Probiotics Who should get Infected?

Probiotics Who should get Infected?
November 27, 2018 Probiotics Who should get Infected? José M. Saavedra, MD Associate Professor of Pediatrics Johns Hopkins University School of Medicine & Medical and Scientific Director, Nestlé Nutrition

November 27, 2018 NO ONE

Objectives Rationale for modulating the intestinal microbiota
Concept of probiotic bacteria Use of probiotic bacteria Misconceptions and mistakes referring to probiotics

Surface Areas of Exposure to the Environment
November 27, 2018 Surface Areas of Exposure to the Environment Skin sq m Respiratory mucosa sq m Intestinal mucosa sq m Gut epithelium mm Bacterial gut content up to 1012 / ml Tightly regulated mucosal immunity is needed to maintain health

Germ-free vs. Colonized Gut

Gut associate lymphoid tissue (GALT)
November 27, 2018 Gut associate lymphoid tissue (GALT) Comprises 70-80% of immunologic cells in the body GALT is the largest immune “organ”

Antigens Microflora Th0 Intestinal Lumen Epithelium Intestinal Mucosa
November 27, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Optional Speaker Notes: This slide, and the three subsequent slides, provide in simplified form a pictorial representation of antigens and intestinal bacteria in the intestinal lumen. Their critical role in the activation of T-cells is described. T-cells are a major component of gut-associated lymphoid tissue (GALT). Undifferentiated (Th0) will normally differentiate, once activated through antigen presentation, into Th1 or Th2 cells. 7

Antigens Microflora Th0 Th2 Th1 Intestinal Lumen Epithelium
November 27, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Th2 Th1 Optional Speaker Notes: Certain types of stimuli from the lumen can activate T cells to differentiate into T-regulatory cells. Both of the Th1 and Th2 cell subsets are critical for maintaining health. However, a balanced response between Th1 and Th2 is critical to decrease the chances that the immune system will overreact. Each subset generates and responds to different types of cytokines, which are cellular mediators of these T-cell subsets. 8

Antigens Microflora Th0 Th2 Th1 TNF-α IL-4 Cytokines IFN-γ IL-5 IL-2
November 27, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 Optional Speaker Notes: Th1 cells, which are associated with TNF-alpha, interferon gamma, and IL2 are critical for cellular immunity and for pathogen protection, including the formation of IgM and IgG. Th2 cells, and their respective cytokines, including interleukins 4, 5 and 10, are important for adequate development of humoral immunity including formation of IgE and IgA antibodies. An exaggerated response of Th1 type cells will lead to autoimmune disorders; examples are Crohn’s disease and type 1 diabetes. Exaggerated response of Th2 will lead to allergic disease. Therefore, a balanced response of the Th1 and Th2 subsets is critical to decrease the chances that the immune system overreacts and lead to one of the more common autoimmune or allergic conditions that we are facing today in modern society. Cytokines Cellular Immunity Pathogen protection IgM, IgG response Humoral Immunity IgE, IgA response Response 9

Microflora Antigens Th0 Th2 Th1 IBD Allergy TNF-α IL-4 IFN-γ IL-5 IL-2
November 27, 2018 Microflora Antigens Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 Over Expression Over Expression IBD Allergy

Microflora Antigens Th0 TReg Th2 Th1 Tolerance IBD Allergy TNF-α IL-4
November 27, 2018 Microflora Antigens Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 TReg Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 TGF-β Over Expression Over Expression Tolerance IBD Allergy

Chronic Disease Prevalence in the last 50 Years
November 27, 2018 Chronic Disease Prevalence in the last 50 Years Decrease in infections is associated with increase immune disorders (Th1) (Th2) Bach JF. N Engl J Med. 2002;347:

Intestinal microflora
November 27, 2018 Gut Barrier and Gut Immune System Intestinal microflora Mucus (Mucin) Keeps pathogens from invading Tight Junctions (Between cells) Decrease gut permeability Immune cells (GALT) 70% of immune cells are located in the GI tract Innate: Macrophages, White cells, Mast cells Adaptive: T and B lymphocytes 13

Bacteria Support Gut Barrier and Modulate Immune Function
November 27, 2018 Bacteria Support Gut Barrier and Modulate Immune Function Gut microflora help support gut barrier function: ↑ Mucin production ↓ Permeability Gut microflora help support the adaptive immune response: Generate IgA activity (humoral) Balance in T helper cell subclasses (cellular) Optional Speaker Notes: Research over the last years has demonstrated that intestinal bacteria help support gut barrier and immune function by mechanisms listed on the slide. A number of clinical trials, as well as review articles have identified and summarized the effects of microflora on gut immunity and barrier function. Data from several of the trials with infants will be presented on subsequent slides. Isolauri E., et al. Am J Clin Nutr 2001;73(suppl):444S-450S. Saavedra JM. Nutr Clin Pract 2007; 22: 14 14

Bacteria and Humans A stormy relationship
Symbiosis Parasitism Commensalism Mutualism

“Modern” Lifestyle Has Decreased Exposure to Bacteria
November 27, 2018 “Modern” Lifestyle Has Decreased Exposure to Bacteria Lower microbial exposure Sterile processed food Decrease in naturally fermented foods Increased hygiene measures Urban life Cesarean sections Antibiotics Altered Intestinal microbiota Inadequate immune response

Chronic Disease Prevalence in the last 50 Years
November 27, 2018 Chronic Disease Prevalence in the last 50 Years Decrease in infections is associated with increase immune disorders Bach JF. N Engl J Med. 2002;347:

IBD: The Case for a Role of Bacteria
Diversion studies Surgical specimen analysis Reduced Lactobacilli in ulcerative colitis specimens Reduced Bifidobacteria in Crohn’s disease specimens Response to antibiotics (metronidazole, ciprofloxacin, clarithromycin , rifamixin) Animal models Clinical features of Crohn’s disease correlate with certain mouse models of colitis (eg, SAMP/Yit, others) Rutgeerts PJ Inflamm Bowel Dis 2001 May;7 Suppl 1:S2-8. Kosiewiczm M M et al. J Clin Invest. 2001 March 15; 107 (6): 667–670. Rivera-Nieves Gastroenterology Apr;124(4):

Food Allergy to egg confirmed by testing at age 1 – 2.
November 27, 2018 Influence of Cesarean Delivery on Relative Risk of Childhood Food Allergy * CI = Adjusted Odds Ratio CI = *P<0.01; adjusted for covariates. Food Allergy to egg confirmed by testing at age 1 – 2. Eggesbo M et al. J Allergy Clin Immunol 2003;112:

Ingestion of Bacteria 3500 BC 2000 BC AD 2000
November 27, 2018 Ingestion of Bacteria Sumerians (Cheese) Celts & Huns (Khefir) Pasteurization Abraham (Milk & curds) Monks (Refine Fermentation) 3500 BC 2000 BC AD 2000 Optional Speaker Notes: Humans have ingested bacteria and have been exposed to a heavy microbial environment for thousands of years. The purposeful use of bacteria for food preservation more than 5000 years ago led to the large consumption of fermented foods. Fermentation of milk for manufacturing of cheese dates back to the earliest times of recorded history. Biblical references refer to the use of milk and fermented curds for human consumption. During the middle ages, the fermentation of fruits and milk led to a refinement in the production of these fermented foods. With the introduction of pasteurization in the last 100 years ingestion of bacteria as part of our food supply has dramatically changed. In the past 50 years, antibiotics became readily available which has also changed not only our microbial environment, but has significantly altered the intestinal flora of individuals, particularly children who consume antibiotics in the first two years of life. In the past 20 years, the reintroduction of potentially beneficial bacteria (the concept of probiotics) has gained attraction as a way to address, at least in part, the microbial imbalance of our current modern society. Antibiotics Probiotics In the last 100 years, we drastically changed our ingestion of microbes and our microbial environment. 20 20

Can we restore GALT stimulus? Can we immuno-modulate GALT?
November 27, 2018 Can we restore GALT stimulus? Can we immuno-modulate GALT?

PROBIOTICS Can we restore GALT stimulus? Can we immuno-modulate GALT?
November 27, 2018 Can we restore GALT stimulus? Can we immuno-modulate GALT? PROBIOTICS Live microbial feed supplement which beneficially affects the host animal by improving its microbial balance. Bifidobacteria Lactobacilli

Intestinal Flora (A Balanced Ecosystem)
November 27, 2018 Intestinal Flora (A Balanced Ecosystem) Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Pseudomonas Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli As mentioned these bacteria in intestine for a complex ecosystem Represnted here major genus of bact Streptococci Eubacteria Bifidobacteria Bacteroides From: Gibson GR. J Nutrition 1995; 125:

Intestinal Flora (A Balanced Ecosystem) Potential Probiotic Bacteria
November 27, 2018 Intestinal Flora (A Balanced Ecosystem) Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Pseudomonas Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli As mentioned these bacteria in intestine for a complex ecosystem Represnted here major genus of bact Streptococci Potential Probiotic Bacteria (When Ingested) Eubacteria Bifidobacteria Bacteroides From: Gibson GR. J Nutrition 1995; 125:

Taxonomy continues to evolve
November 27, 2018 Most Frequently Studied Lactic Acid Bacteria (LAB) for Potential Probiotic Effects BIFIDOBACTERIA B. longum B. lactis (Bb12) B. breve B. bifidum B. animalis B. infantis LACTOBACILLI L. casei L. rhamnosus (GG) L. reuterii L. plantarum L. bulgaricus L. acidophilus (Non-inclusive list) Taxonomy continues to evolve

General Strength of Evidence for a Favorable effect of Specific Probiotic Bacteria
Disease related Treatment of acute diarrhea (pediatric) Prevention of acute diarrhea Antibiotic associated diarrhea (non C difficile) Management of IBD Management of allergic disease Prevention of NEC Management of infection in risk conditions

General Strength of Evidence for a Favorable effect of Specific Probiotic Bacteria
Functional related Maintaining mucosal barrier function Increasing humoral GALT response Maintaining gut permeability Modulating T cell response

Probiotics: Misconceptions
“Probiotics are not uniformly efficacious” “Efficacy an safety of probiotics is not well demonstrated or studied sufficiently” “Probiotics are not ready for prime-time” “The probiotic strain did not show an effect in this study” “There is no benefit shown with probiotics in healthy infants”

Probiotic Concept Facts
Probiotics are number of different organisms, not a single substance Only a specific microorganism which has shown an specific benefit(s) in the population intended can be called a probiotic. If a benefit wasn’t shown in a study- it should not be called a “probiotic” bacteria Efficacy and safety can only be defined for a specific organism(s) – and not for “probiotics” in general

Probiotic Safety Misconceptions
“Probiotics are not uniformly safe- we need to be cautious using it in sick adults or healthy infants.” “Safety of probiotics is not adequately documented because trials are powered for efficacy” “Any bacteria can be pathogenic” ”Sooner or later I think there will be a case of bifidobacteremia”

Probiotic Safety Facts
The safety of specific probiotic bacteria is documented based on multiple sources of evidence Thorough understanding of the organism itself, its bacterial properties, including its full genome: Documentation of bacteremia or infection in controlled or uncontrolled circumstances History of safe use Safety in clinical trials

Example: B. Lactis safety Facts
Thorough understanding of the organism itself, its bacterial properties, including its full genome: No invasive or identifiable pathogenic potential. No undesirable metabolic characteristics (no D- lactate , no toxin production, etc.). Bifidobacteria are the species most commonly found in the GI tract of breast fed infants Bifidobacteria are regularly found in human breast milk in significant amounts

B) Not a single documented bacteremia or infection associated with ingestion of bifidobacteria in general or B. lactis specifically, despite extensive history of use C) History of safe use Hundreds of thousands of metric tons of B. lactis containing products are consumed globally each year Use in infant feeding No adverse effects documented with free use in the food supply nor in infant formula

D) Safety in clinical trials Not a single adverse effect reported in more than 60 controlled human clinical trials with B. animalis and specifically B. animalis sub-species lactis in humans, including premature infants Has GRAS status FDA approval for feeding infants from birth

Probiotic Quality Facts
In the US, a product commercialized to prevent or treat disease is a drug; and can be commercialized even if it produces side effects (as long as the side effects outweigh the benefits) In the US a product commercialized as a food must have NO side effects Thus, in practice, the “safety” bar is in fact higher for a food than for a drug Supplements have different regulation standards

Focus of Probiotic Use Treatment vs not prevention
Disease management vs health maintenance Drug vs food Supplement vs not food Specific bacteria have and will be shown efficacious for managing specific conditions Specific bacteria have and will be shown to have functional immune and digestive health effects

STOP saying “PROBIOTICS” When discussing efficacy or safety !!
Probiotics are a general conceptual term or category of bacteria Efficacy and safety are species- specific

Probiotics: Reported Mechanisms and Clinical Benefits in Pediatrics
November 27, 2018 Probiotics: Reported Mechanisms and Clinical Benefits in Pediatrics Mechanisms Clinical Benefits Balanced intestinal microflora ↓ duration of acute diarrhea ↓ incidence of acute diarrhea ↓ antibiotic associated diarrhea ↓ in severity & incidence of atopic disease ↓ NEC ↓ IBD ↑Ratio of Bifidobacteria & Lactobacilli to pathogens Increased mucin production Enhanced gut permeability Modulation of gut immune response - ↑ humoral immunity (IgA and other antibodies) - Modulation of Th1/Th response towards antigen tolerance Gut Mucosa Gut Lumen GALT PROBIOTICS Saavedra JM NCP, 2007

November 27, 2018 Probiotics in Disease Who should get -specific bacteria- for treatment? A few - for specific conditions

Who should be getting specific bacteria- in the diet? EVERYONE
November 27, 2018 Probiotics in Health Who should be getting specific bacteria- in the diet? EVERYONE

Probiotics jose.saavedra@us.nestle.com José M. Saavedra, MD
November 27, 2018 Probiotics José M. Saavedra, MD Associate Professor of Pediatrics Johns Hopkins University School of Medicine & Medical and Scientific Director, Nestlé Nutrition

Probiotic Concept Facts
INCORRECT: “probiotics are safe” or “we need more studies to show probiotics are safe or efficacious”. CORRECT: This bacteria (genus, species, strain) is a probiotic, with this effect on this population CORRECT: This bacteria has a well (or not well) documented safety record Efficacy and safety are species - specific STOP saying “PROBIOTICS “ If you are discussing safety or a specific effect

Rising Incidence of CD Courtesy of S. Schreiber.

Probiotic Quality Misconceptions
“Probiotics are not well regulated” “Probiotic products don’t always have the bacteria or the amounts they claim” “Once the bottle or can is opened, moisture gets in and probiotic bacteria begin to die, so efficacy can not be guaranteed” “Heat treatment or freeze drying (lyophilizing) decreases viability”

Probiotic Quality Facts
Quality assurance criteria are different for supplements, foods, and drugs Foods are strictly regulated, and infant formula much more so. They contain the species stated in the label in the amounts stated by the manufacturer In the US, a drug is a product specifically commercialized to prevent or treat disease. A supplement is a dietary component that does not replace part of a normal diet

The gut lumen is part of the environment
Diet GUT

November 27, 2018 Meta-analyses of Randomized Clinical Trials on Probiotic Treatment of Acute Diarrhea 95% CI = 95% CI = 95% CI = 95% CI = 8 RCTs 12 RCTs 9 RCTs 18 RCTs Adapted from: Szajewska H., et al. JPGN 2006;42:

RCTs on Prevention of Antibiotic Associated Diarrhea
November 27, 2018 RCTs on Prevention of Antibiotic Associated Diarrhea Reduction in Relative Risk * NS B. lactis L. GG Other CI ( ) CI ( ) * NS CI ( ) % r CI ( ) % Reduction in incidence NS CI ( ) P< p.0.05.

Prevention of NEC with Probiotics in Premature Infants
November 27, 2018 Prevention of NEC with Probiotics in Premature Infants Reduction in Relative Risk * * RR=0.25 CI ( ) RR=0.20 CI ( ) RR=0.50 CI ( ) Optional Speaker Notes: In humans, there are several published studies suggesting that probiotic supplementation can decrease the incidence of NEC. Three prospective studies are identified on this slide. The double-blind study by Dani and colleagues (2002) randomized 585 premature infants from12 NICUs, to receive Lactobacillus GG or placebo from the time of the first feed until discharge. The probiotic supplemented group was found to have a lower incidence of urinary tract infections and NEC than the control group; however, the difference was not statistically significant Bin-Nun and colleagues (2005) randomized 145 very low birth weight neonates (<1500g) to receive either a combination of bifidobacteria (Bifidobacteria infantis, Bifidobacteria bifidus, and Streptococcus thermophilus) or no probiotic supplement. The incidence of NEC was reduced from approximately16.0% in the control infants to 4% in the supplemented infants. Three of the fifteen babies who developed NEC died, and all of the deaths occurred in the non-supplemented group. Lin and others (2005) randomized 367 VLBW infants to either Lactobacillus acidophilus and Bifidobacterium infantis, or placebo from approximately day 7 of life until discharge. Results from this blinded trial provided further support for the potential use of probiotics in reducing the risk of NEC. B. infantis, B. bifidus & S. thermophilus LGG L. acidophilus & B. infantis Bin-Nun A., et al. J Pediatr 2005;147: Dani C., et al. Biol Neonate 2002;82: Lin HC., et al. Pediatrics 2005;115:1-4. *P<0.05 56

Rioux KP 2005 Gastroenterol Clin N Am 2005

Summary Although much remains to be learned, the safety and efficacy documentation to date support the statement that B. lactis, incorporated into the diet, and at the levels found in infant formula in the US, “helps support a healthy immune system” Use of certain probiotic bacteria, including B. lactis, have been shown to have specific prophylactic and therapeutic effects. However, in the US they are not commercialized as drugs (for treatment or prevention of disease )

November 27, 2018 NO ONE

The Modern Sterile Environment and Diet
Dramatic decrease in bacterial stimulus of GALT Greatest effect in small bowel Greatest effect in bacterially deprived circumstances throughout life Prematurity, C section, sterile formula Antibiotics Stool is only a “marker” of altered flora

2) B. lactis: Efficacy Misconceptions and Disinformation
“Efficacy with B. lactis has not been shown” “For healthy infants and children, there is no research that probiotics provide any health benefits.” “Putting probiotics [B.lactis] in the GI tract is like a putting them into a black box”… “we don’t know they work…There are no mechanisms known” “… one doesn’t know how many servings or how much to take for what period of time or for a lifetime,… “

B lactis Efficacy Facts
B. lactis is a specific bacteria: Bifidobacterium animalis, sub-species lactis* B. lactis has been studied in clinical trials and shown to have specific benefits in various populations including , and mostly, in healthy children [See references] as well as in adults [See references] Clinical benefits have been shown during the time of consumption, at the amounts currently found in infant formula in the US. --> B. lactis is a probiotic * Also referred to as B. bifidum and/ or strain Bb12 in certain studies. See attached bibliography

B lactis Efficacy Facts
In vitro studies [See references] and animal trials [See references], provide support and the mechanistic rationale for the findings documented in human clinical trials with B. lactis. The human, animal, and basic science literature [See references] on use of B. lactis specifically supports their beneficial effects on gut barrier function and immune development and modulation during consumption This supports the use of B. lactis an ideal probiotic for the incorporation into routine infant feeding See attached bibliography

Pathogenesis of UC Infection

Colitis in Genetically Susceptible Hosts (Animal models)
Sterile environment No Colitis Bacteria Colitis Genetically Susceptible Host Immune Activation

E. invasive E. coli 131. ROLE OF INDIVIDUAL BACTERIA IN THE PATHOGENESIS OF EXPERIMENTAL COLITIS HLA B27/2 microglobulin transgenic rats raised under sterile (germ-free) conditions do not develop colitis. However, when colonized with normal specific pathogen free commensal cecal bacteria, they develop aggressive disease involving the colon, antrum, and duodenum within one month. When colonized (monoassociated) with a single bacterial strain, Bacteroides vulgatus, these susceptible rats develop moderate colitis but no gastroduodenal disease. However, when similarly mono-associated with E. coli, they exhibit neither disease nor T cell activation. However, some bacteria, including Lactobacillus GG, actually prevent inflammation by attenuating the colitis in HLA B27 transgenic rats colonized with normal cecal bacteria. These results indicate that all commensal bacterial species are not identical in their abilities to induce disease: some are aggressive, such as Bacteroides vulgatus; some are neutral, including E. coli; and some, like Lactobacillus species, are protective. • Rath HC et al. Differential induction of colitis and gastritis in HLA 27 transgenic rats selectively colonized with bacteroides vulgatus and Eschericia coli. Infection and Immunity 1999;67:2969. • Dieleman LA et al. Lactobacillus GG prevents recurrence of colitis in HLA B27 transgenic rats after antibiotic treatment. Gastroenterology 2000;118:A814.

Considerations in assessment of safety of a probiotic
November 27, 2018 Considerations in assessment of safety of a probiotic Individual genus and species History of use Presence in the host Undesirable metabolites (ie D lactate) Interaction with other organisms, plasmids or antibiotic resistance transmission History of use in vulnerable populations Documentation of bacteremia / infection Dose (impossible to “overdose”)

Effect of B. lactis in Preterm Infants:
November 27, 2018 Effect of B. lactis in Preterm Infants: Improved Immune Secretory Function Placebo = 16 Probiotic = 19 * * *P<0.05. Mohan R. et., al. EUROBIO 2006; abstract.

The Case for Bacteria - Probiotics
Active CD (Open label n=25) Lactobacillus salivarius 76% avoided other meds, Mean CDAI fell from 217 to 150 CD maintenance (RCT n=32) Saccharomyces boulardii plus 2g mesalamine 6% relapse/6 months versus 62% relapse with 3g mesalamine Active UC (RCT n=116) 44 (75%) with mesalamine group attained remission 39 (68%) in the E. coli (Nissle 1917) group Pouchitis (RCT maintenance of remission, n=40) 3/20 (15%) relapse on the probiotic preparation, VSL #3 100% with placebo Probiotics may work by effects on immune system (innate and adaptive), barrier function, interaction with other flora. More data in UC and pouchitis than CD McCarthy J. Gut 2001;49(Suppl III):2447. Guslandi M. DDS 2000;45: Rembacken. Lancet. 1999;354:635. Gionchetti. Gastroenterology. 2000;119:305.

Essential Ingredients for IBD
Genes and Bacteria are Essential Ingredients for IBD Genetically engineered mouse IBD Bacterial Colonization Germ-free No IBD

Intestinal Flora of Individuals with IBD Differs from Normal Controls
Increases in E. coli and bacteroides species have been noted in patients with Crohn’s disease vs. controls Giaffer et al, J Med Microbiol 1991 Colonic biopsies from patients with active UC have decreased anaerobic bacteria and lactobacilli compared to inactive UC Fabia et al, Digestion 1993

- Genetic Susceptibility
- Environmental Trigger

November 27, 2018

Surface Areas of Exposure to the Environment
November 27, 2018 Surface Areas of Exposure to the Environment Skin sq m Respiratory mucosa sq m Intestinal mucosa sq m Gut epithelium mm Bacterial gut content up to 1012 / ml Tightly regulated mucosal immunity is needed to maintain health

Rioux KP 2005 Gastroenterol Clin N Am 2005

Intestinal Flora of Individuals with IBD Differs from Normal Controls cont.
Stool of patients with Crohn’s disease have less bifidobacteria compared to healthy controls Favier C et al, Dig Dis Sci 1997 Stool of patients with active pouchitis have less bifidobacteria and lactobacilli and more clostridia Ruseler-van-Embden et al, Gut 1994

Microbiota Dynamics During Life
November 27, 2018 Microbiota Dynamics During Life Bacteroides, Eubacterium, Peptococcaccae Bifidobacterium Escherichia Coli, Streptococcus Lactobacillus Clostridium perfringens From: Mitsuoka T. Nutrition Reviews 1992;50:

Neonatal Response to Colonization
November 27, 2018 Neonatal Response to Colonization 10 20 30 40 50 60 70 80 90 100 110 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 42 44 Age in days Percent of infants with detectable salivary A Later Colonization Initial Colonization J. Pediatr. 1968; 72:685.

*P<0.05; bivariate analysis.
November 27, 2018 Atopic Sensitization and Allergy Symptoms Among Children Living on Farms and Children in Same Rural Community from Non-farming Families * * * * *P<0.05; bivariate analysis. Braun-Fahrlander CH. Clin Exp Allergy 1999;29:28-34.

Increasing Prevalence of Asthma and Atopy
November 27, 2018 Increasing Prevalence of Asthma and Atopy Ninan et al., 1992; BMJ 304:

Prevalence of bifidobacteria in Stools of Atopic and Healthy Infants
*** ** * Optional Speaker Notes: The increased occurrence of allergy in infants has also been associated with differences in intestinal flora. In an attempt to prospectively relate microflora to allergy development, stool samples from 44 newborn infants were analyzed during the first week of life, and at various time periods through the first year. 18 infants had developed atopic dermatitis and/or positive skin prick test results by 2 years of age. The prevalence of colonization with bifidobacteria in infants who developed allergy during the first 2 years of life was significantly less than for those that did not develop atopy. As shown, less than 30% of the atopic infants were colonized with bifidobacteria soon after birth, at 3 months, or at 1 year. Differences in intestinal flora in infants developing allergy suggest that factors in early life (such as sterile formula feeding and birth by C-section) may be a determinant in the later development of immunologic disease, including allergy. *P=0.02; **P=0.03; ***P=0.05 comparing prevalence at a given age Björksten B., et al. J Allergy Clin Immunol 2001;108:

Intestinal Flora (A Balanced Ecosystem)
November 27, 2018 Intestinal Flora (A Balanced Ecosystem) Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Pseudomonas Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli As mentioned these bacteria in intestine for a complex ecosystem Represnted here major genus of bact Streptococci Eubacteria Bifidobacteria Bacteroides From: Gibson GR. J Nutrition 1995; 125:

Intestinal Microbiota
Critical to gut barrier function Critical to balanced immune response Supported by Exposure to bacteria early in life and throughout (particularly bifidobacteria and lactobacilli) Dietary components (Bifidogenic dietary components)

Bifidobacterium lactis
November 27, 2018 Bifidogenic Dietary substances Prebiotics Bifidobacteria Dietary Fibers Inulin Fructo-oligosaccharides Galacto-oligosacccharides Optional Speaker Notes: Bifidobacteria have all the characteristics of a probiotic bacteria. Importantly, the genus Bifidobacterium constitute most of the microflora of breastfed infants. Amongst the bifidobacteria better studied for application as a probiotic is Bifidobacterium lactis. B. lactis is the short name for B. animalis subspecies lactis, previously also called B. bifidum, strain Bb12. Bifidobacterium lactis Nomenclature - B. lactis also: B. animalis sub-species lactis, B. bifidum, strain Bb12 87 87

Microbiota Development
November 27, 2018 Microbiota Development Weaning Bifidobacteria (Anaerobic) Bacteroides Clostridia Anaerobes Rapid accumulation of anaerobic growth Relative Numbers of Anaerobes Coliforms Staphylococcus Klesiella Salmonella Shigella Lactobacillus Streptococci Enterococcus Facultative anaerobes Pseudomonas and M tuberculosis are aerobic Age Day 1-3 Microbiota influenced by: Genetics Mode of delivery Day 4 – Day Microbiota influenced by: Feeding type Environmental exposure Adapted from: Rautava S. et al, JPGN 2004;38:

November 27, 2018 Probiotics & Prebiotics Are these functional ingredients ready for center stage? José M. Saavedra, MD Associate Professor of Pediatrics Johns Hopkins University School of Medicine & Medical and Scientific Director, Nestlé Nutrition Yvette Gaughan, MS, RD, LDN Healthcare Specialist Nestlé Nutrition Deerfield, Illinois

Medical Literature on Probiotics in IBD
Number of publications 1996 to 2004 Fedorak R Inflamm Bowel Dis 2004:10; ; Medline

Microbiota and Crohn Disease
Specific flora appears to be related to initial inflammatory stimulus Specific flora appears to help down-regulate inflammatory process Observations support the use of antibiotics to down-regulate inflammatory response Observations support use of flora modulation Prebiotics Probiotics

Intestinal Flora and Feeding Type (%)
November 27, 2018 Breast fed Formula fed Bifidobacteria Bacteroides Coliforms Harmsen HJM et al.JPGN 2000;30:61

Determinants of Intestinal Flora in Newborns
November 27, 2018 Determinants of Intestinal Flora in Newborns Early diet (Breast Milk vs Formula) Breast milk is not sterile Contains specific species (bifidobacteria) Contains bifidogenic factors (prebiotic oligosaccharides) Birth by C-section (Sterile procedure) Microbial environment, antimicrobials, etc

Bifidobacterial Counts in Breast Milk
Log 10 number of bifidobacteria per ml Optional Speaker Notes: Results from a recent analysis of breast milk samples indicates that breast milk contains bifidobacteria, and specific Bifidobacterium species that may promote a healthy microflora development during infancy. In this study, breast milk samples were collected by manual expression from 20 mothers of one month old breast fed infants. Molecular techniques for analysis (PCR) were applied to samples, in triplicate, prior to calculating a mean value for bifidobacteria species identified. Mothers were interviewed about their previous month’s consumption of probiotic containing preparations. The average bifidobacteria level was 2.64 log cells. In three samples, levels were below the detection limit of the methodology used. However, Bifidobacterium longum was identified in all other samples. Bifidobacterium animalis (of which B. lactis is a sub-species) was the second most prevalent species, detected in 74% of cases. There were no differences between bifidobacteria levels in breast milk samples from mothers that had consumed probiotic containing products (n=13), and those that had not (n=7). Given that bifidobacteria are a strictly anaerobic genus, it is unlikely that bifidobacteria detected were derived from the skin. The authors suggest that these results, in conjunction with other reports of lactic acid bacteria identified in breast milk, point to breast milk, per se, as the source of the bifidobacteria. Mean = 2.64 log cells; 95% CI: Gueimonde M., et al. Neonatology 2007;92:64-66.

Bifidobacterium species in breast milk
November 27, 2018 Bifidobacterium species in breast milk Bifidobacterium species-composition in breast-milk (20 samples by PCR using species-specific primers Gueimonde M et al ESPGHAN 2007

Risk Factors for the Development of Allergies:
November 27, 2018 Risk Factors for the Development of Allergies: Infants C-section Not breastfeeding Exposure to intact cow milk protein 4-fold increase in allergies if given 1st week of life Family hx of allergy Children Urban living Antibiotics first 2 years of life Family hx of allergy Optional Speaker Notes: Changes in our microbial environment, including a lower exposure to bacteria, are considered to be one of the risk factors for immune disease, and specifically for allergy. Birth by sterile C-section procedure and not breastfeeding (feeding of sterile formula) are examples. Exposure to antigens, particularly intact cow milk proteins which can significantly increase risk (especially if given in the first few days of life), and family history are all risk factors for allergy development. In older children, urban verses rural living, the use of antibiotics in the first two years of life, which also drastically modifies intestinal microflora, as well as family history of allergy, have been associated with development of allergic disease. Marini A., et al. Acta Pediatrica Suppl 1996;414:1-22. Braun-Fahrlander CH., et al. Clin Exp Allergy 1999;29:28-34. Eggesbo M., et al. J Allergy Clin Immunol 2003;112: Farooqi IS and Hopkin JM. Thorax 1998;53: Wickens K., et al. Clin Exp Allergy 1999;29: von Mutius E., et al. Clin Exp Allergy 2000; 30: 97 97

Birth Promote Hinder Establishment of “healthy” intestinal flora
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Promote Hinder Establishment of “healthy” intestinal flora Predominance of bifidobacteria Optional Speaker Notes: An infant is born with a sterile gut lumen, which rapidly becomes colonized. Vaginal delivery and exposure of the infant to maternal bacteria during birth helps determine which microorganisms may be the first to colonize the newborn gut. Breastfeeding, which contains both bacteria as well as growth factors (galacto-oligosaccharides) for certain species of bacteria, promotes a balanced and healthy intestinal flora which is predominant in certain species of bacteria, particularly bifidobacteria. Birth by C-section, which is a sterile procedure, feeding of formula that is also sterile, early antibiotic treatment and environments contaminated with microorganisms that would not typically be a part of maternal flora or a home environment, all hinder the establishment of a healthy balance of intestinal flora. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. Development of adequate gut barrier function Development of adequate immune response

Bifidobacterium lactis
November 27, 2018 Bifidobacteria Anaerobic, non motile, gram positive curved rods Produce acids: acetate and lactate Growth inhibited at pH of 5.5 Can survive intestinal digestion and appear in stool Constitute most of the microflora of breastfed infants Optional Speaker Notes: Bifidobacteria have all the characteristics of a probiotic bacteria. Importantly, the genus Bifidobacterium constitute most of the microflora of breastfed infants. Amongst the bifidobacteria better studied for application as a probiotic is Bifidobacterium lactis. B. lactis is the short name for B. animalis subspecies lactis, previously also called B. bifidum, strain Bb12. Bifidobacterium lactis Nomenclature - B. lactis also: B. animalis sub-species lactis, B. bifidum, strain Bb12 99 99

Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria
November 27, 2018 Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria * Optional Speaker Notes: Results from another double-blind, randomized controlled study, in which a number of formula fed infants provided B. lactis realized an increase in stool bifidobacteria, are presented here. In this study, 20 full-term infants were provided formula supplemented with B. lactis, and an equal number provided non-supplemented formula. 14 Breastfed infants served as controls. Results indicated a similar percentage of infants fed B. lactis formula were colonized with bifidobacteria (having > 10 (6) CFU/ml stool) as were the breast milk cohort of infants. A significantly lower percentage of infants were colonized with bifidobacteria after receiving standard formula with no probiotics. In this and other studies, B. lactis was supplemented to infant formula together with Streptococcus thermophilus. S. thermophilus has not typically been found to have a probiotic effect and in some of these formulations it was incorporated for the purposes of helping probiotic viability in the product. *P<0.05 compared to standard formula. Adapted from: Langhendries JP., et al. JPGN 1995;21: 100

Incidence of Diarrhea (%)
November 27, 2018 (p=0.035) Saavedra et al., 1994; The Lancet 344:

Rotaviral Shedding (%)
November 27, 2018 (p=0.025) Saavedra et al., 1994; The Lancet 344:

November 27, 2018 October 1994 Following Lancet Publication 1994

Number of Scientific Publications on Probiotics
November 27, 2018 Number of Scientific Publications on Probiotics Medline 2006

With Extensively Hydrolyzed Formula (EHF) and Probiotics
November 27, 2018 Treatment of Atopic Disease With Extensively Hydrolyzed Formula (EHF) and Probiotics ( ) * * Population: Eczema 4-6 mo on exclusive breast feeding N= 27, results at 2 months of intervention ( ) ( ) *P=0.01 compared to EHF; SCORAD range in parenthesis. Isolauri E., et al. Clin Exp Allergy 2000; 30:

November 27, 2018 Probiotics for the Prevention of Antibiotic Associated Diarrhea (AAD) in Infants * Optional Speaker Notes: The results of the Correa study, one of the studies shown on the previous slide, are highlighted here. In this double-bind, formula controlled study, 80 infants, 6 to 36 months of age were randomized to B. lactis supplemented formula, or placebo at the initiation of antibiotic therapy for 15 days. S. thermophilus, not thought to have a probiotic effect was added to the formula to help maintain viability of the B. lactis. Results indicated a near 2-fold reduction in the incidence of diarrhea in infants receiving the probiotic supplemented formula, compared to control. In most cases, probiotics have been concomitantly used with the prescription of an antibiotic and these have shown a reduction in incidence or duration of loose stools while on antibiotic therapy for various populations of infants and children. *P<0.05 Correa NBO., et al. J Clin Gastroenterol 2005;39: 106

Effect of B. lactis in Preterm Infants: Improved Gut Maturation
November 27, 2018 Effect of B. lactis in Preterm Infants: Improved Gut Maturation Placebo = 32 Probiotic = 37 * ** Significant decrease in calprotectin, a marker of inflamation, in probiotic treated group. The difference in levels between infants also receiving the antibiotic was NS. *P<0.05; **P<0.01 Mohan R. et., al. EUROBIO 2006; abstract.

Birth Promote Promote Hinder Probiotic bifidobacteria
November 27, 2018 Birth C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Probiotic bifidobacteria Promote Promote Hinder Establishment of “healthy” microbiota Predominance of bifidobacteria Development of adequate gut barrier function Development of adequate immune response

Lactic Acid Bacteria, Food and Infant Formula
November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Summerians (Cheese) Celts & Huns (Khefir) Monks Perfect fermentation Domestication of Animals Abraham (Milk & curds) 7,000 BC 3,500 BC 2,000 BC AD 2000

November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Domestication of Animals Summerians (Cheese) Abraham (Milk & curds) Celts & Huns (Khefir) 7,000 BC 2,000 BC AD 3,500 BC 2000 Monks Perfect fermentation

November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Domestication of Animals Summerians (Cheese) Abraham (Milk & curds) Celts & Huns (Khefir) 7,000 BC 2,000 BC AD 3,500 BC 2000 Monks Perfect fermentation Pasteurization Infant Formula 1900 1950 2000

November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Domestication of Animals Summerians (Cheese) Abraham (Milk & curds) Celts & Huns (Khefir) 7,000 BC 2,000 BC AD 3,500 BC 2000 Monks Perfect fermentation Pasteurization Infant Formula Metchnikov 1900 1950 2000

November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Domestication of Animals Summerians (Cheese) Abraham (Milk & curds) Celts & Huns (Khefir) 7,000 BC 2,000 BC AD 3,500 BC 2000 Monks Perfect fermentation Pasteurization Infant Formula Metchnikov Antibiotics 1900 1950 2000

November 27, 2018 Lactic Acid Bacteria, Food and Infant Formula Domestication of Animals Summerians (Cheese) Abraham (Milk & curds) Celts & Huns (Khefir) 7,000 BC 2,000 BC AD 3,500 BC 2000 Monks Perfect fermentation Pasteurization “Probiotics” Foods with Probiotics Infant Formula Metchnikov Antibiotics 1900 1950 2000

Role of Intestinal Microbiota
November 27, 2018 Role of Intestinal Microbiota Germ free animals Atrophic mucosa Germ free animals + normal flora Bacterial translocation leading to death A healthy flora is critical for maintaining GI integrity, barrier function, and survival

Enteral Nutrient supports Enteral Trophisn and Intestinal Flora
Bacteria “train” the GALT Immune effects are manifested systemicaly Antigens activate GALT

Probiotic Characteristics:
November 27, 2018 Probiotic Characteristics: Be nonpathogenic in nature Be resistant to destruction by technical processing Be resistant to destruction by gastric acid and bile Adhere to or transiently colonize intestinal epithelial tissue Provide a measurable benefit to the host Optional Speaker Notes: By definition, a probiotic should be non-pathogenic. Although any organism can be pathogenic in nature, the best-studied probiotics are expected not to cause disease in the host. Probiotics need to be viable in the product in which they are consumed. Therefore, they need to be resistant to the technical processing prior to consumption. Probiotics should remain viable after passage through the stomach and the small bowel, and therefore need to be resistant to gastric acid and bile digestion. Until recently, some considered “being of human origin” as a necessary characteristic for a bacterium to be considered a probiotic. That is no longer the case. In general, to have an effect, a probiotic bacterium needs to transiently colonize the intestinal epithelium. Once the intestinal flora of the host is well-established, the oral intake of bacteria will modify the composition of the flora and may have the various immunologic effects; however once consumption stops, the quantities of the specific probiotic species being ingested will decrease and most often disappear. Therefore, for maintaining its probiotic effects, most of these bacteria need to be consumed regularly and consistently. Most importantly, a probiotic bacterium can only be called a probiotic if a benefit to the host has been demonstrated. Not all bacteria that are ingested and survive digestion are necessarily probiotics. Teitelbaum JE and Walker WA. Ann Rev Nutr 2002;22: 118 118

Bacteria could have positive influence on normal microbiota
November 27, 2018 Bacteria could have positive influence on normal microbiota Hypothesized that lactobacilli were important for human health Metchnikoff E. The prolongation of life: Optimistic Studies. London: G. P. Putnam & Sons, 1907. Elie Metchnikoff ( )

B. lactis Fed to Infants Can Modify Intestinal Microflora:
November 27, 2018 B. lactis Fed to Infants Can Modify Intestinal Microflora: Increase counts of bifidobacteria in infants fed B. lactis Increase short chain fatty acids, and lower stool pH Decrease Clostridia and other Enterobacteriaceae Optional Speaker Notes: Due to its use and viability in the food supply, B. lactis has been shown to increase the counts of bifidobacteria when fed to infants. In a recent double-blind, placebo-controlled clinical trial, Mohan and colleagues (2006) randomized 69 perterm infants to a B. lactis supplemented formula, or unsupplemented formula for their first 21 days of life. Results from analysis of stool samples, collected weekly for 3 weeks, indicated that cell numbers of Bifidobacterium were significantly increased and Enterobacteriaceae and Clostridium significantly decreased in B. lactis supplemented infants, compared to control. In another report of the same cohort of infants (Mohan 2006), fecal pH was significantly lower in the B. lactis supplemented infants compared to control. Low pH facilitates the proliferation of lactic acid bacteria and bifidobacteria and inhibits growth of some pathogenic microorganisms. Reduced pH can result from increased short chain fatty acids, as seen by the increased fecal acetate in the Mohan (2006a) report. An earlier trial by Fukushima and colleagues (1997) evaluated the effect of a follow-up formula containing B. lactis on microflora and fecal metabolites in healthy children age months old. In this prospective trial, 7 toddlers consumed the supplemented formula for 21 days; stool was analyzed prior to supplementation, three times during the intervention, and again one week after cessation of the supplement. Stool analysis results indicated that Clostridia was significantly decreased during intake, and Bifidobacterium, strain Bb 12, was significantly increased, but returned to undetectable levels within one week after the supplementation period. In addition, changes in fecal metabolic products such as significantly decreased ammonia, and significantly increased short chain fatty acids (acetic acid) were found in stool samples during intake, but also returned to levels similar to before intake one week after supplementation. Fukushima Y., et al. Bioscience Microflora 1997;16:65-72. Mohan R., et al. J Clin Microbio 2006;44: Mohan R., et al. JPGN 2006;45:E36-E37(abstr). 120 120

November 27, 2018 Effects of documented with Probiotic Supplementation on Protective Gut Barrier Function and Immune Function Decreased gut permeability Increase d mucin production Increased IgA secreting cells and secretory IgA Increased natural killer cell tumor-killing activity Increased production of macrophages and activated phagocytosis Immune modulation towards antigen tolerancetolerance Optional Speaker Notes: B. lactis has also been shown in various studies to help support gut barrier function and immunity, including decreasing gut permeability, increasing cytokines associated with immunologic tolerance and increasing secretory IgA and IgA secreting cells. To determine the effect of B. lactis on gut mucosal barrier function, a recent case-controlled, blinded trial by Stratiki and colleagues (2007) measured intestinal permeability using the sugar absorption test. Seventy-five premature infants were randomized to a preterm formula supplemented with B. lactis, or to unsupplemented formula. The Lactulose/mannitol ratio in the B. lactis supplemented infants was significantly lower after 30 days than in the control group (p=0.003). Improved intestinal permeability has important health implications, especially in these high risk infants. Another recent double-blind, placebo-controlled trial assessed the influence of probiotic supplementation on cytokines associated with immunologic tolerance. Rautava and colleagues (2006), randomized 81 full term infants that were approximately 1 month old to a probiotic (L. rhamnosus GG and B. lactis) supplemented formula, or to unsupplemented formula. At 12 months, serum concentrations of the soluble innate microbial receptor (sCD14) were significantly higher in the probiotic supplemented infants than the control. Stimulation of CD14 by microbial products has been previously demonstrated to induce TGF-B production in other studies. At 12 months an approximate 6 fold increase in serum TGF-B2 was observed in the supplemented infants. Although not statistically significant, microbial stimulation in the intestine by probiotics could lead to augmented TGF-B2 responses both systemically, and on the mucosa as a result of enhanced signalling through CD14. Another finding in this study was that the number of cow’s milk-specific IgA secreting cells were significantly higher in the probiotic supplemented infants, compared to control. Results from an additional study evaluating B. lactis on IgA levels in young children (Fukushima, 1998) is on the next slide. Fukushima Y., et al. Int J Food Micorbiol 1998;42:39-44. Rautava S., et al. Pediatr Res 2006;60: Stratiki Z., et al. Early Hum Dev 2007 on-line at: 121 121

November 27, 2018 Premature Infants: Prime set-up for an Altered Microbiota and its potential consequences C-section birth Less chances of being breast fed NICU microbes Antibiotics Delayed establishment of microbiota Aberrant composition Optional Speaker Notes: B. lactis has also been shown in various studies to help support gut barrier function and immunity, including decreasing gut permeability, increasing cytokines associated with immunologic tolerance and increasing secretory IgA and IgA secreting cells. To determine the effect of B. lactis on gut mucosal barrier function, a recent case-controlled, blinded trial by Stratiki and colleagues (2007) measured intestinal permeability using the sugar absorption test. Seventy-five premature infants were randomized to a preterm formula supplemented with B. lactis, or to unsupplemented formula. The Lactulose/mannitol ratio in the B. lactis supplemented infants was significantly lower after 30 days than in the control group (p=0.003). Improved intestinal permeability has important health implications, especially in these high risk infants. Another recent double-blind, placebo-controlled trial assessed the influence of probiotic supplementation on cytokines associated with immunologic tolerance. Rautava and colleagues (2006), randomized 81 full term infants that were approximately 1 month old to a probiotic (L. rhamnosus GG and B. lactis) supplemented formula, or to unsupplemented formula. At 12 months, serum concentrations of the soluble innate microbial receptor (sCD14) were significantly higher in the probiotic supplemented infants than the control. Stimulation of CD14 by microbial products has been previously demonstrated to induce TGF-B production in other studies. At 12 months an approximate 6 fold increase in serum TGF-B2 was observed in the supplemented infants. Although not statistically significant, microbial stimulation in the intestine by probiotics could lead to augmented TGF-B2 responses both systemically, and on the mucosa as a result of enhanced signalling through CD14. Another finding in this study was that the number of cow’s milk-specific IgA secreting cells were significantly higher in the probiotic supplemented infants, compared to control. Results from an additional study evaluating B. lactis on IgA levels in young children (Fukushima, 1998) is on the next slide. Inadequate GALT development and maturation Decreased gut barrier (mucin, permeability) Poor humoral and cellular immune response 122 122

Probiotics in Animal IBD Models
Feeding probiotics to mice and rats with IBD-type colitis has been shown to improve or prevent intestinal inflammation So again just like with allergic disase, with the establishment that gut flora is involved in the disease pathogenesis, the prospect of manipulating the flora with the introduction of nonpathogenic probiotic bacteria has of course been looked at in the management of IBD. Again similar to the allergy story these studies are relatively recent and certainly many more need to be done. With the availability of the mouse and rat models of IBD, investigators certainly turned towards these models to see whether probiotics wold modify the disease and they did show that probiotics were effications in improving intestinal inflammation or even prevent it but only when the probiotic manipulation was introduced very early in the life of the animal. Fabia et al, Scand J Gastroenterol 1993 Schultz et al, Gastroenterology 1998 Masden et al, Gastroenterology1999

Microflora Antigens Th0 TReg Th2 Th1 Tolerance IBD Allergy TNF-α IL-4
November 27, 2018 Microflora Antigens Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 TReg Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 TGF-β Over Expression Over Expression Tolerance IBD Allergy

Acquisition of Indigenous Flora by the Newborn
November 27, 2018 Acquisition of Indigenous Flora by the Newborn Factors that influence the development of newborn’s indigenous flora: Gestational age Mode of delivery Type of feeding Antibiotics Optional Speaker Notes: Critical to the development of an adequate immune function of the gut-associated immune tissue is the way in which infants acquire their microflora. Factors that influence the development of microflora are presented on the slide. Very low birth weight infants will have delayed intestinal colonization, and their microflora, particularly when in the NICU, will be different than that of healthy infants born vaginally and whom are breastfed. VLBW infants: delayed intestinal colonization, development of anaerobic flora diminished Infants born by Cesarean Section: delayed intestinal colonization with anaerobic bacteria, gut colonization altered for as long as 6 months after birth Swanson D. Indigenous Flora. In Feigin RD et al., eds. Textbook of Pediatric Infectious Diseases. 5th ed. Philadelphia: WB Saunders;2004: 125

Flora and Immune Response
November 27, 2018 Flora and Immune Response Determinants of Intestinal Flora in newborns are the same as the determinants of exaggerated immune response (allergic reactions).

Re-thinking Early Feeding in Healthy Newborn Infants
November 27, 2018 Re-thinking Early Feeding in Healthy Newborn Infants Continue intact cow proteins? Continue sterile diets?

November 27, 2018 Clinical Benefits and Outcomes Associated with Probiotics in Infants and Children: Modification of intestinal microflora Treatment of acute diarrhea Prevention of acute diarrhea Decrease of antibiotic associated diarrhea Emerging evidence in Treatment and prevention of allergy Prevention of NEC Optional Speaker Notes: Overall, it has been demonstrated that probiotics will modify intestinal microflora towards a more balanced composition, with a greater predominance of bifidobacteria and/or lactobacillus. As shown on the last few slides B. lactis supplementation to infants has resulted in increased counts in stool and increased number of infants being colonized with bifidobacteria. Specific probiotic bacteria have been effective in the treatment and prevention of acute diarrhea, and the greater part of the literature supports these benefits. Specific probiotics may decrease diarrhea associated with the intake of antibiotics, and there is emerging evidence for the benefits of specific probiotics in the treatment and prevention of allergy as well as the prevention of necrotizing enterocolitis. The next several slides discuss the risk factors for allergy, the relationship between allergy, immunity, and microflora and some clinical trials with probiotics in the treatment and prevention of allergy. 128 128

Microbiota Dynamics During Life
November 27, 2018 Microbiota Dynamics During Life First describe the slide!! This is how a normal colonisation occurs... By the time of weaning, the flora is basically acquired and stable. The first bacteria to colonise a previously sterile gut may establish a permanend niche by inducing specific and lasting glycosylation of the glycocalyx as well as by modulating enterocyte gene expression, thus putting later arrivers at competitive disadvantage. Since colostral and breast milk contain numerous factors that favor early colonisation by bifidobacteria and lactobacilli, feeding formulas may alter implantation and long-term colonisation of these organisms. Mitsuoka et al., 1996

The Case for Bacteria - Probiotics
Active CD (Open label n=25) Lactobacillus salivarius 76% avoided other meds, Mean CDAI fell from 217 to 150 CD maintenance (RCT n=32) Saccharomyces boulardii plus 2g mesalamine 6% relapse/6 months versus 62% relapse with 3g mesalamine Active UC (RCT n=116) 44 (75%) with mesalamine group attained remission 39 (68%) in the E. coli (Nissle 1917) group Pouchitis (RCT maintenance of remission, n=40) 3/20 (15%) relapse on the probiotic preparation, VSL #3 100% with placebo Probiotics may work by effects on immune system (innate and adaptive), barrier function, interaction with other flora. More data in UC and pouchitis than CD McCarthy J. Gut 2001;49(Suppl III):2447. Guslandi M. DDS 2000;45: Rembacken. Lancet. 1999;354:635. Gionchetti. Gastroenterology. 2000;119:305.

Medical Literature on Probiotics in IBD
1996 to 2004 Number of publications Fedorak R Inflamm Bowel Dis 2004:10; ; Medline

As we know, There are known knowns. There are things we know we know
As we know, There are known knowns. There are things we know we know. We also know .. There are known unknowns. That is to say We know there are some things We do not know. But there are also unknown unknowns, The ones we don't know We don't know. Donald Rumsfeld Feb. 12, 2002, Department of Defense news briefing

Determinants of Intestinal Flora in Early in Life
November 27, 2018 Determinants of Intestinal Flora in Early in Life Mode of feeding Breast fed vs. Formula Formulas fed infants have less predominance of Bifidobacteria Mode of delivery Vaginal delivery vs. C section C- section infants have less predominance of Bifidobacteria Flora determ by many factors. Two major are .Can we do something about this

November 27, 2018 Influence of Cesarean Delivery on Relative Risk of Childhood Food Allergy * CI ( ) Adjusted Odds Ratio CI ( ) *P<0.01; adjusted for covariates. Food Allergy to egg confirmed by testing at age 1 – 2. Eggesbo M et al. J Allergy Clin Immunol 2003;112:

November 27, 2018 Treatment of Atopic Disease With Extensively Hydrolyzed Formula (EHF) and Probiotics ( ) Population: Eczema 4-6 mo on exclusive breast feeding N= 27, results at 2 months of intervention * * ( ) ( ) *P=0.01 compared to EHF; SCORAD range in parenthesis. Isolauri E., et al. Clin Exp Allergy 2000; 30:

Safety of Probiotics Pros
November 27, 2018 Safety of Probiotics Pros Current use of lactic acid bacteria in food is in thousands of metric tons annually world wide No increases in infections of lactic acid bacteria have been documented with increased consumption No evidence for higher incidence of infections than normal intestinal microbiota

Safety of Probiotics Cons: They are live microorganisms
November 27, 2018 Safety of Probiotics Cons: They are live microorganisms Clearly specific strains interact with gut cellular and metabolic functions Clearly they can translocate - like any other organism

Safety of Probiotics Cons:
November 27, 2018 Safety of Probiotics Cons: Episodes of lacto-bacilemia have been reported in compromised patients No episodes of bifidobacterial infection Bottom line: When referring to efficacy and safety, each species- and likely each individual strain need to be assessed individually

Example of a specific strain ideally suited for use in infant formula
November 27, 2018 B. animals subspecies lactis (Bb12) Example of a specific strain ideally suited for use in infant formula Anaerobic, non motile, Gram + curved rods Produce acetate and lactate Growth inhibited at pH of 5.5 Constitute most of the flora of breastfed infants Can survive intestinal digestion and appear in stool

Considerations in assessment of safety of a probiotic
November 27, 2018 Considerations in assessment of safety of a probiotic Individual genus and species History of use Presence in the host Undesirable metabolites (ie D lactate) Interaction with other organisms, plasmids or antibiotic resistance transmission History of use in vulnerable populations Documentation of bacteremia / infection Dose (impossible to “overdose”)

November 27, 2018 Safety of B. lactis Genus Bifidobacteria: Predominant in breast fed infants No pathogenicity reported B. lactis widely used in foods, including infant weaning foods B. lactis containing yogurt worldwide: ~ 1 Million Tons per year consumed

November 27, 2018 Safety of B. lactis Infant formula with B. lactis: 15 years, 30 countries > 2 million infants More than 15 clinical trials involving more than 1,800 infants, half receiving B. lactis, Including trials in term newborns, infants of HIV mothers, and premature infants Only probiotic bacterium with “GRAS status” for use in infant formula Full genome decoded