Gut Microbiota, Immunity, and Probiotics

Gut Microbiota, Immunity, and Probiotics

Objectives Describe the impact of early infant feeding on immune related disease. Review the role intestinal microbiota in supporting gut barrier function and modulating immune responses. Define probiotics and discuss their potential effect on immunity. Discuss clinical evidence for the use of probiotics in infancy and early childhood.

September 19, 2018 Chronic Disease Prevalence in the Last 50 Years & the Hygiene Hypothesis (Th1) The sharp decrease in infectious diseases in the last 50 years has been associated with a similar increase in various autoimmune or immunologic disorders. Increases in over-expression of Th1 (auto-immune type) and Th2 (allergic type) mediated diseases have been documented. The association between a decrease bacterial exposure and an increase in abnormal immunologic conditions has led to the “hygiene hypothesis” which suggests that the lack of immunologic microbial experience by the host can lead to a disordered immune response, and in turn, to an increase in immune related conditions. One of the fastest increases is in allergic asthma (Th2) A decrease in microbial exposure and infectious diseases is associated with an increase in immune related conditions Bach JF. N Engl J Med. 2002;347: 3 3 3

Increasing Prevalence of Allergic Diseases
15 10 1964 % prevalence (children 8-13 yrs) 1989 5 Aside from asthma, significant increases in allergic rhinitis and AD have also been reported. Asthma Allergic Rhinitis Atopic Dermatitis Ninan, et al., BMJ 1992

Increasing Prevalence of Atopic Dermatitis: Almost 1 in 5 infants develop AD
Atopic dermatitis has been steadily increasing for over the past half century. Today almost one in five infants develop atopic dermatitis. Data collected in 2000 and reported by Selnes found an almost 21% prevalence of atopic dermatitis is Norwegian school children. A study by Eichenfeld of Oregon schoolchildren found a history of AD reported for 17.2% of the children in 1996, a figure that is comparable to the 15.6% prevalence demonstrated in northern European countries. Another recent study of 5- to 9-year-old Japanese schoolchildren assessed that there was evidence of AD in 21% of the children. A cohort study in Britain found the prevalence of AD in children to be 5.1% in those born in 1946, 7.3% in those born in 1958, and 12.2% in those born in In 1994, 20% of children 3 to 11 years of age were reported to have AD. Overall, AD has increased 2- to 3-fold during the past 30 years and currently is believed to affect between 10% and 15% of the population at some point during childhood. Today in the US 17% of infants develop Atopic Dermatitis by 6 months of age* Adapted from Selnes et al. Allergy : 60: 894–899; Eichenfield et al Pediatrics 2003;111: *Moore, et al. Pediatrics 2004; 113(3 Pt 1):

Potential Allergic Reaction
Can We Reduce the Risk? Immune System Potential Allergic Reaction Antigen Antigen

Potential Allergic Reaction
Can We Reduce the Risk? Antigen Immune System Potential Allergic Reaction Antigen Allergenic Proteins: Foods Aero Allergens Immune Modulators: Nutrition Microbes Contributing factors Contributing factors for a healthy infant: Immune Modulators: Nutrition status and microbes Allergenic Proteins: Foods and Aero Allergens May also include medications and hereditary factor

Potential Allergic Reaction Potential interventions
Can We Reduce the Risk? Antigen Immune System Potential Allergic Reaction Allergenic Proteins: Foods Aero Allergens Immune Modulators: Nutrition Microbes Contributing factors Breastfeeding Less allergenic proteins? Oral bacteria- probiotics? Potential interventions to reduce risk

Risk Factors for Allergy at 1 year of Age: Diet
Relative Risk 1 = risk of atopic disease in the general population We are going to talk about two things that can lead to allergy: proteins and the gut microbiota Marini et al Acta Pediatrica 1996, 414:1-22

Can We Reduce the Risk? Antigen Immune System Potential Allergic Reaction Allergenic Proteins: Foods Aero Allergens Immune Modulators: Nutrition Microbes Contributing factors Potential interventions to reduce risk Breastfeeding Less allergenic proteins? Oral bacteria- probiotics?

Allergic Sensitization in Early Life
The first days matter Once sensitized…the immune system remembers “Exclusive breast feeding in the nursery” means: “EXCLUSIVE breast feeding in the nursery” Once you are immunologically sensitized, it can’t be reversed. What happens – particularly in the first days – is very important. Host A, Importance of the first meal on the development of cow's milk allergy and intolerance. Allergy Proc, 1991; 12(4):

Potential for Allergic Reaction
Can We Reduce the Risk of Allergy in Non-Breast Fed Infants? Hydrolyzed proteins may decrease allergenicity and/or “tolerize” the immune system to a protein High Molecular Weight Potential for Allergic Reaction Low Molecular Weight Immune System Hydrolysis In the first few months of life, intact proteins can pass through the gut wall more abundantly, increasing the risk for sensitization. - Since the gut closure has not occurred, it may allow large undigested proteins to pass through more easily - In general, larger and more intact proteins have more antigenic potential and greater potential for sensitization than smaller peptides. As the protein size decreases, the potential for allergic reaction may also decrease. - The larger the protein, the more potentially allergenic it is--especially non- human proteins (like cow’s milk proteins).

Hydrolysis May Reduce Allergenicity of Cow Milk Proteins
Median Molecular Weight of Infant Formulas Daltons One approach to decreasing the allergenicity of cow’s milk proteins is to hydrolyze intact proteins into smaller fractions (peptides). Currently infant formulas can contain intact (whole) casein/whey, partially hydrolyzed whey, or extensively hydrolyzed casein. Dalton weights listed represent median values of the peptides in these infant formulas. Extensively hydrolyzed formulas are therapeutic formulas and contain about 40% free amino acids, have a higher osmolality, and undesirable taste. Partially hydrolyzed whey formula is a routine infant formula with acceptable taste. Intact protein formulas are also routine infant formulas. *It must be noted that, unlike extensively hydrolyzed casein formulas, partially hydrolyzed whey formulas are routine infant formulas and not intended for therapeutic use in infants who have already presented with allergic disease. **Approximate values as reported by major manufacturers.

Cumulative Incidence AD (%)
100% Whey Protein Partially Hydrolyzed and Extensively Hydrolyzed Casein Infant Formulas May Reduce Allergy Risk to 6 Years Cumulative Incidence AD (%) ** * *** * The German Infant Nutrition Intervention Study (GINI) was the largest, longest, independent study assessing the risk of AD with hydrolyzed infant formula with 2252 infants enrolled. This slide represents the 6 year results from the GINI study, which indicated risk reduction of AD at 6 years of life. GINI 6 revealed the important finding that nutritional intervention with hydrolyzed infant formulas in the first 4 months of life may have a long-lasting risk reduction effect on atopic dermatitis in high-risk children. There was not just a delay in the appearance of allergy, but that hydrolyzed formulas may have reduced the risk of ever developing them. The data of the 6-year follow-up of the GINI study showed a persistent risk reduction of eHF-C and pHF-W in the ITT and PP analyses on Allergic Manifestations and Atopic Dermatitis. One formula group in the study, which was not included in this graph is extensively hydrolyzed whey formula, because it is not available in the US. * * *p<0.05 **p=0.021 ***p=0.002 von Berg et al., J Allergy Clin Immunol 2008;121:1442-7

Can We Reduce the Risk? Antigen Immune System Potential Allergic Reaction Allergenic Proteins: Foods Aero Allergens Immune Modulators: Nutrition Microbes Contributing factors Breastfeeding Less allergenic proteins? Oral bacteria - probiotics? Potential interventions to reduce risk

Development of Intestinal Microbiota
The intestine of the newborn at birth is essentially sterile. In the first few hours of life the intestine rapidly becomes colonized by a broad spectrum of bacterial species. 16

Development of Intestinal Microbiota
Coliforms colonize very rapidly – almost immediately during birth. Bifidobacteria are the most common genus of bacteria in human infants, particularly in infants who are breastfed. The bacteria to which infants are exposed in the first hours of life define their intestinal microbiota which is critical in the development of the newborns immune response. CRITICAL PERIOD 17

Bacteria in the GI Tract: Complex Ecosystem Resident and Ingested
September 19, 2018 Bacteria in the GI Tract: Complex Ecosystem Resident and Ingested Stomach 104 CFU/g Candida albicans Helicobacter pylori Lactobacillus Streptococcus 500 – 1000 species Duodenum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Jejunum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Ileum CFU/g Bacteroides Clostridium Enterobacteriaceae Enterococcus Lactobacillus Veillonella Colon CFU/g Bacteroides Bacillus Bifidobacterium Clostridium Enterococcus Eubacterium Fusobacterium Peptostreptococcus Ruminococcus Streptococcus Estimation of culturalable species 18

Role of Intestinal Flora (Microbiota)
September 19, 2018 Role of Intestinal Flora (Microbiota) Gut microbiota help support gut barrier function: ↑ Mucin production ↓ Permeability Gut microbiota help support the adaptive immune response: Generate IgA activity (humoral) Balance in T helper cell subclasses (cellular) 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: 19 19 19

GALT is the largest immune organ
September 19, 2018 GALT Gut associated lymphoid tissue (GALT) comprises 70-80% of immunologic cells in the body GALT is the largest immune organ Most of the immune cells of the infant are associated with the intestine - to help protect the infant from potential pathogens in the intestinal lumen 20

Germ-free vs. Colonized Gut
September 19, 2018 Germ-free vs. Colonized Gut The intestinal mucosa of animals who are born sterile and receive sterile food and are raised in a sterile environment do not develop an intestinal microbiota - and the intestinal mucosa is atrophic, has less cell turnover, less enzyme activity, less secretion of antibodies (IgA in particular), and the mucosa wall is thin. Animals raised in a normal environment and receive non-sterile food, develop a normal intestinal flora; and a normal barrier function, to protect the host. A healthy microbiota is critical to train the immune system to protect the host and decrease the chances for immune over expression (immune related conditions). 21

The Intestine is Sterile at Birth What are the oral sources of bacteria in the newborn’s sources of bacteria? Birth canal Breast feeding Environment

Are there consequences to a sterile birth (c-section)?
Vaginal delivery is NOT a sterile procedure and is a major source of bacteria for the infant Are there consequences to a sterile birth (c-section)?

Cesarean Sections Linked to Increases in
Chronic Disease Odds Ratio CI: CI: CI: * Type 1 Diabetes: All children <19 years hospitalized for Type 1 diabetes (ICD x1, 250.x3) identified (N=1852) from hospital discharge data and linked with their birth certificates. Controls (N=7408) were randomly selected from birth records, frequency matched on year of birth. The increased incidence of Type 1 Diabetes was found in all subjects, but was found significant when analyses were restricted to the 1305 cases aged less than 10 years at hospitalization. Asthma: Methods: Analysis of data from the Oxford record linkage study (ORLS) to generate a retrospective cohort of 248,612 records of births between 1970 and 1989, with follow-up to records of subsequent hospital admission for 4,017 children with asthma up to 1999. Results: Univariate analysis showed significant associations between an increased risk of admission for asthma and later years of birth (reflecting the increase in asthma in the 1970s and 1980s), low social class, asthma in the mother, unmarried mothers, maternal smoking in pregnancy, subsequent births compared with first-born, male sex, low birth weight, short gestational age, caesarean delivery, forceps delivery and not being breastfed.Multivariate analysis, identifying each risk factor that had a significant effect independently of other risk factors, confirmed associations with maternal asthma (odds ratio (OR) 3.1, 95% confidence interval ), male sex (versus female, 1.8, ), low birth weight ( g versus g, 1.2, ), maternal smoking (1.1, ) and delivery by caesarean section (1.2; ). Celiac disease: A retrospective, multicenter, case-control study that included 1950 children. Information on intestinal disease manifestation, together with mode of delivery and gestational age at birth, postnatal complications, and breastfeeding, was collected by the attending physician from children and their parents who were visiting a gastrointestinal outpatient clinic for Crohn disease (CD; 516 cases), ulcerative colitis (250 cases), celiac disease (157 cases), and other gastrointestinal diseases (165 cases) and control subjects who were visiting ophthalmologic, orthodontic, and dental outpatient clinics (862 cases). RESULTS: Whereas the rate of cesarean delivery of children with Crohn disease or ulcerative colitis was similar to that of control subjects, a significantly enhanced likelihood of being born by cesarean delivery was found in children with celiac disease compared with control subjects (odds ratio: 1.8 [95% confidence interval: ]; P = .014). *p< .05 Adapted from: D'Angeli MA, et al. Arch Pediatr Adolesc Med Aug;164(8):732-8. Davidson R, et al. BMC Pulm Med Mar 16;10:14. Decker E, et al. Pediatrics Jun;125(6):e

Influence of Cesarean Delivery on Relative Risk of Childhood Food Allergy
* Adjusted Odds Ratio CI ( ) Several studies have shown a significant association between the occurrence of allergy and infants born by C-section. Data presented in this slide are specific for children with confirmed reactions to egg allergy testing between one and two years of age. Results from this study, of 2803 children from a population-based birth cohort, show the increased odds ratio of allergy among children born vaginally to mothers with no history of allergy, compared to an increase of more than twofold in infants born vaginally to mothers with a history of allergy. A more than sevenfold increase in risk of allergy was reported for children born to mothers with a history of allergy of whom were also delivered by C-section. These relationships remained significant after adjusting for confounding variables including maternal smiling, short-term breast-feeding, older siblings, and others. Results from this study indicate that Cesarean delivery is associated with subsequent food allergy, especially among predisposed children, supporting the theory that factors interfering with colonization may play an important role in the development of food allergy. THE BACTERIA THAT AN INFANT RECEIVES IN THE FIRST FEW MINUTES OF LIFE CAN AFFECT THEIR IMMUNE SYSTEM FOR YEARS CI ( ) *p<0.01; adjusted for covariates Pistiner M et al. J Allergy Clin Immunol 2008; 122(2): Table adapted from: Eggesbo M et al. J Allergy Clin Immunol 2003;112: Food Allergy to egg confirmed by testing at age 1 – 2 y

Quantity of Bacteria in Breast Milk
Plate Counts This slide depicts results from a recent study of 20 mother-infant (full term, vaginally delivered) pairs in Spain. It is particularly important because it provides total contribution of bacteria in breast milk, over time, as well as by species. Total bacteria counts were greatest at day one, and declined steadily over the 3 month testing period. These results are in agreement with others. Solis, G, et al. Anaerobe 2010;16:307-10

Bacteria in Breast Milk
Bacteria generally isolated in breastmilk of healthy women include: Staphylococcus Streptococcus Enterococcus Lactobacillus Bifidobacterium Culture-dependent and independent analyses of breast milk from healthy women identify multiple species of live bacteria and bacterial DNA. Gronlund, MM, et al. Clin Exp Allergy 2007;37: ; Gueimonde M., et al. Neonatology 2007;92:64-66; Solis, G, et al. Anaerobe 2010;16:

Breastfeeding is a source of bacteria
Are there consequences to exclusive formula feeding?

Non-Exclusive Breastfeeding Increases the Risk of Immune Related Disease
p<.05 Odds Ratio Numerous studies have investigated the link between breastfeeding and the decreased risk of various immune related diseases. While the exact mechanisms are unknown, and likely multifaceted, the evidence is overwhelming that early breastfeeding is particularly critical to reduce the risk of chronic disease. A review by Knip found that introducing cow’s milk formula before two months of age led to a relative risk of 6.19 of insulin dependent diabetes. Klement preformed a meta-analysis on a number of studies evaluating the risk of Crohn’s Disease and Ulcerative Colitis and breastfeeding. The data shown here are from an analysis of what he and his group considered the top-tier studies – four in the Crohn’s group and four for UC. p<.05 p<.05 Adapted from: Knip M, et al. Am J Clin Nutr. 2010;91(5):1506S-1513S. Klement E, et al. Am J Clin Nutr. 2004;80(5):

Does Breast Feeding Reduce the Risk of Allergy?
Relative Risk of Atopic Dermatitis Meta Analysis – Infants with a Family History of Atopy Formula Feeding, Risk = 1 Relative Risk 1.00 Study Title: Breast-feeding and the onset of atopic dermatitis in childhood: a systematic review and meta-analysis of prospective studies. The review evaluated the association between exclusive breast-feeding during the first 3 months after birth and atopic dermatitis. Results: Exclusive breast-feeding during the first 3 months of life is associated with lower incidence rates of atopic dermatitis during childhood in children with a family history of atopy. Breast-feeding should be strongly recommended to mothers of infants with a family history of atopy, as a possible means of preventing atopic eczema. Gdalevich M, et al. J Am Acad Derm. 2001;45:

Breast Feeding does not “decrease” risk. Formulas “increase” risk.
Relative Risk of Atopic Dermatitis Meta Analysis – Infants with a Family History of Atopy 1.72 Relative Risk Breast Feeding is THE Standard 1.00 Breastfeeding should be the standard against which formulas is measured. Breastfeeding does not decrease the risk of developing atopic dermatitis – formula feeding increases that risk through the introduction of foreign cow’s milk proteins. Same data: But Converting Breast feeding risk to “1.” Adapted. OR with BF = .58 vs CMF Gdalevich M, et al. J Am Acad Derm. 2001;45:

“Modern” Lifestyle Has Decreased Exposure to Bacteria
September 19, 2018 “Modern” Lifestyle Has Decreased Exposure to Bacteria Lower microbial exposure Cesarean sections Sanitized food supply, including infant formula Decrease in naturally fermented foods Urban life Antibiotics Altered Intestinal microbiota Environmental changes in the industrialized world, as well as changes of life style in western societies, and medicine, have reduced the extent of microbial contact at an early age. These changes affect the initial establishment of the microflora. The hygiene hypothesis, first proposed by Strachan in 1989, offered, in part, an explanation to the increasing prevalence of atopic conditions. The idea was based on epidemiological observations, suggesting an inverse correlation between family size and the prevalence of allergic rhinitis. Today, an extended hygiene hypothesis is emerging that suggests that the modern infant may lack interactions with specific bacteria that promote the development of an anti-inflammatory and tolerogenic immune environment. The initial composition of gut microflora, through exposure to bacteria, may be considered a key determinant in the development and regulation of immune factors. Inadequate immune response Rautava S., et al. JPGN 2004;38: Rook GA. Immunol Today 1998;19: 32

Birth C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Promote Hinder A "healthy” intestinal microbiota modulates important GI functions In review, an infant is born with a sterile gut lumen, which rapidly becomes colonized. A number of factors promote or hinder the establishment of a “healthy” intestinal microbiota in which bifidobacteria predominates. Improved Gut Barrier Function Improved Immune Response 33

Birth Promote Promote Hinder
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Probiotic Supplementation Bifidobacteria / Lactobacilli Vaginal delivery Breast feeding Promote Promote Hinder A "healthy” intestinal microbiota modulates important GI functions In formula fed infants, supplemented with probiotics , we can increase mucosal physiology, increase barrier function, and decrease inflammatory responses safely. Improved Gut Barrier Function Improved Immune Response 34

September 19, 2018 Probiotics Nonpathogenic, live microorganisms in the food supply that, when consumed in adequate amounts, are capable of conferring a health benefit to the host Bifidobacteria Lactobacilli Yeasts (S. boulardii) Douglas LC, Sanders ME. JADA. 2008:108(3): Neu, J. et al. NCP 2007:22: 35 35 35 35

Commonly Used Probiotics
BIFIDOBACTERIA B. longum B. breve B. infantis B. lactis (Bb -12)* LACTOBACILLI L. casei L. rhamnosus (GG) L. plantarum L. reuteri (ATCC 55730, DSM 17938)* This slide indicates some of the most commonly used probiotic genus and species of Bifidobacteria and lactobacilli. Some of the specific clinical areas reviewed in the deck have been evaluated for other strains, such as LGG. Now we’d like to talk about two particular strains that are used in routine formulas, globally: B. lactis and L. reuteri * B. lactis and L. reuteri are the only two probiotics with GRAS status approved for use in term infant formula from day 1.

Bifidobacterium lactis
September 19, 2018 Bifidobacterium lactis Bifidobacteria found in breastmilk Predominant bacteria in the gut of breastfed infants Areas of clinical research showing positive results: Immune system development and modulation Increase in Secretory IgA Acute diarrhea risk reduction Antibiotic diarrhea risk reduction Emerging Evidence in Allergy and NEC GRAS status in term infant formula from day 1 Bifidobacteria have all the characteristics of a probiotic bacteria. Importantly, the genus Bifidobacteria constitute most of the microbiota 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, also called B. bifidum, strain Bb12. 1st bullet: Predominant bacteria in gut of breastfed infants. (Yoshioka 1983) 2nd bullet: Immune system development and modulation. (Fukushima 1998, Mohan 2008, Holscher and Tappenden 2010) 3rd bullet: Balanced intestinal flora (Holscher and Tappenden 2010, Stratiki 2007, Mohan 2006, Langhendries 1995) 4th bullet: Acute diarrhea risk reduction (Saavedra 1994, Mao 2008, Ziegler 2003) 5th bullet: Antibiotic diarrhea risk reduction (Correa 2005) 6th bullet: Allergy (Isolauri 2000, Arvola 2002, Kim 2010) B.lactis, strain Bb-12 Nomenclature - B. lactis also: B. animalis sub-species lactis, B. bifidum 37 37 37

Lactobacillus reuteri
September 19, 2018 Lactobacillus reuteri Isolated from breastmilk Most relevant areas of clinical research showing positive results: Reduced infant colic and crying Improved GI motility Reduced intensity of abdominal pain Regulated bowel movements Improved feeding tolerance in premature infants Diarrhea GRAS status in term infant formula from day 1 lactobacilli have all the characteristics of a probiotic bacteria. Amongst the lactobacilli better studied for application as a probiotics in infants is lactobacillus reuteri. L. reuteri is the short name for Lactobacillus reuteri, strain (ATCC 55730, DSM 17938) 1st bullet: Isolated from breastmilk. (Abramsson 2009) 2nd bullet: Reduced Infant colic and crying. (Savino 2007, 2010) 3rd bullet: Improved GI motility. (Savino 2004, Indrio 2009) 4th bullet: Reduced intensity of abdominal pain. ((Romano 2010) 5th bullet: Regulated bowel movements. (Coccorullo 2010) 6th bullet: Improved feeding tolerance in preterm infants. (Indrio 2008) 7th bullet: Diarrhea. ( Weizman 2005) L. reuteri strain ATCC55730, DSM 17038 Nomenclature – L. reuteri: Parent strain ATCC 55730, cured strain DMS 17938 38 38 38

Differences in Intestinal Microbiota and Feeding Type
September 19, 2018 Differences in Intestinal Microbiota and Feeding Type Bifidobacteria are generally predominant in breast fed infants Percent of Flora This slide represents results of a study by Harmsen et al. from the Netherlands, published in the Journal of Pediatric Gastroenterology and Nutrition in The relative percentage of intestinal bacteria from two representative infants are shown: One infant was breast fed, the other was formula fed for the first 20 days of life. Each had 6 stool samples analyzed for microbial composition. Results obtained from this study confirmed those of other studies in which the colonization of the intestine of the breastfed infant was composed of >60% bifidobacteria within one week after birth. Formula fed infants showed a more diverse flora, with more bacteriodes and coliforms, including E. coli. Breast fed Formula fed Bifidobacteria Bacteroides Coliforms Harmsen HJM et al.JPGN 2000;30:61 39

September 19, 2018 Documented Effects of Probiotic Supplementation on Protective Gut Barrier and Immune Function Decreased gut permeability Increased 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 tolerance 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 signaling 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: 40 40 40

Significantly Increased Fecal IgA in Vaginally Delivered Infants Fed B
Significantly Increased Fecal IgA in Vaginally Delivered Infants Fed B. lactis (Bb-12) Supplemented Infant Formula Methods: Six week-old healthy, full-term infants (n = 172) were enrolled in a prospective, randomized, double-blind, controlled clinical trial with 2 groups studied in parallel to a breastfed comparison group. Formula-fed (FF) infants were randomized to partially hydrolyzed whey formula (CON) or the same formula containing 10(6) colony forming units (CFU) Bb12/g (PRO) for 6 weeks. Fecal secretory IgA (sIgA), calprotectin, lactate, and stool pH were assessed at baseline, 2 weeks, and 6 weeks. Anti-poliovirus-specific IgA and anti-rotavirusspecific IgA were assessed at 2 and 6 weeks. Results: Among vaginally delivered FF infants, PRO consumption increased (P < .05) fecal sIgA compared to CON. Anti-poliovirus-specific IgA concentration increased (P < .05) in all infants consuming PRO, whereas antirotavirus- specific IgA tended to increase (P = .056) with PRO consumption in cesarean-delivered infants. Anthropometrics and tolerance did not differ significantly between FF infants. Conclusions: Infants consuming formula with Bb12 produced feces with detectable presence of Bb12 and augmented sIgA concentration. Furthermore, cesarean-delivered infants consuming Bb12 had heightened immune response, as evidenced by increased anti-rotavirus- and anti-poliovirus-specific IgA following immunization. These results demonstrate that negative immune-related effects of not breastfeeding and cesarean delivery can be mitigated by including Bb12 in infant formula, thereby providing infants a safe, dietary, immune-modulating bacterial introduction. Change from baseline at 6 weeks to 8 weeks Change from baseline at 6 weeks to 12 weeks *p<0.05 Holscher H. J Parenter Enteral Nutr. 2012;36:106S-17S 2012.

Increased Anti-Rotavirus IgA with B
Increased Anti-Rotavirus IgA with B. lactis (Bb12) Supplementation in Infants Born via C-section Probiotics increase anti-rotavirus-specific immunoglobulin A (IgA) in infants delivered by cesarean section. Data are expressed as means ± SEM. CON, control infant formula group; PRO, probiotic infant formula group. PRO infants delivered by cesarean section had greater (P = .056) changes in antirotavirus- specific IgA (fecal) concentrations from V1 (8 weeks of age prior to receiving vaccination) to V2 (12 weeks of age afterreceiving vaccination). *p=0.056 Holscher H. J Parenter Enteral Nutr. 2012;36:106S-17S 2012.

September 19, 2018 Potential Clinical Benefits and Outcomes of Probiotics in Infants and Children Modification of intestinal microbiota Reduced risk and duration of acute diarrhea Reduced risk of atopic dermatitis Reduced risk of NEC Infant colic Overall, it has been demonstrated that certain probiotics can modify intestinal microbiota towards a more balanced composition, with a greater predominance of bifidobacteria and/or lactobacillus. Specific probiotic bacteria have been effective in reducing the risk and duration of acute diarrhea, and the greater part of the literature supports these benefits. Specific probiotics may decrease diarrhea associated with the intake of antibiotics. There is emerging evidence for the potential risk reduction of infant colic, certain allergies, and Necrotizing Enterocolitis. Langhendries JP, et al. J Pediatr Gastroenterol Nutr. 1995; 21(2): Agostoni C, et al. J Pediatr Gastroenterol Nutr. 2004:38: 43 43 43

Probiotics Shown to Reduce the Duration of Acute Diarrhea
Meta-analyses of Randomized Clinical Trials Reduction in Duration (hours) CI: 19 – 42 CI: CI: 14 – 26 12 RCTs 9 RCTs 18 RCTs CI: 10 – 27 8 RCTs This slide depicts results from four meta analyses addressing the use of probiotics for acute diarrhea. As shown, the average reduction in duration in diarrhea varies from approximately fifteen to thirty hours of less duration with probiotics compared to placebo. Most of the clinical trials in these analyses are in pediatric patients; specifically, the meta analysis of Allen 2004 is the only one including adult patients. These meta analyses include studies that used multiple probiotic bacteria in various forms. These include, among others, Lactobacillus rhamnosus GG, L. acidophilus, L. bulgaricus, L. reuteri, and various strains of bifidobacteria. Thus, this meta-analysis provides evidence of the efficacy of probiotic supplements in reducing the duration of acute nonbacterial diarrhea by approximately 1 day. This slides shows you can change the course of an acute, infectious illness by providing oral probiotics. *Probiotic given as a supplement in all studies shown Adapted from: Szajewska H., et al. JPGN 2006;42:

Probiotics and Risk of Acute Diarrhea
Controlled Clinical Trials * * * * * Reduction in incidence (%) This slide depicts individual randomized controlled clinical trials in infants and children using various probiotic bacteria in infant formula (with exception of Ziegler, which gave probiotic as supplement) in the risk reduction of acute enteritis. The specific bacteria which have been most commonly used in these trials include B. lactis, L. rhamnosus GG, and L. reuteri. The reduction in incidence, similar to that seen for treatment of diarrhea, varies significantly among studies, from approximately 15-90%. In most of these studies, the greatest effect has been reported for an effect of probiotics on rotavirus enteritis. *p<0.05 compared to incidence in control of each study

Probiotics and Risk of Antibiotic Associated Diarrhea
Controlled Clinical Trials * RR=0.32 CI ( ) RR=0.29 CI ( ) * RR=0.47 CI ( ) RR=0.52 CI ( ) % Reduction in incidence Several clinical trials have reported efficacy of probiotics for antibiotic-associated diarrhea. This slide summarizes various prospective controlled clinical trials using probiotic bacteria for risk reduction of antibiotic-associated diarrhea. Percent reduction in incidence of antibiotic-associated diarrhea ranged from 4% to 71%. Due to study design and other methodological variables, results have varying levels of statistical significance. Similar to those studies addressing treatment or risk reduction of acute viral diarrhea, no study to date has shown an increase (statistically significant or not) of any type of diarrhea with the use of a probiotic. Jirapinyo: B. infantis and L. acidophilus – probiotic therapies utilized Tankanow: L. acidophilus and L. bulgaricus – probiotic therapies utilized RR=0.96 CI ( ) *P<0.05

Clinical Report on Probiotics and Prebiotics in Pediatrics
Acute Infectious Diarrhea “Results of published randomized controlled trials (RCTs) have indicated that there is modest benefit of giving probiotics in preventing acute gastrointestinal tract infections in healthy infants and children.” “…there is evidence to support the use of probiotics, specifically LGG, early in the course of acute infectious diarrhea to reduce the duration by 1 day.” Antibiotic Associated Diarrhea “…probiotics can be used to reduce the incidence of antibiotic-associated diarrhea.” Thomas D, et al. Pediatrics ;128(6):

Probiotics and Risk of Atopic Dermatitis
B. bifidum, B. lactis, L. acidophilus administered pre- and postnatally significantly reduced the incidence of eczema at 1 year Cumulative Incidence Adj OR=0.24 (CI: 0.075–0.792) In a randomized, double-blind, placebo-controlled trial, 112 pregnant women with a family history of allergic diseases received a once-daily supplement, either a mixture of Bifidobacterium bifidum BGN4, B. lactis AD011, and Lactobacillus acidophilus AD031, or placebo, starting at 4-8 wks before delivery and continuing until 6 months after delivery. Infants were exclusively breast-fed during the first 3 months, and were subsequently fed with breastmilk or cow's milk formula from 4 to 6 months of age. Clinical symptoms of the infants were monitored until 1 yr of age, when the total and specific IgE against common food allergens were measured. A total of 68 infants completed the study. The prevalence of eczema at 1 yr in the probiotic group was significantly lower than in the placebo group (18.2% vs. 40.0%, p=0.048). The cumulative incidence of eczema during the first 12 months was reduced significantly in probiotic group (36.4% vs. 62.9%, p=0.029); however, there was no difference in serum total IgE level or the sensitization against food allergens between the two groups. Prenatal and postnatal supplementation with a mixture of B. bifidum BGN4, B. lactis AD011, and L. acidophilus AD031 is an effective approach in preventing the development of eczema in infants at high risk of allergy during the first year of life. Kim JY. Pediatr Allergy and Immunol Mar;21(2 Pt 2):e

Probiotics and Risk of Atopic Dermatitis
LGG compared to placebo by mothers in last trimester of pregnancy and birth through 6 mo by infants * RR=0.64 CI ( ) * * The occurrence of differences in intestinal flora in infants with allergic disease has led to the use of probiotic agents for the management of atopy. In a double-blind, randomized placebo-controlled trial, probiotics were given prenatally to mothers who had at least one first-degree relative (or partner) with atopic eczema, allergic rhinitis, or asthma, and postnatally for 6 months to their infants. Atopic eczema was subsequently diagnosed in 35% of the 132 children aged 2 years. Results from this study, which has now followed children for more than four years, showed that the consumption of probiotics, in this case Lactobacillus rhamnosus GG, in infants born to families with a history of allergy, significantly decreased atopy diagnosis at the two-year and four-year follow up period. RR=0.51 CI ( ) RR=0.57 CI ( ) Adopted from: Kalliomäki M., et al. Lancet 2001; 357: Kalliomäki M., et al. Lancet 2003; 361: Kalliomäki M., et al. JACI 2007; 119: *P<0.05

Atopic Disease: “Although the results of some studies support the prophylactic use of probiotics during pregnancy and lactation and during the first 6 months of life in infants who are at risk of atopic disorders, further confirmatory evidence is necessary before a recommendation for routine use can be made.”

AAP Clinical Report on Probiotics and Prebiotics in Infants:
Statements on Probiotic Safety “To date, these products (probiotics and prebiotics) seem to be safe for healthy infants and children.” “Probiotics should not be given to children who are seriously or chronically ill until the safety of administration has been established.” “All ingredients used in infant formula must be safe and lawful—that is, food ingredients that are, to date, generally regarded as safe (GRAS)… Prebiotics and probiotics now being added to commercial infant formulas are classified as GRAS.” Thomas D, et al. Pediatrics ;128(6):

Can We Reduce the Risk? Antigen Immune System Potential Allergic Reaction Allergenic Proteins: Foods Aero Allergens Immune Modulators: Nutrition Microbes Contributing factors Breastfeeding Less allergenic proteins: 100% whey partially hydrolyzed Extensively hydrolyzed casein Oral bacteria – B. lactis, L. reuteri Potential interventions to reduce risk

Infant Formulas with Probiotics Currently Available in the US
Product Protein Type Probiotic Routine Infant Formulas with Probiotics GOOD START® Protect Infant Formula 100% whey partially hydrolyzed B. lactis Bb-12 GOOD START® Soothe Infant Formula L. reuteri DSM 17938 Therapeutic Infant Formula with Probiotics Nutramigen® with Enflora™ LGG® Infant Formula Extensively hydrolyzed casein L. rhamnosus GG

Infant Formulas: The goal for non-exclusively breast fed infants
Intestinal microbiota is critical to immune development and function Modern lifestyle has reduced our bacterial experience Certain probiotics added to infant feeding may promote microbiota similar to breastfed infants and support healthy immune function

Ingestion of Bacteria Sumerians (Cheese) Celts & Huns (Khefir)
September 19, 2018 Ingestion of Bacteria Sumerians (Cheese) Celts & Huns (Khefir) Pasteurization Abraham (Milk & curds) Monks (Refine Fermentation) 3500 BC 2000 BC AD 2000 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. 55 55

Intestinal Microbiota (A Balanced Ecosystem)
September 19, 2018 Intestinal Microbiota (A Balanced Ecosystem) Pseudomonas Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Potentially Helpful Bacteria Inhibition of exogenous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli Notes: No single bacteria is necessarily completely harmful or completely helpful. Some bacteria could be potentially harmful and are generally considered “less beneficial;” these include pseudomonas, staphylococci and clostridia. Other bacteria are known to have potentially beneficial effects to the host, including inhibition or competition for potential pathogens, aiding in digestion and absorption, providing trophic factors for the epithelium, involvement in vitamin synthesis, and support of gut barrier function and modulation of the gut immune response of gut-associated lymphoid tissue (GALT). Amongst the potentially beneficial bacteria are multiple species of the genus Lactobacillus and the genus Bifidobacterium. Lactobacilli and bifidobacteria are resident in most human hosts in varying numbers. The potential modification of the intestinal flora by increasing the amount of lactobacilli and bifidobacteria in the intestine, particularly in the small bowel, has led to the concept of probiotics: the ingestion of microorganisms for the purposes of a benefit to the host. Streptococci Eubacteria Bifidobacteria Bacteroides Adapted From: Gibson GR. J Nutrition 1995; 125: 56

Potential Probiotic Bacteria
September 19, 2018 Intestinal Microbiota (A Balanced Ecosystem) Pseudomonas Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Potentially Helpful Bacteria Inhibition of exogenous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli Notes: No single bacteria is necessarily completely harmful or completely helpful. Some bacteria could be potentially harmful and are generally considered “less beneficial;” these include pseudomonas, staphylococci and clostridia. Other bacteria are known to have potentially beneficial effects to the host, including inhibition or competition for potential pathogens, aiding in digestion and absorption, providing trophic factors for the epithelium, involvement in vitamin synthesis, and support of gut barrier function and modulation of the gut immune response of gut-associated lymphoid tissue (GALT). Amongst the potentially beneficial bacteria are multiple species of the genus Lactobacillus and the genus Bifidobacterium. Lactobacilli and bifidobacteria are resident in most human hosts in varying numbers. The potential modification of the intestinal flora by increasing the amount of lactobacilli and bifidobacteria in the intestine, particularly in the small bowel, has led to the concept of probiotics: the ingestion of microorganisms for the purposes of a benefit to the host. Streptococci Potential Probiotic Bacteria (When Ingested) Eubacteria Bifidobacteria Bacteroides Adapted From: Gibson GR. J Nutrition 1995; 125: 57

Probiotic Characteristics
September 19, 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 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: 58 58 58

Number of Scientific Publications on Probiotics
September 19, 2018 Number of Scientific Publications on Probiotics Medline search: Probiotics 59

B. lactis Fed to Infants Can Modify Intestinal Microbiota
September 19, 2018 B. lactis Fed to Infants Can Modify Intestinal Microbiota Increase counts of bifidobacteria in infants fed B. lactis Increase short chain fatty acids, and lower stool pH Decrease clostridia and other enterobacteriaceae 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 preterm 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) 60 60 60

Probiotics for Risk Reduction of Acute Diarrhea
September 19, 2018 Probiotics for Risk Reduction of Acute Diarrhea * Notes: This study was the first to demonstrate a reduction in the incidence of acute diarrhea in a prospective randomized double-blind, placebo-controlled trial with a group of infants chronically hospitalized for management of long term conditions including, respiratory, orthopedic and neurologic problems, but no gastrointestinal disease or chronic use of antibiotics. The 55 enrolled infants, aged 5-24 months, were randomized to receive a standard infant formula or the same formula supplemented with Bifidobacterium bifidum (former nomenclature for B. lactis) and S. thermophilus. During 17 months follow-up, 8 (31%) of the patients who received the control formula and 2 (7%) who received the supplemented formula developed diarrhea during the course of the study (p = 0.035). In this and other studies, B. lactis was supplemented to infant formula together with Streptococcus thermophilus. S. thermophilus was originally added to the formula to support viability of B. lactis. Specific probiotic effects of S. thermophilus have not been further documented. Additional results from this study are presented on the next slide. *P=0.035 Saavedra JM., et al. Lancet 1994;344: 61

Probiotics for Risk Reduction of Acute Diarrhea
September 19, 2018 Probiotics for Risk Reduction of Acute Diarrhea * Notes: The same first study showing a greater than 50% reduction in the incidence of acute diarrhea with B. lactis supplementation also showed a significant reduction in the incidence of shedding rotavirus. The excretion or shedding of rotavirus is a potential marker of the amount of viral replication (severity or duration of infection). The reduction in rotavirus shedding (percent of infants throughout the study that were regularly examined using ELYSSA) was noted independent of the occurrence of clinically evident acute diarrhea by the definition of the study. The reduction of a viral enteritis was a first clinical suggestion that the mechanism of action of probiotics is immune mediated rather than simply an increase in the number of potentially beneficial bifidobacteria versus other potential pathogenic bacteria in the intestinal lumen. Subsequent clinical and animal studies have suggested the improvement related to rotavirus enteritis with probiotics is associated with an increase in anti rotavirus secretory IgA. *P=0.025 Saavedra JM., et al. Lancet 1994;344: 62

Risk Reduction of Atopic Dermatitis with Probiotics
September 19, 2018 Risk Reduction of Atopic Dermatitis with Probiotics L. reuteri compared to placebo by mothers in last month of pregnancy and birth through 12 mo by infants Notes: This was a double-blind, randomized, placebo-controlled trial, which comprised 232 families with allergic disease, of whom 188 completed the study. The mothers received L reuteri (10(8) colony forming units) daily from gestational week 36 until delivery. Their babies then continued with the same product from birth until 12 months of age and were followed up for another year. Primary outcome was allergic disease, with or without positive skin prick test or circulating IgE to food allergens. Results: The cumulative incidence of eczema was similar, 36% in the treated versus 34% in the placebo group. The L. reuteri group had less IgE-associated eczema during the second year, 8% versus 20% (P = .02), however. Skin prick test reactivity was also less common in the treated than in the placebo group, significantly so for infants with mothers with allergies, 14% versus 31% (P = .02). Wheeze and other potentially allergic diseases were not affected. Conclusion: “Although a preventive effect of probiotics on infant eczema was not confirmed, the treated infants had less IgE associated eczema at 2 years of age and therefore possibly run a reduced risk to develop later respiratory allergic disease.” * *p=0.02 Abrahamsson TR., et al. J Allergy Clin Immunol 2007; 119: 63 63 63

Effect of L. reuteri in Infants with Colic
# of responders reporting a 50% reduction in crying from baseline OBJECTIVE: To test the efficacy of Lactobacillus reuteri on infantile colic and to evaluate its relationship to the gut microbiota. STUDY DESIGN: Fifty exclusively breastfed colicky infants, diagnosed according to modified Wessel’s criteria, were randomly assigned to receive either L reuteri DSM (108 colony-forming units) or placebo daily for 21 days. Parental questionnaires monitored daily crying time and adverse effects. Stool samples were collected for microbiologic analysis. RESULTS: Forty-six infants (L reuteri group: 25; placebo group: 21) completed the trial. Daily crying times in minutes/day (median [interquartile range]) were 370 (120) vs 300 (150) (P .127) on day 0 and 35.0 (85) vs 90.0 (148) (P.022) on day 21, in the L reuteri and placebo groups, respectively. Responders (50% reduction in crying time from baseline) were significantly higher in the L reuteri group versus placebo group on days 7 (20 vs 8; P.006), 14 (24 vs 13; P.007), and 21 (24 vs 15; P.036). During the study, there was a significant increase in fecal lactobacilli (P.002) and a reduction in fecal Escherichia coli and ammonia in the L reuteri group only (P .001). There were no differences in weight gain, stooling frequency, or incidence of constipation or regurgitation between groups, and no adverse events related to the supplementation were observed. CONCLUSION: L. reuteri DSM at a dose of 108 colony-forming units per day in early breastfed infants improved symptoms of infantile colic and was well tolerated and safe. Gut microbiota changes induced by the probiotic could be involved in the observed clinical improvement. Pediatrics 2010;126:e526–e533 *p=0.006 **p=0.007 ***p=0.036 Savino F, et al. Pediatrics 2010; 126(3):e526-33

Effect of B. lactis in Preterm Infants: Fecal Bacterial Groups
September 19, 2018 Effect of B. lactis in Preterm Infants: Fecal Bacterial Groups * * * * *p<0.05 Mohan R., et. al., J Clin Microbio 2006;44:

Effect of B. lactis in Preterm Infants:
September 19, 2018 Effect of B. lactis in Preterm Infants: Improved Immune Secretory Function IgA * * Control = 16 Probiotic = 19 *p<0.05 Mohan R. et., al. Pediatr Res. 2008;64:

Effect of B. lactis in Preterm Infants:
September 19, 2018 Effect of B. lactis in Preterm Infants: Improved Weight Gain Weight Gain * Singificantly increased weight gain with B. lactis compared to placebo; the effect is dependent on abx. Control = 32 Probiotic = 37 *p<0.001 Mohan R. et., al. Pediatr Res. 2008;64:

Atopy: “Prebiotics may prove to be beneficial in reducing common infections and atopy in otherwise healthy children. However, confirmatory studies, especially in children fed formula that is not partially hydrolyzed, are needed before any recommendations can be made.” Thomas D, et al. Pediatrics ;128(6):

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 Episodes of lacto-bacilemia have been reported in compromised patients No episodes of bifido-bacterial infection Nevertheless, probiotics are live organisms, with multiple functions; probiotics can clearly, given the right circumstances, lead to translocation and infection, for example in an immuno-compromised host. Approximately 5 well documented episodes of infection with lactobacilli ingestion (as probiotics) have been reported (Michail 2006). There are no reports of spontaneous bifidobacteremia (from bifidobacteria in the flora or the environment), nor are there any well documented reported cases of bifidobacteremia in humans consuming bifidobacteria (various species in foods) or from bifidobacteria shown to have probiotic effects. Clinical trials in infants with diarrhea, prematures, and infants and children on antibiotics, which could be considered immuno-compromised, mentioned earlier in this presentation have not demonstrated any infectious adverse effects.

Safety of Probiotics Pros:
Current use of lactic acid bacteria in food is in thousands of metric tons annually worldwide 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 The genus Lactobacillus and the genus Bifidobacterium (lactic acid bacteria) have been consumed for hundreds of years in the food supply, in very large amounts. There have been sporadic documented infections with lactobacilli; but, the small number of infections do not correlate to ingested lactobacilli. Infection from probiotic ingestion would require translocation from the gut lumen to circulation. The intestine of humans contains 10(10-12) CFU of bacteria, including lactobacilli and bifidobacteria, which can occasionally translocate. In vulnerable populations, such as newborns, certain species from the microflora (e.g., Klebsiella, E. coli, Enterococcus) frequently cause infections and sepsis. Lactobacilli are present in microflora of humans, but are a very rare cause of infection. Bifidobacteria, despite being present (and in many infants they are the predominant species), have not been reported as a pathogen.

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Gut Microbiota, Immunity, and Probiotics

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