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On Lactobacillus plantarum 299v, bacterial translocation and intestinal permeability. Mangell, Peter 2007 Link to publication Citation for published version (APA): Mangell, P. (2007). On Lactobacillus plantarum 299v, bacterial translocation and intestinal permeability. Department of Surgery, Malmö University Hospital, Lund University. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 16. Mar. 2019

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Page 1: On Lactobacillus plantarum 299v, bacterial translocation ...lup.lub.lu.se/search/ws/files/4896174/547953.pdf · Lactobacillus Plantarum Prevents Translocation in Sepsis MANGELL etal

LUND UNIVERSITY

PO Box 117221 00 Lund+46 46-222 00 00

On Lactobacillus plantarum 299v, bacterial translocation and intestinal permeability.

Mangell, Peter

2007

Link to publication

Citation for published version (APA):Mangell, P. (2007). On Lactobacillus plantarum 299v, bacterial translocation and intestinal permeability.Department of Surgery, Malmö University Hospital, Lund University.

General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authorsand/or other copyright owners and it is a condition of accessing publications that users recognise and abide by thelegal requirements associated with these rights.

• Users may download and print one copy of any publication from the public portal for the purpose of private studyor research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portalTake down policyIf you believe that this document breaches copyright please contact us providing details, and we will removeaccess to the work immediately and investigate your claim.

Download date: 16. Mar. 2019

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Paper II

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APMIS 114: 611–8, 2006 C 2006 The AuthorsPrinted in Denmark . All rights reserved

Journal Compilation C 2006 APMIS

ISSN 0903-4641

Adhesive capability of Lactobacillus plantarum299v is important for preventing bacterial translocation

in endotoxemic rats

PETER MANGELL,1 PERNILLA LENNERNAS,2 MEI WANG,3 CRISTER OLSSON,1 SIV AHRNE,3

GORAN MOLIN,3 HENRIK THORLACIUS1 and BENGT JEPPSSON1

1Department of Surgery, Malmö University Hospital, Lund University, Malmö, 2Department for Cell andOrganism Biology, and 3Laboratory of Food Hygiene, Department of Food Technology, Engineering and

Nutrition, Lund University, Lund, Sweden

Mangell P, Lennernäs P, Wang M, Olsson C, Ahrne S, Molin G, Thorlacius H, Jeppsson B. Adhesivecapability of Lactobacillus plantarum 299v is important for preventing bacterial translocation in endo-toxemic rats. APMIS 2006;114:611–8.

The preventive effect of the probiotic Lactobacillus plantarum 299v on bacterial translocation (BT)and the role of adhesion were studied in septic rats. Five groups of rats were pretreated as follows:negative and positive control groups received regular drinking water; the oatmeal group receiveddrinking water mixed with oatmeal; the Lp 299v group received drinking water mixed with oatmealcontaining 109 colony-forming units (CFU) L. plantarum 299v/ml; the Lp 299v-adhª group receiveddrinking water with oatmeal containing 109 CFU/ml of modified L. plantarum 299v (L. plantarum299v-adhª) lacking adhesive properties to enterocytes. On day 8, all rats except the negative controlgroup were given lipopolysaccharide (LPS) intraperitoneally. After 24 h, mesenteric lymph node(MLN), liver and ileum were harvested for culture. Incidence of BT after LPS challenge was 25% and88% in MLN and liver, respectively. BT increased to 75% in MLN and 100% in liver of endotoxemicrats pretreated with oatmeal. Pretreatment with L. plantarum 299v reduced BT to 0% and 12% inMLN and liver, respectively. L. plantarum 299v-adhª did not prevent BT to MLN. Flow cytometryrevealed reduced adherence of these bacteria to intestinal epithelial cells compared to L. plantarum299v. Thus, L. plantarum 299v prevents BT in septic rats, an effect probably dependent on bacterialadherence to the intestinal mucosa. Further, our findings indicate that oatmeal (prebiotics) withoutprobiotics does not prevent BT during sepsis.

Key words: Bacterial translocation; probiotics; prebiotics; sepsis; bacterial adhesion; Lactobacillusplantarum.

Bengt Jeppsson, Department of Surgery, Malmö University Hospital, SE-205 02 Malmö, Sweden. e-mail: bengt.jeppsson/med.lu.se

Bacterial translocation (BT) is considered to bea central pathophysiological process in sepsis(1) and associated with postoperative septiccomplications (2). Therapies aimed at dimin-ishing or abating BT, and thus reducing infec-tious morbidity, include the use of systemic anti-

Received 28 October 2005.Accepted 26 April 2006.

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biotics and gut decontamination. This, however,involves the risk of bacterial multiresistance andsecondary infections. Interest has now shiftedtowards the possibility of stabilizing the gut mi-croflora and thereby preventing potentiallypathogenic bacteria from increasing in numbersand translocating across the intestinal barrier(3). One way to modulate the gut microflora isto administer probiotic bacteria, i.e. viable bac-

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64

Lactobacillus Plantarum Prevents Translocation in SepsisMANGELL et al.

teria, which – when ingested – alter the intesti-nal microflora and thereby affect the host ben-eficially (4). Supplementation with probioticshas proven effective in preventing pancreaticsepsis (5) and postoperative infection in livertransplant patients (6). It has previously beenshown that a specific probiotic strain, Lacto-bacillus plantarum 299v, can reduce intestinalpermeability induced by Escherichia coli (7) andabate bacterial translocation in methotrexate-induced enterocolitis (8) and liver injury (9).

The exact mechanism behind BT is still notknown, but attachment of bacteria to the mu-cosal surface is a prerequisite in the patho-genesis of many infections originating from thegut (10). Also, for a probiotic bacterium to exertany biological effect, adherence to the mucosaseems to be of importance (11, 12). However, itremains to be clarified whether adherence of aprobioticum is a prerequisite for preventing BTof the indigenous microflora.

When supplied, probiotics are sometimesgiven together with prebiotics. These are dietarynon-digestible carbohydrates with the ability toselectively stimulate growth of one or a limitednumber of bacteria (4), often lactobacilli andbifidobacteria, and thereby contribute to en-hancement of the intestinal barrier function.This is also the rationale for providing criticallyill patients with small amounts of enteral nu-trition containing dietary fiber and thus stimu-lating the intestinal mucosa. However, the effectof prebiotics on the indigenous microflora in aseptic state is not known.

The aim of the present study was to examinewhether oral pretreatment with the probiotic L.plantarum 299v may prevent BT to MLN andliver in rats challenged with endotoxin, andwhether such a preventive effect is related to ad-hesive capacity of L. plantarum 299v to the intes-tinal mucosa. Further, the effect of the dietaryfibers of oats on BT during sepsis was studied.

MATERIAL AND METHODS

Experimental designMale Sprague-Dawley rats (Möllegaard, Skensved,

Denmark) weighing 321–423 g were used 7 days afterarrival at the animal facility, where they had free accessto standard rat chow (B & K Universal, Sollentuna,Sweden) and water ad libitum. All experiments were

612

approved by the local Animal Ethics Committee atLund University. A total of 44 rats were divided intofive groups and pretreated one week before the experi-ment as follows: negative (nΩ10) and positive controlgroup (nΩ8) received regular drinking water; the oat-meal group (nΩ8) received regular drinking watermixed with an oatmeal drink containing 18.5 g oat-meal per 100 g drink (13) with an energy content of 325kJ per 100 ml drink (14); the Lp 299v group (nΩ8) re-ceived regular drinking water mixed with oatmeal con-taining 109 colony-forming units (CFU) of L. plan-tarum 299v/ml; the Lp 299v-adhª group (nΩ10) re-ceived regular drinking water mixed with oatmealcontaining 109 CFU of L. plantarum 299v-adhª/ml.This is a strain that originates from L. plantarum 299vbut has lost the ability to adhere to HT 29 cells (11).The twoL. plantarum strains were grown in an oatmealdrink (14), which resulted in a decreased pH (final pHaround 4) and an increased concentration of lactic acidin the drink. In both the Lp 299v and Lp 299v-adhª

groups, 50 ml of the oatmeal drink containing one ofthe bacteria was mixed with 200 ml tap water for eachcage housing two rats. Thus, each pair of rats had ac-cess to 1010 CFU of bacteria per day. Following pre-treatment, the rats were temporarily sedated with CO2and given an intraperitoneal injection of 1 ml sterile0.9% saline with or without 10 mg/kg body weightlipopolysaccharide (LPS) from Escherichia coli sero-type O111:B4 (Sigma, Stockholm, Sweden) (15). After24 h, rats were anesthetized with diethylether andthrough a midline laparotomy under aseptic con-ditions a mesenteric lymph node (MLN) from the ileo-cecal junction was harvested and a tissue specimenwastaken from the liver. Also, 2 cm of distal ileumwas har-vested, split longitudinally, and rinsed in sterile salineto remove fecal matter. All specimens were put intopre-weighed vials containing 3 ml sterile transport me-dium (0.9% sodium chloride, 0.1% peptone, 0.1% Twe-en 80, 0.02% cysteine) (16) and weighed.

Bacterial translocationSamples from MLN and liver were placed in an

ultrasonic bath (Millipore, Sundbyberg, Sweden) for 2min and vortexed on a Chiltern (Thera-Glas, Gothen-burg, Sweden) for 2 min. Viable counts were obtainedby placing 1 ml of each suspended tissue sample onbrain heart infusion agar (BHI; Difco Laboratories,Detroit, MI, USA) and incubated aerobically and an-aerobically (Gas Pack System, Gas Pack, BecktonDickenson Microbiology Systems, Cockeysville, MD,USA) at 37 æC for 3 days, thus yielding total aerobicand anaerobic counts, respectively. The coloniesformed on each plate were counted and the result wasexpressed as CFU per gram tissue.

Intestinal bacteriaDistal ileum samples were placed in an ultrasonic

bath and vortexed as described above. Bacterial vi-able counts were obtained by cultivation on violet red

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Paper IIL. PLANTARUM PREVENTS TRANSLOCATION IN SEPSIS

bile glucose agar (VRBG; Oxoid, Hampshire, Eng-land) incubated aerobically at 37 æC for 24 h (Enter-obacteriaceae counts) and on Rogosa agar (DifcoLaboratories) incubated anaerobically for 3 days(lactobacilli counts).

Flow cytometryThe ability of the two bacterial strains to adhere

to HT-29 cells was analyzed by flow cytometry. Thebacteria were cultured overnight on Rogosa agar forviable counts. The strains were then cultured for 20h in Lactobacillus Carrying Media (LCM) with 1%glucose. After harvesting, centrifugation and washingtwice, 300 ml bacteria (2¿109 CFU/ml) was stainedwith 300 ml 20 mM carboxyfluorescein diacetate(CFDA-SE, Molecular Probes Inc., Eugene, OR,USA). HT-29 cells were grown in culture flasks withDulbecco’s Modified Eagle’s Medium (DMEM,Sigma Aldrich Ltd, Irvine, Ayrshire, UK) supple-mented with 5% fetal bovine serum, 2 mM L-gluta-mine, 100 U/ml penicillin and 100 mg/ml strepto-mycin. After reaching confluence, trypsin was addedand 1¿106 cells were seeded into microtiter plates,which were incubated for 2 days. The CFDA-stainedlactobacilli were added to the wells at a ratio of 150bacteria to one HT-29 cell and incubated for 1 h at37æ C. The medium was then removed and the cell/bacteria suspension fixed with paraformaldehyde.Flow cytometry analysis was performed using aCoulterA EPICSA XL Flowcytometer (Coulter Cor-poration, Miami, FL, USA).

StatisticsData are expressed as median (25th–75th percen-

tile) log10 CFU/g tissue. The incidence of BT wascompared using Fisher’s Exact Test. To compare dif-

Fig. 1. Incidence of positive cultures (%) in I: mesenteric lymph nodes (¤Ωp0.01 vs negative control and §Ωp0.01 vs L. plantarum 299v). II: liver (*Ωp0.001 vs negative control and .Ωp0.05 vs L. plantarum 299v).See text for description of different pretreatment and treatment groups.

613

ferences in number of bacteria between groups, Krus-kal-Wallis ANOVA by ranks was used, followed bymultiple comparison with Dunn’s method. p0.05was considered significant.

RESULTS

All cultures from MLN and liver were sterile inthe negative control group (Fig. 1). Intraperito-neal injection of LPS caused BT to 25% ofMLN’s and 88% of the livers (Fig. 1). This inci-dence of BT was significant in the liver com-pared to the negative control group (p0.001)but did not reach statistical significance in theMLN. Pretreatment with oatmeal alone in-creased LPS-induced BT to 75% of MLN’s (pΩ0.002 vs negative control and pΩ0.007 vs Lp299v) and 100% of the livers (p0.001 vs nega-tive control and pΩ0.001 vs Lp 299v) (Fig. 1).Thus, pretreatment with unfermented oatmealdid not have any preventive effect against BTafter LPS exposure. This sharply contrasts withLPS-challenged rats pretreated with oatmealcontaining L. plantarum 299v (group Lp 299v),in which BT to MLN’s was abolished and a sig-nificant reduction in incidence of BT to livers(12.5%, pΩ0.01 vs positive control) was seen.The incidence of BT is summarized in Table 1.There was no significant difference in bacterialcounts in MLNs or livers between the pretreat-

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Lactobacillus Plantarum Prevents Translocation in SepsisMANGELL et al.

TABLE 1. Incidence of bacterial translocation (%) inthe different treatment groups.

Group Incidence of BT1. Negative control 0/10 (0%)2. Positive control 7/8 (88%)*.3. Oatmeal 8/8 (100%)*.4. L. plantarum 299v 1/8 (12%)5. L. plantarum 299v-adhª 9/10 (90%)*.BTΩbacterial translocation,MLNΩmesenteric lymphnodes. *P0.001 vs negative control. .P0.05 vs L.plantarum 299v.

ment groups (Table 2), but there was a tendencytoward a higher number of bacteria on all thespecimens from septic rats pretreated with oat-meal.

LPS increased the content of Enterobacteria-ceae in the distal ileum (p0.05 vs negative con-trol) (Table 3). A higher count of Enterobacteri-

TABLE 2. Viable count in mesenteric lymph nodes (MLN) and liver in the different treatment groups.

MLN LiverGroup n Aerobe Anaerobe Aerobe AnaerobeNegative control 10 0 0 0 0

0 0 0 0

Positive control 8 0 (0–1.7) 0 (0–1.4) 0.8 (0–1.9) 1.9 (0–2.1)2 2 4 5

Oatmeal 8 3.3 (1.6–3.7) 0 (0–3.3) 2.6 (1.7–3.2) 2.3 (0–3.3)6 3 7 5

LP 299v 8 0 0 0 00 0 1 1

LP 299v-adhª 10 4.1 (0–4.8) 0 (0–3.5) 0 0 (0–1.8)6 3 1 4

Median (25th–75th percentile), log10 CFU/g tissue. Numbers in italics indicate cultures above detection level.

TABLE 3. Intestinal microflora 24 h after lipopolysaccaride (LPS) challenge.Group n Enterobacteriaceae LactobacilliNegative control 6 0 6.3 (6.2 – 6.5)

2 6

Positive control 8 7.1 (4.9–8.4)* 5.9 (5.4–6.1)7 8

Oatmeal 8 5.3 (4.5–6.8) 5.6 (0.0–6.8)8 5

LP 299v 8 5.8 (2.4–7.1) 6.1 (4.9–6.8)6 8

LP 299v-adhª 10 3.8 (2.3–5.0) 6.2 (3.9–6.7)9 10

Median (25th–75th percentile), log10 CFU/g tissue. Numbers in italics indicate cultures above detection level.*P0.05 vs negative control.

614

aceae was also found in the groups pretreatedwith oatmeal with or without L. plantarum299v, but this did not reach statistical signifi-cance. The number of indigenous lactobacilli isrelatively high in the rat and no significantchanges were seen in lactobacilli count whensepsis was induced or with the different pre-treatment.

To study whether any preventive effect of L.plantarum 299v on BT was attributable to theadherence of this bacterium to the intestinalmucosa, rats (group Lp 299v-adhª) were pre-treated with L. plantarum 299v-adhª, a variantof L. plantarum 299v which has lost its abilityto adhere to HT-29 cell (11). The protective ef-fect of L. plantarum 299v on BT was lost whenendotoxemic rats were pretreated with the ad-hesive-deficient L. plantarum 299v-adhª. Thus,BT to MLNs occurred in 70% of the rats (pΩ

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Paper IIMANGELL et al.

TABLE 1. Incidence of bacterial translocation (%) inthe different treatment groups.

Group Incidence of BT1. Negative control 0/10 (0%)2. Positive control 7/8 (88%)*.3. Oatmeal 8/8 (100%)*.4. L. plantarum 299v 1/8 (12%)5. L. plantarum 299v-adhª 9/10 (90%)*.BTΩbacterial translocation,MLNΩmesenteric lymphnodes. *P0.001 vs negative control. .P0.05 vs L.plantarum 299v.

ment groups (Table 2), but there was a tendencytoward a higher number of bacteria on all thespecimens from septic rats pretreated with oat-meal.

LPS increased the content of Enterobacteria-ceae in the distal ileum (p0.05 vs negative con-trol) (Table 3). A higher count of Enterobacteri-

TABLE 2. Viable count in mesenteric lymph nodes (MLN) and liver in the different treatment groups.

MLN LiverGroup n Aerobe Anaerobe Aerobe AnaerobeNegative control 10 0 0 0 0

0 0 0 0

Positive control 8 0 (0–1.7) 0 (0–1.4) 0.8 (0–1.9) 1.9 (0–2.1)2 2 4 5

Oatmeal 8 3.3 (1.6–3.7) 0 (0–3.3) 2.6 (1.7–3.2) 2.3 (0–3.3)6 3 7 5

LP 299v 8 0 0 0 00 0 1 1

LP 299v-adhª 10 4.1 (0–4.8) 0 (0–3.5) 0 0 (0–1.8)6 3 1 4

Median (25th–75th percentile), log10 CFU/g tissue. Numbers in italics indicate cultures above detection level.

TABLE 3. Intestinal microflora 24 h after lipopolysaccaride (LPS) challenge.Group n Enterobacteriaceae LactobacilliNegative control 6 0 6.3 (6.2 – 6.5)

2 6

Positive control 8 7.1 (4.9–8.4)* 5.9 (5.4–6.1)7 8

Oatmeal 8 5.3 (4.5–6.8) 5.6 (0.0–6.8)8 5

LP 299v 8 5.8 (2.4–7.1) 6.1 (4.9–6.8)6 8

LP 299v-adhª 10 3.8 (2.3–5.0) 6.2 (3.9–6.7)9 10

Median (25th–75th percentile), log10 CFU/g tissue. Numbers in italics indicate cultures above detection level.*P0.05 vs negative control.

614

aceae was also found in the groups pretreatedwith oatmeal with or without L. plantarum299v, but this did not reach statistical signifi-cance. The number of indigenous lactobacilli isrelatively high in the rat and no significantchanges were seen in lactobacilli count whensepsis was induced or with the different pre-treatment.

To study whether any preventive effect of L.plantarum 299v on BT was attributable to theadherence of this bacterium to the intestinalmucosa, rats (group Lp 299v-adhª) were pre-treated with L. plantarum 299v-adhª, a variantof L. plantarum 299v which has lost its abilityto adhere to HT-29 cell (11). The protective ef-fect of L. plantarum 299v on BT was lost whenendotoxemic rats were pretreated with the ad-hesive-deficient L. plantarum 299v-adhª. Thus,BT to MLNs occurred in 70% of the rats (pΩ

L. PLANTARUM PREVENTS TRANSLOCATION IN SEPSIS

0.004 vs Lp 299v) (Fig. 1), i.e. the incidence ofBT was significantly higher than when rats werefed regular L. plantarum 299v prior to endotox-emia. An increased incidence of BT to the liverswas also seen as compared to regular L. plan-tarum 299v, but this did not reach statistical sig-nificance.L. plantarum 299v adhered to HT-29 cells

after coincubation, shown as a high fluorescenceintensity with flow cytometry (Fig. 2). When ad-hesion-deficient L. plantarum 299v-adhª wascoincubated with HT-29 cells, a much lowerfluorescence intensity was found, indicating alower rate of adherence.

DISCUSSION

Probiotics is emerging as an attractive means ofpreventing infectious complications after major

Fig. 2. Flow cytometry. L. plantarum 299v and L. plantarum 299v-adhª were stained with CFDA-SE andcoincubated with HT-29 cells to study adherence. Reduced fluorescence intensity with L. plantarum 299v-adhª

indicates lower adherence of these bacteria to HT-29 cells. I: HT-29 cells without bacteria. II: HT-29 cellscoincubated with CFDA-SE-stained L. plantarum 299v. III: HT-29 cells coincubated with CFDA-SE-stainedL. plantarum 299v-adhª. See text for description.

615

surgery (6) and in critical illness (5). In the pres-ent study we found that oral pretreatment withL. plantarum 299v of rats one week before intra-peritoneal injection of LPS significantly reducedthe incidence of bacteria translocating to theMLN and liver. Thus, L. plantarum 299v seemsto prevent BT from the intestine in a septicstate. This preventive effect of L. plantarum299v appears to be associated with its ability toadhere to the intestinal mucosa. Further, thisstudy indicates that pretreatment with oatmealonly, i.e. a prebioticum given without additionof a probioticum, does not prevent BT duringLPS-induced sepsis.

Lactobacilli are part of the commensal micro-flora in both rodents and humans, and havebeneficial effects on the host. There are prob-ably differences in the effects between differentstrains of lactobacilli. In this study we specifi-

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Lactobacillus Plantarum Prevents Translocation in SepsisMANGELL et al.

cally used L. plantarum 299v, which in experi-mental settings has been shown to enhance andinteract with the gut immune system (17, 18),increase mucin production (19), compete withE. coli for colonization (18), reduce translo-cation in enterocolitis (8) and liver injury (9),and prevent E. coli-induced intestinal per-meability (7).

The number of lactobacilli needed to achievecertain biological effects is not known, but thereare no indications that there is an upper limitas to how many bacteria can be given withoutevoking adverse effects. In this study, rats re-ceived an average of 1.1¿1010 CFU of L. plan-tarum 299v per day without any adverse effectssuch as diarrhea or impaired general condition.They drank almost twice the volume of fluidscompared to the rats receiving only regular tapwater, this possibly being due to the fiber con-tent of the oatmeal in which the L. plantarum299v is mixed or perhaps due to the acidity ofthe drink. By mixing the lactobacilli in drinkingwater, the supply of bacteria is more continu-ous, which presumably facilitates colonizationon the intestinal mucosa.

In a septic state, where the mucosal barrierand immune response are compromised, trans-location occurs to such an extent that viablebacteria are able to reach extraintestinal sites,as shown in the positive control group in thepresent study and by others (20, 21). BT toMLN in animal studies varies between 40–100%(15, 22). In this study, BT to MLN was 25% inthe positive control group, which indicates thegreat variation in the rate of translocation be-tween rat species and even between rats of thesame species. Possible reasons for the relativelylow incidence of translocation to MLN in thepositive control group in this study could besampling error or inadequate culturing tech-nique. This is, however, unlikely, as culturesboth of MLN in the oatmeal- and Lp 299vgroups and cultures of liver were positive andstandard culturing techniques were applied. Thereason why the incidence of translocation ishigher to the liver than to the MLN’s is unclear,but might indicate that the hepatic reticulo-endothelial system is affected by sepsis, makingit insufficient to clear the liver from translocatedbacteria, as previously shown in a model withcecal ligation and puncture (23). Care was takento harvest lymph nodes from the ileocecal junc-

616

tion in a standardized manner to detect live bac-teria, indicating BT. Translocating bacteria, ontheir route from the gut to the systemic circula-tion, pass through the lymphatics; it is, however,impossible to determine which group of lymphnodes is ‘‘sentinel’’, i.e. the first to harbor trans-located bacteria and it might be speculated thatthis influences the rate of positive culture ofMLN.

Endotoxin increases the number of Enterob-acteriaceae in the distal ileum, as shown in thepositive control group in this study and byothers (20). Although L. plantarum 299v givento the rats before induction of sepsis decreasedBT, the number of Enterobacteriaceae in the in-testine was not significantly reduced. This indi-cates that the ability of the lactobacilli to inhibittranslocation is not through reduction of thenumber of potentially pathogenic bacteria pres-ent in the intestine. Instead, possible expla-nations might be competition for adhesion siteson the mucosa or that lactobacilli together withthe prebiotic enhance immune surveillance atthe mucosal level, stimulating production ofsIgA by subepithelial B lymphocytes, as shownby Roller et al. (24). Another preventive effectof lactobacilli is the ability to strengthen thephysical barrier, e.g. the mucus layer coveringthe mucosa (19), thus preventing direct contactbetween enterocytes and intestinal bacteria.However, the exact mechanism by which L.plantarum 299v prevents translocation is notknown and further studies are warranted.

Interestingly, the total number of lactobacillion the intestine did not increase with feeding ofL. plantarum 299v. In the rat, lactobacilli consti-tute a significant part of the indigenous micro-flora (25) and the addition of exogenous lacto-bacilli might be difficult to detect in cultures. Ina liver failure model in rats, rectally adminis-tered lactobacilli increased the total number oflactobacilli in the intestine, and were able toabate liver damage and translocation, but alsoin that model, rectally administered L. plan-tarum 299v could not be found in a dominatingnumber on the mucosa (25). It is noteworthythat LPS treatment in the present study did notreduce the number of lactobacilli in any of thetreatment groups. Even if no quantitative differ-ences are noted, there might be important quali-tative differences between the indigenous lacto-bacilli and the exogenously administered L.

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Paper IIL. PLANTARUM PREVENTS TRANSLOCATION IN SEPSIS

plantarum 299v shown by the observation thatL. plantarum 299v given to the rats in the drink-ing water inhibited BT. This indicates that exo-genously supplied bacteria may replace the in-digenous lactobacilli on the intestinal mucosa.

Even though the strain of L. plantarum 299vused in this study is of human origin, it hasbeen shown by randomly amplified polymor-phic DNA (RAPD) to establish itself on the ratmucosa after oral intake (N Osman, personalcommunication) and also on the mucosa ofgnotobiotic rats detected by conventional cul-ture technique (18). The ability of L. plantarum299v to adhere to the intestinal mucosa providesa possible basis for exclusion of other bacteriafrom adhering, one suggested mechanism bywhich translocation is prevented. This is furtheremphasized by the adhesion-deficient L. plan-tarum 299v-adhª used herein, which did notprevent LPS-induced translocation. Flow cyto-metry revealed a reduced ability of L. 299v-adhª to adhere to HT-29 cells compared to L.plantarum 299v. The latter have been shown tocarry a mannose-specific adhesin (26) whichadheres to rat intestine (18), as well as to HT-29cells (27).

In the present study, oatmeal alone had noeffect on the number of lactobacilli on the intes-tinal mucosa (Table 3) and was not able to pre-vent BT to MLN or liver (Fig. 1). However, byadding the probiotic L. plantarum 299v to theprebiotic, BT was inhibited. This is in contrastto some other studies where a prebiotic pre-vented translocation, as in a liver resectionmodel (28). One reason for the difference mightbe the prebiotic used. It may also indicate thatthe positive effects of dietary fibers (prebiotics)can be questioned in severe clinical conditionssuch as sepsis where the mucosa is challengedby toxic substances, reduced blood flow, ormechanically by surgery or trauma. In suchsituations the trophic effect of the dietary fiberson the mucosa might be insufficient to counter-act the negative effects of the trauma. Instead,the prebiotic given alone may even stimulate po-tentially pathogenic bacteria and thus have anegative effect by promoting BT. In contrast,the combination of prebiotics and probioticsnot only strengthens the intestinal barrier butintestinal bacteria trying to translocate are pre-vented from doing so by competitive exclusion,increased production of sIgA, lowering of pH,

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and through upregulation of MUC3 intestinalmucins. The observation in the present studythat the prebiotic not only failed to prevent BTbut even showed a tendency toward increasedBT might be of importance in an intensive caresetting, where septic patients receive part oftheir nutrition through enteral feeding, oftenwith the addition of fiber. It is conceivably ofimportance to combine the enteral formula withboth fiber and a probiotic strain in order to pre-vent BT in those patients.

In conclusion, our data demonstrate thecapability of a probiotic bacterium, L. plan-tarum 299v, to prevent translocation of intesti-nal flora in a septic state and that this effectcorrelates with the ability of L. plantarum 299vto interact with the intestinal mucosa. Also, ourfindings raise the question whether prebioticsgiven in a septic state may be harmful andshould only be given in conjunction with a pro-biotic.

This study was supported by Dir. A. Påhlsson’sFoundation, Gunnar Nilsson’s Foundation, MalmöUniversity Hospital, Apotekare Hedberg’s Founda-tion, Bengt Ihre’s Foundation, Ruth and RichardJuhlin’s Foundation, Einar and Inga Nilsson’sFoundation, and Agne Nilsson’s Foundation.

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