MXPA97007631A - Inhibition of infections by c. difficilemediante oligosacaridos indigestib - Google Patents

Abstract

The present invention relates to the primary etiology of diarrhea associated with antibiotic (also known as pseudomembranous colitis) has been recognized as Costridium difficile. It is believed that the inherent microflora of a healthy individual suppresses C. difficile normally present. However, when the inherent microflora is destroyed (for example, during an antibiotic treatment) the over-growth of C-difficile can occur, causing diarrhea and colitis. The treatment of C. difficile with antibiotics has proven effective, but there are often times of relapse, and dehydration due to diarrhea is an exacerbated problem. It has been suggested that normalization of the microflora will inhibit relapse by C. difficile. Indigestible oligosaccharides have been shown to inhibit C. difficile infection. An oral rehydration solution containing said indigestible oligosaccharides also provides fluid and electrolyte replacement

Description

INHIBITION OF INFECTIONS BY C. DIFFICILE VIA INDIGESTIBLE OLIGOSACARIDOS BACKGROUND OF THE INVENTION This invention relates to a method for using indigestible oligosaccharides for the inhibition of disease states associated with Clostridium difficile, and to oral rehydration solution compositions containing indigestible oligosaccharides. Successful management of acute diarrhea remains a challenge at present. Acute diarrhea leads to the reduction of both fluid and electrolyte. For years, methods for the prevention and treatment of dehydration associated with acute diarrhea have been developed empirically. Prompt administration of an oral solution can prevent the development of dehydration and electrolyte imbalances. Only recently have physicians gained knowledge of the etiology and pathophysiology of diarrhea that allows a more rational basis for treatment. Usually, Clostridium difficile (C. difficile) is involved in diarrhea. The risk of diarrhea increases in overcrowded housing or work conditions, especially when the likelihood of oral transmission of enteric pathogens by fecal waste is high. C. difficile is the main known cause of nosocomal infections. Greenough and others, "Diarrhea in the elderly", PRI NCIPLES OF GERIATRIC MEDICI N AND AND GERONTOLOGY. Hazzard, et al. (Ed) New York, McGraw-Hill, p. 1 168-1 176 (1990); McFarland et al., "A Randomized Placebo-Controlled Trial of Saccharomyces boulardii in Combination with Standard Antibiotics for Clostridium difficile DiseaseP JOURNAL OF TH E AMERICAN MEDICAL ASSOCIATION 271 (24): 1913-1918 (1994) .The greatest risk of obtaining a C. difficile infection usually involves a surgical procedure (usually of a gastrointestinal nature) combined with antibiotic therapy.The diets consumed by such patients are usually not conducive to the establishment or maintenance of normal intestinal bacteria. Difficile can be endemic in long-term care facilities for the elderly, where outbreaks of diarrhea are common Bender and others "Is Clostridium difficile endemic in chronic-care facilities?", TH E LANCET 2: 1 1-13 (1986) There are also economic consequences for infection through this pathogen, since patients who acquire disease is caused by C. difficile require long stays in the hospital, resulting in additional costs for care. Kofski et al., "Clostridium difficile- a common and costly colitis", DISEASES OF TH E COLON AN D RECTUM. 34: 244-248 (1991) noted that C. difficile infections result in an additional $ 2,000 to $ 5,000 per episode at the cost of health care. In addition, in 1991, the cost of inpatient medical care with primary diagnosis of dehydration was $ 446 million. Warren et al., "The Burden and Outcome Associated with Dehydration among U.S. Elderly, 199V, AMERICAN JOURNAL OF PU BLIC H EALTH 84 (8): 1265-1269 (1994) .In addition, patients who have been infected during The hospital stay has shown that they spread the infection to extended care facilities and to their environment, Bender et al., "Is Clostridium difficile endemic in chronic-care facilities?", TH E LANCET 2: 1 1-13 (1986) Bennet et al, "C. difficile diarrhea: A common-and overlooked-nursing home infection GER IATRICS. 45: 77-87 (1990); Kofsky et al., "Clostridium difficile - a common and costly colitis", DISEASES OF TH E COLON AN D RECTUM. 34: 244-248 (1991); Mulligan et al., "Contamination of a Hospital Environment by Clostridium difficile", CURRENT MICROBIOLOGY. 3: 173-175 (1979). Thomas et al., Postantibiotic colonization with Clostridium difficile in nursing home patientsp JOU RNAL OF THE AMERICAN GERIATRICS SOCI ETY. 38: 415-420 (1990), observe that "... C. difficile infection, post-antibiotic serves as a marker of disease in nursing home patients, one that can be differentiated from the risk of antibiotic treatment only C. C. difficile is an obligate anaerobic bacillus, frothing is a component of the normal intestinal flora of approximately 3% to 5% of healthy adults, but can be found in toilets of up to 15% to 20% of adults, who are patients in hospitals, and as much as 50%. % of asymptomatic babies Fekety, "Antibiotic-Associated DiarrheaP DIARRHEAL DISEASES. ed. Field, pp. 293-317 (1991). When established in the colon, the pathogenic strains of C. difficile produce toxins that are the cause of diarrhea and colitis. Banno et al., "Two toxins (D-1 and D-2) of Clostridium difficile causing antibiotic-associated colitis: Purifications and some characterizationP BIOCHEMISTRY INTERNATIONAL 2: 625-635 (1991); Taylor and others, "Comparison of two toxins produced by Clostridium difficile", INFECTION AND IMMUNITY. 34: 1036-1043 (1981). C. difficile produces two large protein exotoxins known as toxin A and toxin B. Toxin A causes fluid secretion, mucosal damage, and intestinal inflammation when injected into the intestine of rodents. Triadafilopolous and others, "Differential effects of Clostridium difficile toxins A and B on rabbit ileumP GASTROENTEROLOGY 93: 273-279 (1987). Toxin B is more potent than toxin A as a cytotoxin in tissue culture, but it is not enterotoxic in animals, Kelly et al., "Clostridium difficile Colitis," The NEW ENGLAND JOURNAL OF MEDICINE, No. 330 (4): 257-262 (1994) Dehydration as a result of diarrhea usually follows infection with C The toxin A (known as Toxin D-2 in Japanese literature) is an endotoxin and is considered to be the primary mediator of the disease associated with C. difficile, it has a molecular weight (MW) of approximately 308,000. B (known as Toxin D-1 in Japanese literature) is a cytotoxin and has a molecular weight of approximately 270,000.Toxin A is believed to cause tissue damage within the gastrointestinal tract and this damage is exacerbated by Toxin B which gives as it turns out Do diarrhea and colitis. Infection with C. difficile manifests itself clinically as uncomplicated diarrhea, non-specific colitis, and more seriously as pseudomembranous colitis (PMC). Death can occur from complications such as dehydration, toxic megacolon or intestinal perforation. McFarland et al., "Nosocomial Acquisition of Clostridium difficile I nfection", TH E NEW ENGLAND D JOUR NAL OF MEDICI. 320: 240-210 (1989): Brown et al. "Risk Factors for Clostridium difficile Toxin-Associated Diarrhea", INFECTION CONTROL HOSPITAL EPIDEMIOLOGY. 1 1 (6): 283-290 (1990). C. difficile colitis occurs at all ages, but is very common in elderly and debilitated patients. It is relatively rare in babies, although the organism is commonly found in their toilets and usually only follows the use of antibiotics. Fekety, "Antibiotic-Associated Diarrhea", DIARRH EAL DISEASES. Field (ed), published by Elsevier, p. 293-317, (1991). C. difficile infection is responsible for all cases of pseudomembranous colitis and up to 20% of cases of diarrhea associated with antibiotics without colitis. Kelly et al., "Clostridium difficile Colitis", TH E N EW ENGLAND D JOU RNAL OF MEDICINE E. 330 (4): 257-262 (1994); McFarland et al., "Risk factors for Clostridium difficile carriage and C. d / f / 'c /' / e-associated diarrhea in a cohort of hospital patients", JOU RNAL OF I N FECTIOUS DISEASES. 162: 678-684 (1990). The persistence of C. difficile as a nosocomic pathogen is facilitated by the ease with which it is transmitted within the hospital environment and by the widespread use of antibiotics. The antibiotics given for the treatment of even minor infections and administered by any route, can precipitate PMC. Virtually every antimicrobial used in the treatment of human infections, including those very commonly used in the treatment of the disease, is known to stimulate PMC. Fekety, "Antibiotic-Associated Diarrhea", DIARR H EAL DISEASES. Field (ed), published by Elsevier, p. 293-317 (1991). PMC can occur during the period immediately after (almost always in 6 months) that the use of antibiotics has been discontinued, during this period, the normal intestinal flora has not yet reached normal levels, this absence of normal flora allows C. Difficile develops at unhealthy population levels. Fekety, "Antibiotic-Associated Diarrhea", DIARRHEAL DISEASES. Field (ed), published by Elsevier, pp. 293-317 (1991). The normal treatment for the disease associated with C. difficile is the administration of antibiotics, vancomycin or metronidazole. None of the new treatments has proven to be effective in clinical analyzes. Fekety et al., "Diagnosis and Treatment of Clostridium difficile colitis", JOU R NAL OF TH AND AMERICAN MEDICAL ASSOCIATION. 269: 71-75 (1993); Kelly and others, "Clostridium difficile Colitis", THE N EW ENGLAND JOURNAL OF MEDICINE. 330 (4): 257-262 (1994). Normal antibiotic therapy is effective in 80% of patients with diseases associated with C. difficile (CDD), but the remaining 20% experience additional episodes of diarrhea or colitis during the allowed period after the antibiotic has been discontinued. Fekety et al., "Treatment of Antibiotic-Associated Clostridium difficile Colitis with Oral Vancomycin Comparison of Two Dosage Regiments", AMERICAN JOURNAL OF MEDICINE. 86: 15-19 (1989); Walters et al., "Relapse of antibiotic associated colitis; endogenous persistence of Clostridium difficile during vancomycin therapy", GUT. 24: 206-212 (1983); Bartlett et al., "Symptomatic Relapse After Oral Vancomycin Therapy of Antibiotic-Associated Pseudomembranous Colitis", GASTROENTEROLOGY. 78: 431-434 (1980). Eleven patients had a recurrence, experienced repeated episodes of disease for several years. Fekety et al., "Diagnosis and Treatment of Clostridium difficile colitis", JOURNAL OF TH AND AMERICAN MEDICAL ASSOCIATION. 269: 71-75 (1993); Kimmey et al., "Prevention of Further Recurrences of Clostridium difficile Colitis with Saccharomyces boulardii", DIGESTIVE DISEASES AN D SCIENCES. 35 (7): 897-901. In a randomized, placebo-controlled analysis, Johnson et al., "Treatment of asymptomatic Clostridium difficile carriers (faecal excretors) with vancomycin or metronidazole", ANNAL OF I NTERNAL MEDICI N. 1 17: 297-302 (1990), compared the efficacy of vancomycin and metronidazole to eradicate faecal excretors as a measure of controlling the nosocomal outbreaks of C. difficile diarrhea. They found that metronidazole was not effective, and although vancomycin was temporarily effective, it was associated with a significantly higher rate of C. difficile carrier, two months after treatment. In their conclusions, they did not recommend antibiotic treatment of asymptomatic patients who excrete C. difficile. However, given the causative role played by antibiotics in CDD, alternatives to antibiotic research may be a more effective strategy to develop new treatments for these conditions of weakness. The significant clinical concern of CDD and the role of antibiotics in causing these diseases emphasize the importance of alternative methods of prevention and treatment development.

COMPENDIUM OF THE INVENTION Thus, in one aspect of the invention, there is provided a method for inhibiting infection of a mammal by Clostridium difficile, said method comprising administering enterally to a mammal, a therapeutically effective amount of an indigestible oligosaccharide. Preferably, the oligosaccharide is a fructo-oligosaccharide, such as 1 -ketose, nistose or 1F-β-fructofuranose or a xylo-oligosaccharide, such as xylobiose, xylotriose or xylotetrosa.

In a second aspect, the invention provides a method for inhibiting Clostridium difficile toxin A, said method comprises enterally administering to a mammal, a therapeutically effective amount of an indigestible oligosaccharide, such as those mentioned above. An important aspect of the management of acute diarrhea is the replacement of fluid and electrolyte. Fluid therapy can be either oral or intravenous, depending on the severity of the dehydration. Oral rehydration solutions are less expensive than intravenous therapy. Once it has been achieved, other therapeutic aspects can be addressed. According to yet another aspect of the invention, an oral rehydration solution is provided which comprises sodium chloride, potassium chloride, a bicarbonate source and an indigestible oligosaccharide, such as those mentioned above. According to a further aspect of the invention, there is provided a method for treating dehydration associated with diarrhea resulting from Clostridium difficile infection, said method comprising enterally administering an oral rehydration solution comprising sodium chloride, potassium chloride, source of bicarbonate, and a therapeutically effective amount of an indigestible oligosaccharide.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A, 1 B and 1 C are representations of the chemical structures of fructo-oligosaccharides which have utility in the practice of the present invention. Figures 2A, 2B and 2C are representations of chemical structures of xylo-oligosaccharides that have utility in the practice of the present invention. Figures 3 and 4 are graphic representations of the results of experiment 1. Figures 5 and 6 are graphic representations of the results of experiment 2. Figures 7 and 8 are graphic representations of the results of experiment 3.

DETAILED DESCRIPTION OF THE INVENTION As used herein and in the claims, "indigestible oligosaccharide" refers to a small portion of carbohydrate (degree of polymerization less than 20 and / or a molecular weight less than 3,600) that is resistant to endogenous digestion in the tract digestive of the human being. The indigestible oligosaccharides which can be employed in preferred embodiments of the invention are fructo-oligosaccharides and xylo-oligosaccharides. The indigestible oligosaccharides which can be used in the highly preferred embodiments of the invention are fructo-oligosaccharides selected from the group consisting of 1 -ketose fructo-oligosaccharides, nistose and 1 F-β-fructofuranosyl-nystase, and xylo-oligosaccharides selected from the group consisting of group consisting of xylo-oligosaccharides of xylobiose, xylotriose and xylotetrosa. As used herein, "inhibit" is understood to refer both to reducing the number of C. difficile and preventing the increase in the number of C. difficile. Fructo-oligosaccharides ("FOS") are carbohydrate polymers that consist of a chain of fructose residues linked by (2 > 1) -β-glycosidic bonds, and usually carry an individual D-glucosyl residue at the end without reduction of the linked chain (1 - »2) -a- as in sucrose. FOS occur in nature in many types of plants including bananas, tomatoes, onions, wheat, barley, honey, asparagus and artichokes. They can also be synthesized from sucrose through the use of transfructosiiation enzymes, such as the enzyme obtained from the fungus Aspergillus niger. Hidaka and others, "Fructooligosaccharides: Enzymatic Preparation and Biofunctions", JOU RNAL OF CARBOHYDRATE CHEMI STRY. 10 (4): 509-522 (1991). The treatment of sucrose with this enzyme results in a mixture of fructo-oligosaccharides containing 2, 3, or 4 fructose residues. The resulting fructo-oligosaccharides are designated respectively as 1 -ketose (G F2), nystase (GF3), and 1 F-β-fructofuranosyl nystase (GF4), the chemical structures of which are depicted in Figure 1. A method for producing FOS industrially is described in the patent of E.U.A. No. 4,681, 771 of Adachi et al. FOS are not hydrolyzed in the small intestine by digestive enzymes of rat or human being, and thus arrive intact to the large intestine. There, many intestinal microorganisms use them. FOS can be used very significantly by bifidobacteria, which are believed to be highly beneficial organisms (Hidaka and others). They can not be used in vitro by certain putrefaction bacteria, of desirable, such as C. perfringes, C. difficile, or E. Coli. Carbohydrates that are not digested in the small intestine can be fermented to short chain fatty acids by microorganisms found in the large intestine. The short chain fatty acids are used as a source of energy for colonocytes and other organs such as the liver. FOS have also been shown to reduce the total serum cholesterol level, probably by lowering the LDL-cholesterol level, and relieve constipation by accelerating peristaltic movement (Hidaka and others). The xylo-oligosaccharides ("XOS") are prepared through enzymatic hydrolysis of corn xylan, sugarcane and cottonseed. The xylans are hydrolyzed by an enzyme xylanase derived from Trichoderma to make XOS. The xylo-oligosaccharides are mainly composed of two, three, and four units of xylose with a β-1 -4 bond, xylobiose, xylotriose and xylotetrosa, respectively, the chemical structures of which are illustrated in Figure 2. The xylobiose, The main component of XOS is relatively abundant in bamboo roots. Imaizumi et al., "Effects of xylooligosaccharides on blood glucose, serum and liver lipids and cecum short-chain fatty acids in diabetic rats", AGRICULTURAL AND BIOLOGICAL CHEMISTRY. 55 (1): 199-205 (1991), quoted from Fujikawa and Okazaki, Food Chemicals (in Japanese) 1:63 (1989). XOS, like FOS, have been shown to be selectively used by in vitro bifidobacteria, Okazaki et al., "Effect of Xylooligosaccharide on the growth of bifidobacteria", BI MICROFLORA FIDOBACTERIA. 9 (2): 77-86 (1990). In addition, clinical studies have shown that XOS, when supplemented to human diets, increase the level of bifidobacteria recovered in feces (Okazaki et al.). In addition, in vitro studies show that potential pathogens such as Staphylococcus, E. coli, C. perfrigens, and C. difficile are not capable of using XOS as a source of energy (Okazaki et al.). It has become enormously obvious that many of the beneficial effects of fermentable carbohydrate are mediated by short chain fatty acids (SCFA) such as acetate, propionate, and butyrate, which are produced during anaerobic fermentation in the colon. The chain fatty acids play a key role in the function of the intestines. The absorption of 100 mmoles of SCFA is associated with the absorption of 360 ml of water. Casparey et al., "Bacterial fermentation of carbohydrates within the gastrointestinal tract", CLINICAL RESEARCH REVIEW. 1: 107-177 (1981). Subsequently, the absence or reduction of SCFA in the colon results in diarrhea. Ramakrishna et al., "Colonic dysfunction in acute diarrhea: the role of luminal short chain fatty acids", GUT. 34: 1215 (1993) found that the production of short-chain fatty acids in faecal waste in patients with acute diarrhea, it was low on the first day of the illness, but it increased during the following 5 days as the condition of the patients improved. In addition, using a rectal dialysis technique in vivo, Ramakrishna et al., "Colonic dysfunction in acute diarrhea: the role of luminal short chain fatty acids", GUT. 34: 1215 (1993) demonstrated that lumenal SCFA could restore the net reabsorption of water and sodium in the rectum of patients with acute diarrhea. In in vivo perfusion studies in healthy patients, they showed the secretion of salt and water in the ascending colon in response to enteral feeding. Bowling et al., "Colonic secretory effect in response to enteral feeding in man", GUT. 34 (Suppl 1): A54 (1993); Bowling et al., "The colonic secretory response to enteral feeding: influence of high strenght diet", CLIN ICAL N UTRITION. 12 (Suppl 2): 23 (1993). Bowling et al., "Reversal by short-chain fatty acids of colonic fluid secretion induces by enteral feeding", LANCET. 342: 1266 (1993) investigated the effect of short chain fatty acids on colonic fluid secretion induced by enteral feeding. The researchers found that the infusion of SCFA directly into the caecum of healthy subjects reversed the secretion of fluid seen in the ascending colon during enteral feeding and it was theorized that these findings could have implications for the management of diarrhea related to enteral feeds. The non-digestibility of indigestible oligosaccharides, such as XOS and FOS, and their selective use by beneficial intestinal bacteria, leads to an increase in the presence of bifidobacteria and results in the production of short-chain fatty acids, which can reduce the pH in the large intestine, and suppress undesirable microorganisms and the substances (for example toxins) they produce. These activities could be beneficial for the health of both humans and animals. Compositions: The diarrhea associated with C. difficile can be treated, inhibiting the level of Toxin A, and also treating the associated dehydration, enterally administering indigestible oligosaccharides in a nutritional composition. Since other nutritional compositions are certainly within the scope of the invention, ideally said nutritional compositions are oral rehydration solutions to help alleviate the dehydration associated with diarrhea. The Oral Rehydration Solution of the World Health Organization (WHO-ORS) is a model ORS developed specifically to treat dehydration associated with diarrhea. It has a specific composition that, unlike water and other common household drinks, facilitates rehydration. The ideal sodium content of an ORS is undecided. The WHO-ORS contains 90 mEq of sodium per liter, but other ORS that contain smaller amounts have been shown to be effective. Two typical, commercially available oral rehydration solutions are described below. A widely used, commercially available oral rehydration solution for adults is EquaLyte ™, which is distributed by Ross Products Division of Abbott Laboratories, Columbus, Ohio, E. U.A.

In 1000 mi, EquaLyte ™ provides: A widely used oral rehydration solution commercially available for infants and children is Pedialyte®, which is distributed by Ross Products Division of Abbott Laboratories, Columbus, Ohio, E. U.A.

In 1000 mi, Pedialyte® provides: The present invention involves the addition of one or more indigestible oligosaccharides to an oral rehydration solution, such as those described above. More specifically, the present invention can be practiced by adding 3-30 g / l (preferably from about 3-18 g / l of fructo-oligosaccharides to an oral rehydration solution, such as Equalyte ™ or Pedialyte®, which is described in the above text, for example, an oral rehydration solution comprising sodium, potassium, chlorine, a source of bicarbonate and a therapeutically effective amount of an indigestible oligosaccharide The indigestible oligosaccharide can be selected from fructo-oligosaccharides (e.g. , 1 -ketose, nystase and 1 F-β-fructofuranosyl nystase) or xylo-oligosaccharides (such as xylobiose, xylotriose and xylotetrosa) Two experiments were conducted to determine the effect of fructo-oligosaccharides (FOS) as a dietary supplement, in mortality in a Syrian hamster model of C. difficile colitis.

ANIMAL MODEL FOR EXPERIMENTS 1 AND 2 The use of the Sirius hamster as a model for C. difficile colitis is widely recognized. Lust et al., "Clindamycin-Induced Enterocolitis in Hamsters," THE JOURNAL OF INFECTIOUS DISEASES. 137, 464-475 (1978), proposed that enterocolitis induced in the hamster by antibiotics is a good model for the investigation of the syndrome in humans. Price and others, "Morphology of experimental antibiotic-associated enterocolitis in the hamster: a model for human pseudomembranous colitis and antibiotic-associated diarrhea" GUT. 20: 467-475 (1979) established the study of the morphology of pseudomembranous colitis associated with experimental antibiotic (PMC). They observed that the hamster model has some morphological differences; however, the bacteriology and toxicology are identical to that of the human being. They concluded that the hamster is a good model to investigate the pathogenesis of PMC and enteropathy associated with antibiotic, in general. In fact, Wilson et al., "Suppression of Clostridium difficile by Normal Hamster Cecal Flora and Prevention of Antibiotic-Associated Cecitis", INFECTION AND IMMUN ITY. 34: 629-628 (1981), observed that studies with the hamster model of colitis associated with antibiotic led to the discovery of C. difficile toxin as a major etiology of colitis associated with antibiotic in humans and for effective treatment with oral vancomycin Wilson et al., "Population Dynamics of Ingested Clostridium Difficile in the Gastrointestinal Tract of the Syrian Hamster," THE JOU RNAL OF INFECTIOUS DISEASES. 151: 355-361 (1985) observed that the best studied animal model of colitis associated with antibiotic was that of the Syrian hamster.

MATERIALS AND METHODS FOR EXPERIMENTS 1 AND 2 Animals: Golden Syrian female hamsters (6-8 weeks of age, 80,120 g) were purchased from Harían Sprague Dawley, Inc., Indianapolis, IN. The animals were randomized into treatment groups and housed at 22 + 2 ° C in the accommodation per group. Free pathogens were obtained and housed in accordance with the standards of the Institutional Animal Care Commitee (IACUC) (Institutional Animal Care Use Committee) and the American Association for the Accreditation of Laboratory Animal Care (AAALAC) (American Association for the Accreditation of Laboratory Animal Care). The animals were fed a special laboratory food diet (PROLAB®, Agway Country Foods, Inc., Syracuse, NY). Antibiotic: Hamsters were medicated daily with ciprofloxacin priming (Miles, I nc., West Haven, CT; 250 mg / kg of body weight / day) from day 0 to day 6. This medication resulted in an "antibiotic sterilization" of the gastrointestinal tract, which allowed the natural infection (overgrowth) of C. difficile. This "natural" infection may be unreliable under laboratory fixation, thus for a consistent degree of infection among animals, additional treatments were added to the experiments to include inoculation (attack) of hamster with C. difficile on day 3 and on day 7. Inoculum: In the studies a human isolate of C. difficile (strain VPI 10463) was used, which showed to produce high quantities of toxin A and B. Wilson and others, "Gnotobiotic models for the study of microbial ecology of Clostridium difficile and Escherichia coli ", TH E JOU RNAL OF IN FECTIOUS DISEASES. 153 (3): 547-551 (1986); Sullivan et al., "Purification and characterization of toxins A and B of Clostridium difficile", INFECTION AND IMMUNITY. 35 (3): 1032-1040 (1982). C. difficile, VPI 10463, was grown in BHI broth (brain heart infusion, Difco Laboratories, Detroit, MI) on a rotating platform under anaerobic conditions. A nocturnal culture (ONC) has a concentration of approximately 109 CFU / ml. The hamsters were inoculated with their respective volume of ONC via priming, on day 3 and day 7.

EXPERIMENTAL DESIGNS EXPERIMENT 1 In the first experiment, 41 golden Syrian hamsters, females, were randomly distributed between 1 to 6 treatment groups. The treatments were arranged at a factor of 2 x 3, with two levels of FOS (0 to 30 g / l in drinking water) and 3 levels of inoculation (approximately 109 CFU / ml) of C. difficile (0, 0.5 or 2.0). my). The hamsters were treated orally with ciprofloxacin (250 mg / kg body weight / day) from day 0 to day 6 and were inoculated with their appropriate volume of a C. difficile overnight culture on day 3 and day 7 .

EXPERIMENT 2 In the second experiment, 63 hamsters were randomly distributed among 1 of 8 treatment groups. The treatments were arranged at a factor of 2 x 2 x 2, with two levels of FOS (0 or 30 g / L in drinking water), 2 levels of C. difficile inoculum (0 or 2.0 ml), and two levels of vancomycin drug (Eli Lilly, Indianapolis, IN) (0 or 50 mg / kg of body weight / day). The level of FOS, the dose of cirpofloxacin and the inoculation protocol were similar to experiment 1. The hamsters in the vancomycin treatment were given 50 mg / kg of body weight / day on days 7 to 13.

COLLECTION OF DATA AND STATISTICS EXPERIMENTS 1 AND 2 After death, the survival time (in days) was recorded (measured from the first day of the administration of the drug ciprofloxacin). The animals were followed until day 24, which was the day in which the experiment was finished. Survival times were summarized as Kaplan-Meier curves (Pocock, CLINICAL TRIALS: A PRACTICAL APPROACH, John Wiley &Sons Ltd., New York, New York, USA, 1983) Animals that did not die at the end of the study , and their survival times were censored on day 24. The differences in FOS minus the control group were tested by log classification statistics, and supplemented with the Wilcoxon generalized test In addition to the global analysis comparing the animals fed with FOS against control (animals that were not given FOS), marginal tests of treatment effect were performed for several subgroups, for example, by the level of C. difficile attack.The test results are reported as two sides, with a statistical significance judged on P less than the 0.05 level.In general, the results of the two statistical tests were similar.The Wilcoxon test (Rosner, FUNDAMENTALS OF BIOSTATISTICS .PWS Publishers, Boston, Massachusetts, E. U.A. (1986)) was designed to test the difference between treatments at early time points, while the log classification test (Neter and Wasserman, APPLIED LINEAR STATISTICAL MODELS, 1974, Richard D. Irwin, Inc., Homewood, Illinois, E UA (1994) was designed to test the difference between treatments at later time points.

RESULTS AND DISCUSSION EXPERIMENT 1 Table 1 shows the mean survival time (MST) for the treatment groups. In general, the dietary supplement with FOS (30 g / l in drinking water) tended to increase the MST (P = .09, log classification statistics) (Table 2). When using the Wilcoxon test, the total effect of FOS was highly significant (P less than .01). If the comparison is made only in those hamsters that were not inoculated (allowing the natural infection to occur), FOS did not improve the MST (P greater than .20). As mentioned above, based on the fact that the natural infection may be inconsistent, in this way the inoculated animals were included in the experiments. When the comparison was made between those hamsters that received C. difficile inoculation (0.5 and 2.0 ml), the FOS had a highly significant effect (P less than 0.1) on MST. In addition, the separation of the inoculation level (0.5 and 2.0 ml) resulted in a higher MST for the hamsters that received the supplementary FOS (P less than .01). Survival curves for hamster inoculated with 0.5 and 2.0 ml of C. difficile are presented in Figures 3 and 4, respectively. These Figures show the improved survival time of hamsters fed supplemental FOS. In general, the FOS tended to increase (P less than .10) the mean survival time (MST). In addition, the MST was increased (P less than .01) for hamsters that consumed FOS, which were inoculated with 0.5 or 2.0 ml of C. difficile.

TABLE 1 Mean Survival Time (MST) of Hamsters Fed with (+/-) 30 g / L of Fructo-Oligosaccharides (FOS) in Drinking Water and Attacked with Variable Levels of Clostridium difficile (2.0, 0.5, or 0 mL) FOS was provided at 30 g / l in drinking water, ad libitum. Approximate water consumption was 8 ml / hamster / day. ONC = nocturnal culture of Clostridium difficile.

TABLE 2 Mean Survival Time (MST) of Hamsters Fed with (+/-) 30 g / L of Fructo-Oligosaccharides (FOS) in Drinking Water and Attacked with Variable Levels of Clostridium difficile (2.0, 0.5, or 0 mL) 1 MST in days 2 0.5 my ONC + 2.0 my ONC 3 ONC = nocturnal culture of C. difficile.

EXPERIMENT 2 Table 3 shows the average survival time for treatment groups. As in the previous experiment, the total effect of the FOS was to increase the hamster MST (P less than .05). In this experiment, there appeared to be an interaction between vancomycin treatment and the level of inoculation (Table 4). Hams supplemented with FOS and inoculated with C. difficile, but not treated with vancomycin, presented an improved MST (P less than .10). However, FOS had no effect (P greater than .20) on inoculated hamsters treated with vancomycin. On the other hand, the FOS supplement increased the MST of uninoculated mice, treated or not treated with vancomycin (P less than .05 and P less than .01, respectively, survival curves, Figures 5 and 6, respectively). Again, survival curves showed the added benefits of FOS. In general, hamsters fed with FOS increased the MST (P less than .05). These data suggest that the dietary supplement with FOS increases the average survival time in a hamster model of colitis due to Clostridium difficile.

TABLE 3 Mean Survival Time (MST) of Hamsters Fed with (+/-) 30 g / L of Fructo-Oligosaccharides (FOS) in Drinking Water and Attacked with Variable Levels of Clostridium difficile, and Treaties (+/-) with Vancomycin Inoculated with a nocturnal culture of Clostridium difficile (2.0ml). 2 FOS was provided at 30 g / l in drinking water, ad libitum. Approximate water consumption was 8 ml / hamster / day.

TABLE 4 Average Survival Time (MST) of Hamsters Fed with (+/-) 30 g / l of Fructo-Oligosaccharides (FOS) in Drinking Water, Attacked (+/-) with Clostridium difficile, and Treaties (+/-) with Vancomycin Group1 30 a / l of FOS CONTROL p-value p-value n MTS2 n MTS2 Wilcoxon classif. log Fully 31 18.0 32 16.0 0.027 0.020 N, Without Vaneo 8 16.5 8 1 3.5 0.003 0.001 N, Vaneo 7 19.0 8 1 7.5 0.021 0.026 1, Without Vaneo 8 14.5 8 1 3.0 0.07 0.054 1, Vaneo 8 1 9.0 8 20.0 0.66 0.95 N 1 5 1 9.0 16 1 5.5 0.007 0.005 i 16 1 6.5 16 16.5 0.43 0.41 Without Vaneo 16 1 5.5 16 13.5 0.001 < 0.001 Vaneo 1 5 1 9.0 16 1 9.0 0.49 0.37 I = Inoculation with a nocturnal culture of C. difficile, N = without inoculation, Vaneo = Vancomycin. MST in days.

In general, the results of Experiments 1 and 2 show that enteral administration of a therapeutically effective amount of an indigestible oligosaccharide (FOS) inhibits infection of a mammal by C. difficile, as shown by the times measured in a model of C. difficile colitis hamster. This has been shown by an increase in survivorship capacity (in certain treatment groups) and a consistent increase in mean survivorship time. Impressively, the improved survival time due to the FOS supplement was above the fiber contained in the diet.

EXPERIMENT 3 The objective of Experiment 3 was to determine the effect of feeding highly fermentable carbohydrate on C. difficile infection in a murine model that included antibiotic administration before attack by pathogens (C. difficile). The model involved the breaking of intestinal microbiota with the antibiotic cefoxitin, followed by an attack with C. difficile (VPI 10463).

MATERIALS AND METHODS Animals: 64 male BALB / c mice (average body weight 19.3 +. 1.6 g) were stored in stainless steel cages. The environmental conditions were controlled to provide a light cycle of 12 h and a constant temperature of 24 ° C. Diets: water and a dietary powder, nutritionally balanced, with a low residue content (Ensure ™), ad libitum were provided. The control mice © received no fiber supplement. Three carbohydrate sources were provided to the remaining groups. The gum arabic (GA, a powder) and the fructo-oligosaccharides (FOS, a powder) were mixed with the Ensure ™ diet (44 mg fiber / g diet powder). The xylo-oligosaccharides (XOS, a syrup) were mixed with drinking water (30 g / l). Strains of Bacteria v Conditions: In this study a known toxigenic strain, Clostridium difficile, was used. Wilson et al., "Gnotobiotic models for the study of microbial ecology of Clostridium difficile and Escherichia coli", JOURNAL OF INFECTIOUS DISEASES. 153 (3): 547-551 (1986); Sullivan et al., "Purification and characterization of toxins A and B of Clostridium difficile", I N FECTION AN D IMMU N ITY. 35 (3): 1032-1040 (1982). The organism was maintained and developed in a reinforced elostridial medium (RCM, Difco, Michigan, USA) and suspensions of normal cells were used as inocula. All diluents and media were prepared using routine anaerobic techniques. Mackie et al., "Enumeration of anaerobic bacterial microflora of the equine gastrointestinal tract", APPLI ED AN D ENVI RONMENTAL MICROBIOLOGY. 54: 2155-2160 (1988); Meyer et al., "Microbiological evaluation of the intraluminal insaeculus digestion technique", APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 51: 622-629 (1986).

EXPERIMENTAL DESIGN The 64 mice consumed Ensure ™ powder for three days. On day four, all mice received 100 μg cefoxitin / g body weight, orogastrically. Cefoxitin (Sigma Chemical Co., St. Louis, Missouri, USA) was dissolved in water and the appropriate dose was delivered to the stomach with a barbed needle (22 ga, 2.54 cm). The next day, the mice were randomly assigned either to the control or to the different fermentable carbohydrate treatments and were fed with their respective diets for five days. At that time, eight mice per diet were inoculated as described above with 200 μl of a suspension of C. difficile, containing 2.8 x 10ß of colony forming units (CFU) / ml. RCM was used to inoculate the remaining 8 mice in each diet. The sacrifice, by asphyxia by CO2, occurred six days after the inoculation. The entire animal protocol was completed in 15 days.

DATA AND STATISTICS COLLECTION Five fecal samples were collected throughout the experiment (days 2, 5, 8, 12, and 15). For clarity, these days of sampling are expressed in relation to the inoculation of C. difficile (day 0) and so are the days -7, -4, -1, 3 and 6. 100 mg of faeces were collected from each animal and immediately suspended in 9.9 ml of anaerobic diluent. Bryant et al., "Culture methods and some characteristic of some of the most numerous groups of bacteria in the bovine rumen," JOURNAL OF DAIRY SCIENCE. 36: 205-217 (1953). Faecal samples were evaluated using the following response criteria: C. difficile numbers on selective cycloserine-cefoxitin-fructose agar. George et al., "Selective and differential medium for isolation of Clostridium difficile", JOURNAL OF CLI N ICAL MICROBIOLOGY. 9: 214-219 (1979); toxin A titration using an enzyme-linked immunosorbent assay (Tox-A-Test, TECH LAB, Blacksburg, Virginia, USA). The treatments were arranged as a randomized complete block design within a division chart. Gilí et al., "Analysis of repetitive measurements of animáis", JOU RNAL OF ANIMAL SCIENCES. 33: 331-336 (1971). The data were subjected to variation analysis according to the procedure of General Linear Models (GLM) (General Linear Models) of SAS (SAS Institute, "SAS User's Guide: Statistics", SAS Institute, Inc. Cary, North Carolina (1988 )). The effects of the treatment were tested using an F statistic and a P value of .05 was chosen as the significant level.

RESULTS AND DISCUSSIONS Fecal C. difficile counts (Table 5) were influenced by a diet x sample interaction (P <.01). At the beginning of the experiment (day 7), there were no detectable counts of C. difficile (<104 CFU / g of faeces) Interestingly, after the dose of cefoxitin (day 6) and three days after inoculation of the pathogen , all mice, including non-inoculated mice, were infected with C. difficile, to a certain degree. For mice fed the control diet, C. difficile was continued to increase throughout the experiment. Six days after inoculation with C. difficile, the control mice had the highest counts of C. difficile (127.5 x 105 CFU / g of feces) and the mice fed with FOS had the lowest counts (0). During the experimental period, toxin A assays were performed on fecal samples, and the incidence (percentage of mice with detectable toxin A) and the toxin A titre are shown (Table 6). Prior to C. difficile inoculation, no toxin A was detected in the stool of any of the mice. Three after inoculation, C. difficile and toxin A were detected virtually in each mouse and those inoculated with C. difficile had the highest titers (104). Six days after inoculation, mice consuming C and GA diets had an incidence of more than 50% of toxin A while mice consuming FOS and XOS that had a lower incidence than 50% toxin A. Only of mice that consumed the C or GA diets and were inoculated with C. difficile developed severe diarrhea (37 and 75%, respectively) or died (12.5 and 50%, respectively).

TABLE 5 Effect of the Diet, Sampling Day, and Inoculation of Clostridium difficile in Fecal Faeces 1 C. difficile counts (x 105 CFU / g of faeces) determined on cycloserine-cefoxitin-egg yolk agar. The data exhibited a diet x sample day interaction; means the column with different superscripts are different (P <.01). 2 Fecal sampling days in relation to pathogen inoculation (day 0). 3 Control (C); gum arabic (GA); fructo-oligosaccharides (FOS); xylo-oligosaccharides (XOS) with (+) or without (-) 200 μl Clostridium difficile inoculation VPI 10463 (2 x 10ß CFU s / ml).

TABLE 6 Percentage of mice having detectable titers of toxin A in feces. 2 Fecal sampling days in relation to pathogen inoculation (day 0). 3 Control (C); gum arabic (GA); fruit-oligosaccharides (FOS); xylo-oligosaccharides (XOS) with (+) or without (-) 200 μl Clostridium difficile inoculation VPI 10463 (2 x 10 * CFUs / ml). 4 Titration of toxin A expressed as the reciprocal aspect of the highest dilution, in which toxin A was detected in fecal samples, nd = not detected. The degrees are emptied through the diets.

CONCLUSION EXPERIMENT 3 Since mice did not develop the same lesions as humans (PMC), a murine model can continue to provide, in vivo, very important data. Studies of microbial interactions coupled with nutritional manipulations may contribute to the understanding of the causative factors in the colonization and infection of C. difficile. In addition, the information provides a better understanding of the ecology of gastrointestinal microbes. In this study, the oligosaccharides suppressed the development of C. difficile and appeared to provide protection to intestinal epithelial tissue.

Claims (1)

1. CLAIMS 1 .- A method to inhibit the infection of a mammal by Clostridium difficile or to inhibit the level of Clostridium difficile toxin A in a mammal, said method comprises enterally administering to said mammal a therapeutically effective amount of an indigestible oligosaccharide. 2. The method according to claim 1, wherein the indigestible oligosaccharide is a fructo-oligosaccharide. 3. The method according to claim 2, wherein the indigestible oligosaccharide is a fructo-oligosaccharide selected from the group consisting of: 1 -ketose, nystase and 1-F-β-fructofuranosyl nystase. 4. The method according to claim 1, wherein the oligosaccharide is indigestible in xylo-oligosaccharide. 5. The method according to claim 4, wherein the indigestible oligosaccharide is a xylo-oligosaccharide selected from the group consisting of xylobiose, xylotriose and xylotetrosa. 6. The method according to claim 1, wherein the indigestible oligosaccharide is administered enterally through a nutritional composition of oral rehydration solution. 7. The method according to claim 6, wherein said oral rehydration solution comprises, in addition to the therapeutically effective amount of an indigestible oligosaccharide, sodium chloride, potassium, and a source of bicarbonate. 8 -. 8 - The method according to claim 7, wherein the indigestible oligosaccharide is a fructo-oligosaccharide selected from the group consisting of: 1 -ketose, nystase and 1 F-β-fructofuranosyl nystase. 9. The method according to claim 7, wherein the indigestible oligosaccharide is a xylo-oligosaccharide selected from the group consisting of xylobiose, xylotriose and xylotetrosa. 10. A method for the treatment of dehydration associated with diarrhea in a person infected with Clostridium difficile, enterally administering an oral rehydration solution comprising sodium chloride, potassium, a source of bicarbonate, and a therapeutically effective amount of a indigestible oligosaccharide. The method according to claim 10, wherein the indigestible oligosaccharide is a fructo-oligosaccharide selected from the group consisting of: 1 -ketose, nystase and 1 F-β-fructofuranosyl nystous. 12. The method according to claim 10, wherein the indigestible oligosaccharide is a xylo-oligosaccharide selected from the group consisting of xylobiose, xylotriose and xylotetrosa. 13. An oral rehydration solution comprising sodium chloride, potassium chloride, a source of bicarbonate and an indigestible oligosaccharide. 14. The solution according to claim 13, wherein the indigestible oligosaccharide is a fructo-oligosaccharide selected from the group consisting of: 1 -ketose, nystase and 1-F-β-fructofuranosyl nystase. 15. The solution according to claim 13, wherein the indigestible oligosaccharide is a xylo-oligosaccharide selected from the group consisting of xylobiose, xylotriose and xylotetrosa. 16. The solution according to claim 13, wherein the indigestible oligosaccharide is present in a concentration of 3-30 g / l of the solution.