Translocation of strictly anaerobic bacteria from the intestinal tract to the mesenteric lymph nodes in gnotobiotic rodents

Ann. ~[nst. Pasteur/Microbiol. 1987, 138, 213-221

(~) ELSEVIER

Paris

1987

TRANSLOCATION OF STRICTLY ANAEROBIC BACTERIA FROM THE iNTESTINAL TRACT TO THE MESENTERIC LYMPH NODES IN GNOTOBIOTIC RODENTS by A. Debure (i), J.C. Rambaud (1), R. Ducluzeau (2), N. Yurdusev and P. Raibaud t2~

(2)

(1) Unitd INSERM U54, HOpital Saint-Lazare, 75010 Paris, and (2) Laboratoire d'Ecologie Microbienne, Institut National de la Recherche Agronomique, Centre de Recherches de Jouy-en-Josas, 78350 Jouy en Josas (France)

SUMMARY

Viable cells of some strictly anaerobic strains belonging to Bacteroides, Clostridium and Fusobacterium genera were present in mesenteric lymph nodes of gnotobiotic rodents harbouring these strains. Various parameters were found to affect the incidence of translocation, including the caecal population level, the length of association with the host and the nature of the strains and host. KEY~WORDS:Gut, Transiocation, Gnotobiotics, Bactetoides, Clostridium, Fusobacterium; Rodents, Mesenteric lymph nodes.

INTRODUCTION

Bacterial translocation has been defined as the passage of viable bacteria from the gastrointestinal tract through the epithelial mucosa to the mesenteric lymph nodes and possibly other organs [2]. In gnotobiotic models, translocation of facultative anaerobic bacteria occurred when their caecal population level was high ~2-5, 12]. 1Several authors, as reviewed by Savage [11], have shown that the predominant bacterial strains of the intestinal flora are strictly anaerobic and that their caecal population level is generally higher than that of faculta~ive!y anaerobic strains. However, translocation of strictly

Submitted November 17, 1986, accepted April 1, 1987.

214

A. D E B U R E A N D COLL.

a n a e r o b i c bacteria was only o b s e r v e d in a t h y m i c ( n u / n u ) m i c e [9]. O u r purpose was to c h e c k w h e t h e r strictly a n a e r o b i c b a c t e r i a w e r e also c a p a b l e o f transloc~.fion in g n o t o b i o t i c n o r m a l r o d e n t s .

MATERIALS AND METHODS Animals. Adult axenic male C3Hej mice (Centre de S41ection des Animaux de Laboratoire, Orl6ans, France) and male Fischer rats were housed in Trexler-type isolators fitted with a rapid transfer system (La Calh6ne, V61izy-Villacoublay, France) and were fed ad libit~m a commercial diet sterilized by y-irradiation at 40 KGy. Bacterial strains. The following strains isolated in our laboratory were used. Strain A of Clostridium perfringens serotype A was isolated from the caecal content of a conventional rat. Strain NN of C. butyricum was isolated from the faeces of a human newborn suffering from necrotizing enterocolitis. Strain FD of C. difficile was isolated from the caecum of a young hare which had died of diarrhoea. Strains C1 of Clostridium sp., B2 of Bacteroides thetaiotaomicron and Fu of Fusobacterium necrogenes were isolated from the faecal predominant flora of a 48-h old piglet. Strain Bs of Bacteroides sp. and strain L of Lactobacillus acidophilus were isolated from the faecal predominant flora of a conventional mouse. Strain EMo of Escherichia coli was of human origin. Inoculation. Inoculations were made with 24- to 48-h non-diluted cultures of the different strains in soft agar medium A containing 1.5 % Bacto peptone, 1.0 % Bacto tryptone, 1.0 % Bacto autolysed yeast (Difco Laboratories, Detroit, Michigan), 0.5 % liver extract (Panmede, Paynes and Byrne, Greenford, Great Britain), 0.5 % glucose and 0.18 % agar at pH 7.7 [10]. One ml containing 5 × 108 to 5 x 109 viable cells per ml was given as drinking water to each animal after overnight wa-~cr deprivation.

Bacterial counts in the small intestine, caecum and faeces of gnotobiotic animals. Bacterial counts were made either from fresh faeces to test for implantation of the different strain~ or from the small intestine and caecum removed as a whole from the autopsied animals to study translocation. Samples from mice monoassociated with B2 and Bs were weighed and immediately introduced into an anaerobic glove box [1]. They were homogenized by means of an (
MLN = mesentericlymph node.

ANAEROBIC

BACTERIA

TRANSLOCATION

IN MICE

215

in the liquid medium of Aranki et al. [1]. Counts were performed by plating O.1 ml of the dilutions on the solid medium of Aranki et al. Samples from mice associated with the other strains were homogenized and serially 10-fold diluted immediately after the autopsy outside the glove box. Medium B' containing 1.5 °70 Bacto peptone, 1 % Bacto tryptone, 1 070 Bacto autolysed yeast, 0.01 070Tween 80 (Touzart et Matignon, Vitry-sur-Seine, France) and 1.0 % agar at pH 6.6 was used for counti::g strainer of Clostridium and Fusobacterium. Solid medium A was used for counting strain L. Both inoculated media were poured into 8- x 400-mm tubes and immediately coo,~ed in running tap water to ensure prompt solidification [10]. Previous experiments bi~ve shown that viable counts were not affected when the described procedure was tlsed for Clostridium and Fusobacterium strains instead of the procedure described/for Bacteroides strains, provided the period of time between autopsy of mice land completion of counting procedure did not exceed one hour. Strain EMo was cou~(lted by plating on Drigalski medium (Institut Pasteur Production, Paris). Incubationiwas carried out for 18 h, for 48 h and for 7 days, respectively, at 37°C for strain EMo, f~r strains L, A, FD, CI, NN and Fu, and for strains B2 and Bs.

Susceptibility of Bacteroides strains to atmospheric oxygen. This was determined by plating 0.1 ml of the I0-s dilution of caecal homogenate from each of 12 mice monoassociated with B2 or Bs on two Petri dishes contahfing the solid medium of Aranki et al. One plate was removed from the anaerobic glove box and exposed for 1 h to the atmospheric oxygen before being reintroduced! into the anaerobic chamber. Both plates were incubated at 37°C inside the glove box for 7 days.

Testiag for translocation. Removal of the middle mesenteric lymph nodes (MLN) draining the jejun!am, ileum, and caecum was carried out according to the technique des,:ribed by Berg and Carlington [2]. The exposed viscera were swabbed with a sterile cotton-tipped applicator stick before and after removal of the MLN to check any bacterial contamination, namely from the intestinal content. Each applicator stick was dipped in a tube of soft agar medium B' or A. In case of bacterial growth in this tube, the subsequent results were discarded. MLN of ,nice monoassoc~tated with B2 and Bs were homogenized inside the anaerobic chamber in 3 ml of sterile water using an <>, plated on the solid medium of Aranki et al. using 0.2 ml of homogenate for each plate and incubated at 37°C inside the anaerobic chamber. MLN of the other gnotobiotic animals were prepared in a similar way, but outside the chamber. Bacterial counts of the whole homogenates were made as previously described using 1 ml for each 8- × 400-mm tube. The incidence of translocatior~ of a given strain to the MLN was defined as the ratio between the number of animals in the MLN of which this strain was recovered and the total number of animals ~ tested. The length of association of animals with the various strains used is given in the tables.

Statistical analyses. The incidence of translocation in our various experimental models was compared using the test of comparison of two percentages in small-sized populations. The population levels of some strains in some models were compared using Student's t test. The paired t test was used to determine the statistical significance of the dif. ference observed in the oxygen susceptibility test.

216

A. DEBURE A N D COLL.

RESULTS Translocation of various strains of Clostridium and of a strain o f L. acidophilus in gnotobiotic mice m o n o a s s o c i a t e d with these strains.

As shown in table I, various strains of Clostridium were found to translocate to the M L N of gnotobiotic mice monoassociated with each strain, as did strain L of L. acidophilus. However, the incidence of translocation and the number of bacterial cells per M L N was lower for Clostridium strains than for the L. acidophilus strain, even though the caecal population levels of strains C1 and L were not statistically different. Strain A of C:. perfringens did not translocate, although its caeeal population level was not statistically different from that of strains CI and NN, which were able to translocate. The number of bacteria was significantly lower (P < 0.001) in the small intestine than in the caecum whatever the strain used (table I).

TABLE I. - - Transiocation of various strains of Clostridium and of a strain of L. acidophilus in gnotobiotic mice moneassociated with each of these strains.

Bacterial strain

Incidence of translocation

Nb of Log10 nb of bacteria/g bacteria/MLN (mean_+ SD) of: (range) small intestine caecum

C. perfringens A

0/9

--

6.8 + 0.7

9.3 + 0.3

C. difficile FD

1/6

22

ND

9.0 + 0.1

Clostridium sp. CI

4/6

5-8

5.1 _+0.7

9.5 + 0.4

C. butyricum NN

6/9

5-15

6.7+0.5

9.2+0.1

L. acidophilus L

6/6

4I-2,400

8.5+0.4

9.7+_0.3

Animals were sacrificed 3 to 6 weeks after inoculation. N D = not determined.

Translocation of various strictly anaerobic Gram-negative strains and o f an

E. coii strain in gnotobiotic mice m o n o a s s o c i a t e d with these strains. W h e n gnotobiotic mice were monoassociated with strain Fu of F.

necrogenes, strain B2 of B. thetaiotaomicron, strain Bs of Bacteroides sp. and strain E M o of E. coli, we observed (table II) that strains B2 and Bs

ANAEROBIC BACTERIA TRANSLOCATION IN MICE

217

TABLE II. - - Transiocation of various Gram-negative striclly auaeJ:obic strains and of an E. coil strain in gnotobiotic rodents monozssociated with each of these strains.

Bacterial strain

Nb of Log~0 nb of bacteria/g Animal Incidenceof bacteria/MLN (mean+_SD) of: host translocation (range) small intestine caecum

F. necrogenes Fu

mice

0/6

--

6.7+0.6

8.8+0.4

B. thetaiotaomicron B2

rat mice

6/12 t2/12

5-12 5-32

ND ND

11.3 + 0.2 11.3+9.1

Bacteroides sp. Bs

mice

7/26

5-121

ND

11.2 _+0.3

E. coli EMo

mice

13/13

3-377

7.4+0.7

9.8+0.2

A n i m a l s were sacrificed 3 to 6 weeks after inoculation. ND = not determined.

translocated to the MLN, as did strain EMo, whereas strain Fu did not. The caecal population level of strain Fu was s~gnificantly lower (P < 0.001) than that of the other Gram-negative strains. Strains B2 and Bs exhibited a statistically different incidence of translocation (P < 0.001), though their caecal population level in monoassociated mice was not significantly different. For strain Bs, the average number of colonies was reduced by half after a 1-h exposure to atmospheric oxygen (240 colonies vs 148, mean for 12 samples; P < 0.02), whereas no susceptibility to oxygen was observed for strain B2 (220 colonies vs 208, mean for 12 samples). The incidence of translocation of strain B2 was significantly lower (P < 0.05) in gnotobiotic rats than in gnotobiotic mice, although there was no difference between the caecal population levels of B2 in the two animals (table II). The number of viable bacteria found in the lymph nodes of mice monoassociated with both strains of Bacteroides was, on an average, lower than that observed in mice monoassociated with E. coli, while the caecal population level of the lat~er was more than 10 times lower (table II). As observed for the Cram-positive strains, the population level of the small intestine was significantly !owe'~ (P < 0.00i) than that of the caecum for the Gram-negative strains Fu and EMo.

Transiocation o f strains B2, Fu and C ! in various diassociated gnotobiotic

mice. When strain Fu of F. necrogenes became established together with strain B2 of B. thetaiotaomicron in diassociated mice, the incidence of translocation of strain Fu and its caecal population level (table III) were significantly

A. DEBURE

218

AND

COLL.

TABLE III. - - Transiocation of strains B2, Fu and C! in vari~,as diassociated gnotobiotic mice. Bacterial strains

Incidence of translocation

Log10 nb e f bacteria/g (mean +_SD) of caecum

B. thetaiotaomicron B2 + F. necrogenes Fu

11/11

10.5 _+0.2

9/11

9.9 +_0.2

B. thetaiotaomicron B2 + Clostridium sp. Cl

6/6

10.5 +_0.1

5/6

9.8 _+0.3

2/6

9.0 _+0.6

0/6

8.9 +_0.3

Clostridium sp. C1 + F. necrogenes Fu

Animals were sacrificed 3 to 6 weeks after inoculation.

higher (P < 0.001) t h a n t h o s e o b s e r v e d in m o n o a s s o c i a t e d m i c e (table II). By contrast, in mice diassociated with strains F u a n d C1, strain F u exhibited the same caecal p o p u l a t i o n level as in m o n o a s s o c i a t e d m i c e (table II) a n d did n o t translocate. T h e incidence o f t r a n s l o c a t i o n o f strain C1 was highest w h e n the caecal p o p u l a t i o n was highest (tables I a n d III). Effect o f the length o f association o f gnotobiotic mice with B2, Fu and C1 u p o n the incidence o f translocation o f these strains. T a b l e IV shows t h a t the incidence o f t r a n s l o c a t i o n o f strain B2 d e c r e a s e d significantly (P < 0.05), t h a t o f strain F u increased significantly (P < 0.01),

TABLE IV. - - Effect of length of association of gnotobiotic mice associated with strains B2, Fu and C! on ~he incidence of translocation of these strains. Bacterial strain

B. thetaiotaomicron B2 F. necrogenes Fu Clostridium sp. C1

Incidence of translocation A

B

12/14 0/14 2/14

7/14 9/14 2/14

LOgl0 nb of bacteria/g of caecum A B 10.5+_0.3 8.6+_0.4 9.3+_0.6

10.5+_0.3 8.6+_0.3 9.4+_0.2

Animals were housed in the same isolator. They were sacrificed2 months (A) or 6 months (B) after inoculation with the 3 strains.

AI~54 tz'ROBIC B A C T E R I A T R A N S L O C A TION I N M I C E

219

and that of strain C1 was not modified when the length of association of the two groups of mice with these strains increased from 2 to 6 months. The caecal population levels of each strain were not significantly different in the two groups of mice.

DISCUSSION Our results clearly showed that a variety of strictly anaerc :~; ~'acteria were able to cross the intestinal mucosa in healthy gnotobiotic ro'~¢nt~, as noted in gnotobiotic mice associated with aerobic bacteria [2, 12]. T ~~se results are apparently in contradiction with those obtained by Berg and Carlington [2]. Using SPF mice, they observed that strictly anaerobic bacteria did not translocate from the intestinal tract to the MLN, although the predominant caecal flora of SPF CD-1 mice was composed of such straiv~s. They also observed that B. fragi/is did not translocate in gnotobiotic CD-i mice diagsociated with this strain a~ad a strain of E. co!i. However, these strains did not belong to the same species as the strains we used. It cannot b~- excluded that straits of B. thetaiotaor.nicron or C. butyricum became established in the predominant intestinal flora of conventional hosts, namely in the human intestine. Our results showed that they eventually can translocate in that case. Howard et al. [8] isolated C. butyricum in the blood of 9 out of 10 infants suffering from necrotizing enterocolitis. From our results, it can be suggested that C. butyricum was present in the predominant intestinal flora of these infants. The route by which anaerobic bacterial strains cross the intestinal tract could be the lyrnphati~cs, where oxygen partial pressure is lower than in the blood vein [7]. This route is also advocated by Steffen and Berg [12]. As observed by other authors [2-5, 1~] for aerobic bacteria, we noticed that translocation of strictly anaerobic bacteria was associated with a high population level in the caecum rather than in the small intestine. However, a high caecal population level of a given strain is not the sole condition for observ;r_g its translocation, since strain Fu had a high incidence of translocation only when it was established for 6 months or more in gnotobiotic mice, whereas its caecal population did not vary. It coul~ be suggested that strain Fu became (~adapted >> to the host intestine as observed by Duval et aL [6] for strain EMo of E. coli, and consequently, newly acquired properties could allow a transmural migration of the <~adapted >>straio Fu. A reverse result was observed for strain B2, since its incidence of translocation decreased when the length of association increased. Increasing stimulation of the mouse immune system by strain B2 could decrease its incidence of translocation. This hypothesis is supported by results obtained with athymic mice [9] and with SPF mice treated with immunosuppressive agents [5]. Similarly, the varying incidence of strain B2 translocation in mice and rats might imply that the response of the immune system is different in the two animals, since there was no significant difference in their caecal population level. The differing incidence of translocation obser~ ved for B2 and Bs strains harbored by gnotobiotic mice at the same caecal

220

A. DEBURE A N D COLL.

popula~::ion level could be due either to a different capacity of transmural migration of the two strains or to the varying susceptibility to oxygen which we observed. This latter hypothesis may be another explanation for the absence of translocatkm of strictly anaerobic bacteria which are predominant in the caecum of 5PF mice. Whatever the mechanisms involved in the translocation of strictly anaerobic strains we used, our results suggest that the caecal popuia~:ion level is not the soie factor governing the extent of translocation, since ttte nature of the host and the length of association of the host with the strain nmy play a role in this complex phenomenon, a notion which is also advocated in human infectious diseases [13].

RESUME TRANSLOCATION DE BACTI~.RIESANAt~ROBIES STRICTEq DE L'INTESTIN VERS LES GANGLIONS MI~SENTERIQUES CHEZ DES RONGEURSGNOTOXI~NIQUES

Des cellules vivantes de certaines souches anadrobies strictes appartenant aux genres Bacteroides, Clostridium et Fusobacterium sont pr6sentes dans les ganglions m~sent6riques de rongeurs gnotox6niques h6bergeant ces souches; nos r6sultats montrent que diff6rents param~tres interviennent dans la translocation tels que le niveau de population dans le caecum, la dur6e de l'association de la souche ~t l'h6te et la nature de la souche et de l'h6te. MOTS-CLES: Intestin, Translocation, Gnotox6nie, Bacteroides, Clostridium, Fusobacterium; Rongeurs, Ganglions m6sent6riques. ACKNOWLEDGMENTS

We thank M. Ladire and J. Rouchette for their helpful technical assistance and A. Bouroche for revision of the manuscript.

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bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. MicrobioL, 1969, 17, 568-576. BERG,R.D. & CARLINGTON,A.W., Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse ~_'node!.infect, lmmun., 1979, 23, 403-411. BER6, R.D. & OWENS, W.E., Inhibition of translocation of viable Escherichia coil from the gastrointestinal tract of mice by bacterial antagonism. Infect. Immun., 1979, 25, 820-827. BER6, R.D., Inhibition of Escherichia coli translocation from the gastrointestinal tract by normal cecal flora in gnotobiotic or antibiotic decontaminated mice. Infect. Immun., 1980, 29, 1073-1081. BERG, R.D., Mechanisms confining indigenous bacteria to the gastrointestinal tract. Amer. J. clin. Nutr., 19S0, 33, 2472-2484.

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[6] DUVAL,Y., RAIBAUD,P. & ROUSSEAU,M., Antagonisms among isogenic strains [7] [8] [9]

[10] [ll] [12] [13]

of Escherichia coli in the digestive tracts of gnotobiotic mice. Infect. Immun., 1981, 34, 957-969. EVEN, P., La respiration, in ~Physiologie Humaine~), 2nd edo (P. Meyer) (p. 1099). F!ammarion M6decine-Sciences, Paris, 1983o HOWARO,F.M., FLYNN,D.M., BEADLEY,J.M., NOONE,P. & SZAWATKOWSKI,M., Outbreak of necrotizing enterocolitis caused by Clostridium buty,'icum. Lancet. !977, IL 1098-1099. OWENS, W.E. & BERG, R.D., Bacterial translocation from the gastrointestinal tract of athymic (nu/nu) mice. Infect. Immun., 1980, 27, 46i-467. RAIBAUO,P., DICKINSON,A., SACQUET,E.~ CHARLIER,H. & MOCQ:~OT,G., La microflore du tube digestif du rat. - - I. Techniques d'6tude et milieux de culture propos6s. Ann. Inst. Pasteur, 1966, 110, 568-590. SAVAGE,D.C., Microbial ecology of the gastrointestinal tract. Ann. Rev. MicrobioL, 1977, 31, 10%I33. STEFFEN,E.K. & BERG, R.D., Relationship between cecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes. Infect. Immun., 1983, 39, 1252-1259. TANCREDE,C. & ANDREMONT, A., Bacterial translocation and Gram-negative bacteremia in patients with hematological malignancies. J. infect. Dis., 1985, 152, 99-103.