Effect of Lactobacillus-Fermented Diets

Digestive Diseases and Sciences,Vol.48,No.6(June2003),pp.1136–1141(C 2003)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 trans-location of gut-derived bacteria,a process promoted by intestinal bacterial overgrowth and disruptionof the gut mucosal barrier.Bacteriotherapy with Lactobacillus has been reported to correct bacterialovergrowth,stabilize mucosal barrier function,and decrease bacterial translocation in rat models ofacute liver injury and failure.In this study we investigated the effect of Lactobacillus-supplementeddiets on intestinalflora and on bacterial translocation rate in portal vein ligated ctobacillus-fermented milk(yogurt)containing at least2×109colony forming units/ml or placebo(water)wasadminstrated by gavage twice daily(2ml)for9days.Portal vein ligation was performed on day7of treatment.Bacterial translocation to mesenteric lymph nodes and quantification of intestinalflora was assessed by standard bacteriological cultures.Bacterial translocation was not significantlydifferent between animals that received yogurt(82%)and those that received placebo(75%).Yogurtdid not induce any significant changes in intestinalflora,whether it was produced with Lactobacillusacidophilus or Lactobacillus GG.In conclusion,in acute prehepatic portal hypertension,bacterio-therapy with Lactobacillus was unable to induce changes in bacterial translocation probably becauseit was unable to induce changes in bacterialflora.KEY WORDS:portal hypertension;bacterial translocation;mesenteric lymph nodes;Lactobacillus,intestinal microflora.Cirrhosis predisposes to the development of severe bacte-rial infections,mainly spontaneous bacteremia and spon-taneous 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 extraintesti-Manuscript received XXXX2002;accepted March7,2003.From the*Hepatic Hemodynamic Laboratory,Veterans Adminis-tration Medical Center,West Haven,Connecticut06516,USA;and †Department of Comparative Medicine and‡Department of Medicine,Yale University School of Medicine,New Haven,Connecticut06510, USA.Supported by grants from the NIDDK(721-H-414776),V A 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 Cen-ter,950Campbell Avenue.West Haven,Connecticut06516,USA.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 por-tal 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 infec-tions in portal hypertension and cirrhosis are,therefore, directed towards eliminating or reducing BT.An imbal-ance 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 over-growth,predominantly of aerobic gram-negative bacteria, especially in severe liver disease(8–10).Anaerobic bac-teria,which represent more than90%of the gutflora,1136Digestive Diseases and Sciences,Vol.48,No.6(June2003)0163-2116/03/0600-1136/0C 2003Plenum Publishing CorporationLactobacillus AND BACTERIAL TRANSLOCATION IN PORTAL HYPERTENSIONdo not translocate readily,while aerobic gram-negative bacilli translocate easily,even across a histologically in-tact intestinal epithelium(11–13).Lactobacilli constitute an integral part of the normal gastrointestinal microecol-ogy and promote growth of gram-positive and anaerobic bacteria while inhibiting the growth of gram-negative bac-teria(14–16).Lactobacilli have been shown to be able to prevent bacterial overgrowth of potentially pathogenic bacteria(15,16).For instance,bacteriotherapy with lac-tobacilli 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 fer-mented with Lactobacillus)on BT and on intestinalflora 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,100mg/kg body wt;Parke,Davis,Avon,Connecticut, USA),After a midline abdominal incision,the portal vein was freed from surrounding tissue.A ligature(silk gut3-0)was placed around a20-gauge blunt-tipped needle lying alongside the portal vein Subsequent removal of the needle yielded a cali-brated 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(100mg/kg), and the abdominal skin was shaved and sterilized with an io-dine solution.All the following surgical procedures were per-formed 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±0.01g.Tissues were then homogenized in a measured amount of saline,and aliquots of0.1ml were plated onto blood,McConkey,and phenylethyl alcohol agar plates(BBL Pre-pared Media,Becton Dickinson Microbiology Systems,Cock-eysville,Maryland,USA).Solid culture media were examined and colonies counted after24and48hr 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 Collec-tion(ATCC)catalog number832,Rockville,Maryland,USA) was cultured in DeMan Rogosa Sharpe(MRS)broth in anerobic conditions for24hr at37◦ctose-free milk was then inocu-lated with approximately108CFU/ml of LA and fermented for 18hr at37◦C to obtain a yogurt.Thefinal number of lactobacilli,determined as viable counts on Rogosa agar after incubation, was tested in separate experiments to be at least1×10CFU/ml yogurt.The yogurt was resuspended(20%)in drinking water to increasefluidity.Two milliliters of this suspension(containing at least4×109viable 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,lactobacilli-fermented milk has been demonstrated to exert antimicrobial acitivity and to enhance the immune response(20–23).This sug-gests 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 Gorbach-Goldbach(GG)(LGG)(Culturelle,CVS Pharmacy,New Haven, Connecticut,USA)was used for preparation of yogurt following the same procedure.Experimental Design.A total of46rats were studied (Table1).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 as2ml of diluted yogurt with at least4×109viable lactobacilli or2ml of drinking water(placebo)by (or water)were ad-ministered for9days prior to the experimental procedure.An investigator separate from the ones performing the experiments, adminstered yogurt or placebo.The investigators performing ex-periments were blinded as to the adminstration of LA supple-ments or water.PVL or sham operation were performed on day 7of treatment;thus,experiments were performed2days 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 addi-tional 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 Lac-tobacillus strains in human and experimental investigations on probiotics(26–29).Determination of Intestinal Microflora.Samples taken from the cecum were collected into sterile tubes containing5ml of transport medium and weighed.Serial dilution in the same transport medium was performedfive times before placing1ml of the sample on brain–heart infusion agar.Total aerobic and anaerobic bacterial counts were made by incubating the plates for3days at37◦C under aerobic or anerobic conditions,re-spectively.After that,the colony forming units(CFU)on each plate were corrected to the weight of the original sample Fur-ther identification of gram-negative anaerobes,lactobacilli,and T ABLE1.A NIMALS S TUDIED IN D IFFERENT G ROUPSGroup PVL ControlsLA-ATCC832118Placebo127LA-GG4Placebo4Total2323Digestive Diseases and Sciences,Vol.48,No.6(June2003)1137WIEST ET ALEnterobacteriaceae were performed using appropriate selective culture media.Statistical Analyses.The main comparisons were made be-tween 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.RESULTSAnimals.There were no significant differences in body weight in the experimental groups.No significant dif-ference in MLN weight used for assessment of BT in the different study groups was noted.PVL rats showed significantly higher spleen weights,expressed as per-centage of body weight,as compared to sham animals (PVL 2.56±0.78vs sham 2.30±0.65mg/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 Lactobacillus -treated rats (9/11or 82%)and in the untreated groups (9/12or 75%)(Ns)(Figure 1).Intestinal Microflora.Results on intestinal total aer-obic 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 nor-mal rats;however,differences did not achieve statistical significance (P =0,11).LA-supplemented diet didnotFig 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.T ABLE 2.I NTESTINAL M ICROFLORA IN T OTAL N UMBERS OF A EROBICAND A NEROBIC B ACTERIA IN F ECAL S PECIMEN FROM PVL AND C ONTROL R ATS T REATED WITH LA (ATCC 832)S UPPLEMENTED D IETOR P LACEBOTotal aerobic ×(107/ml)Total anaerobic (×108/ml)PVLNormal PVL Normal LA 28.0±18.30.54±0.311.2±3.911.0±1.1Placebo21.7±13.00.47±0.314.8±5.411.8±0.8induce 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 composi-tion of intestinal flora regarding different Enterobacteri-aceae subtypes were observed after LA-supplemented diet (Figure 2).Similar results were obtained with LGG in nor-mal 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±1.71107/ml,respectively,anerobes 22.25±4.23108/ml vs 12.5±3.38108/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.DISCUSSIONBT is the main mechanism in the genesis of spontaneous infections in cirrhosis.Therefore,its inhibition is appeal-ing as the optimal means to prevent such infections.In fact,selective intestinal decontamination with norfloxacin has been shown to be useful in preventing bacterial infec-tions in cirrohotic patients with gastrointestinal hemor-rahage and in those with a prior history of spontaneous bacterial peritonitis (SBP)(30).However,in addition to its cost,chronic antibiotic prophylaxis has serious draw-backs,specifically,the emergence of bacteria resistant to quinolones and a change in the spectrum of bacteria re-sponsible for infections (31–34).Therefore,it would be ideal to find an alternative method to antibiotics,a sim-ple,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 im-paired host defenses (3).For each of these pathogenic mechanisms,probiotics such as lactobacilli have been shown to be benefictobacilli constitute an integral part of the normal gastrointestinal microecology and may play the most important role in the preservation of the nat-ural biological equilibrium of the intestinal tract and the growth modulation of other groups of bacteria (23).More-over,lactobacilli have been shown to stabilize gut mucosal1138Digestive Diseases and Sciences,Vol.48,No.6(June 2003)Lactobacillus AND BACTERIAL TRANSLOCATION IN PORTALHYPERTENSIONFig 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 infec-tion (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 2days 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 placebo-treated PVL rats (75%)is consistent with our previous study (4).Unfortunately,the rate of BT was not dif-ferent in PVL animals that received the Lactobacillus -supplemented diet (82%).One explanation is that we were also unable to demonstrate a change in intestinal mi-croflora,despite evidence of intestinal colonization by the organism.While we observed an increase,albeit not sig-nificant,in cecal colonies of aerobic Enterobacteriaceae in PVL rats as compared to sham rats,both aerobic (includ-ing 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 or-ganisms in infections complicating cirrhosis (1,37).It isknown 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 translo-cate 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 in-testinal 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 bac-teria (15,16)via changes in intestinal environmental fac-tors,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 in-stance,stabilization of gut mucosal barrier by Lacto-bacillus has been reported and has been attributed to induction of growth factors (40),maintenance of gastroin-testinal epithelial proliferation and function (41),and in-hibition of adherence and invasion of enterovirulent bac-teria 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 colitisDigestive Diseases and Sciences,Vol.48,No.6(June 2003)1139WIEST ET AL(43).Moreover,lactobacilli have been reported to im-prove 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),enhanc-ing 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 phago-cytic activity and reduced intestinal IgA concentrations as well as deficiencies in serum immunoglobulins,com-plement,and qualitative neutrophil function(1,48–50). Therefore,a potential benefit of lactobacilli in cirrho-sis can not be ruled out.Moreover,in the experimental model of portal vein ligation,in contrast to liver cir-rhosis,development of ascites and bacterial peritonitis is not observed and,hence,results can not be extrapo-lated readily to the clinical situation in patients with liver cirrhosis.In contrast to our results,Lactobacillus dietary supple-ments 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,approximately1×1010 organisms were gavaged per day which,to our knowldge, is the highest dose of lactobacilli administered in rat ex-periments 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 lacto-bacilli(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 possibil-ity that the stated discrepancies may be related to species differences or starin-specific variations in probiotic po-tency.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 Lactobacillus-suplemented diet on BT and intestinal microflora of PVL animals suggests that this 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