Effect of Lactobacillus-Fermented Diets on Bacterial Translocation and Intestinal Flora in Experimental Prehepatic Portal Hypertension

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

C 2003) Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003), pp. 1136–1141 (°

Effect of Lactobacillus-Fermented Diets on Bacterial Translocation and Intestinal Flora in Experimental Prehepatic Portal Hypertension R. WIEST, MD,* F. CHEN, PhD,† G. CADELINA, MS,* R.J. GROSZMANN, MD*‡ and G. GARCIA-TSAO, MD,*‡

Spontaneous bacterial infections in cirrhosis and portal hypertension have been attributed to translocation of gut-derived bacteria, a process promoted by intestinal bacterial overgrowth and disruption of the gut mucosal barrier. Bacteriotherapy with Lactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease bacterial translocation in rat models of acute liver injury and failure. In this study we investigated the effect of Lactobacillus-supplemented diets on intestinal flora and on bacterial translocation rate in portal vein ligated rats. Lactobacillusfermented milk (yogurt) containing at least 2 × 109 colony forming units/ml or placebo (water) was adminstrated by gavage twice daily (2 ml) for 9 days. Portal vein ligation was performed on day 7 of treatment. Bacterial translocation to mesenteric lymph nodes and quantification of intestinal flora was assessed by standard bacteriological cultures. Bacterial translocation was not significantly different between animals that received yogurt (82%) and those that received placebo (75%). Yogurt did not induce any significant changes in intestinal flora, whether it was produced with Lactobacillus acidophilus or Lactobacillus GG. In conclusion, in acute prehepatic portal hypertension, bacteriotherapy with Lactobacillus was unable to induce changes in bacterial translocation probably because it was unable to induce changes in bacterial flora. KEY WORDS: portal hypertension; bacterial translocation; mesenteric lymph nodes; Lactobacillus, intestinal microflora.

Cirrhosis predisposes to the development of severe bacterial infections, mainly spontaneous bacteremia and spontaneous bacterial peritonitis (1, 2). Bacterial translocation (BT), the passage of bacteria from the gastrointestinal tract through the epithelial mucosa into the lamina propria and to mesenteric lymph nodes (MLN) and other extraintestiManuscript received XXXX 2002; accepted March 7, 2003. From the *Hepatic Hemodynamic Laboratory, Veterans Administration Medical Center, West Haven, Connecticut 06516, USA; and †Department of Comparative Medicine and ‡Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA. Supported by grants from the NIDDK (721-H-414776), VA Merit Review Fund, and B. Braun Foundation, Braun-Melsungen, Germany. Address for reprint requests: Dr. Guadalupe Garcia-Tsao, Hepatic Hemodynamic Laboratory/111J, Veterans Administration Medical Center, 950 Campbell Avenue. West Haven, Connecticut 06516, USA.

1136

nal sites (3), has been implicated in the pathogenesis of these spontaneous infections. We have previously reported that BT occurs in a high proportion (∼80%) of rats with acute prehpatic portal hypertension, ie, two days after portal vein ligation (4). It has also been shown to be increased in experimental cirrhosis (5), where it has been related to ascites infections (6, 7). Measures to prevent infections in portal hypertension and cirrhosis are, therefore, directed towards eliminating or reducing BT. An imbalance in intestinal microflora appears to be very important for the process of BT in cirrhosis, as studies performed both in cirrhotic rats and in patients with cirrhosis have demonstrated the presence of small bowel bacterial overgrowth, predominantly of aerobic gram-negative bacteria, especially in severe liver disease (8–10). Anaerobic bacteria, which represent more than 90% of the gut flora, Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

C 2003 Plenum Publishing Corporation 0163-2116/03/0600-1136/0 °

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

Lactobacillus AND BACTERIAL TRANSLOCATION IN PORTAL HYPERTENSION

do not translocate readily, while aerobic gram-negative bacilli translocate easily, even across a histologically intact intestinal epithelium (11–13). Lactobacilli constitute an integral part of the normal gastrointestinal microecology and promote growth of gram-positive and anaerobic bacteria while inhibiting the growth of gram-negative bacteria (14–16). Lactobacilli have been shown to be able to prevent bacterial overgrowth of potentially pathogenic bacteria (15, 16). For instance, bacteriotherapy with lactobacilli has been shown to be effective in preventing BT in experimental models of acute liver injury (17, 18) and liver failure (19). The objective of this study was to investigate, in a placebo-controlled, investigator-blinded study, the effect of dietary supplementation with Lactobacillus (milk fermented with Lactobacillus) on BT and on intestinal flora of an animal model known to have a high rate of BT, the rat with prehepatic portal hypertension. MATERIALS AND METHODS Induction of Portal Hypertension. Portal hypertension was induced surgically by portal vein ligation in aseptic conditions. This model has been extensively studied in our laboratory (4, 5). Briefly, the rats were anesthetized with ketamine hydrochloride (Ketalar, 100 mg/kg body wt; Parke, Davis, Avon, Connecticut, USA), After a midline abdominal incision, the portal vein was freed from surrounding tissue. A ligature (silk gut 3-0) was placed around a 20-gauge blunt-tipped needle lying alongside the portal vein Subsequent removal of the needle yielded a calibrated stenosis of the portal vein. In sham-operated rats, the same operation was performed with the exception that after isolating the portal vein no ligature was placed. After the operation, the animals were housed in plastic cages and allowed free access to rat food and water. Assessment of Bacterial Translocation (BT). On the study day the animals were anesthetized with ketamine (100 mg/kg), and the abdominal skin was shaved and sterilized with an iodine solution. All the following surgical procedures were performed under strict sterile conditions with sterile instruments. The caudal and cranial mesenteric lymph nodes (MLN) were removed and weighed with an Ohaus E400D scale (Ohaus. Corp., Florham Park, New Jersey, USA) with an accuracy of ± 0.01 g. Tissues were then homogenized in a measured amount of saline, and aliquots of 0.1 ml were plated onto blood, McConkey, and phenylethyl alcohol agar plates (BBL Prepared Media, Becton Dickinson Microbiology Systems, Cockeysville, Maryland, USA). Solid culture media were examined and colonies counted after 24 and 48 hr of aerobic incubation at 35◦ C. Any positive MLN cultures were considered indicative of BT from the intestinal lumen. Preparation of Lactobacillus-Supplemented Diet (Yogurt). Lactobacillus acidophilus (LA) [American Type Culture Collection (ATCC) catalog number 832, Rockville, Maryland, USA) was cultured in DeMan Rogosa Sharpe (MRS) broth in anerobic conditions for 24 hr at 37◦ C. Lactose-free milk was then inoculated with approximately 108 CFU/ml of LA and fermented for 18 hr at 37◦ C to obtain a yogurt. The final number of lactobacilli, Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

determined as viable counts on Rogosa agar after incubation, was tested in separate experiments to be at least 1 × 10 CFU/ml yogurt. The yogurt was resuspended (20%) in drinking water to increase fluidity. Two milliliters of this suspension (containing at least 4 × 109 viable lactobacilli) was administered twice daily by gavage in the group randomized to lactobacilli treatment. Rather than administering a concentrate of lactobacilli, we chose to use milk fermented with Lactobacillus acidophilus (yogurt) since, independent of the presence of viable lactobacilli, lactobacillifermented milk has been demonstrated to exert antimicrobial acitivity and to enhance the immune response (20–23). This suggests that the beneficial effect of lactobacilli may also be due to substances produced during the fermentation process such as bacteriocin, organic acids, and other metabolic end products (20–23). In a second group of experiments (see below), a separate strain of Lactobacillus, Lactobacillus casei subtype GorbachGoldbach (GG) (LGG) (Culturelle, CVS Pharmacy, New Haven, Connecticut, USA) was used for preparation of yogurt following the same procedure. Experimental Design. A total of 46 rats were studied (Table 1). In protocol I, Portal Vein Ligated (PVL) (N = 23) and control normal rats (N = 15) were randomly assigned to receive either twice daily dietary supplements with LA as 2 ml of diluted yogurt with at least 4 × 109 viable lactobacilli or 2 ml of drinking water (placebo) by gavage. LA (or water) were administered for 9 days prior to the experimental procedure. An investigator separate from the ones performing the experiments, adminstered yogurt or placebo. The investigators performing experiments were blinded as to the adminstration of LA supplements or water. PVL or sham operation were performed on day 7 of treatment; thus, experiments were performed 2 days after the induction of portal hypertension. In a separate protocol, and once the results with LA were obtained, control rats (N = 8) were given either LGG-fermented milk or placebo. This additional protocol was performed because of reported differences in efficiency of various strains of lactobacilli in different host species and pathological entities (14, 24, 25). LGG was chosen because, after LA, it is one of the most frequently used Lactobacillus strains in human and experimental investigations on probiotics (26–29). Determination of Intestinal Microflora. Samples taken from the cecum were collected into sterile tubes containing 5 ml of transport medium and weighed. Serial dilution in the same transport medium was performed five times before placing 1 ml of the sample on brain–heart infusion agar. Total aerobic and anaerobic bacterial counts were made by incubating the plates for 3 days at 37◦ C under aerobic or anerobic conditions, respectively. After that, the colony forming units (CFU) on each plate were corrected to the weight of the original sample Further identification of gram-negative anaerobes, lactobacilli, and

TABLE 1. ANIMALS STUDIED IN DIFFERENT GROUPS Group LA-ATCC 832 Placebo LA-GG Placebo Total

PVL

Controls

11 12

8 7 4 4 23

23

1137

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

WIEST ET AL Enterobacteriaceae were performed using appropriate selective culture media. Statistical Analyses. The main comparisons were made between Lactobacillius-treated and drinking water-treated groups. Nonparametric statistics, specifically the Mann-Whitney, test were used for comparison of continuous variables. Fisher’s exact test was used for comparison of nominal variables. Results are expressed as medians and ranges.

RESULTS Animals. There were no significant differences in body weight in the experimental groups. No significant difference in MLN weight used for assessment of BT in the different study groups was noted. PVL rats showed significantly higher spleen weights, expressed as percentage of body weight, as compared to sham animals (PVL 2.56 ± 0.78 vs sham 2.30 ± 0.65 mg/kg body wt, P < 0.05). No differences in spleen weight were observed between LA-treated and placebo-treated PVL rats. Bacterial Translocation. BT was not observed in any of the sham-operated or control rats. In PVL rats, BT to MLN was not significantly different in Lactobacillustreated rats (9/11 or 82%) and in the untreated groups (9/12 or 75%) (Ns) (Figure 1). Intestinal Microflora. Results on intestinal total aerobic and anerobic counts for the four groups of animals studied are shown in Table 2. CFU of total aerobic bacteria and Enterobacteriaceae were greater in PVL than in normal rats; however, differences did not achieve statistical significance (P = 0, 11). LA-supplemented diet did not

Fig 1. Rate of bacterial translocation (BT) in PVL rats treated with LA (ATCC 832) supplemented diet or placebo. As can be seen, no difference in occurrence of bacterial translocation was observed between study groups.

1138

TABLE 2. INTESTINAL MICROFLORA IN TOTAL NUMBERS OF AEROBIC AND ANEROBIC BACTERIA IN FECAL SPECIMEN FROM PVL AND CONTROL RATS TREATED WITH LA (ATCC 832) SUPPLEMENTED DIET OR PLACEBO Total aerobic × (107 /ml)

LA Placebo

Total anaerobic (×108 /ml)

PVL

Normal

PVL

Normal

28.0 ± 18.3 21.7 ± 13.0

0.54 ± 0.3 0.47 ± 0.3

11.2 ± 3.9 14.8 ± 5.4

11.0 ± 1.1 11.8 ± 0.8

induce a significant change in the total number, of aerobic or anerobic bacteria in stool specimens of PVL or normal rats. (Table 2). In addition, no changes in the composition of intestinal flora regarding different Enterobacteriaceae subtypes were observed after LA-supplemented diet (Figure 2). Similar results were obtained with LGG in normal rats, that is total fecal aerobic and anerobic counts in LGG-treated (N = 4) and placebo-treated (N = 4) rats were not significantly different (aerobes: 12.65 ± 2.35 × 107 /ml vs 7.58 ± 1.71 107 /ml, respectively, anerobes 22.25 ± 4.23 108 /ml vs 12.5 ± 3.38 108 /ml, respectively) with no changes in composition of intestinal flora (data not shown). LGG colonies could be identified on culture plates of cecum contents of LGG-treated rats but not of placebo-treated animals. DISCUSSION BT is the main mechanism in the genesis of spontaneous infections in cirrhosis. Therefore, its inhibition is appealing as the optimal means to prevent such infections. In fact, selective intestinal decontamination with norfloxacin has been shown to be useful in preventing bacterial infections in cirrohotic patients with gastrointestinal hemorrahage and in those with a prior history of spontaneous bacterial peritonitis (SBP) (30). However, in addition to its cost, chronic antibiotic prophylaxis has serious drawbacks, specifically, the emergence of bacteria resistant to quinolones and a change in the spectrum of bacteria responsible for infections (31–34). Therefore, it would be ideal to find an alternative method to antibiotics, a simple, uncomplicated, and inexpensive way of decreasing BT and the risk of infections. The main mechanisms inplicated in BT are bacterial overgrowth, disruption of the gut mucosal barrier, and impaired host defenses (3). For each of these pathogenic mechanisms, probiotics such as lactobacilli have been shown to be beneficial. Lactobacilli constitute an integral part of the normal gastrointestinal microecology and may play the most important role in the preservation of the natural biological equilibrium of the intestinal tract and the growth modulation of other groups of bacteria (23). Moreover, lactobacilli have been shown to stabilize gut mucosal Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

Lactobacillus AND BACTERIAL TRANSLOCATION IN PORTAL HYPERTENSION

Fig 2. Enterobacteriaceae and subpopulations in PVL rats treated with LA (ATCC 832)-supplemented diet or placebo. As can be seen, Lactobacillus-treated animals (shown on the left side) exhibited an identical fecal composition of Enterobacteriaceae, mainly E. coli and Klebsiella.

barrier (35) and to enhance host resistance against infection (20, 36). Therefore, Lactobacillus-supplemented diet would appear to be a promising alternative to antibiotics in preventing BT. BT has been shown to be increased both in acute portal hypertension and cirrhosis (4–7). Previous studies from our laboratory show that the highest rate of BT occurs in rats with acute portal hypertension, ie, those studied 2 days after the induction of portal hypertension (4, 5). This high incidence of BT makes this model of portal hypertension well suited for investigating the therapeutic potency of Lactobacillus-supplemented diet regarding prevention of BT in portal hypertension. In the present study, the observed rate of BT in placebotreated PVL rats (75%) is consistent with our previous study (4). Unfortunately, the rate of BT was not different in PVL animals that received the Lactobacillussupplemented diet (82%). One explanation is that we were also unable to demonstrate a change in intestinal microflora, despite evidence of intestinal colonization by the organism. While we observed an increase, albeit not significant, in cecal colonies of aerobic Enterobacteriaceae in PVL rats as compared to sham rats, both aerobic (including Enterobacteriaceae) and anerobic organisms were not different in Lactobacillus-treated versus placebo-treated animals. Aerobic gram-negative bacilli, specifically Enterobacteriacea, are the most frequently isolated organisms in infections complicating cirrhosis (1, 37). It is Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

known that different subgroups of intestinal bacteria differ in their ability to pass through the lamina propria into MLN (11, 12, 38). While aerobic gram-negative bacilli translocate easily even across a histologically intact intestinal epithelium, anerobic organisms rarely pass through the mucosa (11–13). A direct relationship between specific strains of cecal organisms and their translocation to MLN has been observed (39). Therefore, any reduction in intestinal aerobic gram-negative bacilli and/or an increase in anerobic organisms should reduce the susceptibility for BT. In fact, the oral administration of lactobacilli has been shown to inhibit the growth of aerobic gram-negative bacteria (15, 16) via changes in intestinal environmental factors, including increased formation of short-chain fatty acids and reduction of colonic pH, which promotes the growth of gram-positive and anerobic bacteria (14–16). The lack of effect on BT also excludes a sufficient action of Lactobacillus on other mechanisms involved in the process of BT in portal hypertension. For instance, stabilization of gut mucosal barrier by Lactobacillus has been reported and has been attributed to induction of growth factors (40), maintenance of gastrointestinal epithelial proliferation and function (41), and inhibition of adherence and invasion of enterovirulent bacteria to intestinal cells (35, 42). However, our data are in accordance with studies in experimental inflammatory bowel disease demonstrating the failure of lactobacilli to improve gut permeability in a model of severe colitis

1139

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

WIEST ET AL

(43). Moreover, lactobacilli have been reported to improve host immunity and resistance against infection (20, 36) by increasing global phagocytic activity (44, 45), induction of interferon and cytokine production, and an increase in killer cells and T lymphocytes (46), enhancing intestinal IgA secretion, and improving removal of toxins (45, 47). These latter effects however, are more likely to be beneficial in preventing BT in cirrhosis, which is known to be associated with decreased host defense mechanisms due to decerased reticuloendothelial phagocytic activity and reduced intestinal IgA concentrations as well as deficiencies in serum immunoglobulins, complement, and qualitative neutrophil function (1, 48–50). Therefore, a potential benefit of lactobacilli in cirrhosis can not be ruled out. Moreover, in the experimental model of portal vein ligation, in contrast to liver cirrhosis, development of ascites and bacterial peritonitis is not observed and, hence, results can not be extrapolated readily to the clinical situation in patients with liver cirrhosis. In contrast to our results, Lactobacillus dietary supplements have been shown to decrease BT in experimental models of acute liver injury (17, 18) and liver failure (19), as well as in different forms of enterocolitis (24, 28). These discrepancies are unlikely to be due to an insufficient dose of lactobacilli since, in our study, approximately 1 × 1010 organisms were gavaged per day which, to our knowldge, is the highest dose of lactobacilli administered in rat experiments investigating changes in intestinal microflora. Nonetheless, it can not be ruled out that even higher doses and/or treatment for a longer period of time could induce favorable changes in bacterial microflora. An ongoing controversy relates to the probiotic potency and colonization efficiency of different strains of lactobacilli (14–17; 24). For this reason, we tested a different type of Lactobacillus, which also failed to alter intestinal microflora. Nonetheless, we cannot exclude the possibility that the stated discrepancies may be related to species differences or starin-specific variations in probiotic potency. Finally, the effect on BT seen in animal models of acute liver failure induced by subtotal liver resection (19) or acute liver injury due to D-galactosamine (17) may be related to different mechanisms of BT acting in these conditions as compared to our model of acute prehepatic portal hypertension. In conclusion, the lack of effect of Lactobacillussuplemented diet on BT and intestinal microflora of PVL animals suggests that this measure is not effective in preventing infections in portal hypertension. However, these results would need to be extended to a cirrhotic anima model in which an effect of Lactobacillus on immunity could be of additional benefit.

1140

ACKNOWLEDGMENTS The authors gratefully acknowledge Maryann Vergato for secretarial assistance and preparation of this manuscript.

REFERENCES 1. Wyke RJ: Problems of bacterial infection in patients with liver disease. Gut 28:623–641, 1987 2. Garcia-Tsao G: Spontaneous bacterial peritonitis. Gastroenterol Clin North Am 21:257–275, 1991 3. Berg RD: Bacterial translocation from the gastrointestinal tract. Adv Exp Med Biol 473:11–30, 1999 4. Garcia-Tsao G, Albillos A, Barden GE, West AB: Bacterial translocation in acute and chronic portal hypertension. Hepatology 17:1081–1085, 1993 5. Garcia-Tsao G, Lee FY, Barden GE, Cartun R, West AB: Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites. Gastroenterology 108:1835–1841, 1995 6. Llovet JM, Bartoli R, Planas R, Cabre E, Jimenez M, Urban A, Ojanguren I, Arnal J, Gassull MA: Bacterial translocation in cirrhotic rats. Its role in the development of spontaneous bacterial peritonitis. Gut 35:1648–1652, 1994 7. Runyon BA, Squier S, Borzio M: Translocation of gut bacteria in rats with cirrhosis to mesenteric lymph nodes partially explains the pathogenesis of spontaneous bacterial peritonitis. J Hepatol 21:792– 796, 1994 8. Guarner C, Runyon BA, Young S, Heck M, Sheikh MY: Intestinal bacterial overgrowth and bacterial translocation in cirrhotic rats with ascites. J Hepatol 26:1372–1378, 1997 9. Lal D, Gorbach S, Levitan R: Intestinal microflora in patients with alcoholic cirrhosis: urea splitting bacteria and neomycin resistance. Gastroenterology 62:275–279, 1972 10. Casafont F, de las Heras G, Martin L, Lopez M, Ledesma F, Pons F: Small bowel bacterial overgrowth in patients with alcoholic cirrhosis. Dig Dis Sci 40:1252–1256, 1995 11. Steffen EK, Berg RD, Deitch EA: Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenterc lymph node. J Infect Dis 157:1032–1038, 1988 12. Wells CL: Relationship between intestinal microecology and the translocation of intestinal bacteria. Antonie Van Leeuwenhoek 58:87–93, 1990 13. Wells CL, Maddaus MA, Reynolds CM, Jechorek RP, Simmons RL: Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria. Infect Immun 55:2689–2694, 1987 14. Johansson ML, Molin G, Jeppsson B, Nobaek S, Ahrne S, Bengmark S: Administration of different Lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effec on the indigenous flora. Appl Environ Microbiol 59:15–20, 1993 15. Stolte M, Kroher G, Meining A, Morgner A, Bayerdorffer E, Bethke B: Modification of the intestinal microflora using probiotics and prebiotics. Scand J Gastroenterol 32(suppl 222):28–31, 1997 16. Salminen S, Salminen E: Lactulose, lactic acid bacteria, intestinal microecology and mucosal protection. Scand J Gastroenterol Suppl 222:45–48, 1997 17. Adawi D, Kasravi FB, Molin G, Jeppsson B: Effect of Lactobacillus supplementation with and without arginine on liver damage and bacterial translocation in an acute liver injury model in the rat. Hepatology 25:642–647, 1997 Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

P1: IBB PP692L-ddas-467457

DDAS.cls

May 9, 2003

12:59

Lactobacillus AND BACTERIAL TRANSLOCATION IN PORTAL HYPERTENSION 18. Kasravi FB, Adawi D, Molin G, Bengmark S, Jeppsson B: Effec of oral supplementation of lactobacilli on bacterial translocation in acute liver injury induced by D-galactosamine. J Hepatol 26:417– 424, 1997 19. Wang XD, Soltesz V, Molin G, Andersson R: The role of oral administration of oatmeal fermented by Lactobacillus reuteri R2LC on bacterial translocation after acute liver failure induced by subtotal liver resection in the rat. Scand J Gastroenterol 30:180–185, 1995 20. Perdigon G, de Macias ME, Alvarez S, Oliver G, de Ruiz Holgado AP: Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei and Lactobacillus acidophilus. Immunology 63:17–23, 1988 21. Tahara T, Oshimura M, Umezawa C, Kanatani K: Isolation, partial characterization, and mode of action of Acidocin J1132, a twocomponent bacteriocin produced by Lactobacillus acidophilus JCM 1132. Appl Environ Microbiol 62:892–897, 1996 22. Bernet-Camard MF, Lievin V, Brassart D, Neeser JR, Servin AL, Hudault S: The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Appl Environ Microbiol 63:2747–2753, 1997 23. Fernandes CF, Shahani K, Armer M: Therapeutic role of dietary lactobacilli and lactobacilli fermented dairy products. FEMS Microbiol Rev 46:343–356, 1987 24. Mao Y, Nobaek S, Kasravi B, Adawi D, Stenram U, Molin G, Jeppsson B: The effects of Lactobacillus starins and oat fiber on methotrexate-induced enterocolitis in rats. Gastroenterology 111:334–344, 1996 25. Fuller R: Probiotics in human medicine. Gut 32:439–442, 1991 26. Salminen S: Functional dairy foods with Lactobacillus strain GG. Nutr Rev 54:S99–101, 1996 27. 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 37:121–128, 1992 28. Gorbach SL, Chang TW, Goldin B: Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG. Lancet (letter) 2:1519, 1987 29. Siitonen S, Vapaatalo H, Salminen S, Gordin A, Saxelin M, Wikberg R, Kirkkola AL: Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated diarrhoea. Ann Med 22:57–59, 1990 30. Rimola A, Garcia-Tsao G, Navasa M, Piddock LJ, Planas R, Bernard B, Inadomi JM: Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consenus document. J Hepatol 32:142– 153, 2000 31. Dupeyron C, Mangeney N, Sedrati L, Campillo B, Fouet P, Leluan G: Rapid emergence of quinolone, resistance in cirrhotic patients treated with norfloxacin to prevent spontaneous bacterial peritonitis. Antimicrob Agents Chemother 38:340–344, 1994 32. Llovet JM, Rodriguez-Iglesias P, Moitinho E, Planas R, Bataller R, Navasa M, Menacho M, Pardo A, Castells A, Cabre E, Arroyo V, Gassull MA, Rodes J: Spontaneous bacterial peritonitis in patients with cirrhosis undergoing selective intestinal decontamination. A retrospective study of 229 spontaneous bacterial peritonitis episodes. J Hepatol 26:88–95, 1997 33. Jackson RJ, Smith SD, Rowe MI: Selective bowel decontamination results in gram-positive translocation. J Surg Res 48(5):444–447, 1990

Digestive Diseases and Sciences, Vol. 48, No. 6 (June 2003)

34. Ortiz J, Vila MC, Sorano G, Minana J, Gana J, Mirelis B, Novella MT, Coll S, Sabat M, Andreu M, Prats G, Sola R, Guarner C: Infections caused by Escherichia coli resistant to norfloxacin in hospitalized cirrhotic patients. Hepatology 29(4):1064–1069, 1999 35. Salminen S, Isolauri E, Salminen E: Clinical uses of probiotics for stabilizing the gut mucosal barrier successful strains and future challenges. Antonie Van Leeuwenhoel 70:347–358, 1996 36. Link H, Rochat F, Saudan KY, Schiffrin E: Immunomodulation of the gnotobiotic mouse through colonization with lactic acid bacteria. Adv Exp Med Biol 371A:465–467, 1995 37. Caly WR, Struss E: A prospective study of bacterial infections in patients with cirrhosis [see comments]. J Hepatol 18:353–358, 1993 38. Wells CL, Maddaus MA, Simmons RL: Proposed mechanisms for the translocation of intestinal bacteria Rev Infect Dis 10:958–979, 1988 39. Steffen EK, Berg RD: Relationship between cecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes. Infect Immun 39:1252–1259, 1983 40. Gorbach SL: Lactic acid bacteria and human health Ann Med 22:37– 41, 1990 41. Goodlad RA, Wright NA: Changes in intestinal cell proliferation, absorptive capacity and structure in young, adult and old rats. J Anat 173:109–118, 1990 42. 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 35:483–489, 1994 43. Kennedy RJ, Hoper M, Deodhar K, Kirk SJ, Gardiner KR: Probiotic therapy fails to improve gut permeability in a hapten model of colitis. Scand J Gastroenterol 35:1266–1271, 2000 44. Schiffrin EJ, Brassart D, Servin AL, Rochat F, Donnet-Hughes A: Immune modulation of blood leukocytes in humans by lactic acid bacteria criteria for strain selection. Am J Clin Nutr 66:515S–520S, 1997 45. Sato K, Saito H, Tomioka H, Yokokura T: Enhancement of host resistance against Listeria infection by Lactobacillus casel: efficacy of cell wall preparation of Lactobacillus casei. Microbiol Immunol 32:1189–1200, 1988 46. Solis-Pereyra B, Aattouri N, Lemonnier D: Role of food in the stimulation of cytokine production Am J Clin Nutr 66:521S–525S, 1997 47. Perdigon G, de Macias ME, Alvarez S, Oliver G, de Ruiz Holgado AA: Effect of perorally administered lactobacilli on macrophage activation in mice. Infect Immun 53:404–410, 1986 48. Rimola A, Soto R, Bory F, Arroyo V, Piera C, Rodes J: Reticuloendothelial system phagocytic activity in cirrhosis and its relation to bacterial infections and prognosis. Hepatology 4:53–58, 1984 49. Rabinovitz M, Gavaler JS, Kumar S, Kajani M, Van Thiel DH: Role of serum complement, immunoglobulins, and cell-mediated immune system in the pathogenesis of spontaneous bacterial peritonitis (SBP) Dig Dis Sci 34:1547–1552, 1989 50. Fierer J, Finley F: Deficient serum bactericidal activity against Escherichia coli in patients with cirrhosis of the liver J Clin Invest 63:912–921, 1979

1141