REVIEW

Bacteria and yeasts in the treatment of acute and chronic infectious diarrhea. Part I. Bacteria
Clin Microbiol Infect 1999; 5: 299-307

Yvan Vandenplas
Academic Children's Hospital, Free University of Brussels, Brussels, Belgium

Treatment of acute infectious gastroenteritis consists mainly of rehydration and rapid realimentation. However, the natural intestinal microflora is disrupted in both acute and chronic infectious diarrhea, resulting in complex interactions possibly aggravating this frequently self-limiting condition. Therefore, additional therapeutic intervention with biotherapeutic agents is worth considering. The results of most randomized prospective double-blind clinical trials with bacterial biotherapeutic agents are disappointing, showing a lack of efficacy, except for Lactobacillus casei strain GG, for which both positive and negative results have been published. Key words: Biotherapeutic agent, diarrhea, gastroenteritis, Lactobacillus, probiotic

INTRODUCTION
In the USA, diarrheal diseases cause an estimated 167 000 hospitalizations and 300 deaths each year among children younger than 5 years of age [1-3]. Treatment is largely symptomatic and involves fluid and electrolyte replacement, and maintenance of nutrition [4-6]. During an episode of gastroenteritis, there is a decrease of protective commensal microflora, followed by an overgrowth of urease-producing pathogenic bacteria [7]. Therefore, normalization of the gastrointestinal microflora during an episode of acute diarrhea may shorten the duration of the diarrhea, make it less severe and improve its outcome. Although the consumption of probiotics, as in fermented milks, dates from pre-biblical times, the theoretical concept of probiotics or biotherapy was first described by the Russian Metchnikov, who was awarded the Nobel Prize for medicine in 1908 for demonstrating that some bacteria could stimulate the growth of Vibrio cholerae, while others inhibited its growth. Lactulose is an example of a prebiotic, a nondigestible ingredient that is beneficial for the host because it enhances growth and/or activity of one or more non-pathogenic bacterial strains in the colon [8]. A biotherapeutic agent or probiotic is a live microbial food supplement which beneficially affects the host by improving the intestinal microbial balance [9]. By definition, these microorganisms need to be prepared, preserved and administered in a viable form. Biotherapeutic agents survive in the intestinal ecosystem. Acidophilus or bifidus yoghurts are probiotic yoghurts. It is proposed to

use probiotic to designate all products fulfilling the above criteria. The term biotherapeutic agent should only be used to designate the medical products (drugs) complying with the above definition. In this review, I shall discuss biotherapeutic agents only, and analyze the literature critically focusing on kinetics, pharmacodynamics, prospective, randomized, placebocontrolled clinical studies testing the efficacy of the biotherapeutic agent against acute and chronic infectious diarrhea, and the side effects of biotherapeutic agents. Unfortunately many review papers consider and attribute identical convincing evidence to case reports, and open and blind studies [10]. Open trials, such as the use of Bacteroides sp. in Clostridium difficile diarrhea [11], are not considered in the discussion.

NATURAL BACTERIAL PROBIOTICS

Fermented milk products such as yoghurt, kefir and buttermilk contain living bacteria, most frequently Bifidobacterium bifidum, Bifidobacterium longum, Lactoba-cillus acidophilus, Lactobacillus bulgaricus, Streptococcus lactis and Streptococcus cremoris [12]. The use of fermented milk products in a clinical setting is difficult because of the need for cold storage and limited shelf-life. Also, it is difficult to persuade sick patients (especially the elderly or children) to consume a sufficiently large quantity (at least three yoghurts daily [13]) of yoghurt or fermented milk to make any clinical impact likely. The resistance of most yoghurt bacteria to bile and acid is poor, although differences between strains have been observed [14,15]. Frequently, this resistance is not mentioned [13]. Lacto-bacillus acidophilus survives the gastric acidity better if administered in milk as vehicle, than with yoghurt or buttermilk [14]. Even if antibiotics such as -lactams, tetracyclines and quinolones are administered intra-venously, the hepatoenteric cycle inactivates these biotherapeutic agents rapidly.

Corresponding author and reprint requests: Yvan Vandenplas, Academic Children's Hospital, Free University of Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium Tel: +32 2 477 57 81 Fax: +32 2 477 57 83 E-mail: pedvsy@az.vub.be Accepted 5 December 1998

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COMMERCIAL PROBIOTIC AGENT FOODS

Some probiotic agents are registered as medicinal products, while others are registered as food. If registered as foods, products are not subjected to the same testing as medical products, and medical terminology cannot be used to describe the claimed effect(s). Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus and Enterococcus faecium SF 68 can be purchased as viable bacteria in dried form as granules or capsules in health food shops, and are thus not available on medical prescription. Probiotic foods have to fulfil the criteria for the food legislation, and not the legislation for medical drugs. The bacteria present in probiotic foods are found in the normal human colonic flora, but are not resistant to most commonly used antibiotics. (An effective probiotic should be naturally resistant to commonly used antibiotics.) The labeling of many probiotic foods may be misleading, in terms of both their microbiological content and their claimed beneficial effects [16,17]. Brewers' yeast (Saccharomyces cerevisiae) is sold as a food supplement for human consumption for constipation, but is not viable. The regulations on probiotic agent preparations and the marketing of dairy foods containing probiotic bacteria must give better guarantees for the consumer on the quality of these expensive products [17].

PHARMACODYNAMICS OF THE PHARMACEUTICAL BACTERIALBIOTHERAPEUTICAGENTS

Colonization resistance Colonization resistance is the ability of the normal colonic flora to protect the host against colonization by pathogens, and is achieved via a complex interaction between the various strains. A biotherapeutic agent is intended to inhibit the proliferation of pathogens during a period when the normal colonic microflora is disturbed [9]. At present, no single bacterium or combination of bacteria is capable of inducing colonization resistance to pathogens in a similar way to the natural colonic microflora [9]. Production of antibacterial substances In vitro, Lactobacillus casei strain GG inhibits the growth of Gram-positive and Gram-negative bacteria, and produces hydrogen peroxide, which has a bactericidal effect, together with other inhibitory metabolic products [9]. However, it is not clear whether these antibacterial substances are also effective in vivo, although they are reported to be so in animal models with Lactobacillus acidophilus strain LA1 and Lactobacillus casei strain GG [9,20-22]. In vitro, yoghurt with Streptococcus thermophilus and Lactobacillus bulgaricus is bactericidal for Clostridium difficile within 2h [20]. However, in vivo, hamsters are not protected from death by Clostridium difficile-induced colitis, even if very large amounts of yoghurt have been administered [20]. Competition for nutrients Growth of Clostridium difficile depends on the availability of monosaccharides. Increased competition in vivo for monosaccharides inhibits Clostridium difficile growth, while an increased supply of them stimulates its growth [23]. Little is known about the influence of bacterial biotherapeutic agents on nutrient competition. Competitive inhibition at bacterial adhesion sites Competitive inhibition by the biotherapeutic agent at the adhesion sites for bacterial pathogens is another interesting possible mechanism of action [9]. Certain Lactobacillus acidophilus strains, such as LA1, compete in vitro for cell adhesion to and/or invasion of enterocyte-like cells with enteropathogenic Escherichia coli, Salmonella typhimurium and Yersinia pseudotuberculosis [24]. The Lactobacillus acidophilus LB strain produces antibacterial activity in vitro against Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa and Enterobacter spp. [21]. In contrast, there is no inhibition of lactobacilli and bifidobacteria [21]. However, if Escherichia coli is administered before the Lactobacillus acidophilus (as occurs when acute gastroenteritis is treated with a biotherapeutic agent), the protection by the Lactobacillus strain is severely reduced, because of non-specific steric hindrance of the receptor sites [21]. Heat-killed Lactobacillus acidophilus was demonstrated in vitro to retain adhesion capacity, although it was not viable [25,26].

RECTAL BIOTHERAPY
Although theoretically interesting in patients with colitis, rectal biotherapy has only been investigated, over a period of more than 40 years, in uncontrolled studies and in small numbers of patients (n < 30) [12,18]. The rectal route is difficult to manage for doctors and patients, and the (theoretical) risk of a spread of undetected pathogens cannot be ignored.

KINETICS OF THE PHARMACEUTICAL BACTERIAL BIOTHERAPEUTIC AGENTS

A biotherapeutic agent has to be detectable alive in the entire gastrointestinal tract (thus also in feces), even when administered simultaneously with antibiotics, in numbers high enough to be effective. A biotherapeutic agent can only be effective if it survives contact with gastric and pancreatic secretions, including acid [9,12]. It is essential for a biotherapeutic agent that its natural resistance to antibiotics cannot be transferred to the natural colonic flora or to pathogens present in the host, including in the gastrointestinal tract [9], and that the biotherapeutic agent does not acquire the resistance of some commensal or pathogenic microorganism and subsequently transfer it to other commensal or pathogenic microorganisms. Most lactobacilli are killed by acid and/or bile [9,12]. Only 1.5% and 37.5% of ingested Lactobacillus acidophilus and bifidobacteria, respectively, reach the ileum [19].

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However, even in vitro, 10 times the amount of heat-killed Lactobacillus acidophilus than of the living strain was required to obtain the same prevention of adhesion of enterotoxic Escherichia coli [25]. Even more unrealistic concentrations are needed to obtain a preventive effect in vivo [25]. Viable lactobacilli failed to prevent or change the duration or symptoms of enterotoxic Escherichia coli diarrhea in adults [27]. Viable Lactobacillus acidophilus preparations are not effective in the prevention of travelers' diarrhea [28,29], although enterotoxic Escherichia coli is the most important (40%) causal organism [30]. Lactobacillus salivarius inhibits colonization of the stomach with Helicobacter pylori in gnotobiotic BALB/c and germ-free mice [31]. As a consequence, the relevance of competitive inhibition for adhesion sites is restricted to prevention, since in therapeutic conditions the adhesion of the pathogen has already taken place. The clinical relevance of the in vitro model, in which the preventive administration of lactobacilli competes with the adhesion of enteropathogens, and especially of enterotoxic Escherichia coli, is thrown into question by the disappointing clinical results. Enhancement of the immune defense system Children with rotavirus diarrhea secreted significantly more rotavirus-specific IgA cells during the convalescence period if a living rather than a heat-killed Lactobacillus casei strain GG was administered [32]. During the diarrhea period itself, rotavirus-specific antibodies were similar and very low in both groups, and there was no difference in the duration of diarrhea observed [32]. The increased specific antibody production occurred at a later stage, and is (probably) relevant in the prevention of reinfection [32]. These data have been confirmed in a similar study, showing that Lactobacillus casei strain GG is more effective than Lactobacillus casei rhamnosus or a combined lactobacillus preparation [33]. The enhancement of the immune system by the administration of lactobacilli during a rotavirus infection might be relevant in the prevention of reinfection, but does not influence the acute phase. Lactobacillus casei strain GG has an immunostimulating effect on oral rotavirus vaccination [34]. Lactobacillus casei strain GG has the potential to increase the gut IgA immune response and promotes the gut immunologic barrier in patients with Crohn's disease [35]. Lactobacillus reuteri prevents Cryptosporidium parvum infection in immunodeficient mice [36].

Lactobacillus bulgaricus had been administered prophylactically 36 h before the Escherichia coli [27]. The same combination also failed to show any effect in the prevention of travelers' diarrhea, antibiotic-associated diarrhea and enteral feeding-associated diarrhea [28,37,38]. Viable Lactobacillus acidophilus was tested, without success, in the prevention of travelers' diarrhea [29,39]. A heat-killed Lactobacillus acidophilus strain was evaluated in a double-blind placebo-controlled study in the treatment of acute diarrhea [40]. All these studies failed to demonstrate any significant difference between the Lactobacillus acidophilus and the placebo groups. Lactobacillus casei strain GG Lactobacillus casei strain GG was evaluated in a prospective randomized placebo-controlled study in 880 volunteers for the prevention of travelers' diarrhea, showing a non-significant effective trend in the Lactobacillus casei strain GG group (p=0.07) [41]. In a subgroup of less than 200 patients visiting a particular area of Turkey, significant protection was observed, albeit only during the first week [41]. In contrast, at another location, a higher incidence of diarrhea was observed in the Lactobacillus casei strain GG group, making it difficult to draw firm conclusions [41]. The etiology of the diarrhea at the various destinations was not investigated [41]. Lactobacillus casei strain GG was shown to be effective in recovery from acute rotavirus diarrhea in children [42,43] and in non-bloody diarrhea of undetermined etiology [44], by decreasing the duration of diarrhea. There was no effect of Lactobacillus casei strain GG on the clinical outcome of premature infants who were enterally fed [45], although Lactobacillus casei strain GG did colonize the intestine [45,46]. The administration of Lactobacillus casei strain GG in oral rehydration solution tends to reduce the duration of diarrhea, although this effect seems to be limited to rotavirus-positive gastroenteritis [47], and could not be confirmed [48]. Lactobacillus casei strain GG may also decrease the duration of antibioticassociated diarrhea [49]. Lactobacillus casei strain GG has also been reported to be effective in Clostridium dffiicile colitis [50-52]. Finally the addition of Lactobacillus casei strain GG to a cows' milk elimination diet in children with atopic dermatitis and food allergy promotes endogenous barrier mechanisms by alleviating intestinal inflammation, and results in a significant additional decrease of the eczema [53]. Lactobacillus fermentum (KLD strain) Lactobacillus fermentum is a bacterium belonging to the natural colonic microflora, resistant to acid, and adhering to enterocytes [39]. However, no protection against travelers' diarrhea could be demonstrated [39]. Also, there was no effect on the hydrogen breath test, symptom score and number of defecations in patients with bacterial overgrowth [54]. Enterococcus faecium SF68 Several randomized double-blind studies have been carried out with Enterococcus faecium SF68 [55,56]. Enterococcus faecium SF68 was effective in the treatment of acute diarrhea of unspecified etiology [55]. A preventive effect of Enterococcus faecium on antibiotic-associated diarrhea was suggested, but the number of patients included was too small (45) to demonstrate a significant difference (p=0.11) [55]. In a large population of adult patients with proven 3/13

RANDOMIZED DOUBLE-BLIND STUDIES WITH PHARMACEUTICAL BACTERIAL BIOTHERAPEUTIC AGENTS (TABLE 1)

Lactobacillus acidophilus Different types of Lactobacillus acidophilus preparation have been tested in double-blind placebo-controlled studies. Although Lactobacillus rhamnosus is present in some products, it has not been clinically evaluated. A dried (freeze-dried?) combination of Lactobacillus acidophilus and Lactobacillus bulgaricus has been tested for the prevention of Escherichia coli diarrhea, of travelers' diarrhea, of amoxycillin-associated diarrhea and of diarrhea during enteral feeding [27,28,37]. The symptoms and duration of diarrhea with enterotoxic Escherichia coli remained unchanged, even if Lactobacillus acidophilus and

Table 1 Randomized double-blind studies with bacterial biotherapeutic agents Reference Study population 94 Indication Biotherapeutic agent Clinical outcome p-value NS

95

Acute diarrhea in infants

27 28 38 37 29 39 40 41 42 45 47

48 50 38 35 319 202 71 820 71 20 287 101 186 32 16 39 17 123 39 26 181 14 45 78 114 39 97 40 55 30 143 11

Diarrhea in ETEC-infected volunteers Travelers' diarrhea Amoxycillin-associated diarrhea Enteral feeding-associated diarrhea Travelers' diarrhea Travelers' diarrhea Acute diarrhea in children Travelers' diarrhea Acute diarrhea Enteral feeding in prematures Duration of diarrhea in children Duration of diarrhea if rotavirus positive Duration of diarrhea if rotavirus negative Non-bloody acute diarrhea Erythromycin-associated diarrhea Clostridium difficile colitis Atopic dermatitis with elimination diet Acute diarrhea Acute diarrhea in children Non-bloody diarrhea Travelers' diarrhea Bacterial overgrowth Antibiotic-associated diarrhea Acute diarrhea Duration of Vibria cholerae diarrhea Duration of Escherichia coli diarrhea Duration of rotavirus diarrhea Duration of rotavirus diarrhea Prevention of diarrhea Prevention of diarrhea Prevention of diarrhea Chronic abdominal pain

44 49 51 53 48 96 39 54 55 55 56 56 57 58 59 60 61 97

Streptococcus thermophilus Streptococcus lactis Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus bulgaricus Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus addophilus Lactobacillus bulgaricus Lactobacillus addophilus Lactobacillus acidophilus Lactobacillus acidophilus (heat Hied) Lactobacillus acidophilus Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus GG Lactobacillus fermentum Lactobacillus fermentum Enterococcus faecium SF 68 Enterococcus faecium SF 68 Enterococcus faecium SF 68 Enterococcus faecium SF 68 Lactobacillus reuteri Lactobacillus reuteri Streptococcus thermophilus Bifidobacterium bifidum Bifidobacterium bifidum Lactobacillus plantarum 299 V Lactobacillus plantarum 299 V

NS NS NS NS NS NS NS NS <0.001 NS NS <0.05 NS <0.01 <0.05 <0.001 <0.05 NS NS 0.055 NS NS NS < 0.01 NS NS NS NS 0.035 NS NS NS

ETEC, enterotoxic Escherichia coli; NS, not statistically significant. Vibrio cholerae or enterotoxic Escherichia coli diarrhea, no effect on the volume and duration of diarrhea was found [56]. A possible explanation for the absence of any effect may be that Enterococcus faecium SF68 is unable to inhibit the growth of both pathogens in the gastrointestinal tract [56]. Lactobacillus reuteri Lactobacillus reuteri in high dose (1010 or 1011 colonyforming units) or low dose (107 colony-forming units), or placebo (lactose), was administered to children with rotavirus diarrhea [57,58]. Although the number of children with watery stools on day 2 was significantly reduced in the Lactobacillus reuteri group, there was no effect on the duration of the diarrheal episode [58]. The main effect of Lactobacillus reuteri was on the duration of the diarrhea, with a correlation between the dosage and the clinical effect [57]. However, it should be questioned whether lactose should be given as placebo, because of the possible secondary deficiency in lactase activity [57]. Prepared milk food with Streptococcus thermophilus and/or Bifidobacterium bifidum, or Lactobacillus plantarium 299V A locally prepared infant formula with Streptococcus thermophilus and Bifidobacterium bifidum had a statistically significant effect on the prevention of diarrhea occurring in a ward with chronically sick children, 5-24 months old [59]. The incidence of rotavirus diarrhea 4/13

also tended to be reduced [59]. However, in a similarly designed study including only Bifidobacterium bifidum as biotherapeutic agent, there was no difference in the number of infants with diarrhea or in the number of diarrhea episodes [60]. In this study the duration of diarrhea was less in the group receiving the biotherapeutic agent (1.2 versus 2.3 days) [60]. Lactobacillus plantarum 299 V given daily in a fermented oatmeal powder failed to reduce the incidence of diarrhea in a day-care facility [61]. Bacillus cereus IP 5832 and Bacillus subtilis Although only a few case reports can be found in the literature between 1950 and today Bacillus cereus IP 5832 and Bacillus subtilis have been widely used in France and Belgium, without any proof of benefit [62]. A combination of four Bacillus subtilis strains, resistant to most antibiotics, has been developed, but has not been thoroughly evaluated [63].

Nosocomial epidemics with vancomycin-resistant Enterococcus faecium have been reported in oncologic patients [80,81], resulting in a mortality of 73% because of the absence of any therapeutic possibility [80]. Bacillus subtilis bacteremia occurred in four of 20 (oncologic) patients, but was also reported in other severely sick patients [62,82,83]. An unusual case of food poisoning with Staphylococcus aureus in combination with Bacillus cereus and Bacillus subtilis has been described. It has been speculated that very large numbers of bacteria may accelerate the manifestations of food poisoning [84]. As there is no evidence that Bacillus subtilis tablets are effective, their use must be restricted [62]. Although the incidence of infections remains extremely low, the idea arises that lactic acid bacteria may become pathogenic under certain circumstances [76]. Resistance or plasmid transfer If a bacterial biotherapeutic agent is used simultaneously with an antibiotic, the biotherapeutic agent must be resistant to the antibiotic. However, transfer of genetic material from biotherapeutic agents to the commensal or pathogenic flora would have catastrophic consequences [9]. Gene transfer has been reported between enterococci in the gastrointestinal tract of experimental rats [85]. Plasmid transfer from Lactobacillus reuteri to Enterococcus faecium and from Enterococcus faecium to Enterococcus faecalis has been reported [9]. Many Enterococcus faecium strains have plasmids which are resistant to multiple antibiotics, including vancomycin [9]. In 1994, a biotherapeutic agent containing Enterococcus faecium SF 68 was taken off the Belgian market, because of reported nosocomial vancomycin-resistant infections. The induction of lactamase production in anaerobic bacteria was reported as a complication of the prevention of diarrhea associated with the administration of ceftriaxone (in other words, the resistance to the antibiotic was taken over by the anaerobic bacteria) [86]. Resistance to cephalosporins and macrolides was induced in a combination of four strains of Bacillus subtilis bacteria [63]. Azide-resistant Bacillus subtilis mutants have been reported [86]. Older publications report multiresistant Bacillus cereus [87] and Enterococcus faecium [55]. At least one plasmid can be isolated from 80% of the Bacillus cereus strains, and can be transferred to Bacillus subtilis, where it is retained [88]. A transfer of macrolide-lincosamide-streptogramin B resistance between Clostridium difficile and Bacillus subtilis, and vice versa, has been described without plasmid DNA being found [89]. An account of an epidemic of multiresistant Enterococcus faecium with transferable vancomycin-resistance was recently published [81]. The increase in vancomycinresistant enterococci may be associated with the possible use of bacterial preparations which are administered orally to animals and humans. The association of enterococci (even if they are not intrinsically vancomycin resistant) with lactobacilli (which are intrinsically vancomycinresistant) makes it difficult to detect vancomycin-resistant enterococci in biotherapeutic agents [89]. The vancomycin resistance of lactobacilli is chromosomal and not plasmid mediated, and, therefore, the risk of transmission to other organisms is small [10]. Enterococci are commensal organisms with a potential virulence [90]. The safety and the long-term effects of bacterial biotherapeutic agents on antibiotic resistance must be thoroughly investigated before large-scale use can be 5/13

SIDE EFFECTS OF PHARMACEUTICAL BACTERIAL BIOTHERAPEUTIC AGENTS

Malabsorption and metabolic acidosis The addition of viable Lactobacillus acidophilus to milk does not improve the digestion of lactose: milk to which Lactobacillus acidophilus is added causes the same degree of malabsorption in lactase-deficient consumers as ordinary milk [64,65]. After ingestion of lactobacilli in lactasedeficient patients, large numbers of the microorganisms are discovered in the colonic flora, but without any effect on lactose malabsorption [65,66]. Some lactobacillus preparations contain small amounts of lactose; administration of these products is not indicated in lactose intolerance. It seems undesirable to administer small amounts of extra lactose in the treatment of acute infectious gastroenteritis, a disease possibly compromising the lactase activity. Disturbances in the subtle balance between Gram-positive and Gram-negative bacteria in the natural colonic flora may be induced by the administration of Gram-positive bacteria such as lactobacilli and bifidobacteria, which grow more rapidly than Gram-negative bacteria. This disequilibrium may result in metabolic D-lactate acidosis, as a consequence of the bacterial carbohydrate metabolism [67]. Gram-positive overgrowth may occur in many situations, including anatomic or functional short bowel, antibiotic treatment, exclusive feeding with dairy products, excessive presence of carbohydrates in the diet, and/or the use of oral Lactobacillus acidophilus tablets [67-71]. Bacteremia Endocarditis, meningitis, pneumonia and sepsis have been reported with lactobacilli [33,72,73]. Also, indigenous bacteria, not those introduced by a biotherapeutic agent, have been identified as causative factors in endocarditis [74,75]. In case reports, lactic acid bacteria are also mentioned as causing local infections such as chest infections, digestive tract infections, urinary tract infections and meningitis [76]. In Finland, among 3317 blood culture isolates, lactobacilli were identified in eight patients [77]. None of them was caused by Lactobacillus casei strain GG [77,78]. Although the pathogenic potential of bacterial biotherapeutic agents is low, it does exist [77]. Enterococci are a known cause of sepsis in premature infants [79].

recommended [9]. Because of these side effects, the benefit/risk ratio of pharmaceutical bacterial biotherapeutic agents in the prevention of antibiotic-associated diarrhea should be carefully considered. Induced arthritis and immunodepressive effect of the peptidoglycan present in the cell membrane of Gram-positive bacteria Cell membranes of streptococci and lactobacilli may cause chronic polyarthritis in men [91]. A Gram-positive intestinal flora, including bifidobacteria, Lactobacillus casei strain GG and Lactobacillus acidophilus, aggravates the consequences of such an induced arthritis in rats [92]. This is presumably due to peptidoglycan, an essential component of the Gram-positive bacterial cell wall that may cause an immunopathologic process [91,92]. Peptidoglycan contains D-glutamine and D-alanine, amino acids which do not exist as such in nature. Mammals do not have proteases which can break down polypeptides containing D-amino acids. This can cause an irreversible Bcell inactivation because of saturation of the membrane receptors. Reactive arthritis (Reiter's syndrome) has been described in Shigella, Salmonella and Campylobacter infections via an indirect mechanism [93]. Lipopolysaccharides of Salmonella spp. were detected in the joints of patients with reactive arthritis after a Salmonella infection [94]. This finding may possibly limit the use of Gram-positive biotherapeutic agents in the treatment of diarrhea caused by bacteria which increase the permeability of the intestinal wall, such as salmonellae. Administration of heat-killed Lactobacillus acidophilus is contraindicated in salmonellosis.

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CONCLUSIONS
Biotherapeutic agents offer a large number of therapeutic benefits in the management of acute and chronic infectious diarrhea. A critical evaluation of their efficacy is a challenge, because many different micro-organisms have been used in different presentations and in different combinations. Most studies can be criticized (open, controlled without a placebo, too few patients, etc.). Although some in vitro studies provide very interesting results, the clinical utility of many bacterial biotherapeutic agents in vivo could frequently not be demonstrated. Some studies using Lactobacillus casei strain GG have provided promising data, although other studies provide contradictory results. All bacterial biotherapeutic agents still need to have their efficacy demonstrated in welldesigned randomized double-blind studies before they can be recommended on a large scale. If bacterial biotherapeutic agents are to be used on a large scale in the future, the eventual risk of transfer of their intrinsic resistance to most antibiotics should be thoroughly investigated.

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23. Hudault S, Livin V, Bernet-Camard MF, Servin AL. Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection. Appl Environ Microbiol 1997; 63: 513-18. 24. Michelich VJ, Nunez-Montiel O, Schuster GS, Thompson F, Dowell VR. Diet as a coadjuvant for development of antibiotic-associated diarrhea in hamsters. Lab Anim Sci 1981; 31: 259-62. 25. Bernet MF, Brassart D, Neeser JR, Servin AL. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 1994; 35: 483-9. 26. Chauvire G, Coconnier MH, Kermes S, Darfeuille-Michaud A, Joly B, Servin AL. Competitive exclusion of diarrheagenic Escherichia coli ETEC from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus. FEMS Microbiol Lett 1992; 91: 213-18. 27. Coconnier MH, Franoise B, Chauvire G, Servin AL. Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells. J Diarrhoeal Dis Res 1993; 11: 235-42. 28. Clements ML, Levine MM, Black RE, et al. Lactobacillus prophylaxis for diarrhea due to enterotoxigenic Escherichia coli. Antimicrob Agents Chemother 1981; 20: 104-8. 29. DeDios-Pozo-Olano JD, Warram JH, Gomez RG, Cavazos MG. Effect of a lactobacilli preparation on traveller's diarrhea: a randomized double-blind clinical trial. Gastroenterology 1978; 74: 829-30. 30. Kollaritsch HH, Kremsner P, Wiedermann G, Schemer O. Prevention of traveller's diarrhoea. Comparison of different non-antibiotic preparations. Travel Med Int 1989; 11: 9-17. 31. Peetermans W Reizigersdiarree. Tijdschr Geneesk 1991; 47: 895-903. 32. Kabier G, Aiba Y, Takagi A, Kamiya S, Miwa T, Koga Y Prevention of Helicobacter pylori infection by lactobacilli in a gnotobiotic murine model. Gut 1997; 41: 49-55 33. Kaila M, Isolauri E, Saxelin M, Arvilommi H, Vesikari T Viable versus inactivated Lactobacillus strain GG in acute rotavirus diarrhoea. Arch Dis Child 1995; 72: 51-3. 34. Majamaa H, Isolauri E, Saxelin M, Vesikari T. Lactic acid bacteria in the treatment of acute rotavirus gastroenteritis. J Pediatr Gastroenterol Nutt 1995; 20: 333-8. 35. Isolauri E, Joensuu J, Suomalainen H, Luomala M, Vesikari T. Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine 1995; 13: 310-12. 36. Malin M, Suomalainen H, Saxelin M, Isolauri E. Promotion of IgA immune response in patients with Crohn's disease by oral bacteriotherapy with Lactobacillus GG. Ann Nutr Metab 1996; 40: 137-45 37. Alak JI, Wolf BW, Mdurvwa EG, Pimentel-Smith GE, Adeyemo O. Effect of Lactobacillus reuteri on intestinal resistance to Cryptosporidium parvum infection in a murine model of acquired immunodeficiency syndrome. J Infect Dis 1997; 175: 218-21. 38. Heimburger DC, Sockwell DG, Geels WJ. Diarrhea with enteral feeding: prospective reappraisal of putative causes. Nutrition 1994; 10: 392-6. 39. Tankanow RM, Ross MB, Ertel IJ, Dickinson DG, McCormick LS, Garfinkel JF. A double-blind, placebocontrolled study on the efficacy of Lactinex in the prophylaxis of amoxillin-induced diarrhea. DICP 1990; 24: 382-4. 40. Katelaris PH, Salam I, Farthing MJG. Lactobacilli to prevent traveller's diarrhea? N Engl J Med 1995; 333: 1360-1. 41. Boulloche J, Mouterde O, Mallet E. Traitement des diarrhes aigus chez le nourisson et le jeune enfant. Ann Pdiatr 1994; 41: 457-63. 42. Oksanen PJ, Salminen S, Saxelin M, et al. Prevention of traveler's diarrhoea by Lactobacillus GG. Ann Med 1990; 22: 53-6.

43. Isolauri E, Juntunen M, Rautanen T, Sillanaukee P, Koivula T A human Lactobacillus strain (Lactobacillus casei sp. strain GG) promotes recovery from acute diarrhea in children. Pediatrics 1991; 88: 90-7. 44. Kaila M, Isolauri E, Soppi E, Virtanen E, Laine S, Arvilommi H. Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr Res 1992; 32: 141-4. 45. Raza S, Graham SM, Allen SJ, Sultana S, Cuevas L, Hart CA. Lactobacillus GG promotes recovery from acute nonbloody diarrhea in Pakistan. Pediatr Infect Dis 1995; 14: 107-11 46. Millar MR, Bacon C, Smith SL, Walker V, Hall MA. Enteral feeding of premature infants with Lactobacillus GG. Arch Dis Child 1993; 69: 483-7. 47. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S. Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig Dis Sci 1992; 37: 121-8. 48. The ESPGHAN Working Group on Acute Diarrhoea. Lactobacillus GG administered in oral rehydration solution to children with acute diarrhea: a multicenter European trial. J Pediatr Gastroenterol Nutr 1998; 26: 547(A). 49. Rautanen T, Isolauri E, Salo E, Vesikari T. Management of acute diarrhoea with low osmolarity oral rehydration solutions and Lactobacillus strain GG. Arch Dis Child 1998; 79: 157-60. 50. Siitonen S, Vapaatalo H, Salminen S, et al. Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated diarrhoea. Ann Med 1990; 22: 57-9. 51. Loret de Mola O, Gonzalez-Valina R, Munos W, ReevesGarcia J, Acosta J. Recovery of chronic Clostridium difficile colitis with the use of Lactobacillus GG. J Pediatr Gastroenterol Nutr 1998; 26: 542(A). 52. Gorbach SL, Chang TW, Goldin B. Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG. Lancet 1987; ii: 1519. 53. Biller JA, Katz AJ, Flores AF, Buie TM, Gorbach SL. Treatment of recurrent Clostridium difficile colitis with Lactobacillus GG. J Pediatr Gastroenterol Nutr 1995; 21: 224-6 54. Majamaa H, Isolauri E. Probiotics: a novel approach in the management of food allergy. J Allergy Clin Immunol 1997; 99: 179-85. 55. Stotzer PO, Blomberg L, Conway PL, Henriksson A, Abrahamsson H. Probiotic treatment of small intestinal bacterial overgrowth by Lactobacillus fermentum KLD. Scand J Infect Dis 1996; 28: 615-19. 56. Wunderlich PF, Braun L, Fumagalli I, et al. Double blind report on the efficacy of lactic acid-producing Enterococcus SF68 in the prevention of antibiotic-associated diarrhoea and in the treatment of acute diarrhoea. J Int Med Res 1989; 17: 333-8. 57. Mitra AK, Rabbani GH. A double-blind, controlled trial of Bioflorin (Streptococcus faecium SF68) in adults with acute diarrhea due to vibrio cholerae and enterotoxigenic Escherichia coli. Gastroenterology 1990; 99: 1149-52. 58. Shornikova AV, Casas AI, Mykknen H, Salo A, Vesikari T. Bacteriotherapy with Lactobacillus reuteri in rotavirus gastro-enteritis. Pediatr Infect Dis J 1997; 16: 1103-7. 59. Shornikova AV, Casas IA, Isolauri E, Mykknen H, Vesikari T. Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. J Pediatr Gastroenterol Nutr 1997; 24: 399-404. 60. Saavedra JM, Bauman NA, Oung I, Perman JA, Yolken RH. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and shedding of rotavirus. Lancet 1994; 344: 1046-9. 61. Chouraqui JP, Van Egroo LD, Fichot MC. Prevention of diarrhea by feeding infants with an acidified milk formula containing Bifidobacterium bifidum. J Pediatr Gastroenterol Nutr 1998; 26: 539(A). 62. Ribeim H, Vanderhoof JA. Reduction of diarrheal illness following administration of Lactobacillus plantarium 299V

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81. Edmond MB, Ober JF, Weibaum DL, et al. Vancomycinresistant Enterococcus faecium bacteremia: risk factors for infection. Clin Infect Dis 1995; 20: 1126-31. 82. Boyce JM, Opal SM, Chow JW, et al. Outbreak of multidrugresistant Enterococcus faecium with transferable B class vancomycin resistance. J Clin Microbiol 1994; 32: 1148-53. 83. Lanco S, Guillot M, Eckart P, et al. Infections systmiques svres Bacille cereus: aspects actuels de la pathognicit des bactries du genre Bacillus. Arch Pediatr 1997: 4: 114455. 84. Oggioni MR, Pozzi G, Valensin PE, Galieni P, Bigazzi C. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol 1998; 36: 325-6. 85. Fenton PA, Dobson KW, Eyre A, McKendrick MW. Unusually severe food poisoning from vanilla slices. J Hyg Lond 1984; 93: 377-80. 86. Lonard F, Andremont A, Leclerq B, Labia R, Tancrde C. Use of -lactamase-producing anaerobes to prevent ceftriaxone from degrading intestinal resistance to colonization. J Infect Dis 1989; 160: 274-80. 87. Klein M, Hofmann B, Klose M, Freudl R. Isolation and characterization of a Bacillus subtilis secA mutant allele conferring resistance to sodium azide. FEMS Microbiol Lett 1994; 124: 393-4. 88. Ganczarski A, Srocziski K, Brozik H, et al. Modifications de la flore intestinale par le Bacillus subtilis (Sp. 5832). Gaz Med France 1960; 67: 2115-25. 89. Bernhard K, Schrempf H, Goebel W. Bacteriocin and antibiotic xesistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol 1978; 133: 897-903. 90. Murray BE. What can we do about vancomycin-resistant Enterococci? Clin Infect Dis 1995; 20: 1134-6. 91. Jett BD, Huycke MM, Gilmore MS. Virulence of enterococci. Clin Microbiol Rev 1994; 7: 462-8. 92. Mills JA. Do bacteria cause chronic polyarthritis? N Engl J Med 1989; 320: 245-56. 93. Kohashi O, Kohashi Y, Takashi T, Ozawa A, Shigematsu N. Reverse effect of Gram-positive bacteria vs. Gram-negative bacteria on adjuvant-induced arthritis in germfree rats. Microbiol Immunol 1985; 29: 487-97. 94. Hagiage M. La flore intestinale. De lquilibre au dsquilibre. Paris: Vigot, 1994. 95. Granfors K, Jalkanen S, Lindberg AA., et al. Salmonella lipo-polysaccharide in synovial cells from patients with reactive arthritis. Lancet 1990; 335: 685-8. 96. Pearce JL, Hamilton JR. Controlled trial of orally administered lactobacilli in acute infantile diarrhea. J Pediatr 1974; 84: 261-2. 97. Pant AR, Graham SM, Allen SJ, et al. Lactobacillus GG and acute diarrhoea in young children in the tropics. J Trop Pediatr 1996; 42: 162-5. 98. Young RJ, Vanderhoof JA. Successful probiotic therapy of chronic recurrent abdominal pain in children. Gastroenterology 1997; 112: A856.

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REVIEW

Bacteria and yeasts in the treatment of acute and chronic infectious diarrhea. Part II: Yeasts
Clin Microbiol Infect 1999; 5: 389-395

Yvan Vandenplas
Academic Children's Hospital, Free University of Brussels, Brussels, Belgium Key words: Biotherapeutic agent, diarrhea, gastroenteritis, probiotic, Saccharomyces boulardii yeast

INTRODUCTION
In the first of these two linked articles [1], the clinical importance of diarrheal diseases, the accompanying changes in gut flora and the mainstays of treatment were briefly described. It was demonstrated that, although the idea of using bacteria as biotherapeutic agents is theoretically interesting, the results of in vivo randomized, placebo-controlled, clinical trials have been disappointing, with the exception of Laclobacillus GG. In this second part, the use of yeasts will be discussed. The only yeast with which double-blind studies have been carried out is Saccharomyces boulardii.

administration in rats of Saccharomyces boulardii with clindamycin and ampicillin, which are active against anaerobic bacteria, increases the number of yeast cells recovered in the feces [8]. This was confirmed with ampicillin in human volunteers [5]. However, nystatin completely eliminates Saccharomyces boulardii from the gastrointestinal tract, although Saccharomyces boulardii can be administered 4-6 h after fluconazole without any effect on its viability in the intestine [9].

PHARMACODYNAMICS OF SACCHAROMYCES BOULARDII

The pharmacodynamics of Saccharomyces boulardii involve three different aspects: a direct antagonistic effect, an antisecretory effect, by acting specifically on the binding of toxins to intestinal receptors, and a trophic effect, by stimulating enzymatic activities and intestinal defense mechanisms. Oral Saccharomyces boulardii induces several cellular and humoral immunologic changes in the peripheral blood of healthy volunteers [10]. Saccharomyces boulardii activates C2, C3, C3a and C5a [11]. Antagonistic effect In vitro, Saccharomyces boulardii reduces the growth of Candida albicans, Escherichia coli, Shigella species, Salmonella typhi and other microorganisms [12]. Saccharomyces boulardii has been shown to inhibit the growth of Pseudomonas aeruginosa and Staphylococcus aureus in an enteral nutrient liquid into which these pathogens had been inoculated [13]. In vitro, Saccharomyces boulardii significantly reduces the number of red blood cells which adhere to Entamoeba histolytica, and the number of Entamoeba histolytica organisms in which red blood cells are found [14]. A significant reduction in the number and severity of signs of disease was observed in rats inoculated with Entamoeba histolytica if Saccharomyces boulardii was administered [15]. Substances produced by Saccharomyces boulardii compete with the receptors of the target cells for Entamoeba histolytica [14]. In vivo, Saccharomyces boulardii reduces the Candida albicans population in mice 10-50-fold. This antagonistic effect is curative and preventive, and is reported for Candida krusei and Candida pseudotropicalis, but not for Candida tropicalis [15]. In immunodepressed mice (antibiotic decontamination and prednisolone 9/13

SACCHAROMYCES BOULARDII
Saccharomyces boulardii is a non-pathogenic yeast which was isolated from lychees in Indochina, and which grows at the unusually high temperature of 37C [2]. It is commercially available as a freeze-dried viable preparation, and is the only yeast with which double-blind studies have been carried out. Saccharomyces boulardii differs from baker's yeast Saccharomyces cerevisiae in a number of taxonomic, metabolic and molecular parameters [2].

PHARMACOKINETICS OF SACCHAROMYCES BOULARDII

Saccharomyces boulardii survives gastric acid and bile and can be detected alive throughout the entire digestive system if it is given daily in its freeze-dried form [3-6]. Two to five days after administration, the yeast becomes undetectable in the feces [3,7]. Saccharomyces boulardii is intrinsically resistant to antibacterial antibiotics. Simultaneous

Corresponding author and reprint requests: Yvan Vandenplas, Academic Children's Hospital, Free University of Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium Fax: +32 2 477 57 83 E-mail: pedvsy@az.vub.ac.be Accepted 5 December 1998

injections), the administration of Saccharomyces boulardii significantly reduces the incidence of translocation of Candida albicans to the mesenteric lymph glands, liver and kidneys [16]. The addition of Saccharomyces boulardii to tetracycline treatment in severe salmonella enteritis reduces the mortality rate significantly [17]. Similarly, Saccharomyces boulardii was shown to increase the survival ratio in Shigella flexneri infection and Staphylococcus aureus infection (significantly) and in Salmonella typhimurium infection (trend) [18,19]. However, the protective effect of Saccharomyces boulardii in the reduction of the mortality and its effect at histologic level are not due to a reduction of the bacterial population in the intestine; the precise pathophysiologic mechanism is unknown [20] . Antisecretory effect by acting specifically on the binding of bacterial toxins Two different mechanisms for activity against bacterial toxins have been discovered. Saccharomyces boulardii produces two proteins, one of 54 kDa and another of 120 kDa. The 120-kDa protein has no proteolytic activity, and specifically competes with the hypersecretion induced by Vibrio cholerae and enterotoxic Escherichia coli by reducing the formation of cyclic AMP in the intestinal cells [21]. Intestinal lesions caused by Vibrio cholerae remain absent or are less severe in rats given Saccharomyces boulardii [22]. In addition, Saccharomyces boulardii was shown to reduce significantly sodium and water secretion in intestinal loops of rats which had been inoculated with V. cholerae toxin [23]. A similar reduction in hypersecretion was found in mice inoculated with enterotoxic and enteropathogenic Escherichia coli if Saccharomyces boulardii was added [24-26]. The second protein produced is a 54-kDa protease which acts on toxin A of Clostridium difficile [26]. Saccharomyces boulardii significantly reduces the liquid secretion and permeability caused by toxin A in rat ileum [26,27]. In vitro, it has been demonstrated that the protease of 54 kDa digests both toxin A and the receptor on the enterocyte [28]. In vivo, Saccharomyces boulardii decreases the mortality rate, the quantity of toxin A and the intestinal lesions caused by Clostridium difficile [29,30].

Trophic effect on the enterocyte with stimulation of enzymatic expression and intestinal defense mechanisms Polyamines, such as spermine and spermidine, cause significant increases in length and weight of the intestine [31], accelerated maturation of the microvillous enzymes (lactase, sucrase, maltase and aminopeptidase) and increases in the immunoglobulin A secretion in villi and crypt cells in young test animals [32-36]. Polyamines increase the number of glucose carriers in the membrane of the enterocyte, and are essential to achieve maximal glucose absorption in the enterocyte [37]. Saccharomyces boulardii contains polyamines (spermine, spermidine) and has similar trophic effect on the intestinal mucosa, with an increase in disaccharidase activity, as an equivalent amount of spermine and spermidine given to test animals [38]. Whether there is a stimulating effect on intestinal adaptation in short bowel syndrome is not clear, since contradictory results have been reported [39,40].

RANDOMIZED DOUBLE-BLIND STUDIES WITH SACCHAROMYCES BOULARDII (TABLE 1)

Prevention of antibiotic-associated diarrhea The efficacy of Saccharomyces boulardii in the prevention of antibiotic-associated diarrhea has been demonstrated in several clinical trials [41-43]. The preventive effect of a low dosage was demonstrated in patients treated with tetracycline or -lactam antibiotics because of an upper respiratory tract infection [41]. Significant efficacy was demonstrated in the prevention of diarrhea in a hospital with a high incidence of antibiotic-associated diarrhea and Clostridium difficile [42,43]. Saccharomyces boulardii shortens the duration of antibiotic-associated diarrhea, with no relapse during a follow-up period of 6 weeks. In the Saccharomyces boulardii group there was significantly less intestinal gas production and fever than in the control group. Prevention of diarrhea during enteral feeding Saccharomyces boulardii significantly reduced the number of days with diarrhea in patients hospitalized in an intensive care unit and in patients with severe burns [44-46]. A higher caloric intake by enteral feeding was tolerated in patients treated with Saccharomyces boulardii, without symptoms of intolerance (diarrhea, vomiting, nausea).

Table 1 Randomized placebo-controlled double-blind studies with Saccharomyces boulardii References Study Indication population Prevention of antibiotic-associated diarrhea 193 7 Prevention of antibiotic-associated diarrhea 388 41 Prevention of antibiotic-associated diarrhea 180 42 Prevention of enteral feeding-associated diarrhea 40 44 Prevention of enteral feeding-associated diarrhea 128 46 Prevention of enteral feeding-associated diarrhea in 20 45 patients with severe burns Prevention of travelers' diarrhea 1231 47 Recurrence of Clostridium difficile colitis 124 43 Treatment of acute diarrhea in adults 92 53 Treatment of acute diarrhea in children 130 54 Treatment of chronic diarrhea in children 40 57 (35/40: Giardia lamblia) Treatment of chronic diarrhea in AIDS patients 35 58

Clinical outcome (p value) < 0.05 < 0.001 < 0.05 < 0.001 0.002 < 0.001 < 0.002 0.05 < 0.05 < 0.01 < 0.001 < 0.002 10/13

Prevention of travelers' diarrhea The effect of Saccharomyces boulardii in the prevention of travelers' diarrhea was demonstrated in 1231 people traveling to several destinations all over the world [47]. Participants were divided into three groups: a placebo group, a group receiving 250 mg/day Saccharomyces boulardii, and another group receiving 500 mg/day. The incidence of travelers' diarrhea was 42.6%, 33.6%, and 31.8%, respectively (p<0.002). Regional differences in the prevention of travelers' diarrhea were observed. The preventive effect was highest in North and West Africa (58% and 59% reduction; p<0.01), and in various tropical islands (40% reduction; p<0.05) [47]. Treatment of (recurrent) chronic Clostridium difficile diarrhea Saccharomyces boulard has a protective effect against toxin A, an enterotoxin, and toxin B, a cytotoxin, of Clostridium difficile [48]. The addition of Saccharomyces boulardii for 1 month to standard antibiotic therapy (vancomycin and/or metronidazole) for Clostridium difficile infection significantly reduced the number of relapses [43]. The death rate in hamsters infected with Clostridium difficile treated with clindamycin was significantly reduced if Saccharomyces boulardii was added to the treatment [49]. The effect was greatest in patients who relapsed at least once. In the end, the number of patients still positive for Clostridium difficile or its toxin was significantly reduced in the Saccharomyces boulardii group (8.6% versus 26.8%) [43]. This randomized double-blind study confirmed the earlier results obtained with Saccharomyces boulardii in two open studies in 14 patients with multiple Clostridium difficile relapses [50,51]. The effect of Saccharomyces boulardii was investigated in an open study in 19 children, with a mean age, of 8 months, who had at least 2 weeks of chronic diarrhea without symptoms of colitis and in whom Clostridium difficile with toxin B was detected as the single pathogen [52]. After 1 week of treatment, the symptoms (defecation frequency, duration and frequency of colic) had disappeared in 95% of the patients. In 85%, toxin B had disappeared after 2 weeks, and after 1 month Clostridium difficile was no longer detectable in the feces in 73% of the patients [52]. Treatment of acute diarrhea in children and adults The efficacy of Saccharomyces boulardii was investigated in a randomized double-blind study in 92 adults with acute diarrhea. After 48 h, a significant reduction of the diarrhea score (defecation frequency and consistency) was noticed [53]. The effect of Saccharomyces boulardii was demonstrated in a double-blind placebo-controlled study in 130 children, 3 months to 3 years old, with acute diarrhea [54]. After 48 and 96 h, significantly more children were clinically cured (less than four normal stools a day). The return to a normal stool consistency was significantly enhanced. Milk-based feeding could be continued in the Saccharomyces boulardii group without any ride effects [54]. Two controlled studies evaluating the efficacy of Saccharomyces boulardii in the treatment of diarrhea in young children confirm these data: reduction in defecation frequency and improved consistency of feces, better weight gain, and rapid normalization of the gastrointestinal transit tune [55,56].

Treatment of chronic diarrhea (giardiasis, shigellosis) The effect of Saccharomyces boulardii was investigated in 40 children with chronic diarrhea caused by Giardia lamblia (35/40) or, Shigella species (4/40), or of unknown etiology (1/40) [57]. Patients with G. lamblia were treated for 5 days with tinidazole, and those with Shigella spp. were given trimethroprim-sulfamethoxazole; Saccharomyces boulardii or placebo was administered in addition to the antibiotic. After 1 month, the number of stools per day was significantly (p=0.006) reduced in the Saccharomyces boulardii group. In addition, endoscopic intestinal lesions were reduced significantly (p=0.009). The overall histologic and clinical evaluation improved in 70% of the Saccharomyces boulardii treated group, compared to 10% in the placebo group (p=0.0007) [57]. Treatment of AIDS diarrhea In a randomized double-blind study in 35 AIDS patients with chronic diarrhea, 61% were found to be diarrhea-free after 1 week of treatment with Saccharomyces boulardii, compared to 12% in the placebo group (p<0.002) [58]. After one week, weight in the Saccharomyces boulardii group had increased by 2 kg, whereas the placebo group had lost a mean of 3.1 kg [58]. These results confirm an earlier open study in 17 AIDS patients with chronic diarrhea for more than 1 month. Saccharomyces boulardii normalized defecation frequency after 15 days (from nine stools per day to 2.1 per day), normalised stool consistency and improved weight gain (mean of 4 kg in 2 weeks) [62]. Pilot studies and open studies Open studies with significant results favoring Saccharomyces boulardii have been carried out in persistent travelers' diarrhea, with ineffective prior treatment in two-thirds of the patients [60], in children with short bowel syndrome with bacterial overgrowth [59], and in patients with Crohn's disease [61]. A positive effect was demonstrated in all.

SIDE EFFECTS OF SACCHAROMYCES BOULARDII

The commercial preparation of Saccharomyces boulardii contains minute, and thus clinically irrelevant, amounts of lactose, which will, in any case, be digested by the disaccharidase-stimulating properties of Saccharomyces. The safety of Saccharomyces boulardii has been investigated in animal models and in randomized doubleblind studies. In mice given 5% Saccharomyces boulardii for 70 days in their drinking water, no translocation from the gastrointestinal tract was observed. Saccharomyces boulardii could not be detected in the organs (liver, kidneys, lungs and heart) or in the mesenteric lymph glands [2]. In an immunodepressed animal model (prednisolone and antibiotic decontamination), Saccharomyces boulardii could only be detected at very low concentrations in the mesenteric lymph glands; Saccharomyces boulardii could not be detected in the liver, spleen or kidneys [16]. Saccharomyces boulardii was given to more than 40 AIDS patients, without any serious side effects being reported [58,62]. Over a period of 10 years, during which millions of Saccharomyces boulardii treatments have been prescribed, a few cases with Saccharomyces boulardii fungemia have 11/13

been reported [63-70]. All the patients thus far reported with Saccharomyces boulardii fungemia have had intravenous lines, with a majority of deep venous central catheters. In most patients a high dosage of Saccharomyces boulardii was given. All patients, except one, recovered after antimycotic treatment without any further problems. Careful deployment of Saccharomyces boulardii in patients with central venous lines is recommended. In one study, constipation and increased thirst was reported to occur [43]. Other side effects have not been reported [71].

CONCLUSIONS
Biotherapeutic agents offer a large number of therapeutic benefits in the prevention or treatment of several gastrointestinal disorders, including the management of acute and chronic infectious diarrhea, antibiotic-associated diarrhea and travelers' diarrhea [71]. The critical evaluation of their efficacy is a challenge because many different microorganisms have been used in different preparations and in different combinations. Although some in vitro studies provide very interesting results, the clinical utility of many bacterial biotherapeutic agents in vivo could frequently not be demonstrated. Some studies using Lactobacillus casei strain GG have produced promising data, although other studies provide contradictory results. Saccharomyces boulardii is a non-bacterial biotherapeutic agent, and is the only biotherapeutic agent with systematic convincing data from double-blind studies. Results show significant efficacy in the prevention and treatment of acute diarrhea. The WHO considers Saccharomyces boulardii to be a possible treatment for recurrent Clostridium difficile colitis [4].

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