INFECTION AND IMMUNITY, Nov. 1994, P. 4768-47740019-9567/94/$04.00+0Copyright X) 1994, American Society for Microbiology

Composition and Diversity of Intestinal Coliform Flora InfluenceBacterial Translocation in Rats after Hemorrhagic Stress

M. KATOULI,l* T. BARK,2 0. LJUNGQVIST,2 T. SVENBERG,2 AND R. MOLLBY'Laboratory for Bacteriology, Microbiology and Tumorbiology Center, Karolinska Institute, S-171 77 Stockholm,1

and Department of Surgery, Karolinska Hospital, S-1 71 76 Stockholm, 2 Sweden

Received 29 March 1994/Returned for modification 17 May 1994/Accepted 9 August 1994

Coliform bacteria are the most frequently reported bacteria to translocate after hemorrhage. We investigatedthe correlation between composition and diversity of the cecal coliform flora and the degree of translocationin a rat model of hemorrhagic stress. Two groups of nine rats each were bled to 60 and 50 mm Hg mean arterialblood pressure, respectively. A sham-operated group without bleeding (n = 9) and a noninstrumented group

(n = 6) served as controls. From each rat, 40 coliform isolates from the cecum and up to 16 from positivemesenteric lymph node (MLN) cultures were tested with an automated biochemical fingerprinting method. Thephenotypic diversity of coliforms in each cecal sample was calculated as Simpson's diversity index (DI), andsimilarities between bacterial types in different samples were calculated as population similarity coefficients.Three rats in the sham-operated group and seven in each of the bled groups showed bacterial translocation.Of the different biochemical phenotypes (BPTs) found in the cecum of bled rats (mean, 6.5 BPTs), only a fewwere detected in MLNs (mean, 1.9 BPTs per MLN), with Escherichia coli being the dominant species. Thetranslocating E. coli strains were mainly of two BPTs. Rats showing no translocation either did not carry thesestrains or had a high diversity of coliforms in the cecum. Furthermore, translocation of these coliform typeswas independent of their proportion in the cecum. In bled rats, the diversity of coliforms (mean DI, 0.53) was

significantly higher than that in control groups (mean DI, 0.30; P = 0.004), suggesting that hemorrhagestimulates an increase in diversity of cecal coliforms. Rats with similar coliform flora and subjected to the sametreatment showed similar patterns of translocation. Our results suggest that the composition of the coliformflora is an important factor in translocation and that certain coliform strains have the ability to translocateand survive in MLNs more easily than others.

An important function of the intestinal mucosal barrier is toprevent bacteria colonizing the gut from invading systemicorgans and tissues. However, under certain conditions, indig-enous bacteria cross this barrier and appear in mesentericlymph nodes (MLNs) and possibly other organs, a processtermed bacterial translocation (5, 19, 42).

Translocation of enteric bacteria depends on disturbance ofthe intestinal microecology with subsequent bacterial over-

growth (7, 10, 11, 36), impaired host immunity (4, 6, 20), or

physical disruption of the gut mucosal barrier (29, 32). Accu-mulating evidence from human and animal studies suggeststhat factors such as trauma (18, 34) endotoxemia (13, 15),hemorrhage (1, 2, 26), and thermal injury (12, 14, 30) promotebacterial translocation. Less-studied factors in bacterial trans-location are the composition and diversity of the intestinalmicroflora. This flora develops as a result of the interactionbetween the intestinal physiology and bacteria that contami-nate the body (16). The normal flora varies between animalspecies and alters as the host ages. It has been postulated thatmore than 500 species of bacteria are present in the intestineand almost 99% of them are obligate anaerobes (17). Althoughthese bacteria outnumber coliforms by a factor of 100:1 to1,000:1, their degree of translocation (if any) is very low (39,40).A high diversity of the normal flora is believed to stabilize

the intestinal microecosystem (22). If this stability is disturbed,e.g., by administration of antibiotics, the endogenous as well

* Corresponding author. Mailing address: Laboratory for Bacteriol-ogy, Microbiology and Tumorbiology Center, Karolinska Institute,S-171 77 Stockholm, Sweden. Phone: (46) 8 728 7159. Fax: (46) 8 33 1547.

as exogenous bacteria may colonize the intestine and over-

grow, resulting in a low diversity. Therefore, evaluation of thediversity of the normal flora should be useful for understandingthe functional status of the intestine under conditions inducingbacterial translocation. However, studying the diversity of a

bacterial population by conventional methods requires culturalanalysis of a large number of bacteria from each sample, whichis laborious and time consuming. We previously reported on a

computerized typing method based on biochemical fingerprint-ing of bacteria (the Phene Plate or PhP system) (24, 25, 31).This system measures kinetics of bacterial metabolism on

highly discriminatory substrates in microtiter plates and yields,for each isolate, a set of quantitative data (biochemical finger-prints). A personal computer program calculates similaritiesamong the tested strains as a dendrogram. These data are alsoused to calculate diversities of and similarities between inves-tigated floras. We have recently evaluated the PhP system forstudying diversity and stability of the intestinal coliforms inpiglets during their first 3 months of life (27, 28). The systemproved to be very discriminatory and was able to monitor thespread of bacterial phenotypes between different samples.While hemorrhage is reported to induce bacterial transloca-

tion in animal models and in traumatized patients, studies on

the role of the diversity of coliform flora under such conditionsare lacking. We previously demonstrated an increased level ofcoliform translocation after hemorrhage in rats (3). The PhPsystem was used to compare biochemical phenotypes (BPTs)of bacterial isolates found in MLNs and in the intestines of theanimals by analyzing a relatively large number of coliformsfrom each sample (3). Since the PhP system is easy to use anda large number of bacterial isolates can be tested within a very

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short time, it is a suitable tool to monitor changes of intestinalbacterial populations under different conditions.

In the present study, we used this system to investigate thecorrelation between the composition and diversity of theintestinal coliforms and the degree of translocation afterhemorrhagic stress in rats.

MATERIALS AND METHODS

Animals. All experiments were approved by the local Ani-mal Care Committee and performed according to the guide-lines of the National Institutes of Health. Thirty-three maleSprague-Dawley rats (Fl hybrid; Harlan Olac Ltd., Oxon,England), weighing 338 ± 4 g, were used. The animals weredelivered from one breeder (Alab, Sollentuna, Sweden) atleast 1 week before the experiments. They were housed inindividual wire cages in a light- and temperature-controlledenvironment and fed a complete enteral diet, calculated tomeet their nutritional requirements. Food consumption andweight gain did not differ significantly between the groups.Water was provided ad libitum before and after the experi-ment, but food was withheld from the animals the morning ofthe experiment onwards.

Experimental procedure. Hemorrhage and sampling proce-dures have been described before (3). In brief, animals in twogroups of nine rats each (groups M and H) were subjected tofemoral catheterization for bleeding and continuous measure-ment of mean arterial blood pressure. The hemorrhage fol-lowed a curve with decreasing blood pressure to 60 (group M)and 50 (group H) mm Hg mean arterial blood pressure after 60min. Blood was not reinfused. After completion of hemor-rhage, the catheters were removed, the blood vessel wasligated, the wounds were closed, and the animals were placedin separate cages. Twenty-four hours later, MLNs were asep-tically excised and collected for bacteriological analysis. Bloodsamples were obtained by aseptic transthoracic heart puncture,and cecal contents were collected directly from the cecum. Asham-operated control group (group S; n = 9) was subjected tothe same experimental procedures except bleeding. To assessthe influence of catheterization and handling per se on studyparameters in this breed of animals, a second control groupwithout any instrumentation (group N; n = 6) was included.

Bacteriological analysis. MLNs were washed free of bloodin sterile saline and homogenized in 2 ml of brain heartinfusion broth (Difco Laboratories, Detroit, Mich.) with sterileTeflon-coated tissue grinding rods as described before (3).Aliquots (0.5 ml) of tissue homogenate were placed on 7%horse blood and MacConkey agar (Difco) plates. Cecal spec-imens were subcultured on the same media as MLN samples.Plates were incubated at 37°C for 24 h, and bacterial isolateswere identified with an API 20E system (API System S.A., LaBalme les Grottes, France). Blood samples (2 ml) werecultured in bottles containing brain heart infusion broth (Bio-Hospital AB, Kopparberg, Sweden), incubated for 1 week at37°C, and subcultured on blood and MacConkey agar platesafter 48 h and 7 days.

Biochemical fingerprinting. An automated system for bio-chemical fingerprinting, the Phene Plate or PhP system, wasused to identify BPTs of coliforms in the ceca and MLNs andto compare the diversity of bacteria in different samples (27,31). To study composition and diversity of cecal coliforms, weused the PhP-RS plates, specifically developed for this purpose(28). Reagents used in the PhP-RS plates and the method forcomparing bacterial populations have been described before(27, 28). In brief, each microplate contains eight parallel sets of11 dehydrated reagents, specifically chosen to differentiate

coliform bacteria. To the first well in each row, not containingany reagent, 375 ,ul of growth medium (0.1% [wt/vol] proteosepeptone, 0.01% [wt/vol] bromothymol blue) was added. Toeach of the other wells, 125 p.l of the medium was dispensed.Forty colonies from each cecal sample and up to 16 from eachpositive MLN culture were picked directly from MacConkeyagar plates with a 1-,ul plastic inoculation loop, and eachcolony was suspended into the first well of a row. By using amultichannel pipette, 25 p.l of the homogenized bacterialsuspension from the first well in each row was transferred toeach one of the other wells in the same row. The inoculatedplates were incubated at 37°C, and the absorbance value (A620)of each well was measured after 16, 40, and 64 h with amicroplate reader and automatically transferred to a personalcomputer.

Calculations. An identity level of 0.975 based on reproduc-ibility of the system as described before (24, 25, 31) was set.Isolates showing correlation coefficients higher than the iden-tity level were assigned to the same BPTs. BPTs with morethan one isolate were called common BPTs, and those withonly one isolate were called single BPTs. The phenotypicdiversity of bacterial populations was calculated as Simpson'sdiversity index (DI) (23). A high DI (maximum, 1) indicatesthat the tested isolates are evenly distributed into differentBPTs, whereas a low DI (minimum, 0) indicates that one orfew BPTs dominate the population. The phenotypic similaritybetween different bacterial populations in two samples wascalculated as the population similarity (Sp) coefficient asdescribed before (27). The Sp coefficient calculates the pro-portion of isolates that are identical in two compared bacterialpopulations. If two populations contain similar dominatingBPTs, the Sp value is high (maximum, 1), and if the popula-tions contain different BPTs, it is low (minimum, 0). Coliformpopulations from different samples were compared, and ma-trices of obtained Sp coefficients were clustered by the un-weighted pair group method with arithmetic averages to yielda dendrogram (35). In the dendrogram, each cecal sampleconsisting of 40 isolates is represented on the vertical axis, andsimilarities between samples calculated as Sp coefficients arerepresented on the horizontal axis. All data handling, includingoptical readings, and calculations of correlation coefficientsand diversity indices as well as clustering and printing ofdendrograms, was performed by using the PhP software (Bio-Sys inova, Stockholm, Sweden).

Statistical analysis. Fisher's two-tail exact test, Student's ttest, and the least-square regression method were used.

RESULTS

Three sham-operated rats (group S) and seven in each of thehemorrhage groups (M and H) showed bacterial translocation(Table 1). Each animal carried several BPTs of coliforms in thececum, some of which dominated the population (Table 1).However, cecal samples from rats subjected to hemorrhagecontained more BPTs of coliform bacteria than samples fromthe control rats (P = 0.027) (Table 2).More than one BPT was also detected among translocating

strains in MLNs. To verify the origin of these strains, oneisolate from each BPT (n = 32) (Table 1) was selected andcompared with the BPTs found in the ceca of correspondingrats. Of these, only 19 coliform BPTs (all strains designated inTable 1 with subscripts Cl, C2, S1, S2, and S3) were detectedin cecal samples. One isolate from each of these BPTs (19isolates) was selected and identified to the species level.Eighteen were Escherichia coli, and one was Proteuls mirabilis;the latter was also isolated from a blood culture (rat M3).

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TABLE 1. Pattern of translocation, diversity, and the BPTs of coliforms found in the ceca and MLNs of rats in different groups

BPTs found/40 colonies tested Translocation No of BPTs found in MLNsRatno.DI~~~~~~~~~~~~~~~~~~~~~~~~~N.oRat no. DI Present colonies

No. BPTa or absent CFU/MLN tested/MLN No. BPTa

Group NNI 0.582 5 20, 17, 1C,, 1, 1 -

N2 0.000 1 40cON3 0.009 3 38cl 1, 1 -

N4 0.685 6 13, 18, 6C2, 1, 1, 1 -

N5 0.354 4 32cr, 4c2, 2, 2N6 0.145 3 37, 2, 1c2

Group SSi 0.279 6 34, 2C2 1, 1, 1,1 -

S2 0.233 4 35cl, 3, 1, 1 + 12 12 1 12c,S3 0.747 9 17cl, 11, 2, 2, 2, 3C2, 1, 1, 1 -

S4 0.629 4 22, 7, 9, 2 -

S5 0.236 5 35, 2, 1, 1, 1 -

S6 0.232 3 35, 3, 2 -

S7 0.146 4 37cl, 1, 1C2 1 + 16 16 3 6CI, 9C29 1S8 0.145 3 37cl, 2, 1 + 16 16 2 15cI, 1S9 0.145 3 37, 2,lc-

Group MMl 0.571 5 25, 8, 1, 4, 2M2 0.576 6 23C2' 13S2, 1, 1, 1, 1 + 36 16 2 13S2, 3M3 0.669 5 20C2' llcl, 3, 5S3 1 + >500 16 2 13cjq 3S3M4 0.191 4 36, 2cl,1, 1 + 46 NTV NTM5 0.792 11 5, 8, 4, 2, 15cl, 1, 1, 1, 1, 1, 1 + 11 11 1 llcM6 0.753 7 7, 12, 2, 16cl 1C2, 1, 1M7 0.635 7 23, 7c,, 2, 1, 1, 1, 5 + 240 16 3 13cl, 2, 1M8 0.603 9 25, 2, 3cj, 3, 1, 1, 1, 1, 3 + 41 16 1 16c,M9 0.801 6 8C2, 4cj, 12, 10, 1, 5 + 26 16 2 14cl, 2

Group HHi 0.746 6 15, 9C2' 11, 2, 2, 1 + 238 16 2 11C2, 5H2 0.663 6 21C2, 10, 2sj, 4, 2, 1 + 150 16 4 5s1, 9C29 1

1H3 0.758 12 19C2,4, 3, 4c, 3, 1, 1, 1, 1, 1, 1, 1H4 0.695 5 15, 15cl, 8, 1, 1 + 320 16 3 13cl, 2, 1H5 0.000 1 40c, + 316 16 1 16c,H6 0.495 6 28C2, 5, 4, 1, 1, 1 + 20 16 2 13C29 3H7 0.915 15 2, 9C2,4c,, 5, 2, 2, 2, 3, 4, 2, 1, 1, 1, 1, 1 -H8 0.142 2 37c,q 3 + 300 16 1 16c,H9 0.377 4 31C2 7c 1, 1 + 166 16 2 12c, 4

a BPTs with subscripts Cl, C2, S1, S2, and S3 denote identical BPTs found in the ceca and MLNs. BPTS3 was also found in a blood sample. For details regardingsignificance of subscripts, see Results.

b NT, not tested.

When these 18 E. coli strains from MLNs were compared witheach other, two major BPT groups (Cl and C2) along with twosingle strains (Si and S2) that were highly similar to BPT Clwere detected (Fig. 1). Cl, C2, or both were detected in cecalsamples from 29 of all 33 rats (Table 1). Often, these strainswere not the most common types in cecal samples, and

although their relative amounts were always higher in MLNs(Table 1), there was not a strong relationship between theproportion of these strains in cecal samples and their degree oftranslocation (Fig. 2a). Rats not carrying Cl or C2 in theirceca, i.e., three rats in group S and one rat in group M, did notshow translocation. Animals subjected to hemorrhage had a

TABLE 2. Comparison of coliform diversities, expressed as DI, and the number of BPTs found in the cecaand MLNs of bled and control rats

DI of cecal coliforms No. of BPTs (mean ± SD) in:Treatment group No. of rats (mean ± SD) Ceca MLNs

Noninstrumented group (N) 6 0.29 ± 0.26 3.8 ± 1.5Sham-operated group (S) 9 0.31 ± 0.20 4.7 ± 1.7 2.0 ± 0.8Sum of control groups (N+S) 15 0.30 ± 0.23a 4.2 ± 1.7" 2.0 ± 0.8Hemorrhage group, 60 mm Hg (M) 9 0.62 ± 0.17 6.6 ± 2.0 1.8 ± 0.7Hemorrhage group, 50 mm Hg (H) 9 0.53 ± 0.28 6.3 ± 4.2 2.1 ± 1.0Sum of hemorrhage groups (M+H) 18 0.57 ± 0.24a 6.5 ± 3.3" 2.0 ± 0.9

a (N+S) versus (M+H), P = 0.004; Student's t test.b (N+S) versus (M+H), P = 0.027; Fisher's two-tail exact test.

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Correlation coefficientID-

0 90 level

Ratno.

BPT az

Eato tR

o00

HI113H6S7 cI

M719118

H2 SI

112 S2H2H4H8S2 C2

H5S8H9S7M13 S3

FIG. 1. Dendrogram showing similarities between BPTs of 19coliform isolates found in MLNs of rats. Each isolate represents acoliform BPT that was found in the MLN as well as in the cecum of thesame rat. ID-level; identity level.

higher diversity of cecal coliforms than controls (P = 0.004)(Table 2). There was also a weak but significant negativecorrelation (P = 0.04) between diversity of coliforms in thesegroups of rats and the degree of translocation (Fig. 2b).The coliform floras from different rats were compared with

each other, and corresponding Sp values were calculated.Figure 3 shows dendrograms derived from clustering of the Spvalue matrices for rats in different groups by the unweightedpair group method with arithmetic averages. Different popu-lations were observed within each group, while some rats hadhigh Sp values, indicating that they carried very similar coli-form bacteria (Fig. 3). When coliform populations of all ratswere compared, two major phenotypic clusters (A and B) werefound (Fig. 4). Among instrumented rats (groups S, M, and H)with cecal coliform populations of cluster A, 10 of 11 (91%)showed bacterial translocation. In contrast, only 7 of 16 (44%)instrumented rats in cluster B showed translocation (P =

0.018) (Fig. 4). Indeed, among sham-operated animals (groupS), only those carrying coliform flora belonging to cluster Ashowed translocation. In general, rats subjected to the sametreatment and with similar cecal coliform populations showedsimilar patterns of translocation (Fig. 4).

DISCUSSIONIn the present study, we extended our previous observations

on the effect of nonlethal hemorrhagic shock on bacterialtranslocation in rats (3) and used a biochemical fingerprintingmethod, the PhP system, to investigate correlations betweenthe composition and diversity of cecal coliform floras andtranslocation after hemorrhage. Rats normally carried coli-form bacteria of different BPTs in their ceca, but only a few ofthese BPTs were found in MLN cultures. Intestinal coliformsare the most frequently reported bacteria to translocate afterhemorrhage (26, 37). These bacteria are part of the normalflora and are easily killed after phagocytosis, surviving onlyunder circumstances that impair host defenses (41). Our

110 -100 -

90 -

80 -

70 -60 -

SO -

40 -

30 -

20 -10 -0-

C

II T IlI *-1 l-I

D 10 20 30 40 50 60 70 80 90 100 110

Proportion in cecal sample(%)

bZ 390

P 290

c

0 1900._

C)0 90

go

F- -lo

-10 190 390 590 790 990

Diversity Index (10-3)FIG. 2. Correlation between the proportion of translocating strains

in cecal samples and their degree of translocation (a) and the diversityof cecal coliforms and the degree of translocation, expressed asnumber of CFU per MLN (b), in hemorrhaged rats (groups M and H).The least-square regression method was used for statistical analysis oflog CFU values. Rat M3 was not included in the calculations becauseof overgrowth of P. mirabilis.

finding that only certain BPTs of the coliform flora (mainly E.coli strains) were found in MLNs of rats indicates that thesestrains have properties which enable them to translocate andsurvive in MLNs more easily than others. Interestingly, amajority of these bacteria were of two BPTs. Strains of thesetwo BPTs were evidently a part of the indigenous coliformfloras in our supply of locally purchased rats since they werefound in 29 of 33 rats tested. Wells et al. (38) noted a high rateof spontaneous translocation of an indigenous streptomycin-sensitive E. coli strain in their supplied batches of rats; theorganism survived in the MLNs of the animals at least 48 hafter translocation. In our study, spontaneous translocation didnot occur in noninstrumented control animals, although all ofthem carried either Cl or C2 strains or both in their ceca. Ratsnot carrying these BPTs, i.e., three in group S and one in groupM, did not show translocation. These results suggest that underhemorrhagic stress, translocation of coliforms is promoted by

s~~~so

E

I I I

I 10~~~~~~~~~~~~~~~~~~~~E-coli

P-mirabilis

-I

I

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4772 KATOULI ET AL.

Ratno.

S NI

N4

N6

N2

N3

N5

Ratno.

MIme

M4M5M6

M7M2M3

M9

Sp-val ue0.3 0.5

M

SIS5

S6

S9S4

S2S7SoS3

Ratno-

HIH4H5HOH2H3H6H9H7

FIG. 3. Dendrograms showing Sp coefficients between coliformfloras of rats in groups N, S, M, and H.

Sp-value Ratno.

the presence of certain bacterial types in the cecum and thatthese bacteria may be not necessarily dominant types amongthe population. By using gnotobiotic or conventionally rearedanimals, Berg (8) and Berg and Owens (9) showed that thenumber of viable bacteria recovered from MLNs was directlyrelated to increased numbers of the intestinal bacteria. In thepresent study, we did not estimate the actual population size ofcoliforms in the ceca of rats, but our results showed that theproportion of translocating BPTs in cecal samples was poorlyrelated to their degree of translocation (Fig. 2a), suggestingthat the predominance of a coliform type in the cecum is not animportant factor for bacterial translocation.

Strains of some BPTs in MLN cultures were not detected incorresponding cecal samples. These strains were normallypresent in small numbers, and some had a high similarity to thedominating BPTs in the same material, suggesting that theymight be a phenotypic variant of these BPTs. It is also possiblethat they originated from other parts of the intestinal tract ortheir numbers in the cecum were small enough to escapedetection. In the present study, we considered translocatingstrains as those found in positive MLN cultures and alsodetected in the cecum of the same rat.

Bacterial translocation involves complex interactions be-tween host defense mechanisms and abilities of bacteria totranslocate across mucosal barrier and survive in the hostileenvironment of host tissues. Under normal conditions, with anintact immune system and normal ecology of the intestinalflora, none or few enteric bacteria translocate. In most controlrats (groups N and S), coliform floras were dominated by onebacterial type, resulting in a low diversity of coliforms in these

Traug-DI locat ion Treatmemnt

0.5820 .6850.8010.000O .0090.3540.2330.1460.1450.0000.1420.7580.5760.6690.7460.6950.1450.2790.2360.2320.1450.1910.6030.6290.4950.7530.6350.6630.7920.5710.7580.3770.915

- CControl (N.I)- Cnbuttrol(N.I)+ Hemorrhage (60 mm. Hg)- Control (N.I)- Cobutrol (N.I)- Control (N.1)+ Sham-opert aed+ Sham-operated+ Sham-operated+ Hemorrhage (50 mm.Hg)+ Hemorrhage (50 mm.Hg)- Shams-operated+ Hemorrhage (60 mm.Hg)+ Hemorrhage (60 m"m.Hg)+ Hemorrhage (50 mm.Hg)+ Hemorrhage (50 mm.Hg)- Control (N.I)- bShams-operated- hShams-operated- Shams-operated- Sham-operated+ Hemorrhage (60 mm.Hg)+ Hemorrhage (60 mm.Hg)- Sham-operated+ Hemorrhage (50 mm.Hg)+ Hemorrhage (60 mm.Hg)+ Hemorrhage (60 mm.Hg)+ Hemorrhage (50 mm.Hg)+ Hemorrhage (60 mm.Hg)- Hemorrhage (60 mm.Hg)- Hemorrhage (50 mm.Hg)+ Hemorrhage (50 mm.Hg)- Hemorrhage (50 mm.Hg)

FIG. 4. Dendrogram showing correlation between composition of coliform floras and pattern of translocation in rats of groups N, S, M, andH. N.I, noninstrumented. The major phenotypic clusters A and B are indicated.

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animals. In contrast, bled rats had a higher diversity of cecalcoliforms. This may be an effect of hemorrhagic stress on theecology of coliform floras, resulting in an increase in therelative number of some coliform types which existed in lownumber. The mechanism(s) behind this finding remains to beelucidated. It has been shown that hemorrhage in the presentmodel reduces intestinal blood flow by 40% (33). Inadequategut oxygenation may, in turn, reduce the intramucosal pH (21),which may affect the gut flora.

Rats with a high diversity of coliforms in their ceca either didnot show translocation or demonstrated very low numbers ofbacteria in their MLNs (Table 1). In contrast, rats with lowcoliform diversity showed a high degree of bacterial transloca-tion provided that they carried common BPTs. In the presentstudy, we found a weak but significant (P = 0.04) correlationbetween the diversity of coliforms in the cecum and the degreeof translocation of the coliforms (Fig. 2b). We tested anothertwo groups of rats treated in the same way as groups M and H;similar results were obtained, and the degree of significancewas again the same (data not shown). These results indicatethat under hemorrhagic stress, translocation of coliforms may

be adversely affected by their high diversity in the cecum. Itshould be noted, however, that the populations of translocatingstrains may be smaller in ecosystems of greater diversity than inthose of lesser diversity. If this is the case, the true determinantof translocation might be the population size of the translo-cating strains rather than the diversity of the ecosystem. Thishypothesis, however, is not fully supported by our findings inthis study, where there was no direct correlation between theproportion of translocating strains and the degree of translo-cation.

Rats used in the present study were all obtained from one

supplier, although they were housed separately in cages beforeand after the experiments. Under normal animal husbandry,where animals are in contact with each other, the chance ofacquiring bacteria from other animals is high. It is thusexpected that large numbers of identical bacterial strains andthus high Sp coefficients between bacterial populations ofdifferent rats will be found. By using a population similarityassay, we found similar coliform floras in different rats in eachgroup. Generally, there were two different types of coliformpopulations in all rats. Those with high Sp coefficients yieldedsimilar patterns of translocation upon similar treatments (Fig.4), suggesting that the composition of the cecal coliform florais important for translocation after hemorrhage.

In conclusion, the PhP system for biochemical fingerprintingof coliforms is a useful and reliable technique to trace andidentify the origin of translocating strains. Of several types ofcoliforms found in the cecum of each rat, only a few were

detected in MLNs after hemorrhage, indicating that certaincoliforms translocate more easily than others. Only rats carry-

ing these coliforms showed translocation. Furthermore, trans-

location of these coliform types was independent of theirrelative proportion in the cecum. Coliform diversities differedbetween controls and bled groups, implying that hemorrhagestimulates an increase in diversity of cecal coliforms. Rats withhigh Sp coefficients yielded similar patterns of translocationupon similar treatments, suggesting that the type and compo-

sition of the coliform flora are important for translocation. Wealso conclude that the population similarity assay is a usefulmethod to study similarities of the intestinal coliform popula-tions under conditions which induce translocation.

ACKNOWLEDGMENTS

We thank Birgitta Karlsson at the Karolinska Institute and BeritHeilborn at the Karolinska Hospital for skillful technical assistance.

This work was supported by grants 584/89 L139 and 0777/89 D326from the Swedish Council for Forestry and Agricultural Research, theKarolinska Institute Funds, the Swedish Medical Research Council(no. 09101), Stockholm, Sweden, and the Laerdal Foundation forAcute Medicine, Stavanger, Norway.

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2. Baker, J. W., E. A. Deitch, L. Ma, and R. Berg. 1988. Hemorrhagicshock promotes the systemic translocation of bacteria from thegut. J. Trauma 28:896-906.

3. Bark, T., M. Katouli, 0. Ljungqvist, R. Mollby, and T. Svenberg.1993. Bacterial translocation after non-lethal hemorrhage in therat. Circ. Shock 41:60-65.

4. Berg, R. D. 1983. Bacterial translocation from the gastrointestinaltracts of mice receiving immunosuppressive chemotherapeuticagents. Curr. Microbiol. 8:285-292.

5. Berg, R. D. 1983. Translocation of the indigenous bacteria fromthe intestinal tract, p. 333-352. In D. Hentage (ed.), The intestinalmicroflora in health and disease. Academic Press, Inc., New York.

6. Berg, R. D. 1989. Bacterial translocation in the immunocompro-mised host. Microecol. Ther. 18:43-49.

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