnancy are some of the drawbacks of antibiotic administration. In vivo experiments involving intravaginal Staphylococcosis in mice triggered by Staphylococcus aureus reported the anti-staphylococcal activity of specific strains of B. animalis VKL and VKB along with Lactobacillus strains (Babenko et al. 2012). The probiotic strains used not only assists in the elimination of S. aureus but also in the establishment of a normal and stable vaginal microflora. Further research should be targeted towards developing such probiotic formulations for clinical applications. Reduction of risks of preterm delivery, obesity and stress management, treatment of oral plaque and improvement of symptoms related to depressive disorders and Schizophrenia (Akkasheh et al. 2015; Minami et al. 2015; Nishihira et al. 2014; Toiviainen et al. 2015; Tomasik et al. 2015; Myhre et al. 2011) are some of the numerous beneficial effects offered by bifidobacteria. Detrimental effects on immune activation, GI tract with inflammation caused by HIV infection are not completely restituted by combined antiretroviral therapy (cART). Recent study documents promising results in improving HIV induced damages by supplementing cART therapy with probiotic B. breve, B. infantis and B. longum strains along with L. acidophilus, L. plantarum, L. casei, L. delbrueckii ssp. bulgaricus and Streptococcus faecium (d’Ettorre et al. 2015). While certain species of bifidobacteria and lactobacilli have demonstrated oxalate degrading abilities thus opening scope for their application in treatment of recurrent calcium oxalate-based kidney stones (Kullin et al. 2016; Kirkali et. al 2015). Although strain specific effects are observed, yet these clinical results indicate boundless prospect of genus Bifidobacterium towards amelioration of human health. 2.2. Adverse effects. Though studies investigating anaerobic microbes in clinical isolates are rare, yet sporadic cases of adverse effects related to bifidobacteria have been reported. Bush et al. (2014) suggested that these apparently probiotic and benign organisms can also have adverse effect on human health. Their case study reports the sepsis of prosthetic joint post total hip arthroplasty. Clinical examination of fluid from joint revealed the presence of Bifidobacterium, possibly from intake of probiotic formulation (containing strains of B. bifidum, B. breve and B. longum along with Lactobacillus and Streptococcus) ingested by the patient. While, B. dentium,

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B. denticolens, B. inopinatum forms a class of bifidobacteria commonly associated with dental caries (Leahy et al. 2005; Mahlen and Clarridge 2009). For the first time occurrence of sepsis by B. longum in adult has been documented, while bacteremia in preterm infants has also been reported (Ha et al. 1999; Bourne et al. 1978; Weber et al. 2015). Certain strains of Bifidobacterium longum subsp. infantis, originating from administered probiotics have been identified by Bertelli et al. (2015) in a separate study as causative agents in multiple cases of bacteremia in preterm infants. Besides bacteremia, meningitis, endocarditis, peritonitis, intra-abdominal, enterocolitis, gynecologic and pulmonary infections involving Bifidobacterium spp. as causative agent has also been reported (Hata et al. 1988; Bourne et al. 1978; Weber et al. 2015; Wilson et al. 1972). Studies also attribute the occurrence of bacterial vaginosis to the increase in population of bifidobacteria along with other anaerobic microorganisms such as Bacterioides spp. (Schwiertz et al. 2015). Bifidobacteria mediated infections also include abdominal abscesses, obstetric and gynecologic and wounds (Brook and Frazier 1993). While specific B. breve strain has been identified as a causative agent of septicaemia in neonates, administered with probiotic formulations post operative procedures (Ohishi et al. 2010). Although such clinical incidences are often attributed to immune compromise in patients and prematurely born neonates rather than bifidobacteria itself (Margolles et al. 2011; Ohishi et al. 2010), it is nevertheless important to unambiguously establish the probiotic property of a particular strain prior to its clinical use. Report suggest that B. animalis subsp. animalis ATCC 25527T and B. infantis ATCC 15697T strains are capable of inducing duodenal and colonic inflammation in germ free IL10 deficient mice (Moran et al 2009). The authors suggest possible pathogenicity of these bifidobacteria strains in a susceptible host. Hence it is recommended that new probiotic strains for consumption should be identified from the commensal flora of humans devoid of any intrinsic antibiotic resistance, so that any rare probiotic infection could be eliminated (Borriello et al 2003). Although the number of bifidobacteria mediated infections has not raised much compared to its general consumption and clinical usage, a thorough understanding of their risks and benefits is unavoidable. Mechanisms of translocation of probiotic species from gastrointestinal to extraintestinal sites, their bloodstream survival, mucin degradation activity and possible infectivity should be considered carefully prior to its administration, especially to patients with challenged immune systems (Borriello et al 2003; Boyle et al 2006).

3. Modes of probiotic action Bifidogenic effects are largely known, yet the underlying mechanism is poorly understood. Bifidobacteria exerts probiotic effect against conditions ranging from IBS, ulcerative colitis, allergic diseases, Immunoglobulin E

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(IgE) associated diseases, atopic dermatitis (AD) to oral plaque. Thus it is quite unlikely that all probiotic bifidobacteria strains operate using same single mechanism. Of the numerous literature reports available, Table 1 selectively lists some, highlighting the possible underlying mechanisms of bifidogenic activity with a schematic overview in Fig. 1. Each of these actions involves detailed mechanistic pathways which have been discussed here. 3.1. Adhesion to the gut epithelium followed by colonisation. Adhesion to the intestinal mucosa is the primordial need for bacteria for prolonged persistence to exert their health promoting effects. This may be perceived though several mechanisms exploiting host cell surface glycan and lectin receptor/motifs to bacterial capsular polysaccharides (Esko and Sharon 2009; Fanning et al. 2012; Hidalgo-Cantabrana et al. 2015). Studies indicate strain specific interplay of lectins, lipoproteins, adhesion molecules, pili/appendages or exopolysaccharides assisted multifactorial binding to the gut mucosa (Gleinser et al. 2012; Guglielmetti et al. 2008; Kavanaugh et al. 2013; Roger et al. 2010). Successful sequencing of a large number of bifidobacterial genomes has enabled identification and prediction of genes encoding adhesion factors. Probiotic-mediated modulation of multi-faceted adhesion factors involving transcription, chaperone proteins and glycoside hydrolases have been attributed to the adherence of B. longum subsp. infantis ATCC 15697 to HT-29 and Caco2 intestinal cells (Kavanaugh et al. 2013).

In vitro studies show that cell wall surface lipoprotein, BopA,

functions as adhesion promoter in B. bifidum MIMBb75 to Caco-2 cells with its adhesion directly correlated to the increase in the lipoprotein expression (Gleinser et al. 2012; Guglielmetti et al. 2008). Later, studies readdressed the role of BopA mediator. Antiserum against BopA demonstrated the adhesion of B. bifidum to human colonic mucin to be BopA independent (Kainulainen et al. 2013). Such contradictory results suggest the need for sustained efforts to establish mechanisms involving protein mediated adhesions. Glycolytic enzymes are known to mediate cross-talk between human plasminogen and B. bifidum S16, B. breve BBSF, B. longum S123 and B. lactis BI07. Study show that the enolase contains pattern recognition receptors for the human plasminogen with high binding affinity (Candela et al. 2009). Electron microscopy and mucin binding assays attribute the adhesion of certain B. bifidum strains to human intestinal cell line HT29 to the surface associated Transaldolase (González-Rodríguez et al. 2012). It is suggested that such enzymes could also promote colonisation of GI epithelial tissues post adhesion. Recent study predicts the possibility of fimbrial attachment of B. longum subsp. longum to the GI epithelium. Putative fimbrial His-BL0675 A type protein (among the variants B, C, D and E) showed strong binding affinity to the surface oligosaccharides on porcine colonic mucine (Suzuki et al. 2016). The identified

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gene cluster in the bacterial genome are designated as major (FimA or FimP) and minor (FimB and/or FimQ) pilin subunit encoding genes (Foroni et al. 2011). Other studies involve identification of gene clusters responsible for pili biosynthesis in specific B. breve and B. bifidum strains (Motherway et al. 2011; Turroni et al. 2013). Type IVb tight adherence (Tad) pilus-encoding gene cluster tad2003 is identified for efficient gut colonisation of B. breve UCC2003 in murines. Transmission electron microscopy and atomic force microscopy also confirms the presence of pili structures on bacterial cells (Foroni et al. 2011; González-Rodríguez et al. 2012). Other than indigenous factors, exogenous contributions from prebiotic and human milk oligosaccharides (HMO) play key role in bacterial adhesion and colonisation (Bouhnik et al. 2004; Chichlowski et al. 2012; Hidalgo-Cantabrana et al. 2014; Kavanaugh et al. 2013; Salazar et al. 2015; Wickramasinghe et al. 2015). They not only serve as substrates for bifidobacterial metabolism, but have been reported to increase the adhesion and efficacy of Bifidobacterium spp (Kailasapathy and Chin 2000; Osman et al. 2006). This is possibly due to exogenous protective effects of the prebiotics, enhancing the survival rate of the probiotic cells within the gastrointestinal tract and stimulating growth and activity of the ingested probiotics along with other species residing within the gut microbiota (Kailasapathy and Chin 2000). Another interesting mechanism involves exopolysaccharide mediated bifidobacterial adhesion onto human intestinal mucosal surface (Inturri et al. 2014; López et al. 2012; Ranadheera et al. 2014; Salazar et al. 2014). Recent studies have identified gene cluster responsible for EPS production from genomes of most Bifidobacterium strains (Hidalgo-Cantabrana et al. 2014). Comparative study between B. breve A28 and B. bifidum A10, shows that the former strongly adheres to the intestinal cell lines due to higher production of EPS than the latter (Alp et al. 2010). 3.2. Production of metabolites and lowering of pH. Metabolism of dietary constituents (such as fructans and undigested carbohydrates) by intestinal probiotic community can generate molecules of physiological importance (Puertollano et al. 2014; Yasmin et al. 2015). Enzymes such as α, β-glucosidase, galactosidases and fructofuranosidases involving breakdown of the dietary carbohydrate residues has been reported to be expressed by probiotic strains in the intestine. Although the activity of the enzymes are dependent upon the carbon source available in the growth medium, yet B. lactis BB-12 is reported to show glucosidase and galactosidase activities irrespective of the media used. However reported diversity exists between different species of Bifidobacterium genus in terms of their inulin metabolic activity (Vuyst and Leroy 2011). The metabolites produced via heterofermentative pathways such as short chain fatty acids (SCFAs), polyunsaturated fatty acid (PUFA) derivatives,

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hydrogen peroxides, diacetyls and carbon dioxides are known to exert inhibitory effects on the growth of pathogens (Kailasapathy and Chin 2000; Matsuki et al. 2013; Sugahara et al. 2015; Vuyst and Leroy 2011). SCFA includes lactates, formats, acetates, propionates, butyrates and pimelates (Asarat et al. 2015; Sugahara et al. 2015; Tabasco et al. 2014). These and other organic acids play a key role in reducing intestinal pH, preventing the growth and colonisation by acid sensitive and putrefactive pathogens (Fukuda et al. 2011). Murine based in vivo experiments demonstrated the protective role of bifidobacterial acetate against enterohaemorrhagic Escherichia coli O157:H7, while lactate and acetates from B. breve DN-156 007 participate in regulating epithelial proliferation in the gut, contributing towards gut homeostasis (Fukuda et al. 2011; Matsuki et al. 2013). Antimutagenic effects of probiotics such as bifidobacteria have increased the use of such strains for reduction in levels of toxic pro-carcinogenic enzymes and colorectal cancers. The metabolites such as propionates and butyrates are known to exert antagonistic effect especially on colon carcinoma cell proliferation inducing apoptosis of carcinoma cells and transformed cell lines in humans (Bindels et al. 2012; Gioia et al. 2014; Lee et al. 2008). Other biosynthetic pathways of bifidobacteria include synthesis of Vitamin B12 and folates, conferring protection against cancer (Gioia et al. 2014; Rossi et al. 2011). The PUFAs are particularly interesting due to the anti-carcinogenic, anti-inflammatory, anti-atherogenic and anti-diabetic properties (Gioia et al. 2014). Thus fermentation of dietary carbohydrate components by probiotic species confers a range of health beneficial effects. 3.3. Release of bacteriocins. One of the popularly suggested mechanisms of probiotic effect of bifidobacteria is through the release of compounds (molecular masses ranging from ~ 3-130 kDa) known as bacteriocins (Martinez et al. 2013; Poltavska and Kovalenko 2012). These ribosomally synthesised antimicrobial compounds were first reported in 1980 (Martinez et al. 2013). They exhibit strain specific activity against intra-genus (narrow spectrum) or inter-genus (wide range) bacterial strains. Since their identification and characterisation, bacteriocins have been classified into four categories based on their molecular masses, thermal stability and amino acid composition (Klaenhammer 1993). These compounds generally exhibit broad range activity against some species of the genera Bacillus, Enterococcus, E. coli, Salmonella, Clostridium, Shigella, Staphylococcus and Streptococcus (Collado et al. 2005; Martinez et al. 2013). The bacteriocins commonly promote antimicrobial activity through pore formation/permeabilisation leading to cell lysis and preventing cell wall biosynthesis (Bajaj et al. 2015; Liu et al. 2015; Martinez et al. 2013). Their proteinaceous nature makes them resistant to the actions of enzymes such as amylase and lipase but susceptible towards proteinases (Collado et al. 2005). The maximum inhibitory effects of these bacteriocins have been found to be between 8-16 hours of cell

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culture (Fliss et al. 2010; Poltavska and Kovalenko 2012). This explains the maximum antimicrobial activity of specific bifidobacteria strains in their late logarithmic phase of growth. Bifidocin B from B. bifidum NCFB 1454 exhibits pH dependent absorption onto cell surface receptors of gram positive bacteria, leading to cell death (Klaenhammer 1993). Similarly Bifidocin A isolated from B. animalis BB04 exhibits wide range of bactericidal activity (against gram positive and negative bacteria and some yeasts) through cell lysis of pathogens, while acidocin B isolated from Bifidobacterium sp. inhibits Clostridium sp. in fermented food products (Bali et al. 2014; Liu et al. 2015). Antibiotic sensitive and resistant strains of H. pylori are inhibited by thermally stable bifidobacteria bacteriocins (at high temperatures of 80 and 100 °C) (Collado et al. 2005). Other bacteriocins produced by genus Bifidobacterium include bifidin I (from B. bifidum), bifilact (from B. lactis), thermophilicin (from B. thermophilum), bifilong and bisin (from B. longum) (Liu et al. 2015). These bacteriocins in general possess stability in a wide pH range of 3-10 and also towards the action of weak organic solvents. They have significant thermal stability and are resistant towards refrigeration, freezing, action of salts and enzymes. A more detailed understanding of their mechanism of action could promote them as potential candidates for application in food processing and preservation, heath care, prophylactic and pharmaceutical industries. 3.4. Enhancement of the Epithelial Barrier. Human GI tract is covered with a lining of epithelium decorated with a layer of protruding villi which isolates interior of the body from the external environment. The space in between is sealed with tight junctions which regulate though trans-membrane proteins and actin cytoskeleton (Ulluwishewa et al. 2011). The dynamic yet tight epithelial junctions between the villi prevent the entry of pathogens and undesirable metabolites into the blood stream while allowing essential nutrient to pass through, upon stimulation. However certain pathogens like Vibrio cholerae release endogenous toxins, increasing the permeability of the epithelial junctions (Arrieta et al. 2006). This often leads to serious chronic disorders known as Leaky gut syndrome (LGS) and IBS. Symptoms of LGS include inflammation and irritation allowing uninterrupted influx of pathogenic toxins into the blood stream compromising the endocrine and lymphatic system (Saggioro et al. 2014). Other than pathogen invasions, toxins, pro-inflammatory cytokines, inflammatory mediators, mast cells, dietary conditions, oxidative stress and production of hydrogen peroxide are some of the factors contributing to the lowering of epithelial barrier function of the human gut. Probiotic bifidobacteria operates through strain and dose dependent enhancement of gut epithelial barrier by several different ways. They stimulate the secretion of thick mucus, a layer that is being constantly replaced, thus preventing the strong adhesion of pathogens. Trans-

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epithelial electrical resistance measurements show prevention of tumor necrosis factor (TNF-α) mediated disruption of epithelial barrier by certain bifidobacteria species (Hsieh et al. 2015; Ulluwishewa et al. 2011). Studies also report bifidobacteria mediated wound repair of Caco-2 cell monolayer pre-treated with TNF-α (Sultana et al. 2013). The enhancement of the barrier with increase in tight junction function is also attributed to the modulation of associated trans-membrane proteins (Hsieh et al. 2015). 3.5. Immunomodulatory effects. Many of the immunomodulatory bioactive signalling molecules of probiotics are microbe-associated molecular patterns (MAMPs) which interact with trans-membrane host pattern recognition receptors (PRRs) like toll-like receptors (TLRs) (Tomoda et al. 2015). The exact mechanism of interaction in bifidobacteria is still largely unknown, however may be mediated CHAP domain of TgaA or by MAMP-TLR2 mapping (Guglielmetti et al. 2014; Ventura et al. 2014). Bifidobacteria is reported to exhibit not just species but strain specific immunomodulatory potency. B. adolescentis DB-2458 and B. longum subsp. infantis GB-1496 isolated from human breast milk exerts strong immunomodulatory effects through Th1/Th2 associated cytokine regulation (Chiu et al. 2014). This increases the scope of their incorporation in probiotic formulation for infants. A dose dependent regulation of Th1/Th2 cytokine levels is observed in mitogenstimulated human peripheral blood mononuclear cells (PBMCs) by B. bifidus and B. infantis strains in combinations with L. acidophilus (Li et al. 2011). Dendritic cells (DCs) form the gateway to our immune system. B. breve CNCM1-4035 and its culture supernatant is capable of improving innate immune responses of human intestinal DCs against pathogenic Salmonella enterica serovar Typhi through TLR signalling pathways (Bermudez-Brito et al. 2013). The increase in anti-inflammatory cytokine IL-10 compared to pro-inflammatory cytokines and chemokines, TNF-α, is crucial in maintaining homeostasis against Salmonella inflammation. Upregulation of TLR9 gene transcription by both intact live cells and cell free culture supernatant of B. breve indicate that TLR9 signalling is a pathway for anti-inflammatory effects (Bermudez-Brito et al. 2013). While B. animalis subsp. lactis BB-12 is shown to down-regulate the expression of TLR-2 on PBMC and corresponding pro-inflammatory cytokine secretion in adults (Meng et al. 2015). Modulation of immune responses in vitro by induction of IL-10 production has also been reported for some members of B. adolescentis, B. longum and B. bifidum species (Vieira et al. 2015; Vitaliti et al. 2014). A combination of L. rhamnosus and B. breve M-16V is reported to supress the immune responses induced by cigarette smoke. In vitro expression of IL-1β, IL-6, IL-10, IL-23, TNFα, CXCL-8 and HMGB1 release in human THP-1 macrophages along with cigarette smoke-induced activation of TLR4, TLR9 and NF-κB signalling pathways were reported to be suppressed by the probiotic microorganisms (Mortaz et al. 2015).

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Conjointly B. breve BR03 and L. salivarius LS01 show clinical efficacy in alleviating symptoms of allergic asthma, improving the functioning of lung. They increase the release of IL-10, an inhibitor of proinflammatory cytokines IL-13, IL-17 and TGF-β and rebalance Th1/Th2 levels (Drago et al. 2015). Serine protease forms an important class of effector molecule encoded by genes found in several bifidobacterial species. They inhibit the action of human proteases like neutrophils and pancreatic elastases, which are found at the site of intestinal inflammation (Ventura et al. 2014). 3.6. Competitive exclusion of pathogens. The gut microbiome is a natural environment harbouring a diverse collection of microbes. The key to existence of individual species is space for growth, binding receptor sites and access to nutritional resources within the GI tract. Hence, competition between individual species determines the homeostatic composition of the gut microbiota. Probiotic bacteria operate through strain specific antagonistic mechanisms for competitive exclusion of pathogens (Gueimonde et al. 2007). B. breve CNCM I-4035 is found to exhibit inhibitory effects on the growth of enterotoxigenic (ETEC) and enteropathogenic (EPEC) bacteria, while other species were found to inhibit adhesion of pathogens onto the intestinal mucus (Collado et al. 2006; Muñoz-Quezada et al. 2013; Uraipan et al. 2015). MAMP-PRR interactions play key role in association of pathogens with the intestinal epithelia. Probiotics also express molecular patterns which recognise the same trans-membrane receptors as the pathogens, thereby blocking the sites for pathogenic access by competitive exclusion and sometimes displacing already attached pathogens (Madsen 2012). Prebiotics often assists the competitive exclusion of enteric pathogen by competing for pathogen binding sites (Becerra et al. 2015; Chichlowski et al. 2012). The ability of bifidobacteria to efficiently sequester iron in the gut is a mechanism of competition for nutrients, causing iron starvation in EPEC bacteria (Vazquez-Gutierrez et al. 2015).

4. Roles of surface associated structural components. Research interest at present focuses on understanding the probiotic activity of cell wall components, to devise structural component based advanced therapeutics, replacing the use of intact cells. However the molecular basis of the strain-specific probiotic actions mediated by these effector molecules need to be thoroughly investigated. Of the different components associated, notable interest lies with exopolysaccharides, peptidoglycans and lipoteichoic acids. 4.1. Exopolysaccharides and biofilms. Densely packed communities of microbes surround themselves in slimy exopolymeric matrices known as biofilms. Polysaccharides being a major constituent in these matrices, referred to as exopolysacharides (EPS) have thus gained extensive attention. In this regard, works by Ruas-Madiedo,

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Reyes-Gavilán and Sinderen are particularly noteworthy (Fanning et al. 2012; Hidalgo-Cantabrana et al. 2013 and 2014; Ruas-Madiedo et al. 2009; Salazar et al. 2008). Bifidobacteria strains are popularly associated with such exopolymeric substances, forming an interfacial layer separating the bacteria from its surrounding environment. Mechanistic investigation of mode of action of intact bifidobacteria cells has convincingly shown the assistance of exopolysaccharides in its probiotic activity. Thus efforts have been made to sequence genomes and identify genes related to EPS biosynthesis. Chemical identification of monosaccharide composition of purified EPS has been achieved for several Bifidobacterium spp. strains. These studies indicate variability in structural constitution and related gene clusters among species/subspecies within Bifidobacterium genus. Primordial challenge faced in devising probiotic supplement is the viability of the microorganism under physiological conditions of GI tract and retaining its probiotic property. Inspite of strain specific viability of bacteria, presence of EPS layer is attributed to its enhanced survival in human gut. Numerous direct and indirect experiments show that presence of EPS provides long term resistance to bifidobacteria towards the action of bile salts and acids (Alp et al. 2010; Fanning et al. 2012; Sánchez et al. 2014; Wickramasinghe et al. 2015). In fact reports suggest that bile induces/affects the production of EPS in certain strains of B. animalis and B. longum (Alp et al. 2010; Hidalgo-Cantabrana et al. 2013; Salazar et al. 2008; Sánchez et al. 2014). Recently through combined transcriptome and physiological approach, it was showed that enhanced acid tolerance of B. breve BB8dpH compared to its parent B. breve BB8 is related to expression of genes involved in biosynthesis of cell wall components, namely, peptidoglycan and EPS, along with those involved in carbohydrate transport, metabolism and energy production pathways (Yang et al. 2015). Similarly upregulation of several genes related to EPS synthesis in B. longum subsp. longum BBMN 68 was observed post acid adaptation (Jin et al. 2015). Along with EPS induced adhesion of bifidobacteria to intestinal epithelia (Inturri et al. 2014; López et al. 2012; Ventura et al. 2014), large number of studies have been aimed at its contribution in inhibition/exclusion of pathogens. EPS isolated from B. bifidum WBIN03, facilitates the growth of lactobacilli along with other anaerobic bacteria while simultaneously inhibits the growth of enterobacteria, enterococci and Bacteroides fragilis. 80μg/ml of EPS from B. longum BCRC 14634 is reported to inhibit food spoilage and pathogenic bacteria (Wu et al. 2010). The probiotic activity of EPS is attributed to its interference with the cell division process of the pathogens rather than cell lysis. While B. bifidum PRL2010 isolated from infant faeces were found to adhere onto human intestinal Caco-2 and HT-29 cell monolayers, thus preventing the adhesion of pathogenic E. coli and Cronobacter sakazakii (Serafini et al. 2013). It was also shown that EPS in EPS+B. breve

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UCC2003 is responsible for its persistence in murine gut and colon and assisted in reducing colonisation by pathogenic C. rodentium (Fanning et al. 2012). Although no specific host receptors have been identified in bifidobacterial EPS recognition, EPS has been suggested as an effector in cross-talk between probiotic microorganism and host immune system (Hidalgo-Cantabrana et al. 2014; Turroni et al. 2014). EPS+B. breve UCC2003 treated mice when infected with C. rodentium shows lower levels of pro-inflammatory cytokines compared to the ones treated with EPS-B. breve UCC2003 strains, which has higher levels of IL-12, IFN-γ TNFα (Fanning et al. 2012). While treatment of murine macrophage cell line J77A.1 enhances the IL-10 levels and suppresses lipopolysaccharide (LPS) activity and TNF-α levels (Wu et al. 2010). An increase in suppressorregulatory TGF-β cytokine level is observed in Wistar rats fed with EPS producing B. animalis subsp. lactis IPLA R1 and B. longum IPLA E44 strains (Salazar et al.2014). 4.2. Petidoglycan. These biopolymers are one of the major components of the bifidobacteria cell walls and have been associated to its acid tolerance and immunomodulatory effects. In fact resistance of Bifidobacterium to acid stress is known to improve after pre-stressing cells at low pH. The acid tolerance response (ATR) at significantly low pH has been shown to increase the protein abundance related in peptidoglycan synthesis and other metabolic processes of B. longum subsp. longum BBMN68 (Jin et al. 2015; Zhang et al. 2015). The changes in EPS, peptidoglycan and cyclopropane fatty acid content of bifidobacteria induced by acid adaptation results in the strengthening of the cell wall, thereby blocking the entry of H + (Jin et al. 2012; Sánchez et al. 2014). A3α-peptidoglycan from Bifidobacterium cell wall is seen to enhance immune response promoting health in sea cucumber, Apostichopus japonicas, against Vibrio splendidus infection (Zhang et al. 2015). While peptidoglycan from B. bifidum induces apoptosis in colorectal carcinoma cells in mice (Li-sheng et al. 1999). 4.3. Lipoteichoic acids and lipoglycans. Cell surface associated lipoteichoic acid (LTA) often serves as major virulence factors in gram positive bacteria. These complex glycolipid structures (lipofuranan linked to monoglycerophospate groups with D-alanine substitutions) mediate interaction with host receptors, triggering signalling pathways, resulting in probiotic/pathogenic effects (Lebeer et al. 2010; Lee et al. 2013; Lynch et al. 2004). Along with LTA, closely resembling structures of macroamphiphilic lipoglycans have been reported in bifidobacteria, having L-alanine substituted single glycerol phosphate monomeric side chains attached to the glycan backbone by phosphodiester linkages (Lebeer et al. 2012; Fischer et al. 1987). These lipoglycans are also classified as type V LTA (Schneewind et al. 2014). Immunomodulatory role of LTA in probiotic Lactobacillus through TLR2 recognition is well established. Although these biopolymers have long been identified in bifidobacteria, their roles in PRR mediated interaction in host have not yet been properly studied. However Guo

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et al. (2015) to our best knowledge first showed bifidobacterial LTA in combination with 5-fluorouracil to exhibit anti-tumor effects which alleviate chemotherapeutic side effects by immune modulatory activity (Xie et al. 2012). Nevertheless, there is clear need for more research in this direction along with in vivo demonstration of LTA mediated biological activities. As seen from discussion, clinical reports show that administration of bifidobacteria along with bacteria belonging to other probiotic genera to exhibit combinatorial effects in a host (Guo et al 2015; Isidro et al. 2015; Mortaz et al 2015). Bifidobacteria have been widely reported to exhibit strain specific probiotic activity through immune modulation, pathogen exclusion, production of metabolites and bacteriocins and surface associated biopolymers. Such differential behaviour could be ascribed to the strain-dependent molecular mechanism of operation. Enhanced efficacy of multi-strain/combinatorial therapy reported in pathogen combat possibly arises due to a combination of several probiotic mechanisms, with each strain making their own contributions. Although such therapy has been widely put to use, underlying mechanisms attributed to each component has not been much analysed. Careful selection of strains based on their probiotic mechanism could help in developing targeted formulations against different pathogens.

7. Conclusion The increased studies aimed at establishing probiotic mechanisms and determining clinical efficacy in the past decade indicates the promising future of bifidobacteria in replacing partially/completely antibiotics in the treatment of several pathological conditions. Probiotics have bestowed beneficial health effects in long term therapies especially in immune-mediated and atopic diseases almost in all age groups. Simultaneously infrequent studies report contradictory findings challenge the use of the probiotic microorganism, prompting the need for in-depth study prior to their administration to patients. Successful combinatorial therapy calls for analysis of strain-dependent probiotic mechanisms involved. Such efforts could help in selecting strains for customised formulations with synergistic effects against specific pathogens. Intake of intact cells along with beneficial health effects might pose risks of mutations and intrinsic resistance leading to antibiotic resistant phenotypes. The advancement in the field of probiotics has begun the quest for developing new era probiotics which aim to replace the use of live intact microorganisms by their heat inactivated counterparts or structural components responsible for their probiotic activity. Purified cell-surface components such as EPS, LTA along with metabolites and bifidocins might play a pivotal role in replacing intact cell therapy. Inspite of the promising prospect of bifidobacteria, corroborative studies are needed for

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understanding the role of bifidobacteria in altering gut microbial populations and associated physiological effects in humans.

Conflict of Interest: There is no conflict of authorship and both the authors approve the final version of the manuscript.

Acknowledgement. The authors would like to express their gratitude towards The University of Manchester. Both the authors are currently Research Associates at The University of Manchester. Dr. Amrita Sarkar is the submission’s guarantor. The topic for the review is conceived by her. She has been involved in the major literature study, data streamlining, graphic designing and manuscript writing. Dr. Santanu Mandal has assisted in scientific discussions and editing the manuscript.

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Zhang CY, Chen GF, Wang YY, Xu Z, Wang

YG, Song XL. (2015) A3α-peptidoglycan extracted

from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicas. Aquacult Nutr 21, 679-689. Zihler A, Blay GL, Chassard C, Braegger CP, Lacroix C. (2014) Bifidobacterium thermophilum RBL67 Inhibits Salmonella enterica Serovar Typhimurium in an In vitro Intestinal Fermentation Model. J Food Nutr Disor S1003, 1-10.

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Fig. 1. Schematic representation of protective mechanism of bifidobacteria displayed against pathogen invasion and in immunomodulation in host intestinal epithelium.

31

Table 1. Selective list of studies reporting different mechanisms of probiotic action of bifidobacteria documented through in vitro and in vivo experiments. Bacteria strains

Analysis

Mechanism of probiotic action

Ref.

Probiotic: (180 isolates analysed) B.

Intact cells, in vitro,

7 strains (B. longum L25, B. pseudocatenulatum

longum, B. pseudocatenulatum, B. bifidum,

Caco-2 cells, agar spot

C63, H34, B. catenulatum L21, L48, L51, B.

et al

B. adolescentis, B. catenulatum, B. dentium,

test

animalis E43) were chosen on basis of bile, acid,

2008

B. animalis

antibiotic resistance, enzymatic activities and

Pathogenic: E. coli wt 555, Salmonella

growth in culture medium.

enterica var. Typhimurium LT2UK1,

(1) Bile (45% of 180 isolates) and low pH (20% of

Staphylococcus aureus ATCC 1448, Listeria

total isolates, pH 3.5) tolerant

monocytogenes 1078, Bacillus cereus NCIB

(2) Strain dependent adhesion to Caco-2 intestinal

1771

cells lines, with L48 and L51 showing better

Delgado

adhesion compared L21. L25 and H43 showed lowest adhesion (3) All 7 strains inhibited pathogenic attachment to Caco-2 cell line (except Bacillus cereus) (4) E43 weakly inhibited only E. coli and L. monocytogenes.

Probiotic: B. breve UCC2003

Intact, in vivo (mice

(1) B. breve-EPS related (a) increased tolerance to

Fanning

Pathogenic: Citrobacter rodentium ICC180

model), flow

adverse GI tract conditions (b) increased

et al.

cytometry,

persistence in gut and (c) reduced colonisation of

2012

bioluminescent

pathogen (2) Reduction of levels of pro-

imaging, agglutination

inflammatory cytokines (3) IFN-γ–, TNF-α–, IL-

assay

12–positive T cells, and IL-12–positive B cells were significantly attenuated.

Probiotic: B. infantis BCRC 14602 Pathogenic and other strains: B. infantis BCRC 14602, B. adolescentis BCRC 14606,

Agar well diffusion,

(1) Antimicrobial activity of B. infantis BCRC

Cheikhy

cell free culture

14602 by production of proteinaceous bacteriocins

oussef et

supernatant (CFS),

(~ 3.0 kDa) against Salmonella, Shigella, E. coli,

al. 2009

32

B. bifidum BCRC 14615, B. longum BCRC

kinetic study of

Clostridium, Bacillus, Staphylococcus certain

14634, L. acidophilus SBDC, L. plantarum

bacteriocins

Lactobacillus and intragenus strains (except

BCRC 11697, isolate from Chinese Tibet

production, Bradford

producer strain) (2) Bacteriocin activity was

Koumiss, L. delbrueckii subsp. delbrueckii

assay, Tricine-SDS-

inactivated upon treatment with proteolytic

MLCC, L. paracasei subsp. paracasei

PAGE

enzymes, but not with catalase, α-amylase and

MLCC, L. casi W2 SBDC, L. fermentum

lipase (3) Bacteriocin exhibited high temperature

ATCC 9338, L. helveticus BCRC 14097, L.

stability up to 121 C for 15 min pH stability in the

lactis ATCC 11454, Pediococcus

range of 4-10

pentosaceus ATCC 33316, Salmonella sp. MLCC, Salmonella enteritidis CGMCC (B) 50041, Salmonella typhimurium ATCC 29631, Salmonella enterica ssp. enterica ATCC 13076, E. coli TG1 MLCC, E. coli GH5α MLCC, E. coli AS 1.543 MLCC, Staphylococcus aureus AS 1.72 MLCC, Bacillus subtilis MLCC, Bacillus cereus ATCC 14574, Clostridium butyricum MLCC, Shigella dysenteriae CGMCC (B) 51387, Sacchromyces cerevisiae ATCC 40075

Probiotic: B. thermophilum RBL67

In vitro intestinal

(1) Growth in intestinal environment (2) Inhibition

Zihler et

Pathogenic: Salmonella enterica subsp.

fermentation system

of pathogen colonisation (3) Bacteriocin-like

al. 2014

enterica serovar Typhimurium M557

with immobilized

inhibitory compounds (4) Rebalancing metabolic

faecal microbiota, plate

activity of gut microbiota post antibiotic therapy (5)

counts, FISH flow

Possible competition for nutrient and growth in co-

cytometry

cultures leading to competitive exclusion and antagonism by organic acid

Probiotic: B. bifidum CECT 7366

Intact cells and culture

(1) Resistance to adverse GI tract conditions (2)

Chenoll

Pathogenic: Helicobacter pylori NCTC

supernatant, in vitro

Pathogen inhibition upto 81.94% for supernatant

et al.

33

11637, NCTC 11638 and 6 human isolates

and in vivo (mice

and 94.77% for purified supernatant (3) Adhesion

model), agar diffusion

to intestinal mucus (4) Partial relief of pathogenic

assay, broth inhibition

damage to gastric tissues (5) Proteinaceous

assay

antagonistic compounds in supernatant exhibiting

2011

pathogen inhibition

Probiotic: B. bifidum NCIMB 30179, B.

Intact cells, culture

(1) B. breve NCIMB 30180 showed highest

Tejero-

breve NCIMB 30180, B. infantis NCIMB

supernatants, in vitro,

antimicrobial activity while B. infantis NCIMB

Sariñena

30181, B. longum NCIMB 30182 along with

agar spot method, agar

30181 showed almost no effect. (2) B. breve

et al.

strains belonging to Lactobacillus,

diffusion assay

showed highest inhibition of pathogen (3)

2012

Lactococcus, Streptococcus and Bacillus

Production of organic acids (mainly lactic and

genera

acetic acids) from glucose fermentation and

Pathogenic: Escherichia coli NCFB 1989,

consequent lowering of culture pH is the main

S. Typhimurium LT2, Staphylococcus

inhibitory mechanism. B. longum NCIMB 30182

aureus ssp. aureus NCTC 8532,

produced highest amount of lactic acid while B.

Enterococcus faecalis NCTC 775,

infantis NCIMB 30181 produced greater amount of

Clostridium difficile ATCC 43594

acetic acid (4) The observed antimicrobial activity is mainly genus specific with significant various among species.

Probiotic: B. longum BCRC 14634

Intact cells, EPS, LPS,

(1) BCRC 14634-EPS is a mild immune modulator

Pathogenic: E. coli BCRC 10239, S.

in vitro (murine

for macrophages inducing IL-10 secretion in

Typhimurium ATCC 14028, Staphylococcus

macrophage cell line),

J774A.1 cells. EPS also induced lower levels of

aureus ATCC 6538P, Bacillus subtilis

J774A.1 cells,

TNF-α secretion and slight changes in the

ATCC 21778, Pseudomonas aeruginosa

sandwich enzyme

morphology of J774A.1 cells (2) EPS pre-treatment

IFO 3898, Vibrio parhaemolyticus ATCC

immunoassay, phase-

prevented LPS-induced release of TNF-α from

17802, Bacillus cereus ATCC 10361

contrast microscope,

J774A.1 cells. EPS may act as an LPS blocker

Petroff-Hausser

(both have similar structure and might interact with

counting chamber

same receptors) (3) Pathogens were inhibited by 80 ppm EPS (4) EPS treatment did not lyse the pathogens, but rather impaired their division

34

Wu et al. 2010

Probiotic: B. longum BB536, B. animalis

In vitro, metabolites,

(1) SCFA metabolites butyrate and propionate

Asarat et

subsp. lactis BB12, B. lactis LAFTI B94,

LPS, Pour plate

produced in addition to lactate and acetate after

al. 2015

along with 3 strains belonging to

technique, HPLC

culturing in skim milk containing inulin, hi-maize

Lactobacillus genus

or β-glucan, with BB12 producing high amounts of

Pathogenic: LPS component from

SCFAs (2) SCFA show regulatory effects on

Escherichia coli O111:B4

cytokines released by LPS stimulated human PBMCs. The addition of 2 mM butyrate with LPS to PBMCs significantly inhibited release of proinflammatory cytokines (TNF-a, IL-12, TGF-b1 and IFN-g) (3) Butyrate and acetate induce higher production of anti-inflammatory cytokines (IL-4 and IL-10) than propionate at the same concentration

Probiotic: 2 isogenic strains of B.

Intact cells, EPS, in

(1) Mucoid EPS provides a higher resistance to GI

Hidalgo-

animalis ssp. lactis DSM10140 differing in

vitro, ex vivo,

conditions and increased adhesion to human

Cantabra

their EPS-producing phenotype

immunohistochemistry

enterocytes (epithelial intestinal cell line HT29) (2)

na et al.

Pathogenic: none

assay, Transmission

Cytokine profile of human PBMCs and ex

Electron Microscopy,

vivo colon tissues, suggests that EPS producing

Cryo-Scanning

strain could have a higher anti-inflammatory

Electron Microscopy,

activity

2015

human colonic mucosa organ culture

Probiotic: 313 Lactic acid bacteria and 17

Intact cells, cell free

(1) NIF7AN3, NIF7AN5, NIF7AN10 showed

Bifidobacterium (5 suitable strains identified

culture supernatant, in

strong adhesion to mucin. (2) The 5 strains studied

et al.

for study) B. longum subsp. longum

vitro, antibacterial

showed tolerance towards gastric acid and bile

2015

NIF3AN3, NIF7AN1, B. bifidum NIF7AN2,

activity assay, adhesion

salts. NIF7AN3, NIF7AN5, NIF7AN10 showed

NIF7AN5, NIF7AN10

assay using porcine

increase in number post treatment with acid and

Pathogenic: E. coli TISTR 780,

gastric mucin type III

bile. Possibly due to set of adaptive mechanism in

Staphylococcus aureus TISTR 1466,

response to various environmental stresses (3) All

35

Uraipan

Shigella sonnei, S. flexneri, Salmonella

showed high competitive exclusion of food borne

enterica ssp. enteric serovar Typhimurium

pathogens

SA2093, S. paratyphi A

Probiotic: B. bifidum (S17, PRL2010,

Intact cells, in vitro,

(1) B. bifidum showed highest adhesion to all tested

NCIMB41171), B. longum ssp. longum

adhesion assay,

intestinal epithelial cell (IEC) lines. (2)

et al.

(NCC2705, DJO10A, BBMN68, F8,

Northern blot, Southern

Proteinaceous cell surface component mediated

2012

JCM1217, JDM301, KACC 91563), B.

blot, Western blot

adhesion of B. bifidum (S17) to IECs. (3) B.

longum ssp. infantis (157 F, ATCC

bifidum-specific protein, BopA, mediated adhesion

15697), B. breve (UCC2003, DSM20213,

to IECs. B. bifidum, B. longum and B. infantis

ACS-071-V-Sch8b), B. adolescentis

strains overexpressing bopA showed enhanced

(ATCC15703 , L2-32), B. dentium (Bd1,

adhesion to IECs.

Gleinser

ATCC 27678, ATCC 27679, JCVIHMP022), B. animalis ssp. lactis (AD011, BB-12, BLC1, Bl-04, DSM10140, V9), B. pseudocatenulatum DSM20438, B. catenulatum DSM16992, B. angulatum DSM20098, B. gallicum DSM20093 Pathogenic: none

Probiotic: B. bifidum PRL2010 (with

Intact cells, in vitro and

(1) Host induced upregulation of genes cluster that

Turroni

Lactococcus lactis (NZ9000)-pil2 and L.

in vivo (mice model),

encode sortase-dependent pili (2) Initial adhesion of

et al.

lactis-pil3 clones)

Western blot, Cloning

B. bifidum cells to the enterocytes by extending

2013

Pathogenic: none

Pili-encoding B.

their pili toward the apical surface of the host

bifidum genes in L.

epithelial cells (3) L. lactis-pil3 clones induced

lactis, adhesion assay

higher TNF-α response in mouse model, compared

using Caco-2 cell line,

with non-piliated L. lactis-pil3 clones, indicating B.

adhesion inhibition

bifidum pili to influence mucosal immune

assay using anti-pili

responses.

antibodies, immunoassay human

36

macrophage-like cell line U937, AFM, light microscopy

Probiotic: B. breve CNCM I-4035

Intact cells, cell free

(1) Live CNCM I-4035 and CFS induces innate

Bermude

Pathogenic: Salmonella enterica

CFS, in vitro,

immune responses by activating TLR signalling

z-Brito et

serovar Typhi CECT 725

Immunoassay, Reverse

pathway (2) Intact CNCM I-4035 cells and CFS

al. 2013

transcriptase reaction

upregulated TLR9 gene transcription in presence of

and polymerase chain

S. typhi, with CFS having more potential (3)

reaction

CNCM I-4035 affects intestinal immune response, whereas its supernatant exerts anti-inflammatory effects via DCs (4) In presence of S. typhi, CFS decreased pro-inflammatory cytokines, chemokines and restored TGF-β levels in human intestinal DCs challenged while CNCM I-4035 induced proinflammatory TNF-α, IL-8 and RANTES, as well as anti-inflammatory IL-10

Probiotic: B. breve Yakult strain, B. bifidum

Intact cells, in vitro, in

(1) B. breve activates intestinal CD103+ DCs to

Jeon et

Yakult strain YIT10347, B. adolescentis

vivo (mice model),

produce IL-10 and IL-27 via TLR2/MyD88

al. 2012

ATCC15703, B. longum ATCC 15707 and

Flow cytometry,

signalling pathway thus inducing IL-10-producing

L. casei strain Shirota

intracellular cytokine

Tr1 cells in the large intestine (2) B. breve helps in

Pathogenic: none

staining, real time

maintaining homeostasis by inducing intestinal IL-

polymerase chain

10-producing Tr1 cells. (3) B. longum activated

reaction, T-cell-

DCs moderately induces IL-10-producing T cells

mediated colitis model,

(4) While B. adolescentis or B. bifidum did not

histopathological

induce IL-10 production from co-cultured CD4+ T

analysis

cells

37