Vol. 25, No. 2INFECTION AND IMMUNITY, Aug. 1979, p. 685-6930019-9567/79/08-0685/09$02.00/0

Capacity of Anaerobic Bacteria from Necrotic Dental Pulps toInduce Purulent Infections

GORAN K. SUNDQVIST,1 2* MATS I. ECKERBOM,' AKE P. LARSSON,3 AND ULF T. SJOGREN'

Departments of Endodontics' and Oral Microbiology,' University of Umed, S-901 87 Umed, and Departmentof Oral Pathology, University of Lund, S-221 01 Lund,3 Sweden

Received for publication 30 April 1979

Combinations of bacteria isolated from the root canals of teeth with necroticpulps and periapical bone destruction were tested for their capacity to induceabscess formation and transmissible infections when inoculated subcutaneouslyinto guinea pigs. Transmissible infections could be induced with combinationsobtained from teeth with purulent apical inflammation, but not with combinationsfrom symptomless teeth with chronic apical inflammation. All combinationswhich gave transmissible infections contained strains of Bacteroides melanino-genicus or B. asaccharolyticus (formerly B. melaninogenicus subsp. asaccha-rolyticus). The results suggest that purulent inflammation in the apical region incertain cases may be induced by specific combinations of bacteria in the rootcanal and that the presence of B. melaninogenicus or B. asaccharolyticus in suchcombinations is essential. However, with one exception, the strains needed thesupport of additional microorganisms to achieve pathogenicity. The results indi-cate that Peptostreptococcus micros was also essential. Histological sections ofthe lesions in the guinea pigs showed that all bacterial combinations inducedacute inflammation with an accumulation of polymorphonuclear leukocytes andthe formation of an abscess. However, the presence of B. melaninogenicus or B.asaccharolyticus in the combinations resulted in a failure of abscess resolution,with a gradually increasing accumulation of polymorphonuclear leukocytes.

A bacterial infection of necrotic dental pulptissue induces inflammation in the tissues at theapex of the tooth (42). This inflammation isusually chronic and asymptomatic, but can alsobe acute (32). Instrumentation of an infectedroot canal may produce an acute exacerbationof a chronic apical inflammation (20, 36). Sinceexacerbation may also occur spontaneously, ithas been suggested that this may be a result ofan increased virulence of microorganisms in theroot canal or may be due to a decreased hostdefence (3). No specific microorganisms have,however, been identified as the causative agents(1).In a recent study it was shown that the root

canals ofteeth with necrotic pulps and periapicalbone destruction and symptoms of swelling andtenderness harbored a larger number of bacteriaand a more complex anaerobic bacterial florathan did the root canals of teeth with necroticpulps and periapical bone destruction but with-out clinical symptoms (42). The clinical symp-toms, swelling and tenderness, were invariablyassociated with purulent inflammation, and cer-tain bacteria were found more frequently in theroot canals of teeth with evidence of pus for-

mation than in teeth without such evidence. Thepurulent apical inflammation of pulpal originmay thus be caused by specific microorganismsor combinations of microorganisms.The aim of the present study was to investi-

gate the capacity of specific combinations ofbacteria isolated from necrotic pulp tissue toinduce dermal purulent inflammations andtransmissible infections in guinea pigs (28).

MATERIALS AND METHODSMicroorganisms. A total of 88 bacterial strains, 85

of which were anaerobic, were tested. The bacteriahad been isolated from the root canals of teeth withperiapical bone destruction (42). Many of the strainsdid not fit into recognized species, but numerical tax-onomic analysis (42) showed that such strains couldbe put into distinct groups. The strains were used intheir originally isolated combinations but also in othercombinations. Seven of the combinations were fromteeth with acute symptoms (combinations D, G, H, P,AB, UJB, BN), and the remaining 11 combinationswere from symptomless teeth. The number of strainsin the various bacterial combinations ranged between1 and 12 (Tables 1 and 2).

Bacteriological media and culture conditions.PY-glucose broth and PRAS-dilution blanks were pre-

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TABLE 1. Composition of the bacterial flora in combinations not causing transmissibleinfections in guinea pigs

Combina- Organism(s) Combination Organism(s)tion

B Bacteroides ochraceus AC Eubacterium alactolyticumEubacterium lentum

C P. anaerobius P. microsEubacterium lentum Lactobacillus sp. group la

Eubacterium alactolyticumFusobacterium sp. group 20 BA Selenomonas sputigenaLactobacillus sp. group 30 F. nucleatumPeptococcus sp. group 10 Lactobacillus sp. group 10

Fusobacterium sp. group 10E S. mitis P. micros

Eubacterium sp. group 40Hb F. nucleatum Anaerobic Vibrio

Eubacterium sp. group 10 Actinomyces naeslundiiLactobacillus sp. group 3aFusobacterium sp. group 20 IN Bacteroides ochraceusB. asaccharolyticusAnaerobic Vibrio UJA F. nucleatum

Bacteroides oralisM S. mitis

EL F. nucleatumU Arachnia propionica

X Actinomyces naeslundiiPropionibacterium acnes

a These strains do not fit into recognized species and were classified by numerical taxonomy (42).b Combination H was derived from a tooth with purulent apical inflammation.

TABLE 2. Bacteria recovered after the infection was transferred four times in guinea pigs% of count with bacterial combinations

Bacterial species recoveredD G P AB" UJB BNh

F. nucleatum 17 (9-35)c 6 (2-13) 5 (0-24) 6 (3-10)Fusobacterium sp. group 1 or NRe NR NR

2dB. melaninogenicus 65 (62-78) 42 (20-52) 20 (1-25) 32 (26-37) 76 (38-84)B. asaccharolyticus 51 (42-75)Bacteroides oralis 6 (0-21)P. anaerobius NR 6 (3-23) 8 (5-29) 5 (0-24)P. micros 28 (18-33) 49 (37-67) 32 (14-50) 14 (2-23) 20 (10-47)Peptococcus sp. group0'O (0-2) NR NR NRAnaerobic Vibrio 3 (2-10) 0 (0-3) 0 (0-3) NREubacterium sp. group I' 4 (3-11) 0 (0-5)Eubacterium sp. group 2 or 4d NR 0 (0-5) NR 10 (5-13)Eubacterium alactolyticum 0 (0-7)Lactobacillus sp. group 1 or 7 (2-15) 15 (5-21) 5 (0-19) 8 (6-20) 12 (0-21)

3dL. catenaforme 4 (3-9)Veillonella sp. 5 (0-12) 10 (0-19)Selenomonas sputigena 0 (0-5)Actinomyces sp. 0 (0-3) 7 (0-11) NRArachnia propionica NRS. mitis 29 (18-44)

a Two strains included in Lactobacillus sp. group.'Two strains included in Fusobacterium sp. group.' Median value and range expressed as percentage of total viable count in the purulent exudate.d These strains do not fit into recognized species and were classified by numerical taxonomy (42).e NR, Inoculated but not recovered.

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pared as described by Holdeman and Moore (18).Horse blood (Statens Bakteriologiska Laboratorium,Stockholm, Sweden) was hemolysed by freeze-thawingbefore being used in brain heart infusion agar medium(18). Agar media were prepared in room atmosphereand were stored for at least 24 h in an anaerobic boxbefore use. The anaerobic box had an atmosphere of10% hydrogen and 5% carbon dioxide in nitrogen (42).Each of the different strains was cultivated on bloodagar slants for 3 to 4 days at 370C. Colonies of eachstrain in a combination were suspended in PY-glucosebroth, and all suspensions were transferred to one tubewith a final amount of 5 ml of PY-glucose broth. Thecell suspension was homogenized in a glass mortar andtransferred to a syringe in the anaerobic box. Theconcentration of the different bacterial strains wasdetermined by viable count on blood agar. The averageconcentration of bacteria in the suspension was 108cells per ml.

Testing of pathogenicity. According to a stand-ardized procedure, 1 ml of the suspension was inocu-lated into the subcutaneous tissue of the groin of 200-to 250-g guinea pigs (Axells Djurfarm, Sollentuna,Sweden). As a control, PY-glucose broth without bac-teria was inoculated. The animals were examined dailyfor the presence of developing lesions. Hard, nodular,caseous swellings or rapid healing was interpreted aspoor pathogenicity of the combination, whereas thedeath of the animal, the spreading of a necrotic lesion,or localized abscess formation was seen as evidence ofhigh virulence. In the case of abscess formation, thecontents of the abscess were aspirated, and 0.5 ml wasinoculated into another guinea pig; this procedure wasrepeated each time a purulent infection was estab-lished. A bacterial combination was considered to in-duce a transmissible infection when the procedurecould be repeated four times. After four transmissionsthe contents of the abscess were analyzed; 0.1 ml ofthe abscess contents from the fifth animal was addedto 5 ml of PY-glucose broth. To avoid oxidation of thebroth, the sampling tube was flushed with oxygen-freegas (97% carbon dioxide, 3% hydrogen). From thebroth 10-fold serial dilutions were made in the anaer-obic box, and samples were cultivated on blood agarto estimate the concentration of viable bacteria andthe relative numbers of the various bacterial strains.Some strains could be recognized by their colonialmorphology. Other strains had to be identified afterGrain staining and determination of their fermentationproducts (42). Each combination of bacteria was in-oculated into four guinea pigs on three different oc-casions.

Histological procedures. Infected tissues wereexcised from various animals at different times, thuspermitting the development of the lesions to be stud-ied either at daily intervals for a total of 5 days or atlonger intervals for a total period of 3 weeks. Thespecimens were fixed for 48 h in neutral bufferedFormalin at room temperature, dehydrated, andembedded in paraffin. Sections (5 [im) were takenthrough the midportion of the lesions and stained withhematoxylin and eosin. The Brown-Brenn stainingprocedure was used for the demonstration of the pres-ence of bacteria.

Antibody determination. Blood was obtainedfrom the guinea pigs by cardiac puncture before inoc-ulation and 2 weeks thereafter. Sera were prepared,inactivated at 560C for 30 min, frozen, and stored at-80'C until required. The presence of antibodies wasdemonstrated by: (i) the indirect fluorescent antibodytechnique, (ii) the double diffusion in gel method ofOuchterlony, and (iii), for hemagglutinating strains(Fusobacterium nucleatum), the hemagglutination in-hibition test. The strains were grown in PY-glucosebroth, washed three times in phosphate-buffered sa-line, pH 7.2, suspended in phosphate-buffered saline,and kept frozen at -80'C until required. The indirectfluorescent antibody (FA) technique was performedby the method of Williams et al. (48). Fluoresceinisothiocyanate-labeled rabbit anti-guinea pig immu-noglobulin G (IgG) serum and the same serum unla-beled were obtained from Behringswerke, Marburg,West Germany. When fluorescence occurred, its spec-ificity was tested by blocking the fluorescence (i) byabsorption of the tested serum with the strain underinvestigation, (ii) by adding unlabeled anti-IgG serum,and (iii) by omission of the test serum before addingthe labeled anti-IgG serum (41).A nonimmune reaction with the Fc part of the IgG

molecules which gives fluorescence with the FA tech-nique has been reported (9, 25). Species of staphylo-cocci (10) and streptococci (22) have this nonimmunereactivity with IgG, but the presence of similar struc-tures in other microbial species has been investigatedonly to a limited extent, and the possibility could notbe ruled out that the fluorescence with the strains inthis study was due to a non-immunological reaction.The Fc binding capacity of the strains with positiveFA reactions was therefore tested with a radioimmu-nological assay (6) and by their ability to agglutinatesensitized sheep erythrocytes (5). These tests weremade by P. Christensen, Lund, Sweden. The doublediffusion in gel test was performed with the modifica-tions described by Wadsworth (46). Bacterial intracel-lular material was prepared as described by Holm (19).The hemagglutination inhibition test was carried outby the method of Kwapinski (23).

RESULTS

Pathogenicity. Inoculation of PY-glucosebroth without microorganisms produced no vis-ible macroscopic reactions. When the combina-tions of bacteria isolated from 11 symptomlessteeth with chronic apical inflammation were in-oculated, no transmissible infection developed.In contrast, six of the seven combinations de-rived from teeth with purulent apical inflam-mation induced transmissible infections inguinea-pigs (Table 3). Thus, there was a corre-lation between the ability ofthese bacterial com-binations to induce purulent apical inflamma-tions in humans and their ability to producetransmissible infections when inoculated subcu-taneously into guinea pigs.Bacteriological findings. All of the combi-

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TABLE 3. Induction of transmissible infection inguinea pigs by bacterial combinations isolated from

necrotic dental pulpsaNo. of series in

Proportion of infected ani- which trans-mals at the first inoculation missible infec-

in a series tion was estab-Bacterial lishedbcombina-

tion Complete Bacte- Only Com- OnlyCopeeroides Ony plete Bac-combina- Bacte- pleteB-ex- .ccombi- ter-tion cluded' roides nation oides'

D 8/12 0/4 0/4 4G 10/12 0/4 1/4 10 0H 0/12 0/4 0/4P 12/12 0/4 0/4 9AB 12/12 0/4 1/4 12 0UJB 10/12 0/4 4/4 10 4BN 8/12 0/4 0/4 5a Twelve animal series were tested for each com-

plete combination and four were tested for each re-duced combination.

b A bacterial combination was considered to inducea transmissible infection when the infection in the firstanimal in a series could be serially transferred to fourother animals.

'B. melaninogenicus or B. asaccharolyticus.

nations of bacteria which produced transmissi-ble infections contained strains of Bacteriodesmelaninogenicus or B. asaccharolyticus. Thecombinations D, G, P, AB, and UJB containedB. melaninogenicus subsp. intermedius, and thecombination BN contained B. asaccharolyticus(formerly B. melaninogenicus subsp. asaccha-rolyticus) (8). Combination H also contained thelatter species but did not induce transmissibleinfections. When the B. melaninogenicus or B.asaccharolyticus strains were excluded from thecombinations, no transmissible infections devel-oped in the animals. One strain of B. melanin-ogenicus subsp. intermedius (UJB13-c) was ableto produce transmissible infection in pure cul-ture (Table 3). These results suggested that pu-rulent inflammation in the apical region may insome cases be induced by specific combinationsof bacteria in the root canal and that the pres-ence of B. melaninogenicus or B. asaccharolyt-icus in these combinations was essential.Most of the inoculated strains could be iso-

lated after the infection had been transferredfour times (Table 2). The distribution of thebacterial strains in the exudates (Table 2) sug-gests, however, that in addition to B. melanin-ogenicus or B. asaccharolyticus, Peptostrepto-coccus micros may be essential for pathogenic-ity. Streptococcus mitis may play a similar rolein combination G. The inoculum had to contain10' bacteria per ml to induce transmissible infec-

INFECT. IMMUN.

tion. The density of the bacteria in the abscessfluid was 109 to 1010 cells per ml.Antibody determination. The levels of an-

tibodies in the guinea pig sera are shown inTable 4. Sera collected before the inoculationreacted not only with the B. melaninogenicus orB. asaccharolyticus strains, but also with strainsof F. nucleatum, Actinomyces species, Pepto-streptococcus anaerobius, and Lactobacilluscatenaforme. The strains giving positive FA re-actions had no Fc binding capacity with IgG,and the results indicate therefore that guineapigs have natural antibodies against thesestrains. Generally, no significant increase in thetiters could be detected after the abscesses haddeveloped. However, in combinations D and BNone animal showed a significant increase in thelevel of antibodies.Histopathological observations. At 1 day

after inoculation all bacterial combinationscaused a lesion that exhibited the classic featuresof acute inflammation, with accumulation of pol-ymorphonuclear leukocytes (PMNs) and fluidand with loss of collagen fibers. The degree oftissue reaction did, however, vary. When B. mel-aninogenicus or B. asaccharolyticus was pres-ent, a large abscess formed, with a heavy centralaccumulation of degenerating PMNs and micro-organisms. These portions were walled off by aprominent layer of PMNs, followed by edema-tous connective tissue diffusely sprinkled withPMNs and showing considerable loss of collagenarchitecture (Fig. 1).

In contrast, in the absence of B. melaninogen-icus or B. asaccharolyticus, a smaller and morecircumscribed abscess formed, in which denselypacked microorganisms were walled off by adense layer of PMNs, with moderate signs ofdegeneration and connective tissue edema.Thus, the number of PMNs in association withthe microorganisms, as well as the degree ofPMN degeneration and connective tissueedema, seemed to increase in the presence of B.melaninogenicus or B. asaccharolyticus.With bacterial combinations containing B.

melaninogenicus or B. asaccharolyticus, the in-itial abscess increased in size during the first 5days. Large numbers of necrotic and degenerat-ing PMNs filled the abscess cavity, which waslined by a thick layer of PMNs showing signs ofgradually increased degeneration toward thecavity (Fig. 2). Peripheral to this layer, fibro-blastic proliferation was evident, with a moder-ate production of collagen. Diffusely spreadPMNs were observed among the fibroblasts,which also proliferated into the superficial mus-cular layer.

In the sections stained by the Brown-Brenn

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TABLE 4. Presence of antibodies in guinea pigs in which transmissible infection was inducedPresence of antibodies by:

Bacterial StnOuchter- FA technique Hemagglutination inhibi-combination

Strain lonymethod(test se- Normal seruMb Test serum Normal se- Test serumrum)' rum

D Actinomyces sp. - +(4-8)c +(4-128)P. anaerobius - +(4-32) +(4-128)B. melaninogenicus - +(4-16) +(4-512)

G Bacteroides oralis - - +(4-16)B. melaninogenicus - +(4-32) +(4-32)

P F. nucleatum + - +(4-16) - +(4)B. melaninogenicus - +(4) +(4-16)

AB F. nucleatum - +(4) +(4-16) +(8) +(8)L. catenaforme - +(4) +(4-16)B. melaninogenicus - +(4-16) +(4-16)

BN F. nucleatum (BN11a-d) + +(4-16) +(4-16)B. asaccharolyticus - +(4) +(4-32)F. nucleatum (BN9a-1) + +(4-16) +(4-256) +(16) +(16)F. nucleatum (BN9a-m) + - +(4-64) - +(8)

UJB B. melaninogenicus - +(4-32) +(4-32)Selenomonas sputigena - - +(4)F. nucleatum - - +(4-16) - +(8)

a Test serum was collected 7 to 10 days after an abscess had developed.b Normal serum was collected before the inoculation.'Numbers in parentheses represent the reciprocal of the highest dilution of serum giving fluorescence or

hemagglutination inhibition.

procedure, a number of PMNs at the junctionbetween the dense PMN layer and the surround-ing fibroblastic connective tissue showed posi-tive cytoplasmic staining, which also corre-sponded well with a periodic acid-Schiff stainingof the cells. Apparently, these cells were activelyengaged in phagocytosis of microorganisms (Fig.3).

After a 5-day exposure to the combinationswithout B. melaninogenicus or B. asaccharo-lyticus, a small and localized abscess was seen inthe animals. The abscess showed some centralnecrosis, which was walled off by a layer ofPMNs. Reactive fibroblastic connective tissuesurrounded the abscess, and adjacent to thistissue a number of PMNs showed the same stainreaction as described above, i.e. signs of phago-cytosed microorganisms.Among animals exposed to the bacterial com-

bination for longer periods of time, extensivespread of the infection, including perforation ofthe skin and the peritoneal cavity, was observedin animals infected with combinations contain-ing B. melaninogenicus or B. asaccharolyticus.In animals infected with combinations withoutthese strains, however, a gradual resolution tookplace, and after 3 weeks only a few inflammatorycells could be recognized in the connective tissueat the site of infection.

DISCUSSION

Most periapical inflammations caused by in-fected dental root canals are chronic and developinsidiously and without symptoms (36). Acuteexacerbations, which may occur spontaneouslyor as a sequel to endodontic treatment, have notbeen associated with the occurrence of certainmicroorganisms (1, 13).

In the present study, combinations of bacteriaisolated from the root canals of pulpless teethwith or without symptoms but with periapicalbone destruction were tested for their capacityto induce transmissible infections in guinea pigs.It was repeatedly found that abscesses could beproduced. However, persistent abscesses onlydeveloped as a result of infection with bacterialcombinations from teeth with clinical symptomsand evidence of pus formation. The presence ofB. melaninogenicus or B. asaccharolyticus wasfound to be essential for inducing this transmis-sible infection. This is in accordance with earlierobservations (27, 39, 43), but the association ofB. melaninogenicus and B. asaccharolyticuswith the purulent inflammation of pulpless teethhas not been reported before.

Recently, B. asaccharolyticus, formerly con-sidered a subspecies of B. melaninogenicus (18),has been elevated to species rank (8). In the

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690 SUNDQVIST ET AL.

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1 4,

a 0 -tt Ads ? i * ;

Ilk,~~~~~~~~

' A'^ - -,f,,,' ,

<',.''IIIs

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FIG. 1. Portion of skin excised 1 day after inoculation with the bacterial combination UJB, B. melanino-genicus included, showing edge of abscess cavity (A) in dermis walled off by a dense layer of PMNs. Thepreparation was stained with hematoxylin and eosin. x40.

FIG. 2. Wall of dermal abscess at 5 days after inoculation with the same bacterial combination describedin the legend to Fig. 1. Abscess cavity (A) is filled with necrotic material and walled off by PMNs (W) andfibroblastic connective tissue (CT). The preparation was stained with hematoxylin and eosin. x 75. See Fig. 3for further details.

FIG. 3. Higher magnification of abscess wall corresponding to that shown in Fig. 2. In Brown-Brenn- aswell as periodic acid-Schiff-stained sections, the PMNs within a well-defined area of the wall (*) exhibited apositive stain reaction, interpreted as evidence ofphagocytosed microorganisms. CT, Fibroblastic connectivetissue; A, abscess cavity. The preparation was stained with periodic acid-Schiff stain. x130.

majority of previous reports concerning B. mel-aninogenicus, no identification to subspecieslevel was made and B. asaccharolyticus wasreported as B. melaninogenicus. B. melanino-genicus, including B. asaccharolyticus, has beenisolated from various human infections (24) andis a common inhabitant of the oral cavity in

humans (4). It is among the predominant speciesin the gingival crevice, making up approximately5% of the viable counts (12, 40). Slots (37) hasreported recently that B. melaninogenicus con-stitutes more than 50% of the cultivable flora inthe gingival pocket in patients with advancedmarginal periodontitis. Only recently, however,

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ANAEROBIC BACTERIA INDUCE PURULENT INFECTIONS 691

has the organism been isolated regularly fromroot canal samples of pulpless teeth (2, 21, 42,49).

B. melaninogenicus, including B. asaccharo-lyticus, contains endotoxin (16, 30) and is ableto hydrolyze collagen (11, 14, 15), fibrin (47), andother proteins (27). It also produces metabolitesthat may be of importance in the infective proc-ess (26). The collagenolytic activity of B. mela-ninogenicus is of special interest, since tissuedestruction may be the principal pathogenic fac-tor of the organism (38). Both B. melaninogen-icus and B. asaccharolyticus exhibit collagen-olytic activity and also the capacity to inducetransmissible infection. The similarity betweenB. asaccharolyticus and the subspecies of B.melaninogenicus has been shown, by numericaltaxonomic analysis, to be only 63% (42). Thissuggests that one or more specific propertiesmay be of particular importance in abscess for-mation, for example the collagenolytic capacity.

In the present study, transmissibility of the B.melaninogenicus or B. asaccharolyticus infec-tion could be demonstrated as occurring despitea massive immigration of PMNs to the site ofinfection. This indicates that the virulence maybe partly related to an ability to resist ingestionor intracellular killing once ingested. It is ofinterest that Okuda and Takazoe (33) found thata highly infective strain of B. melaninogenicus,which had a capsule, was not readily phagocy-tosed and killed. It has also been found that oralstrains of the species may have a capsule andthat encapsulated strains produce experimentalabscesses, in contrast to non-encapsulatedstrains (44). Recently, strains of B. asaccharo-lyticus have also been shown to be encapsulated(29).Moreover, we found that almost all additional

strains in each combination containing B. mel-aninogenicus or B. asaccharolyticus survivedin the tissues, which is suggestive of impairedfunction among the accumulated PMNs. Thismay be due to a leukocidal action of B. melanin-ogenicus and B. asaccharolyticus. In ovine footabscesses, which are also caused by a synergisticmixed infection, one of the organisms involved(Fusobacterium necrophorum) produces a leu-kocidal toxin which protects both this and otherorganisms from being phagocytosed (34). Therole of B. melaninogenicus and B. asaccharo-lyticus may be the same in other polymicrobialpyogenic infections of the oral cavity. In fact,Okuda and Takazoe (33) have demonstratedthat capsular material from B. melaninogenicusinhibits phagocytosis and the phagocytic killingof another microorganism in an in vitro system.

In a polymicrobial infection, the pathogenicity

expressed by the bacteria is the result of syner-gistic actions in the tissue (35). B. melaninogen-icus and B. asaccharolyticus are usually unableto induce abscesses when inoculated subcuta-neously in pure culture into guinea pigs (27).Additional organisms are required in the inocu-lum to achieve pathogenicity. However, differentstrains of B. melaninogenicus vary as to thedegree of pathogenicity. Macdonald et al. (27)were able to produce abscesses with one strainof B. melaninogenicus (CR2A) without the sup-port of additional organisms. In the presentstudy, the strain UJB13-c exhibited the samecapacity. It is also clear that not all organisms inthe pathogenic combinations of the presentstudy are essential for transmissible infection tooccur. The bacteria recovered from the abscessesafter four subsequent transfers in the animals(Table 2) suggest that, in addition to B. mela-ninogenicus and B. asaccharolyticus, the an-aerobic P. micros and the facultatively anaero-bic S. mitis could be essential organisms. Ourfailure to produce transmissible infection withcombination H (Table 3), which was derivedfrom a tooth with signs of purulent inflamma-tion, may be due to a failure to isolate an essen-tial organism in the original sample.We could detect an increased level of antibod-

ies to B. melaninogenicus, B. asaccharolyticus,and F. nucleatum in sera collected after an ab-scess had formed, but we also found that theguinea pigs had naturally occurring antibodiesto some of the bacterial species. In humans lowlevels of antibodies to B. melaninogenicus (7,17), F. nucleatum (17), and most other gingivalcrevice microorganisms (31, 48) can be demon-strated. This is consistent with what is usuallyobserved in the case of indigenous bacteria.Thus, it is possible that the reactions observedin the apical tissues in patients and in the skinof the guinea pigs may have been aggravated byimmunological reactions. The lack of a consist-ent increase in the antibody titer after abscessformation in the guinea pigs suggests, however,that the immunological contribution to the re-action could only have been small.The mechanical instrumentation of the in-

fected root canal is an important part of endo-dontic treatment. Usually irrigating solutionsare employed to facilitate this procedure. It hasbeen shown that the contents of root canals areextruded at the apical foramen when irrigatingsolutions are used (45). If the extruded materialis infected, it may induce an acute exacerbation(20, 36, 49). As irrigation is regularly used, it islikely that microorganisms are often forced outperiapically, but the experience is that acuteexacerbation occurs in relatively few cases. The

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finding that certain acute exacerbations may becaused by polymicrobial infections in which spe-cial microorganisms attain pathogenicity by syn-ergism may explain why it does not occur moreoften.

LITERATURE CITED

1. Bartels, H. A., I. J. Naidorf, and H. Blechman. 1968.A study of some factors associated with endodontic"flare-ups." Oral Surg. Oral Med. Oral Pathol. 25:255-261.

2. Bergenholtz, G. 1974. Micro-organisms from necroticpulp of traumatized teeth. Odontol. Revy 25:347-358.

3. Blechman, H. 1973. Infection of the pulp and periapicaltissues, p. 237-239. In W. A. Nolte (ed.), Oral microbi-ology, 2nd ed. C. V. Mosby Co., St. Louis.

4. Burdon, K. L. 1928. Bacterium melaninogenicus fromnormal and pathogenic tissues. J. Infect. Dis. 42:161-171.

5. Christensen, P., and G. Kronwall. 1974. Capacity ofgroup A, B, C, D and G streptococci to agglutinatesensitized sheep red cells. Acta Pathol. Microbiol.Scand. 82:19-24.

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