INFECTION AND IMMUNITy, Feb. 1981, p. 592-597 Vol. 31, No. 20019-9567/81/020592-06$02.00/0

Animal Model for Anaerobic Lung AbscessDON W. KANNANGARA, HARAGOPAL THADEPALLI,* VINH T. BACH, AND DAVID WEBB

Division of Infectious Diseases, Department of Internal Medicine, Charles R. Drew Postgraduate School ofMedicine, Martin Luther King, Jr. General Hospital, Los Angeles, California 90059 and University of

California-Los Angeles School ofMedicine, Los Angeles, California 90024

There are no satisfactory animal models for the study of anaerobic lung abscess.Aspiration of food, gastric mucin, or hydrochloric acid, or any combination ofthese, along with oropharyngeal bacteria, is commonly believed to cause aspira-tion pneumonia and lung abscess. In the animal model described, none of theseadjuvants was effective in producing anaerobic lung abscesses. Anaerobic bacteriaderived from dental scrapings of a healthy adult (Peptococcus morbillorum,Fusobacterium nucleatum, Eubacterium lentum, and Bacteroides fragilis), wheninoculated transtracheally without any adjuvants into New Zealand male whiterabbits, consistently produced lung abscesses. Neither B. fragilis by itself nor amixture of P. morbillorum, F. nucleatum, and E. lentum without the addition ofB. fragilis produced lung abscesses. The bacterial isolates used in this study werestored in prereduced chopped-meat-glucose medium and subcultured severaltimes and were found effective in reproducing anaerobic lung abscesses repeatedly.This animal model is suitable for the study of pathogenesis, diagnosis, andtreatment of B. fragilis-associated anaerobic lung abscess.

Anaerobic bacteria are nornal flora of themouth (11), and presumably aspiration of oro-pharyngeal secretions leads to the developmentof a lung abscess (2, 3). On this basis, Smith (12,13) attempted to produce lung abscesses in mice,guinea pigs, and rabbits by intratracheal inoc-ulation of material obtained from the teeth ofpatients with pyorrhea. In his studies, the inoc-ulum was undefined and the fatality rate washigh, making it unsuitable for controlled studies.Additional factors, such as gastric hydrochloricacid (4), mucin, or food material, are said to actas adjuvants in the development of lung abscess.It is not known whether aerobes promote anaer-obic lung abscess formation by virtue of syner-gism as they do in intra-abdominal abscesses(10, 16). A bacteriologically well-defined animalmodel is obviously needed to elucidate the path-ogenic mechanisms of anaerobic lung abscess.

(This work was presented at the 11th Inter-national Congress of Chemotherapy-19th Inter-science Conference on Antimicrobial Agents andChemotherapy, Boston, Mass., October, 1979.)

MATERIALS AND METHODSAnimals. New Zealand white male rabbits, each

weighing nearly 2.5 kg (LIT Rabbitry, Aptos, Calif.)were used throughout the study, with the exception ofone batch of 2-week-old rabbits used to assess theeffect of age on the development of lung abscess.

Bacterial strains. Clinical isolates of stock cul-tures of anaerobic bacteria, Peptostreptococcus ana-erobius, Fusobacterium nucleatum, Bacteroides fra-gilis, and Bacteroides melaninogenicus subsp. mela-

ninogenicus, were used in the beginning of the study.A strain of Fusobacterium necrophorum, ATCC27852, was commercially obtained for use in this study.The Escherichia coli and Pseudomonas aeruginosain this study were human blood culture isolates.

Subsequently, a set of bacteria cultured from thedental scrapings of a healthy male adult free of dentalinfection was used, namely, group C Streptococcus,Peptococcus morbillorum, F. nucleatum, Eubacte-rium lentum, and B. fragilis.

Preparation of inocula. Bacteria. All anaerobiccultures were stocked at room temperature in prere-duced, anaerobically sterilized chopped-meat-glucosemedium, then transferred to prereduced, anaerobicallysterilized peptone-yeast-glucose medium the day be-fore the experiment and incubated at 37°C. Brothcultures of each organism were diluted with the pep-tone-yeast-glucose medium to contain 108 viable cellsper ml as determined by the nephelometric methodagainst McFarland barium sulfate standard no. 1.Equal volumes of each bacterial species were mixed toprepare the final inoculum immediately before inoc-ulation.

Additives. Additives included sterile hydrochloricacid, sterile chopped-meat-glucose medium, meltedbrain heart infusion agar, freshly drawn rabbit blood,house dust, and rabbit chow in tap water. Viablebacterial counts were done in the final inoculum afterthe addition of the above additives. In addition, aero-bic bacteria, such as P. aeruginosa, E. coli, and En-terococcus, were inoculated in order to study bacterialsynergism. Later, group C Streptococcus was usedalong with dental isolates for the same purpose. Thedetails ofvarious inocula and the additives used in thisstudy were as shown in Table 1.Experimental design. The rabbits were anesthe-

tized with ether, and the ventral portion of the neck

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ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 593

TABLE 1. Results of initial experimentsOutcome

Expt No. of InocuIUMa Additive Autopsy culture resultsnos. rabbits noculuAPneumodtia Lung ab- (no. of rabbits)

scess

i-7 40 P. anaerobius, F. CMG or BHIb or 1 (empyema) None Lung tissue sterile inFetdagro

11-12 15 P. anaerobius, B.fragilis, S. fae-calis, ± F. nuclea-tum

13 10 P. anaerobius, F.necrophorum, B.fragilis

14 5 E. coli only

15 5 E. coli, P. anaero-

bius, F. necropho-rum, B. fragilis

16-17 19 P. anaerobius, F.necrophorum, B.fragilis, B. mela-ninogenicussubsp. melanino-genicus

melted agar orrabbit blood orHCI pH 1 or pH2.3 (1 ml each)

HCI, pH 1 (1, 1.5, or3.5 ml)

all

11d (6 died)

HCI, pH 1 (1 or 2 2ml)

Sterilized housedust in tap water,0.2 g/18 ml (1 ml)

(1 died)

House dust as (2 died)above

House dust as Noneabove

0.5% or 1% mucin Noneand sterilizedrabbit chow intap water, 0.2 g/18 ml (1 ml)

None P. aeruginosa (4); B.fragilis (2); P.aeruginosa, B. fra-gilis, and P. anaero-bius (3); sterile (11)

None P. anaerobius (1); B.fragilis (1); sterile(13)

1 B. fragilis (2; also hadpositive blood cul-tures); sterile (8)

1 E. coli (3); sterile (2)

1 E. coli, B. fragilis, andP. anaerobius (1)

None Lung tissue sterile inall

a 0l colony-forming units per ml. F. necrophorum isolate was strain ATCC 27852.b CMG, Chopped-meat-glucose medium; BHI, brain heart infusion broth.c Six young rabbits, 2 weeks old, ca. 1 kg each.d Five cases were in the young rabbits.

was shaved and cleaned with povidone iodine (Beta-dine; Purdue, Norwalk, Conn.). The trachea was ex-

posed by a midline vertical incision in the neck, andan 8-in. (ca. 20.3-cm), 22 gauge catheter with a 19gauge needle (Deseret Pharmaceuticals, Sandy, Utah)was introduced by a single puncture through the uppertracheal rings. The catheter was then introduced intothe tracheobronchial tree as far as possible, and theneedle was withdrawn with the catheter left in thetrachea. About 2 ml of the bacterial inoculum wasintroduced through the trachea with or without theadditives as shown in Table 1. The catheter was thenremoved, and the incision was closed with metallicskin clips. The animals recovered from the anesthesiasoon after the procedure and were transferred to theircages.

Evaluation. (i) Fluoroscopy. All rabbits were ex-amined under a fluoroscopy screen every other day for3 weeks, and "spot films" were taken when any pul-monary lesions were seen. The roentgenograms were

later correlated with the lesions seen at autopsy.(ii) Gallium-67 citrate scanning. To monitor the

inflammatory activity of the lesions, 1.5 mCi of 67Gawas injected through the marginal vein of the ear, andrabbits were scanned 48 and 72 h later for evidence ofincreased isotope uptake in the lungs. The 67Ga scanswere repeated in all animals that showed increaseduptake in the lungs to assess the progress of infection.

Autopsy. All animals were autopsied at the end ofthe study, and the lungs were aseptically removed andsectioned with sterile instruments. A portion of theaffected lung was cultured for reisolation of the bac-teria used in the inoculum, and the rest was fixed inFormalin for histopathological examination. Hematox-ylin-eosin-stained sections of the affected lung werestudied for histological changes. The Brown and Brennmodifications of Gram stain were used for the detec-tion of bacterial invasion of the tissues. A lung abscesswas defined as gangrenous destruction of the lungaccompanied by pus formation and fibrous tissue re-

nucleatum, B. fra-gilis, or single iso-lates of same

8-10 20 (6)C P. anaerobius, B.fragilis, P. aerugi-nosa, ± F. nuclea-tum

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594 KANNANGARA ET AL.

action around the necrotic cavities of the lung. Whenno such destructive changes were seen, consolidationalone was interpreted as pneumonia. Aerobic and an-aerobic cultures were obtained from the lung abscess,areas of pneumonia, and apparently nornal areas atautopsy. Blood cultures were drawn into Trypticasesoy and thioglycolate media (BBL Microbiology Sys-tems, Cockeysville, Md.).

Cultures and identification of bacteria. Thespecimens were cultured for aerobic bacteria in bloodagar and MacConkey agar and cultured for anaerobicbacteria in freshly prepared blood agar, supplementedwith menadione and incubated at 37°C. Incubationand isolation of all anaerobic bacteria were achievedin the anaerobic glove box of Aranki et al. (1). Finalidentification of the anaerobic bacteria was done bygas-liquid chromatography and biochemical reactionsin the prereduced, anaerobically sterilized media. Onlyquantitative bacteriology cultures were done; the bac-teria from the infected sites were not quantitated.

RESULTS

Preliminary experiments (experiments 1to 17, Table 1). Initially many different com-

binations of anaerobic and aerobic bacteria andvarious adjuvants were inoculated transtra-cheally into rabbits in an attempt to induce lungabscess fornation. The anaerobic bacteria used

included laboratory stock cultures of P. anaero-bius, F. nucleatum, F. necrophorum, B. fragilis,and B. melaninogenicus. These were tried singlyor in combinations with aerobic bacteria such as

P. aeruginosa, E. coli, and Streptococcus fae-calis in the presence of different adjuvants,namely, hydrochloric acid, chopped-meat-glu-cose medium, brain heart infusion broth, meltedagar, rabbit blood, sterilized house dust, mucin,and sterilized rabbit chow. All blood cultureswere negative except in two rabbits in experi-ment no. 13. The experiments were found to betime-consuming and laborious but not produc-tive of useful results. To save space, these are

summarized in Table 1.Successful experiments (Table 2). Stock

cultures of anaerobic bacteria used during thefirst 17 experiments were inconsistent in produc-ing lung lesions. Hence, it was decided to use

fresh isolates. Dental scrapings from a patientwith no apparent dental infection were culturedovernight, and the individual isolates were iden-tified as P. morbillorum, F. nucleatum, E. len-

tum, B. fragilis, and group C Streptococcus.These isolates were then mixed with sterile rab-bit chow (0.2 g with 18 ml of water) and injected

TABLE 2. Experiments with inoculation ofhuman dental isolates

no. No. of Outcome Autopsy and cultureExpt n.rabbits Inoculum' AdditivereutPneumonia Lung abscess results

18 8 P. morbillorum, F. Rabbit chow in None 7b (1 died) All animals at autopsynucleatum, E. len- tap water, 0.2 developed lung ab-tum, B. fragilis, g/18 ml (1 ml) scess. Culture ofpusgroup C Strepto- yielded B. fragilis,coccus E. lentum, P. mor-

billorum, F. nuclea-tum, and group CStreptococcus

19,20 15 P. morbillorum, F. None 2 13 (2 died) "7Ga uptake increasednucleatum, E. len- in all 13 survivorstum, B. fragilisc on day 14. Autopsy:

Multiple lung ab-scesses, 0.5 to 2 cmin diameter, seen inboth lungs, more inthe right lung. Cul-ture: B. fragilis, F.nucleatum, P. mor-billorum, and E.lentum

21 6 B. fragilis None 3 None B. fragilis (3 rabbits)

22 6 P. morbillorum, F. None None None None isolatednucleatum, E. len-tum

a lo' colony-forming units per ml.b Multiple lung abscesses, mainly in the right lung.7Ga was injected on day 12.

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ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 595

transtracheally into eight rabbits. One rabbitdied on day 3, was left overnight in the freezer,and was unsuitable for autopsy. The seven re-maining animals were sacrificed on day 12. Allseven had multiple lung abscesses, mostly in theright lung; five of them had empyema in addi-tion. These lung abscesses, on culture, yieldedB. fragilis and group C Streptococcus in allseven. In addition, Peptococcus and E. lentumwere isolated in three animals, and Fusobacte-rium was isolated in two.

In the above experiment, bacterial isolatesfrom dental scrapings with sterile rabbit chowwere successful in the development of lung ab-scess. Hence, the next experiment was designedto determine the role of group C Streptococcusand rabbit chow.

In this experiment, eight rabbits were inocu-lated with a mixture of pure cultures of P. mor-billorum, F. nucleatum, F. lentum, and B. fra-gilis, but neither group C Streptococcus nor theadditive rabbit chow was administered. One rab-bit died of pneumonia on day 3, and the remain-ing seven developed lung abscesses, as demon-strated by 67Ga citrate scan (see Fig. 1). Theywere sacrificed on day 14, and all had multiplelung abscesses. The blood cultures were nega-tive. Culture of the pus from the lung abscessesrevealed B. fragilis in seven, E. lentum in five,P. morbillorum in three, and F. nucleatum intwo. This experiment suggested that no addi-tives are essential and that anaerobes alone canproduce lung abscess. The experiment was re-peated subsequently in a batch of 20 rabbits, allof which developed lung abscesses. The ab-scesses were more common in the right lungthan the left, but sometimes occupied both sides.Empyema was noted frequently, and the ab-scesses were often multiple. The initial lesionwas pneumonia, which was best seen around day4. By day 7, abscesses could be easily recognizedat autopsy.

Six rabbits were injected with pure culture ofB. fragilis (experiment no. 21). None of theseanimals developed lung abscess. Similarly, P.morbillorum, F. nucleatum, and E. lentum incombination did not produce lung abscess in sixrabbits (experiment no. 22). B. fragilis appearsto play an important role in the pathogenesis oflung abscess. It is incapable of producing lungabscesses by itself, but is able to do so in com-bination with other anaerobic bacteria.

In summary, stock cultures of anaerobic bac-teria were ineffective in producing lung abscessin experimental animals. Food particles, such asmeat and rabbit chow, or hydrochloric acid andgastric mucin failed to potentiate the formationof anaerobic lung abscess. Other additives, suchas house dust, blood, melted agar, and brain

heart infusion broth, were also ineffective. F.necrophorum ATCC 27852, known to be patho-genic in mice and rabbits (17), failed to causelung abscess. Neither a mixture of P. morbillo-rum, F. nucleatum, and E. lentum without B.fragilis, nor pure cultures of B. fragilis, wascapable of producing lung abscess, but thesecultures produced lung abscess consistently inrabbits when combined.

DISCUSSION

Lack ofa bacteriologically well-defined animalmodel is a major impediment to the study of thepathogenesis and therapy of anaerobic lung ab-scess. Smith (12, 13) inoculated pyorrhea mate-rial and pus from Vincent's angina from humansinto the exteriorized trachea of rabbits to inducelung abscess formation. Varney (14) did similarstudies in dogs by inoculating pus obtained froma case of lung abscess in a human. Althoughanaerobic bacteria were noted in the pus, theinocula were not defined, as the bacteria werenot identified. Furthermore, these models unfor-tunately carried a high mortality rate and weretherefore unsuitable.

Several other factors are mentioned as pro-moting the development of lung abscess. Amongthem, aspiration of gastric contents, i.e., hydro-chloric acid (4), gastric mucin, and food, is con-sidered to lead the way to chemical pneumonitisand tissue necrosis, followed by bacterial infec-tion causing lung abscess. In the initial experi-ments, we attempted to mimic the naturalcourse by inoculating hydrochloric acid, gastricmucin, and food (rabbit chow), but failed toproduce lung abscess. This failure may in partbe related to the use of laboratory stock culturesof anaerobic bacteria in the inoculum. In manyof the animal models described for anaerobicinfections, suppurative lesions were produced byemploying such adjuvants as chopped-meat-car-bohydrate medium, semisolid agar cultures (15),blood and hemostatic agents (6), and gentamicinpretreatment (C. A. Rotilie, R. J. Fass, and R.L. Perkins, Clin. Res. 22:452A, 1974). Unfortu-nately, adaptation of these methods did not im-prove development of lung abscesses. A finalattempt utilizing sterile house dust, which re-sults in a high incidence of pneumonia in pigs(8), also failed to serve the purpose.Lung abscesses were consistently produced in

rabbits when a mixture of fresh isolates of an-aerobes from dental scrapings of an apparentlyhealthy male was injected transtracheally.These abscesses were histologically character-ized as gangrenous destruction of the lung sub-stance, filled with purulent material surroundedby fibrous tissue. Fluoroscopic examination sug-

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FIG. 1. Experimental anaerobic lung abscess in a rabbit (A) Multilocular lung abscess. (B) Normal WGacitrate scan in a rabbit shown for comparison. (C) 67Ga scan done on day 14 of infection. Note the denseincreased uptake of "Ga in the right lung.

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ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 597

gested that the majority of rabbits developedpneumonia and pleural effusions by day 4 andabscess formation at the end of the first week.These abscesses were documented by obtainingchest roentgenograms and positive 67Ga scans,

by histopathological examination, and by cul-ture of the purulent material. By these parame-ters, the clinical course ofthe disease was similarto lung abscess in humans. There are severalother pathogenic mechanisms involved in thedevelopment of lung abscess in humans; septi-cemia and bronchial obstruction, for example,also cause lung abscess. The present model mim-ics only lung abscess developed after aspiration.

B. fragilis is not an uncommon isolate in casesof lung abscess. It was found in 9 of 40 cases

(22%) reported by Finegold (5). In his experi-ence, B. fragilis is found in 15 to 20% of all

anaerobic pleuropulmonary infections. In mostinstances, B. fragilis is found along with otheranaerobic bacteria. B. fragilis is associated withnearly 15 to 20% of all dental infections (9). Ourexperimental studies also suggest that, althoughB. fragilis alone may be incapable of causinglung abscess, it can do so in collaboration withother anaerobic bacteria (7).The animal model described is bacteriologi-

cally well defined, reproducible, and consistent.By utilizing this modeL the effects of environ-mental factors on the pathogenesis of anaerobiclung infections can be studied. Furthermore, thismodel may also provide clues to study the lungdefense mechanisms in anaerobic lung infec-tions. This model may also be used to evaluatethe therapeutic efficacy of various antibiotics inanaerobic lung infections. The lung lesions couldbe followed up by fluoroscopy, roentgenograms,and 67Ga scans. The animals need not be sacri-ficed in order to document the lesions, as inother animal models for anaerobic infections.The disadvantage of this model is that it cannotbe produced without the addition of B. fragilis,which is found in only 15 to 20% of anaerobiclung infections.For the first time, we now have a bacteriolog-

ically well-defined model for the study of anaer-obic lung abscess, and we hope that it will beused by other investigators to answer some ofthe questions related to anaerobic lung abscess.

ACKNOWLEDGMENT

This work was supported by Public Health Service grantHL-20963-01 from the Airways Diseases Branch, Division ofLung Diseases, National Heart and Lung Institute, Bethesda,Md.

LITERATURE CITED

1. Aranki, A., S. A. Syed, E. B. Kenny, and R. Freter.1969. Isolation of anaerobic bacteria from gingiva andmouse cecum by means of a simplified glove box pro-cedure. Appl. Microbiol. 17:568-572.

2. Bartlett, J. G., and S. M. Finegold. 1972. Anaerobicpleuropulmonary infections. Medicine 51:413-450.

3. Bartlett, J. G., S. L Gorbach, and S. M. Finegold.1974. The bacteriology of aspiration pneumonia. Am. J.Med. 56:202-207.

4. Exarhos, N. D., W. D. Logan, 0. A. Abbott, and C. R.Hatcher. 1965. The importance of pH and volume intracheobronchial aspiration. Dis. Chest 47:167-169.

5. Finegold, S. M. 1977. Anaerobic bacteria in human dis-ease, p. 239. Academic Press, Inc., New York.

6. Hill G. B. 1978. Enhancement of experimental anaerobicinfections by blood, hemoglobin, and hemostatic agents.Infect. Immun. 19:443 449.

7. Hite, K. E., M. Locke, and H. C. Hesseltine. 1948.Synergism in experimental infections with nonsporulat-ing anaerobic bacteria. J. Infect. Dis. 84:1-9.

8. Jericho, K. W. F., and N. Harries. 1975. Dusty feed andacute respiratory disease in pigs. Can. Vet. J. 16:360-366.

9. Kannangara, D. W., H. Thadepalli, and J. L Mc-Quirter. 1980. Bacteriology and treatment of dentalinfections. Oral Surg. Oral Med. Oral Pathol. 50:103-109.

10. Onderdonk, A. B., J. G. Bartlett, T. Louie, N. Sulli-van-Seigler, and S. L Gorbach. 1976. Microbial syn-ergy in experimental intra-abdominal abscess. Infect.Immun. 13:22-26.

11. Rosebury, T. 1962. Microorganisms indigenous to man.McGraw-Hill, New York.

12. Smith, D. T. 1927. Experimental aspiratory abscess. Arch.Surg. 14:231-239.

13. Smith, D. T. 1930. Fusospirochetal disease of the lungsproduced with cultures from Vincent's angina. J. Infect.Dis. 46:303-309.

14. Varney, P. L 1929. The bacterial flora of treated anduntreated abscess of the lung. Arch. Surg. 19:1602-1617.

15. Walker, C. B., and T. D. Wilkins. 1976. Use of semisolidagar for initiation of pure Bacteroides fragilis infectionin mice. Infect. Immun. 14:721-725.

16. Weinstein, W. M., A. B. Onderdonk, J. G. Bartlett,and S. L. Gorbach. 1974. Experimental intra-abdomi-nal abscess in rats: development of an experimentalmodel. Infect. Immun. 10:1250-1255.

17. Wilkins, T. D., andL D. S. Smith. 1974. Chemotherapyof an experimental Fusobacteruum (Sphaerophorus)necrophorum infection in mice. Antimicrob. AgentsChemother. 5:658-662.

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