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A quantitative study of aerobic and anaerobic bacteria in chronic suppurative otitis media
Journal of Infection (i982) 5, 47-55
A quantitative study of aerobic and anaerobic bacteria in
chronic suppurative otitis media Grace Sweeney, Gerard L. P i c o z z i and George G. B r o w n i n g
Departments of Bacteriology and Otolaryngology, Royal h~firmary, Glasgow G4 oSF Summary During a period of 2I months, specimens from the ears of I3O patients with chronic suppurative otitis media ( C S O M ) were cultured qualitatively and quantitatively for aerobic and anaerobic bacteria. Bacteria were cultured from 95 per cent of these specimens. Proteus species, the most common aerobic isolate was grown from 6I per cent of the patients. Using an appropriate method it was possible to isolate anaerobic bacteria from 44 per cent of specimens and Bacteroides species predominated: B. melaninogenicusand B.fragilis were equally common. T h e main bacterial species - both aerobic and anaerobic - were present in the ear in counts which averaged lO 9 organisms per ml. Pseudornonasspecies were an exception, however, and regularly showed higher counts of IO 11 bacilli per ml. T h e high bacterial counts may be one of the reasons for the poor results of antibiotic therapy.
Introduction T h e m a n a g e m e n t o f c h r o n i c s u p p u r a t i v e otitis m e d i a ( C S O M ) is a m a j o r clinical p r o b l e m in b o t h g e n e r a l a n d h o s p i t a l p r a c t i c e . A l t h o u g h antibiotics are o f t e n p r e s c r i b e d to t r e a t t h e f o u l - s m e l l i n g d i s c h a r g e , t h e r e a p p e a r s to be n o scientific e v i d e n c e to justify t h e i r use in this c o n d i t i o n . C u l t u r e o f t h e e x u d a t e is n o t always c a r r i e d o u t b u t e v e n w h e n this is d o n e t h e r e l e v a n c e o f the results to p a t i e n t m a n a g e m e n t is u n c e r t a i n . I n a B r i t i s h s u r v e y 1 Staphylococcus aureus a n d v a r i o u s species o f G r a m - n e g a t i v e bacilli w e r e g r o w n f r e q u e n t l y f r o m s u p p u r a t i n g ears b u t c u l t u r e f o r a n a e r o b i c b a c t e r i a was n o t p e r f o r m e d . O t h e r studies 2,3.4 h a v e r e c o r d e d an i n c i d e n c e o f a n a e r o b e s in a d u l t C S O M b e t w e e n I ' I p e r c e n t a n d 33 p e r cent. A l m o s t c e r t a i n l y the i n c r e a s e in t h e p r o p o r t i o n o f s p e c i m e n s f o u n d to c o n t a i n a n a e r o b e s is d u e to r e c e n t i m p r o v e m e n t s in t h e m e t h o d s u s e d for t h e i r isolation. I t was d e c i d e d to investigate p a t i e n t s w i t h active C S O M a n d t h e b a c t e r i o l o g i c a l results are p r e s e n t e d h e r e : t h e clinical aspects will b e r e p o r t e d e l s e w h e r e ( B r o w n i n g , P i c o z z i , C a l d e r a n d S w e e n e y , in p r e p a r a t i o n ) .
Materials and methods Patients O n e h u n d r e d a n d t h i r t y p a t i e n t s w i t h C S O M w h o a t t e n d e d an o u t - p a t i e n t clinic b e t w e e n J a n u a r y I 9 7 9 a n d S e p t e m b e r 198o w e r e s t u d i e d . T h e i r h i s t o r y o f ear disease v a r i e d f r o m t w o to 50 years a n d the p a t i e n t s ' ages r a n g e d f r o m Address for correspondence: Bacteriology Department, Wolfson Building, Glasgow Royal Maternity Hospital, Glasgow G4 oNA. oi63-4453/82/040047+o9 $02.00/0
~ I982 The Brltish Society for the Study of Infection
48
G. S W E E N E Y ,
G. P I C O Z Z I
A N D G. G. B R O W N I N G
I7 to 70 years. All patients had active disease detectable b y the secretion of copious pus in association with hyperaemia and oedema of the middle ear mucosa, and none had received antimicrobial therapy, local or systemic, in the four weeks preceding their initial hospital visit. W h e n the patients attended the follow-up clinic to assess response to treatment, serial specimens were collected: the results of these specimens will be r e p o r t e d with the clinical aspects of the study. Eleven patients w h o presented with m u c o i d n o n - p u r u l e n t discharge in the presence of hyperaemia, were investigated as a control group : these patients had not been treated with antibiotics in the preceding four weeks. Specimen
collection
A loopful (I #1) of exudate was collected u n d e r direct microscopic vision t h r o u g h an aural s p e c u l u m from the most clinically active area in the ear of each patient. T h e bacteriologist (GS) was present at the clinic to make an initial dilution of the sample which was then taken to the laboratory and cultured with minimal delay. In this s t u d y specimens were r a n d o m l y selected for culture from only one ear of patients w h o presented with bilateral disease. Aerobic culture
A loopful of exudate was diluted with either p e p t o n e water or brain heart infusion b r o t h ( B H I ) and quantitative counts were made b y a modified Miles Misra method. Five plates of C o l u m b i a b l o o d agar were inoculated with the io -4, io -6, io -7, IO-s and Io -9 dilutions and in addition a single plate of C L E D agar with primary (Io -~) dilution. Anaerobic culture
D u r i n g the early part of the s t u d y (i.e. the first 73 patients) dilutions o f a loopful of exudate were m a d e in u n s u p p l e m e n t e d thioglycollate b r o t h ~,6 b u t later, it was recognised that thioglycollate was inhibitory to some anaerobes 7 and B H I b r o t h was substituted. T h e broths were steamed before making the dilutions and the counts were carried out on five plates of freshly-prepared, gentamicin b l o o d agar. After inoculation the cultures were incubated for a continuous period of 96 hours u n d e r anaerobic conditions generated b y the Gaspak System (BBL). Identification of aerobes
Standard laboratory m e t h o d s were "used. Coliform bacilli were identified b y the A P I z o E system (API L a b o r a t o r y Products). Corynebacteria were recognised b y their colonial m o r p h o l o g y and appearance on a G r a m - s t a i n e d film. Streptococci were g r o u p e d by the P h a d e b a c t m e t h o d (Pharmacia). Antibiotic sensitivity tests to I2 different drugs were carried out b y disc diffusion on isosensitest agar (Oxoid). Identification of anaerobes
Bacteria unable to grow in air with IO per cent carbon dioxide and sensitive to metronidazole were considered obligate anaerobes. 8 G r a m - n e g a t i v e anaerobic bacilli were provisionally assigned to one of three groups : Bacteroides fragilis if they grew on agar containing bile and failed to p r o d u c e pigment,
Bacteria in C S O M
49
T a b l e I Results of culture of specimens from the ears of patients with chronic
suppurative otitis media No. of patients
Culture Aerobic organisms cnly Aerobic and anaerobic organism~ No growth Total
(%) 83 41
(64) (32)
6
(5)
I3o
B. melaninogenicus if their g r o w t h was inhibited b y bile and they expressed pigment, and a heterogenous n o n - p i g m e n t e d group of unidentified Bacteroides/ Fusobacteria spp. which showed sensitivity to bile. D u r i n g the early part of the study, detailed identification of these organisms was n o t a t t e m p t e d because this information was not considered relevant to the m a n a g e m e n t of the patient. L a t e r it b e c a m e apparent that identification to species level might be of interest and an a t t e m p t was m a d e to recover and identify isolates collected from the 57 patients in the latter part of the trial. G r a m - n e g a t i v e anaerobic bacilli were cultured from 25 o f these patients and stored in s k i m m e d milk at - 2 0 °C. H o w e v e r , strains from only I7 of the 25 patients were recoverable and these were s u b s e q u e n t l y characterized b y a scheme based on biochemical and tolerance tests. 9 0 b l i g a t e l y anaerobic cocci were identified b y their sensitivity in vitro to metronidazole. 1° Results
T a b l e I shows that bacteria were cultured from ear specimens from 95 per cent of patients with chronic suppurative otitis media w h o had p u r u l e n t aural discharge. T h e aerobic organisms cultured from this group are listed in T a b l e II. Proteus spp. were b y far the c o m m o n e s t isolate (61 per cent of patients) with Proteus mirabilis the most c o m m o n single species. O f the other aerobic G r a m - n e g a t i v e bacilli isolated, Pseudomonas spp. were the most frequent. Staphylococcus aureus was cultured from 25 per cent and coagulase-negative staphylococci from 18 per cent of the patients. M a n y of the discharges contained anaerobic bacteria and during the early part of the s t u d y anaerobes were cultured from z7 of 73 patients (23 per cent). W h e n brain heart infusion was substituted for thioglycollate b r o t h as diluent there was a significant increase in the rate of isolation of anaerobes to 44 per cent (25 of 57 patients). T h e species of anaerobic bacteria cultured from patients with p u r u l e n t discharge are listed in T a b l e III. This shows that the main species isolated were B. melaninogenicus and B. fragilis. T h e results of detailed identification of the 26 anaerobes g r o w n from z 7 patients are recorded in T a b l e IV. Very large n u m b e r s of bacteria were isolated from the ears of patients with
G. SWEENEY~
5° Table
II
G. PICOZZI
AND
G. G. BROWNING
Aerobic bacteria isolated f r o m patients with chronic suppurative otitis media N o . of isolates
Proteus spp. Staphylococcus aureus Pseudomonas spp.
79 33 25 24 2I 8
C o a g u l a s e - n e g a t i v e staphylococci ' C o l i f o r m ' bacilli* Corynebacterium spp.
Escherichia coli Streptococcus faecalis Streptococcus pyogenes
7 5 I
Others
2
Total
205
* Inc•udesgeneraandspecies•faer•bicGram-negativebaci••i•therthanth•se1istedhere,e•g.K•ebsiella• Enterobacter etc. where the number of isolates was less than three: a few could not be identified by the API system.
Table
III
Anaerobic bacteria isolated f r o m patients with chronic suppurative otitis media No. of isolates
Species
B. melaninogenicus B. fragilis Bacteroides/Fusobacteria spp.
28 26 9 7 I
A n a e r o b i c cocci Clostridium spp. Total
Table
IV
71
Detailed identification of anaerobic isolates f r o m 1 7 patients
Species
B. melaninogenicus B. assacharolyticus B. fragilis B. vulgatus Fusobacterium spp. A n a e r o b i c cocci Total
No. of isolates
5 2 5 2 2 io 26
Bacteria in C S O M
5I
T a b l e V Results of culture of specimens from patients with mucoid otitis media
Culture Aerobic organisms Anaerobic organisms No growth Total
No. of patients (%) 5 (45) o (o) 6 (55) II
a p u r u l e n t discharge. Pseudomonas spp. for example, were present in mean concentrations of IO 11 organisms per ml. T h e mean counts of Proteus spp., Staph. aureus, coagulase-negative staphylococci and Bacteroides spp. were IO 9 per ml. As a rule more than one bacterial species was isolated and this was the case in 88 of the I3o patients (68 per cent). H o w e v e r , in 36 of the 83 patients (43 per cent) w h e r e culture of the specimen grew aerobes only (Table I), a single infecting species was present and Staph. aureus (I3 isolations) was more c o m m o n than Proteus species (I I isolations). In ~6 of the 4I patients (63 per cent) with b o t h aerobes and anaerobes, two or more different anaerobes were detected in the p u r u l e n t discharge. In contrast to the results in patients with suppurative otitis media anaerobes were not isolated from patients with m u c o i d discharge. Although this control group was too small to allow valid comparison, specimens from six of the I I patients studied showed no g r o w t h (Table V). T h e seven isolates of aerobic bacteria from patients in this group were three strains of coagulase-negative staphylococci and one each of Proteus spp., Klebsiella spp., Staph. aureus, and a n o n - h a e m o l y t i c streptococcus: counts averaged IO 7 organisms per ml. Anaerobic isolates are of course always sensitive to metronidazole b u t the aerobic bacteria had a variety of patterns of antibiotic sensitivity (Table VI). Discussion
A l t h o u g h it is widely accepted that bacteria play a major role in chronic suppurative otitis media the response to antimicrobial chemotherapy is notoriously poor. O u r o w n observations to be reported elsewhere (Browning, Picozzi, Calder and Sweeney, in preparation) s u p p o r t this in that 75 per cent of ears are still actively discharging after four weeks of appropriate systemic or topical antimicrobial therapy. In the study described here specimens were taken with particular attention to avoid contamination from the external ear and examined by quantitative culture in the laboratory with minimal delay. T h e r e are no previous reports in which the n u m b e r s of bacteria in the exudate have been counted. T h e results showed that, in patients with purulent aural discharge, bacteria were present at a concentration which averaged I O 9 organisms per ml. As previous workers have reported 5,6 Gram-negative bacilli were f o u n d to dominate the aerobic bacterial flora, although where only
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Bacteria in C S O M
53
a single species was present, Staph. aureus was the most common pathogen. However, in this study Proteus spp. (mostly Proteus mirabilis) were isolated more often than Pseudomonas spp. When present, Pseudomonas spp. were generally found in greater numbers than the other bacteria and counts of this genus averaged Io 11 organisms per ml. T h e high counts obtained with all bacterial species may have contributed to the disappointing results of antimicrobial therapy since it has been separately reported that Staphylococcus aureus in skin lesions in counts of more than ~o6 organisms per cm 2 impaired the healing process. 12 In agreement with previous investigations by the Finnish group 3,4 anaerobes were never isolated alone but only as part of a mixed aerobic-anaerobic flora. T h e fact that anaerobic bacteria are often present may account for the foetid discharge characteristic of chronic otitis media. T h e results also showed that anaerobic bacteria, almost all of which were Bacteroides spp. were p r e s e n t also in large n u m b e r s - in up to 44 per cent of the patients. This is higher than reported by the Finnish workers 3,11who found anaerobic bacteria in 33 per cent of their patients. On the other hand investigators in the U.S.A. 6'13 have reported an incidence of anaerobes of 5o and 66 per cent respectively, in two studies on a series of children with otitis media. In contrast to our investigation however, their patients were predominantly children with intact tympanic membranes. T h e majority of the anaerobic isolates reported by the American workers were peptococci and other Gram-positive anaerobic bacteria quite different from those cultured by us from adults with perforated ear drums in which Bacteroides spp. were by far the most frequent anaerobic organisms. In this investigation B. melaninogenicus and B. fragilis were equally common. This finding is of some interest, since although B. melaninogenicus is almost invariably present in the normal flora of the oropharynx, B. fragilis is relatively rare. T M T h e frequency of B. fragilis in chronic ear infection probably reflects its greater pathogenic potential. Bacteroides spp. were reported to inhibit the phagocytosis in vitro of coliform b a c i l l i - particularly Proteus spp. 1~ Anaerobes may play a similar role in vivo and in protecting aerobic organisms from host defence mechanisms in conditions like C S O M , contribute to the unsatisfactory results of treatment. Mixed infections may also give rise to a more purulent response than infection with aerobes alone. This 'pathological synergy' has been demonstrated in guineapigs in which Escherichia coli and B. fragilis were inoculated separately and together into freshly made superficial incisions. 16 When inoculated together, mixtures of Io 4 E. coli and IO4 B.fragilis produced copious pus whereas inocula containing the same total n u m b e r of cells of each organism alone did not. In C S O M where aerobes and anaerobes co-exist, each at concentrations considerably greater than the threshold levels of ro ~ and IO~ bacterial per ml, the characteristic formation of pus with the rapid destruction of tissue may represent manifestation in vivo of this synergism. T h e bacteria in the ears of patients with purulent disease may have gained entry to the middle ear from the oropharynx. However, the predominance of aerobic Gram-negative bacilli in the condition is perhaps surprising since these are rare inhabitants of the oropharynx; the alternative route of infection in patients who have perforated drums is via the external ear.
54
G. SWEENEY~ G. PICOZZI AND G. G. BROWNING
Antibiotic sensitivity tests showed that the topical aminoglycosides gentamicin and n e o m y c i n were active in vitro against the principal aerobic isolates. C h l o r a m p h e n i c o l w h i c h is often used topically and the systemic antibiotic cephalexin also a p p e a r e d effective on disc testing against aerobes with the e x c e p t i o n o f Pseudomonas spp. As m i g h t be e x p e c t e d the majority of strains of S t a p h . aureus expressed fl-lactamase. T h e results o f this bacteriological investigation o f c h r o n i c s u p p u r a t i v e otitis media do not allow conclusions to be d r a w n a b o u t the p a t h o g e n i c role o f the p r o f u s e bacterial flora w h i c h was d e m o n s t r a t e d in almost all the patients. T h e n o t o r i o u s difficulty in treating the condition m i g h t m e a n that bacteria were s e c o n d a r y invaders or m e r e l y passengers in a c o m p l e x pathological process largely d e t e r m i n e d by o t h e r factors. Alternatively, systemic or topical antibiotic t h e r a p y a l t h o u g h in w i d e s p r e a d use, m a y not be able to reach the sites of infection due to tissue d e s t r u c t i o n and the collection o f p u r u l e n t discharge in the meatus and the b o n y tissues s u r r o u n d i n g the m i d d l e ear. A l t h o u g h the p r e s e n c e of m e t r o n i d a z o l e was d e m o n s t r a t e d in tissue taken frotn the m i d d l e ear o f patients who had been given the d r u g p r e - o p e r a t i v e l y , 4 in o u r study antibiotic activity was not detected in pus collected f r o m patients receiving oral cephalexin. T h i s finding m a y be due either to lack of d r u g p e n e t r a t i o n or to cephalexin d e s t r u c t i o n by fl-lactarnases of the G r a m - n e g a t i v e bacteria in the exudate. T h e observation that high counts o f recognised p a t h o g e n s correlate with p u r u l e n t discharge suggests that the bacteria p r e s e n t have almost certainly c o n t r i b u t e d to the pathological process. (We are indebted to the consultants of the Department of Otolaryngology, Glasgow Royal Infirmary, for allowing their patients to be included in this study.)
References
I. Rees T. Bacteria from the chronic ear. Br J Clin Pract I97I; 25: 453-454. 2. Palva T, Karja J, Palva A. Bacterial analyses in chronic otitis media. ORL Digest I97I ; 33: 19-26. 3. Jokipii AMM, Karma P, Ojala J, Jokipii L. Anaerobic bacteria in chronic otitis media. Arch Otolaryngol I977; *o3: 278-~8o. 4- Jokippi L, Karma P, Jokippi AMM. Access of metronidazole into the chronically inflamed middle ear with reference to anaerobic bacterial infections. Arch Otorhinolaryngol 1978; 220 : 167-174. 5- Palva T, Hallstrom O. Bacteriology of chronic otitis media. Arch Otolaryngol 1965; 82: 359-364. 6. Brook I, Finegold SM. Bacteriology of chronic otitis media. J A M A I979; 241 : 487-488. 7. Sutter VL, Citron CM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. London : CV Mosby, I98O: 81. 8. Prince HN, Gruneberg E, Titsworth E, de Lorenzo WF. Effects of I-(2 Nitro- I-imidazolyl)3-methoxy-2-propanol and 2-methyl-5-nitroimidazole-I-ethanol against anaerobic and aerobic bacteria and protozoa. Appl Microbiol 1969; I8: 728-730. 9. Duerden B. The isolation and identification of Bacteroides spp. from the normal human gingival flora. J Med Microbiol I98O; *3: 89-IOI. I o. Watt B, Jack EP. What are anaerobic cocci? J Med Microbiol I971 ; I o: 461-468. I I. Karma P, Jokipii L, Ojala K, Jokipii AMM. Bacteriology of the chronically discharging middle ear. Acta Otolaryngol I978; 86 : I io-, I4. Iz. Kligman AM, Leyden JJ, McGinley KJ. Bacteriology. J Invest Dermatol I976; 67 : 16o-I68.
B a c t e r i a in C S O M
55
13. Brook I. Anaerobic isolates in chronic recurrent suppurative otitis media. Infection 1979; 7:247-251-
14. Bartlett JG, Gorbach SL. Anaerobic infections of the head and neck. Otolaryngol Clin North Am 1976; 9:655-678. 15. Ingham HR, Sisson PR, Tharagonnet D, Selkon JB, Codd AA. Inhibition of phagocytosis in vitro by obligate anaerobes. Lancet 1977; 2: 1252-1254. 16. Kelly MJ. The quantitative and histological demonstration of pathogenic synergy between Escherichia coff and Bacteroides fragilis in guinea-pig wounds. J Med Microbiol 1978 ; I I : 513-523.
A quantitative study of aerobic and anaerobic bacteria in
chronic suppurative otitis media Grace Sweeney, Gerard L. P i c o z z i and George G. B r o w n i n g
Departments of Bacteriology and Otolaryngology, Royal h~firmary, Glasgow G4 oSF Summary During a period of 2I months, specimens from the ears of I3O patients with chronic suppurative otitis media ( C S O M ) were cultured qualitatively and quantitatively for aerobic and anaerobic bacteria. Bacteria were cultured from 95 per cent of these specimens. Proteus species, the most common aerobic isolate was grown from 6I per cent of the patients. Using an appropriate method it was possible to isolate anaerobic bacteria from 44 per cent of specimens and Bacteroides species predominated: B. melaninogenicusand B.fragilis were equally common. T h e main bacterial species - both aerobic and anaerobic - were present in the ear in counts which averaged lO 9 organisms per ml. Pseudornonasspecies were an exception, however, and regularly showed higher counts of IO 11 bacilli per ml. T h e high bacterial counts may be one of the reasons for the poor results of antibiotic therapy.
Introduction T h e m a n a g e m e n t o f c h r o n i c s u p p u r a t i v e otitis m e d i a ( C S O M ) is a m a j o r clinical p r o b l e m in b o t h g e n e r a l a n d h o s p i t a l p r a c t i c e . A l t h o u g h antibiotics are o f t e n p r e s c r i b e d to t r e a t t h e f o u l - s m e l l i n g d i s c h a r g e , t h e r e a p p e a r s to be n o scientific e v i d e n c e to justify t h e i r use in this c o n d i t i o n . C u l t u r e o f t h e e x u d a t e is n o t always c a r r i e d o u t b u t e v e n w h e n this is d o n e t h e r e l e v a n c e o f the results to p a t i e n t m a n a g e m e n t is u n c e r t a i n . I n a B r i t i s h s u r v e y 1 Staphylococcus aureus a n d v a r i o u s species o f G r a m - n e g a t i v e bacilli w e r e g r o w n f r e q u e n t l y f r o m s u p p u r a t i n g ears b u t c u l t u r e f o r a n a e r o b i c b a c t e r i a was n o t p e r f o r m e d . O t h e r studies 2,3.4 h a v e r e c o r d e d an i n c i d e n c e o f a n a e r o b e s in a d u l t C S O M b e t w e e n I ' I p e r c e n t a n d 33 p e r cent. A l m o s t c e r t a i n l y the i n c r e a s e in t h e p r o p o r t i o n o f s p e c i m e n s f o u n d to c o n t a i n a n a e r o b e s is d u e to r e c e n t i m p r o v e m e n t s in t h e m e t h o d s u s e d for t h e i r isolation. I t was d e c i d e d to investigate p a t i e n t s w i t h active C S O M a n d t h e b a c t e r i o l o g i c a l results are p r e s e n t e d h e r e : t h e clinical aspects will b e r e p o r t e d e l s e w h e r e ( B r o w n i n g , P i c o z z i , C a l d e r a n d S w e e n e y , in p r e p a r a t i o n ) .
Materials and methods Patients O n e h u n d r e d a n d t h i r t y p a t i e n t s w i t h C S O M w h o a t t e n d e d an o u t - p a t i e n t clinic b e t w e e n J a n u a r y I 9 7 9 a n d S e p t e m b e r 198o w e r e s t u d i e d . T h e i r h i s t o r y o f ear disease v a r i e d f r o m t w o to 50 years a n d the p a t i e n t s ' ages r a n g e d f r o m Address for correspondence: Bacteriology Department, Wolfson Building, Glasgow Royal Maternity Hospital, Glasgow G4 oNA. oi63-4453/82/040047+o9 $02.00/0
~ I982 The Brltish Society for the Study of Infection
48
G. S W E E N E Y ,
G. P I C O Z Z I
A N D G. G. B R O W N I N G
I7 to 70 years. All patients had active disease detectable b y the secretion of copious pus in association with hyperaemia and oedema of the middle ear mucosa, and none had received antimicrobial therapy, local or systemic, in the four weeks preceding their initial hospital visit. W h e n the patients attended the follow-up clinic to assess response to treatment, serial specimens were collected: the results of these specimens will be r e p o r t e d with the clinical aspects of the study. Eleven patients w h o presented with m u c o i d n o n - p u r u l e n t discharge in the presence of hyperaemia, were investigated as a control group : these patients had not been treated with antibiotics in the preceding four weeks. Specimen
collection
A loopful (I #1) of exudate was collected u n d e r direct microscopic vision t h r o u g h an aural s p e c u l u m from the most clinically active area in the ear of each patient. T h e bacteriologist (GS) was present at the clinic to make an initial dilution of the sample which was then taken to the laboratory and cultured with minimal delay. In this s t u d y specimens were r a n d o m l y selected for culture from only one ear of patients w h o presented with bilateral disease. Aerobic culture
A loopful of exudate was diluted with either p e p t o n e water or brain heart infusion b r o t h ( B H I ) and quantitative counts were made b y a modified Miles Misra method. Five plates of C o l u m b i a b l o o d agar were inoculated with the io -4, io -6, io -7, IO-s and Io -9 dilutions and in addition a single plate of C L E D agar with primary (Io -~) dilution. Anaerobic culture
D u r i n g the early part of the s t u d y (i.e. the first 73 patients) dilutions o f a loopful of exudate were m a d e in u n s u p p l e m e n t e d thioglycollate b r o t h ~,6 b u t later, it was recognised that thioglycollate was inhibitory to some anaerobes 7 and B H I b r o t h was substituted. T h e broths were steamed before making the dilutions and the counts were carried out on five plates of freshly-prepared, gentamicin b l o o d agar. After inoculation the cultures were incubated for a continuous period of 96 hours u n d e r anaerobic conditions generated b y the Gaspak System (BBL). Identification of aerobes
Standard laboratory m e t h o d s were "used. Coliform bacilli were identified b y the A P I z o E system (API L a b o r a t o r y Products). Corynebacteria were recognised b y their colonial m o r p h o l o g y and appearance on a G r a m - s t a i n e d film. Streptococci were g r o u p e d by the P h a d e b a c t m e t h o d (Pharmacia). Antibiotic sensitivity tests to I2 different drugs were carried out b y disc diffusion on isosensitest agar (Oxoid). Identification of anaerobes
Bacteria unable to grow in air with IO per cent carbon dioxide and sensitive to metronidazole were considered obligate anaerobes. 8 G r a m - n e g a t i v e anaerobic bacilli were provisionally assigned to one of three groups : Bacteroides fragilis if they grew on agar containing bile and failed to p r o d u c e pigment,
Bacteria in C S O M
49
T a b l e I Results of culture of specimens from the ears of patients with chronic
suppurative otitis media No. of patients
Culture Aerobic organisms cnly Aerobic and anaerobic organism~ No growth Total
(%) 83 41
(64) (32)
6
(5)
I3o
B. melaninogenicus if their g r o w t h was inhibited b y bile and they expressed pigment, and a heterogenous n o n - p i g m e n t e d group of unidentified Bacteroides/ Fusobacteria spp. which showed sensitivity to bile. D u r i n g the early part of the study, detailed identification of these organisms was n o t a t t e m p t e d because this information was not considered relevant to the m a n a g e m e n t of the patient. L a t e r it b e c a m e apparent that identification to species level might be of interest and an a t t e m p t was m a d e to recover and identify isolates collected from the 57 patients in the latter part of the trial. G r a m - n e g a t i v e anaerobic bacilli were cultured from 25 o f these patients and stored in s k i m m e d milk at - 2 0 °C. H o w e v e r , strains from only I7 of the 25 patients were recoverable and these were s u b s e q u e n t l y characterized b y a scheme based on biochemical and tolerance tests. 9 0 b l i g a t e l y anaerobic cocci were identified b y their sensitivity in vitro to metronidazole. 1° Results
T a b l e I shows that bacteria were cultured from ear specimens from 95 per cent of patients with chronic suppurative otitis media w h o had p u r u l e n t aural discharge. T h e aerobic organisms cultured from this group are listed in T a b l e II. Proteus spp. were b y far the c o m m o n e s t isolate (61 per cent of patients) with Proteus mirabilis the most c o m m o n single species. O f the other aerobic G r a m - n e g a t i v e bacilli isolated, Pseudomonas spp. were the most frequent. Staphylococcus aureus was cultured from 25 per cent and coagulase-negative staphylococci from 18 per cent of the patients. M a n y of the discharges contained anaerobic bacteria and during the early part of the s t u d y anaerobes were cultured from z7 of 73 patients (23 per cent). W h e n brain heart infusion was substituted for thioglycollate b r o t h as diluent there was a significant increase in the rate of isolation of anaerobes to 44 per cent (25 of 57 patients). T h e species of anaerobic bacteria cultured from patients with p u r u l e n t discharge are listed in T a b l e III. This shows that the main species isolated were B. melaninogenicus and B. fragilis. T h e results of detailed identification of the 26 anaerobes g r o w n from z 7 patients are recorded in T a b l e IV. Very large n u m b e r s of bacteria were isolated from the ears of patients with
G. SWEENEY~
5° Table
II
G. PICOZZI
AND
G. G. BROWNING
Aerobic bacteria isolated f r o m patients with chronic suppurative otitis media N o . of isolates
Proteus spp. Staphylococcus aureus Pseudomonas spp.
79 33 25 24 2I 8
C o a g u l a s e - n e g a t i v e staphylococci ' C o l i f o r m ' bacilli* Corynebacterium spp.
Escherichia coli Streptococcus faecalis Streptococcus pyogenes
7 5 I
Others
2
Total
205
* Inc•udesgeneraandspecies•faer•bicGram-negativebaci••i•therthanth•se1istedhere,e•g.K•ebsiella• Enterobacter etc. where the number of isolates was less than three: a few could not be identified by the API system.
Table
III
Anaerobic bacteria isolated f r o m patients with chronic suppurative otitis media No. of isolates
Species
B. melaninogenicus B. fragilis Bacteroides/Fusobacteria spp.
28 26 9 7 I
A n a e r o b i c cocci Clostridium spp. Total
Table
IV
71
Detailed identification of anaerobic isolates f r o m 1 7 patients
Species
B. melaninogenicus B. assacharolyticus B. fragilis B. vulgatus Fusobacterium spp. A n a e r o b i c cocci Total
No. of isolates
5 2 5 2 2 io 26
Bacteria in C S O M
5I
T a b l e V Results of culture of specimens from patients with mucoid otitis media
Culture Aerobic organisms Anaerobic organisms No growth Total
No. of patients (%) 5 (45) o (o) 6 (55) II
a p u r u l e n t discharge. Pseudomonas spp. for example, were present in mean concentrations of IO 11 organisms per ml. T h e mean counts of Proteus spp., Staph. aureus, coagulase-negative staphylococci and Bacteroides spp. were IO 9 per ml. As a rule more than one bacterial species was isolated and this was the case in 88 of the I3o patients (68 per cent). H o w e v e r , in 36 of the 83 patients (43 per cent) w h e r e culture of the specimen grew aerobes only (Table I), a single infecting species was present and Staph. aureus (I3 isolations) was more c o m m o n than Proteus species (I I isolations). In ~6 of the 4I patients (63 per cent) with b o t h aerobes and anaerobes, two or more different anaerobes were detected in the p u r u l e n t discharge. In contrast to the results in patients with suppurative otitis media anaerobes were not isolated from patients with m u c o i d discharge. Although this control group was too small to allow valid comparison, specimens from six of the I I patients studied showed no g r o w t h (Table V). T h e seven isolates of aerobic bacteria from patients in this group were three strains of coagulase-negative staphylococci and one each of Proteus spp., Klebsiella spp., Staph. aureus, and a n o n - h a e m o l y t i c streptococcus: counts averaged IO 7 organisms per ml. Anaerobic isolates are of course always sensitive to metronidazole b u t the aerobic bacteria had a variety of patterns of antibiotic sensitivity (Table VI). Discussion
A l t h o u g h it is widely accepted that bacteria play a major role in chronic suppurative otitis media the response to antimicrobial chemotherapy is notoriously poor. O u r o w n observations to be reported elsewhere (Browning, Picozzi, Calder and Sweeney, in preparation) s u p p o r t this in that 75 per cent of ears are still actively discharging after four weeks of appropriate systemic or topical antimicrobial therapy. In the study described here specimens were taken with particular attention to avoid contamination from the external ear and examined by quantitative culture in the laboratory with minimal delay. T h e r e are no previous reports in which the n u m b e r s of bacteria in the exudate have been counted. T h e results showed that, in patients with purulent aural discharge, bacteria were present at a concentration which averaged I O 9 organisms per ml. As previous workers have reported 5,6 Gram-negative bacilli were f o u n d to dominate the aerobic bacterial flora, although where only
~2
G. SWEENEY~
G. PICOZZI
AND
G. G. BROWNING
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Bacteria in C S O M
53
a single species was present, Staph. aureus was the most common pathogen. However, in this study Proteus spp. (mostly Proteus mirabilis) were isolated more often than Pseudomonas spp. When present, Pseudomonas spp. were generally found in greater numbers than the other bacteria and counts of this genus averaged Io 11 organisms per ml. T h e high counts obtained with all bacterial species may have contributed to the disappointing results of antimicrobial therapy since it has been separately reported that Staphylococcus aureus in skin lesions in counts of more than ~o6 organisms per cm 2 impaired the healing process. 12 In agreement with previous investigations by the Finnish group 3,4 anaerobes were never isolated alone but only as part of a mixed aerobic-anaerobic flora. T h e fact that anaerobic bacteria are often present may account for the foetid discharge characteristic of chronic otitis media. T h e results also showed that anaerobic bacteria, almost all of which were Bacteroides spp. were p r e s e n t also in large n u m b e r s - in up to 44 per cent of the patients. This is higher than reported by the Finnish workers 3,11who found anaerobic bacteria in 33 per cent of their patients. On the other hand investigators in the U.S.A. 6'13 have reported an incidence of anaerobes of 5o and 66 per cent respectively, in two studies on a series of children with otitis media. In contrast to our investigation however, their patients were predominantly children with intact tympanic membranes. T h e majority of the anaerobic isolates reported by the American workers were peptococci and other Gram-positive anaerobic bacteria quite different from those cultured by us from adults with perforated ear drums in which Bacteroides spp. were by far the most frequent anaerobic organisms. In this investigation B. melaninogenicus and B. fragilis were equally common. This finding is of some interest, since although B. melaninogenicus is almost invariably present in the normal flora of the oropharynx, B. fragilis is relatively rare. T M T h e frequency of B. fragilis in chronic ear infection probably reflects its greater pathogenic potential. Bacteroides spp. were reported to inhibit the phagocytosis in vitro of coliform b a c i l l i - particularly Proteus spp. 1~ Anaerobes may play a similar role in vivo and in protecting aerobic organisms from host defence mechanisms in conditions like C S O M , contribute to the unsatisfactory results of treatment. Mixed infections may also give rise to a more purulent response than infection with aerobes alone. This 'pathological synergy' has been demonstrated in guineapigs in which Escherichia coli and B. fragilis were inoculated separately and together into freshly made superficial incisions. 16 When inoculated together, mixtures of Io 4 E. coli and IO4 B.fragilis produced copious pus whereas inocula containing the same total n u m b e r of cells of each organism alone did not. In C S O M where aerobes and anaerobes co-exist, each at concentrations considerably greater than the threshold levels of ro ~ and IO~ bacterial per ml, the characteristic formation of pus with the rapid destruction of tissue may represent manifestation in vivo of this synergism. T h e bacteria in the ears of patients with purulent disease may have gained entry to the middle ear from the oropharynx. However, the predominance of aerobic Gram-negative bacilli in the condition is perhaps surprising since these are rare inhabitants of the oropharynx; the alternative route of infection in patients who have perforated drums is via the external ear.
54
G. SWEENEY~ G. PICOZZI AND G. G. BROWNING
Antibiotic sensitivity tests showed that the topical aminoglycosides gentamicin and n e o m y c i n were active in vitro against the principal aerobic isolates. C h l o r a m p h e n i c o l w h i c h is often used topically and the systemic antibiotic cephalexin also a p p e a r e d effective on disc testing against aerobes with the e x c e p t i o n o f Pseudomonas spp. As m i g h t be e x p e c t e d the majority of strains of S t a p h . aureus expressed fl-lactamase. T h e results o f this bacteriological investigation o f c h r o n i c s u p p u r a t i v e otitis media do not allow conclusions to be d r a w n a b o u t the p a t h o g e n i c role o f the p r o f u s e bacterial flora w h i c h was d e m o n s t r a t e d in almost all the patients. T h e n o t o r i o u s difficulty in treating the condition m i g h t m e a n that bacteria were s e c o n d a r y invaders or m e r e l y passengers in a c o m p l e x pathological process largely d e t e r m i n e d by o t h e r factors. Alternatively, systemic or topical antibiotic t h e r a p y a l t h o u g h in w i d e s p r e a d use, m a y not be able to reach the sites of infection due to tissue d e s t r u c t i o n and the collection o f p u r u l e n t discharge in the meatus and the b o n y tissues s u r r o u n d i n g the m i d d l e ear. A l t h o u g h the p r e s e n c e of m e t r o n i d a z o l e was d e m o n s t r a t e d in tissue taken frotn the m i d d l e ear o f patients who had been given the d r u g p r e - o p e r a t i v e l y , 4 in o u r study antibiotic activity was not detected in pus collected f r o m patients receiving oral cephalexin. T h i s finding m a y be due either to lack of d r u g p e n e t r a t i o n or to cephalexin d e s t r u c t i o n by fl-lactarnases of the G r a m - n e g a t i v e bacteria in the exudate. T h e observation that high counts o f recognised p a t h o g e n s correlate with p u r u l e n t discharge suggests that the bacteria p r e s e n t have almost certainly c o n t r i b u t e d to the pathological process. (We are indebted to the consultants of the Department of Otolaryngology, Glasgow Royal Infirmary, for allowing their patients to be included in this study.)
References
I. Rees T. Bacteria from the chronic ear. Br J Clin Pract I97I; 25: 453-454. 2. Palva T, Karja J, Palva A. Bacterial analyses in chronic otitis media. ORL Digest I97I ; 33: 19-26. 3. Jokipii AMM, Karma P, Ojala J, Jokipii L. Anaerobic bacteria in chronic otitis media. Arch Otolaryngol I977; *o3: 278-~8o. 4- Jokippi L, Karma P, Jokippi AMM. Access of metronidazole into the chronically inflamed middle ear with reference to anaerobic bacterial infections. Arch Otorhinolaryngol 1978; 220 : 167-174. 5- Palva T, Hallstrom O. Bacteriology of chronic otitis media. Arch Otolaryngol 1965; 82: 359-364. 6. Brook I, Finegold SM. Bacteriology of chronic otitis media. J A M A I979; 241 : 487-488. 7. Sutter VL, Citron CM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. London : CV Mosby, I98O: 81. 8. Prince HN, Gruneberg E, Titsworth E, de Lorenzo WF. Effects of I-(2 Nitro- I-imidazolyl)3-methoxy-2-propanol and 2-methyl-5-nitroimidazole-I-ethanol against anaerobic and aerobic bacteria and protozoa. Appl Microbiol 1969; I8: 728-730. 9. Duerden B. The isolation and identification of Bacteroides spp. from the normal human gingival flora. J Med Microbiol I98O; *3: 89-IOI. I o. Watt B, Jack EP. What are anaerobic cocci? J Med Microbiol I971 ; I o: 461-468. I I. Karma P, Jokipii L, Ojala K, Jokipii AMM. Bacteriology of the chronically discharging middle ear. Acta Otolaryngol I978; 86 : I io-, I4. Iz. Kligman AM, Leyden JJ, McGinley KJ. Bacteriology. J Invest Dermatol I976; 67 : 16o-I68.
B a c t e r i a in C S O M
55
13. Brook I. Anaerobic isolates in chronic recurrent suppurative otitis media. Infection 1979; 7:247-251-
14. Bartlett JG, Gorbach SL. Anaerobic infections of the head and neck. Otolaryngol Clin North Am 1976; 9:655-678. 15. Ingham HR, Sisson PR, Tharagonnet D, Selkon JB, Codd AA. Inhibition of phagocytosis in vitro by obligate anaerobes. Lancet 1977; 2: 1252-1254. 16. Kelly MJ. The quantitative and histological demonstration of pathogenic synergy between Escherichia coff and Bacteroides fragilis in guinea-pig wounds. J Med Microbiol 1978 ; I I : 513-523.