INFECTION AND IMMUNITY, Sept. 1981, p. 854-861 Vol. 33, No. 30019-9567/81/090854-08$02.00/0

Promotion of the Translocation of Enteric Bacteria from theGastrointestinal Tracts of Mice by Oral Treatment with

Penicillin, Clindamycin, or MetronidazoleRODNEY D. BERG

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

Received 16 March 1981/Accepted 5 June 1981

Specific pathogen-free mice were treated orally with antibiotics to determinewhether the resulting disruption of the normal flora ecology would allow certaingram-negative enteric bacteria to overpopulate the ceca, thereby promoting thetranslocation of these bacteria from the gastrointestinal tract. The mice weretreated orally with penicillin, clindamycin, or metronidazole for 4 days, and thenthe antibiotic was discontinued. The mice were tested at various intervals forviable enteric bacilli translocating from the gastrointestinal tract to the mesentericlymph nodes. Penicillin treatment decreased the total anaerobe population levelsin the ceca an average of 1,000-fold, whereas clindamycin treatment decreasedthese anaerobe levels only 10-fold, and metronidazole treatment slightly increasedthe anaerobe levels. Penicillin or metronidazole treatment increased the entericbacilli populations in the ceca an average of 1,000-fold, whereas clindamycintreatment increased the enteric bacilli populations 100,000-fold. The peak inci-dence of translocation of the enteric bacilli to the mesenteric lymph nodesaveraged 100% after penicillin treatment, 97% after clindamycin treatment, and62% after metronidazole treatment. Thus, oral treatment of mice with penicillin,clindamycin, or metronidazole for only 4 days disrupts the normal flora ecology,allowing an overgrowth in the ceca of the gram-negative enteric bacilli andpromoting their translocation to the mesenteric lymph nodes.

Berg and Garlington (6) reported that bacteriaof the indigenous flora are confined to the gas-trointestinal (GI) tract and are not present inthe mesenteric lymph nodes, spleens, or livers ofspecific pathogen-free (SPF) mice. However,certain types of these indigenous bacteria, suchas Escherichia coli, are cultured from the mes-enteric lymph nodes of gnotobiotic mice inocu-lated intragastrically with the whole cecal mi-croflora from SPF mice (6). This passage ofviable bacteria from the GI tract to the mesen-teric lymph nodes and other organs is defined asbacterial translocation (6). The incidence oftranslocation of indigenous E. coli from the GItract to the mesenteric lymph nodes increasesdramatically in gnotobiotic mice monoasso-ciated with E. coli, as compared with the inci-dence of E. coli translocation in gnotobiotescolonized with the entire cecal microflora (4, 7).E. coli populations of 1010 to 1011 organisms perg of cecum are maintained in monoassociatedgnotobiotes, and there is a 100% incidence oftranslocation of E. coli to the mesenteric lymphnodes (8). If these monoassociated gnotobioticmice are then colonized with a whole cecal mi-

croflora, the population levels of E. coli decreaserapidly to approximately 107 organisms per g ofcecum, with a concomitant reduction in the in-cidence of translocation of E. coli to the mes-enteric lymph nodes (4). E. coli also translocatesfrom the GI tract to the mesenteric lymph nodesof SPF mice decontaminated with bacitracinplus streptomycin and subsequently inoculatedorally with E. coli (4). The abnormally highpopulation levels of E. coli in the ceca of mon-oassociated gnotobiotic or antibiotic-decontam-inated mice promote the translocation of viableE. coli from the GI tract to the mesentericlymph nodes and other organs. Thus, bacterialantagonism of the GI population levels of certainindigenous bacteria, such as E. coli, by othernormal flora bacteria is one defense mechanismconfining these indigenous bacteria to the GItract in conventional mice (5).Dubos et al. (9) found that 100 mg of penicillin

per liter in the drinking water of mice of theNCS strain causes a rapid disappearance of lac-tobacilli from the feces, followed by an increasein the numbers of enterococci and gram-negativebacilli. These NCS mice do not usually contain

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BACTERIAL TRANSLOCATION 855

lactose-fermenting coliform bacteria in theirfeces (17) but do exhibit large numbers of E. coliwhen given oral penicillin for 1 week (9). Inanother study, Savage and Dubos (16) culturedprimarily lactose-fermenting coliforms, Klebsi-ella-Enterobacter types, enterococci, and clos-tridia from the ceca of NCS mice receiving 300mg of penicillin per liter orally but no lactoba-cili, group N streptococci, or fusiform-shapedbacteria. Penicillin treatment had a greater dis-ruptive effect on the bacterial flora than didtreatment with terramycin (16), kanamycin (16),or chloramphenicol (9).

Oral antibiotic treatment disrupts the normalecology of the gastrointestinal tract, allowingcertain indigenous bacteria to overpopulate. Ourprevious studies (4, 6, 7) demonstrated that ab-normally high population levels of certain bac-teria in the GI tract promote translocation ofthese bacteria from the GI tract to the mesen-teric lymph nodes and possibly other organs.Thus, treatment of mice with oral antibiotics,such as penicillin, should increase the populationlevels of certain indigenous bacteria and pro-mote translocation of these bacteria from the GItract. This paper describes the increase in theincidence of translocation of certain enteric ba-cilli from the GI tract to the mesenteric lymphnodes of SPF mice after oral treatment withpenicillin, clindamycin, or metronidazole.

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

Charles River Breeding Laboratories, Inc., Wilming-ton, Mass. A breeding colony of these SPF CD-1 micewas established under barrier-sustained conditions.The SPF mice were kept in autoclaved polypropylenecages (Maryland Plastics, New York, N.Y.) with stain-less steel lids covered with individual filter tops (Sci-entific Products, Inc., Grand Prairie, Tex.). The micewere fed Purina Laboratory Chow (Ralston PurinaCo., Inc., St. Louis, Mo.) and given acidified water(0.001 N HCI) ad libitum. Bedding consisted of San-I-Cel laboratory animal bedding (Paxton Processing Co.,Inc., Paxton, Ill.).

Antibiotic treatment of SPF mice. SPF CD-1mice, 8 weeks old, were given penicillin G (500 U/ml;Pfizer Inc., New York, N.Y.), clindamycin (0.5 mg/ml;The Upjohn Co., Kalamazoo, Mich.), or metronidazole(1.0 mg/ml; Searle and Co., Chicago, Ill.) for 4 days adlibitum in their dfinking water. Groups of mice weretested at various intervals for the translocation ofviable enteric bacteria to the mesenteric lymph nodes.The numbers of strictly anaerobic, aerobic, and facul-tatively anaerobic bacteria also were determined pergram of cecum.

Testing for translocation of enteric bacteria.Mice were killed by cervical dislocation and placed inan anaerobic glove box (Coy Manufacturing Co., AnnArbor, Mich.) (1) maintained at less than 10 parts ofoxygen per 106 parts of an atmosphere consisting of

5% carbon dioxide, 10% hydrogen, and 85% nitrogen.The oxygen level inside the anaerobic glove box wasmonitored daily with a Trace Oxygen Analyzer (Lock-wood and McLorie, Inc., Horsham, Pa.). The cecawere removed aseptically, homogenized with Teflongrinders (Tri-R Instruments, Rockville Center, N.Y.),and cultured on prereduced enriched tryptic soy agar(1) (Difco Laboratories, Detroit, Mich.) inside theanaerobic glove box for strictly anaerobic bacteria.The mice were then removed from the anaerobic glovebox and tested for the translocation of enteric bacteriato the mesenteric lymph nodes as previously described(6). The exposed viscera were swabbed with a sterile,cotton-tipped applicator stick which was then placedin a tube of sterile, prereduced tryptic soy broth(Difco) and incubated aerobically to test for any bac-terial contamination of the viscera. No bacterial con-tamination of the viscera was detected in these exper-iments. The mesenteric lymph nodes were placed ingrinding tubes containing 3.0 ml of brain heart infusion(Difco). The nodes were homogenized with Teflongrinders, and the homogenate was incubated at 37°Cfor 24 h. The homogenate was Gram stained andcultured aerobically on Tergitol-7 agar (Difco) to de-tect any enteric bacilli. Hypothetically, only one viablebacterium in the mesenteric lymph node will producea positive culture after incubation by these culturingprocedures. The enteric bacteria were identified withthe API 20E system (Analytab Products, Plainview,N.Y.).

Determination of cecal population levels. Cecalpopulation levels of strictly anaerobic bacteria weredetermined in the anaerobic glove box. The ceca wereremoved aseptically from antibiotic-treated and con-trol mice, weighed, and homogenized in prereduced,enriched tryptic soy broth. Dilutions of the homoge-nate were cultured on enriched tryptic soy agar con-taining 1 mg of polymyxin B (Pfizer) per ml to inhibitthe growth of facultatively anaerobic enteric bacillisuch as E. coli. The enriched tryptic soy agar plateswere incubated at 37°C in the anaerobic glove box forat least 4 days. The numbers of strictly anaerobicbacteria were calculated per gram of cecum.

Aerobic and facultatively anaerobic bacteria werealso cultured from the ceca of these mice. Variousdilutions of homogenized ceca were removed from theanaerobic glove box and plated on Tergitol-7 agar.The plates were incubated aerobically at 37°C for 24h. The numbers of enteric bacilli were calculated pergram of cecum.

RESULTSPenicillin treatment. SPF mice were given

500 U of penicillin G per ml in their drinkingwater for 4 days, and then the antibiotic wasdiscontinued. Ten mice were sacrificed at var-ious intervals from day 0 through day 22, andthe ceca were cultured for viable aerobic, strictlyanaerobic, and facultatively anaerobic bacteria.The mesenteric lymph nodes were also testedfor the presence of viable aerobic and faculta-tively anaerobic enteric bacilli. The numbers ofanaerobic bacteria per gram of cecum decreased

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nearly 100-fold after 4 days of penicillin treat-ment (Fig. 1A). The numbers of enteric bacilliper gram of cecum increased 10-fold during thissame 4-day period. Translocation of viable en-teric bacilli to the mesenteric lymph nodes in-creased from 10% on day 0 to 100% by day 4.These bacteria were identified as Enterobactercloacae, Enterobacter aerogenes, Klebsiellapneumoniae, E. coli, Proteus morganii, Proteusmirabilis, and Pseudomonas aeruginosa. Atthis point, the penicillin treatment was discon-tinued. The cecal population levels of the anaer-obic bacteria then increased back to the originallevels by day 7, and the cecal population levelsof the enteric bacilli began to decrease. In addi-tion, by day 7 the enteric populations had de-creased to the original level of 107 organisms perg of cecum, with a concomitant decrease in theincidence of the translocation of enteric bacillito the mesenteric lymph nodes to 70% at day 7and to 30% by day 22.

This experiment was repeated two more timesfor the following reasons: 107 enteric bacilli perg of cecum seemed an unusually high level forthe control mice at day 0, and penicillin treat-ment produced only a 10-fold increase in theenteric populations. The population levels ofenteric bacilli in these experiments were only 105to 106 organisms per g of cecum on day 0, andthe anaerobe population level was 109 organismsper g of cecum (Fig. 1B and C). There was adramatic decrease (10,000-fold in Fig. 1B and1,000-fold in Fig. 1C) in the population levels ofanaerobic bacteria by day 4 of the penicillintreatment. Also, there was a dramatic increasein the enteric populations from day 0 to day 4(1,000-fold in Fig. 1B and 100,000-fold in Fig.1C), as compared with only a 10-fold increase inthe entric populations in the previous experi-ment (Fig. 1A). Translocation of the entericbacilli to the mesenteric lymph nodes againreached peak incidences of 100%. Even thoughthere were some differences in the results ofthese three experiments, penicillin treatment for4 days produced a general pattern of a decreasein the cecal populations of anaerobic bacteriaand an increase in the cecal populations of en-teric bacteria, with a concomitant increase inthe incidence of the translocation of the entericbacilli to the mesenteric lymph nodes.Clindamycin treatment. It was of interest

to determine if antibiotics that primarily affectanaerobic bacteria would also reduce the cecalpopulations of anaerobes, allow the enterics tomultiply in the cecum, and promote the trans-location of the enteric bacilli from the GI tract.SPF mice were given 0.5 mg of cindamycin perml in their drinking water for 4 days. Ten mice

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FIG. 1. Bacterial translocation to the mesentericlymph nodes of mice treated orally with penicillin.Each point represents the mean, and the verticallines represent the standard errors. Ten mice weretested per point. Symbols: A, mean log,o populationlevels of anaerobic bacteria per gram of cecum; 0,mean logl0 population levels ofgram-negative entericbacilli per gram of cecum; , incidence of transloca-tion of the gram-negative enteric bacilli to the mes-enteric lymph nodes, expressed as the percentage ofthe mesenteric lymph nodes exhibiting positive cul-tures.

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BACTERIAL TRANSLOCATION 857

were sacrificed at various intervals and testedfor the translocation of viable enteric bacilli tothe mesenteric lymph nodes. The cecal popula-tion levels were also determined for strictly an-aerobic, aerobic, and facultatively anaerobicbacteria. The anaerobe levels per gram ofcecumdecreased 100-fold by day 2 but then returnedto the original levels by day 4 (Fig. 2A). Theenteric bacilli levels increased more than100,000-fold by day 7. Translocation of the en-teric bacilli to the mesenteric lymph nodesreached a 100% incidence on day 4, similar tothe incidence for the penicillin treatment exper-iments (Fig. 1A and C). The anaerobe popula-tions unexpectedly decreased between days 7and 11 but then increased again by day 25.This experiment was repeated because of the

unusually high population levels of enteric bac-teria in the ceca of the clindamycin-treated mice(1010 to 1011 organisms per g of cecum) (Fig. 2A),as compared with levels of 108 organisms per gof cecum in the penicillin-treated mice (Fig. 1Aand B). Unexpectedly, the total anaerobe levelsin the ceca did not decrease during the 4 days ofclindamycin treatment in this experiment, butthe enteric bacilli levels increased 1,000-fold(Fig. 2B). This increase in enteric populations tonearly 1010 organisms per g of cecum is muchgreater than the enteric population increasesobserved in any of the penicillin treatment ex-periments. The translocation incidence of theenteric bacilli to the mesenteric lymph nodesincreased to 90% by day 4.The total anaerobe levels did not decrease

during this second clindamycin treatment exper-iment (Fig. 2B), whereas they decreased ca. 100-fold in the first clindamycin treatment experi-ment (Fig. 2A). Consequently, the clindamycintreatment experiment was repeated a third time,and the results are shown in Fig. 2C. In thisexperiment, the anaerobe levels decreased 10-fold during clindamycin treatment, whereas theenteric bacilli levels increased 100,000-fold. Thispattern is similar to that of the first clindamycintreatment experiment (Fig. 2A), in which theanaerobes decreased 100-fold and the entericbacilli increased 100,000-fold during the clinda-mycin treatment. Translocation of the entericbacilli to the mesenteric lymph nodes againreached an incidence of 100%, as in the firstclindamycin treatment experiment (Fig. 2A).The total anaerobe population levels stabilizedat 109 to 1010 organisms per g of cecum afterclindamycin was discontinued, as in the secondclindamycin treatment experiment (Fig. 2B),rather than fluctuating as in the first clindamy-cin treatment experiment (Fig. 2A).Metronidazole treatment. SPF mice were

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given 1.0 mg of metronidazole per ml in theirdrinking water for 4 days to determine whetherthis antibiotic would disrupt the normal floraecology to the same degree as did penicillin and

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clindamycin and promote translocation of theenteric bacilli to the mesenteric lymph nodes.Interestingly, the total anaerobe levels in thececa actually increased 10-fold during the met-ronidazole treatment (Fig. 3A), rather than de-creasing 10- to 1,000-fold as with the penicillinor clindamycin treatment. Nonetheless, the en-teric population levels per gram of cecum in-creased nearly 10,000-fold during the 4 days ofmetronidazole treatment. The translocation in-cidence of the enteric bacilli to the mesentericlymph nodes, however, reached a maximum ofonly 45%.This experiment was repeated for the follow-

ing reasons: the total anaerobe levels in the cecadid not decrease as in the penicillin and clinda-mycin treatment experiments, and the enterictranslocation incidence peaked at only 45%, ascompared with a peak incidence of 100% in boththe penicillin and clindamycin treatment exper-iments. Again, the total anaerobe levels actuallyincreased during the metronidazole treatment(Fig. 3B). The enteric bacilli levels increased1,000-fold, and translocation to the mesentericlymph nodes reached a maximum incidence of90% by day 4.The metronidazole treatment experiment was

repeated a third time because the translocationincidence of the enteric bacilli to the mesentericlymph nodes was 90% in the second metronida-zole treatment experiment (Fig. 3B) but only45% in the first metronidazole treatment exper-iment (Fig. 3A). The enteric translocation ratereached a maximum of only 50% in this thirdexperiment (Fig. 3C). The enteric bacilli levelsincreased more than 100-fold during the metro-nidazole treatment. The total anaerobe popula-tion in the ceca decreased slightly (Fig. 3C),whereas in the first (Fig. 3A) and second (Fig.3B) metronidazole treatment experiments, thetotal anaerobe populations actually increased.Each of the antibiotics produced characteris-

tic disruptions in the normal flora ecology, eventhough there were differences among experi-mental results with the same antibiotic. Thechanges in ecology were as follows: (i) penicillintreatment decreased the total anaerobe popula-tion levels in the ceca an average of 1,000-fold,whereas clindamycin treatment decreased theseanaerobe levels only 10-fold, and metronidazoletreatment slightly increased these anaerobelevels; (ii) penicillin or metronidazole treatmentincreased the enteric bacilli populations in thececa an average of 1,000-fold, whereas clinda-mycin treatment increased these enteric bacillilevels 100,000-fold; and (iii) the peak incidenceof translocation of the enteric bacilli to the mes-enteric lymph nodes averaged 100% after peni-

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cillin treatment, 97% after clindamycin treat-ment, and 62% after metronidazole treatment.Thus, oral treatment of mice with penicillin,clindamycin, or metronidazole for only 4 daysdisrupts the normal flora ecology, allowing anovergrowth in the ceca of the gram-negativeenteric bacilli and promoting their translocationto the mesenteric lymph nodes.

DISCUSSIONDubos et al. (9) observed a rapid disappear-

ance of lactobacilli followed by an increase inthe numbers of enterococci and gram-negative,lactose-fermenting bacilli in the fecal flora ofmice given penicillin. Savage and Dubos (16)observed a decrease in the populations of anaer-obic bacteria and an increase in coliform levelsin penicillin-treated mice. Leigh and Simmons(14) found a decrease in total Bacteroides pop-ulations in the feces of patients receiving chin-damycin, whereas coliform numbers increased.Hartley et al. (11) also detected an increase inthe numbers of coliform bacteria in the feces ofhumans given oral clindamycin for 5 days. Clin-damycin is active primarily against gram-posi-tive cocci, Bacteroides species, and certain Clos-tridium species (15). Metronidazole is thoughtto be reduced within anaerobically metabolizingcells to a reactive intermediate, perhaps hydrox-ylamine, that reacts with the cellular deoxyri-bonucleic acid to stop nucleic acid synthesis (2).Metronidazole is unique in that it is active onlyagainst obligately anaerobic organisms but notagainst obligately aerobic, facultatively anaero-bic, or microaerophilic bacteria (8). Treatmentwith oral penicillin, clindamycin, or metronida-zole will most likely decrease the numbers ofcertain anaerobic bacteria in the GI tract butnot directly affect the numbers ofgram-negative,facultatively anaerobic organisms. Thus, it is notsurprising that the oral antibiotic treatmentsdescribed in this paper disrupted the delicateecological balance of the normal flora in the GItract, allowing certain bacteria to overpopulate.The important finding is that the disruption ofthe normal GI flora after oral treatment withthese antibiotics promotes the translocation ofviable enteric bacilli from the GI tract to themesenteric lymph nodes.

Indigenous E. coli reach population levels ofapproximately 109 to 1010 organisms per g ofcecum in monoassociated gnotobiotic or anti-biotic-decontaminated mice and translocate ata 100% incidence to the mesenteric lymph nodes(4). Intragastric inoculation of these mice witha whole cecal microflora from SPF mice reducesthe population levels of E. coli 1,000-fold, andtranslocation of E. coli to the mesenteric lymph

nodes ceases (4). Thus, antagonism of the cecalpopulation levels of E. coli by other members ofthe normal flora is a mechanism confining E.coli to the GI tract. Several studies suggest thatthe anaerobic bacteria of the normal flora antag-onize E. coli populations. Lee and Gemmell (13)found a relationship between the decline in thenumbers of coliforms and the appearance of thestrictly anaerobic bacteria that occurs in thececa of newbom mice at ca. 3 weeks after birth.Freter and Abrams (10) reported that a collec-tion of 95 species of anaerobic bacteria inocu-lated intragastrically into gnotobiotic mice mon-associated with E. coli reduced the E. coli pop-ulations to near the normal level found in con-ventional mice. Volatile fatty acids, such as bu-tyric acid, produced by certain anaerobic bacte-ria may inhibit the growth of the coliforms (13).Thus, it is not unexpected that a decrease in thenumbers of anaerobic bacteria in the ceca ofmice treated with certain antibiotics allowsgram-negative enteric bacilli, such as E. coli, tooverpopulate the ceca. This is the pattern thatoccurred after oral treatment with either peni-cillin or clindamycin. The total anaerobe popu-lations in the ceca did not decrease after met-ronidazole treatment, even though the levels ofenteric bacilli increased. The mice did not drinkthe metronidazole-treated water as readily asthey drank the penicillin- or clindamycin-treatedwater. It also is possible that certain strains ofanaerobic bacteria that antagonize the entericbacilli decrease in numbers, but the total anaer-obe population levels in the ceca still do notdecrease significantly. Other anaerobic strainsthat do not antagonize the enteric bacilli mightincrease in numbers and fill the ecological nichesleft by the decreasing populations of the anaer-obic strains that do antagonize the enteric ba-cili.

Interestingly, although metronidazole is se-creted in the bile and absorbed through the wallsof the GI tract, it normally is not detected in thelumen of the large bowel in the high concentra-tions that it reaches in the bowel mucosa (2).This may be the result of its inactivation bycertain aerobic or facultatively anaerobic bacte-ria (8). Thus, when metronidazole is adminis-tered for short periods, it exerts only a slightantibacterial activity on the anaerobic flora (2).Certain strictly anaerobic bacteria compete withand antagonize the cecal population levels ofother strictly anaerobic bacteria of the normalflora (3). Perhaps metronidazole treatment elim-inates the strains of anaerobic bacteria that areassociated closely with the gut mucosa and notthe anaerobic bacteria in the gut lumen. In thiscase, particular anaerobic bacterial strains as-

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sociated with the mucosa might be eliminatedby metronidazole, allowing the enteric bacilli tooverpopulate without causing a decrease in thetotal anaerobe population levels.

Antibiotic decontamination of mice withstreptomycin plus bacitracin followed by inoc-ulation with E. coli (4), K. pneumoniae (18), orP. aeruginosa (8) promotes the translocation ofthese bacteria to the mesenteric lymph nodes.However, the whole bacterial flora is eliminatedfrom the GI tract after treatment with the com-bination of streptomycin plus bacitracin (4, 18).Thus, antibiotic-decontaminated mice resemblegermfree mice in this regard, and colonizationwith only one bacterial type allows that orga-nism to reach abnormally high levels in thececum, with concomitant translocation from theGI tract. Treatment of mice with penicillin orclindamycin decreased the cecal populationlevels of the anaerobic bacteria but did not elim-inate them entirely. Furthermore, treatmentwith metronidazole did not decrease the totalanaerobe population levels in the cecum, yet itallowed an increase in the numbers of entericbacilli. Therefore, treatment of mice with peni-cillin, clindamycin, or metronidazole disruptsthe ecological balance of the normal GI flora,promoting translocation of the gram-negativeenteric bacilli, but does not simulate the germ-free state as does antibiotic decontaminationwith streptomycin plus bacitracin.Savage and Dubos (16) detected a decrease in

the numbers of total anaerobes and an increasein the numbers of coliforms in the ceca of micegiven oral penicillin. They did not observe asignificant effect on the histology of the cecalmucosa after penicillin treatment. Therefore,the translocation of E. coli and other gram-neg-ative enteric bacilli to the mesenteric lymphnodes after penicillin treatment and probablyclindamycin and metronidazole treatment aswell is likely due to the overgrowth of theseorganisms in the ceca and not to any damage tothe mucosa caused by the antibiotic treatment.Even though penicillin, clindamycin, and met-

ronidazole all eliminated certain anaerobic bac-teria, they each exerted characteristic antibac-terial effects. Thus, oral penicillin treatment de-creased the total population levels of anaerobesin the ceca 1,000-fold, whereas clindamycintreatment decreased the total anaerobe levels anaverage of only 10-fold, and metronidazole treat-ment did not decrease the total anaerobe levelsat all. However, the numbers of enteric bacilli inthe ceca increased only 1,000-fold in penicillin-or metronidazole-treated mice but increased anaverage of 100,000-fold in clindamycin-treatedmice. The translocation incidence of the enteric

bacilli to the mesenteric lymph nodes was 100%after treatment with penicillin when the entericpopulations reached 1010 organisms per g ofcecum (Fig. 1A and B) and 90% when the entericpopulations reached a maximum of 10l1 orga-nisms per g of cecum in the metronidazole treat-ment experiment (Fig. 3B), but the translocationincidences were only 45 and 56%, respectively,in the metronidazole treatment experimentswhen the numbers of enteric bacilli reached lessthan 1010 organisms per g of cecum (Fig. 3A andC). In previous experiments (8), translocation ofE. coli to the mesenteric lymph nodes of gnoto-biotic mice ceased when the E. coli populationlevels decreased to 106 to 107 organisms per g ofcecum. Apparently, translocation of enteric ba-cilli from the GI tract occurs when the levels ofenteric bacilli reach between 107 and i08 orga-nisms per g of cecum, although the precise pop-ulation level required to promote the transloca-tion of these bacteria is not known.

Bacterial translocation from the GI tract pos-sibly could be a first step in the production ofdisease by certain opportunistic bacteria of thenormal GI flora. Debilitated patients, such asthose with leukemia (12), may be especially vul-nerable to infections caused by the bacteria oftheir own flora. These patients, of course, receivevarious antibiotics to control bacterial infec-tions. The results presented in this paper suggestthat oral antibiotic treatment can disrupt theecological balance of the normal GI flora, allow-ing certain bacteria to overpopulate the boweland thereby promote translocation of these bac-teria from the GI tract. Consequently, a betterunderstanding of the effects of antibiotics on theecology of the normal GI flora and the mecha-nisms operating to prevent bacteria from trans-locating from the GI tract may provide a basisfor more logical treatment of particular patients.

ACKNOWLEDGMENTSI gratefully acknowledge the technical assistance of Ellen

Wommack Bernard and Theresa Sprenger Dunn.This investgation was supported by Public Health Service

grants AI 14235 and AI 15826 from the National Institute ofAllergy and Infectious Diseases and by American CancerSociety grant PDT-135.

LITERATURE CITED1. Aranki, A., and R. Freter. 1972. Use of anaerobic glove

boxes for the cultivation of strictly anaerobic bacteria.Am. J. Clin. Nutr. 25:1329-1334.

2. Baines, E. J. 1978. Metronidazole: its past, present andfuture. J. Antimicrob. Chemother. 4:97-111.

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VOL. 33, 1981

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