Probiotics.

Disease-a-Month ] (2015) ]]]–]]]

Contents lists available at ScienceDirect

Disease-a-Month journal homepage: www.elsevier.com/locate/disamonth

Probiotics Barry A. Mizock, MD, FACP, FCCM

Definitions Commensal bacteria (indigenous microbiota) These are the microorganisms that are present on body surfaces that are covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). Commensal bacteria are considered as self by the hostʼs immune system. Pathobiont These are the bacteria that are normally symbiotic but have the potential to be pathologic under certain conditions. Intestinal dysbiosis An unnatural shift in the composition of the gut microbiota that may result from diet (e.g., high fat), psychological or physical stress, antibiotics, or radiation that is associated with an imbalance between protective and harmful bacteria. Human microbiota (microflora) Refers to the 10–100 trillion microbial cells harbored by each person, primarily in the gut. Human microbiome The entire collection of genes found in all the microbes associated with a particular host. Human metagenome A metagenome is comprised of all the genetic elements of the host and all those of all the microorganisms (microbiome) that live in or on that host. http://dx.doi.org/10.1016/j.disamonth.2015.03.011 0011-5029/& 2015 Mosby, Inc. All rights reserved.

B.A. Mizock / Disease-a-Month ] (2015) ]]]–]]]

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Metagenomics Metagenomics is the study of the collective genomes of the members of a microbial community. It involves cloning and analyzing the genomes without culturing the organisms in the community, thereby offering the opportunity to describe the hosts diverse microbial inhabitants, many of which cannot yet be cultured. Functional metagenomics A technique directed toward connecting specific microbial phyla to specific functions in the environment. Prebiotic (functional fiber) A selectively fermented ingredient that allows specific changes both in the composition and activity in the gastrointestinal microflora that confers benefits upon host well-being and health. Probiotic Live microorganisms that when administered in adequate amounts confer a health benefit on the host. Synbiotic Nutritional supplements combining probiotics and prebiotics in a form of synergism. Medical food A food administered under the supervision of a physician and intended for the specific dietary management of a disease for which distinctive nutritional requirements are established. Live cultures Microbes associated with foods as food fermentation agents or starter cultures.

History of probiotics Recognition of the relationship between gut health and human disease may be traced back to Hippocrates (460–370 BC) who stated: “All diseases begin in the gut.” The Old Testament provided some of the earliest evidence suggesting that ingested bacteria could have a beneficial effect on health; it was stated that Abraham owed his longevity to the consumption of sour milk. Research in the modern era began with Theodor Escherich, who in 1886 described the relationship of intestinal bacteria to the physiology of digestion in the infant. In 1892, Ludwig Doderlein proposed that microorganisms (lactobacilli) could be used to treat vaginal infections. Eli Metchnikoff is considered the father of the probiotic concept. In his 1907 book—The Prolongation of life—he proposed that colonic bacteria played a role in aging and adverse health in adults (“death begins in the colon”). This theory grew of the observation that Bulgarian peasants who consumed fermented milk foods had remarkable longevity; they had an average life span of 87 years, with four of every 1000 living past 100 years. He postulated that the body was slowly poisoned (“autointoxicated”) by toxins produced by proteolytic microbes in the intestine that were responsible for aging. He further hypothesized that aging could be prevented by modifying the gut flora with “useful microbes” obtained by consumption of sour milk and


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lactic acid-producing bacteria. Metchnikoffʼs experiments led him to believe that this bacterial strain (which he called the “Bulgarian bacillus”) could successfully establish itself in the gut and decrease the number of “putrefactive” bacteria, thereby delaying the aging process. To test this hypothesis, he drank sour milk every day until his death at the age of 71 years. The organism investigated by Metchnikoff was previously isolated by Stamen Grigoroff who demonstrated how healthy bacteria in yogurt helped digestion and improved the immune system. This organism was subsequently renamed Lactobacillus delbrueckii, subspecies bulgaricus. Unfortunately, there is little evidence that the probiotic concept was received with any enthusiasm in the Western world. In 1908, Henry Tissier, a pediatrician working at the Pasteur Institute in Paris, first reported the isolation of Y-shaped bacteria (which he named Bifidus) from the stool of a breast-fed infant. He observed that Bifidus was found in significant numbers in the stool of healthy infants, whereas children with diarrhea had low concentrations of this organism. Tissier proposed that this organism could be used to treat infant diarrhea by displacing proteolytic bacteria from the gut. Bifidus was subsequently renamed Bacillus acidophilus because of its acid tolerance. In 1917, during World War I, Alfred Nissle isolated a nonpathogenic strain of Escherichia coli from the stool of a soldier, who was one of a few who did not develop enterocolitis during a severe outbreak of Shigellosis. This strain was named E. coli Nissle 1917 and was subsequently used to treat gastrointestinal salmonellosis and shigellosis. Minoru Shirota recognized the therapeutic potential of using bacteria to modulate gastrointestinal microflora. In 1930, he succeeded in isolating and culturing a Lactobacillus strain capable of surviving the passage through the gastrointestinal tract. This bacterium was named Lactobacillus casei strain shirota. Several individuals have been credited with proposing the term probiotic. Werner Kollath in 1953 used the term Probiotika to describe active substances that are essential for a healthy development of life. In 1964, Lilly and Stillwell used the term probiotic (“for life”) to describe “substances secreted by one microorganism that stimulate the growth of another.” In 1989, Roy Fuller proposed a definition that removed the term “substances,” which could have included antibiotics. His definition of probiotic: “a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance,” emphasized that probiotics must be viable organisms. The most current definition of probiotic was proposed by the joint Food and Agriculture Organization/World Health Organization Working Group in 2002. Their definition of probiotic: “live microorganisms that when administered in adequate amounts confer a health benefit on the host” is currently favored. The term “functional food” was first used in Japan in the 1980s, where there is a government approval process called Foods for Specified Health Use. Functional foods can be considered as those, which are given a function beyond basic nutrition, often related to health promotion or disease prevention, by adding new ingredients or more of existing ingredients. Yogurt and other fermented milks containing probiotics (e.g., kefir) may be considered among the first functional foods. The term prebiotic is credited to Gibson and Roberfroid who defined a prebiotic as “a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one of a limited number of bacteria in the colon.” This definition shares characteristics with that of dietary fiber. The term synbiotic is used when the functional food contains both probiotics and prebiotics.

Ecology of gut microbiota The healthy human intestinal microbiota is estimated at 1014 organisms, which collectively encode 3–4 million genes, or approximately 150 times more than the human genome. This microbial genome enables the microbiota to perform diverse metabolic activities that are not encoded in the human genome and that are of benefit to the host. These include extracting energy and nutrients from food, vitamin biosynthesis, bile salt transformation, developing innate and adaptive immunity, maintaining gut epithelial integrity, functioning as a barrier to

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colonization by microbial pathogens, and metabolism of drugs. Food degradation products that humans cannot digest (e.g., cellulose or oligosaccharides) can be fermented into short-chain fatty acids by enteric organisms, where they may be used as an energy source or have other beneficial effects. Fermentation is therefore a process that offers symbiotic benefit to the host by enabling utilization of a wider variety of foodstuffs. Alterations in composition of the gut microbiota that are unfavorable to the host (dysbiosis) have been linked to a number of diseases including inflammatory bowel disease, Clostridium difficile infections, diabetes and obesity. These will be discussed in further detail in a subsequent section. The ecology of the gut microbiota changes with age. At birth in vaginally delivered infants, the gut becomes colonized with organisms similar to its motherʼs vaginal flora. In contrast, infants delivered by Cesarean section harbor bacterial communities that resemble those of the skin such as Staphylococcus, Corynebacterium, and Propionibacterium species. The bacterial inoculum provided by passage through the motherʼs vagina during delivery helps to colonize the infantʼs gut and establishes a microbiome that promotes the development of innate and adaptive immunity. The lack of initial acquisition of this natural inoculum in babies delivered by Cesarean section might account for observations of subsequent alterations in immunity in this population that places them at higher risk for infection or atopic diseases. Beneficial bacteria such as Bifidobacterium are also transferred to the infant from the mother during breast-feeding and serve to colonize the infant gut. During the first few years of life, diversity of the gut microbiome increases rapidly in response to diet and illness. Administration of antibiotics in infants appears to diminish the diversity of gut flora that in turn could have negative effects on long-term health such as increasing the risk of developing asthma, allergy, and obesity.2 In adulthood, the microbiome may continue to change, albeit at a slower rate than in childhood. The microbiome of healthy elderly individuals is similar to younger persons; however, that of frail elderly may be substantially different.1,3 The distribution of bacteria in the gut varies based on location. Bacterial density is low in the stomach and duodenum, due to the presence of gastric acid and pancreatic enzymes. The density increases in the distal small intestine (a mix of aerobes and anaerobes) and is greatest in the large intestine where bacterial concentration rises to estimated 1011–1013 bacteria per gram of colonic content, of which 99.99% are anaerobes. It had been previously thought that the human intestinal microbiome contained approximately 5000 species based on stool culture results. However, determining this population using culture has significant limitations. Development of molecular profiling techniques such as 16S rRNA gene sequence analysis indicated that approximately 60–80% of sequences did not conform to known cultured species of bacteria.4 Thus, a significant proportion of the numerically most abundant bacteria within the gut remain unstudied or unnamed. In addition, stool analysis does not provide information about localized changes in the small bowel and colon and is also not representative of organisms in the mucosal layer adjacent to the intestinal epithelium. Although 16S rRNA gene sequence analysis is useful in identifying bacterial species, it provides no information about bacterial physiology or metabolism. Newer techniques involving genome sequencing of community DNA may be useful in this regard, because they enable identification of certain bacterial genes that code for metabolic function. This has given rise to the analytic technique called functional metagenomics.5 The presence of a “core microbiota” has been proposed by Arumagam et al.6 in which the human gut microbiome falls into three distinct types or “enterotypes.” Each of the three enterotypes is identifiable by variation in the levels of one of the three main genera: Bacteroides, (enterotype 1), Prevotella (enterotype 2), and Ruminococcus (enterotype 3). It was subsequently proposed that “fecotypes” might be a more accurate descriptor than enterotypes due to variation in the population and abundance throughout the gastrointestinal tract. In 2011, Wu et al.7 concluded that only enterotypes 1 and 2 were strongly supported by the data and that the evidence for the Ruminococcus enterotype was lacking. They also observed that these two enterotypes were associated with long-term diets; enterotype 1 (Bacteroides) with diets rich in protein and animal fat, and Prevotella with simple carbohydrates. However, the existence and biological significance of distinct enterotypes has been the subject of much debate with some


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investigators, favoring the concept of a continuum of species functionality rather than discrete types.8 Studies in humans have demonstrated that gut microbial communities are highly variable over time. A longitudinal study of four body sites in two individuals indicated marked variability across months, weeks, and even days.9 This suggested that no core temporal microbiome existed at high abundance. Rather than categorizing the intestinal microbiome based on morphologic or culture criteria, it may be more informative to link microbial composition to function. Therefore, although the population of individual bacterial species may exhibit temporal variation, it is possible that groups of bacteria sharing a common metabolic function (“metabolome”) are more consistently maintained.10 Thus, the functional attributes of a bacterial community could be preserved despite changes in constituent species. A healthy diet encourages the growth of microbes that are beneficial to the host. One of the essential ingredients of a “gut healthy” diet is that it is rich in certain substances that “feed” desirable microbes in the colon. Important members of this food class include those containing fiber and resistant starch such as found in fruits, vegetables, legumes, or garlic. These foods resist multiple digestive enzymes that are produced in salivary, gastric, and pancreatic secretions. Consequently, they are not digested in the small bowel but undergo fermentation and further metabolism in the cecum and proximal colon. Fermentable substances such as fructooligosaccharides or inulin are also available as dietary supplements or added to enteral feeding formulas. Metabolic cross-feeding is a phenomenon in which one species lives off the products of another species in a synbiotic relationship.11 Soluble (e.g., oligosaccharides) and non-soluble fiber (e.g., cellulose) and resistant starch escape digestion in the small intestinal but are fermented by saccharolytic bacteria such as Lactobacillus or Bifidobacterium in the colon to form pyruvate and lactate (Fig. 1). Butyrate and other short-chain fatty acids (SCFA) are synthesized from pyruvate by anaerobic bacteria, with the major butyrate producers being Clostridium clusters XIVa and IV (e.g., Roseburia and Faecalibacterium prausnitzii).12 Thus, direct stimulation of fermentation by certain foods and prebiotic supplements may lead indirectly to increased butyrate formation. In humans, butyrate is the preferred energy source for colonic epithelial cells. Butyrate also influences gene expression and inhibits the activation of NFkB, leading to decreased expression of proinflammatory cytokines and a consequent anti-inflammatory effect.13 Butyrate enemas have been used therapeutically in patients with predominately distal ulcerative colitis.14

Intestinal dysbiosis, obesity, and diabetes Intestinal dysbiosis can be defined as an unnatural shift in the composition of the gut microbiota that is associated with an imbalance between protective and harmful bacteria. A number of factors can promote intestinal dysbiosis including diet, underlying illness, antibiotics, or radiation. Recent evidence suggests that alterations in the intestinal microbiota could play an important role in the pathogenesis of obesity and its related diseases such as the metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease. Analysis of the gut microbiota in obese mice has revealed differences at the phylum level when compared to lean animals, with greater numbers of Firmicutes than Bacteroidetes and less diversity overall.15 Similar observations have been noted in obese humans.16 However, these associations have not been found consistently, resulting in some uncertainty as to their significance. An important question is whether the microbiota common to the obese phenotype actually contributes to the development or maintenance of obesity. Turnbaugh et al.17 investigated the effect of diet on intestinal microbiome of “humanized” mice in which fecal microbiota from healthy adult humans was transplanted into germ-free animals. The mice were initially fed a low-fat plant polysaccharide-rich diet and then switched to a Western diet high in sugar and fat. The Western diet induced changes in the gut microbiome within a single day that was characterized by an overgrowth of bacteria within the Firmicutes phylum as well as a significant reduction in several Bacterioidetes species. In addition, mice that were fed by the

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Fig. 1. Intestinal bacterial fermentation and butyrate production.

Western diet had a significant increase in adiposity within 2 weeks of feeding, compared to ageand gender-matched mice, consuming the low-fat polysaccharide-rich diet. Although alterations in the gut microbiota have been implicated as a cause of obesity and the metabolic syndrome, a high-fat diet may alter the intestinal microbiota independent of obesity.18 In addition, the specific type of dietary fatty acid may be more important than total calories from fat. Studies in mice have indicated that diets rich in omega-6 polyunsaturated fatty acids (PUFAs) promote growth of pathobionts (resident microbes with pathogenic potential), whereas diets supplemented with omega-3 PUFA can reverse such alterations.19 Various mechanisms have been proposed to explain the ability of the gut microbiome to influence obesity. They include (1) alterations of intestinal permeability with increased translocation of bacterial products such as lipopolysaccharide (LPS), resulting in a low level of chronic systemic inflammation; (2) the ability of SCFA such as acetate and propionate to signal via intestinal epithelial receptors; and (3) caloric salvage by some microbiota being more efficient at making calories available from food. In 2007, Cani et al.20 proposed that the gut microbiota plays a key role in the onset of lowgrade inflammation associated with obesity and insulin resistance. Consumption of a high-fat diet promotes translocation of lipopolysaccharide (via chylomicrons), originating from enteric


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gram-negative bacteria to target tissues. This in turn triggers the secretion of proinflammatory cytokines (e.g., tumor necrosis factor, interleukin 1, and interleukin 6) from immune cells that cause insulin resistance, hyperinsulinemia, and excessive hepatic and adipose tissue lipid storage. This process was termed “metabolic endotoxemia.” With overfeeding or obese conditions, a constant stimulus from nutrient intake results in a more consistently active inflammatory response. As the consequence of progressive activation of the systemic immune response, insulin resistance becomes more prominent, and frank diabetes may result. In contrast to the classical systemic inflammatory response that is triggered by infection, this low-grade chronic inflammation occurs in the absence of infection. The term metainflammation (i.e., metabolically triggered infection) has been proposed to describe this process.21 There are relatively few studies that couple changes in microbiota composition upon probiotic supplementation with anti-obesity functions. In healthy overweight subjects, administration of Lactobacillus gasseri SBT2055 resulted in reduction of abdominal visceral and subcutatneous fat.22 The concept of using probiotics to treat obesity is intriguing, but requires further confirmation.

Types of probiotics Probiotics must be identified by their genus, species, and strain level. The majority of probiotics in clinical use are species from three genera: Lactobacillus, Bifidobacterium, and Saccharomyces. Both Lactobacilli and Bifidobacteria are saccharolytic bacteria that can ferment carbohydrates to lactic acid that inhibits growth of pathogenic bacteria. In addition, pyruvate produced from fermentation can be utilized by certain colonic anaerobes to produce beneficial SCFA. Lactobacilli are normally found in healthy gut but are present in relatively low numbers even in individuals consuming probiotics. Lactobacilli are also found in the vaginal secretions of healthy women. Some of the Lactobacilli commonly found in yogurt and probiotic supplements include L. acidophilus, L. acidophilus DDS-1, L. bulgaricus, L. rhamnosus GG, L. plantarum, L. reuteri, L. salivarius, L. casei, L. johnsonii, and L. gasseri. Bifidobacteria are the constituents of normal gut flora and can also be found in the vagina and oral cavity. Bifidobacteria that are used as probiotics include B. bifidum, B. lactis, B. longum, B. breve, B. infantis, B. thermophilum, and B. pseudolongum. Saccharomyces boulardii is the only yeast probiotic. It was previously identified as a unique species of yeast but is now believed to be a strain of Saccharomyces cerevisiae (bakerʼs yeast). Commercially marketed probiotics are available either as single species or combination of multiple species. Some of the more commonly used probiotics in the US are listed in the Table.

Probiotic mechanisms of action There are a number of mechanisms of action for probiotics that confer potential beneficial effects on the gut23 (Fig. 2). By transiently colonizing the gastrointestinal tract, probiotics serve to correct dysbiosis that contributes to underlying disease. This may occur as the consequence of colonization resistance, a term that refers to the ability of certain bacteria to interact with gut epithelial cells to prevent adherence of enteric pathogens to binding sites on the epithelial surface. Colonizing probiotics may also exert a direct antimicrobial effect by secreting products such as bacteriocins that inhibit the growth and virulence of enteric pathogens. Certain probiotics have been demonstrated to increase the release of antibacterial peptides called defensins from Paneth cells (a type of epithelial cell present in the crypts of the small intestine). Lactic acid-producing probiotics (e.g., Lactobacillus spp. and Bifidobacterium spp.) may exert an antimicrobial effect on pathogens by reducing the local pH of the gut lumen. Some strains of probiotics have been discovered to interfere with quorum sensing, a mechanism by which bacteria enhance their virulence.24 Probiotics have also shown the ability to enhance the production of mucins from gut epithelial cells. Mucins serve as an antibacterial barrier that prevent binding of pathogens. Probiotics promote the production of secretory IgA in gut that binds to pathogens; they also exert an anti-inflammatory effect in the gut


8 Table Common probiotic products. Product

Contents

Activia yogurt (Dannon, Canada)

B. lactis DN-173 010 Z 100 Million live bacteria/gram

Align (Procter & Gamble)

Bi. infantis 35624 1 Billion CFU/capsule

Bacid (Erfa, Canada Inc.)

L. rhamnosus 1 Billion bacteria per capsule

Bio-K þ (Bio-K international)

L. acidophilus CL 1285, L. casei LBC80R 12.5 Billion/capsule (regular strength) 25 Billion/capsule (extra strength) 50 Billion/98 g bottle beverage

Culturelle (ConAgra Foods)

LGG 10 Billion bacteria þ 200 mg insulin/capsule

DanActive (Dannon, Canada)

L. bulgaricus, S. thermophiles, L casei 4 10 Billion L. casei/bottle

Florajen (American Lifetime, Inc., US)

L. acidophilus 20 Billion bacteria/capsule

Florastor (Biocodex)

S. boulardii 250 mg/capsule or packet

Howaru, Howaru Protect (Danisco)

L. acidophilus NCFM, B. lactis Bi-07 10 Billion bacteria/capsule or stick 5 Billion bacteria/tablet

Jamieson Probiotic Sticks (Jamieson Natural Sources, Canada)

L. helveticus and B. longum 1 Billion CFU/powder stick

Kefir (Lifeway)

L. rhamnosis, L. plantarum, L. casei, L. acidophilus, L. reuteri, Leuconostoc cremoris, Strep diacetylactis, S. florentinus, B. longum, B. brevis, and B. lactis 7–10 Billion CFU/cup

Lactinex (Benton, Dickinson & Co.)

L. acidophilus and L. bulgaricus 1 Million CFU/tablet, 100 million CFU/packet

VSL#3 (Sigma-Tau Pharmaceuticals)

L. acidophilus, L. plantarum, L. paracasei, L. bulgaricus, B. breve, B. infantis, B. longum, and Strep thermophilus 450 Billion bacteria/packet 225 Billion per two capsules

Yakult (Yakult USA, Inc.)

L. casei Shirota 8 Billion active bacteria per 80 ml bottle

Adapted with permission from Pharmacistʼs Letter/Prescriberʼs Letter July 2012.

by preventing the activation of NFκB (a proinflammatory transcription factor) and IL-8 (a neutrophil chemoattractant). Finally, it has been noted that some probiotics may activate opioid and cannabinoid receptors in gut that could prove useful in ameliorating visceral pain, which is a prominent feature in some patients with irritable bowel syndrome.

Therapeutic use of probiotics in specific diseases A large number of studies have assessed the utility of probiotics in the prevention or treatment of certain clinical conditions. However, at this point in time the results have been


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Fig. 2. Probiotic mechanisms of action.

mixed. The diseases that currently have the strongest positive evidence are: antibioticassociated diarrhea, infectious childhood diarrhea, inflammatory bowel disease (especially for maintenance of remission in ulcerative colitis), and pouchitis. These conditions will be discussed below; entities in which probiotic therapy is of potential value will also be reviewed. Current consensus and societal practice guidelines for probiotic therapy will be summarized. Diarrheal illnesses Diarrheal illnesses are perhaps the best documented indication for probiotic therapy, particularly in the pediatric population. The following discussion will focus on clinical trials and meta-analyses that support the utility of probiotics in the prevention and/or treatment of diarrhea. These clinical settings include Clostridium- and non-Clostridium-associated antibiotic diarrhea, acute infectious diarrhea in children, and travelerʼs diarrhea. Antibiotic-associated diarrhea Diarrhea is a common side effect of antibiotic use, ranging from 10% to 30% in the outpatient setting to as high as 39% in hospitalized patients. Some authorities lump Clostridium-associated antibiotic diarrhea (CDAD) with non-Clostridium difficile-associated antibiotic diarrhea (NCDAD), while others classify these as separate entities. NCDAD has been defined as a benign, self-limited diarrhea typically following the use of oral antimicrobials (especially amoxicillin/clavulanate). Symptoms may begin as early as 24 h after the first dose. Typically, no pathogens are identified and the diarrhea is caused by changes in the composition and the function of the intestinal flora. Most patients respond to supportive measures and discontinuation of antibiotics. In contrast, CDAD diarrhea refers to a wide spectrum of diarrheal illnesses caused by the toxins produced by

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this organism, including cases of severe colitis with or without the presence of pseudomembranes. CDAD accounts for 10–25% of AAD, and the presence of a hypervirulent strain (NAP1/ 027/B1) additionally increases the severity of the disease. The pathophysiology of CDAD and colitis results from a disruption of the normal bacterial flora of the colon, colonization with C. difficile, and the release of toxins that lead to mucosal damage and inflammation. CDAD may begin during antibiotic therapy or 5–10 days after the antibiotic is stopped. The antibiotics most commonly associated with CDAD are the fluoroquinolones, cephalosporins, carbapenems, or clindamycin. The most significant studies of probiotics in prophylaxis of AAD have been in children, with L. rhamnosus GG (LGG) and S. boulardii being the most effective agents. Vanderhoof et al.25 found that administration of LGG concurrent with an oral antibiotic in children significantly reduced stool frequency and increased stool consistency during antibiotic therapy by the 10th day compared with placebo. Kotowska et al.26 observed that when S. boulardii was administered in conjunction with antibiotics for upper respiratory infection in children (aged 6 months to 14 years), 8% in the treatment group developed diarrhea versus 23% in the control group. A Cochrane Database System Review in 2011 assessed the efficacy and safety of probiotics (any strain or dose) used for the prevention of NCAD in children.27 Overall, 16 trials were analyzed that included treatment with Bacillus spp., Bifidobacterium, Lactobacilli, Saccharomyces, or Streptococcus, either alone or in combination. Results from 15/16 trials showed a large benefit from probiotics compared to placebo or no treatment control. The incidence of diarrhea in the probiotic group was 9% compared with 18% in the control group (RR ¼ 0.81). Subgroup analysis showed high dose ( Z5 billion CFUs/day) is more effective than low dose. The results of probiotic prophylaxis of AAD in adults have been somewhat mixed. Positive results were found by Gao et al.28 who performed a study in adults assessing the dose-response efficacy of L. acidophilus and L. casei for prophylaxis of NCDAD and CDAD. A total of 225 patients were randomized to one of the three groups: two probiotic capsules/day, one probiotic and one placebo/day, or two placebo capsules/day. Prophylaxis began within 36 h of initial antibiotic administration and continued for 5 days after the last antibiotic dose. Patients were followed up for an additional 21 days. Patients getting two probiotic capsules/day had the lowest incidence of diarrhea. In addition, each probiotic group had a lower diarrhea incidence versus placebo. Ojetti et al.29 studied the impact of L. reuteri supplementation in patients undergoing anti-Helicobacter pylori (HP) therapy. The incidence of diarrhea was reduced in the probiotic-supplemented group relative to those who did not receive the probiotic. Armuzzi et al.30 also found the benefit of oral administration of the probiotic LGG on antibiotic-associated gastrointestinal side effects during HP eradication therapy. A Cochrane review published in 2013 studied probiotics for the prevention of CDAD in adults and children.31 A total of 23 trials were entered. The results demonstrated that probiotics significantly reduced the risk of diarrhea by 64% (2% incidence in the probiotic group compared to 5.5% in the placebo or no treatment control group). It was concluded that moderate quality evidence suggests that probiotics are both safe and effective for preventing CDAD. Regarding treatment of CDAD, there is no good evidence that probiotics are efficacious either as a primary therapy for CDAD or as an adjunct to standard antibiotic therapy for initial therapy. A Cochrane Database System review in 2008 examined four studies that met inclusion criteria.32 Probiotics were administered in conjunction with conventional antibiotics for the treatment of CD colitis in adults. Four studies were entered but were small and had methodological problems. It was concluded that insufficient evidence existed to recommend probiotic therapy either as an adjunct to antibiotic therapy or used alone for the treatment of C. difficile colitis. Recurrent CDAD is defined as a return of signs and symptoms of CDAD along with a positive stool test within a month of a successful treatment of an initial episode. Recurrent CDAD is extremely difficult to treat and one recurrence increases the likelihood of further recurrences. Treatment with oral metronidazole or vancomycin is increasingly associated with treatment failures, and recurrence has been observed in up to 30% of patients after their first episode and in up to 60% after two or more recurrences. The emergence of a virulent strain of the organism (NAP1/027/B1) has been associated with even higher rates of treatment failure. Probiotics have


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shown promise as adjunctive therapy for preventing recurrent CDAD; clinical trials using S. boulardii have provided the best evidence for efficacy. One of the largest trials was done by McFarland et al.33 who studied 124 patients with active CDAD (64 patients had an initial episode of CDAD and 60 had a history of at least one prior CDAD episode). Patients received standard antibiotics and either S. boulardii (1 g/day) or placebo for 4 weeks and were followed up for an additional 4 weeks after therapy. Effectiveness was based on the recurrence rate of CDAD. The patients with CDAD who received standard antibiotics plus S. boulardii had a significantly lowerrisk ratio of recurrence (RR ¼ 0.43) compared to placebo. The efficacy of probiotic therapy was significant in those with recurrent CDAD (19% versus 24%), but not in patients with the initial episode of CDAD. Practice guidelines for the prevention and treatment of AAD were provided by the Yale/ Harvard workshop in 2014.34 Probiotics were given a level A recommendation for effectiveness in prevention of AAD. Specific strains of organisms most effective for this condition included S. boulardii, LGG and a combination of L. casei DN114 G01, L. bulgaricus, and S. thermophilus. For prevention of recurrent CDAD, S. boulardii, LGG and fecal bacteriotherapy (stool transplantation) were given a level B/C rating. For prevention of CDAD, probiotics were given a level B/C rating; LGG and S. boulardii are the most effective organisms. The 2010 recommendations on probiotic therapy from the American Academy of Pediatrics state: “there is some evidence to support the use of probiotics to prevent AAD but no evidence that it is beneficial for treatment.”35 A guideline from the AAP in 2013 stated: “Because there is a lack of controlled studies in children, probiotics are not recommended for either the prevention or the treatment of C. difficile infection.” Prevention of travelerʼs diarrhea Up to 35% of the individuals who travel to developing countries may experience bouts of diarrhea. Most cases occur within the first 2 weeks of travel and last about 4 days. Regions with the highest risk are Africa, South Asia, Latin America, and the Middle East. Bacteria are the most frequent cause of travelerʼs diarrhea (TD), and enterotoxigenic E. coli is the most common pathogen. Up to 10% of the travelers who suffer from TD go on to develop persistent diarrhea and about 10% eventually evolve to suffer from post-infectious irritable bowel syndrome. Clinical data confirming the effectiveness of probiotics in preventing TD is relatively limited. Nevertheless, prophylaxis of TD constitutes one of the largest markets for probiotics in Europe. McFarland et al.36 performed a meta-analysis of 12 studies and found that probiotics significantly prevented TD (RR ¼ 0.85, p o 0.001). The yeast S. boulardii and a mixture of Lactobacillus acidophilus and Bifidobacterium bifidum appeared to have the greatest efficacy. In contrast, a meta-analysis by Takahashi et al.37 did not show that probiotics were effective in preventing TD. No practice guidelines for the use of probiotics in prophylaxis of TD are currently available. Treatment of acute infectious diarrhea in children A few probiotic strains have shown good evidence of efficacy in the treatment of acute-onset, infectious diarrhea in children. This appeared to be particularly true for the treatment of rotavirus-induced diarrhea, where early administration of probiotics showed efficacy. In contrast, data demonstrating efficacy of probiotics in adults are sparse. A 2010 Cochrane review examined studies that compared a specified probiotic agent with a placebo or no probiotic in individuals with documented or suspected acute infectious diarrhea.38 A total of 63 randomized clinical trials met inclusion criteria; of these 56 recruited infants and young children. Probiotics reduced the duration of diarrhea, although the size of the effect varied considerably between studies. The significant beneficial effects related to the mean duration of diarrhea (mean difference ¼ 24.8 h), reduction of diarrhea lasting Z 4 days, and reduced stool frequency on day 2. It was noted that the difference in effect size between studies was not explained by study quality, probiotic strain, the number of different strains, the viability of the organism, dosage of organism, the causes of diarrhea, severity of diarrhea, or country where the

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study was performed. No adverse events were attributed to the probiotic intervention. The review concluded “When used in conjunction with rehydration therapy, probiotics appeared to be safe and have clear beneficial effects in shortening the duration and reducing stool frequency in acute infectious diarrhea.” Szajewska et al.39 performed a meta-analysis of LGG for treating acute gastroenteritis in children (an update of their 2007 meta-analysis). A total of 11 randomized clinical trials (RCT) were entered. LGG significantly reduced the duration of diarrhea compared with placebo or no treatment (mean difference ¼ 1.05 days). Notably, LGG was more effective when used at a daily dose Z 1010 CFU/day. Despite the fact that probiotics appear to be beneficial in acute infectious diarrhea, several caveats should be mentioned: (1) the clinical relevance of such effect is moderate, because no more than one day of reduction of diarrhea is to be expected, (2) only a few specific strains have evidence-based proof of efficacy (e.g., LGG and S. boulardii), and (3) dosage is crucial; only doses for LGG and S. boulardii mentioned above seem to be effective. The 2014 Yale/Harvard workshop gave probiotics a level A rating for the treatment of infectious childhood diarrhea in children (S. boulardii, LGG, and L. reuteri SD2112) and a level B rating for prevention of infection (S. boulardii, LGG, and L. reuteri SD2112).34 The 2010 recommendations on probiotics and prebiotics from the American Academy of Pediatrics state: “there is some evidence in otherwise healthy infants and young children to support the use of probiotics early in the course of diarrhea from acute viral gastroenteritis and that use of probiotics reduces its duration by one day. However, the available evidence does not support the routine use of probiotics to prevent infectious diarrhea unless there are special circumstances.”35

Inflammatory bowel disease Inflammatory bowel disease (IBD) consists of two disorders: ulcerative colitis (UC) and Crohnʼs disease (CD). The key feature of IBD is chronic, uncontrolled inflammation of the gut mucosa. The central characteristic that distinguishes IBD from inflammatory responses seen in the normal gut is an inability to downregulate those responses, resulting in a chronically inflamed gut. Pathologically, CD is characterized by focal transmural inflammatory lesions and ulcerations that can be present anywhere in the gastrointestinal tract, whereas UC is more superficial and limited to the colon, beginning in the rectum with proximal continuous extension. Genetics, dietary factors, and alterations in the gut microbiome play a pathogenetic role in IBD. Evidence supporting a genetic basis for IBD includes familial clustering and racial and ethnic differences in risk. A family history is present in 10–20% of those afflicted with IBD. Certain genetic variants correlate with an increased risk of disease. Some of these variants (e.g., NOD2) are involved in bacterial recognition as well as in innate and adaptive immunity. Variants associated with IBD lead to disordered immune responses that in turn contribute to loss of intestinal homeostasis. A dramatic rise in the incidence and prevalence of IBD has been noted over the past 2 decades in Western Europe and North America. Epidemiologic studies have noted a predominance of CD over UC in developed nations, notably in children. The typical Western diet that is high in fat and simple sugars has been associated with IBD, due in part to effects on the intestinal microbiome. Increased consumption of omega-6 fatty acids (e.g., corn oil) and decreased intake of omega-3 fatty acids may also play a role. Breast-feeding may have a protective effect on the development of IBD in childhood due in part on its beneficial effects on the developing microbiome.40 Although intestinal dysbiosis is generally recognized as playing a central role in the pathogenesis of IBD, it remains to be determined whether it is the cause or consequence of IBD. It is thought that the chronic inflammatory state associated with IBD selects against beneficial commensal microbes in favor of those that are able to flourish in this environment. Many of these opportunistic bacteria have properties that are highly proinflammatory. The term


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pathobiont was introduced by Chow and Mazmanian41 to define bacteria that are normally symbiotic but have the potential to be pathologic under certain conditions, including IBD. Human studies have indicated that patients with IBD have reduced diversity of the phylum Firmicutes with a notable reduction in organisms that synthesize the anti-inflammatory SCFA butyrate (e.g., Clostridial groups IV and XIVa).42 An abundance of sulfite-reducing bacteria have also been observed in humans with IBD, and their metabolic product hydrogen sulfide is toxic to the colonic epithelium. Metagenomics studies have demonstrated functional alterations in IBD that could also play a pathogenic role.43 Based on some of the alterations described above, an ideal probiotic would need to be a good intestinal colonizer that produces high levels of butyrate levels at the site of action. Additionally, utilizing a combination of a probiotic along with a prebiotic that stimulates butyrate production might be more beneficial than either agent alone.

Probiotics for induction and maintenance of remission in UC Both small anecdotal trials and larger randomized controlled trials have shown efficacy with two specific probiotics: VSL#3 (a combination probiotic containing B. breve, B. longum, B. infantis, L. acidophilus, L. planetarium, L. paracasei, L. bulgaricus, and S. thermophilus) and E. coli Nissle 1917. A trial conducted by Sood et al.44 was one of the first to show benefit of a probiotic in inducing remission in UC. VSL#3 or placebo was administered to patients with mild to moderate active UC. At week 6, more subjects randomized to VSL#3 had at least a 50% decrease in the UC Disease Activity Index (UCDAI) compared to placebo. By week 12, more subjects randomized to VSL#3 achieved remission (43%) compared to placebo (16%). Furthermore significantly more patients given VSL#3 responded with a decrease in their UCDAI by at least three points compared to placebo, and a greater proportion of subjects in the VSL#3 group achieved mucosal healing (32%) compared to the placebo group (15%) by week 12. The authors speculated that since the vast majority of VSL#3 users were concurrently on mesalamine, it is possible that VSL#3 effectiveness is maximized when these agents are given together. Tursi et al.45 performed a study of similar design but only 8 weeks duration. Subjects on concomitant therapy (usually with mesalamine) with mildly to moderately active UC were randomized to VSL#3 or placebo. The number of patients with a decrease in UCDAI scores of 50% or more was greater in the VSL#3 group (63%) than in the placebo group (41%). However, outcomes on endoscopic scores were not different between the groups. A trend toward greater remission at 8 weeks was noted relative to placebo but did not reach statistical significance. Bibiloni et al.46 in an open-label study found that VSL#3 induces remission in patients with active UC. A total of 34 patients were entered who received VSL#3 twice daily for 6 weeks. Intent to treat analysis demonstrated remission in 53%, response in 24%, no response in 9%, worsening in 9%, and failure to complete in 5%. No biochemical or adverse clinical events were noted. A combined induction of remission/response rate of 77% was found and no adverse events were reported. Miele et al.47 investigated the effect of VSL#3 on induction and maintenance of remission in children with UC. Overall, 29 patients, aged 1.7–16 years with newly diagnosed UC were randomized to receive VSL#3 or placebo, in conjunction with steroid induction and mesalamine maintenance treatment. On induction of remission, patients continued to receive concomitant therapy of VSL#3 and mesalamine, or mesalamine and placebo for 1 year or until relapse. All 29 patients responded to induction therapy. Remission was achieved in 13 patients (92.8%) treated with VSL#3 and mesalamine, and in four patients (36.4%) treated with placebo and mesalamine. A total of 21% of patients treated with VLS#3 relapsed within 1 year while 73.3% treated with placebo relapsed. Kruis et al.48 compared E. coli Nissle 1917 with mesalamine regarding the ability to maintain remission in UC. Over the course of 12 weeks, the relapse rate of the two agents was not significantly different (14% versus 16%). The same group subsequently performed a similar but larger study lasting 12 months and again found a comparable clinical relapse rate (36% versus 34%).

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Shen et al.49 performed a meta-analysis of 23 RCT of UC. A significantly higher remission rate in patients with active UC who were treated with probiotics was noted when compared to placebo. Only VSL#3 significantly increased the remission rates compared with controls in patients with active UC. No significant difference in adverse effects were detected between probiotic and control subjects. It was concluded that maintaining remission of UC with probiotics was as effective as with mesalamine and superior to placebo. Fujiya et al.50 performed a meta-analysis that investigated the effects of probiotics on the induction or maintenance of remission in IBD (UC and CD). A total of 20 RCTs were entered. In patients with UC, probiotics decreased the disease activity, increased the remission induction rate, and prevented relapse of disease as effectively as mesalamine. In addition, probiotic therapy decreased the development of pouchitis in post-operative UC patients. Mardini et al.51 performed a meta-analysis of 5 studies of patients with mild-moderately active UC who were treated with VSL#3. A greater than 50% decrease in the UC disease activity index was achieved in 44.6% of the VSL#3-treated patients versus 25.1% of the patients given placebo (OR ¼ 2.8, NNT: 4–5). The response rate was 53.4% in VSL#3-treated patients versus 29.8% in patients given placebo (OR ¼ 2.4, NNT: 4–5). No serious side effects were reported. It was concluded that VSL#3 (at a daily dose of 3.6 1012 CFU/day) added to conventional therapy is safe and more effective than conventional therapy alone in achieving higher response and remission rates in mild to moderately active UC. Despite these promising findings, a Cochrane analysis performed in 2011 concluded that there was insufficient evidence to make conclusions about the efficacy of probiotics for maintenance of remission in UC.52

Pouchitis The surgical treatment of choice for patients with refractory UC is proctocolectomy with ileal pouch-anal anastomosis (IPAA). The most common long-term complication after IPAA for UC is inflammation of the ileal reservoir, called pouchitis. Symptomatic manifestations of pouchitis include increased stool frequency, rectal bleeding, urgency, tenesmus, incontinence, and abdominal pain. The clinical diagnosis of pouchitis is usually confirmed by endoscopy. A pouchitis disease activity index (PDAI) based on clinical symptoms and endoscopic and histologic findings has been developed to grade the severity of pouchitis. About half of the patients who undergo IPAA will have at least one episode of pouchitis. About 60% of patients suffer one or more recurrences, and 5–32% of them develop chronic pouchitis, which in turn may require pouch excision in 10% of cases. Pouchitis typically occurs only after the diverting ileostomy is closed and usually responds to treatment with antibiotics. These observations suggest that alterations in the gut microbiome play a pathogenic role that in turn could be amenable to treatment with probiotics. Dysbiosis in pouchitis is characterized by a decreased ratio of anaerobic to aerobic bacteria and reduced fecal concentrations of lactobacilli and bifidobacteria.53 Several small RCT and a meta-analysis have provided evidence that probiotics are effective for the prevention of pouchitis. Among the various probiotic agents, VSL#3 has been the most extensively studied probiotic for prophylaxis. Gionchetti et al.54 performed a double-blind study of 40 patients to compare the efficacy of VSL#3 with placebo in the maintenance treatment of chronic pouchitis. At 9 months of follow-up, all 20 patients treated with placebo relapsed, while 17 of 20 patients treated with VSL#3 were still in remission. However, all of the 17 patients relapsed within 4 months of suspension of active treatment. The same group performed a double-blind RCT on the effectiveness of VSL#3 (at a daily dose of 1.8 1013 CFU) in the maintenance of antibiotic-induced remission in patients with refractory or recurrent pouchitis and found similar results.55 In a third study, treatment with high-dose VSL#3 (3.6 1012 CFU/ day) was administered for 4 weeks in patients with mild pouchitis (PDAI 7-12).56 At the end of the study period, 69% of patients were in remission after treatment. This suggested that the high doses of VSL#3 are effective in the treatment of mild pouchitis. Nevertheless, antibiotics remain the treatment of choice for acute and chronic pouchitis.


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Mimura et al.57 studied 36 patients with at least two episodes of pouchitis in the previous year who were randomized while in remission to either VSL#3 or placebo. Remission was maintained in 85% of the VSL#3 group and in one patient (6%) in the placebo group. Shen et al.58 studied 31 patients with antibiotic-dependent pouchitis. All patients received 2 weeks of ciprofloxacin followed by VSL#3 3.6 g/day for 8 months. At the 8-month follow-up, six patients were still on VSL#3 therapy and the remaining 25 had discontinued the therapy, due to either recurrence of symptoms while on treatment or development of adverse effects. All the six patients who completed the 8-month course had repeat clinical and endoscopic evaluation; these patients had a mean PDAI and endoscopy scores that were not statistically different from baseline. Thus in this study, only a minority of patients with antibiotic-dependent pouchitis remained on the probiotic therapy and in symptomatic remission after 8 months. Elahi et al.59 performed a meta-analysis investigating the benefit of probiotics in preventing pouchitis in the patients who underwent IPAA; 5 RCT were included. The primary outcome was a PDAI Z 7. The results yielded an OR of 0.04 with a 95% CI of 0.01–0.14 (p o 0.0001) in the treatment group compared to placebo, confirming the benefit of probiotics in management of pouchitis after IPAA. The meta-analysis by Fujiya et al.50 found that probiotic treatment was useful for relieving the disease activity, increasing the remission induction rate and preventing the relapse of disease in UC patients as effectively as mesalamine and could decrease the development of pouchitis in post-operative UC patients. It appears therefore that the major benefit for probiotics in pouchitis is in prevention and maintenance of remission, while evidence for benefit in treating acute pouchitis is less compelling.

Crohnʼs disease In contrast to UC, there is a paucity of studies assessing the utility of probiotics for induction of remission in CD. A 2006 Cochrane review concluded that there was no evidence to suggest that probiotics are beneficial for the maintenance of remission in CD.60 A subsequent Cochrane review in 2008 also concluded that there was no evidence to support the use of probiotics for induction of remission in CD.61 A number of studies have assessed the utility of probiotics in maintaining remission in CD. Prantera et al.62 randomized 37 patients who had undergone ileocecal resection to treatment with LGG or placebo. No difference in endoscopic or clinical recurrence was found. Marteau et al.63 studied 98 patients with CD who had undergone resection within the prior 3 weeks. Patients were randomized to either L. johnsonii or placebo. No significant difference in endoscopic appearance at 6 months was found. In a study by Van Gossum et al.,64 70 patients undergoing ileocecal resection of CD were randomized postoperatively to either L. johnsonii or placebo. At 12 weeks, no difference in endoscopic recurrence rates was found. A meta-analysis by Fujiya et al.50 entered 20 RCTs that investigated the effects of probiotics on the induction or maintenance of remission in IBD. No significant effect of probiotic treatments were shown in either the induction or maintenance of remission in CD. Ghouri et al.65 performed a meta-analysis to assess the therapeutic effect of probiotics, prebiotics, and synbiotics relative to placebo in patients with IBD. No significant difference in clinical outcomes in patients with CD was found. The authors concluded that there is insufficient data to recommend probiotics for use in CD. Updated practice guidelines for the use of probiotic therapy in patients with IBD were provided by the Yale/Harvard Workshop in 2014.34 Probiotics had B-level evidence for inducing remission in UC, and A-level evidence for maintenance of remission in UC. The best-studied organisms were E. coli Nissle 1917 and VSL#3. Probiotic therapy of pouchitis in UC received an A-level rating for preventing and maintaining remission, and a C-level rating for inducing remission (VSL#3 was the most often studied probiotic agent). Since the majority of studies

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failed to show a significant difference for probiotics for maintenance of remission in CD, a C-level rating was given for this indication. The 2011 World Gastroenterology Organization guidelines gave level 1b recommendations for three clinical settings in IBD: (1) for maintenance of remission in UC (E. coli Nissle 1917—dose 5 1010 viable bacteria twice daily), (2) for treatment of mildly active UC or pouchitis (VSL#3— dose 2–9 1011 CFU twice daily), and (3) for prevention and maintenance of remission in pouchitis (VSL#3—dose of 2–4.5 1011 CFU twice daily).66

Irritable bowel syndrome The Irritable bowel syndrome (IBS) is one of the more common disorders encountered by clinicians. Prevalence estimates in North America range from 5% to 15%, with peak prevalence occurring between 20 and 39 years of age. IBS affects 1.5 times more women than that of men and is more common in lower socioeconomic populations. Although IBS does not predispose patients to severe illness, it can profoundly affect the quality of life. IBS is defined as an abdominal discomfort or pain associated with altered bowel habits for at least three days per month in the previous three months, with the absence of organic disease. IBS cannot be diagnosed by an objective test or biomarker. Therefore, diagnosis currently rests entirely on clinical grounds. Symptoms relate to disordered bowel motility producing spasm, visceral hypersensitivity, alterations in brain processing of pain, stress, and underlying psychiatric disorders (e.g., anxiety). Low-grade mucosal inflammation may also play a role in the pathogenesis of visceral hypersensitivity and altered motility in IBS. Patients are divided clinically into three symptom subtypes: diarrhea-predominant, constipation-predominant, and a mixed presentation with alternating diarrhea and constipation. A prior history of an episode of acute infectious gastroenteritis is associated with an almost four-fold increase in the odds of developing IBS within the next 2 years.67 Alterations in the gut microbiome have been noted in patients with IBS based on fecal cultures.68 The most consistent finding was instability of the fecal microbiota reflecting a loss of homeostasis in which the host is unable to maintain a “healthy” intestinal microbiota. Advances in microbial analysis using gene-cloning techniques have provided valuable information regarding the microbiome in IBS.68 Studies utilizing this methodology revealed different alterations of microbial populations in the symptom subtypes. Those individuals with the diarrhea-predominant subtype manifest the greatest deviation from healthy control, while the constipation-predominant subtype has the least deviation. Functional analysis will likely provide further insight into the interaction between microbiota and symptoms. For example, organisms that produce large quantities of organic acids or sulfides may be more likely to cause abdominal pain and bloating.68,69 Animal studies have suggested that probiotics may influence both visceral hypersensitivity and dysmotility through a direct action by bacterial metabolites on sensitive nerve endings in the gut mucosa or via indirect pathways that inhibit mucosal immune activation and visceral sensitization.70 Trials of probiotics in IBS began in the early 2000s, and a large number of clinical investigations followed. However, drawing conclusions from these studies has been confounded by differences in study design, dose, and strain of probiotics. In addition, interpreting results is difficult due to a large placebo response that often develops during the study. Therefore, clear evidence of consistent benefit is lacking. A number of meta-analyses of probiotics in patients with IBS have been performed. Most provide evidence for a beneficial effect on abdominal pain and flatulence, whereas probiotics have an equivocal effect on bloating. Moayyedi et al.71 found a statistically significant effect in favor of probiotics over placebo in improving pain scores. Many of the trials entered utilized combinations of probiotics, most often Bifidobacteria and Lactobacilli. The meta-analysis suggested that Bifidobacteria constituted the active agent in the probiotic combinations studied. A meta-analysis performed by Brenner et al.72 found that B. infantis 35624 promoted significant improvement in the composite score for abdominal pain/discomfort, bloating/


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distention, and/or bowel movement difficulty compared with placebo. No other probiotic showed significant improvement in IBS symptoms. The dose of B. infantis may be important; administration of at least 108 CFU/day is required for a therapeutic response. A 2014 monograph published by the American College of Gastroenterology concluded that probiotics improve global symptoms, bloating, and flatulence in IBS.73 However, the recommendation was deemed to be weak based on low quality of evidence. Additionally, it is likely that only some patients will respond. Regarding prebiotics or synbiotics, it was concluded that there is insufficient evidence to recommend these agents in IBS. The 2014 Yale/Harvard Workshop on probiotic use gave Bifidobacterium infantis B35624 and VSL#3 a level B rating in IBS, while Bifidobacterium animalis and Lactobacillus plantarum 299V were given a level C rating.34 The 2010 guidelines from the American Academy of Pediatrics stated: “The sustained or longterm benefit of using probiotics to treat disorders such as irritable bowel syndrome requires further study; currently, use is not recommended in children.”35 Allergic diseases The clinical classification of allergy includes entities such as atopic dermatitis, allergic asthma, allergic rhinitis, and food allergies. These diseases are quite common in children and in a substantial proportion of adults as well. The International Study of Asthma and Allergies in Childhood published in 2013 found that the global prevalence for current asthma, rhinoconjunctivitis, and eczema in the 13–14 year age group was 14.1%, 14.6%, and 7.3% respectively, while in the 6–7 year age group the prevalence was 11.7%, 8.5%, and 7.9%.74 Evidence for an inverse association between infections in early life and allergic disease has led to interest in the hygiene hypothesis as proposed by Strachan75 in 1989. In this hypothesis, reduced microbial exposure due to improved sanitary conditions has made children more vulnerable to develop atopic sensitization. In contrast, children with more exposure to microbes in their environment (e.g., those who attend daycare centers or live on farms) have a lower prevalence of allergic disease. It is believed that microbial exposures at an early age may activate innate immune pathways, which in turn suppresses Th2 lymphocyte expansion and development of IgE antibodies. However, others have suggested that the hygiene hypothesis is unlikely to be the sole explanation for the allergic diseases since asthma prevalence has begun to decline in both children and adults in Western countries, but there is little evidence that sanitary conditions in these countries has worsened. In addition, the decline in prevalence of asthma in Western countries is not matched by a similar change in atopic eczema and food allergy, where the prevalence continues to increase.76 A pediatric study indicated that children who later developed atopic sensitization had fewer Bifidobacteria, more Clostridia, and different bacterial cellular fatty acid profiles in stool during the first few weeks of life.77 The gastrointestinal tract is typically the route for the initial allergic responses, and food allergy is common in infants with atopic eczema. Breast-feeding exclusively during the first few months of life (o 4 months) has been shown to reduce the risk and severity of eczema and asthma.78,79 It has been suggested that this effect relates to passage of commensal bacteria from mother to infant through breast milk that in turn helps to establish and maintain gut barrier integrity. By blunting the “leaky gut,” which results from a local inflammatory response to dietary antigens in predisposed individual, these bacteria reduce intestinal absorption of sensitizing substances and promote immune tolerance. Probiotics have been investigated both in prevention of immune sensitization (i.e., primary prevention) and as a means to reduce symptoms after sensitization (secondary prevention). Probiotics for primary prevention Kalliomaki et al.80 studied 132 pregnant women with a family history of atopic disease. The subjects were randomized to receive two capsules of either LGG or placebo daily for 2–4 weeks prior to expected delivery. The mothers then either continued the treatment while breast-feeding

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or administered the probiotic or placebo to their infants for 6 months. The primary endpoint was chronic recurring atopic eczema. At 2 years of age, the frequency of atopic eczema of children in the probiotic group was half that of the placebo group with a number needed to treat (NNT) of 4.5. A significant reduction in the rate of atopic eczema was also seen in the infants who were never directly given probiotics for the first 3 months but were breast-fed (15% in probiotic group versus 47% in the placebo group). It was concluded that LGG was effective in prevention of early atopic disease in children at high risk. In contrast, a study by Kopp et al.81 failed to find benefit of supplementation with LGG during pregnancy (4–6 weeks before expected delivery) and early infancy (6 months) on the incidence of atopic dermatitis. In addition, children treated with probiotics had an increased rate of recurrent episodes of wheezing bronchitis. Tang et al.82 performed a meta-analysis of studies that evaluated combined prenatal and post-natal treatment and found a significant protective effect with probiotic/prebiotic treatment for both IgE- and non-IgE-associated eczema. Treatment with L. reuteri or a combination of B. lactis BB12 and LGG both significantly reduced sensitization in infants of allergic or sensitized mothers. L. reuteri was also associated with a trend to reduced sensitization for all infants in the treatment group. Administration of probiotics exclusively in the post-natal period appeared to be much less effective than when administered prenatally or pre-and postnatally.83,84 Foolad et al.85 performed a meta-analysis of studies examining nutritional supplementation in prevention and amelioration of atopic dermatitis among children younger than 3 years of age. A total of 21 studies were analyzed. Nutritional supplementation was shown to be an effective method to prevent atopic dermatitis or decreasing its severity. The best evidence for probiotics appeared to be for supplementation with LGG to mothers and infants as a means to prevent the development and reducing severity of atopic dermatitis. A 2007 Cochrane meta-analysis included 12 studies that investigated probiotics administered to infants for prevention of allergic disease and food hypersensitivity.86 The subjects who were given probiotics had a significant reduction in development of eczema (RR ¼ 0.82) but not food hypersensitivity, asthma, or allergic rhinitis. However, due to significant heterogeneity in the studies, it was concluded that there was insufficient evidence to recommend the addition to probiotics to infant feeds for prevention of allergic disease or food hypersensitivity. Another Cochrane meta-analysis found that there was insufficient evidence to determine the role of prebiotic supplementation of infant formula for prevention of allergic disease and food hypersensitivity.87

Probiotics for secondary prevention Attempts to treat allergic disease once manifested using probiotics have been less successful than when used for primary prevention. Nevertheless, positive results were found in infants with atopic eczema who received LGG.88 A 2008 Cochrane review of pediatric patients treated with probiotics for eczema included 12 studies.89 No significant difference was found that would confirm the efficacy of probiotic therapy in improving symptom scores or eczema severity. Subgroup analysis did not identify a population with different treatment outcomes. It was concluded that probiotics are not an effective treatment for eczema. In addition, probiotic treatment carries a small risk of adverse events (infections and bowel ischemia). It was concluded that effective treatment of allergic disease is ideally initiated during infancy. The American Academy of Pediatrics guidelines on probiotics concluded that 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 the infants who are at risk of atopic disorders, further confirmatory evidence is necessary before a recommendation for routine use can be made.35 The AAP recommendations also stated: “Prebiotics and probiotics are generally recognized as safe for healthy infants and children. However, probiotics should not be given to children who are seriously or chronically ill until the safety of administration has been established. Long-term health benefits of probiotics in the prevention of allergy beyond early


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infancy remain to be proven. Questions remain regarding the optimal duration of probiotic administration as well as preferred microbial dose and species. It also remains to be established whether there is significant biological benefit in the administration of probiotics during pregnancy and lactation, with direct comparison to potential biological benefit derived from probiotic-containing infant formulas.”35 The 2014 Yale/Harvard workshop gave probiotic therapy to treat atopic eczema associated with cowʼs milk allergy a grade A rating (LGG or B. lactis are the most effective agents).34 Atherosclerosis Atherosclerosis is the most important cause of cardiovascular diseases, and among the leading causes of death worldwide. Pathologically, atherosclerosis is characterized by accumulation of cholesterol and macrophage recruitment to the arterial wall. It can therefore be considered both a metabolic and an inflammatory response in the in the vascular endothelium. The pathogenesis of atherosclerotic vascular disease is not entirely known. An intriguing hypothesis involves a central role of the gut microbiome in metabolizing elements contained in high-fat foods to an end product that is highly atherogenic90 (Fig. 3). The key features of this hypothesis includes the following elements: (1) high-fat foods contain increased quantities of phosphatidylcholine, choline, and L-carnitine, which are substrates for synthesis of trimethyamine (TMA); (2) the gut microbiome of the individuals who chronically consume high-fat diet is altered, favoring species that more efficiently produce TMA from these substrates; (3) TMA subsequently undergoes hepatic metabolism to trimethylamine-N-oxide (TMAO); and

Fig. 3. Pathway linking high-fat diet to atherosclerotic cardiovascular disease. TMA: trimethylamine, TMAO: trimethylamine-N-oxide.

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(4) elevated plasma TMAO levels are associated with an increased risk of incident major adverse cardiovascular events in humans. The atherogenic mechanism of TMAO appears to involve its ability to inhibit macrophage reverse cholesterol transport.90 Probiotics may prove effective in preventing atherosclerosis by targeting this pathway. Animal data has shown that administration of L. rhamnosis or L. paracasei probiotics decreased TMAO levels.91 Some of the more impressive clinical data of probiotics in cardiovascular disease were published by Jones et al.92 who evaluated the cholesterol-lowering efficacy of a yogurt formulation containing microencapsulated bile salt hydrolase-active L. reuteri NCIMBB 30242 taken twice daily over 6 weeks in hypercholesterolemic adults. Overall, 114 subjects completed the double-blind RCT. Over the intervention period, subjects consuming the probiotic yogurt attained significant reductions in LDL-C of 8.92%, total cholesterol of 4.8%, and non-HDL cholesterol of 6% compared to placebo. A drop in the atherogenic lipoprotein apoB-100 of 0.19 mmol/L was also observed. A second study performed by Jones et al.93 investigated the cholesterol-lowering efficacy and mechanism of action of L. reuteri NCIMBB 30242 capsules (200 mg/day) in hypercholesterolemic adults. A total of 127 subjects completed the study. L. reuteri reduced LDL cholesterol by 11.6%, total cholesterol by 9.14%, non-HDL cholesterol by 11.3%, and apoB-100 by 8.4% relative to placebo. Triglyerides and HDL were unchanged. Highsensitivity C-reactive protein and fibrinogen were reduced by 1.05 mg/L and 14.25%, respectively. This study also explored potential mechanisms of action of L. reuteri. One mechanism involves the ability of L. reuteri to deconjugate bile via bile salt hydrolase (BSH). This in turn results in increased fecal excretion of deconjugated bile salts with a compensatory increase in the utilization of cholesterol to produce new bile acids. Mean plasma deconjugated bile acids were found to be increased by 1.0 nmol/L relative to placebo in this study. DiRienzo94 reviewed clinical trials that examined the effects of probiotics on LDL-C in order to explore their potential as a therapeutic agent. A total of 26 clinical studies and two metaanalyses were reviewed. Of the probiotics examined, L. reuteri NCIMB 30242 was found to best meet therapeutic lifestyle change dietary requirements by: (1) significantly reducing LDL-C and total cholesterol with robustness similar to that of existing TLC (therapeutic lifestyle changes, dietary options). (2) improving other coronary heart disease risk factors such as inflammatory biomarkers, and (3) being generally recognized as safe. Based on these results, the authors concluded that L. reuteri NCIMB 30242 is a viable candidate for both future dietary studies and as a potential option for inclusion in dietary recommendations in patients with hypercholesterolemia. Although probiotics hold promise in prevention and treatment of cardiovascular disease, there are currently no societal guidelines supporting their use.

Bacterial vaginosis and vulvovaginal candidiasis The normal vaginal mucosa is colonized mainly by Lactobacillus species (e.g., L. crispatus and L. jensenii) but other bacteria may also be present, albeit in lower numbers. However, the population of the vaginal microbiota is not constant and may undergo a variety of changes depending in age, pregnancy, sexual activity, and medications such as antibiotics. This in turn may result in “vaginal dysbiosis,” resulting in clinical conditions such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). BV refers to overgrowth of one of the several types of bacteria normally present in the vagina, thereby upsetting the natural balance (vaginal dysbiosis). Species such as Gardnerella and Atopobium have been implicated in BV. BV is predominately seen in premenopausal women, especially during pregnancy. BV differs from VVC in that there is no inflammatory reaction with the former. Nevertheless, there is often an overlap in symptoms between the two conditions. The reported incidence of BV ranges from 5% to 50%. Common local symptoms include vaginal discharge, dysuria, and pruritus. More serious complications include ascending infections (e.g., pelvic inflammatory disease), obstetrical complications (e.g., chorioamnionitis and premature birth), as well as with urinary tract infection. BV is usually treated with oral or intravaginal


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antibiotics (e.g., metronidazole) but is often associated with treatment failure and high rates of recurrence, especially over the long term. BV recurs in up to 40% within 3 months and in up to 50% within 6 months after initiation of antibiotic therapy. VVC (vaginal thrush) is the excessive growth of yeast in the vagina that results in irritation. Both acute and recurrent VVC are common. The prevalence of VVC is difficult to determine because the clinical diagnosis is often based on symptoms and not confirmed by microscopic examination or culture. Many women have an identifiable cause of VVC such as antibiotics, pregnancy, uncontrolled diabetes, or immunosuppression. However, one-third to half of the recurrent VVC cases have no obvious cause.95 Observations that the women who suffer from BV or VVC have depleted levels of Lactobacillus species prompted trials, investing the use of oral or intravaginal probiotic Lactobacillus strains for treatment and prevention. The mechanism by which orally consumed probiotics are effective is thought to involve entry into the vagina, following excretion from the rectum. Intravaginal administration of probiotic suppositories enables direct replacement of the unhealthy vaginal by probiotic bacteria. The mechanism of action of probiotics is multifactorial and appears to involve production of lactic acid (which maintains low pH), bacteriocins, and hydrogen peroxide. Two approaches for the treatment of BV with probiotics have been used: treatment with probiotics alone or administration of probiotics following a conventional regimen of antibiotic treatment (adjuvant therapy). Most of the studies using probiotics for treatment administered a vaginal suppository containing various Lactobacillus species, most commonly L. acidophilus, L. rhamnosus, or L. reuteri RC-14, given from 5 days to 4 weeks. The best results occurred when probiotics were used as adjuvant therapy, where better cure rates of symptomatic BV were found as well as diminished frequency of recurrences during 1–6 months of follow-up.96 In contrast to BV, there is a lack of good studies supporting the effectiveness of lactobacillus in treating VVC. Ehrstrom et al.97 performed a RCT assessing the efficacy of vaginal colonization with probiotics and effects on clinical outcome. A total of 45 women with diagnosed VVC were entered. Patients were randomized to vaginal capsules containing multiple Lactobacillus species or placebo for 5 days, following completion of conventional treatment. Lactobacillus strains were present 2–3 days after administration in 89% of women receiving the probiotic capsule. Less recurrences and less malodorous discharge were noted. Hilton et al.98 performed an unblinded crossover trial of probiotic yogurt in 33 women with a history of VVC. Daily consumption of probiotic yogurt reduced recurrences. However, interpretation of the results was limited by small size and high dropout rate. In summary, it appears that in BV and VVC, the preferred route of administration of probiotics is intravaginal. Although the evidence supporting the use of probiotics in VVC is not as strong as BV, the empirical use of probiotics may be considered in women with frequent recurrence of VVC or in those with contraindications to antifungal agents. The 2014 Yale Workshop on probiotic use gave a Level C recommendation for the use of probiotics (L. acidophilus, LGG, and L. reuteri) for the treatment of BV.34 The 2012 UK guidelines for the treatment of BV suggest that probiotics can be considered for the treatment of relapsed BV but no recommendations could be made.99

Colorectal cancer The incidence of colorectal cancer (CRC) is increasing in all Western countries and in many developing countries. CRC cancer is found in 5% of population in the US and ranks second only to lung cancer as a cause of mortality. Certain types of CRC have an inherited basis. However, sporadic CRC is much more common, representing 85–90% of all forms of CRC. It appears that environmental factors such as high intake of animal fat (especially red meat and processed meat) and a low intake of fiber and fish play a role in the pathogenesis of CRC. Recent research has suggested an association between intestinal dysbiosis and colorectal carcinoma, thus raising the question, “could colon cancer be considered a bacteria-related disease?”100 Bacteria that have been found to be more abundant in stools of patients with CRC compared with healthy

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controls include anaerobes such as Bacteroides and Clostridium species and sulfur-reducing bacteria. Enterococcus, Escherichia, Shigella, Klebsiella, Streptococcus, and Peptostreptococcus have also been reported to be present in increased quantities in stool of patients with CRC.101 High levels of fecal bile acids have also been associated with higher CRC risk in humans. Bacterial genome sequencing techniques have added further evidence, strengthening the association of CRC with alterations in the normal gut microbiome. Tjalsma et al.102 have proposed that CRC can be initiated by “driver” bacteria that are eventually replaced by “passenger” bacteria that promote tumorigenesis. Bacterial drivers in CRC can be defined as intestinal bacterial with pro-carcinogenic features that may initiate CRC development by producing carcinogenic factors such as DNA-damaging compounds. Candidate drivers include members of Enterobacteriaceae. Bacterial passengers are defined as gut bacteria that are relatively poor colonizers of a healthy intestinal tract but have a competitive advantage in the tumor microenvironment, allowing them to outcompete bacterial drivers of CRC. The initial drivers of this process may therefore disappear with time as they are outcompeted by passenger species. Fusobacterium spp. and other potentially pathogenic bacteria such as Salmonella, Citrobacter, and Shigella are considered as passenger bacteria. The importance of diet in promoting CRC is well recognized. Schulz et al.103 demonstrated that administration of a high-fat diet to mice caused dysbiosis that promoted intestinal carcinogenesis that was independent of obesity. High saturated fat intake appears to alter the intestinal microbiota that in turn favors the production of bacterial metabolites (e.g., hydrogen sulfide, ammonia, and reactive oxygen species) that are genotoxic and promote mutations. Numerous species of lactic acid-producing bacteria have been shown to possess cancerpreventing attributes. Animal studies have investigated mechanisms by which lactic acid bacteria prevent or slow progression of CRC. Potential mechanisms include a reduction in aberrant crypt foci, enhanced apoptosis of damaged cells, inactivation of carcinogenic compounds, competition with pathogenic microbiota, improvement of the hostʼs immune response, fermentation of undigested food, inhibition of tyrosine kinase signaling pathways, and antioxidant effects.104 L. reuteri may prevent CRC by downregulating NFκB-dependent gene products that regulate cell proliferation and survival.105 Lactobacillus and Bifidobacterium contain bile salt hydrolases that blunt the carcinogenic effects of bile acids produced as the consequence of a high-fat diet. Animal models of CRC have indicated that the efficacy of probiotics in preventing tumorigenesis varies, depending on the time of administration.106 The optimal effects occur if the probiotic is given prior to the carcinogen or early following administration. Dietary fiber and prebiotics (especially inulin and oligofructose) also play a role in protection against development of CRC.106 Proposed mechanisms include a reduction in transit time of feces in the gut, absorption of bile salts and bacterial toxins, and anticarcinogenic effects of the prebiotic metabolite, butyrate. Human data regarding the effect of probiotics in preventing or treating CRC are more difficult to interpret due to heterogeneity between studies. Supportive evidence has been reported in clinical studies where probiotics or synbiotics have been used to prevent CRC, reduce postoperative complications, decrease toxicity related to therapy, and improve quality of life. Rafter et al.107 investigated the effects of dietary synbiotics on cancer risk factors in polypectomized and CRC patients. Administration of a synbiotic preparation containing L. rhamnosus, B. lactis, and inulin resulted in a significant change in fecal flora with a decrease in C. perfringens. This in turn reduced colorectal cellular proliferation and improved epithelial barrier function in polypectomized patients. Genotoxic assays of biopsy samples also indicated favorable changes. The Italian Yogurt trial investigated the potential beneficial effects of yogurt in prevention of CRC.108 This trial was a large prospective study of 45,000 volunteers of the EPIC-Italy cohort who completed a dietary questionnaire, including questions on yogurt intake. The investigators found that yogurt intake was inversely associated with CRC risk. The hazard ratio for CRC in the highest versus the lowest tertile of yogurt intake was 0.62. The protective effect of yogurt was noted to be stronger in men than that of women. It was concluded that high yogurt intake was significantly associated with decreased CRC risk, suggesting that yogurt should be part of a diet to prevent the disease.


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Redman et al.109 performed a meta-analysis of the efficacy and safety of probiotics in people with cancer. A total of 11 studies were included. Probiotics significantly reduced the incidence of common toxicity criteria (CTC) grade Z 2 diarrhea, reduced the incidence of CTC grade Z 3 diarrhea, and may have reduced the average frequency of daily bowel movements and need for anti-diarrheal medication. However, the authors cautioned that most of the evidence was not clinically convincing. In summary, there is considerable animal and human evidence that suggests that dietinduced alterations in the gut microbiome contribute to the development of CRC. Furthermore, in vitro studies and animal models demonstrate that probiotics, prebiotics, and synbotics have therapeutic potential in preventing CRC. However, the data obtained from human studies is not strong enough to justify a recommendation that these agents should be used in prevention or therapy of CRC or other gastrointestinal malignancies. To date, there are no existing societal guidelines that recommend the use of probiotics in the prevention or treatment of CRC.

Critical illness Critical illness especially in the setting of ischemia, tissue injury, or antibiotic therapy promotes intestinal dysbiosis; as a result, the protective effect of beneficial commensal bacteria on the gut epithelium (colonization resistance) is diminished. These alterations in the gut microbiome may be linked to increased morbidity and mortality. Under conditions of extreme stress and nutrient deprivation, activators of virulence gene expression (quorum sensing) are “turned on” in opportunistic pathogens such as Pseudomonas aeruginosa.110 Binding of pathogenic bacteria to the intestinal epithelium activate a local inflammatory response with release of pro-inflammatory mediators. This in turn promotes increased permeability of the intestinal epithelium due to alterations in the tight junctions between enterocytes. The resulting enteritis facilitates the passage of luminal antigens into the portal circulation that activates hepatic macrophages and promotes a systemic inflammatory response that can lead to multiple system organ failure. Meakins and Marshall111 characterized the inflammatory response in gut as the “motor” of the multiple organ failure syndrome. Administration of probiotics has been investigated as a means of improving outcome (especially by reducing hospital-acquired infections) in the setting of organ transplantation, major abdominal surgery, and severe trauma. Administration of probiotics is potentially beneficial by improving barrier function and restoring normal intestinal permeability. Probiotics may also be beneficial by producing antimicrobial molecules, by lowering intestinal luminal pH through formation of lactic acid, or by interfering with quorum sensing in bacterial pathogens. Several probiotics have been investigated in the setting of critical illness, with the majority of studies utilizing various species of Lactobacillus. However, it is unclear whether one species is superior to another since existing studies are often of small sample size, comprised of differing ICU populations, and have utilized a variety of administration techniques. One area in which probiotics have been extensively investigated is prevention of respiratory infection in mechanically ventilated patients. Ventilator-associated pneumonia (VAP) complicates the clinical course in up to 30% of critically ill patients and is a major cause for increased length of ICU stay, as well as increased costs. Randomized controlled studies of probiotics as a preventative measure in VAP have used a variety of probiotics but most contained Lactobacillus species (e.g., L. plantarum, L. rhamnosus, and L. casei). The majority of the trials showed a trend for decreased incidence of VAP in the probiotic group but a significant difference was found only in a few.112–114 Meta-analyses also have provided variable results. Siempos et al.115 performed a metaanalysis of five randomized controlled trials and found that probiotic therapy was beneficial in terms of reduced incidence of VAP (OR 0.61), length of ICU stay, and colonization of the respiratory tract with Pseudomonas aeruginosa. However, no difference was found regarding ICU mortality, in-hospital mortality, or duration of mechanical ventilation. Gu et al.117 performed a

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meta-analysis of seven trials and found that probiotics did not significantly decrease the incidence of VAP.116 However, the authors cautioned that firm conclusions from the results could not be made due to heterogeneity among study designs. In a subsequent study, the same authors performed a meta-analysis of effects of probiotics on outcomes of trauma. The use of probiotics was associated with a reduction in the incidence of nosocomial infections (RR ¼ 0.65), VAP, and length of ICU stay. No effect on reducing mortality was seen. Petrof et al.118 performed a systematic review of 23 RCTs and found that probiotics were associated with reduced infectious complications in 11 trials. Seven trials reported data on VAP and the VAP rate was also significantly reduced with probiotics. Probiotics had no effect on ICU or hospital length of stay. As with the previous meta-analyses, clinical and statistical heterogeneity precluded firm recommendations. Probiotics have also been investigated as a means of preventing post-operative infection. The most promising results were in patients undergoing liver transplantation or elective upper gastrointestinal surgery.119 Perioperative provision of probiotics and synbiotics resulted in a three-fold reduction in post-operative infection. Despite the fact that probiotics have been proven to be safe in healthy patients, concern has been raised regarding the safety of probiotics in critically ill patients, immunocompromised patients (including transplant), and premature neonates. Theoretical risks include the risk of dissemination and systemic infection, as well as the potential to transfer antibiotic resistance genes. Issues regarding administration of probiotics to patients with acute pancreatitis will be discussed below (see Therapeutic considerations). The Canadian Clinical Practice Guidelines recommended that the use of probiotics should be considered in critically ill patients.120 However, no recommendation for dose or a particular type of probiotic could be made with the exception of S. boulardii, which should not be used as it is considered unsafe in ICU patients. The Canadian guidelines also stated that addition of probiotics to enteral nutrition is associated with a reduction in overall infectious complications and a trend toward a reduction in the incidence of VAP. Guidelines provided in 2009 by The Society of Critical Care Medicine and American Society for Parenteral and Enteral Nutrition stated that administration of probiotic agents has been shown to improve outcome, most consistently by decreasing infection in specific critically ill patient populations such as transplantation, major abdominal surgery, and severe trauma (grade C level of evidence).121 However, no recommendation can currently be made for the use of probiotics in the general ICU population due to lack of consistent effect on outcome. The guidelines further added that no recommendation can currently be made for use of probiotics in patients with severe acute necrotizing pancreatitis, based on the disparity of evidence in the literature and the heterogeneity of the bacterial strains utilized.

Non-alcoholic fatty liver disease Non-alcoholic fatty liver disease (NAFLD) represents a spectrum ranging from noninflammatory accumulation of fat in hepatocytes to hepatic steatosis with inflammation (steatohepatitis) that may have associated fibrosis. It is now the most common cause of liver disease in Western countries. The prevalence of NAFLD has risen rapidly in parallel with the increase in diabetes and obesity, and accumulating evidence supports an association with the metabolic syndrome. The reported prevalence of NAFLD varies between 20% and 30%, with values approaching 90% in patients with morbid obesity. The pathogenesis of NAFLD relates in large part to obesity and insulin resistance in which an increased hepatic influx of free fatty acids (FFA) occurs. These FFA form triglycerides, leading to hepatic fat accumulation. FFA can be hepatotoxic by increasing oxidative stress and activating inflammatory pathways thereby causing steatohepatitis. Several studies have provided evidence that supports the relationship between obesity and associated changes in the intestinal microbiome with insulin resistance and NAFLD. Experimental evidence has suggested that dysbiosis leads to increased permeability of the


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intestinal epithelial barrier due to direct effects on tight junctions as well as loss of protective commensals such as Bifidobacterium. This in turn results in increased flux of proinflammatory molecules via the portal circulation to the liver. Hepatic inflammation appears to be mediated by inflammasomes, which are intracellular protein complexes expressed in hepatic cells that regulate responses to pathogens and stress. Inflammasomes and IL-18 appear to influence the progression from NAFLD to steatohepatitis; they also induce changes in the gut microbiome that promote development of the metabolic syndrome.122 Regulation of intestinal microbiome by using probiotics and prebiotics has been explored as a treatment of NAFLD. The rationale for the use of probiotics stems from their ability to maintain gut epithelial barrier integrity and reduce the production of proinflammatory cytokines. Loguercio et al.123 studied patients with either NAFLD or alcoholic liver cirrhosis and compared them to HCV positive patients with chronic hepatitis (with or without cirrhosis). All of the subjects were treated with VSL#3 for 3 months followed by another month of washout in an open-label fashion without controls. Plasma levels of transaminases improved significantly in all subjects after treatment with VSL#3, with the effect being maintained after washout in the NAFLD, chronic hepatitis, and alcoholic cirrhosis groups. Eslamparast et al.124 studied the effects of a synbotic supplementation in patients with NAFLD. The subjects received either a twice-daily supplement of a synbiotic containing multiple probiotics plus fructooligosaccharide or placebo for 28 weeks. At the end of the study, alanine aminotransferase (ALT) decreased in both groups, with the reduction being significantly greater in the synbiotic group. Tumor necrosis factor, NF-kappaB (NFκB), and a fibrosis score were also lower in the synbiotic group. Vajro et al.125 performed a study of probiotics in obese children with NAFLD. Subjects were treated with LGG or placebo for 8 weeks. A significant decrease in ALT with normalization was observed in 80% of subjects in the probiotic group. Aller et al.126 studied 30 adult patients with NAFLD (diagnosed by liver biopsy) who were either given L. bulgaricus, S. thermophilus, or placebo for 3 months. They found that probiotic therapy resulted in improved liver aminotransferases (ALT, AST, and gammaglutamine transferase) in patients with NAFLD. Malaguarnera et al.127 studied the effect of administration of synbiotic therapy in patients with non-alcoholic steatohepatitis. A total of 66 patients were randomly divided into two groups receiving either B. longum with fructooligosaccharide and lifestyle modification (diet and exercise) versus lifestyle modification alone. Liver biopsies were performed at entry and repeated after 24 weeks of treatment. At the end of study, the synbiotic group showed significant differences in AST, LDL cholesterol, CRP, TNF, insulin resistance, steatosis, and the steatohepatitis (NASH) activity index. Ma et al.128 performed a meta-analysis of effects of probiotics on NAFLD. Four randomized trials involving 134 NAFLD/NASH patients were included. The results showed that probiotic therapy significantly decreased ALT, AST, total cholesterol, HDL, TNF, and insulin resistance. The use of probiotics was not associated with changes in BMI, glucose, and LDL. The authors concluded that probiotic therapies can reduce liver aminotransferases, total cholesterol, TNF, and improve insulin resistance in NAFLD patients. The 2012 practice guideline by the American Association for the Study of Liver Diseases, the American College of Gastroenterology, and the American Gastroenterological Association made no mention of probiotics as a therapeutic agent in NAFLD.129 It is therefore difficult to make firm therapeutic recommendations regarding the utility of probiotics in NAFLD. Future confirmatory studies may strengthen the recommendations for probiotics as a therapeutic agent in NAFLD.

Radiation enteritis Pelvic malignancies are commonly treated with radical modes of radiation therapy (RT). Patients at risk include those with malignancies of the rectum, bladder, cervix, pancreas, prostate, vulva, and uterus. Acute gastrointestinal side effects occur in over 70% of patients, while up to 30% of patients experience chronic effects, notably diarrhea.

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RT causes intestinal dysbiosis, as well as decreased absorptive surface for nutrients, and decreased gut transit time; the consequence is significant malabsorption of various nutrients and malnutrition. A study of pelvic RT in gynecological cancer utilized pyrosequencing (a method of DNA sequencing) to assess the effects of RT on the overall composition and alterations of the gut microbiota.130 Significant differences were found between the RT patients and healthy controls. RT markedly reduced the numbers of species-level taxa as well as the abundance of each community. Notably, the phyla Firmicutes and Fusobacteria were significantly reduced, and dysbiosis was linked to health status. There have been a number of recent RCTs investigating the utility of probiotics in amelioration of radiation-induced small bowel disease.131–133 These studies assessed the effect of probiotics on the acute rather than chronic effects of pelvic RT. Probiotic agents that have shown promise include VSL#3, LGG, and a combination of L. acidophilus and B. bifidum. In addition to the type and dose of probiotic, the time of initiation (e.g., 1–2 weeks before RT) and duration of treatment may prove to be important in establishing and maintaining a favorable gut microbiome, thereby mitigating adverse effect of RT on the gut. The major benefit of probiotic therapy appears to be an improvement in diarrhea and decreased use of anti-diarrheal medication. Several meta-analyses have been performed to assess the efficacy of probiotics in prophylaxis of radiation enteritis. All were limited by significant heterogeneity between studies. The metaanalysis by Wedlake et al.134 assessed the efficacy of nutritional interventions (elemental, low fat, high fiber, low lactose, and probiotics) to counteract acute gastrointestinal toxicity during therapeutic pelvic RT. The authors concluded that although probiotics offered promise, insufficient high-grade evidence existed to recommend nutritional intervention during pelvic RT. A meta-analysis performed by the Mucositis Study Group reviewed studies of agents for the management of gastrointestinal mucositis in cancer patients.135 The panel concluded that probiotic treatment containing Lactobacillus species might be beneficial for the prevention of chemotherapy and RT-induced diarrhea in patients with pelvic malignancies. Hamad et al.136 performed a meta-analysis of probiotics for prevention of radiation-induced bowel disease after RT. Four outcomes were analyzed: incidence of diarrhea, loperamide use, watery, and soft stools. The pooled OR for incidence of diarrhea significantly favored the use of probiotics over control. They concluded that probiotic supplementation demonstrates a probable beneficial effect in prevention, and a possible benefit in the treatment of radiation-induced diarrhea. The 2014 recommendations of the Yale/Harvard Workshop gave probiotics a level C rating for prophylaxis of radiation enteritis (preferred agents: VSL#3, L. acidophilus).34

Therapeutic considerations for probiotic use Risks of probiotic therapy Currently marketed probiotics have a long track record of safety. However, there may be minimal risk in certain situations that relates to the quality of the product rather than to the probiotic per se. One of the more commonly mentioned safety concerns of probiotic therapy is sepsis. The cases of sepsis that have been associated with probiotics containing Lactobacilli or Bifidobacteria have been reported but are rare. Administration of probiotics to immunosuppressed (e.g., HIV, premature infants, the elderly, and inflammatory bowel disease) has been a topic of concern, but even in this setting, the incidence of sepsis is quite low. Avoidance of S. boulardii probiotics in patients with central venous catheters or synthetic cardiac valve replacement has been recommended based on several reports of fungemia.137 Another concern relates to the potential for transfer of antibiotic resistance from probiotics to the indigenous gut microbiota. However, the risk appears to be minimal with currently available probiotics. Much concern relating to probiotic therapy was generated by publication of the PROPATRIA trial published in Lancet in 2008.138 This study investigated the effect of probiotic prophylaxis in


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predicted severe acute pancreatitis. An increased mortality was observed in the patients who received the multispecies probiotic (Ecologic 641) relative to placebo that was attributed to bowel ischemia. Another possible explanation for this outcome is that more patients in the probiotic group had established organ failure at the time treatment began. The journal subsequently published an expression of concern related to “major shortcomings in the design, approval, and conduct” of the study. Nevertheless, the results should provoke caution when probiotic administration is being considered in certain groups of critically ill patients. Probiotics for health The consumption of food and supplements containing live bacteria for general health purposes has long been popular in Northern Europe, perhaps due to the tradition of consuming fermented foods such as yogurt. The consumption of probiotics drinks is also very popular in Japan. In the US, interest in probiotic supplements is increasing due in part to the use of probiotics to treat common disease such as IBS as well as media ads promoting the health benefits of consuming probiotic yogurt. Probiotics hold interest for two main groups: those with underlying disease where data supporting the therapeutic effect of specific probiotics is available (e.g., UC) and a second group that is interested in probiotics to improve their general health (e.g., to improve gut health and immune function). In 1948, the World Health Organization defined health as “a state of complete physical mental and social well-being and not merely the absence of disease or infirmity.” This definition that has yet to be revised and has been criticized because it leaves no room for improvement. It would therefore be impossible for probiotics to have any significant beneficial effect in those who are already healthy. Since there are no clear markers of health, it is difficult to provide hard evidence documenting beneficial effects of probiotics in improving general health status. Nevertheless, some manufacturers have made claims regarding their products that are unsubstantiated by clinical research, and national health agencies often demand retraction of these claims. Fortunately for the consumer, probiotics are considered “generally recognized as safe” by the FDA, so there is little risk of adverse effects in healthy individuals. The individual who consumes probiotics for general health should therefore take comfort in the fact that probiotics are not likely to be harmful and may confer unspecified beneficial benefits on digestive or immune health. However, they should also be aware that the effects of probiotics are strain specific and any positive effects depend on the preparation chosen and also on the dose. In addition, a reputable brand should be selected since some probiotics currently available do not contain adequate amounts of viable bacteria or may contain other bacteria that are not listed on the label. Consultation with their primary provider or a dietician may facilitate choosing the optimal product. Microencapsulated probiotics One of the major problems associated with delivery of viable probiotic bacteria to the gut is surviving transit through the hostile environment of the stomach. The technique of microencapsulation has been effective in reducing probiotic destruction by gastric acid.139 This process involves surrounding probiotic bacteria by a coating to produce microcapsules, which are resistant to degradation by gastric acid, thereby improving the ability to colonize the gut. Microencapsulated probiotic bacteria have been shown to be five times more efficient than nonencapsulated strains in colonizing the gut.139 In addition, microencapsulation may be effective in improving shelf-live stability of probiotic strains. Clostridium probiotics One of the problems that probiotic bacteria encounter is surviving in the anaerobic conditions of the colon, while remaining stable in the aerobic conditions of the environment

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(e.g., “on the shelf”). This may be the reason many probiotic products are consumed in association with fermented and dairy food products. Certain Clostridial bacteria (e.g., C. coccoides and C. leptum groups) populate the healthy human gut and are able to form spores that enable survival in diverse environments. Spores would be stable “on the shelf” and also would be likely to survive transit through the stomach. Once they reach the colon, they germinate and evolve into metabolically active vegetative cells that can survive in the anaerobic environment of the colon. Unfortunately, there is little data in humans, although the probiotic C. butyricum MIYAIRI 588 has been approved for use in Europe as a supplement to animal feed.140 Experimental data has shown this probiotic to be protective against AAD and to antagonize pathogenic effects of E. coli 0157:H7, and C. difficile.141–143 Certain Clostridial species have anti-inflammatory effects that could be effective in treatment of IBS.140

Engineered probiotics Bioengineered probiotics are usually lactic acid bacteria that have been genetically modified to serve as delivery vectors for therapeutic proteins. One of the mechanisms underlying the pathogenesis of IBD involves abnormally high proteolytic activity. Research in animals has indicated that L. lactis or L. casei can be programmed to deliver protease inhibitors (e.g., elafin) to the gut mucosa, thereby inhibiting local inflammation.144 These bacteria have also been programmed to deliver antioxidant enzymes to the gut mucosa, thereby reducing oxidative stress.145 Engineered probiotics have also been used to improve self-tolerance in autoimmune diseases such as type 1 diabetes by delivering autoantigenic peptides through the intestinal mucosa.146 E. coli Nissle 1917 has been modified to produce a lipid compound normally synthesized in the small intestine in response to feeding that reduces food intake and weight gain.147 Probiotic bacteria could also be engineered to function as a vaccine or as a delivery vehicle for therapeutic drugs. References 1. Dominguez-Bello MG, Blaser MJ, Ley RE, et al. Development of the human gastrointestinal microbiota and insights from high-throughput sequencing. Gastroenterology. 2011;140:1713–1719. 2. Fouhy F, Guinane CM, Hussey S, et al. High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin. Antimicrob Agents Chemother. 2012;56:5811–5820. 3. Claesson MJ, Jeffery IB, Conde S, et al. Gut microbiota composition correlates with diet and health in the elderly. Nature. 2012;488:178–184. 4. Suau A, Bonnet R, Sutren M, et al. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol. 1999;65:4799–4807. 5. Uchiyanma T, Miyazaki K. Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr Opin Biotechnol. 2009;20:616–622. 6. Arumagam M, Raes J, Pelletier E, et al. Enterotypes of the human gut microbiome. Nature. 2011;473:174–180. 7. Wu GD, Chen J, Hoffmann C, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science. 2011;334:105–108. 8. Jeffery IB, Claesson MJ, OʼToole PW. Categorization of the gut microbiota: enterotypes or gradients? Nat Rev. 2012;10:591–592. 9. Caporaso JG, Lauber CL, Costello EK, et al. Moving pictures of the human microbiome. Genome Biol. 2011;12:R50. 10. Ursell LK, Haiser HJ, Van Truen W, et al. The intestinal metabolome: an intersection between microbiota and host. Gastroenterology. 2014;146:1470–1476. 11. De Vuyst L, Leroy F. Cross-feeding between bifidobacteria and butyrate-producing colon bacteria explains bifidobacterial competitiveness, butyrate production, and gas production. Int J Food Microbiol. 2011;149:73–80. 12. Pryde SE, Duncan SH, Hold GL. The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett. 2002;217:133–139. 13. Macfarlane GT, Macfarlane S, Blackett KL. Fermentation and the effects of probiotics on host metabolism. In: Floch MH, Kim AS, eds. Probiotics: A Clinical Guide. Thorofare, NJ: Slack Incorporated; 2010:83–94. 14. Steinhart AH, Hiruki T, Brzezinski A, et al. Treatment of left-sided ulcerative colitis with butyrate enemas: a controlled trial. Aliment Pharmacol Ther. 1996;10:729–736. 15. Cani PD, Delzenne NM. The role of gut microbiota in energy metabolism and metabolic disease. Curr Pharm Des. 2009;15:1546–1558. 16. Ley RE, Turnbaugh PJ, Klein S, et al. Microbial ecology: human gut microbes associated with obesity. Nature. 2006;444:1022–1023.


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Probiotics.

Editorial: Probiotics.

Probiotics revisited.

Probiotics and necrotizing enterocolitis.

Probiotics and Prebiotics.

Probiotics in human medicine.

Beneficial Properties of Probiotics.

Probiotics: an update.

Probiotics in pediatrics.

Clinical Uses of Probiotics.

Probiotics and microbiota composition.

Dairy Propionibacteria: Versatile Probiotics.

Probiotics and liver disease.

Probiotics in obstetrics and gynaecology.

Probiotics in transition: novel strategies.

Probiotics and antibiotic-associated diarrhoea.

Probiotics in critically ill children.

Probiotics: A review for NPs.

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