Animal Model for Anaerobic Lung Abscess

Transcription

1 INFECTION AND IMMUNITy, Feb. 1981, p Vol. 31, No /81/ $02.00/0 Animal Model for Anaerobic Lung Abscess DON W. KANNANGARA, HARAGOPAL THADEPALLI,* VINH T. BACH, AND DAVID WEBB Division of Infectious Diseases, Department of Internal Medicine, Charles R. Drew Postgraduate School of Medicine, Martin Luther King, Jr. General Hospital, Los Angeles, California and University of California-Los Angeles School ofmedicine, Los Angeles, California There are no satisfactory animal models for the study of anaerobic lung abscess. Aspiration of food, gastric mucin, or hydrochloric acid, or any combination of these, along with oropharyngeal bacteria, is commonly believed to cause aspiration pneumonia and lung abscess. In the animal model described, none of these adjuvants was effective in producing anaerobic lung abscesses. Anaerobic bacteria derived from dental scrapings of a healthy adult (Peptococcus morbillorum, Fusobacterium nucleatum, Eubacterium lentum, and Bacteroides fragilis), when inoculated transtracheally without any adjuvants into New Zealand male white rabbits, consistently produced lung abscesses. Neither B. fragilis by itself nor a mixture of P. morbillorum, F. nucleatum, and E. lentum without the addition of B. fragilis produced lung abscesses. The bacterial isolates used in this study were stored in prereduced chopped-meat-glucose medium and subcultured several times and were found effective in reproducing anaerobic lung abscesses repeatedly. This animal model is suitable for the study of pathogenesis, diagnosis, and treatment of B. fragilis-associated anaerobic lung abscess. Anaerobic bacteria are nornal flora of the mouth (11), and presumably aspiration of oropharyngeal secretions leads to the development of a lung abscess (2, 3). On this basis, Smith (12, 13) attempted to produce lung abscesses in mice, guinea pigs, and rabbits by intratracheal inoculation of material obtained from the teeth of patients with pyorrhea. In his studies, the inoculum was undefined and the fatality rate was high, making it unsuitable for controlled studies. Additional factors, such as gastric hydrochloric acid (4), mucin, or food material, are said to act as adjuvants in the development of lung abscess. It is not known whether aerobes promote anaerobic lung abscess formation by virtue of synergism as they do in intra-abdominal abscesses (10, 16). A bacteriologically well-defined animal model is obviously needed to elucidate the pathogenic mechanisms of anaerobic lung abscess. (This work was presented at the 11th International Congress of Chemotherapy-19th Interscience Conference on Antimicrobial Agents and Chemotherapy, Boston, Mass., October, 1979.) MATERIALS AND METHODS Animals. New Zealand white male rabbits, each weighing nearly 2.5 kg (LIT Rabbitry, Aptos, Calif.) were used throughout the study, with the exception of one batch of 2-week-old rabbits used to assess the effect of age on the development of lung abscess. Bacterial strains. Clinical isolates of stock cultures of anaerobic bacteria, Peptostreptococcus anaerobius, Fusobacterium nucleatum, Bacteroides fragilis, and Bacteroides melaninogenicus subsp. melaninogenicus, were used in the beginning of the study. A strain of Fusobacterium necrophorum, ATCC 27852, was commercially obtained for use in this study. The Escherichia coli and Pseudomonas aeruginosa in this study were human blood culture isolates. Subsequently, a set of bacteria cultured from the dental scrapings of a healthy male adult free of dental infection was used, namely, group C Streptococcus, Peptococcus morbillorum, F. nucleatum, Eubacterium lentum, and B. fragilis. Preparation of inocula. Bacteria. All anaerobic cultures were stocked at room temperature in prereduced, anaerobically sterilized chopped-meat-glucose medium, then transferred to prereduced, anaerobically sterilized peptone-yeast-glucose medium the day before the experiment and incubated at 37 C. Broth cultures of each organism were diluted with the peptone-yeast-glucose medium to contain 108 viable cells per ml as determined by the nephelometric method against McFarland barium sulfate standard no. 1. Equal volumes of each bacterial species were mixed to prepare the final inoculum immediately before inoculation. Additives. Additives included sterile hydrochloric acid, sterile chopped-meat-glucose medium, melted brain heart infusion agar, freshly drawn rabbit blood, house dust, and rabbit chow in tap water. Viable bacterial counts were done in the final inoculum after the addition of the above additives. In addition, aerobic bacteria, such as P. aeruginosa, E. coli, and Enterococcus, were inoculated in order to study bacterial synergism. Later, group C Streptococcus was used along with dental isolates for the same purpose. The details of various inocula and the additives used in this study were as shown in Table 1. Experimental design. The rabbits were anesthetized with ether, and the ventral portion of the neck 592

2 VOL. 31, 1981 ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 593 TABLE 1. Results of initial experiments Outcome Expt No. of InocuIUMa Additive Autopsy culture results nos. rabbits noculuapneumodtia Lung ab- (no. of rabbits) scess 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 67Ga was injected through the marginal vein of the ear, and rabbits were scanned 48 and 72 h later for evidence of increased isotope uptake in the lungs. The 67Ga scans were repeated in all animals that showed increased uptake in the lungs to assess the progress of infection. Autopsy. All animals were autopsied at the end of the study, and the lungs were aseptically removed and sectioned with sterile instruments. A portion of the affected lung was cultured for reisolation of the bacteria used in the inoculum, and the rest was fixed in Formalin for histopathological examination. Hematoxylin-eosin-stained sections of the affected lung were studied for histological changes. The Brown and Brenn modifications of Gram stain were used for the detection of bacterial invasion of the tissues. A lung abscess was defined as gangrenous destruction of the lung accompanied by pus formation and fibrous tissue re- i-7 40 P. anaerobius, F. CMG or BHIb or 1 (empyema) None Lung tissue sterile in nucleatum, B. fragilis, or single iso- rabbit blood or Fetdagro melted agar or all lates of same HCI ph 1 or ph 2.3 (1 ml each) (6)C P. anaerobius, B. fragilis, P. aeruginosa, ± F. nucleatum P. anaerobius, B. fragilis, S. faecalis, ± F. nucleatum P. anaerobius, F. necrophorum, B. fragilis 14 5 E. coli only 15 5 E. coli, P. anaerobius, F. necrophorum, B. fragilis P. anaerobius, F. necrophorum, B. fragilis, B. melaninogenicus subsp. melaninogenicus HCI, ph 1 (1, 1.5, or 3.5 ml) HCI, ph 1 (1 or 2 2 ml) Sterilized house dust in tap water, 0.2 g/18 ml (1 ml) 11d (6 died) (1 died) House dust as (2 died) above House dust as None above 0.5% or 1% mucin None and sterilized rabbit chow in tap water, 0.2 g/ 18 ml (1 ml) a 0l colony-forming units per ml. F. necrophorum isolate was strain ATCC 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. None P. aeruginosa (4); B. fragilis (2); P. aeruginosa, B. fragilis, and P. anaerobius (3); sterile (11) None P. anaerobius (1); B. fragilis (1); sterile (13) 1 B. fragilis (2; also had positive blood cultures); sterile (8) 1 E. coli (3); sterile (2) 1 E. coli, B. fragilis, and P. anaerobius (1) None Lung tissue sterile in all was shaved and cleaned with povidone iodine (Betadine; Purdue, Norwalk, Conn.). The trachea was exposed by a midline vertical incision in the neck, and an 8-in. (ca cm), 22 gauge catheter with a 19 gauge needle (Deseret Pharmaceuticals, Sandy, Utah) was introduced by a single puncture through the upper tracheal rings. The catheter was then introduced into the tracheobronchial tree as far as possible, and the needle was withdrawn with the catheter left in the trachea. About 2 ml of the bacterial inoculum was introduced through the trachea with or without the additives as shown in Table 1. The catheter was then removed, and the incision was closed with metallic skin clips. The animals recovered from the anesthesia soon after the procedure and were transferred to their cages. Evaluation. (i) Fluoroscopy. All rabbits were examined under a fluoroscopy screen every other day for 3 weeks, and "spot films" were taken when any pulmonary lesions were seen. The roentgenograms were

3 594 KANNANGARA ET AL. action around the necrotic cavities of the lung. When no such destructive changes were seen, consolidation alone was interpreted as pneumonia. Aerobic and anaerobic cultures were obtained from the lung abscess, areas of pneumonia, and apparently nornal areas at autopsy. Blood cultures were drawn into Trypticase soy and thioglycolate media (BBL Microbiology Systems, Cockeysville, Md.). Cultures and identification of bacteria. The specimens were cultured for aerobic bacteria in blood agar and MacConkey agar and cultured for anaerobic bacteria in freshly prepared blood agar, supplemented with menadione and incubated at 37 C. Incubation and isolation of all anaerobic bacteria were achieved in the anaerobic glove box of Aranki et al. (1). Final identification of the anaerobic bacteria was done by gas-liquid chromatography and biochemical reactions in the prereduced, anaerobically sterilized media. Only quantitative bacteriology cultures were done; the bacteria from the infected sites were not quantitated. RESULTS Preliminary experiments (experiments 1 to 17, Table 1). Initially many different combinations of anaerobic and aerobic bacteria and various adjuvants were inoculated transtracheally into rabbits in an attempt to induce lung abscess fornation. The anaerobic bacteria used included laboratory stock cultures of P. anaerobius, F. nucleatum, F. necrophorum, B. fragilis, and B. melaninogenicus. These were tried singly or in combinations with aerobic bacteria such as P. aeruginosa, E. coli, and Streptococcus faecalis in the presence of different adjuvants, namely, hydrochloric acid, chopped-meat-glucose medium, brain heart infusion broth, melted agar, rabbit blood, sterilized house dust, mucin, and sterilized rabbit chow. All blood cultures were negative except in two rabbits in experiment no. 13. The experiments were found to be time-consuming and laborious but not productive of useful results. To save space, these are summarized in Table 1. Successful experiments (Table 2). Stock cultures of anaerobic bacteria used during the first 17 experiments were inconsistent in producing lung lesions. Hence, it was decided to use fresh isolates. Dental scrapings from a patient with no apparent dental infection were cultured overnight, and the individual isolates were identified as P. morbillorum, F. nucleatum, E. lentum, B. fragilis, and group C Streptococcus. These isolates were then mixed with sterile rabbit chow (0.2 g with 18 ml of water) and injected TABLE 2. Experiments with inoculation of human dental isolates Expt no. n.rabbits No. of Inoculum' Outcome Autopsy and culture Additivereut Pneumonia Lung abscess results 18 8 P. morbillorum, F. Rabbit chow in None 7b (1 died) All animals at autopsy nucleatum, E. len- tap water, 0.2 developed lung abtum, B. fragilis, g/18 ml (1 ml) scess. Culture of pus group C Strepto- yielded B. fragilis, coccus E. lentum, P. morbillorum, F. nucleatum, and group C Streptococcus 19,20 15 P. morbillorum, F. None 2 13 (2 died) "7Ga uptake increased nucleatum, E. len- in all 13 survivors tum, B. fragilisc on day 14. Autopsy: Multiple lung abscesses, 0.5 to 2 cm in diameter, seen in both lungs, more in the right lung. Culture: B. fragilis, F. nucleatum, P. morbillorum, and E. lentum 21 6 B. fragilis None 3 None B. fragilis (3 rabbits) 22 6 P. morbillorum, F. None None None None isolated nucleatum, E. lentum a b lo' colony-forming units per ml. Multiple lung abscesses, mainly in the right lung. 7Ga was injected on day 12. INFECT. IMMUN.

4 VOL. 31, 1981 transtracheally into eight rabbits. One rabbit died on day 3, was left overnight in the freezer, and was unsuitable for autopsy. The seven remaining animals were sacrificed on day 12. All seven had multiple lung abscesses, mostly in the right lung; five of them had empyema in addition. These lung abscesses, on culture, yielded B. fragilis and group C Streptococcus in all seven. In addition, Peptococcus and E. lentum were isolated in three animals, and Fusobacterium was isolated in two. In the above experiment, bacterial isolates from dental scrapings with sterile rabbit chow were successful in the development of lung abscess. Hence, the next experiment was designed to determine the role of group C Streptococcus and rabbit chow. In this experiment, eight rabbits were inoculated with a mixture of pure cultures of P. morbillorum, F. nucleatum, F. lentum, and B. fragilis, but neither group C Streptococcus nor the additive rabbit chow was administered. One rabbit died of pneumonia on day 3, and the remaining seven developed lung abscesses, as demonstrated by 67Ga citrate scan (see Fig. 1). They were sacrificed on day 14, and all had multiple lung abscesses. The blood cultures were negative. Culture of the pus from the lung abscesses revealed B. fragilis in seven, E. lentum in five, P. morbillorum in three, and F. nucleatum in two. This experiment suggested that no additives are essential and that anaerobes alone can produce lung abscess. The experiment was repeated subsequently in a batch of 20 rabbits, all of which developed lung abscesses. The abscesses were more common in the right lung than the left, but sometimes occupied both sides. Empyema was noted frequently, and the abscesses were often multiple. The initial lesion was pneumonia, which was best seen around day 4. By day 7, abscesses could be easily recognized at autopsy. Six rabbits were injected with pure culture of B. fragilis (experiment no. 21). None of these animals developed lung abscess. Similarly, P. morbillorum, F. nucleatum, and E. lentum in combination did not produce lung abscess in six rabbits (experiment no. 22). B. fragilis appears to play an important role in the pathogenesis of lung abscess. It is incapable of producing lung abscesses by itself, but is able to do so in combination with other anaerobic bacteria. In summary, stock cultures of anaerobic bacteria were ineffective in producing lung abscess in experimental animals. Food particles, such as meat and rabbit chow, or hydrochloric acid and gastric mucin failed to potentiate the formation of anaerobic lung abscess. Other additives, such as house dust, blood, melted agar, and brain ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 595 heart infusion broth, were also ineffective. F. necrophorum ATCC 27852, known to be pathogenic in mice and rabbits (17), failed to cause lung abscess. Neither a mixture of P. morbillorum, F. nucleatum, and E. lentum without B. fragilis, nor pure cultures of B. fragilis, was capable of producing lung abscess, but these cultures produced lung abscess consistently in rabbits when combined. DISCUSSION Lack ofa bacteriologically well-defined animal model is a major impediment to the study of the pathogenesis and therapy of anaerobic lung abscess. Smith (12, 13) inoculated pyorrhea material and pus from Vincent's angina from humans into the exteriorized trachea of rabbits to induce lung abscess formation. Varney (14) did similar studies in dogs by inoculating pus obtained from a case of lung abscess in a human. Although anaerobic bacteria were noted in the pus, the inocula were not defined, as the bacteria were not identified. Furthermore, these models unfortunately carried a high mortality rate and were therefore unsuitable. Several other factors are mentioned as promoting the development of lung abscess. Among them, aspiration of gastric contents, i.e., hydrochloric acid (4), gastric mucin, and food, is considered to lead the way to chemical pneumonitis and tissue necrosis, followed by bacterial infection causing lung abscess. In the initial experiments, we attempted to mimic the natural course by inoculating hydrochloric acid, gastric mucin, and food (rabbit chow), but failed to produce lung abscess. This failure may in part be related to the use of laboratory stock cultures of anaerobic bacteria in the inoculum. In many of the animal models described for anaerobic infections, suppurative lesions were produced by employing such adjuvants as chopped-meat-carbohydrate medium, semisolid agar cultures (15), blood and hemostatic agents (6), and gentamicin pretreatment (C. A. Rotilie, R. J. Fass, and R. L. Perkins, Clin. Res. 22:452A, 1974). Unfortunately, adaptation of these methods did not improve development of lung abscesses. A final attempt utilizing sterile house dust, which results 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 anaerobes from dental scrapings of an apparently healthy male was injected transtracheally. These abscesses were histologically characterized as gangrenous destruction of the lung substance, filled with purulent material surrounded by fibrous tissue. Fluoroscopic examination sug-

5 .v:.n%.9 I......i so A..200Q rid B * C...v ar.= if.* #* t xp: -'m :,.:- o4: A,-.:1 t R FIG. 1. Experimental anaerobic lung abscess in a rabbit (A) Multilocular lung abscess. (B) Normal WGa citrate scan in a rabbit shown for comparison. (C) 67Ga scan done on day 14 of infection. Note the dense increased uptake of "Ga in the right lung. 596

6 VOL. 31, 1981 gested that the majority of rabbits developed pneumonia and pleural effusions by day 4 and abscess formation at the end of the first week. These abscesses were documented by obtaining chest roentgenograms and positive 67Ga scans, by histopathological examination, and by culture of the purulent material. By these parameters, the clinical course of the disease was similar to lung abscess in humans. There are several other pathogenic mechanisms involved in the development of lung abscess in humans; septicemia and bronchial obstruction, for example, also cause lung abscess. The present model mimics only lung abscess developed after aspiration. B. fragilis is not an uncommon isolate in cases of lung abscess. It was found in 9 of 40 cases (22%) reported by Finegold (5). In his experience, B. fragilis is found in 15 to 20% of all anaerobic pleuropulmonary infections. In most instances, B. fragilis is found along with other anaerobic bacteria. B. fragilis is associated with nearly 15 to 20% of all dental infections (9). Our experimental studies also suggest that, although B. fragilis alone may be incapable of causing lung abscess, it can do so in collaboration with other anaerobic bacteria (7). The animal model described is bacteriologically well defined, reproducible, and consistent. By utilizing this model the effects of environmental factors on the pathogenesis of anaerobic lung infections can be studied. Furthermore, this model may also provide clues to study the lung defense mechanisms in anaerobic lung infections. This model may also be used to evaluate the therapeutic efficacy of various antibiotics in anaerobic lung infections. The lung lesions could be followed up by fluoroscopy, roentgenograms, and 67Ga scans. The animals need not be sacrificed in order to document the lesions, as in other animal models for anaerobic infections. The disadvantage of this model is that it cannot be produced without the addition of B. fragilis, which is found in only 15 to 20% of anaerobic lung infections. For the first time, we now have a bacteriologically well-defined model for the study of anaerobic lung abscess, and we hope that it will be used by other investigators to answer some of the questions related to anaerobic lung abscess. ANIMAL MODEL FOR ANAEROBIC LUNG ABSCESS 597 ACKNOWLEDGMENT This work was supported by Public Health Service grant HL from the Airways Diseases Branch, Division of Lung Diseases, National Heart and Lung Institute, Bethesda, Md. LITERATURE CITED 1. Aranki, A., S. A. Syed, E. B. Kenny, and R. Freter Isolation of anaerobic bacteria from gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17: Bartlett, J. G., and S. M. Finegold Anaerobic pleuropulmonary infections. Medicine 51: Bartlett, J. G., S. L Gorbach, and S. M. Finegold The bacteriology of aspiration pneumonia. Am. J. Med. 56: Exarhos, N. D., W. D. Logan, 0. A. Abbott, and C. R. Hatcher The importance of ph and volume in tracheobronchial aspiration. Dis. Chest 47: Finegold, S. M Anaerobic bacteria in human disease, p Academic Press, Inc., New York. 6. Hill G. B Enhancement of experimental anaerobic infections by blood, hemoglobin, and hemostatic agents. Infect. Immun. 19: Hite, K. E., M. Locke, and H. C. Hesseltine Synergism in experimental infections with nonsporulating anaerobic bacteria. J. Infect. Dis. 84: Jericho, K. W. F., and N. Harries Dusty feed and acute respiratory disease in pigs. Can. Vet. J. 16: Kannangara, D. W., H. Thadepalli, and J. L Mc- Quirter Bacteriology and treatment of dental infections. Oral Surg. Oral Med. Oral Pathol. 50: Onderdonk, A. B., J. G. Bartlett, T. Louie, N. Sullivan-Seigler, and S. L Gorbach Microbial synergy in experimental intra-abdominal abscess. Infect. Immun. 13: Rosebury, T Microorganisms indigenous to man. McGraw-Hill, New York. 12. Smith, D. T Experimental aspiratory abscess. Arch. Surg. 14: Smith, D. T Fusospirochetal disease of the lungs produced with cultures from Vincent's angina. J. Infect. Dis. 46: Varney, P. L The bacterial flora of treated and untreated abscess of the lung. Arch. Surg. 19: Walker, C. B., and T. D. Wilkins Use of semisolid agar for initiation of pure Bacteroides fragilis infection in mice. Infect. Immun. 14: Weinstein, W. M., A. B. Onderdonk, J. G. Bartlett, and S. L. Gorbach Experimental intra-abdominal abscess in rats: development of an experimental model. Infect. Immun. 10: Wilkins, T. D., and L D. S. Smith Chemotherapy of an experimental Fusobacteruum (Sphaerophorus) necrophorum infection in mice. Antimicrob. Agents Chemother. 5: