INFECTION AND IMMUNITY, Sept. 1979, p. 820-827 Vol. 25, No. 30019-9567/79/09-0820/08 $02.00/0

Inhibition of Translocation of Viable Escherichia coli fromthe Gastrointestinal Tract of Mice by Bacterial Antagonism

RODNEY D. BERG* AND WILLIAM E. OWENS

Department of Microbiology and Immunology, Louisiana State University Medical Center, School ofMedicine in Shreveport, Shreveport, Louisiana 71130

Received for publication 24 May 1979

The incidence of translocation of viable Escherichia coli C25 from the gastroin-testinal tract to the mesenteric lymph nodes was compared in gnotobiotic micecolonized with only E. coli C25 and in gnotobiotic mice colonized with E. coli C25plus the whole cecal flora from specific pathogen-free mice. The population levelsof E. coli C25 in the ilea and ceca of these mice also were compared. E. coli C25maintained high population levels in the gastrointestinal tracts of the monoasso-ciated gnotobiotes, and the incidence of translocation to the mesenteric lymphnodes was 100%. The gastrointestinal population levels of E. coli C25 werereduced drastically in the gnotobiotes associated with both E. coli C25 and a cecalflora with a concomitant reduction in the incidence of translocation of E. coli C25from 100 to 0%. A decrease in the numbers of viable E. coli C25 per mesentericlymph node also accompanied the decrease in E. coli C25 population levels in thegastrointestinal tracts of these mice. Thus, high population levels of E. coli C25in the gastrointestinal tracts of monoassociated gnotobiotic mice appear topromote translocation of viable E. coli C25 to the mesenteric lymph nodes.Bacterial antagonism of E. coli population levels in conventional mice, therefore,could be one mechanism whereby viable E. coli are confined to the gastrointes-tinal tract.

Bacterial translocation is defined as the pas-sage of viable bacteria from the gastrointestinaltract through the epithelial mucosa into thelamina propria and then to the mesentericlymph nodes and possibly other organs. Patho-genic bacteria, such as certain Salmonella spe-cies, readily penetrate the gastrointestinal epi-thelia of mice and appear in the mesentericlymph nodes (8, 21, 22). Other bacteria consid-ered to be allochthonous (17) or transient mem-bers of the gastrointestinal flora also have beenobserved to pass from the gastrointestinal tractto the mesenteric lymph nodes and other organsin certain experimental mice (7, 9, 14, 23). Thereis little information available, however, as towhether or not indigenous or autochthonous (17)bacteria of the gastrointestinal tract translocatefrom the gastrointestinal lumen to other organs.We recently reported that viable bacteria of theindigenous gastrointestinal flora of specific path-ogen-free (SPF) mice could not be cultured fromthe mesenteric lymph nodes, spleens, or livers ofthese mice (4). Certain types of these indigenousbacteria, however, were cultured from the mes-enteric lymph nodes of gnotobiotic mice inocu-lated intragastrically with the whole cecal mi-croflora from SPF mice. No viable bacteria werecultured from these organs of control SPF mice

also inoculated with a whole cecal flora. Indige-nous Escherichia coli was cultured from themesenteric lymph nodes of 96% of gnotobioticmice monoassociated with this organism but innone of the mesenteric lymph nodes of SPFcontrol mice also inoculated with indigenous E.coli. Indigenous Lactobacillus acidophilus alsotranslocated to the mesenteric lymph nodes ofgnotobiotic mice monoassociated with L. aci-dophilus. Thus, there are mechanisms active inadult SPF mice which inhibit certain viableindigenous or autochthonous microbes fromtranslocating from the gastrointestinal tract tothe mesenteric lymph nodes, spleens, or livers,whereas these mechanisms are either absent orreduced in gnotobiotic mice.

It is well known that many bacteria, such asE. coli, maintain much higher population levelsin the gastrointestinal tracts of gnotobiotic micemonoassociated with one of these bacteria thanin the gastrointestinal tracts of SPF or conven-tional mice harboring an antagonistic normalmicroflora (5, 6, 13). Thus, the population levelobtained in the gastrointestinal tract by a par-ticular bacterial species might be one of thefactors determining whether or not this bacterialspecies will translocate to the mesenteric lymphnodes. This paper describes experiments testing

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BACTERIAL TRANSLOCATION 821

the hypothesis that high population levels ofcertain bacteria in the gastrointestinal tracts ofgnotobiotic mice promote the translocation ofthese bacteria to the mesenteric lymph nodes.On the other hand, bacterial antagonism by theindigenous flora reduces the gastrointestinalpopulation levels of certain bacteria and mayinhibit translocation of these bacteria to themesenteric lymph nodes.

MATERIALS AND METHODSAnimals. SPF (CD-1) mice were purchased from

Charles River Breeding Laboratories, Wilmington,Mass. The mice were housed under barrier-sustainedconditions with automatically controlled temperature,humidity, and light conditions. The SPF mice werekept in autoclaved polystyrene cages (Maryland Plas-tics, New York, N.Y.) with stainless steel lids coveredwith individual filter tops (Econ-filter cover, ScientificProducts, Grand Prairie, Tex.). The mice were main-tained with Purina Laboratory Chow (Ralston PurinaCo., Inc., St. Louis, Mo.) and acidified water (0.001 NHCI) given ad libitum. Bedding consisted of San-I-Cellaboratory animal bedding (Paxton Processing Co.,Inc., Paxton, Ill.).

Germfree and gnotobiotic mice (CD-1 strain,Charles River Breeding Laboratories) were housed inautoclaved polystyrene cages with stainless steel wirelids inside Trexler-type flexible vinyl isolators (Germ-free Supply Division, Standard Safety Equipment Co.,Palatine, Ill.) sterilized with 2% peracetic acid (FMCCorp., Buffalo, N. Y.) containing 0.1% Bio-Soft-N-300(TEA linear alkylate sulfonate, 60% active, StepanChemical Co., Northfield, Ill.). The mice were fedautoclavable Purina Laboratory Chow 5010 (RalstonPurina Co.). Their cages contained San-I-Cel labora-tory animal bedding. The food, water, and beddingwere vacuum sterilized in a bulk sterilizer chamber(Hoeltge, Inc., Cincinnati, Ohio) with a 28-inch (ca.70-cm) vacuum cycle in an automatic sterilizeradapted with a vacuum pump (American SterilizerCo., Erie, Pa.).

Bacteria. The indigenous E. coli was isolated fromthe mesenteric lymph nodes of gnotobiotic mice inoc-ulated intragastrically with a suspension of cecal con-tents from SPF,-CD-1 mice as previously described(3). Nonindigenous, streptomycin-resistant E. coli0127:B8 was obtained from the American Type Cul-ture Collection (ATCC 12740) (Rockville, Md.). Non-indigenous, streptomycin-resistant E. coli C25 wasisolated originally from the feces of a healthy human(11, 16). The E. coli strains were cultured overnight inbrain heart infusion broth, centrifuged, and resus-pended to the desired concentrations in sterile normalsaline. The bacterial suspensions were transferred toglass tubes, and the outsides were sterilized with 2%peracetic acid and transferred into the vinyl germfreeisolators. Germfree mice were inoculated with theseE. coli strains by placing the viable cultures on theirfood and in their drinking water.The mouse cecal microflora for inoculating the gno-

tobiotic mice monoassociated with E. coli C25 wasobtained from SPF, CD-1 mice. Three SPF mice were

killed by cervical dislocation and placed in an anaer-obic glove box (Coy Manufacturing Co., Ann Arbor,Mich.) (2) maintained at less than 10 parts of oxygenper 106 parts of an atmosphere consisting of 5% carbondioxide, 10% hydrogen, and 85% nitrogen. The oxygenlevel inside the anaerobic glove box was monitoreddaily with a Trace Oxygen Analyzer (Lockwood andMcLorie, Inc., Horsham, Pa.). The mouse ceca wereremoved aseptically, cut into small pieces, and placedin 50 ml of sterile prereduced tryptic soy broth (DifcoLaboratories, Detroit, Mich.) prepared with 0.3 Mphosphate buffer (pH 7.5) containing 0.05% dithio-threitol (Sigma Chemical Co., St. Louis, Mo.). Thececal suspension was mixed vigorously on a VortexGenie mixer (Scientific Products) and allowed to set-tle. The supernatant containing the cecal microflorawas transferred inside the anaerobic glove box tosterile glass tubes and stoppered with sterile soft rub-ber stoppers. The stoppered tubes containing the sus-pensions of cecal contents then were removed fromthe anaerobic glove box, the outsides were sterilizedwith peracetic acid, and the tubes were placed into thevinyl isolators containing the gnotobiotic mice mon-oassociated with E. coli C25. The mice were inoculatedintragastrically with the cecal contents with 2.5-inch(ca. 6.35-cm), 22-gauge stainless steel feeding needleswith 2-mm stainless steel bulbs on their tips (Popperand Sons, Inc., New Hyde Park, N. Y.) as describedpreviously (3).Testing for translocation of E. coli strains.

Gnotobiotic mice were killed by cervical dislocationand placed in the anaerobic glove box. Their abdomenswere soaked with 70% alcohol, an incision was madethrough the skin with sterile scissors, and the skincovering the abdomen was reflected. An incision thenwas made through the peritoneum with another pairof sterile scissors. The abdominal wall was reflected,exposing the peritoneal cavity. The exposed viscerawere swabbed with a sterile cotton-tipped applicatorstick which then was placed in a tube of sterile brainheart infusion and incubated aerobically to test forany bacterial contamination of the viscera. The middlemesenteric lymph node was located in the mesenteryof the ascending colon and excised with another set ofsterile instruments. The mesenteric lymph node wasplaced in a sterile grinding tube containing 3.0 ml oftryptic soy broth with or without 1 mg of streptomycinsulfate per ml. The nodes were homogenized withTeflon grinders (Tri-R Instruments, Rockville Center,N. Y.) and removed from the anaerobic glove box.Dilutions of the homogenates were cultured on Ter-gitol-7 agar (5, 6, 19) containing 1 mg of streptomycinsulfate per ml and incubated aerobically at 37°C for24 h to detect E. coli C25 or E. coli 0127:B8. In someexperiments indigenous E. coli was detected by cul-turing the homogenates on Tergitol-7 agar withoutstreptomycin. The remaining lymph node homogenatealso was incubated at 37°C for 24 h and then Gramstained and cultured on Tergitol-7 agar containingstreptomycin to detect the nonindigenous E. colistrains and on Tergitol-7 agar without streptomycin todetect the indigenous E. coli. Hypothetically, as fewas one viable E. coli organism in the mesenteric lymphnode will produce a positive culture after incubationwith these culturing procedures. The entire mesenteric

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822 BERG AND OWENS

lymph node was homogenized in 0.3 ml of tryptic soybroth and plated on Tergitol-7 agar plates to deter-mine the numbers of viable E. coli C25 per mesentericlymph node.

Bacterial population levels. Gnotobiotic micewere killed and placed in the anaerobic glove box todetermine the population levels of strictly anaerobicbacteria. The ilea and ceca were removed asepticallyand weighed individually. The organs were homoge-nized with Teflon grinders in tubes containing brainheart infusion. Dilutions then were made in tubes ofprereduced normal saline. Portions of the various di-lutions (0.1 ml) and 0.1 ml of the remaining organhomogenate were plated on prereduced, enriched tryp-tic soy agar (3, 13) containing polymyxin B to inhibitE. coli growth. After at least 3 days of incubation at370C, the population levels were determined as logoof viable anaerobes per gram of tissue.The dilution tubes were removed from the anaero-

bic glove box, and 0.1 ml of each dilution was platedon Tergitol-7 agar to determine the population levelsof the various E. coli strains. Nonindigenous E. coliC25 and E. coli 0127:B8 are both streptomycin-resist-ant, whereas the indigenous E. coli isolate is strepto-mycin-sensitive. Streptomycin sulfate (1 mg/ml) wasadded to the Tergitol-7 agar to inhibit growth of theindigenous E. coli and thereby select for E. coli C25or E. coli 0127:B8. Indigenous E. coli were culturedon Tergitol-7 agar plates without streptomycin. TheTergitol-7 agar plates were incubated aerobically at370C for 24 h. The viable E. coli were computed as

logo numbers per gram of tissue.

RESULTS

Germfree and SPF mice were inoculated witheither indigenous E. coli or nonindigenous E.coli 0127:B8 by placing viable cultures of theseorganisms on their food pellets and in theirdrinking water. Earlier experiments comparing

intragastric and oral inoculations demonstratedthat germfree mice are colonized as easily withthese E. coli strains by oral inoculations as byintragastric inoculations. The population levelsof E. coli strains in the gnotobiotic mice, theinoculated SPF mice, and control, uninoculatedSPF mice were determined 3 and 7 days afterthe inoculations. The mesenteric lymph nodesof these mice also were cultured to determinethe incidence of translocation of E. coli from thegastrointestinal tract. Translocation of both in-digenous E. coli and nonindigenous E. coli 0127:B8 to the mesenteric lymph nodes occurred in95% of the gnotobiotic mice, but in none of theinoculated SPF mice or control SPF mice (Table1). The cecal population levels of indigenous E.coli in the gnotobiotic mice were nearly 104 timesgreater than their cecal population levels in theinoculated SPF mice, and the cecal populationlevels of nonindigenous E. coli 0127:B8 were

more than 106 times greater in the gnotobioticmice compared with their population levels inthe inoculated SPF mice. Thus, indigenous E.coli and nonindigenous E. coli 0127:B8 appearto translocate to the mesenteric lymph nodes ofthese gnotobiotic mice but not of the SPF micebecause they maintain much higher populationlevels in the gastrointestinal tracts of the gno-tobiotic mice compared with their populationlevels in the SPF mice.Monoassociated gnotobiotic mice and SPF

mice differ in many characteristics other thanthe gastrointestinal population levels of E. coli.For example, the lamina propria of these gno-

tobiotes contains fewer lymphoid cells than thelamina propria of SPF mice. Consequently, we

TABLE 1. Cecal population levels of indigenous E. coli and nonindigenous E. coli 0127:B8 compared withtranslocation of these bacteria to the mesenteric lymph nodes ofgnotobiotic and SPF mice

Indigenous A: co/i Nonindigenou..s / (l 01 27 B:

NumberMice a of bacteria 3 )ays 7 D)ays 3 I)as 7 D)as..

placed on Incidence of (ecal Incidence of C-Calt Iidence of ( real Incidence to (Cr almouse food trantlocation population trannb'atnion population transloaloIaon popoulatiou fi-an.4oatponulaeItio

to A1la level to %I L' level tn %I.', lesnd to %u.N t.enet

(Gnolobiotes 00b 14/1SC 98d | 14/15 9.9 15'15 96 14 15 98(9.3-10.31 1 9.2-10.3) 4.8-10.2) S. 1-100)

Specific pathogen-free 10.0 0O5 5.9 0/5 5.9 0 5 3. 0 5 3. 3(5.4-6.1 (5.6-6.2) (2.5-3.4 3.0-36)

Specific pathogen-free None 045 4.5 0,5 4.5 0/5 0 0 5 0

(4.34.7) (4.3-4.7)

Germfree mice were colonized with the E. coli strains by placing viable bacteria on the mouse food. Control,specific pathogen-free mice also received food inoculated with the E. coli strains.

b Logo viable E. coli placed on the mouse food.'Number of mesenteric lymph nodes exhibiting viable E. coli compared with number of mice tested; MLN

denotes mesenteric lymph node.d Mean log1o viable E. coli per gram of cecum cultured at 3 or 7 days following inoculation of the food with

viable E. coli; ranges in parentheses.

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decided to attempt to relate the decrease in theincidence of translocation of E. coli to the mes-

enteric lymph nodes to a decrease in gastroin-testinal population levels of E. coli in the sameanimal model, the gnotobiotic mouse, ratherthan comparing two different animal modelssuch as gnotobiotic and SPF mice. Gnotobioticmice monoassociated with E. coli should exhibita decrease in E. coli population levels wheninoculated with the whole cecal flora from SPFmice containing an antagonistic microflora. Thisdecrease in E. coli population levels then shouldbe accompanied by a decrease in the incidenceof translocation of this E. coli strain to themesenteric lymph nodes if there is a relationshipbetween population levels and incidence oftranslocation. The indigenous E. coli straintested in the first experiment could not be usedin this experiment since it could not be distin-guished on culture media from the other indig-enous E. coli present in the whole cecal flora.Nonindigenous E. coli 0127:B8 is streptomycin-resistant and therefore can be cultured sepa-

rately from the streptomycin-sensitive E. colistrains indigenous to these mice. However, thececal population levels of nonindigenous E. coli

BACTERIAL TRANSLOCATION 823

0127:B8 were nearly 10 times less than thececal population levels of the indigenous E. colistrain (Table 1). Consequently, we used strep-tomycin-resistant E. coli C25 in the followingexperiments since previous work in our labora-tory had demonstrated that E. coli C25 main-tains a higher cecal population level similar tothat of the indigenous E. coli.Two groups of gnotobiotic mice were monoas-

sociated with E. coli C25. After 1 week, one

group of monoassociated gnotobiotes was inoc-ulated intragastrically with the cecal contentsfrom three SPF mice. The cecal contents were

prepared in an anaerobic glove box and trans-ferred to the germfree isolator in tightly stop-pered glass tubes to insure the viability of thestrictly anaerobic bacteria. The other group ofgnotobiotes monoassociated with E. coli C25served as controls. At various intervals from 1 to14 days after the inoculations with cecal contentsfrom SPF mice, both experimental and controlgnotobiotic mice were killed, and the populationlevels of E. coli C25 were determined in boththe ileum and cecum (Table 2). The populationlevels of total anaerobes also were determinedfor both ileum and cecum to demonstrate colo-

TABLE 2. Reduction in the gastrointestinal population levels of E. coli C25 by bacterial antagonism withconcomitant reduction in the incidence of translocation of viable E. coli C25 to the mesenteric lymph nodes

ofgnotobiotic mice

Days Gnotobiotes inoculate( Gnotobiotes inoculated with E. coli C25following with only E. co/i C25 plus cecal contents from SPF mice

inoculation Population levels of Population levels Population levels ofof gnotobiotes F. coli C25 Incidence of of anaerobes E. coli C25 Incidence ot

with cecal translocation Iranslocationcontents Ileum Cecum of E. co/i C25 Ileum Cecum Ileum Cecum of E. coli C25

1 5.4 9.5b 5/5c 9.6 1.X 6.1 9.7 6 7

(5.3-5.51 (8.9-9.8) (8.8-9.9) (11.6-12.1 (5.3-6.4( 19.3-10.0)

2 5.5 9.4 5/5 8.1 10.4 5.7 7.6 7 7

(5.3-5.8) (9.2-9.5) (7.7-8.3) (9.3-10.8) (4.7-6.3) (6.9-7.9)

3 5.4 9.5 5/5 7.4 9.5 5.2 6.8 4,i 7

15.1-5.6) (9.3-9.6) (7.1-7.8) (9.2-9.9) (4.8-5.4) i6.0-7.3)

4 6.5 9.6 5/S 7.4 9.7 5.5 7.4 0! 7

(5.9-6.8) (9.3-9.9) (7.1-7.8) (9.5-9.9) (5.1-6.0) (6.6-7.3)

7 6.2 9.8 S/5 ND 9.5 ND 6.5 017(4.6-7.8) (9.6-10.3) (8.8-9.6) (6.0-7.1

14 6.7 9.5 5/5 ND 9.8 ND 6.3 0/7(6.0-7.9) (9.3-9.8) j (8.8-9.9) (6.0-6.4)

a Germfree mice were monoassociated with E. coli C25 for 1 week prior to inoculation with cecal contentsfrom SPF mice.

b Mean logo viable bacteria per gram of tissue; ranges in parentheses.e Number of mesenteric lymph nodes exhibiting viable E. coli C25 compared with number of mice tested.

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824 BERG AND OWENS

nization by these organisms. These mice alsowere tested for viable E. coli C25 in the mesen-teric lymph nodes. The E. coli C25 populationlevels in the ceca of the control, monoassociatedgnotobiotes remained between 109 and 10'0/g ofcecum throughout the experiment. The E. coliC25 population levels in the ceca of the gnoto-biotes inoculated with both E. coli C25 and thewhole cecal flora from SPF mice, however, de-creased rapidly from 109 to 1010/g of cecum to106 to 107/g of cecum. The ileal population levelsof E. coli C25 did not decrease significantly by4 days in the monoassociated gnotobiotes inoc-ulated with both E. coli C25 and the whole cecalflora. The incidence of translocation of E. coliC25 to the mesenteric lymph nodes in the gno-tobiotes inoculated with the cecal microfloradecreased to 56% (4/7) by day 3 after inoculationand to 0% (0/7) by day 4 after inoculation. Theincidence of translocation of E. coli C25 in thecontrol group of monoassociated gnotobiotes re-mained at 100% (5/5) throughout the 14-day testperiod. The incidence of translocation of E. coliC25 and the cecal population levels of E. coliC25 in both the control, monoassociated gnoto-biotes and the gnotobiotes colonized with E. coliC25 plus the whole cecal flora are plotted in Fig.1 to illustrate this relationship more clearly. Thececal population levels of E. coli C25 decreasedramatically by day 2 after inoculation with thececal contents from SPF mice and stabilize be-tween 106 to 107/g of cecum by day 3 afterinoculation. There is a delay of 1 day after thedecrease in cecal population levels of E. coliC25, and then the incidence of translocation ofE. coli C25 to the mesenteric lymph nodes alsodecreases. These results suggest that the inci-dence of translocation of E. coli C25 to themesenteric lymph nodes is related to the popu-lation levels attained by E. coli C25 in the cecaof the gnotobiotic mice.The decrease in cecal population levels of E.

coli C25 occurred by 3 days after inoculation ofgnotobiotic mice with an antagonistic cecal flora.The translocation of viable E. coli C25 to themesenteric lymph nodes ceased by 4 days afterinoculation with the cecal flora. Consequently,this experiment was repeated with the testingperiods reduced from 24 to 12 h in an attempt tocharacterize better the relation between gas-trointestinal population levels of E. coli C25 andtranslocation to the mesenteric lymph nodes.Furthermore, the numbers of viable E. coli C25per mesenteric lymph node were determined toascertain whether or not the numbers of viableE. coli C25 per mesenteric lymph node alsodecreased concomitant with the decrease innumbers of viable E. coli C25 in the ceca andilea of the gnotobiotic mice. Again, the decrease

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1 3 5 7 9 11 13 15

DAYS FOLLOWING INOCULATION WITH CECAL CONTENTS

FIG. 1. Reduction in the cecal population levels ofE. coli C25 by bacterial antagonism inhibits trans-location of viable E. coli C25 to the mesenteric Iymphnodes of gnotobiotic mice. Germfree mice were mon-

oassociated with E. coli C25 for 1 week before inoc-ulation with the cecal contents from SPF mice. Thedata were obtained from these mice at various daysafter inoculation with the cecal contents from SPFmice. The solid lines represent gnotobiotic mice mon-oassociated with E. coli C25 throughout the entiretest period. The dashed lines represent gnotobioticmice monoassociated with E. coli C25 for 1 weekfollowed by inoculation with the cecal contents fromSPF mice. The solid circles represent the mean num-

bers of viable E. coli C25 per gram of cecum. Theopen circles represent the incidence of translocationof viable E. coli C25 to the mesenteric lymph nodesdetermined by the percentage of mice exhibiting via-

ble E. coli C25 in their mesenteric lymph nodes.

in the incidence of translocation of viable E. coliC25 to the mesenteric lymph nodes closely par-alleled the decrease in the population levels ofE. coli C25 in both the ceca and ilea of thesemice (Table 3). This decrease in incidence oftranslocation to the mesenteric lymph nodesoccurred between 3 and 4 days after inoculationwith the cecal contents from SPF mice. Thenumbers of viable E. coli C25 per mesentericlymph node, however, decreased even earlier,between 24 and 36 h, after inoculation with thececal contents. Very few viable E. coli C25 were

detected in the mesenteric lymph nodes afterinoculation with the cecal contents, and only 1

viable E. coli C25 was cultured from the mes-

enteric lymph nodes of five mice 84 h afterinoculation. These results add support to therelationship between the population levels at-

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-105

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BACTERIAL TRANSLOCATION 825

TABLE 3. Reduction in the gastrointestinal population levels of E. coli C25 by bacterial antagonism with aconcomitant reduction in the incidence of translocation and reduction in the numbers of E. coli C25

translocating to the mesenteric lymph nodes ofgnotobiotic mice

Gnotiobioitc mice inoculated Gnolobiolic mice inoculated aith /I coh (254Housr with oniv F. coh C25 t)lu% cecal ciintents frotm SP1 nice %ii onitvr'.

fihlitwino T_______ ---_ _ _ __--. il/I io/i (2in Wulati;o Poptiulatioin levels if Incidence Population levels if Poipulation levels f litletti ter 511%

of onittoti0oles f!-C'S_5 *nly of E. cofi C25 anaerobic bacteria IE iiil (25 isn I uofi o5taolliioi/ed Inicewith cecal translication per MLNd Iraiitlocatfito poer IL'. cOipare(l *iihciintentsa IleoIt Cecumn if E. coil C25 lleum Cecuttt Ileuti Ceim'i fit coli ('25 cittntritl ttivt

24 96b 18 5/5c 700d 8.6 11 9 7.2 1I53 155.0 22149.5-9.84 11.4-12.41 50-150 | 18.1-9.81 111.4-12.34' 52-8.01 1091164)8-200

.36 9.1 12.0 4/4 27.5 7.7 11.4 7.5 10.0 45 4.0 14.5i8.7-9.74 11.9-12.0 4112-59) 17.1-8.71 11.2-11.71 16.-7.91 49.4-11.5 i 0-21\

48 9.5 11.2 4,4 101.5 8.5 11.4 x 2 9.1 6.0 5.9(9.2-9.8) 10.8-11.74 48-160 7.8-9.24 11.1-lI5 6.4-10.0) 4.4-9.5i 42-12)

60 8.5 11.7 414 50.5 7.4 11.0 6.5 8.6 4 5 5.0 9 9

f7.6-9.3) 10.2-12.2) (40-1504 17.2-8.01 110.5-11.6) 6.0-7.34 47.7-9.14 40-1 1

72 9.4 11.8 5/5 88.0 7.9 11.1 6.4 7.1 4 5 2.0 2.3

19.0-9.71 111.3-12.0) 112-170) 47.3-8.9) 410.6-11.3) 45.9-7.14 6.7-7.9) (0-5)

84 8.1 11.6 4/4 38.0 7.4 10.1 5.0 6.5 I 5 0 0

47.4-8.8) 411.3-12.0) (30-42i f6.9-7.9) 49.4-11.14 44.9-5.1) 5.4-7.14 0-1

96 7.2 11.4 5/5 19.5 7.9 10.6 5.6 6.5 5 0 0

46.8-7.5) 111.1-12.1) 410-251 47.5-9.04 49.8-11.54 45.1-6.1 45.9-704 | 40-1

aGermfree mice were monoassociated with E. coli C25 for 1 week prior to inoculation with cecal contentsfrom SPF mice.'Mean logio viable bacteria per gram of tissue; ranges in parentheses.'Number of mesenteric lymph nodes exhibiting viable E. coli C25 compared with number of mice tested.d Median E. coli C25 per MLN; ranges in parentheses. MLN denotes mesenteric lymph nodes.'Percent of numbers of viable E. coli C25 per MLN in gnotobiotic mice associated with E. coli C25 plus cecal

contents from SPF mice compared with gnotobiotic mice monoassociated with E. coli C25.

tained by E. coli C25 in the gastrointestinaltracts of mice and translocation of these viablebacteria to the mesenteric lymph nodes. In SPFmice or gontobiotic mice colonized with thewhole flora, the population levels of E. coli aremaintained at low levels due to bacterial antag-onism by the normal flora, and these E. colitranslocate only infrequently to the mesentericlymph nodes. Conversely, in monoassociatedgnotobiotes E. coli reach much higher gastroin-testinal population levels, and translocation tothe mesenteric lymph nodes occurs more readily.

DISCUSSIONNew groups of microorganisms populate the

habitat of the gastrointestinal epithelia of thenewborn mouse at weekly intervals after birthin a characteristic succession (17). At the end ofweek 1 after birth, facultative anaerobes such asthe coliforms and enterococci colonize the intes-tinal tract at high population levels. These pop-ulation levels remain high until about midwaythrough week 3 after birth when they decline tomuch lower levels characteristic of their levelsin adult mice. Before this decline, coliforms andenterococci are cultured in numbers as high as109 to 10'0/g of large intestine and cecum (18).Histological sections of these organs reveal mi-

crocolonies of tiny gram-negative rods morpho-logically identical to E. coli embedded in themucous layer of the epithelium. Subsequent im-munofluorescence studies by Davis et al. (10)confirmed that these microcolonies in the mucinon the epithelium of the large bowel indeedconsisted of coliforms and enterococci. Duringweek 3 after birth when the coliform populationhas declined to low levels, the coliform micro-colonies in the mucous layer are observed onlyinfrequently by immunofluorescence. The fusi-form-shaped anaerobes apparently displace thecoliforms and enterococci from the mucous layerin these young mice. Lee and Gemmell (15) alsodemonstrated a relationship between the declinein numbers of coliforms in the gastrointestinaltract of newborn mice and the appearance of thestrictly anaerobic bacteria. Thus, the gnotobioticmouse model described in this paper with a cecalmicroflora consisting of strict anaerobes to an-tagonize the E. coli C25 population resemblesthe antagonism of E. coli by anaerobes thatoccurs during the natural development of thegastrointestinal bacterial flora in the conven-tional mouse.Lee and Gemmell (15) observed that the in-

crease in numbers of strict anaerobes and thedecrease in coliform population levels are pre-

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826 BERG AND OWENS

ceded by ingestion of solid food particles by theinfant mice. Presumably, the ingestion of solidfood changes the local microenvironments in thegastrointestinal tract, allowing the strict anaer-obes to establish in large numbers. They suggestthat this could be due to an addition of newnutrients or more likely the creation of highlyreduced conditions. They also suggest that cer-tain volatile fatty acids, especially butyric acid,produced by these anaerobic fusiform-shapedbacteria may inhibit the growth of these coli-forms. Syed et al. (20) reported that E. coli C25population levels in monoassociated gnotobioticmice fed a refined diet are reduced to the levelsfound in normal mice after the inoculation ofthese gnotobiotes with a collection of 50 strictlyanaerobic bacteria and 80 facultative anaerobicbacteria. They suggest that the population levelof E. coli C25 in the gastrointestinal tract of themouse is a function of E. coli multiplication inthe cecum, and perhaps the lower ileum, andthat the size of the E. coli population is con-trolled primarily by a limiting nutrient. In laterwork, Freter and Abrams (13) discovered that asecond collection of 95 anaerobes inoculated intognotobiotic mice monoassociated with E. coliC25 and fed a crude diet was necessary to reducethe E. coli population levels to that found innormal mice. It appears that a different set ofmicroorganisms is antagonistic to E. coli C25,depending upon the diet given the animals. TheSPF and gnotobiotic mice received differentdiets in our experiments. Nonetheless, we foundpreviously that translocation of indigenous bac-teria did not occur in SPF mice receiving auto-clavable Purina Laboratory Chow 5010 andhoused in germfree vinyl isolators (4). However,the possibility of a dietary effect cannot be ruledout completely at this time. Several complexmechanisms can operate to reduce the E. colipopulation levels with a certain inhibitory mech-anism predominating in a particular microenvi-ronment. Freter (12) has found that in compe-tition for substrates, inhibitory fatty acids at lowpH, and a labile volatile inhibitor, each servesunder certain conditions to reduce the E. coliC25 population levels in vitro in anaerobic con-tinuous-flow cultures.One possibility is that the strictly anaerobic

bacteria of the cecal flora inoculum form a layerin the mucin on the mucosal epithelia whichphysically inhibits the E. coli C25 from ap-proaching and penetrating the mucosal epithe-lia. As already mentioned, Davis et al. (10) foundthat the strictly anaerobic bacteria replace mi-crocolonies of indigenous E. coli in the mucosalayer during the development of the intestinalmicroflora of the infant mouse. Freter (13), how-ever, employing histological sections frozen in

methylcellulose as described by Davis et al. (10),could not detect any layering of E. coli C25 onthe mucosal epithelia of gnotobiotic mice colo-nized with E. coli C25 for 30 days. It is notknown where in the bowel E. coli C25 passesthrough the mucosal epithelium. The middlemesenteric lymph node drains the jejunum,ileum, cecum, and ascending colon (8). The pri-mary site of penetration by Salmonella typhi-murium is the distal ileum, although S. typhi-murium also appears to penetrate the cecum ininoculated gnotobiotic mice (8). Thus, E. coliC25 might translocate across the ileal epitheliumwhere there is not an extensive layer of strictanaerobes on the mucosa (17). It is not possibleat this time to assess the effect of anaerobicbacteria forming a layer on the mucosal epitheliain our bacterial translocation model.Abrams and Bishop (1) reported that signifi-

cantly more S. typhimurium translocate to themesenteric lymph nodes of gnotobiotic mice in-oculated intragastrically with this organism thanto the mesenteric lymph nodes of conventionalmice similarly inoculated. S. typhimurium alsoreach much higher population levels in the smallintestines of these monoassociated gnotobiotesthan in the small intestines of the inoculatedconventional mice. Ligation of the small intes-tines of the conventional mice, however, resultsin a decrease in peristalsis, thereby allowing theS. typhimurium to reach high population levelsin the small intestine of these conventional micesimilar to the levels found in the monoassociatedgnotobiotic mice. Thus, when intestinal empty-ing is prevented in conventional mice by ilealligation, both the intestinal population levelsand the numbers of S. typhimurium translocat-ing to the mesenteric lymph nodes are similar inboth gnotobiotic and conventional mice. Theyconclude that the presence of the normal intes-tinal flora does not influence directly the resist-ance of the intestinal mucosa to translocation byS. typhimurium. Instead, the normal flora in-creases peristalsis, thereby decreasing the pop-ulation levels of S. typhimurium and inhibitingtranslocation.Our gnotobiotic mouse model is related more

clearly to the natural development of the normalbacterial flora of the mouse. Antagonism of in-digenous E. coli strains by the anaerobic bacte-ria is a consequence of the sequential develop-ment of the normal bacterial flora in the mousegastrointestinal tract. In our experiments, antag-onism by the anaerobic bacteria also reduces theintestinal population levels of E. coli C25. Thedecrease in the incidence and numbers of E. coliC25 translocating to the mesenteric lymph nodeconcomitant with the decrease in intestinal pop-ulation levels of E. coli C25 supports the sugges-

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tion that unusually high intestinal populationlevels of certain bacteria promote their translo-cation to the mesenteric lymph nodes. This re-lationship may apply to E. coli and other speciesof bacteria indigenous to the mouse gastrointes-tinal tract in addition to certain pathogenic bac-teria, such as S. typhimurium, that easily pene-trate the gastrointestinal mucosa.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of EllenW. Bernard and Paul L. Schuetze. This investigation wassupported in part by Public Health Service grant AI 14235from the National Institutes of Allergy and Infectious Diseasesand American Cancer Society grant PDT-135.

LITERATURE CITED

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2. Aranki, A., and R. Freter. 1972. Use of anaerobic gloveboxes for the cultivation of strictly anaerobic bacteria.Am. J. Clin. Nutr. 25:1329-1334.

3. Berg, R. D. 1978. Antagonism among the normal anaer-obic bacteria of the mouse gastrointestinal tract deter-mined by immunofluorescence. Appl. Environ. Micro-biol. 35:1066-1073.

4. Berg, R. D., and A. W. Garlington. 1979. Translocationof certain indigenous bacteria from the gastrointestinaltract to the mesenteric lymph nodes and other organsin a gnotobiotic mouse model. Infect. Immun. 23:403-411.

5. Berg, R. D., and D. C. Savage. 1972. Immunologicalresponses and microorganisms indigenous to the gas-trointestinal tract. Am. J. Clin. Nutr. 25:1364-1371.

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9. Cypress, R. H., D. W. Swidwa, J. F. Kenny, and R. B.Yee. 1974. Influence of a metozoan infection in the

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15. Lee, A., and E. Gemmell. 1972. Changes in the mouseintestinal microflora during weaning: role of volatilefatty acids. Infect. Immun. 5:1-7.

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