Ann 0107 86: 197i
ANAEROBIC BACTERIA IN OTITIS MEDIA
R. S.
FULGHUM, PHD
GREE:s'VILLE, NORTH CAROLINA
H.
J.
DANIEL,
III,
J.
PHD
G.
YARBOROUGH,
BS
CHAPEL HILL, NORTH CAROLINA
GREE:s'VILLE, NORTH CAROLINA
SUMMARY - Anaerobic bacteria, Peptostreptococcus intermedius and Propionibacterium acnes, were found in mixed culture specimens from four of ten tested cases of chronic secretory otitis media. These anaerobic bacteria were in a mixed infection flora with aerobic bacteria, most often Staphylococcus epulermidis and Corynebacterium sp. which do not fit any established species. The finding of anaerobic bacteria in otitis media is consistent with the sporadic reports of the involvement of anaerobic bacteria in otitis media in the literature since 1898.
The etiology of otitis media is complex and remains essentially unknown for some forms of the disease.' Microbial etiological agents, some alone and some in combination (polymicrobic) are known. I A proportion of the present medical treatment procedures are destined to continued failure until the etiological agents and their effects are delineated. Antibiotic treatment not aimed at the specific microbial agent( s) involved will be futile in a proportion of cases and may ultimately become the most common cause of serious hearing problems originating from middle ear inflammation. The earliest references to the involvement of obligately anaerobic bacteria in otitis media are found in the thesis by Rist 2 in 1898, who studied suppurative disease of the mastoid and middle ear, and septicemia of otitic origin. Rist concluded that anaerobic bacteria of numerous types were involved in these infections. He described a number of cases including the bacteriology of organisms isolated in his laboratory. Unfortunately the methods used by Rist and colleagues in the French school
were not suitable for clinical laboratories, their work was forgotten, and bacteriologists and clinicians remained unaware of the important role of anaerobic bacteria in infection. In 1913 Krumwiede and Pratt" isolated anaerobic fusiform bacilli from discharging ears and reported fusiforms mixed with spirochetes. Earlier work by these authors! noted that fusiform bacilli were often found within a mixed infection flora. Italian microbiologists also reported finding anaerobic bacteria in otitis media. Busacca" in 1923 described ten cases of chronic suppurative otitis media, showing by description and photomicrography the similarity of some of the infection florae to that of oral Vincent's (fuso-spirochetal) infection. Other organisms were also found in these mixed infection florae. Brisotto" reported anaerobic bacteria from 10 of 25 cases involving otorrhea. He found not only spiral organisms and fusiform bacilli but also other bacilli of varying morphologies such as coryneforms and diplobacilli. He also reported
From the Department of Microbiology and Immunology, East Carolina University School of Medicine, and the Department of Speech, Language and Auditory Pathology, and Department of Anatomy, Greenville, North Carolina, and the University of North Carolina at Chapel Hill, North Carolina. This study was supported in part by the North Carolina United Way, and in part by a grant from Sigma Xi, the Scientific Research Society of North America.
196
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
197
The literature on the microbiology of finding anaerobic cocci. In four cases of mastoiditis he found numerous an- middle ear effusions abounds with reaerobic organisms including cocci, ba- ports of sterile specimens from otitis cilli, spirals and filamentous forms, most media. Undoubtedly many of these of these as mixed infections involving were sterile; however, in view of the numerous reports of anaerobic bacterial anaerobic bacteria. More recently Dietrich et a17 reported involvement in up to 40% of the cases of three otitis media cases yielding Bac- otitis media examined for anaerobic teroides sp. as a monoinfection, one bacteria, it would appear that many of otitis media case yielding anaerobic the so-called "sterile" specimens were cocci as a monoinfection, one case of improperly handled and/or inapproprimastoiditis yielding both Bacteroides sp. ately cultured for the detection of and anaerobic cocci, one case of otitis strictly anaerobic bacteria. It is now specimen colmedia yielding mixed cultures contain- well-known that proper 12-15 as well as use and transport lecting ing several anaerobes, and one case of chronic otitis media containing a mixed of strict anaerobic methodologies12.14.15 culture infection flora yielding both are of utmost importance in routinely anaerobic and aerobic bacteria. Bulin- detecting anaerobic bacteria from the ska and Kalowski" recovered anaerobic disease lesions. bacteria from 8 of 21 cases of otitis Some treatment failures could be atmedia, most having mixed infection tributed to the involvement of anaeroflora including Bacteroides sp., B. me- bic bacteria in otitis media. It is now laninogenicus and Corynebacterium sp. known that many anaerobic bacteria as well as aerobic organisms. Lin and are resistant or are developing resisArcala? recently reported a case in tance to antibiotics. Many strains of which Fusobacterium necrophorum was Bacteroides fragilis, for example, are isolated from the blood of an infant known to produce potent penicillinases hospitalized with respiratory distress when growing in mixed culture lesions. and a profuse, fetid, suppurative dis- Organisms such as B. fragilis may not charge from the left ear resulting from only be resistant to penicillin drugs but otitis media and mastoiditis.. Although also protect the other members of the the organism was not isolated from ear infection flora from penicillin and simdischarge nor were Gram stained ilar drugs resulting in treatment failure smears reported on specimens from the of infections containing B. fragilis as a discharging ear, the authors believed member of the infection flora.16 the F. necrophorum to be the causative Careful studies in the past decagent. Liu 10 reported, among many aerobic bacteria isolated from middle ade utilizing strict anaerobic techear effusions, the finding of one Gram niques12.14.15 have clearly shown annegative, rod-shaped anaerobic bac- aerobic bacteria to be involved in many infectious processes.Ui'" At least 50% terium. of infections of endogenous origin yield Otitis media with involvement by anaerobic bacteria as a part of the inanaerobic bacteria can be the initial fection flora with anywhere from two focus of serious infection metastasizing to seven or more species involved.F" to other tissues. For example, Heineman The fact that some anaerobes in mixed and Braude-! showed that chronic otitis culture with other anaerobes or aerobes media involving anaerobic bacteria was have a synergistic ability to cause disthe most probable origin of 8 (3 fatal) ease is now well-known.F's" of 18 cases of brain abscesses containing anaerobic bacteria since these paSeveral groups21-24 have recently detients had a history of mastoiditis, lineated the most common of the aerobic chronic otorrhea or chronic otitis media. organisms involved in otitis media. .. Moore WECo Personal communication, 1970.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
FULGHUM ET AL.
198
These, discussed at length in recent national and international meetings and conferences, are well-known and will not be reviewed here. It is the intent of this communication to present results of a pilot study of the anaerobic bacteriology of chronic otitis media in laboratory animals and in human patients, and to develop another hypothesis regarding the etiology of otitis media. METHODS AND MATERIALS
Technique. For aerobic and facultatively anaerobic organisms, common bacteriology laboratory practices were used. For anaerobic bacteria, the strict anaerobic technique of Hungate 25 as modified by the Virginia Polytechnic Institute and State University (VPISU) Anaerobe Laboratory12.26 was utilized for specimen collection, transport and isolation of organisms. Organisms were identified by using the information and diagnostic keys in several manuals and publications.12.27.29 Media. Anaerobic cultures were isolated and characterized in prereduced anaerobically sterilized (PRAS) media utilizing the deep anaerobic techniques of Hungate25 as modified by Moore and associates. 12.26 Essentially, these techniques combine chemically reduced media with a system of media preparation, inoculation, etc, in which media and organisms are protected from air by a constant flow of oxygen-free gas purging media vessels, tubes, etc. Tubes are sealed with butyl rubber stoppers after manipulations. Roll tubes are stoppered tubes, the inside surface of which is coated with a PRAS agar medium and which contain an oxygen-free gas phase. 12.26 PRAS media formulations are those of the VPISU Anaerobe Laboratory12 except for chopped meat media in which we substitute cooked meat medium dehydrated meat particles" for freshly ground meat. Commercially prepared media for characterizing aerobic isolates were purchased, prepared from dehydrated media, or made up from individual components according to directions found in methodology manuals. 27,28 Specimens. Specimens of cerumen were taken from the external auditory canals of three normal human subjects. This was done prior to beginning the study of the human cases of chronic secretory otitis media as a baseline study of normal external auditory canal flora detectable with our methods. Cases of chronic otitis media in laboratory rats were selected for pilot studies utilizing an
animal model. Rats were sacrificed and autopsied. Heads were removed, skinned and surface disinfected with Betadine®..... Cranial cavities were aseptically opened, brains removed, and the middle ear entered through tegmen tympani. Middle ear fluid specimens were taken and immediately inoculated into appropriate media (see Isolation, below), Human cases of chronic secretory otitis media presenting for myringotomy and drainage were utilized in the pilot studies of humans. All cases had intact tympanic membranes. None of these patients were on antibiotic therapy during this episode. A sterile speculum was used in tympanocentesis and a sterile 5.08 em 20 gauge needle on a 3 ml syringe was inserted through the sterile speculum into the middle ear cavity. Middle ear fluid was aspirated, usually yielding 0.5 to 1.0 ml of fluid. The needle was removed, disconnected, and a fresh sterile needle was attached to the syringe. Air was expelled and the specimen inoculated into a serum stoppered bottle of PRAS chopped meat medium. After transport to the bacteriology laboratory, a small amount of fluid from the chopped meat medium was removed via syringe and needle from the serum stoppered bottle for inoculation of media. Isolation of Bacteria from Specimens. For primary isolation specimens were streaked from the unincubated PRAS chopped meat transport vial to Columbia blood agar, chocolate agar supplemented with hemoglobin, and "IsoVitale X,t and brain heart infusion agar supplemented with yeast extract, vitamin K, and hemin. These plates were incubated aerobically at 37 C. To isolate anaerobic organisms, PRAS brain heart infusion supplementedt- agar roll tubes were streaked with inoculum from the transport vial. Incubation was at 37 C. Colonies developing on these media were checked for purity, restreaked. and transferred to media (PRAS chopped meat for anaerobes; Trypticase soy agart slants for aerobes) for holding as stock cultures. All isolated organisms were tested for ability to grow under both aerobic and anaerobic conditions. Identity of isolates was determined by comparing characteristics of isolates with published descriptions. 12.29
Assay of Pathogenic Potential. Methods similar to those of Hill et al30 were used to assay mixtures of cultures for pathogenicity. RESULTS
Animal Pilot Experiment. Four Holtzman rats with bilateral otitis media and two Long-Evans hooded rats free of the disease were examined. No growth was
.. Difco Laboratories, Detroit, MI. .... The Purdue Frederick Co., Norwalk, CT. t Baltimore Biological Laboratories, Cockeysville, MD.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
199
OTITIS MEDIA TABLE I MICROORGANISMS ISOLATED FROM TEN EARS WITH CHRONIC OTITIS MEDIA AT MYRINGOTOMY No,
Organisms Aerobic
8
Staph. epidermidis Corynebacterium sp.
7
Gram positive rod Actinomycete" Fungus"
2 1 3
Anaerobic 1
Peptostrep, intermedius Propionibacterium acnes Peptostrep. intermedius I
3 1
Gram positive oval"
1
" Not identified
found in the media utilized to culture specimens from the well Long-Evans rats. Growth was obtained in two of the eight specimens from the diseased Holtzman rats. The anaerobic organisms isolated were found to be Propionibacterium acnes. No attempt was made in the animal pilot experiment to isolate or characterize aerobic or facultatively anaerobic species.
Normal Human External Auditory Canal. Specimens of cerumen, taken from the external auditory canals of three normal human subjects, were examined anaerobically and aerobically. Of eleven isolates, nine were Gram positive cocci identified as Staphylococcus epidermidis and two were short Gram negative rods, not identifiable. Two other isolates, lost on subculture, appeared to have been coryneform bacteria. Human Pilot Experiment. Ten specimens of middle ear fluid from human cases of chronic otitis media were examined. Four of these were found to contain one or two species of obligately anaerobic bacteria. In addition, S. epidermidis was found in eight of the ten specimens. Seven of the ten specimens yielded Corynebacterium sp that do not fit the established criteria28 ,29 for any well-recognized species. These may be new species and, if so, will be described
elsewhere. Two specimens contained fungi and one contained an aerobic actinomycrete. Table I lists the organisms isolated from each specimen. In most cases multiple isolates were made of each organism utilizing the different media employed. Mixtures of the anaerobic and aerobic isolates reconstituting the infection flora from each of two patients caused intrahepatic abscesses in one of four healthy mice inoculated with 1 ml of mixed inoculum. DISCUSSION
The finding of anaerobic bacteria in four of the ten cases of chronic otitis media in humans warrants further investigation of the role of anaerobic bacteria in the pathogenicity of otitis media. It is now well-understood that proper specimen collection and transport can be the most important factor in successful detection of anaerobic bacteria from clinical specimens.P'P While there is still disagreement as to the best methods of transport some workers have found that transport media are less than optimum because they often have a higher oxidation-reduction potential than the lesion from which the specimen was collected.P Also, if the transport medium can support growth of one or more species from the specimen, overgrowth of these at the expense of losinz or having isolation made more difficult of other important species in the specimen may occur. Thus, the present study was made more difficult by our choice of PRAS-CM in serum stoppered vials as a transport medium. Anaerobic bacteria, by definition, thrive in areas of low oxvaen concentration or in areas devoid of oxygen. All of the anaerobic bacteria now known together present a spectrum of oxygen sensitivity ranging from organisms such as Clostridium tertium and C. carnis, which can grow slowly and poorly on the surface of appropriate media exposed to air, to very strictly anaerobic bacteria such as C. haemolyticum, C. novyi type B, Butyrivibrio
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
200
FULGHUM ET AL.
fibrisolvens, Treponema macrodentium, and B. ruminicola brevis. Many organisms such a~ P. acnes are .i~t~r mediate in their oxygen sensitivity between the extremes of the above examples while other species such as Peptostreptococcus intermedius contain both aerotolerant and obligately anaerobic strains. In addition, many other factors may be involved in the expression by a bacterial species of a requirement for anaerobic conditions in vitro. Among these are ability to produce peroxidases, catalase, superoxide dismutase, ability to transfer electrons metabolically at various oxidation-reduction potentials and sensitivity to oxidized organic compounds in bacteriological media exposed to air, as summarized by Smith.P The point of this discussion is that one cannot refute a claim of finding anaerobes from an area containing some gaseous oxygen. Nor can one expect, because of such oxygen, to never find anaerobes. Thus we take exception to the statement by Ingelstedt et aPI "Nor seems the milieu favorable for anaerobic bacteria." The facts that oxygen tension is reduced, that there is tissue damage which may involve release of enzymes that further reduce oxygen tension in the liquid phase (middle ear Ruid) and. enzym:s su?h as tissue catalase and superoxide dismutase that would protect some anaerobic bacteria, and that there are aerobic bacteria depleting the dissolved oxygen in the middle ear Ruid of a number of cases of otitis media, all clearly point to the fact that middle ear Ruid in chronic otitis media could very well be an excellent milieu for the growth of and contribution to chronicity by anaerobic bacteria. Such an. hypothesis is supported by the .findmg ?f clinically significant anaerobic bactena in some cases of chronic otitis media historically as well as in the present studv. The reports of Ingelstedt et alBI and Elner,32 who both reported lowered oxygen concentrations in the middle ~~r cavities of patients with secretory otitis media. and the theory of Sade,33 who feels the lack of ventilation to be the single most important factor in the
chronicity of secretory otitis media, should be reconsidered in light of our findings of clinically significant anaerobic bacteria in cases of otitis media. We do not mean to imply from the present study that we consider anaerobic bacteria to be a primary cause of otitis media, nor even to contribute to chronicity in the chronic otitis media, although they may possibly do either or both. We are, however, proposing a scientific hypothesis to stimulate further study regarding the role of anaerobic bacteria in otitis media since they are found in the disease and must have some role in the ecology of the microflora-microflora and host-microflora relationships. To this end, we consider it to be of utmost importance to undertake careful studies of both the aerobic and anaerobic bacteriology in the following areas: 1) animal model normal flora: the normal flora of the middle ear should be delineated and, where necessary, characterized, 2) animal models of otitis media, both in naturally acquired and in induced ot~tis media should be developed and studied microbiologically, 3) human normal Hora of the middle ear, if any, should be carefully evaluated and all species delineated and characterized, 4) a full scale clinical trial should carefully document the presence, the species, an~ the antibiotic susceptibilities, of all microorganisms found in the middle ear of patients with otitis media. The finding of Corynebacterium sp. and S. epidermidis, the latter of whic.h is now considered to have pathogemc potential'" in most specimens, as well as of the anaerobic bacteria, P. acnes and P. (Streptococcus) interme~ius rai~es the question of the pathogemc potential of each alone and in combination. Roberts 20,34 has shown synergy between the anaerobe, Fusiformis necrophorus and C. pyogenes. Fusiformis nec:opho~us produced a leukocidal exotoxm which protected the C. pyogenes, allowing it to become invasive. Other examples of pathogenic synergy and other ecological syntrophisms in anaerobic infe~ti?ns are found in the Iiterature.l" Thus It IS well-
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
known that mixed culture infections can be produced by organisms usually not pathogenic in pure culture. It also appears that many different mixtures of anaerobic organisms may produce a given disease syndrome. In the present study, we have been able to demonstrate only a sporadic and weak pathogenicity of these isolates for healthy mice using methods similar to those of Hill et al.30 Animal models of otitic infection by these organisms need to be developed in order to fulfill Koch's postulates.
Propionibacterium aenes, formerly known as C. acnes, is a member of the "normal" flora of the human skin mouth, and hair. It is also found in ~ wide variety of clinical specimens where its pro~u.ction of enzym~s, including neurarninidases, hyaluromdase, lipase, are most likely contributory to pathogenicity. Diseases from which clinically significant isolations of P. aenes have been made include maliznancies reticuloendotheliosis, endoc;rditis, s'epticemia, adenitis, arthritis, osteomyelitis, leukorrhea, lung abscesses, purulent meningitis, brain abscesses and erythema. Smith!" has reviewed the salient features of this organism which is known to cause infections alone or in combination with other organisms. P. aenes is also the most frequently isolated anaerobic contaminant in anaerobic studies because of its ubiquity in the sebaceous glands of the human skin. In assigning clinical significance to P. aenes, findings of this organism should be supported by reisolations from the same patient. Peptostreptoeoeeus intermedius is a species composed of several biotypes, some strains of which are more tolerant of oxygen than others. It is a part of the microflora of the nose mouth, throat, and intestine of most humans. It is also often isolated from clinical specimens'? although alone it is not pathogenic for laboratory animals and probably only slightly so for man.l" It has been isolated from the external auditory canals of 2 of 22 subjects.l"
201
The organism is also now considered to be a member of the genus Streptoeoeeus. 3 ;; ,3 G Our isolates of P. intermedius appeared to be obligately anaerobic and never grew on any aerobically incubated plating medium; however, when our results were being confirmed," slight growth occurred aerobically on their blood agar plating medium, which was a different formulation from ours. The species is generally more easily isolated under anaerobic conditions and may become more aerotolerant upon laboratory subculture, although strains that remain obligately anaerobic are known. 12 •18 In brain abscesses, a large proportion of which are thought to originate from otitic infections, anaerobic streptococci (not further identified), anaerobic coryneform bacteria (not further identified), and other anaerobic bacteria were found.F Such literature reviews lend credence to our finding and our hypotheses. Therefore, we hypothesize that chronic otitis media may be caused by, or become chronic in part due to, a number of organisms in various combinations including anaerobes. This makes it imperative to isolate, identify and report antibiotic susceptibilities of all members of these infection florae. The reviewed literaturet!' suggests that a wider variety of specimens from the various types of otitis media as well as more detailed laboratory studies would reveal a wider spectrum of anaerobic bacterial involvement in middle ear disease than found in this study. Considering the past findings of anaerobic bacterial participation in mixed culture infections 12 ,13 ,1 7 ,18 as well as single species infections, we would not be surprised to find a variety of different species of anaerobes playing a role in otitis media. While many of these might be considered opportunistic pathogens or organisms supporting in various ways 16,19.20,34 the maintenance and/or extension of the lesion by other bacteria, it is imperative that we discover their role and learn to deal with the total in-
.. VPISU Anaerobe Laboratory.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
202
FULGHUM ET AL.
fection flora in order to improve patient care, shorten treatment time and expense, and to reduce permanent damage to the auditory apparatus in our patients. All of the organisms isolated in this study are members of the nasal-oral and/or skin flora. The numbers of organisms found point toward growth of these species in the middle ear fluid rather than contamination by normal flora during; myringotomy. While most cases of chronic otitis media have or have had tympanic membrane rupture, the cases utilized here had intact membranes at the time of myringotomy. These findings typify one general type of anaerobic infection, the chronic state associated with anaerobes in the infection flora following tissue damage whether it be mechanical, viral, mycoplasmal, bacterial, or a combination thereof. More often than not such anaerobic infections are of endogenous origin. Endogenous or not, the problems of and the inimical effects of anaerobic infections require laboratory study by appropriate anaerobic techniques 12 ,14 ,1 5 to ensure proper treatment and to shorten treatment time. Considering the increasing chronicity of otitis media and its often debilitating
sequelae, the study of the role of anaerobic bacteria in chronic otitis media could be a major contribution. Finally, we point to the animal model investigations of Drs. H. J. Daniel and P. J. Loesche" and Means et al38 reporting the nonsusceptibility of otitis media in the Mongolian gerbil (Meriones unguiculatus). The hypertrophied and hyperventilated middle ear bullae of this species is theorized to be related to its low disease incidence. In contrast the high incidence of otitis media in the laboratory rat39 ,40 may be related to that species' small, underventilated, and poorly drained middle ear cavities. If structural and functional differences in middle ear cavities allow for differences in size, ventilating capacity and drainage, then these differences may well be related to the ability of anaerobic bacteria to thrive. If so, therapeutic regimens should, in part. continue to be directed toward ventilation and drainage of this system to increase oxygenation and to reduce the accumulation of fluids in which bacteria including anaerobic bacteria may become established. Nature's method of allowing ventilation and drainage by means of membrane rupture may have didactic import.
REFERENCES 1. Glorig A, Gerwin KS: Otitis Media, pro-
ceedings of the National Conference. Springfield, III, Charles C. Thomas, 1972 2. Rist E: Etudles Bacteriolouiques sur Les Infections D'Oriuine Otique, these pour Ie Doctorate in Medecine. Faculte de Medicine de Paris, 1898 3. Krumwiede C Jr, Pratt JS: Fusiform bacilli: cultural characteristics. J Infect Dis 12:438-441, 1913 4. Krumwide C Jr, Pratt JS: Fusiform bacilli: isolation and cultivation. J Infect Dis 12: 199-201, 1913 5. Busacca G: Alcune recerche suI reperto batteriologico delIa otite cronica suppurata, con speciale rigvardo alIa presenza di spirochete e di bacilli fusiformi. Tentativi di cum col neosalvarsan. Arch Ital Otol Rhinol Laryngol 34:414-426, 1923 6. Brisotto P: Dei germi anaerobi in otologia e di alcune richerche batteriologiche
sull'ozena. Riforma Med 39:769-77,5, 1923 7. Dietrich H, Klein W, Nickel H: Uber auftreten und resistenzbestimrnung anaerober sporenloser infektionserreger. Zbl Bakt 197: 515-526, 1965 8. Bulinska H, Kalowski M: Bakterie beztlenowo w patologii przewleklego zapalenia ucha srodkowego, Otolaryngol Pol 28:301305, 1974 9. Lin RG, Arcala AE: Fusobacterium septicemia with otitis media and mastoiditis. Postgrad Med 57: 159-160, 1975 10. Liu Y, Lang R, Lim D, et al: Microorganisms in chronic otitis media with effusions. Ann Otol Rhinol Laryngol 85 (Suppl 25:245-247) 1976 II. Heineman HS, Braude AI: Anaerobic infection of the brain. Am J Med 35:682-697, 1963 12. Holdeman LV, Moore \VEC: Anaerobe Laboratory Manual. Virginia Polytechnic In-
" Unpublished study presented at the American Association of Laboratory Animal Science Annual Meeting in Boston, Massachusetts, 1975.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
stitute and State University Anaerobe Laboratory, Blacksburg, Virginia, 1972 13. Finegold SM, Rosenblatt JE: Practical aspects of anaerobic sepsis. Medicine 52:311322,197,'3 14. Dowell VR Jr, Hawkins TM: Laboratory Methods in Anaerobic Bacteriology. DHEW Publication No. (CDC) 74-8272. U.S. Government Printing Office, Washington, DC, 1974 15. Sutter VL, Vargo VL, Finegold SM: Wadsworth Anaerobic Bacteriology Manual, ed 2. University of California, Los Angeles, 1975 16. Hackman AS, Wilkins TD: In vivo protection of Fusobacterium necrophorum from penicillin by Bacteroides fragilis. Antimicrob Agents Chemother 7 :698-703, 1975 17. Balows A (ed): Anaerobic Bacteria: Role in Disease. Springfield, Ill, Charles C. Thomas, 1974 18. Smith LDS: The Pathogenic Anaerobic Bacteria. Springfield, Ill, Charles C. Thomas, 1975 19. MacDonald JB: On the pathogenesis of mixed anaerobic infections of mucous membranes. Ann R CoIl Surg Engl 31:361-378, 1962 20. Roberts DS: The pathogenicity of Fusiformus necrophorus and Corynebacterium pyogenes. Br J Exp Pathol 48:665-673, 1967 21. Howie VM, Ploussard JH: Bacterial etiology and antimicrobial treatment of exudative otitis media. South Med J 64:233-239, 1971 22. Liu YS, Lim DJ, Lang RW, et al: Chronic middle ear effusions. Arch Otolaryngol 101:278-286, 1975 23. Berry YJ, Rose RM: Otitis media: obstruction and a clinical approach. Forum on Infection 1:1-13, 1974 24. Feigin RD, Chang MJ: Treating otitis media. Drug Therapy, 4(9):116-125,1974 25. Hungate RE: The anaerobic mesophilic cellulolytic bacteria. Bacteriol Rev 14:1-49, 1950 26. Moore WEC: Techniques for routine culture of fastidious anaerobes. Int J Syst BacterioI16:173-190,1966 27. Bailey WR, Scott EG: Diagnostic Mi-
crohiology, ed 4. St. Louis, C. V. Mosby, Co, 1974 28. Lennette EH, Spaulding EH, Truant JP (eds): Manual of Clinical Microbiology, ed 2. Washington, DC, American Society for Microbiology, 1974 29. Buchanan RE, Gibbons NE ( eds ) : Bergey's Manual of Determinative Bacteriology, ed 8. Baltimore, Williams and Wilkins Co, 1974 30. Hill GB, Osterhout S, Pratt PC: Liver abscess production by non-sporeforrning anaerobic bacteria in a mouse model. Infect Immun 9:599-603, 1974 31. Ingelstedt S, Johnson B, Rudcrantz H: Gas tension and pH in middle ear effusion. Ann Otol Rhinol Laryngol 84: 198-202, 1975 32. Elner A: Normal gas exchange in the human ear. Ann Otol Rhinol Laryngol 85 (SuppI25:161-164) 1976 33. SaM J: The biopathology of secretory otitis media. Ann Otol Rhinol Laryngol 83: 54-70, 1974 34. Roberts DS: Synergistic mechanisms in certain mixed infections. J Infect Dis 120:720724, 1969 35. Rogosa M: Peptococcaceae, a new family to include the gram positive, anaerobic cocci of the genera Peptococcus, Peptostrepiococcus, and Ruminococcus. Int J Syst Bacteriol 21 :234-237, 1971 36. Holdeman LV, Moore WEC: New genus, Coprococcus, twelve new species and emended descriptions of four previously described species of bacteria from human feces. Int J Syst Bacterial 24:260-277, 1974 37. Swartz MN, Karchmer AW: Infections of the central nervous system, in Ballows A ( ed ): Anaerobic Bacteria: Role in Disease. Springfield, Ill, Charles C. Thomas, 1974 38. Means LW, Daniel HJ, Jordan LH, et al, Nonsusceptibility to otitis media of the laboratory gerbil, Meriones unguiculatus. Physiol Psychol 3:229-230, 1975 39. Daniel HJ, Means LW, Dressel ME, et al: Otitis media in laboratory rats. Physiol Psychol 1:7-8, 1973 40. Daniel HI, Carmine FH, Cook RA: Otitis media in two strains of laboratory rats. J Aud Res 11:276-278, 1971
ACKNOWLEDGMENTS-We are grateful to the University of North Carolina School of Medicine for providing Mr. Yarborough with a summer stipend for medical students. Dr. W. S. Bost of Greenville and Drs. N. D. Fischer and W. P. Biggers of the U.N.C. School of Medicine at Chapel Hill kindly allowed us to collect specimens from patients at myringotomy. Dr. J. J, Crawford allowed us to use his facilities in preparation of specimens for transport to Greenville. In our laboratory, Mr. John Worthington prepared anaerobic media and characterized the cultures of anaerobic bacteria. Dr. Lillian V. Holdeman of the Virginia Polytechnic Institute and State Laboratory kindly confirmed the identity of most of our anaerobic isolates. Dr. Sydney Finegold provided references numbered 2-7 of the literature review regarding anaerobic bacteria in otitis media. Ms. Diane Gardner carried out the study on otitis media in rats. REPRINTS-R. S. Fulghum, PhD, Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
ANAEROBIC BACTERIA IN OTITIS MEDIA
R. S.
FULGHUM, PHD
GREE:s'VILLE, NORTH CAROLINA
H.
J.
DANIEL,
III,
J.
PHD
G.
YARBOROUGH,
BS
CHAPEL HILL, NORTH CAROLINA
GREE:s'VILLE, NORTH CAROLINA
SUMMARY - Anaerobic bacteria, Peptostreptococcus intermedius and Propionibacterium acnes, were found in mixed culture specimens from four of ten tested cases of chronic secretory otitis media. These anaerobic bacteria were in a mixed infection flora with aerobic bacteria, most often Staphylococcus epulermidis and Corynebacterium sp. which do not fit any established species. The finding of anaerobic bacteria in otitis media is consistent with the sporadic reports of the involvement of anaerobic bacteria in otitis media in the literature since 1898.
The etiology of otitis media is complex and remains essentially unknown for some forms of the disease.' Microbial etiological agents, some alone and some in combination (polymicrobic) are known. I A proportion of the present medical treatment procedures are destined to continued failure until the etiological agents and their effects are delineated. Antibiotic treatment not aimed at the specific microbial agent( s) involved will be futile in a proportion of cases and may ultimately become the most common cause of serious hearing problems originating from middle ear inflammation. The earliest references to the involvement of obligately anaerobic bacteria in otitis media are found in the thesis by Rist 2 in 1898, who studied suppurative disease of the mastoid and middle ear, and septicemia of otitic origin. Rist concluded that anaerobic bacteria of numerous types were involved in these infections. He described a number of cases including the bacteriology of organisms isolated in his laboratory. Unfortunately the methods used by Rist and colleagues in the French school
were not suitable for clinical laboratories, their work was forgotten, and bacteriologists and clinicians remained unaware of the important role of anaerobic bacteria in infection. In 1913 Krumwiede and Pratt" isolated anaerobic fusiform bacilli from discharging ears and reported fusiforms mixed with spirochetes. Earlier work by these authors! noted that fusiform bacilli were often found within a mixed infection flora. Italian microbiologists also reported finding anaerobic bacteria in otitis media. Busacca" in 1923 described ten cases of chronic suppurative otitis media, showing by description and photomicrography the similarity of some of the infection florae to that of oral Vincent's (fuso-spirochetal) infection. Other organisms were also found in these mixed infection florae. Brisotto" reported anaerobic bacteria from 10 of 25 cases involving otorrhea. He found not only spiral organisms and fusiform bacilli but also other bacilli of varying morphologies such as coryneforms and diplobacilli. He also reported
From the Department of Microbiology and Immunology, East Carolina University School of Medicine, and the Department of Speech, Language and Auditory Pathology, and Department of Anatomy, Greenville, North Carolina, and the University of North Carolina at Chapel Hill, North Carolina. This study was supported in part by the North Carolina United Way, and in part by a grant from Sigma Xi, the Scientific Research Society of North America.
196
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
197
The literature on the microbiology of finding anaerobic cocci. In four cases of mastoiditis he found numerous an- middle ear effusions abounds with reaerobic organisms including cocci, ba- ports of sterile specimens from otitis cilli, spirals and filamentous forms, most media. Undoubtedly many of these of these as mixed infections involving were sterile; however, in view of the numerous reports of anaerobic bacterial anaerobic bacteria. More recently Dietrich et a17 reported involvement in up to 40% of the cases of three otitis media cases yielding Bac- otitis media examined for anaerobic teroides sp. as a monoinfection, one bacteria, it would appear that many of otitis media case yielding anaerobic the so-called "sterile" specimens were cocci as a monoinfection, one case of improperly handled and/or inapproprimastoiditis yielding both Bacteroides sp. ately cultured for the detection of and anaerobic cocci, one case of otitis strictly anaerobic bacteria. It is now specimen colmedia yielding mixed cultures contain- well-known that proper 12-15 as well as use and transport lecting ing several anaerobes, and one case of chronic otitis media containing a mixed of strict anaerobic methodologies12.14.15 culture infection flora yielding both are of utmost importance in routinely anaerobic and aerobic bacteria. Bulin- detecting anaerobic bacteria from the ska and Kalowski" recovered anaerobic disease lesions. bacteria from 8 of 21 cases of otitis Some treatment failures could be atmedia, most having mixed infection tributed to the involvement of anaeroflora including Bacteroides sp., B. me- bic bacteria in otitis media. It is now laninogenicus and Corynebacterium sp. known that many anaerobic bacteria as well as aerobic organisms. Lin and are resistant or are developing resisArcala? recently reported a case in tance to antibiotics. Many strains of which Fusobacterium necrophorum was Bacteroides fragilis, for example, are isolated from the blood of an infant known to produce potent penicillinases hospitalized with respiratory distress when growing in mixed culture lesions. and a profuse, fetid, suppurative dis- Organisms such as B. fragilis may not charge from the left ear resulting from only be resistant to penicillin drugs but otitis media and mastoiditis.. Although also protect the other members of the the organism was not isolated from ear infection flora from penicillin and simdischarge nor were Gram stained ilar drugs resulting in treatment failure smears reported on specimens from the of infections containing B. fragilis as a discharging ear, the authors believed member of the infection flora.16 the F. necrophorum to be the causative Careful studies in the past decagent. Liu 10 reported, among many aerobic bacteria isolated from middle ade utilizing strict anaerobic techear effusions, the finding of one Gram niques12.14.15 have clearly shown annegative, rod-shaped anaerobic bac- aerobic bacteria to be involved in many infectious processes.Ui'" At least 50% terium. of infections of endogenous origin yield Otitis media with involvement by anaerobic bacteria as a part of the inanaerobic bacteria can be the initial fection flora with anywhere from two focus of serious infection metastasizing to seven or more species involved.F" to other tissues. For example, Heineman The fact that some anaerobes in mixed and Braude-! showed that chronic otitis culture with other anaerobes or aerobes media involving anaerobic bacteria was have a synergistic ability to cause disthe most probable origin of 8 (3 fatal) ease is now well-known.F's" of 18 cases of brain abscesses containing anaerobic bacteria since these paSeveral groups21-24 have recently detients had a history of mastoiditis, lineated the most common of the aerobic chronic otorrhea or chronic otitis media. organisms involved in otitis media. .. Moore WECo Personal communication, 1970.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
FULGHUM ET AL.
198
These, discussed at length in recent national and international meetings and conferences, are well-known and will not be reviewed here. It is the intent of this communication to present results of a pilot study of the anaerobic bacteriology of chronic otitis media in laboratory animals and in human patients, and to develop another hypothesis regarding the etiology of otitis media. METHODS AND MATERIALS
Technique. For aerobic and facultatively anaerobic organisms, common bacteriology laboratory practices were used. For anaerobic bacteria, the strict anaerobic technique of Hungate 25 as modified by the Virginia Polytechnic Institute and State University (VPISU) Anaerobe Laboratory12.26 was utilized for specimen collection, transport and isolation of organisms. Organisms were identified by using the information and diagnostic keys in several manuals and publications.12.27.29 Media. Anaerobic cultures were isolated and characterized in prereduced anaerobically sterilized (PRAS) media utilizing the deep anaerobic techniques of Hungate25 as modified by Moore and associates. 12.26 Essentially, these techniques combine chemically reduced media with a system of media preparation, inoculation, etc, in which media and organisms are protected from air by a constant flow of oxygen-free gas purging media vessels, tubes, etc. Tubes are sealed with butyl rubber stoppers after manipulations. Roll tubes are stoppered tubes, the inside surface of which is coated with a PRAS agar medium and which contain an oxygen-free gas phase. 12.26 PRAS media formulations are those of the VPISU Anaerobe Laboratory12 except for chopped meat media in which we substitute cooked meat medium dehydrated meat particles" for freshly ground meat. Commercially prepared media for characterizing aerobic isolates were purchased, prepared from dehydrated media, or made up from individual components according to directions found in methodology manuals. 27,28 Specimens. Specimens of cerumen were taken from the external auditory canals of three normal human subjects. This was done prior to beginning the study of the human cases of chronic secretory otitis media as a baseline study of normal external auditory canal flora detectable with our methods. Cases of chronic otitis media in laboratory rats were selected for pilot studies utilizing an
animal model. Rats were sacrificed and autopsied. Heads were removed, skinned and surface disinfected with Betadine®..... Cranial cavities were aseptically opened, brains removed, and the middle ear entered through tegmen tympani. Middle ear fluid specimens were taken and immediately inoculated into appropriate media (see Isolation, below), Human cases of chronic secretory otitis media presenting for myringotomy and drainage were utilized in the pilot studies of humans. All cases had intact tympanic membranes. None of these patients were on antibiotic therapy during this episode. A sterile speculum was used in tympanocentesis and a sterile 5.08 em 20 gauge needle on a 3 ml syringe was inserted through the sterile speculum into the middle ear cavity. Middle ear fluid was aspirated, usually yielding 0.5 to 1.0 ml of fluid. The needle was removed, disconnected, and a fresh sterile needle was attached to the syringe. Air was expelled and the specimen inoculated into a serum stoppered bottle of PRAS chopped meat medium. After transport to the bacteriology laboratory, a small amount of fluid from the chopped meat medium was removed via syringe and needle from the serum stoppered bottle for inoculation of media. Isolation of Bacteria from Specimens. For primary isolation specimens were streaked from the unincubated PRAS chopped meat transport vial to Columbia blood agar, chocolate agar supplemented with hemoglobin, and "IsoVitale X,t and brain heart infusion agar supplemented with yeast extract, vitamin K, and hemin. These plates were incubated aerobically at 37 C. To isolate anaerobic organisms, PRAS brain heart infusion supplementedt- agar roll tubes were streaked with inoculum from the transport vial. Incubation was at 37 C. Colonies developing on these media were checked for purity, restreaked. and transferred to media (PRAS chopped meat for anaerobes; Trypticase soy agart slants for aerobes) for holding as stock cultures. All isolated organisms were tested for ability to grow under both aerobic and anaerobic conditions. Identity of isolates was determined by comparing characteristics of isolates with published descriptions. 12.29
Assay of Pathogenic Potential. Methods similar to those of Hill et al30 were used to assay mixtures of cultures for pathogenicity. RESULTS
Animal Pilot Experiment. Four Holtzman rats with bilateral otitis media and two Long-Evans hooded rats free of the disease were examined. No growth was
.. Difco Laboratories, Detroit, MI. .... The Purdue Frederick Co., Norwalk, CT. t Baltimore Biological Laboratories, Cockeysville, MD.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
199
OTITIS MEDIA TABLE I MICROORGANISMS ISOLATED FROM TEN EARS WITH CHRONIC OTITIS MEDIA AT MYRINGOTOMY No,
Organisms Aerobic
8
Staph. epidermidis Corynebacterium sp.
7
Gram positive rod Actinomycete" Fungus"
2 1 3
Anaerobic 1
Peptostrep, intermedius Propionibacterium acnes Peptostrep. intermedius I
3 1
Gram positive oval"
1
" Not identified
found in the media utilized to culture specimens from the well Long-Evans rats. Growth was obtained in two of the eight specimens from the diseased Holtzman rats. The anaerobic organisms isolated were found to be Propionibacterium acnes. No attempt was made in the animal pilot experiment to isolate or characterize aerobic or facultatively anaerobic species.
Normal Human External Auditory Canal. Specimens of cerumen, taken from the external auditory canals of three normal human subjects, were examined anaerobically and aerobically. Of eleven isolates, nine were Gram positive cocci identified as Staphylococcus epidermidis and two were short Gram negative rods, not identifiable. Two other isolates, lost on subculture, appeared to have been coryneform bacteria. Human Pilot Experiment. Ten specimens of middle ear fluid from human cases of chronic otitis media were examined. Four of these were found to contain one or two species of obligately anaerobic bacteria. In addition, S. epidermidis was found in eight of the ten specimens. Seven of the ten specimens yielded Corynebacterium sp that do not fit the established criteria28 ,29 for any well-recognized species. These may be new species and, if so, will be described
elsewhere. Two specimens contained fungi and one contained an aerobic actinomycrete. Table I lists the organisms isolated from each specimen. In most cases multiple isolates were made of each organism utilizing the different media employed. Mixtures of the anaerobic and aerobic isolates reconstituting the infection flora from each of two patients caused intrahepatic abscesses in one of four healthy mice inoculated with 1 ml of mixed inoculum. DISCUSSION
The finding of anaerobic bacteria in four of the ten cases of chronic otitis media in humans warrants further investigation of the role of anaerobic bacteria in the pathogenicity of otitis media. It is now well-understood that proper specimen collection and transport can be the most important factor in successful detection of anaerobic bacteria from clinical specimens.P'P While there is still disagreement as to the best methods of transport some workers have found that transport media are less than optimum because they often have a higher oxidation-reduction potential than the lesion from which the specimen was collected.P Also, if the transport medium can support growth of one or more species from the specimen, overgrowth of these at the expense of losinz or having isolation made more difficult of other important species in the specimen may occur. Thus, the present study was made more difficult by our choice of PRAS-CM in serum stoppered vials as a transport medium. Anaerobic bacteria, by definition, thrive in areas of low oxvaen concentration or in areas devoid of oxygen. All of the anaerobic bacteria now known together present a spectrum of oxygen sensitivity ranging from organisms such as Clostridium tertium and C. carnis, which can grow slowly and poorly on the surface of appropriate media exposed to air, to very strictly anaerobic bacteria such as C. haemolyticum, C. novyi type B, Butyrivibrio
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
200
FULGHUM ET AL.
fibrisolvens, Treponema macrodentium, and B. ruminicola brevis. Many organisms such a~ P. acnes are .i~t~r mediate in their oxygen sensitivity between the extremes of the above examples while other species such as Peptostreptococcus intermedius contain both aerotolerant and obligately anaerobic strains. In addition, many other factors may be involved in the expression by a bacterial species of a requirement for anaerobic conditions in vitro. Among these are ability to produce peroxidases, catalase, superoxide dismutase, ability to transfer electrons metabolically at various oxidation-reduction potentials and sensitivity to oxidized organic compounds in bacteriological media exposed to air, as summarized by Smith.P The point of this discussion is that one cannot refute a claim of finding anaerobes from an area containing some gaseous oxygen. Nor can one expect, because of such oxygen, to never find anaerobes. Thus we take exception to the statement by Ingelstedt et aPI "Nor seems the milieu favorable for anaerobic bacteria." The facts that oxygen tension is reduced, that there is tissue damage which may involve release of enzymes that further reduce oxygen tension in the liquid phase (middle ear Ruid) and. enzym:s su?h as tissue catalase and superoxide dismutase that would protect some anaerobic bacteria, and that there are aerobic bacteria depleting the dissolved oxygen in the middle ear Ruid of a number of cases of otitis media, all clearly point to the fact that middle ear Ruid in chronic otitis media could very well be an excellent milieu for the growth of and contribution to chronicity by anaerobic bacteria. Such an. hypothesis is supported by the .findmg ?f clinically significant anaerobic bactena in some cases of chronic otitis media historically as well as in the present studv. The reports of Ingelstedt et alBI and Elner,32 who both reported lowered oxygen concentrations in the middle ~~r cavities of patients with secretory otitis media. and the theory of Sade,33 who feels the lack of ventilation to be the single most important factor in the
chronicity of secretory otitis media, should be reconsidered in light of our findings of clinically significant anaerobic bacteria in cases of otitis media. We do not mean to imply from the present study that we consider anaerobic bacteria to be a primary cause of otitis media, nor even to contribute to chronicity in the chronic otitis media, although they may possibly do either or both. We are, however, proposing a scientific hypothesis to stimulate further study regarding the role of anaerobic bacteria in otitis media since they are found in the disease and must have some role in the ecology of the microflora-microflora and host-microflora relationships. To this end, we consider it to be of utmost importance to undertake careful studies of both the aerobic and anaerobic bacteriology in the following areas: 1) animal model normal flora: the normal flora of the middle ear should be delineated and, where necessary, characterized, 2) animal models of otitis media, both in naturally acquired and in induced ot~tis media should be developed and studied microbiologically, 3) human normal Hora of the middle ear, if any, should be carefully evaluated and all species delineated and characterized, 4) a full scale clinical trial should carefully document the presence, the species, an~ the antibiotic susceptibilities, of all microorganisms found in the middle ear of patients with otitis media. The finding of Corynebacterium sp. and S. epidermidis, the latter of whic.h is now considered to have pathogemc potential'" in most specimens, as well as of the anaerobic bacteria, P. acnes and P. (Streptococcus) interme~ius rai~es the question of the pathogemc potential of each alone and in combination. Roberts 20,34 has shown synergy between the anaerobe, Fusiformis necrophorus and C. pyogenes. Fusiformis nec:opho~us produced a leukocidal exotoxm which protected the C. pyogenes, allowing it to become invasive. Other examples of pathogenic synergy and other ecological syntrophisms in anaerobic infe~ti?ns are found in the Iiterature.l" Thus It IS well-
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
known that mixed culture infections can be produced by organisms usually not pathogenic in pure culture. It also appears that many different mixtures of anaerobic organisms may produce a given disease syndrome. In the present study, we have been able to demonstrate only a sporadic and weak pathogenicity of these isolates for healthy mice using methods similar to those of Hill et al.30 Animal models of otitic infection by these organisms need to be developed in order to fulfill Koch's postulates.
Propionibacterium aenes, formerly known as C. acnes, is a member of the "normal" flora of the human skin mouth, and hair. It is also found in ~ wide variety of clinical specimens where its pro~u.ction of enzym~s, including neurarninidases, hyaluromdase, lipase, are most likely contributory to pathogenicity. Diseases from which clinically significant isolations of P. aenes have been made include maliznancies reticuloendotheliosis, endoc;rditis, s'epticemia, adenitis, arthritis, osteomyelitis, leukorrhea, lung abscesses, purulent meningitis, brain abscesses and erythema. Smith!" has reviewed the salient features of this organism which is known to cause infections alone or in combination with other organisms. P. aenes is also the most frequently isolated anaerobic contaminant in anaerobic studies because of its ubiquity in the sebaceous glands of the human skin. In assigning clinical significance to P. aenes, findings of this organism should be supported by reisolations from the same patient. Peptostreptoeoeeus intermedius is a species composed of several biotypes, some strains of which are more tolerant of oxygen than others. It is a part of the microflora of the nose mouth, throat, and intestine of most humans. It is also often isolated from clinical specimens'? although alone it is not pathogenic for laboratory animals and probably only slightly so for man.l" It has been isolated from the external auditory canals of 2 of 22 subjects.l"
201
The organism is also now considered to be a member of the genus Streptoeoeeus. 3 ;; ,3 G Our isolates of P. intermedius appeared to be obligately anaerobic and never grew on any aerobically incubated plating medium; however, when our results were being confirmed," slight growth occurred aerobically on their blood agar plating medium, which was a different formulation from ours. The species is generally more easily isolated under anaerobic conditions and may become more aerotolerant upon laboratory subculture, although strains that remain obligately anaerobic are known. 12 •18 In brain abscesses, a large proportion of which are thought to originate from otitic infections, anaerobic streptococci (not further identified), anaerobic coryneform bacteria (not further identified), and other anaerobic bacteria were found.F Such literature reviews lend credence to our finding and our hypotheses. Therefore, we hypothesize that chronic otitis media may be caused by, or become chronic in part due to, a number of organisms in various combinations including anaerobes. This makes it imperative to isolate, identify and report antibiotic susceptibilities of all members of these infection florae. The reviewed literaturet!' suggests that a wider variety of specimens from the various types of otitis media as well as more detailed laboratory studies would reveal a wider spectrum of anaerobic bacterial involvement in middle ear disease than found in this study. Considering the past findings of anaerobic bacterial participation in mixed culture infections 12 ,13 ,1 7 ,18 as well as single species infections, we would not be surprised to find a variety of different species of anaerobes playing a role in otitis media. While many of these might be considered opportunistic pathogens or organisms supporting in various ways 16,19.20,34 the maintenance and/or extension of the lesion by other bacteria, it is imperative that we discover their role and learn to deal with the total in-
.. VPISU Anaerobe Laboratory.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
202
FULGHUM ET AL.
fection flora in order to improve patient care, shorten treatment time and expense, and to reduce permanent damage to the auditory apparatus in our patients. All of the organisms isolated in this study are members of the nasal-oral and/or skin flora. The numbers of organisms found point toward growth of these species in the middle ear fluid rather than contamination by normal flora during; myringotomy. While most cases of chronic otitis media have or have had tympanic membrane rupture, the cases utilized here had intact membranes at the time of myringotomy. These findings typify one general type of anaerobic infection, the chronic state associated with anaerobes in the infection flora following tissue damage whether it be mechanical, viral, mycoplasmal, bacterial, or a combination thereof. More often than not such anaerobic infections are of endogenous origin. Endogenous or not, the problems of and the inimical effects of anaerobic infections require laboratory study by appropriate anaerobic techniques 12 ,14 ,1 5 to ensure proper treatment and to shorten treatment time. Considering the increasing chronicity of otitis media and its often debilitating
sequelae, the study of the role of anaerobic bacteria in chronic otitis media could be a major contribution. Finally, we point to the animal model investigations of Drs. H. J. Daniel and P. J. Loesche" and Means et al38 reporting the nonsusceptibility of otitis media in the Mongolian gerbil (Meriones unguiculatus). The hypertrophied and hyperventilated middle ear bullae of this species is theorized to be related to its low disease incidence. In contrast the high incidence of otitis media in the laboratory rat39 ,40 may be related to that species' small, underventilated, and poorly drained middle ear cavities. If structural and functional differences in middle ear cavities allow for differences in size, ventilating capacity and drainage, then these differences may well be related to the ability of anaerobic bacteria to thrive. If so, therapeutic regimens should, in part. continue to be directed toward ventilation and drainage of this system to increase oxygenation and to reduce the accumulation of fluids in which bacteria including anaerobic bacteria may become established. Nature's method of allowing ventilation and drainage by means of membrane rupture may have didactic import.
REFERENCES 1. Glorig A, Gerwin KS: Otitis Media, pro-
ceedings of the National Conference. Springfield, III, Charles C. Thomas, 1972 2. Rist E: Etudles Bacteriolouiques sur Les Infections D'Oriuine Otique, these pour Ie Doctorate in Medecine. Faculte de Medicine de Paris, 1898 3. Krumwiede C Jr, Pratt JS: Fusiform bacilli: cultural characteristics. J Infect Dis 12:438-441, 1913 4. Krumwide C Jr, Pratt JS: Fusiform bacilli: isolation and cultivation. J Infect Dis 12: 199-201, 1913 5. Busacca G: Alcune recerche suI reperto batteriologico delIa otite cronica suppurata, con speciale rigvardo alIa presenza di spirochete e di bacilli fusiformi. Tentativi di cum col neosalvarsan. Arch Ital Otol Rhinol Laryngol 34:414-426, 1923 6. Brisotto P: Dei germi anaerobi in otologia e di alcune richerche batteriologiche
sull'ozena. Riforma Med 39:769-77,5, 1923 7. Dietrich H, Klein W, Nickel H: Uber auftreten und resistenzbestimrnung anaerober sporenloser infektionserreger. Zbl Bakt 197: 515-526, 1965 8. Bulinska H, Kalowski M: Bakterie beztlenowo w patologii przewleklego zapalenia ucha srodkowego, Otolaryngol Pol 28:301305, 1974 9. Lin RG, Arcala AE: Fusobacterium septicemia with otitis media and mastoiditis. Postgrad Med 57: 159-160, 1975 10. Liu Y, Lang R, Lim D, et al: Microorganisms in chronic otitis media with effusions. Ann Otol Rhinol Laryngol 85 (Suppl 25:245-247) 1976 II. Heineman HS, Braude AI: Anaerobic infection of the brain. Am J Med 35:682-697, 1963 12. Holdeman LV, Moore \VEC: Anaerobe Laboratory Manual. Virginia Polytechnic In-
" Unpublished study presented at the American Association of Laboratory Animal Science Annual Meeting in Boston, Massachusetts, 1975.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
OTITIS MEDIA
stitute and State University Anaerobe Laboratory, Blacksburg, Virginia, 1972 13. Finegold SM, Rosenblatt JE: Practical aspects of anaerobic sepsis. Medicine 52:311322,197,'3 14. Dowell VR Jr, Hawkins TM: Laboratory Methods in Anaerobic Bacteriology. DHEW Publication No. (CDC) 74-8272. U.S. Government Printing Office, Washington, DC, 1974 15. Sutter VL, Vargo VL, Finegold SM: Wadsworth Anaerobic Bacteriology Manual, ed 2. University of California, Los Angeles, 1975 16. Hackman AS, Wilkins TD: In vivo protection of Fusobacterium necrophorum from penicillin by Bacteroides fragilis. Antimicrob Agents Chemother 7 :698-703, 1975 17. Balows A (ed): Anaerobic Bacteria: Role in Disease. Springfield, Ill, Charles C. Thomas, 1974 18. Smith LDS: The Pathogenic Anaerobic Bacteria. Springfield, Ill, Charles C. Thomas, 1975 19. MacDonald JB: On the pathogenesis of mixed anaerobic infections of mucous membranes. Ann R CoIl Surg Engl 31:361-378, 1962 20. Roberts DS: The pathogenicity of Fusiformus necrophorus and Corynebacterium pyogenes. Br J Exp Pathol 48:665-673, 1967 21. Howie VM, Ploussard JH: Bacterial etiology and antimicrobial treatment of exudative otitis media. South Med J 64:233-239, 1971 22. Liu YS, Lim DJ, Lang RW, et al: Chronic middle ear effusions. Arch Otolaryngol 101:278-286, 1975 23. Berry YJ, Rose RM: Otitis media: obstruction and a clinical approach. Forum on Infection 1:1-13, 1974 24. Feigin RD, Chang MJ: Treating otitis media. Drug Therapy, 4(9):116-125,1974 25. Hungate RE: The anaerobic mesophilic cellulolytic bacteria. Bacteriol Rev 14:1-49, 1950 26. Moore WEC: Techniques for routine culture of fastidious anaerobes. Int J Syst BacterioI16:173-190,1966 27. Bailey WR, Scott EG: Diagnostic Mi-
crohiology, ed 4. St. Louis, C. V. Mosby, Co, 1974 28. Lennette EH, Spaulding EH, Truant JP (eds): Manual of Clinical Microbiology, ed 2. Washington, DC, American Society for Microbiology, 1974 29. Buchanan RE, Gibbons NE ( eds ) : Bergey's Manual of Determinative Bacteriology, ed 8. Baltimore, Williams and Wilkins Co, 1974 30. Hill GB, Osterhout S, Pratt PC: Liver abscess production by non-sporeforrning anaerobic bacteria in a mouse model. Infect Immun 9:599-603, 1974 31. Ingelstedt S, Johnson B, Rudcrantz H: Gas tension and pH in middle ear effusion. Ann Otol Rhinol Laryngol 84: 198-202, 1975 32. Elner A: Normal gas exchange in the human ear. Ann Otol Rhinol Laryngol 85 (SuppI25:161-164) 1976 33. SaM J: The biopathology of secretory otitis media. Ann Otol Rhinol Laryngol 83: 54-70, 1974 34. Roberts DS: Synergistic mechanisms in certain mixed infections. J Infect Dis 120:720724, 1969 35. Rogosa M: Peptococcaceae, a new family to include the gram positive, anaerobic cocci of the genera Peptococcus, Peptostrepiococcus, and Ruminococcus. Int J Syst Bacteriol 21 :234-237, 1971 36. Holdeman LV, Moore WEC: New genus, Coprococcus, twelve new species and emended descriptions of four previously described species of bacteria from human feces. Int J Syst Bacterial 24:260-277, 1974 37. Swartz MN, Karchmer AW: Infections of the central nervous system, in Ballows A ( ed ): Anaerobic Bacteria: Role in Disease. Springfield, Ill, Charles C. Thomas, 1974 38. Means LW, Daniel HJ, Jordan LH, et al, Nonsusceptibility to otitis media of the laboratory gerbil, Meriones unguiculatus. Physiol Psychol 3:229-230, 1975 39. Daniel HJ, Means LW, Dressel ME, et al: Otitis media in laboratory rats. Physiol Psychol 1:7-8, 1973 40. Daniel HI, Carmine FH, Cook RA: Otitis media in two strains of laboratory rats. J Aud Res 11:276-278, 1971
ACKNOWLEDGMENTS-We are grateful to the University of North Carolina School of Medicine for providing Mr. Yarborough with a summer stipend for medical students. Dr. W. S. Bost of Greenville and Drs. N. D. Fischer and W. P. Biggers of the U.N.C. School of Medicine at Chapel Hill kindly allowed us to collect specimens from patients at myringotomy. Dr. J. J, Crawford allowed us to use his facilities in preparation of specimens for transport to Greenville. In our laboratory, Mr. John Worthington prepared anaerobic media and characterized the cultures of anaerobic bacteria. Dr. Lillian V. Holdeman of the Virginia Polytechnic Institute and State Laboratory kindly confirmed the identity of most of our anaerobic isolates. Dr. Sydney Finegold provided references numbered 2-7 of the literature review regarding anaerobic bacteria in otitis media. Ms. Diane Gardner carried out the study on otitis media in rats. REPRINTS-R. S. Fulghum, PhD, Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834.
Downloaded from aor.sagepub.com at FLORIDA ATLANTIC UNIV on September 4, 2015
