Intestinal flora contain pathogenic micro-organisms, primarily in the large bowel; but most are benign, and some have advantageous effects. Altering enteric flora to modulate a gastrointestinal disease process is a concept that is gaining in popularity throughout the world. Several studies have recently been completed or are currently underway to evaluate the potential of this concept. This review summarizes the progress made in using probiotic bacteria in the treatment or prevention of pediatric gastrointestinal disorders.
At birth, the gastrointestinal tract is sterile. Enteric flora are acquired during the newborn period and remain relatively stable throughout life. The primary source is maternal vaginal and fecal floras that are usually ingested at the time of delivery (1). There may also be some environmental acquisition, especially in infants born by cesarean delivery who do not have an opportunity to ingest maternal flora at delivery. Consequently, there may be some delay in acquisition of permanent flora by these infants (2).
Once enteric bacterial flora have been established, it is fairly difficult to modify or change them permanently. Temporarily, flora may be modified negatively through the use of antibiotics or positively, through the introduction of desirable bacterial species into the gastrointestinal tract-that is, through the use of probiotics. Alternatively, nutrients(prebiotics) that stimulate the growth of certain favorable organisms, may be given to change flora in a positive way (3-6). Probiotics are defined as microorganisms, which, when ingested, may have a positive effect in the prevention and treatment of a specific pathologic condition (7). In contrast, Prebiotics are substances that, when ingested, promote the growth and establishment of beneficial micro-organisms (8,9).
PROBIOTIC ORGANISMS
Lactobacilli are perhaps among the most attractive of probiotics because of their longstanding use in fermentation of foods and historical record of safety (10,11). Lactobacillus is a diverse group of organisms that is part of a larger group of lactic acid bacteria-nonpathogenic gram-positive bacteria that produce lactic acid as a primary metabolic end product. Lactic acid bacteria include lactobacillus, lactococcus, streptococcus, and others. Lactobacilli, themselves, vary greatly from one species to another, often with different genetic makeup and different adherence and colonization patterns (12). Consequently, use of the term "lactobacillus" is meaningless when classifying probiotics; each particular lactobacillus species must be studied individually for its probiotic properties and ultimately for its beneficial clinical effects.
In addition to lactobacillus, several other organisms have shown therapeutic potential, including a few species of Saccharomyces, a yeast, and additional bacteria, Bifidobacterium, andStreptococcus thermophilus (Table 1)(13). Many of the inconsistencies in studies in which probiotic organisms are evaluated for beneficial clinical effects are caused by the lack of specificity in selecting the species for study or by the inadequate identification or reporting of the species involved.
A probiotic should be innocuous. Broad consumption obviates the opportunity for distribution of potentially pathogenic organisms. Probiotics should act against pathogens by multiple mechanisms and should elicit minimal resistance to its effects (14). The organisms should also be capable of marshalling host defenses against potential pathogens. The onset of beneficial effects should be rapid in comparison with the several days to weeks required for a vaccine to become fully protective(15,16). Probiotics should function whether administered alone or with antibiotics.
Most of the studies on probiotics have been performed using whole, live organisms (17). However, killed organisms or even components of organisms may be effective in stimulating immunologic reactions or even in inducing binding with certain compounds or structures within the gastrointestinal tract (18). In addition, probiotics may function by synthesizing certain compounds or producing metabolic by-products that could be protective or induce a positive effect. In this situation, these metabolic by-products may be used therapeutically(19,20).
MECHANISMS OF ACTION
Probiotics have several mechanisms of action (Table 2). For example, Lactobacillus casei subsp rhamnosus(Lactobacillus GG) produces compounds that inhibit the growth of several gram-positive and gram-negative bacteria(21,22). Examples include hydrogen peroxide and pyroglutamate (21). A few other lactobacilli are capable of producing similar substances. Short chain fatty acids are commonly produced (23). These lower the colonic pH, which favors the growth of organisms with less pathogenicity. Most of these data have been derived from studies in vitro, however; confirmation of these effects in vivo would be difficult.
Binding to enteric epithelium is one of the determinants in establishing the efficacy of a probiotic (24). Colonization resistance occurs through this binding, competitively inhibiting adhesion of pathogenic bacteria (25-27). For example, Lactobacillus GG and Lactobacillus plantarum 299V (LP299V) competitively inhibit the attachment of Escherichia coli 0157H7 to HT-29 cells (28,29). Other lactobacillus strains have been shown to compete with enteropathic E. coli for attachment to mucus in pig ileum (30).Saccharomyces boulardii inhibits the attachment ofEntamoeba histolytica trophozoites to erythrocytes in vitro(31). An effect on barrier function with a lactobacillus strain has been demonstrated by decreased mucosal permeability to mannitol in germ-free rats (32).
Probiotics may also compete for nutrients otherwise consumed by pathogenic organisms. For example, consumption of monosaccharides by a probiotic may reduce the growth of Clostridium difficile, which is dependent on monosaccharides for growth (33). Probiotics may also modify toxin receptors through an enzymatic mechanism, which has been shown with S. boulardii through its effect on the C. difficile toxin A receptor in the rabbit ileum(34). Similar effects have been postulated for the cholera toxin receptor. Saccharomyces, through its production of polyamines, may also have a trophic effect on the small intestine(35).
Another possible mechanism for the positive effects of a probiotic is its effect on generalized immune enhancement, most likely through its ability to bind to epithelial cells. Lactobacillus GG stimulates antibody production against rotavirus (36,37).S. boulardii is capable of activating complement and the reticuloendothelial system (38,39).
A major factor in determining the effectiveness of a probiotic is its ability to survive the digestive process and thrive in the gastrointestinal tract. Difficulties in establishing colonization are the focus of most investigations in search of effective probiotics (18,26). Colonization must be routinely assessed through intestinal biopsy and fecal sampling. Many bacteria are killed by the acid pH in the stomach or the effects of bile in the duodenum.Lactobacillus GG has been shown to persist in the intestinal tract after cessation of administration for at least 7 days, although levels decrease significantly once administration is discontinued (40-42). Once the organism survives gut transit, its efficacy as a probiotic is primarily determined by its ability to bind with receptors and inhibit the attachment of pathogenic organisms, its synthesis of antimicrobial substances, and its metabolism of available nutrients for pathogens in the intestinal lumen (13,14,43).
ANTIBIOTIC DIARRHEA
One of the most obvious clinical indications for the use of probiotics is the treatment or prevention of antibiotic-associated diarrhea, a common problem occurring in approximately 20% of hospitalized patients who are administered antibiotics (44,45). One third of incidences of antibiotic-associated diarrhea appear to be caused by C. difficile (46). The cause of the remaining two thirds is uncertain but is thought to be disturbances in bacterial metabolism that affect short chain fatty acid production in the large intestine. Presumably, antibiotics kill the organisms responsible for producing the short chain fatty acids that are normally absorbed in the colon. Then, somewhat larger molecules accumulate, are not adequately absorbed, and exert an osmotic effect in the colon, resulting in diarrhea.
Several species of organisms have been studied as possible therapeutic agents in the treatment or prevention of antibiotic-associated diarrhea. These include S. boulardii, a few lactobacilli, andBifidobacterium. Bifidobacterium longum, when administered in yogurt, caused a significant reduction in stool weight, reduction in stool frequency, and lessening of abdominal discomfort in patients treated with antibiotics (47). Reduction in symptoms was minimal, however. Enterococcus faecium SF68, in a multicenter, double-blind trial, reduced the incidence of antibiotic-associated diarrhea from 27% to 9%, when compared with placebo (48).Lactobacillus GG was administered to eight volunteers who were treated with erythromycin. Eight control subjects received pasteurized yogurt. Lactobacillus GG-treated patients had an average of 2 days of diarrhea versus 8 days in those patients treated with placebo(49). Recently, Lactobacillus GG reduced antibiotic-associated diarrhea in 188 pediatric outpatients treated with a wide variety of antibiotics for common childhood infections(50). Lactobacillus acidophilus andLactobacillus bulgaricus, when administered together, also reduce the incidence of antibiotic diarrhea from nearly 30% to almost zero(51).
In three large studies, investigators have evaluated the efficacy ofS. boulardii in antibiotic-induced diarrhea. In a study including 388 patients, Adam et al. (52) evaluated coadministration of S. boulardii and antibiotics. They demonstrated an incidence of antibiotic-associated diarrhea of 4.5% versus 17.5% in the control group. Surawicz et al. (53) and McFarland et al.(54) described similar reductions of diarrhea from groups of 180 and 193 patients respectively who were administered antibiotics, when compared with placebo.
There are negative studies suggesting that not all probiotics are efficacious in the treatment of antibiotic-associated diarrhea. L. acidophilus and L. bulgaricus, given in the form of Lactinex(Becton Dickinson, Franklin Lake, N.J., U.S.A.), failed to reduce the incidence of antibiotic-associated diarrhea in one study(55). The same combination is ineffective in reducing diarrhea associated with enterotoxigenic E. coli and "traveler's" diarrhea (56,57).
CLOSTRIDIUM DIFFICILE
Approximately one third of cases of antibiotic-associated diarrhea appear to be caused by C. difficile (46,58). Standard therapy consists of a 10-day course of metronidazole or vancomycin. Relapses are common after therapy. Initial attempts at probiotic therapy consists of fecal infusions from healthy volunteers to reestablish normal flora (59,60). Although this from of therapy is moderately successful in some patients, it does not enjoy widespread aesthetic appeal. Results of studies by Gorbach et al. (61) and Tvede and Rask-Madsen (62) in adults have suggested that orally administered Lactobacillus GG significantly reduces relapse after therapy for C. difficile-associated antibiotic-induced diarrhea. Comparable findings have been described by Biller et al.(63). In these studies, typical doses consisted of 1010 colony forming units per day for 1 to 2 weeks. S. boulardii inhibits binding of C. difficile to gut mucosa in a rat model (34). S. boulardii was also effective, when combined with vancomycin, in a large, placebo-controlled study in 124 adults in reducing relapse from 46% to 23% after incidences ofC. difficile-associated diarrhea (64). Buts et al. (65) demonstrated the efficacy of S. boulardii in an open-label, uncontrolled trial of 19 infants with enteropathies who had C. difficile-positive stools.
INFECTIOUS DIARRHEA
Probiotics have been used in the treatment of infectious diarrhea as well. A slight reduction in diarrhea in patients with enteric infections was observed by Wunderlich et al. (48) using E. faecium SF68. S. boulardii was used in the treatment of patients with AIDS-associated infectious diarrhea, reducing stools in 58% of treated patients versus only 6% of placebo patients(66). Mitra and Rabbani (67) could not show a beneficial effect, primarily in patients with cholera and pathogenic E. coli infection.
Isolauri et al. (68,69) in placebo-controlled studies in children hospitalized because of acute diarrhea, demonstrated reduction in duration of diarrhea from 2.4 days to 1.4 days versus placebo, using Lactobacillus GG in powder or fermented milk. The predominant causative diarrheal agent in the study of Isolauri et al. was rotavirus, and when these patients were analyzed independently, the effect was even more significant. Similar findings withLactobacillus GG were reported by Raza et al.(70) in 40 Pakistani children. There was significant reduction in watery diarrhea, presumably viral in origin, but the probiotic did not appear to be effective in the treatment of patients with bloody diarrhea, most likely of bacterial origin. Majamaa et al.(71) reported a small, but significant, reduction in duration of rotavirus-induced diarrhea with Lactobacillus GG. They used alternative probiotics, including Lactobacillus rhamnosus, Lactobacillus delbrueckii, and S. thermophilus as controls. Similarly, Pant et al. (72) conducted a study in whichLactobacillus GG shortened the duration of watery diarrhea but not of bloody diarrhea in children in Thailand. These effects ofLactobacillus GG appear to be highly species-specific, in that other lactobacilli, when studied similarly, have not been effective(73). Sepp et al. (74) demonstrated effectiveness of Lactobacillus GG alone versus TMP-SMX in the treatment of children with shigellosis. Sheen et al.(75) also demonstrated the effectiveness ofLactobacillus GG in a diarrhea control program in a community of Peruvian children.
Similar to Lactobacillus GG, S. boulardii has been effective in the treatment of acute diarrhea in children. S. boulardii (0.5 g/day for 5 days) was added to a standard oral rehydration solution in 38 children with acute diarrhea (76). There was a significant reduction in stool weight and an increased transit time in patients treated with Saccharomyces. Another large (n = 130), placebo-controlled, double-blind study in children with acute diarrhea showed that Saccharomyces treatment reduced the number of stools(77). Comparable findings have been shown in a multicenter, double-blind study of 92 adult outpatients with acute diarrhea(78).
TRAVELER'S DIARRHEA
Several studies have been conducted to assess probiotic prevention of traveler's diarrhea (79,80). In most of these,Lactobacillus GG has been used and has significantly reduced the incidence of traveler's diarrhea. At least one study showed that the effectiveness of treatment varies with the destination of the traveler(81). These studies have been limited, in relating success or failure with indigenous organisms, by lack of assessment of the actual cause of the diarrhea. In one double-blind, placebo-controlled trial with S. boulardii, travelers taking the active yeast preparation experienced a modest but statistically significant reduction in the incidence of diarrhea (82).
PREVENTION OF INFECTIOUS DIARRHEA
Studies have also been conducted to determine the efficacy of probiotics in the prevention of infantile diarrhea. Saavedra et al.(83) demonstrated that bifidobacteria and S. thermophilus, when administered together, decrease the incidence of diarrhea from 31% to 7% in a group of hospitalized infants, when compared with placebo (83). Rotavirus shedding also decreased from 39% to 10% in the treated group. Two studies have demonstrated that colonization with Lactobacillus GG did not reduce the fecal reservoir of potential pathogens and therefore, had no positive effect(84,85). Because the object of probiotic therapy is not to change the fecal reservoir but to prevent attachment of pathogens, the results may not be relevant to probiotic efficacy.
PROBIOTIC EFFECTS ON IMMUNE FUNCTION
From the standpoint of mechanism, many studies have been conducted in which the effect of probiotics on stimulation of the immune system has been examined (17,18). Isolauri et al.(86) demonstrated that Lactobacillus GG increases the presence of immunoglobulin (Ig) M-secreting cells after administration of rotavirus vaccine. Immunoglobulin M seroconversion was not affected, but IgA seroconversion was improved. Majamaa et al.(71) also demonstrated that Lactobacillus GG enhances the IgA response to rotavirus.
INTESTINAL ALLERGY
Probiotics appear to have some effect on ameliorating intestinal allergy. They may reduce translocation of foreign proteins by tightening the mucosal barrier or by affecting the induction of tolerance(87). Majamaa et al. demonstrated that the addition ofLactobacillus GG to a hydrolyzed whey formula results in a significant improvement in a clinical allergy score when compared with placebo (88). Fecal tumor necrosis factor-α, which is associated with allergy-induced intestinal inflammation, also decreases after Lactobacillus GG administration.
VAGINITIS
Chronic vaginitis, especially that caused by Candida, can be treated with probiotics. Investigators using Lactobacillus GG have demonstrated that, when administered directly, the organism is efficacious in preventing recurrence (89-91). Hilton et al. (92,93) conducted studies using an orally administered strain of L. acidophilus and recorded a significant reduction in vaginal Candida colonization. Because vaginal contamination occurs from fecal flora, administration of oral probiotics with subsequent fecal colonization would seem to be a rational approach to the patient with chronic, recurrent vaginitis. Lactobacillus vaginal suppositories are also effective in reducing the incidence of recurrent urinary tract infections (94-96).
IRRITABLE BOWEL SYNDROME
One of the more interesting potential uses of probiotics is as a therapy for irritable bowel syndrome. A small study has been conducted in Poland using LP299V in adults (97). The results demonstrated a reduction in symptoms of abdominal pain and constipation when compared with placebo. A small, double-blind, placebo-controlled crossover study in children with recalcitrant, chronic, recurrent abdominal pain of childhood also showed a slight, but statistically significant, reduction in the severity of abdominal pain in LP299V-treated patients compared with results in control subjects (98). It is difficult to speculate why this particular probiotic may be effective in the treatment of patients with irritable bowel syndrome. L. plantarum 299V is known to synthesize nitric oxide by at least three pathways, and nitric oxide is an important mediator of gut motility (15). In addition, LP299V may displace the attachment of other organisms from the intestinal mucosa, which themselves may have a modulatory effect on gut motility. Because it has been previously shown to reduce inflammation in an animal model, LP299V may also reduce any inflammatory component associated with the disease (99).
LACTOSE INTOLERANCE AND CHOLESTEROL REDUCTION
The treatment of lactose intolerance with probiotics would seem to make sense in some cases, although presently there are other effective ways to treat lactose intolerance. Yogurt reduces symptoms of lactose intolerance when compared with a similar lactose load in the form of milk(100-102).
The potential use of probiotics in cholesterol reduction has been proposed, because certain bacteria are capable of enzymatic modification of the cholesterol molecule (103). However, because colonization of the proximal small intestine would be necessary for this to work, the potential seems limited.
INFLAMMATORY DISORDERS
Certain probiotics appear to reduce intestinal inflammation. In methotrexate-induced mucosal injury in the rat, LP299V reduces inflammation, reduces bacterial translocation, and increases mucosal regeneration(104). The potential use of probiotics in inflammatory diseases suggests many possibilities for probiotic therapy. Infants with intestinal inflammation secondary to formula protein intolerance may benefit. Tolerance of resident intestinal flora is broken down in active inflammatory bowel disease (105) and affected patients may be candidates for probiotic therapy. L. plantarum 299V has been efficacious in the reduction of multiple organ failure (106,107). In the form of fermented oatmeal soup, LP299V was administered to a group of patients after surgery as part of an enteral feeding formula in Sweden. This resulted in a marked reduction in the incidence of multiple organ failure and liver injury (106,108). Probiotics have also been suggested for treatment or prevention of Helicobacter pylori colonization(109). Lactobacillus GG has recently been shown to reduce fecal urease activity in children with chronic arthritis, a correlate of disease activity in patients with arthritis(110).
CANCER PREVENTION
Probiotics have even been suggested as a means of reducing the risk of development of cancer (111). Participation of intestinal microflora in toxic events resulting in activation or detoxification of mutagens, carcinogens, and tumor promoters has been well documented (15). For example, inhibition of bacterial enzymes involved in the synthesis of colonic carcinogens may be a beneficial mechanism. Such enzymes as glucuronidase, azoreductase, and nitroreductase could be inhibited by a mechanism so simple as a reduction in intracolonic pH. Some intestinal flora may themselves be carcinogenic, and simple modification of the flora may be helpful. In general, species ofBifidobacterium and lactobacillus have low levels of carcinogenic enzyme activity in the gut in comparison with activities of other anaerobes, such as bacteriods and clostridia (112). Although some data exist from small clinical and animal studies, extensive investigation must be conducted before probiotics can be considered for cancer prevention.
SAFETY ISSUES
For probiotics to be widely used in the prevention or treatment of gastrointestinal disorders, they must be safe. Lactobacilli and bifidobacteria raise little concern because they normally reside in the gastrointestinal tract of healthy people. They have been taken in large doses for years in many countries and have a long history of safe use(9). For example, widespread screening of large populations in Finland taking Lactobacillus GG failed to find a single case of Lactobacillus GG infection (113,114). Lactobacillus GG has also been administered to preterm infants without evidence of adverse effects(115). Some strains of lactobacilli have been identified as causative factors in endocarditis, but these were indigenous bacteria, not those introduced by a probiotic (116,117). New experimental studies have demonstrated gene transfer between enterococci in the gastrointestinal tracts of experimental rats (118). These are not usually considered to be human probiotic organisms, however, and this effect has not been demonstrated with lactobacillus. Although most lactobacilli are vancomycin-resistant, this particular form of vancomycin resistance is chromosomal and not plasmid mediated and therefore, the risk of transmission of vancomycin resistance to other organisms, especially enterococci or staphylococci, is minimal. Some concern has been raised about the safety of S. boulardii. This probiotic has induced development of disseminated fungicemia in a few patients, although all were successfully treated with fungicidal therapy(119). However, S. boulardii has been administered to numerous patients with acquired immune deficiency syndrome, Crohn's disease, and enteritis without development of infection(120).
Recently, concern has been raised regarding the viability of organisms sold in probiotic preparations. In a recent survey of several commercially available probiotic products, few actually contained the number or type of live organisms stated on the package. In some cases, the number of live organisms actually present was markedly less than advertised(121). Consequently, if probiotics are to be used for medical purposes, careful viability and colonization studies must be performed periodically, and clinicians must make certain that only viable products from reputable companies are recommended.
SUMMARY
Probiotics appear to be useful in the prevention or treatment of several gastrointestinal disorders, including infectious diarrhea, antibiotic diarrhea, and traveler's diarrhea (Table 3). Results of preliminary human and animal studies suggest that patients with inflammatory diseases, and even irritable bowel syndrome, may benefit from probiotic therapy. Probiotics represent an exciting therapeutic advance, although much investigation must be undertaken before their role in gastroenterology is clearly delineated. Questions related to probiotic origin, survivability, and adherence are all important considerations for further study. More important, each probiotic proposed must be studied individually and extensively to determine its efficacy and safety in each disorder for which its use may be considered.
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