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The role of anaerobic bacteria in otitis media: Microbiology, pathogenesis, and implications on therapy
Clinical Reviews
8:10g--117, 1987
The Role of Anaerobic Bacteria in Otitis Media: Microbiology, Pathogenesis, and Implications on Therapy ITZttAK BROOK, MD, MSc The current knowledge of the recovery of anaerobic bacteria from cases of acute and chronic otitis media is reviewed. Although techniques for cultivation of anaerobes were used in the studies presented, the methodologies of collection, transportation, and cultivation of the specimens varied. Consequently, there were differences in the rates of recovery of these organisms and the species isolated. Anaerobes, mainly gram-positive cocci, were recovered from a quarter of ear aspirates in acute otitis media in about one third mixed with aerobic and facultative bacteria. In a study of otitis media with effusion, anaerobic bacteria were recovered in 12% of the culture-positive aspirates. The predominant anaerobes were gram-positive cocci and Bacteroides melaninogenicus. Several studies reported the recovery of anaerobes in about half of the patients with chronic otitis media and in those with cholesteatoma. The predominant anaerobes were gram-positive cocci, Bacteroides sp, and Fusobacteria sp. Many of these organisms produced ~-Iactamase and might have contributed to the failure of these patients to respond to penicillins. The potential synergistic relationships between the various aerobic and anaerobic bacteria and the role of the capsule of anaerobic bacteria as a virulence factor are discussed.
Otitis media frequently occurs in childhood and may cause serious morbidity, such as permanent ear damage, decreases in hearing, and sometimes serious sequelae such as extension of the infection to the intracranial space. Recent developments in microbiologic methodologies have increased the awareness of the important role of anaerobic bacteria in ear infections. The predominance of anaerobic organisms in the oral flora, where they outnumber the aerobic bacteria by a ratio of 10 to one, explains their involvement in otitis media. Recognition of the role of anaerobic organisms in these infections has important implications for their management, since some of these organisms are resistant to various antimicrobials.
The recovery of anaerobic bacteria depends on the utilization of proper and adequate techniques of collection, transportation and inoculation of the specimens? In collection of specimens through the ear canal, effort should be made to avoid the normal flora at that site by prior cleansing and sterilization of the external ear canal. The exposure of specimen to oxygen should be reduced by using minimal suctioning, preferably by a syringe and needle, and avoiding forceful and constant negativepressure suctioning such as when Senturia ear specimen collection is used. Transportation to the laboratory should be prompt, and if delayed, adequate transport media should be used to insure anaerobic environment. After expulsion of the air bubbles a corked syringe and needle is the simplest mode of transportation of liquid specimens. Swabs may be placed in prereduced tubes or in special commercially available transport media. Specimens should be inoculated into prereduced culture media that are supportive for the growth of anaerobic organisms and inoculated in an anaerobic environment for sufficient time to allow the growth of fastidious organisms.
Received June 27, 1986, from the Naval MedicalResearch Institute, National Naval Medical Center, Bethesda, Maryland. Acceptedfor publication October 16, 1986. The opinions and assertions contained herein are the private ones of the writer and are not to be construedas official or reflectingthe views of the Navy Department or the Naval Service at large. Address reprint requests to Dr. Brook: Armed Forces RadiobiologyResearch Institute,Bethesda, MD 20814. 109
ANAEROBES IN OTITIS MEDIA
This review summarizes the recent developments in the understanding of the role of anaerobic bacteria in acute and chronic otitis media and discusses the synergy between these organisms and their aerobic and facultative counterparts, and the importance of encapsulation of these pathogens as a virulence factor. ACUTE OTITIS MEDIA
Acute otitis media (AOM) is one of the most common diseases of early childhood. 2 In many cases, recurrent AOM is followed by chronic inflammation.
Microbiology
America n
Journal of Otolaryngology 110
Streptococcus pneumoniae and Haemophilus influenzae are the principal etiological agents in AOM2 -7 Of the two, S pneumoniae was recovered more often irrespective of age, and its predominance increases with age. H influenzae, an infrequent cause of AOM in older children, shows increased resistance to ampicillin. Other causative agents include group A [3-hemolytic streptococcus (GABHS), Branhamella catarrhalis, Staphylococcus aureus, and gram-negative bacilli. 7 Gram-negative bacilli and staphylococci are generally implicated in AOM in neonates. However, S pneumoniae and H influenzae are the most common etiologic agents among neonates. In most past studies, microorganisms were recovered in about two thirds of aspirates, while the other third generally showed no growth. 3-6 However, no attempts were made in these studies to culture for anaerobic bacteria by proper and prompt transportation of the specimens, by inoculation into adequate media, and by incubation in anaerobic environment. Two recent studies 8'~ used techniques for cultivation of anaerobic as well as aerobic bacteria. In the larger of these, g 186 children with AOM were studied using a technique that minimized the exposure of the aspirate to oxygen./~ Aerobic bacteria alone, predominantly S pneumoniae and H influenzae, were isolated from 118 (63%) patients, and anaerobic organisms alone, most anaerobic cocci, from 24 (13%1 patients. Twenty-six (14%) aspirates yielded mixtures of aerobes and anaerobes, and several had multiple aerobic or anaerobic organisms. Anaerobes were recovered from a total of 50 (27%) of the patients. No bacterial growth was noted in 18 (9.7%) patients, a rate lower than
that obtained in previous studies in which techniques for recovery of anaerobes were not used2 -71 The predominant aerobic organisms included S pneumoniae (62 patients, 37%), H influenzae (52 patients, 30%), and S aureus (15 patients, 8%). The predominant anaerobes were Peptostreptococcus sp (39 patients, 21%) recovered alone in 15 cases and mixed with other bacteria, in the rest of the cases. Propionibacterium acnes were identified in 12 patients (7%). Other anaerobic organisms were one of each of Veillonella sp, Bifidobacterium sp, Eubacterium sp, Clostridium ramosum, and microaerophilic streptococci. Since antisepsis of the external auditory canal prior to tympanocentesis was not done in these studies, some of the anaerobic species may represent contamination of the middle ear aspirate by the external ear canal flora and the tympanic membrane surface. However, this may not be the case in the majority of the cases, since gram-stain preparation of the ear aspirates conformed with the recovered anaerobic organisms in most cases. In another study employing techniques for cultivation of anaerobes, the external ear canal and the tympanic membrane surface were sterilized prior to myringotomy. 11 Three anaerobes were recovered from the 28 studied infants: two isolates of Clostridium sp and one isolate of Peptococcus magnus. The isolation of anaerobic bacteria from the middle ear even after antisepsis of the external auditory canal suggests that these bacteria occasionally may play a direct or ancillary role in the pathogenesis of AOM. Additional support for the role of anaerobic bacteria came from a study of the bacterial flora of the external auditory canal of 72 healthy children. 12 The most common aerobic isolates were Staphylococcus epidermidis, a-hemolytic streptococcus, and Pseudomonas aeruginosa, The two anaerobic species recovered were P acnes (13 isolates) and Peptostreptococcus sp (two isolates). Thus, Peptostreptococcus sp recovered from 21% of middle ear aspirates obtained from children with AOM 1~ was isolated from only 3% of external ear canal specimens. The differences in the isolation rate of this organism in the external ear canal and the middle ear further supports its possible role in acute and chronic otitis media. P acnes, on the other hand, is a component of the skin flora and is a rare pathogen. 1 However, the rate of isolation of this organism from the external ear canal was 18%, which is higher than its recovery rate from middle ear aspirates in in-
BROOK
fected patients, which was 7% in acute and 6% in chronic otitis media. Because of the lack of a large-scale study where proper sterilization of the ear canal has been performed, the role of anaerobes in AOM has to be further substantiated.
Antimicrobial Therapy Because the most common offending organisms are S pneumoniae and H influenzae, most patients respond favorably to ampicillin or amoxicillin. In patients who are allergic to penicillin, erythromycin and/or sulfa drugs may be prescribed. ~ Cefaclor also has been effective in the treatment of this disease, ~3 mostly because of its anti-Haemophilus activity. Combination therapy has also gained popularity, largely because of the growing number of ampicillin-resistant H influenzae. The combinations successfully used are penicillin and sulfonamide, ~4 trimethoprimsulfamethoxazole, 15 erythromycin-sulfisoxazole, 16 and, most recently, amoxicillin and clavulanic acid (a ~-lactamase inhibitor). ~7 Because the anaerobes recovered in AOM are susceptible to penicillins and other antimicrobials commonly used to treat AOM, no change in the recommended therapy is advocated. However, the combination of trimethoprim-sulfamethoxazole is effective against only 50% of anaerobic gram-positive cocci (AGPC). OTITIS MEDIA WITH EFFUSION Otitis media with effusion (OME) is a common cause of mild hearing loss in children, most often between the ages of 2 and 7 years. Persistent middle ear effusion was found for at least 1 month in up to 40% of children who had suffered from AOM and for at least 3 months in 10% of the children. 18
Microbiology Because no organisms were recovered in persistent or chronic middle ear effusions, they were assumed for many years to be sterile. 19,2~However, in 1958 Senturia et al. 21 examined 130 patients with OME and identified bacteria in 33% of ears with serous effusions, in 25% of ears with mucoid effusions, in 51% of ears with mucopurulent effusions, and in 29% of ears with purulent effusions, In 1974 Kokko 22 cultured bacteria in 22% of ear effusions studied, Healy and Teele, 23 who investigated 57 children, reported that 35 of
96 specimens (36%) had bacteria that closely resembled those found in AOM. Riding et al. 24 studied the ear aspirates of 274 children and found that 45% of the ears contained bacteria. In 1976 Liu et al. 25 examined 102 patients with OME and found organisms in 66% of ears with serous effusions and 36% of the ears with mucoid effusions. None of these studies employed techniques for transportation and cultivation of anaerobes, and the external canal was not sterilized. Adlington and Davies 26 found no evidence supporting the role of viruses in OME. Klein and Teele, 27 however, isolated viruses in 29 of 663 patients (4.4%); 22 of the viruses were respiratory syncytial virus. Attempts to recover anaerobes in OME have been conducted since 1979. Giebink et al., 28 who studied 144 serous and mucoid effusions, recovered aerobic bacteria in 20% of the effusions. H influenzae was isolated predominantly from serous effusions and S epidermidis predominantly from mucoid samples. One effusion each yielded a virus (Herpesvirus heminis) and an anaerobe (Propionibacterium sp). However, the anaerobic techniques used were inappropriate for recovery of fastidious organisms, and only liquid medium was used for the initial culturing for anaerobes. Teele et al,, 29 w h o studied 20 serous effusions, did not recover anaerobes. The lack of recovery of anaerobes may have been due to the delay (up to 6 hours) in processing the effusions. Sipila et el., 3~who studied 110 middle ear aspirates, found aerobic bacteria in 35 (32%) of the aspirates. An anaerobic organism was recovered in one instance. Although the specimens were plated without delay, the culture media used for cultivation of anaerobes is not specified and might have been inadequate for cultivation of fastidious organisms. Brook et al. 31 recovered bacteria from 23 of 57 (41%) of their patients. Anaerobes were the only isolates in 17% of the culture-positive aspirates, and in an additional 26%, they were present mixed with aerobes. Aerobic organisms only were recovered in 13 aspirates (57%). A total of 45 bacterial isolates were recovered, accounting for two isolates per specimen (1.4 aerobes and 0.6 anaerobes). The predominant aerobic isolates were H influenzae, S aureus, and S pneumoniae. The anaerobes recovered were AGPC (six isolates), Bacteroides melaniogenicus (five isolates), and P aches (three isolates). It is of interest that the
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ANAEROBES IN OTITIS MEDIA
anaerobes recovered from these patients were similar to those previously recovered from children with acute 7'8 and chronic 32 otitis media. Nine ~-lactamase-producing organisms (BLPOs) were recovered from eight patients (35%). These included all five isolates of S aureus, three of the five B melaninogenicus, and one of eight H influenzae.
Antimicrobial Therapy The role of bacteria in the pathogenesis of OME is not yet clear; however, antimicrobial agents often are used in an attempt to clear the ear effusion from microorganisms. The presence of anaerobic as well as aerobic bacteria, many of which are BLPOs, in serous ear aspirates raises the question whether antibiotics effective against these BLPOs should be used. Controlled studies are needed to define the value of antimicrobial treatment in patients with OME and to clarify the role of bacteria in the pathogenesis of the inflammatory process. CHRONIC SUPPURATIVE OTITIS MEDIA AND CHOLESTEATOMA Chronic suppurative otitis media (CSOM) can be insidious, persistent, and very often destructive, with sometimes irreversible sequelae. In many cases of chronic otitis media, a cholesteatoma can develop; CSOM with cholesteatoma tends to be persistent and progressive.
Microbiology Although past studies reported the recovery of anaerobes from many cases of CSOM, aerobes (mainly S aureus and gram-negative enteric be-
cilli) were considered to be the major pathogens. 3a Several recent studies (Table 1) reaffirmed the role of anaerobes in CSOM. 34-4~ The variability in the rate of recovery of anaerobes in these studies may be due to differences in geographic locations and laboratory techniques. In several of the studies, the delays in cultivation was extensive, and the length of incubation was inadequate for anaerobic bacteria. The predominant anaerobic organisms recovered in these studies were AGPC and B melaninogenicus group. In a carefully done study, Fulghum et el. 34 recovered Peptostreptococcus intermedius and P aches from four of 10 cases of COM. These authors aspirated unperforated tympanic membranes. It is not clear, however, whether they have sterilized the auditory canal prior to aspiration. In another report using proper method for isolation of anaerobes, Karma et al. 3~ obtained aspirates through perforation of the ear drum and recovered anaerobes from one third of 1.14 patients. Bacteroides sp accounted for 50% of the anaerobes (29 isolates) and AGPC for 25% (15 isolates). Aygagari et al. aa recovered anaerobes in 68 of 115 (59%) patients. Anaerobic organisms only were present in 11 (10%) patients, aerobic organisms only in 40 (35%), and mixed infection was present in 57 (50%). The predominant anaerobes were Bacteroides sp and AGPC, and the major aerobes were P aeruginosa, S aureus, and Proteus sp. The delay in inoculation of the specimens is not discussed. Sugita et al. a7 recovered anaerobes in 62 of 76 (8%) cases. This low recovery rate of anaerobes may be due to the use of a medium that did not support anaerobic growth and to unspecified delays in inoculating the specimens. Sweeney et al. 3s isolated anaerobes from 52 of
TABLE 1. Frequency of Recovery of Anaerobic and Aerobic Organisms Recovered in Chronic Supparative Otitis Media
AUTHOR
American Journal
of Otolaryngology 112
Karma at a125 Sugita et al? 7 Aygagari et al? 6 Brook 4~ S w e e n e y et al? ~ Constable and Butler 3"
No. OF CASES WHERE ANAEROBES WEIIE RECOVERED/ TOTAL NO. OF AEROBIC Bacteroides CASES COCCI SP 38/114 62/760 68/115 35/68 52/130 20/100
(33%) (8%) (59%) (51%} (44%) (20%)
15 38 33 31 7 9
29 18 43 21 54 7
Fuse-
Bacterium Clastridium
-
sP
sP
2 6 7 4 9
2 2 6 3 1
-
-
-
Staphylococcus aureus
Psuedomonas sP
OTHER GRAMNECAT~VE RoDs
32 8 22 15 33 29
16 12 29 33 25 15
37 31 32 31 86 34
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130 (44%) patients. These authors did not specify the length of delay in inoculation and admitted that the unsupplemented thioglycolate media that was used in the first 73 patients was inhibitory for anaerobic growth. Constable and Butler 39 found anaerobes in 20 of 100 (20%) aspirates. Specimens were sent in an anaerobic transport media and were processed within I hour. However, the media used was not enriched, and the plates were incubated for only 24 hours before exposure to room air. All of these factors might have reduced the number of anaerobes isolated in these studies. In none of these studies was there any attempt to differentiate between organisms that reside in the ear canal and those recovered only from the inner ear, It is possible that some of the isolates were not true pathogens but colonizers of the ear canal, Brook 4~recovered anaerobic bacteria from 51% of ear aspirates of children suffering from CSOM. The ear aspirates were inoculated immediately into enriched media supportive for the growth of anaerobes, and the media were incubated for 14 days, thus allowing the slow-growing organisms sufficient time to grow. The majority of the anaerobic isolates were AGPC, Bacteroides sp (including B fragilis and B melaninogenicus groups), and Fusobacterium nucleatum. The predominant aerobic bacteria isolated were enteric gramnegative rods (mostly P aeruginosa) and S aureus. Anaerobic isolates usually were mixed with other bacteria, and the number of isolates ranged between two and four per specimen. A comparison also was made between the bacteria recovered in the middle ear and those present in the external ear canal. Only half of the bacteria recovered from the middle ear were present also in the external auditory canal. Furthermore, external ear canal culture yielded in many cases bacteria that were not present in the middle ear. These findings demonstrate that cultures collected from the external auditory canal prior to its sterilization can be misleading. This is particularly important in relationship to P aeruginasa, which is recovered more frequently in the ear canal than in the middle ear. Although this organism is a common inhabitant of the ear canal, 11 it can be recovered also from the middle ear. Direct middle-ear aspirates through the perforation in the eardrum are therefore more reliable in establishing the bacteriology and selection of therapy for CSOM. The pathogenicity of anaerobic bacteria is supported by their higher recovery rate from the mid-
dle ear only compared with their recovery from the external canal. Thirty-eight anaerobic strains were recovered from the middle ear only, as campared with seven in the external canal. In a recent study, we evaluated the presence of BLPOs in 48 children with CSOM. 41 The ear canal was sterilized and specimens were inoculated without delay and incubated for 14 days. Eighty-three aerobic and 93 anaerobic isolates were recovered. Aerobic bacteria only were involved in 22 (46%) patients, and anaerobic organisms only in five (12%). Mixed aerobic and anaerobic isolates were recovered 21 cases (44%). Beta-lactamase-producing organisms (24 anaerobes and 16 aerobes) were recovered from 31 (65%) patients. These included all isolates each of the S aureus and B fragilis groups, 11 of 19 of the B melaninogenicus group, three of six of Baeteroides oralis, four of six of H influenzae, two of three S epidermidis, and two of four of B catarrhalis. The recovery of BLPOs is not surprising, because most of our patients received multiple courses of penicillins, which might have isolated the resistant organisms. Furthermore, we were able to detect the enzyme in 84% of the ear aspirates that contained BLPOs in excess of 104 CFU/ml. The bacteriology of cholesteatomas present in chronically infected ears provides further support for the role of anaerobes in chronic ear infection. Cholesteatoma specimens were obtained by Brook 42 from patients undergoing surgery for CSOM and cholesteatoma. All cultures were processed for aerobic and anaerobic bacteria and were obtained from surgical specimens, which excludes any possibility of contamination by skin flora. A total of 74 isolates (40 aerobes and 34 anaerobes) was recovered in 24 specimens. Aerobes alone were isolated in eight (33%) specimens, anaerobes only in four (27%), and aerobic and anaerobic bacteria in 12 (50%). Fifty isolates (27 aerobes and 23 anaerobes) were present in numbers greater than 100 CFU/gm. The most commonly isolated aerobic organisms were P aeroginisa (nine isolates), Proteus sp (seven isolates), K pneumoniae (five isolates), S aureus (five isolates), and Escherichia call (four isolates). The anaerobic bacteria most commonly isolated were AGPC (12 isolates), Baeteroides sp (12 isolates, including five B fragilis group), Clostridium sp (three isolates), and Bifidobacterium sp (three isolates). These findings indicate the polymicrobial aerobic and anaerobic bacteriology of cholesteatoma in chronically infected ears. Similar
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data were found by Iino et a l . , 43 who also detected organic volatile acids (a product of the anaerobic bacteria metabolism) in the cholesteatoma.
Pathogenesis Support for the importance of anaerobes in CSOM is p r o v i d e d by their recovery from most infectious complications of this infection. Anaerobes similar to those recovered in CSOM were recovered from 23 of 24 (96%) specimens from patients with chronic mastoiditis. 44 Anaerobes were recovered from most of the patients with intracranial abscesses complicating CSOM. 1,33,45 The high number of negative cultures of chronic suppurative middle ear effusions in certain studies m a y be due to inappropriate methods for recovery of anaerobes. In o n e s t u d y , 46 bacteria were seen on direct smears in 80% of middle ear effusions, although only 40% of the effusions yielded bacterial growth. Foul-smelling pus in the middle ear suggests the presence of anaerobic bacteria in m a n y of the patients. Cholesteatoma that a c c o m p a n y CSOM can induce the absorption of bone. Various theories attempt to explain the factors involved in the process of expansion of cholesteatoma and the collagen degradation that occurs in its vicinity. 47'48 The production of organic acids by anaerobic bacteria m a y be involved in the destructive p r o c e s s . 43 Because cholesteatoma associated with CSOM contains bacteria similar to those recovered from chronically infected ears, the cholesteatoma may serve as a nidus of the chronic infection.
Antimicrobial Therapy
American Journal of Otolaryngology 114
Attempts to treat CSOM using antimicrobial therapy alone generally are not successful. The organisms u s u a l l y treated are the aerobic isolates, mainly S aureus and the gram-negative enteric bacilli. In an o p e n study, Brook 49 used parenteral carbenicillin or clindamycin to treat CSOM. Combined therapy with gentamicin was used w h e n aerobic rods also were recovered. Although therapy was successful in only half of the patients, this s t u d y demonstrated that therapy directed against the organisms isolated from patients' effusion c o u l d eradicate the infection in many instances. Kenna et al. 5~were able to achieve an i m p r o v e m e n t in 32 of 36 (89%) patients with CSOM with parenteral antimicrobial agents and daily aural toilet. Although the authors did not
obtain cultures of anaerobic bacteria, many of the antimicrobial agents they used were effective against anaerobic bacteria. Controlled studies are needed to further evaluate the role of antimicrobial therapy with or without surgery. Until recently, most of the anaerobes recovered from respiratory tract and orofacial infections were susceptible to penicillin. The S aureus and B fragilis groups are k n o w n to resist penicillin through production of ~ lactamase. However, an alarming number of Bacteroides sp (mostly B melaninogenicus group and Bacteroides buccalis), formerly susceptible to penicillins, are currently showing increasing resistance to these drugs by virtue of production of the enzyme ~ lactamase. 51 The appearance of penicillin resistance among Bacteroides sp has important implications for chemotherapy. Such organisms can release the enzyme and degrade penicillins or cephalospotins in the area of the infection. In this way, they can protect not only themselves but also penicillin-sensitive pathogens. Penicillin therapy directed against a susceptible pathogen might be rendered ineffective by the presence of a penicillinase-producing organism. 52 Several in vitro, in vivo, and patient data provide support for this theory. 53 A 200-fold increase in resistance of GABHS to penicillin was observed when it was inoculated with S aureus, 54 and a 8,500-fold increase of the organism's resistance occurred when it was inoculated with B fragilis. ~5 The same phenomenon was demonstrated in animals. Hackman and Wilkins 56 showed that penicillin-resistant strains of Bacteroides sp protected penicillin-sensitive F nucleatum from penicillin therapy in mice. Brook et al., 52 using a subcutaneous abscess model in mice, demonstrated protection of GABHS from penicillin by ~-lactamase-producing Bacteroides sp. Two recent reports ~7,58 described five adults and 75 children (including 19 with CSOM) with clinical failures after penicillin therapy associated with the isolation of aerobic and anaerobic BLPO. Most of these patients did not respond to various penicillin therapies and may represent clinical failures due to BLPOs, The isolation of BLPOs from two thirds of chronically inflamed ears and the ability to detect the free enzyme in the ear fluid 41 raise the question of whether the treatment of CSOM with penicillins is adequate in all instances and whether therapy should be directed at the eradication of BLPOs whenever they are present. Surgical drainage is still the therapy of choice, However,
BROOK
the presence of penicillin-resistant aerobic or anaerobic bacteria may warrant also the administratior, of appropriate antimicrobial agents effective against these organisms, e. g., clindamycin, metronidazole, chloramphenicol, carbenicillin, cefoxitin, or the combination of clavulanic acid and amoxicillin or ticarcillin. This should be considered especially if the infection persists despite surgical drainage. PATHOGENIC ROLE OF ANAEROBIC ORGANISMS IN OTITIS MEDIA The polymicrobial nature of CSOM is apparent as the number of isolates in most ear aspirates varies between two and five. Several isolates in each aspirate were recovered also from up to one third of patients with AOM and OME. 8,4~ The recovery of mixed aerobic and anaerobic flora in otitis media is not unique to this infection and occurs in many other infections involving anaerobic bacteria. 1'33 The synergistic relationship between the anaerobic and the aerobic and facultative flora has been studied and provides further support for the pathogenic role of anaerobic bacteria in otitis media.
Synergy Between Anaerobic and Aerobic or Facultative Flora in Otitis Media Polymicrobic infections are known to be more pathogenic for experimental animals than those involving single organisms. 59 Altemeier 6~ showed that individual isolates recovered from mixed infection were relatively innocuous to animals, but combinations of facultative and anaerobic strains had increased virulence. Similar observations were reported by Meleney et al. 61 and Hite et al. 62 Brook et al. 6a,64 evaluated the synergistic potential between aerobic and anaerobic bacteria recovered in CSOM. Each bacterium was injected subcutaneously into mice alone or mixed with another organism, and synergistic effects were determined by observing animal mortality, abscess formation, 63 and enhancement of bacterial growth. 64 The bacteria tested included AGPC, Bacteroides sp, and Fusobacterium sp. Facultarive and anaerobic bacteria included S aureus, P aeruginosa, E coli, K pneumoniae, and P mirabilis. Bacterial synergy was demonstrated between the aerobic organisms and the Baeteroides sp and AGPC; it was especially apparent between P aeruginosa and S aureus and the anaerobes. These
findings are of particular relevance to the pathologic role of t h e s e organisms in CSOM, since the combination of P aeruginosa and AGPC was isolated from 40% of patients with CSOM, and S aureus and anaerobic cocci were recovered in 9% of these patients. 4~ The demonstration of synergy between the anaerobic and aerobic bacteria commonly recovered in ear infections further indicates their pathogenic role in these infections. Several hypotheses have b e e n proposed to explain microbial synergy. W h e n this p h e n o m e n o n occurs in mixtures of aerobic and anaerobic flora, it may be caused by protection from phagocytosis and intracellular killing, us production of essential growth factors, 6~ and lowering of oxidation-reduction potentials in host tissuesJ .7
Role of Encapsulated Anaerobes in Otitis Media An important virulence factor of anaerobes in the possession of capsule. Onderdonk et al. 68 demonstrated the pathogenicity of encapsulated B fragilis by its ability to induce abscesses alone. Simon eta]. 69 described decreased phagocytosis of the encapsulated B fragilis. Capsular material from B melaninogenieus also inhibits phagocytosis and phagocytic killing of other microorganisms in an in vitro system. 7~ We evaluated the importance of encapsulated strains recovered from 13 clinical samples 71 by subcutaneously injecting into mice each of the 35 isolates (30 anaerobes and five aerobes) alone or in all possible combinations with the other isolates recovered from the same abscess and observed their ability to induce and/or survive in a subcutaneous abscess. Most of the organisms that were encapsulated were able to cause abscesses by themselves and were recovered from the abscesses even w h e n inoculated alone. This increased virulence of encapsulated strains was found in Bacteroides sp, AGPC, Clostridium sp, and E coll. We recently s t u d i e d the importance of encapsulated Bacteroides sp and AGPC as judged by their ability to cause subcutaneous abscesses in mice, 72-74 With f e w exceptions, possession of a capsule enabled these organisms to contribute more to the infectious process than their aerobic counterparts. A l t h o u g h unencapsulated organisms did not i n d u c e abscesses, many of the isolates that contained few encapsulated organisms (less than 1%), w h e n inoculated mixed with other abscess-forming aerobic and anaerobic bacteria,
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survived in the abscess and became heavily encapsulated after 10 to 14 days. Thereafter, these heavily encapsulated isolates were able to induce abscesses when injected alone. This phenomenon may explain how nonpathogenic organisms that are part of the normal oral flora become pathogens in CSOM. The mechanisms responsible for the phenomenon is yet unknown and may be due to either genetic transformation or a process of selection. Detection of a capsule in a clinical isolate may suggest a pathogenic role for the organism in the infection. A recent study supports the importance of encapsulated anaerobic organisms in respiratory infections, including otitis media. 7a The presence of encapsulated Bacteroides sp and AGPC was investigated in 48 patients with CSOM and in the pharynx of 26 individuals without inflammation. Forty-five of the 60 (75%) isolates of the B melaninogenicus and B fragilis groups, B oralis, and AGPC were found to be encapsulated in the patients with CSOM, as compared to only 34 of 96 (35%) of the controls (P < 0.001). The recovery of encapsulated anaerobic organisms in most patients with CSOM, as well as other upper respiratory tract infections, 7a supports the pathogenic role of these organisms. Early and vigorous antimicrobial therapy, directed at both aerobic and anaerobic bacteria, may abort the infection prior to the emergence of the encapsulated bacterial strains that contribute to the chronicity of the infection, CONCLUSIONS
Recent studies pointed to the role of anaerobic bacteria in otitis media. The recovery of anaerobic bacteria depends on adequate collection, transportation and inoculation of specimens. The increased recovery rate of [Mactamase-producing organisms in these infections warrants the administration of appropriate antimicrobia] agents directed also against these organisms. The synergistic relationship between the various aerobic and anaerobic bacteria recovered from patients with otitis media and the presence of encapsulated anaerobic organisms in this infection support their pathogenic role,
American Journal of Otolaryngology 116
References 1, Brook I: Anaerobic Infections in childhood. Boston, GK Hall, 1983 2. Howie VM, Ploussard JH: The otitis prone condition. Am J Dis Child 1975;129:675-678
3. Howie VM, Ploussard JH, Sloyer I: Otitis media: A clinical and bacteriological correlation. J Pediatr
1970;45:29-35 4. Kamme C, Lundgren K, Mardh PA: The etiology of acute otitis media in children. Scand J Infect Dis 1971;3:217-223 5. MeLinn SE, Daly IF, Jones JE: Cephalexin monahydrate suspension. Treatment of otitis media. JAMA
1975;234:171-173 6. Schwartz REI, Rodriguez WJ, Khan WN, et al: The increasing incidence of ampicillin-resistant Haemaphi/us influenzae. A cause of otitis media. JAMA 1978;239:320-323 7. Schwartz RH, Brook I: Gram-negative rod bacteria as a cause of acute otitis media in children. Ear Nose Throat ] 1981;60:9-11 8. Brook I, Anthony BF, Finegold SM: Aerobic and anaerobic bacteriology of acute atitis media in children, J pediatr 1978;92:13-15. 9. Brook I: Otitis media in children: A prospective study of aerobic and anaerobic bacteriology. Laryngoscope 1979;89:992-997 10. Brook h A practical technique for tympamnocentesis for culturing aerobic and anaerobic bacteria. Pediatrics 1980;65:626-627 11. Brook I, Schwartz R: Anaerobic bacteria in acute otitis media. Acta Otolaryngol 1981;91:111-114 12. Brook I: Microbiological studies of the bacterial flora of the external auditory canal in children. Acta OtalaryngoI 1981;91:285-287 13. Schwartz RH, Schwartz OM: Acute atitis media: diagnosis and drug therapy. Drugs 1980;19:107-118 14. Syriapou]ou V, Sheifele D, Howie UM, etah Incidence of ampicillin-resistant Haemaphilus influenzae in otitis media. J Pediatr 1976;89:838-841 15. Schwartz RH, Rodriguez WJ, Mann R, et al: The nasapharyngeal culture in acute otitis media: A reappraisal of its usefulness. JAMA 1979;241;2170-2173 16. Howard IE, Nelson JD, Clahsen J, etal: Otitis media of infancy and early childhood: A double blind study of four treatment regimens. Am J Dis Child
1976;139:965-970 17. Odio CM, Kusmiesz H, Shelton S, e t a h Comparative treatment trial of Augmentin versus cefaclar for acute atitis media with effusion. J Pediatr 1985;75: 819-826 18. Teele DW, Klein JO, Rosner BA: Epidemialogy of otitis media in children. Ann Otol Rhinol Laryngol 1980;89 (suppl);5-6 19. Robinson JM, Nicholas HO: Catarrhal atitis media with effusion--A disease of a retropharyngeal and lympathic system. South Med ] 1951;44:777-789 20. Siirala U, Vuari M: The problem of sterile otitis media. Prac Ore Rhino Laryngol, 1956;19:159-169 21. Senturia BH, Gessert CF, Carr CD, e t a h Studies concerned with tubotympanitis. Ann Otol Rhinol Laryngol
1958;67:440.-467 22. Kokko E: Chronic secretory otitis media in children: A clinical study. Acta Otolaryngol [Suppl] 1974;327:7---44 23. Healy GB, Teele DW: The microbiology of chronic middie ear effusions in children. Laryngoscope
1977;87:1472-1478 24. Riding KH, Bluestone CD, Michaels RH, et ah Microbiology of chronic and recurrent otitis media with effusion. J Pediatr 1978;93:739-743 25. Liu Y, Lira D, Lang R, et al: Chronic middle ear effusions. Immunochemical and bacterial investigation. Arch Otolaryngol 1976; 101:278-286 26. Adlington P, Davies JE: Virus studies in secretory otitis media. J Laryngol Oral 1969;83:161-173 27. Klein JO, Teele DW: Isolation of viruses and mycoplasmas from middle ear effusion', A review. Ann Otol Rhinol Laryngol 1976;85(suppl 25}:140-144
BROOK 28. Giebink GS, Mills L, Huff JS: The microbiology of serous and mucoid otitis media. Pediatrics 1979;63: 915-919 29. Teele DW, Healy GB, Tally FP: Persistent effusions of the middle ear: Cultures for anaerobic bacteria. Ann Oral Rhinol Laryngol 1980;83(suppl):102-103 30. Sipila P, Jokipii MM, Jokipii L, et ah Bacteria in the middle ear and ear canal of patients witKmon-inflammed ears. Acta Otolaryngol 1981;92:123-120 31. Brook I, Yocum P, Shah K, etah Aerobic and anaerobic bacteriological features of serous otitis media in children. Am J Otolaryngol 1983;4:389-392 32. Brook I, Finegold SM: Bacteriology of chronic etitis media. ]AMA 1979;24I:487-488 33. Finegold SM: Anaerobic Bacteria in Human Disease. New York, Academic Press, 1977 34. Fulghum RS, Daniel RJ, Yarborough JG: Anaerobic bacteria in otitis media. Ann Otolaryngol 1977;80:96-203 35. Karma P, Jokipii L, Ojala K, et al: Bacteriology of the chronically discharging middle ear. Acta Otolaryngol 1978;86:110--114 36. Aygagari A, Pancholi VK, Pandi SC, at al: Anaerobic bacteria in chronic suppurative otitis media. Indian J Mad Res 1981;73:869-864 37. Sugita R, Kawamura S, Ichikawa C, etah Studies of anaerobic bacteria in chronic otitis media. Laryngoscope 1981;9:816-821 38. Sweeney G, Picozzi GL, Browning GG: A quantitative study of aerobic and anaerobic bacteria in chronic suppurative otitis media. J Infect 1982;5:47-55 39. Constable L, Butler I: Microbial flora in chronic otitis media, J Infect 1982;5:57-60 40. Brookh Chranic attila media in children: Microbiological studies. Am ] Dis Child 1980;134:580-564 41. Brook I; Prevalence of beta-lactamase-producingbacteria in chronic otitis media, Am J Dis Child 1985;139',280-283 42. Brook I: Aerobic and anaerobic bacteriology of cholesteatoma. Laryngoscope 1981;91:250-253 43. Iino Y, Hoshimi E, Tomioka S, etah Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac, Ann Oral Rhinol Laryngl 1983;92:91-96 44, Brook I: Aerobic and anaerobic bacteriology of chronic mastoiditis in children. Am J Dis Child 1981;135',478-479 45. Brook I: Bacteriology of intracranial abscess in children, J Neurosurg 1981;54:484-488 46. Liu YS, Lim DJ, Lang R, et al: Microorganisms in chronic otitis media with effusion, Ann Otol Rhinol Laryngol 1976;85',245-249 47. Fernandez C, Lindsay ]R, Moskowitz M: Some observation on the pathagenesis of the middle ear cholesteatoma. Arch_ Otolaryngol 1952;69:537-548 48, Juers AL: Cholesteatoma genesis. Arch Otolaryngol 1965;81:5-8 49. Brook h Bacteriology and treatment of chronic otitis media in children. Laryngoscope 1979;89:1129-1139 50. Kenna MA, Bluestone CD, Reilly JS, etah Medical management of chronic suppruative atitis media without cholesteatoma i n children. Laryngoscope 1980;96:148-151 51. Brook i, Calhoun L, Yocum P: Beta-lactamase-producing isolates of Bacteraides species of children. Antimicrob Agents Chemother 1980;18:164-166 52. Brook I, Pazzaglia G, Coolbaugh IC, etah In viva protection of group A beta hemolytic streptococci from penicillin by beta-lactamase-producing Bacteroides species, I Antimicrab Chemother 1983;12:599-696 53. Brook h The role of beta-lactamase-producingbacteria in the persistence of streptococcal tonsillar infection, Roy Infect Dis 1984;6',601-607 54. Simon HI, Sukai W: Staphylococcal antagonism to penicillin-G therapy of hemolytic streptococcal pharyn-
55.
56,
57,
58, 59, 60. 61, 62. 63.
64, 65. 66. 67. 68.
69,
70. 71. 72.
73. 74, 75, 76,
gem infection: Effect of axacillin. Pediatrics 1963;31:463--469 Brook I, Yokum P: In vitro protection of group A beta hemolytic streptococci from penicillin and cephalathin by Bacteroides fragilis. Chemotherapy 1983;29:18-23 Hackman AS, Wilkins TD: Influence of penicillinase production by strains of Bocteroides melaninogenicus and Bacteraides oralis on penicillin therapy of an experimental mixed anaerobic infection in mice. Arch Oral Biol 1976;21:385-389 Heimdahl A, Van Konow L, Nard CE: Isolation of betalactamase-producing Bacteroides strains associated with clinical failures with penicillin treatment of human orofacial infection. Arch Oral Biol 1980;25:689-692 Brook I: Beta-lactamase-producing bacteria recovered after clinical failures with various penicillin therapy. Arch Otolaryngyol 1984;110;228-231 Meleney FL: Bacterial synergy in disease processes. Ann Surg 1931;22:961-981 Alternator WA: The pathogenicity of the bacteria of appendicitis. Surgery. 1942;11:374-378 Meleney F, Olpp S, Harvey HD, et el', Peritonitis, [[. Synergism of bacteria commonly found in peritoneal exudates. Arch Surg 1932;25:709-721 Hire KE, Locke M, Hesseltine HC: Synergism in experimental infections with nonsporulating anaerobic bacteria, J Infect Dis 1949;84:1-9 Brook I, Hunter V, Walker RI: Synergistic effects of anaerobic cocci, Bacteroides, ClostridiG Fusobacteria, and aerobic bacteria on mouse mortality and induction of subcutaneous abscess, ] infect Dis 1984;149:924-928 Brook h Enhancement of growth of aerobic and fecultatire bacteria in mixed infections with Bocteroids sp Infect Immunol 1985;50:929-931 Inhgam HR, Tharag0nnet D, Sisson PR, etah Inhibition of phagocytosis in vitro by obligate anaerobes. Lancet 1977;1:252-254 Lay M, Krudell KC, Milford AF: Succinate as a growth factor for Baeteroides melaninogenicus. J Bacterial 1971;108:175-178 Mergenhagen SE, Thonard ]C, Scherp HW: Studies on synergistic infections. I. Experimental infections with anaerobic streptococci. J Infect Dis 1958;i03:a3-44 Onderdonk AB, Cisneros DL, Bartlett JG: The capsular palysaccharide of Bacteroides fragilis as a virulence factor: Comparison to the pathogenic potential of encapsulated strains. J Infect Dis 1977:136:82-89 Simon GL, Klempnar MS, Kasper DL, at ah Alterations in opsonophagocytic killing by neutrophils of Bacteraides fragilis associated with animal and laboratory passage: Effect of capsular polysaccharide. J Infect Dis 1082;145:72--79 Okuda K, Takazoe I: Antiphagocytic effects of the capsular structure of a pathogenic strain of Bacteraides melaninogenicus. Bull Tokyo Den Coil 1973;14:99-104 Brook I, Walker R[: Infectivity of organisms recovered from polymicrabial abscesses. Infect Immunol 1983;41:986-989 Brook I, Gillmore ]D, Coolbaugh JC, et al: Pathogenicity of encapsulated Bacteroides melaninogenlcus group, Bacteroides oralis and Bacteroides ruminieola in abscesses in mice. J Infect 1983;7',218-226 Brook I, Walker RI: Significance of encapsulated Bacteraides meloninogenicus and Bacteroidesfragilis groups in mixed infections. Infect Immunol 1984;44:12-15 Brook I, Walker Rh Pathogenicity of anaerobic gram-positive cocci. Infect Immunol 1984;45:320-326 Brook I: Encapsulated anaerobic bacteria in upper respiratory tract infections. J Mad Microb 1986;22:171-174 Brook I, Gober AE: Baeteroides melaninogenicus: Its recovery from tonsils of children with acute tonsillitis. Arch Otolaryngol 1984;109;818-820
Volume 8 Number 2 March 1987 117
8:10g--117, 1987
The Role of Anaerobic Bacteria in Otitis Media: Microbiology, Pathogenesis, and Implications on Therapy ITZttAK BROOK, MD, MSc The current knowledge of the recovery of anaerobic bacteria from cases of acute and chronic otitis media is reviewed. Although techniques for cultivation of anaerobes were used in the studies presented, the methodologies of collection, transportation, and cultivation of the specimens varied. Consequently, there were differences in the rates of recovery of these organisms and the species isolated. Anaerobes, mainly gram-positive cocci, were recovered from a quarter of ear aspirates in acute otitis media in about one third mixed with aerobic and facultative bacteria. In a study of otitis media with effusion, anaerobic bacteria were recovered in 12% of the culture-positive aspirates. The predominant anaerobes were gram-positive cocci and Bacteroides melaninogenicus. Several studies reported the recovery of anaerobes in about half of the patients with chronic otitis media and in those with cholesteatoma. The predominant anaerobes were gram-positive cocci, Bacteroides sp, and Fusobacteria sp. Many of these organisms produced ~-Iactamase and might have contributed to the failure of these patients to respond to penicillins. The potential synergistic relationships between the various aerobic and anaerobic bacteria and the role of the capsule of anaerobic bacteria as a virulence factor are discussed.
Otitis media frequently occurs in childhood and may cause serious morbidity, such as permanent ear damage, decreases in hearing, and sometimes serious sequelae such as extension of the infection to the intracranial space. Recent developments in microbiologic methodologies have increased the awareness of the important role of anaerobic bacteria in ear infections. The predominance of anaerobic organisms in the oral flora, where they outnumber the aerobic bacteria by a ratio of 10 to one, explains their involvement in otitis media. Recognition of the role of anaerobic organisms in these infections has important implications for their management, since some of these organisms are resistant to various antimicrobials.
The recovery of anaerobic bacteria depends on the utilization of proper and adequate techniques of collection, transportation and inoculation of the specimens? In collection of specimens through the ear canal, effort should be made to avoid the normal flora at that site by prior cleansing and sterilization of the external ear canal. The exposure of specimen to oxygen should be reduced by using minimal suctioning, preferably by a syringe and needle, and avoiding forceful and constant negativepressure suctioning such as when Senturia ear specimen collection is used. Transportation to the laboratory should be prompt, and if delayed, adequate transport media should be used to insure anaerobic environment. After expulsion of the air bubbles a corked syringe and needle is the simplest mode of transportation of liquid specimens. Swabs may be placed in prereduced tubes or in special commercially available transport media. Specimens should be inoculated into prereduced culture media that are supportive for the growth of anaerobic organisms and inoculated in an anaerobic environment for sufficient time to allow the growth of fastidious organisms.
Received June 27, 1986, from the Naval MedicalResearch Institute, National Naval Medical Center, Bethesda, Maryland. Acceptedfor publication October 16, 1986. The opinions and assertions contained herein are the private ones of the writer and are not to be construedas official or reflectingthe views of the Navy Department or the Naval Service at large. Address reprint requests to Dr. Brook: Armed Forces RadiobiologyResearch Institute,Bethesda, MD 20814. 109
ANAEROBES IN OTITIS MEDIA
This review summarizes the recent developments in the understanding of the role of anaerobic bacteria in acute and chronic otitis media and discusses the synergy between these organisms and their aerobic and facultative counterparts, and the importance of encapsulation of these pathogens as a virulence factor. ACUTE OTITIS MEDIA
Acute otitis media (AOM) is one of the most common diseases of early childhood. 2 In many cases, recurrent AOM is followed by chronic inflammation.
Microbiology
America n
Journal of Otolaryngology 110
Streptococcus pneumoniae and Haemophilus influenzae are the principal etiological agents in AOM2 -7 Of the two, S pneumoniae was recovered more often irrespective of age, and its predominance increases with age. H influenzae, an infrequent cause of AOM in older children, shows increased resistance to ampicillin. Other causative agents include group A [3-hemolytic streptococcus (GABHS), Branhamella catarrhalis, Staphylococcus aureus, and gram-negative bacilli. 7 Gram-negative bacilli and staphylococci are generally implicated in AOM in neonates. However, S pneumoniae and H influenzae are the most common etiologic agents among neonates. In most past studies, microorganisms were recovered in about two thirds of aspirates, while the other third generally showed no growth. 3-6 However, no attempts were made in these studies to culture for anaerobic bacteria by proper and prompt transportation of the specimens, by inoculation into adequate media, and by incubation in anaerobic environment. Two recent studies 8'~ used techniques for cultivation of anaerobic as well as aerobic bacteria. In the larger of these, g 186 children with AOM were studied using a technique that minimized the exposure of the aspirate to oxygen./~ Aerobic bacteria alone, predominantly S pneumoniae and H influenzae, were isolated from 118 (63%) patients, and anaerobic organisms alone, most anaerobic cocci, from 24 (13%1 patients. Twenty-six (14%) aspirates yielded mixtures of aerobes and anaerobes, and several had multiple aerobic or anaerobic organisms. Anaerobes were recovered from a total of 50 (27%) of the patients. No bacterial growth was noted in 18 (9.7%) patients, a rate lower than
that obtained in previous studies in which techniques for recovery of anaerobes were not used2 -71 The predominant aerobic organisms included S pneumoniae (62 patients, 37%), H influenzae (52 patients, 30%), and S aureus (15 patients, 8%). The predominant anaerobes were Peptostreptococcus sp (39 patients, 21%) recovered alone in 15 cases and mixed with other bacteria, in the rest of the cases. Propionibacterium acnes were identified in 12 patients (7%). Other anaerobic organisms were one of each of Veillonella sp, Bifidobacterium sp, Eubacterium sp, Clostridium ramosum, and microaerophilic streptococci. Since antisepsis of the external auditory canal prior to tympanocentesis was not done in these studies, some of the anaerobic species may represent contamination of the middle ear aspirate by the external ear canal flora and the tympanic membrane surface. However, this may not be the case in the majority of the cases, since gram-stain preparation of the ear aspirates conformed with the recovered anaerobic organisms in most cases. In another study employing techniques for cultivation of anaerobes, the external ear canal and the tympanic membrane surface were sterilized prior to myringotomy. 11 Three anaerobes were recovered from the 28 studied infants: two isolates of Clostridium sp and one isolate of Peptococcus magnus. The isolation of anaerobic bacteria from the middle ear even after antisepsis of the external auditory canal suggests that these bacteria occasionally may play a direct or ancillary role in the pathogenesis of AOM. Additional support for the role of anaerobic bacteria came from a study of the bacterial flora of the external auditory canal of 72 healthy children. 12 The most common aerobic isolates were Staphylococcus epidermidis, a-hemolytic streptococcus, and Pseudomonas aeruginosa, The two anaerobic species recovered were P acnes (13 isolates) and Peptostreptococcus sp (two isolates). Thus, Peptostreptococcus sp recovered from 21% of middle ear aspirates obtained from children with AOM 1~ was isolated from only 3% of external ear canal specimens. The differences in the isolation rate of this organism in the external ear canal and the middle ear further supports its possible role in acute and chronic otitis media. P acnes, on the other hand, is a component of the skin flora and is a rare pathogen. 1 However, the rate of isolation of this organism from the external ear canal was 18%, which is higher than its recovery rate from middle ear aspirates in in-
BROOK
fected patients, which was 7% in acute and 6% in chronic otitis media. Because of the lack of a large-scale study where proper sterilization of the ear canal has been performed, the role of anaerobes in AOM has to be further substantiated.
Antimicrobial Therapy Because the most common offending organisms are S pneumoniae and H influenzae, most patients respond favorably to ampicillin or amoxicillin. In patients who are allergic to penicillin, erythromycin and/or sulfa drugs may be prescribed. ~ Cefaclor also has been effective in the treatment of this disease, ~3 mostly because of its anti-Haemophilus activity. Combination therapy has also gained popularity, largely because of the growing number of ampicillin-resistant H influenzae. The combinations successfully used are penicillin and sulfonamide, ~4 trimethoprimsulfamethoxazole, 15 erythromycin-sulfisoxazole, 16 and, most recently, amoxicillin and clavulanic acid (a ~-lactamase inhibitor). ~7 Because the anaerobes recovered in AOM are susceptible to penicillins and other antimicrobials commonly used to treat AOM, no change in the recommended therapy is advocated. However, the combination of trimethoprim-sulfamethoxazole is effective against only 50% of anaerobic gram-positive cocci (AGPC). OTITIS MEDIA WITH EFFUSION Otitis media with effusion (OME) is a common cause of mild hearing loss in children, most often between the ages of 2 and 7 years. Persistent middle ear effusion was found for at least 1 month in up to 40% of children who had suffered from AOM and for at least 3 months in 10% of the children. 18
Microbiology Because no organisms were recovered in persistent or chronic middle ear effusions, they were assumed for many years to be sterile. 19,2~However, in 1958 Senturia et al. 21 examined 130 patients with OME and identified bacteria in 33% of ears with serous effusions, in 25% of ears with mucoid effusions, in 51% of ears with mucopurulent effusions, and in 29% of ears with purulent effusions, In 1974 Kokko 22 cultured bacteria in 22% of ear effusions studied, Healy and Teele, 23 who investigated 57 children, reported that 35 of
96 specimens (36%) had bacteria that closely resembled those found in AOM. Riding et al. 24 studied the ear aspirates of 274 children and found that 45% of the ears contained bacteria. In 1976 Liu et al. 25 examined 102 patients with OME and found organisms in 66% of ears with serous effusions and 36% of the ears with mucoid effusions. None of these studies employed techniques for transportation and cultivation of anaerobes, and the external canal was not sterilized. Adlington and Davies 26 found no evidence supporting the role of viruses in OME. Klein and Teele, 27 however, isolated viruses in 29 of 663 patients (4.4%); 22 of the viruses were respiratory syncytial virus. Attempts to recover anaerobes in OME have been conducted since 1979. Giebink et al., 28 who studied 144 serous and mucoid effusions, recovered aerobic bacteria in 20% of the effusions. H influenzae was isolated predominantly from serous effusions and S epidermidis predominantly from mucoid samples. One effusion each yielded a virus (Herpesvirus heminis) and an anaerobe (Propionibacterium sp). However, the anaerobic techniques used were inappropriate for recovery of fastidious organisms, and only liquid medium was used for the initial culturing for anaerobes. Teele et al,, 29 w h o studied 20 serous effusions, did not recover anaerobes. The lack of recovery of anaerobes may have been due to the delay (up to 6 hours) in processing the effusions. Sipila et el., 3~who studied 110 middle ear aspirates, found aerobic bacteria in 35 (32%) of the aspirates. An anaerobic organism was recovered in one instance. Although the specimens were plated without delay, the culture media used for cultivation of anaerobes is not specified and might have been inadequate for cultivation of fastidious organisms. Brook et al. 31 recovered bacteria from 23 of 57 (41%) of their patients. Anaerobes were the only isolates in 17% of the culture-positive aspirates, and in an additional 26%, they were present mixed with aerobes. Aerobic organisms only were recovered in 13 aspirates (57%). A total of 45 bacterial isolates were recovered, accounting for two isolates per specimen (1.4 aerobes and 0.6 anaerobes). The predominant aerobic isolates were H influenzae, S aureus, and S pneumoniae. The anaerobes recovered were AGPC (six isolates), Bacteroides melaniogenicus (five isolates), and P aches (three isolates). It is of interest that the
Volume 8 Number 2 March 1987 iii
ANAEROBES IN OTITIS MEDIA
anaerobes recovered from these patients were similar to those previously recovered from children with acute 7'8 and chronic 32 otitis media. Nine ~-lactamase-producing organisms (BLPOs) were recovered from eight patients (35%). These included all five isolates of S aureus, three of the five B melaninogenicus, and one of eight H influenzae.
Antimicrobial Therapy The role of bacteria in the pathogenesis of OME is not yet clear; however, antimicrobial agents often are used in an attempt to clear the ear effusion from microorganisms. The presence of anaerobic as well as aerobic bacteria, many of which are BLPOs, in serous ear aspirates raises the question whether antibiotics effective against these BLPOs should be used. Controlled studies are needed to define the value of antimicrobial treatment in patients with OME and to clarify the role of bacteria in the pathogenesis of the inflammatory process. CHRONIC SUPPURATIVE OTITIS MEDIA AND CHOLESTEATOMA Chronic suppurative otitis media (CSOM) can be insidious, persistent, and very often destructive, with sometimes irreversible sequelae. In many cases of chronic otitis media, a cholesteatoma can develop; CSOM with cholesteatoma tends to be persistent and progressive.
Microbiology Although past studies reported the recovery of anaerobes from many cases of CSOM, aerobes (mainly S aureus and gram-negative enteric be-
cilli) were considered to be the major pathogens. 3a Several recent studies (Table 1) reaffirmed the role of anaerobes in CSOM. 34-4~ The variability in the rate of recovery of anaerobes in these studies may be due to differences in geographic locations and laboratory techniques. In several of the studies, the delays in cultivation was extensive, and the length of incubation was inadequate for anaerobic bacteria. The predominant anaerobic organisms recovered in these studies were AGPC and B melaninogenicus group. In a carefully done study, Fulghum et el. 34 recovered Peptostreptococcus intermedius and P aches from four of 10 cases of COM. These authors aspirated unperforated tympanic membranes. It is not clear, however, whether they have sterilized the auditory canal prior to aspiration. In another report using proper method for isolation of anaerobes, Karma et al. 3~ obtained aspirates through perforation of the ear drum and recovered anaerobes from one third of 1.14 patients. Bacteroides sp accounted for 50% of the anaerobes (29 isolates) and AGPC for 25% (15 isolates). Aygagari et al. aa recovered anaerobes in 68 of 115 (59%) patients. Anaerobic organisms only were present in 11 (10%) patients, aerobic organisms only in 40 (35%), and mixed infection was present in 57 (50%). The predominant anaerobes were Bacteroides sp and AGPC, and the major aerobes were P aeruginosa, S aureus, and Proteus sp. The delay in inoculation of the specimens is not discussed. Sugita et al. a7 recovered anaerobes in 62 of 76 (8%) cases. This low recovery rate of anaerobes may be due to the use of a medium that did not support anaerobic growth and to unspecified delays in inoculating the specimens. Sweeney et al. 3s isolated anaerobes from 52 of
TABLE 1. Frequency of Recovery of Anaerobic and Aerobic Organisms Recovered in Chronic Supparative Otitis Media
AUTHOR
American Journal
of Otolaryngology 112
Karma at a125 Sugita et al? 7 Aygagari et al? 6 Brook 4~ S w e e n e y et al? ~ Constable and Butler 3"
No. OF CASES WHERE ANAEROBES WEIIE RECOVERED/ TOTAL NO. OF AEROBIC Bacteroides CASES COCCI SP 38/114 62/760 68/115 35/68 52/130 20/100
(33%) (8%) (59%) (51%} (44%) (20%)
15 38 33 31 7 9
29 18 43 21 54 7
Fuse-
Bacterium Clastridium
-
sP
sP
2 6 7 4 9
2 2 6 3 1
-
-
-
Staphylococcus aureus
Psuedomonas sP
OTHER GRAMNECAT~VE RoDs
32 8 22 15 33 29
16 12 29 33 25 15
37 31 32 31 86 34
BROOK
130 (44%) patients. These authors did not specify the length of delay in inoculation and admitted that the unsupplemented thioglycolate media that was used in the first 73 patients was inhibitory for anaerobic growth. Constable and Butler 39 found anaerobes in 20 of 100 (20%) aspirates. Specimens were sent in an anaerobic transport media and were processed within I hour. However, the media used was not enriched, and the plates were incubated for only 24 hours before exposure to room air. All of these factors might have reduced the number of anaerobes isolated in these studies. In none of these studies was there any attempt to differentiate between organisms that reside in the ear canal and those recovered only from the inner ear, It is possible that some of the isolates were not true pathogens but colonizers of the ear canal, Brook 4~recovered anaerobic bacteria from 51% of ear aspirates of children suffering from CSOM. The ear aspirates were inoculated immediately into enriched media supportive for the growth of anaerobes, and the media were incubated for 14 days, thus allowing the slow-growing organisms sufficient time to grow. The majority of the anaerobic isolates were AGPC, Bacteroides sp (including B fragilis and B melaninogenicus groups), and Fusobacterium nucleatum. The predominant aerobic bacteria isolated were enteric gramnegative rods (mostly P aeruginosa) and S aureus. Anaerobic isolates usually were mixed with other bacteria, and the number of isolates ranged between two and four per specimen. A comparison also was made between the bacteria recovered in the middle ear and those present in the external ear canal. Only half of the bacteria recovered from the middle ear were present also in the external auditory canal. Furthermore, external ear canal culture yielded in many cases bacteria that were not present in the middle ear. These findings demonstrate that cultures collected from the external auditory canal prior to its sterilization can be misleading. This is particularly important in relationship to P aeruginasa, which is recovered more frequently in the ear canal than in the middle ear. Although this organism is a common inhabitant of the ear canal, 11 it can be recovered also from the middle ear. Direct middle-ear aspirates through the perforation in the eardrum are therefore more reliable in establishing the bacteriology and selection of therapy for CSOM. The pathogenicity of anaerobic bacteria is supported by their higher recovery rate from the mid-
dle ear only compared with their recovery from the external canal. Thirty-eight anaerobic strains were recovered from the middle ear only, as campared with seven in the external canal. In a recent study, we evaluated the presence of BLPOs in 48 children with CSOM. 41 The ear canal was sterilized and specimens were inoculated without delay and incubated for 14 days. Eighty-three aerobic and 93 anaerobic isolates were recovered. Aerobic bacteria only were involved in 22 (46%) patients, and anaerobic organisms only in five (12%). Mixed aerobic and anaerobic isolates were recovered 21 cases (44%). Beta-lactamase-producing organisms (24 anaerobes and 16 aerobes) were recovered from 31 (65%) patients. These included all isolates each of the S aureus and B fragilis groups, 11 of 19 of the B melaninogenicus group, three of six of Baeteroides oralis, four of six of H influenzae, two of three S epidermidis, and two of four of B catarrhalis. The recovery of BLPOs is not surprising, because most of our patients received multiple courses of penicillins, which might have isolated the resistant organisms. Furthermore, we were able to detect the enzyme in 84% of the ear aspirates that contained BLPOs in excess of 104 CFU/ml. The bacteriology of cholesteatomas present in chronically infected ears provides further support for the role of anaerobes in chronic ear infection. Cholesteatoma specimens were obtained by Brook 42 from patients undergoing surgery for CSOM and cholesteatoma. All cultures were processed for aerobic and anaerobic bacteria and were obtained from surgical specimens, which excludes any possibility of contamination by skin flora. A total of 74 isolates (40 aerobes and 34 anaerobes) was recovered in 24 specimens. Aerobes alone were isolated in eight (33%) specimens, anaerobes only in four (27%), and aerobic and anaerobic bacteria in 12 (50%). Fifty isolates (27 aerobes and 23 anaerobes) were present in numbers greater than 100 CFU/gm. The most commonly isolated aerobic organisms were P aeroginisa (nine isolates), Proteus sp (seven isolates), K pneumoniae (five isolates), S aureus (five isolates), and Escherichia call (four isolates). The anaerobic bacteria most commonly isolated were AGPC (12 isolates), Baeteroides sp (12 isolates, including five B fragilis group), Clostridium sp (three isolates), and Bifidobacterium sp (three isolates). These findings indicate the polymicrobial aerobic and anaerobic bacteriology of cholesteatoma in chronically infected ears. Similar
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ANAEROBES IN OTITIS MEDIA
data were found by Iino et a l . , 43 who also detected organic volatile acids (a product of the anaerobic bacteria metabolism) in the cholesteatoma.
Pathogenesis Support for the importance of anaerobes in CSOM is p r o v i d e d by their recovery from most infectious complications of this infection. Anaerobes similar to those recovered in CSOM were recovered from 23 of 24 (96%) specimens from patients with chronic mastoiditis. 44 Anaerobes were recovered from most of the patients with intracranial abscesses complicating CSOM. 1,33,45 The high number of negative cultures of chronic suppurative middle ear effusions in certain studies m a y be due to inappropriate methods for recovery of anaerobes. In o n e s t u d y , 46 bacteria were seen on direct smears in 80% of middle ear effusions, although only 40% of the effusions yielded bacterial growth. Foul-smelling pus in the middle ear suggests the presence of anaerobic bacteria in m a n y of the patients. Cholesteatoma that a c c o m p a n y CSOM can induce the absorption of bone. Various theories attempt to explain the factors involved in the process of expansion of cholesteatoma and the collagen degradation that occurs in its vicinity. 47'48 The production of organic acids by anaerobic bacteria m a y be involved in the destructive p r o c e s s . 43 Because cholesteatoma associated with CSOM contains bacteria similar to those recovered from chronically infected ears, the cholesteatoma may serve as a nidus of the chronic infection.
Antimicrobial Therapy
American Journal of Otolaryngology 114
Attempts to treat CSOM using antimicrobial therapy alone generally are not successful. The organisms u s u a l l y treated are the aerobic isolates, mainly S aureus and the gram-negative enteric bacilli. In an o p e n study, Brook 49 used parenteral carbenicillin or clindamycin to treat CSOM. Combined therapy with gentamicin was used w h e n aerobic rods also were recovered. Although therapy was successful in only half of the patients, this s t u d y demonstrated that therapy directed against the organisms isolated from patients' effusion c o u l d eradicate the infection in many instances. Kenna et al. 5~were able to achieve an i m p r o v e m e n t in 32 of 36 (89%) patients with CSOM with parenteral antimicrobial agents and daily aural toilet. Although the authors did not
obtain cultures of anaerobic bacteria, many of the antimicrobial agents they used were effective against anaerobic bacteria. Controlled studies are needed to further evaluate the role of antimicrobial therapy with or without surgery. Until recently, most of the anaerobes recovered from respiratory tract and orofacial infections were susceptible to penicillin. The S aureus and B fragilis groups are k n o w n to resist penicillin through production of ~ lactamase. However, an alarming number of Bacteroides sp (mostly B melaninogenicus group and Bacteroides buccalis), formerly susceptible to penicillins, are currently showing increasing resistance to these drugs by virtue of production of the enzyme ~ lactamase. 51 The appearance of penicillin resistance among Bacteroides sp has important implications for chemotherapy. Such organisms can release the enzyme and degrade penicillins or cephalospotins in the area of the infection. In this way, they can protect not only themselves but also penicillin-sensitive pathogens. Penicillin therapy directed against a susceptible pathogen might be rendered ineffective by the presence of a penicillinase-producing organism. 52 Several in vitro, in vivo, and patient data provide support for this theory. 53 A 200-fold increase in resistance of GABHS to penicillin was observed when it was inoculated with S aureus, 54 and a 8,500-fold increase of the organism's resistance occurred when it was inoculated with B fragilis. ~5 The same phenomenon was demonstrated in animals. Hackman and Wilkins 56 showed that penicillin-resistant strains of Bacteroides sp protected penicillin-sensitive F nucleatum from penicillin therapy in mice. Brook et al., 52 using a subcutaneous abscess model in mice, demonstrated protection of GABHS from penicillin by ~-lactamase-producing Bacteroides sp. Two recent reports ~7,58 described five adults and 75 children (including 19 with CSOM) with clinical failures after penicillin therapy associated with the isolation of aerobic and anaerobic BLPO. Most of these patients did not respond to various penicillin therapies and may represent clinical failures due to BLPOs, The isolation of BLPOs from two thirds of chronically inflamed ears and the ability to detect the free enzyme in the ear fluid 41 raise the question of whether the treatment of CSOM with penicillins is adequate in all instances and whether therapy should be directed at the eradication of BLPOs whenever they are present. Surgical drainage is still the therapy of choice, However,
BROOK
the presence of penicillin-resistant aerobic or anaerobic bacteria may warrant also the administratior, of appropriate antimicrobial agents effective against these organisms, e. g., clindamycin, metronidazole, chloramphenicol, carbenicillin, cefoxitin, or the combination of clavulanic acid and amoxicillin or ticarcillin. This should be considered especially if the infection persists despite surgical drainage. PATHOGENIC ROLE OF ANAEROBIC ORGANISMS IN OTITIS MEDIA The polymicrobial nature of CSOM is apparent as the number of isolates in most ear aspirates varies between two and five. Several isolates in each aspirate were recovered also from up to one third of patients with AOM and OME. 8,4~ The recovery of mixed aerobic and anaerobic flora in otitis media is not unique to this infection and occurs in many other infections involving anaerobic bacteria. 1'33 The synergistic relationship between the anaerobic and the aerobic and facultative flora has been studied and provides further support for the pathogenic role of anaerobic bacteria in otitis media.
Synergy Between Anaerobic and Aerobic or Facultative Flora in Otitis Media Polymicrobic infections are known to be more pathogenic for experimental animals than those involving single organisms. 59 Altemeier 6~ showed that individual isolates recovered from mixed infection were relatively innocuous to animals, but combinations of facultative and anaerobic strains had increased virulence. Similar observations were reported by Meleney et al. 61 and Hite et al. 62 Brook et al. 6a,64 evaluated the synergistic potential between aerobic and anaerobic bacteria recovered in CSOM. Each bacterium was injected subcutaneously into mice alone or mixed with another organism, and synergistic effects were determined by observing animal mortality, abscess formation, 63 and enhancement of bacterial growth. 64 The bacteria tested included AGPC, Bacteroides sp, and Fusobacterium sp. Facultarive and anaerobic bacteria included S aureus, P aeruginosa, E coli, K pneumoniae, and P mirabilis. Bacterial synergy was demonstrated between the aerobic organisms and the Baeteroides sp and AGPC; it was especially apparent between P aeruginosa and S aureus and the anaerobes. These
findings are of particular relevance to the pathologic role of t h e s e organisms in CSOM, since the combination of P aeruginosa and AGPC was isolated from 40% of patients with CSOM, and S aureus and anaerobic cocci were recovered in 9% of these patients. 4~ The demonstration of synergy between the anaerobic and aerobic bacteria commonly recovered in ear infections further indicates their pathogenic role in these infections. Several hypotheses have b e e n proposed to explain microbial synergy. W h e n this p h e n o m e n o n occurs in mixtures of aerobic and anaerobic flora, it may be caused by protection from phagocytosis and intracellular killing, us production of essential growth factors, 6~ and lowering of oxidation-reduction potentials in host tissuesJ .7
Role of Encapsulated Anaerobes in Otitis Media An important virulence factor of anaerobes in the possession of capsule. Onderdonk et al. 68 demonstrated the pathogenicity of encapsulated B fragilis by its ability to induce abscesses alone. Simon eta]. 69 described decreased phagocytosis of the encapsulated B fragilis. Capsular material from B melaninogenieus also inhibits phagocytosis and phagocytic killing of other microorganisms in an in vitro system. 7~ We evaluated the importance of encapsulated strains recovered from 13 clinical samples 71 by subcutaneously injecting into mice each of the 35 isolates (30 anaerobes and five aerobes) alone or in all possible combinations with the other isolates recovered from the same abscess and observed their ability to induce and/or survive in a subcutaneous abscess. Most of the organisms that were encapsulated were able to cause abscesses by themselves and were recovered from the abscesses even w h e n inoculated alone. This increased virulence of encapsulated strains was found in Bacteroides sp, AGPC, Clostridium sp, and E coll. We recently s t u d i e d the importance of encapsulated Bacteroides sp and AGPC as judged by their ability to cause subcutaneous abscesses in mice, 72-74 With f e w exceptions, possession of a capsule enabled these organisms to contribute more to the infectious process than their aerobic counterparts. A l t h o u g h unencapsulated organisms did not i n d u c e abscesses, many of the isolates that contained few encapsulated organisms (less than 1%), w h e n inoculated mixed with other abscess-forming aerobic and anaerobic bacteria,
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ANAEROBES IN OTITIS MEDIA
survived in the abscess and became heavily encapsulated after 10 to 14 days. Thereafter, these heavily encapsulated isolates were able to induce abscesses when injected alone. This phenomenon may explain how nonpathogenic organisms that are part of the normal oral flora become pathogens in CSOM. The mechanisms responsible for the phenomenon is yet unknown and may be due to either genetic transformation or a process of selection. Detection of a capsule in a clinical isolate may suggest a pathogenic role for the organism in the infection. A recent study supports the importance of encapsulated anaerobic organisms in respiratory infections, including otitis media. 7a The presence of encapsulated Bacteroides sp and AGPC was investigated in 48 patients with CSOM and in the pharynx of 26 individuals without inflammation. Forty-five of the 60 (75%) isolates of the B melaninogenicus and B fragilis groups, B oralis, and AGPC were found to be encapsulated in the patients with CSOM, as compared to only 34 of 96 (35%) of the controls (P < 0.001). The recovery of encapsulated anaerobic organisms in most patients with CSOM, as well as other upper respiratory tract infections, 7a supports the pathogenic role of these organisms. Early and vigorous antimicrobial therapy, directed at both aerobic and anaerobic bacteria, may abort the infection prior to the emergence of the encapsulated bacterial strains that contribute to the chronicity of the infection, CONCLUSIONS
Recent studies pointed to the role of anaerobic bacteria in otitis media. The recovery of anaerobic bacteria depends on adequate collection, transportation and inoculation of specimens. The increased recovery rate of [Mactamase-producing organisms in these infections warrants the administration of appropriate antimicrobia] agents directed also against these organisms. The synergistic relationship between the various aerobic and anaerobic bacteria recovered from patients with otitis media and the presence of encapsulated anaerobic organisms in this infection support their pathogenic role,
American Journal of Otolaryngology 116
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1975;234:171-173 6. Schwartz REI, Rodriguez WJ, Khan WN, et al: The increasing incidence of ampicillin-resistant Haemaphi/us influenzae. A cause of otitis media. JAMA 1978;239:320-323 7. Schwartz RH, Brook I: Gram-negative rod bacteria as a cause of acute otitis media in children. Ear Nose Throat ] 1981;60:9-11 8. Brook I, Anthony BF, Finegold SM: Aerobic and anaerobic bacteriology of acute atitis media in children, J pediatr 1978;92:13-15. 9. Brook I: Otitis media in children: A prospective study of aerobic and anaerobic bacteriology. Laryngoscope 1979;89:992-997 10. Brook h A practical technique for tympamnocentesis for culturing aerobic and anaerobic bacteria. Pediatrics 1980;65:626-627 11. Brook I, Schwartz R: Anaerobic bacteria in acute otitis media. Acta Otolaryngol 1981;91:111-114 12. Brook I: Microbiological studies of the bacterial flora of the external auditory canal in children. Acta OtalaryngoI 1981;91:285-287 13. Schwartz RH, Schwartz OM: Acute atitis media: diagnosis and drug therapy. Drugs 1980;19:107-118 14. Syriapou]ou V, Sheifele D, Howie UM, etah Incidence of ampicillin-resistant Haemaphilus influenzae in otitis media. J Pediatr 1976;89:838-841 15. Schwartz RH, Rodriguez WJ, Mann R, et al: The nasapharyngeal culture in acute otitis media: A reappraisal of its usefulness. JAMA 1979;241;2170-2173 16. Howard IE, Nelson JD, Clahsen J, etal: Otitis media of infancy and early childhood: A double blind study of four treatment regimens. Am J Dis Child
1976;139:965-970 17. Odio CM, Kusmiesz H, Shelton S, e t a h Comparative treatment trial of Augmentin versus cefaclar for acute atitis media with effusion. J Pediatr 1985;75: 819-826 18. Teele DW, Klein JO, Rosner BA: Epidemialogy of otitis media in children. Ann Otol Rhinol Laryngol 1980;89 (suppl);5-6 19. Robinson JM, Nicholas HO: Catarrhal atitis media with effusion--A disease of a retropharyngeal and lympathic system. South Med ] 1951;44:777-789 20. Siirala U, Vuari M: The problem of sterile otitis media. Prac Ore Rhino Laryngol, 1956;19:159-169 21. Senturia BH, Gessert CF, Carr CD, e t a h Studies concerned with tubotympanitis. Ann Otol Rhinol Laryngol
1958;67:440.-467 22. Kokko E: Chronic secretory otitis media in children: A clinical study. Acta Otolaryngol [Suppl] 1974;327:7---44 23. Healy GB, Teele DW: The microbiology of chronic middie ear effusions in children. Laryngoscope
1977;87:1472-1478 24. Riding KH, Bluestone CD, Michaels RH, et ah Microbiology of chronic and recurrent otitis media with effusion. J Pediatr 1978;93:739-743 25. Liu Y, Lira D, Lang R, et al: Chronic middle ear effusions. Immunochemical and bacterial investigation. Arch Otolaryngol 1976; 101:278-286 26. Adlington P, Davies JE: Virus studies in secretory otitis media. J Laryngol Oral 1969;83:161-173 27. Klein JO, Teele DW: Isolation of viruses and mycoplasmas from middle ear effusion', A review. Ann Otol Rhinol Laryngol 1976;85(suppl 25}:140-144
BROOK 28. Giebink GS, Mills L, Huff JS: The microbiology of serous and mucoid otitis media. Pediatrics 1979;63: 915-919 29. Teele DW, Healy GB, Tally FP: Persistent effusions of the middle ear: Cultures for anaerobic bacteria. Ann Oral Rhinol Laryngol 1980;83(suppl):102-103 30. Sipila P, Jokipii MM, Jokipii L, et ah Bacteria in the middle ear and ear canal of patients witKmon-inflammed ears. Acta Otolaryngol 1981;92:123-120 31. Brook I, Yocum P, Shah K, etah Aerobic and anaerobic bacteriological features of serous otitis media in children. Am J Otolaryngol 1983;4:389-392 32. Brook I, Finegold SM: Bacteriology of chronic etitis media. ]AMA 1979;24I:487-488 33. Finegold SM: Anaerobic Bacteria in Human Disease. New York, Academic Press, 1977 34. Fulghum RS, Daniel RJ, Yarborough JG: Anaerobic bacteria in otitis media. Ann Otolaryngol 1977;80:96-203 35. Karma P, Jokipii L, Ojala K, et al: Bacteriology of the chronically discharging middle ear. Acta Otolaryngol 1978;86:110--114 36. Aygagari A, Pancholi VK, Pandi SC, at al: Anaerobic bacteria in chronic suppurative otitis media. Indian J Mad Res 1981;73:869-864 37. Sugita R, Kawamura S, Ichikawa C, etah Studies of anaerobic bacteria in chronic otitis media. Laryngoscope 1981;9:816-821 38. Sweeney G, Picozzi GL, Browning GG: A quantitative study of aerobic and anaerobic bacteria in chronic suppurative otitis media. J Infect 1982;5:47-55 39. Constable L, Butler I: Microbial flora in chronic otitis media, J Infect 1982;5:57-60 40. Brookh Chranic attila media in children: Microbiological studies. Am ] Dis Child 1980;134:580-564 41. Brook I; Prevalence of beta-lactamase-producingbacteria in chronic otitis media, Am J Dis Child 1985;139',280-283 42. Brook I: Aerobic and anaerobic bacteriology of cholesteatoma. Laryngoscope 1981;91:250-253 43. Iino Y, Hoshimi E, Tomioka S, etah Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac, Ann Oral Rhinol Laryngl 1983;92:91-96 44, Brook I: Aerobic and anaerobic bacteriology of chronic mastoiditis in children. Am J Dis Child 1981;135',478-479 45. Brook I: Bacteriology of intracranial abscess in children, J Neurosurg 1981;54:484-488 46. Liu YS, Lim DJ, Lang R, et al: Microorganisms in chronic otitis media with effusion, Ann Otol Rhinol Laryngol 1976;85',245-249 47. Fernandez C, Lindsay ]R, Moskowitz M: Some observation on the pathagenesis of the middle ear cholesteatoma. Arch_ Otolaryngol 1952;69:537-548 48, Juers AL: Cholesteatoma genesis. Arch Otolaryngol 1965;81:5-8 49. Brook h Bacteriology and treatment of chronic otitis media in children. Laryngoscope 1979;89:1129-1139 50. Kenna MA, Bluestone CD, Reilly JS, etah Medical management of chronic suppruative atitis media without cholesteatoma i n children. Laryngoscope 1980;96:148-151 51. Brook i, Calhoun L, Yocum P: Beta-lactamase-producing isolates of Bacteraides species of children. Antimicrob Agents Chemother 1980;18:164-166 52. Brook I, Pazzaglia G, Coolbaugh IC, etah In viva protection of group A beta hemolytic streptococci from penicillin by beta-lactamase-producing Bacteroides species, I Antimicrab Chemother 1983;12:599-696 53. Brook h The role of beta-lactamase-producingbacteria in the persistence of streptococcal tonsillar infection, Roy Infect Dis 1984;6',601-607 54. Simon HI, Sukai W: Staphylococcal antagonism to penicillin-G therapy of hemolytic streptococcal pharyn-
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gem infection: Effect of axacillin. Pediatrics 1963;31:463--469 Brook I, Yokum P: In vitro protection of group A beta hemolytic streptococci from penicillin and cephalathin by Bacteroides fragilis. Chemotherapy 1983;29:18-23 Hackman AS, Wilkins TD: Influence of penicillinase production by strains of Bocteroides melaninogenicus and Bacteraides oralis on penicillin therapy of an experimental mixed anaerobic infection in mice. Arch Oral Biol 1976;21:385-389 Heimdahl A, Van Konow L, Nard CE: Isolation of betalactamase-producing Bacteroides strains associated with clinical failures with penicillin treatment of human orofacial infection. Arch Oral Biol 1980;25:689-692 Brook I: Beta-lactamase-producing bacteria recovered after clinical failures with various penicillin therapy. Arch Otolaryngyol 1984;110;228-231 Meleney FL: Bacterial synergy in disease processes. Ann Surg 1931;22:961-981 Alternator WA: The pathogenicity of the bacteria of appendicitis. Surgery. 1942;11:374-378 Meleney F, Olpp S, Harvey HD, et el', Peritonitis, [[. Synergism of bacteria commonly found in peritoneal exudates. Arch Surg 1932;25:709-721 Hire KE, Locke M, Hesseltine HC: Synergism in experimental infections with nonsporulating anaerobic bacteria, J Infect Dis 1949;84:1-9 Brook I, Hunter V, Walker RI: Synergistic effects of anaerobic cocci, Bacteroides, ClostridiG Fusobacteria, and aerobic bacteria on mouse mortality and induction of subcutaneous abscess, ] infect Dis 1984;149:924-928 Brook h Enhancement of growth of aerobic and fecultatire bacteria in mixed infections with Bocteroids sp Infect Immunol 1985;50:929-931 Inhgam HR, Tharag0nnet D, Sisson PR, etah Inhibition of phagocytosis in vitro by obligate anaerobes. Lancet 1977;1:252-254 Lay M, Krudell KC, Milford AF: Succinate as a growth factor for Baeteroides melaninogenicus. J Bacterial 1971;108:175-178 Mergenhagen SE, Thonard ]C, Scherp HW: Studies on synergistic infections. I. Experimental infections with anaerobic streptococci. J Infect Dis 1958;i03:a3-44 Onderdonk AB, Cisneros DL, Bartlett JG: The capsular palysaccharide of Bacteroides fragilis as a virulence factor: Comparison to the pathogenic potential of encapsulated strains. J Infect Dis 1977:136:82-89 Simon GL, Klempnar MS, Kasper DL, at ah Alterations in opsonophagocytic killing by neutrophils of Bacteraides fragilis associated with animal and laboratory passage: Effect of capsular polysaccharide. J Infect Dis 1082;145:72--79 Okuda K, Takazoe I: Antiphagocytic effects of the capsular structure of a pathogenic strain of Bacteraides melaninogenicus. Bull Tokyo Den Coil 1973;14:99-104 Brook I, Walker R[: Infectivity of organisms recovered from polymicrabial abscesses. Infect Immunol 1983;41:986-989 Brook I, Gillmore ]D, Coolbaugh JC, et al: Pathogenicity of encapsulated Bacteroides melaninogenlcus group, Bacteroides oralis and Bacteroides ruminieola in abscesses in mice. J Infect 1983;7',218-226 Brook I, Walker RI: Significance of encapsulated Bacteraides meloninogenicus and Bacteroidesfragilis groups in mixed infections. Infect Immunol 1984;44:12-15 Brook I, Walker Rh Pathogenicity of anaerobic gram-positive cocci. Infect Immunol 1984;45:320-326 Brook I: Encapsulated anaerobic bacteria in upper respiratory tract infections. J Mad Microb 1986;22:171-174 Brook I, Gober AE: Baeteroides melaninogenicus: Its recovery from tonsils of children with acute tonsillitis. Arch Otolaryngol 1984;109;818-820
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