Inrernahnal Journal of Pediatric Otorhinolaryyngology,21 (1991) 155-161 C 1991 Elsevier Science Publishers B.V. 0165-5876/91/$03.50
PEDOT
155
00702
Beta-lactamase production and bacterial tolerance in recurrent acute otitis media Kristian
Roos
i, Lena
Lind
* and Stig E. Holm
3
’ ENT Department, Lundby Hosprtal, Giiteborg (Sweden), ’ Department of Clinical Bactenolom, Gijteborg (Sweden) and -’ Department of Clinrcal Bacteriology, Limed (Sweden)
Key words: Treatment
failure;
Otitis media;
/3-Lactamase;
Tolerance
Abstract Different reasons for treatment failure or relapse of acute otitis media (AOM) have been suggested. In this study 38 children (8 treatment failures, 13 relapses of AOM within one month and 17 with a new AOM) were compared to 25 matching healthy children, regarding fi-lactamase producing bacteria and tolerance to penicillin V and ampicillin. Branhamella catarrhalis was the most common bacteria found in the nasopharynx and was isolated in 60% of children with AOM and in 48% of the control group. Fifty-two percent of the children classified as treatment failure or relapse of AOM had aerobic /?-lactamase producing bacteria in NPH. No bacteria tolerant to penicillin or ampicillin were found. Thus, P-lactamase-producing bacteria seem to play a decisive role in treatment failure and relapse of AOM. On the other hand, tolerance to penicillin V or ampicillin does not seem to have any impact on healing of AOM.
Introduction Treatment failure and relapse of acute otitis media (AOM) are common problems especially in young children. Treatment failure is often defined as continued infection in spite of antibiotic treatment or a recurrence within a few days after treatment is finished. Relapse is often regarded as a new infection from bacteria persisting in the nasopharynx (NPH). Pneumococci (Pn) account for about 30-50% of the episodes of AOM, while Haemophilus influenzae (H.i.) is suggested as the causative agent in 20%. In Sweden, about 8% of H.i. isolates are fi-lactamase
Correspondence: K. Roos, ENT Department,
Sweden.
Lundby
Hospital,
Wieselgrensplatsen
2A, S-417 17 Giiteborg.
156
producers [7]. Branhamella catarrhalis (B.C.) is believed to cause about 10% of AOM episodes in children. The number of P-lactamase-producing strains of this species have increased from 4% in 1977 [12] to 40-45% in 1984 [4] in Sweden. Group A streptococci (GAS) are only found in about 5% of children with AOM [8]. The overall risk for infection with a j?-lactamase-producing strain has been estimated to be about 4-5% among consecutive cases of AOM [lo]. Tolerance to penicillin V, defined as the MBC/MIC ratio 2 16 or 2 32 has been suggested as one reason for treatment failure in various types of infections such as streptococcal tonsillitis [5] and endocarditis [l]. In a study from Canada, tolerance was reported in 5% of H.i. isolates [2]. Penicillin V tolerance has occasionally been demonstrated in isolates of pneumococci in e.g. South Africa [ll]. The aim of the present study was to investigate whether p-lactamase production or tolerance were correlated to treatment failure or relapse of AOM.
Materials and methods Patients Thirty-eight children, 17 girls and 21 boys, with a mean age of 2.9 years, sought medical help in an open care unit due to acute purulent otitis media. Twenty-two of the patients were less than 2 years of age. The study went on during January 1986-April 1987. Eight children had completed treatment with antibiotics due to acute otitis media less than one week earlier. These patients were regarded as treatment failures. Thirteen children had had an infection between one week and one month earlier and were regarded as relapses of AOM. Another 17 children had had otitis media more than one month earlier. All children were looked upon by an ENT-specialist with otomicroscopy. The patients had, prior to entering the study, been treated with penicillin V (17 patients) ampicillin (2), trimethoprim (3), cefaclor (10) or erythromycin (4). Two patients did not know what antibiotic they had previously been given. Twenty-five children, without clinical signs of upper respiratory tract infection and who had not been treated with antibiotics during the preceding month, served as controls. They matched in age and sex and entered the study during the same time of the year as the patients studied. Treatment Patients with treatment failure or relapse of AOM were treated with cefaclor or trimethoprim. Patients with a primary AOM were given penicillin V or, if allergic to penicillin, erythromycin. The 38 children, who got antibiotic treatment, had a follow-up at 4-week intervals. Prior to the present AOM 6 of the 8 children classified as treatment failure had recently had another AOM before this last failure. These 6 children were treated with paracentesis during the following days under general anesthesia and were simultaneously given antibiotics.
157
Sampling Nasopharyngeal (NPH) samples were taken with a swab, via the nasal route, both at the initial visit and at the first control visit after 1 month. The swab was stored in 1 ml TY-medium [6] and immediately sent for bacteriological examination. The middle-ear secretions from the 6 children with a new treatment failure were aspirated with a 2 ml syringe and the secretion was immediately transported to the bacteriological laboratory. Cultivation The NPH sample in TY-medium was stirred in a Vortex mixer for 20 s after arrival at the bacteriological laboratory. An amount of 0.003 ml was spread on Colombia blood agar, streptoagar (containing human blood, Gentiana violet and inhibitors of gram-negative rods) and McLeod agar plates. The TY-medium was then diluted to 10-l in fresh TY-medium. From this dilution 0.1 ml was spread on the same plates as mentioned above. The plates were incubated in CO, atmosphere for 24 h and 48 h. The bacterial growth on the plates was counted and the bacteria typed by conventional techniques. The secretions from the middle ears were cultivated on Colombia blood agar. streptoagar and McLeod agar. The plates were incubated in CO, atmosphere for 24 h and 48 h. The bacterial colonies were counted and typed by conventional techniques. P-Lactamase production was tested by the nitrocefin method [14]. Tolerance Two different methods were used to test if the bacterial strain was tolerant (MBC/MIC 2 16) to penicillin V or ampicillin. (1) The bacteria were cultivated in TY-medium at 37°C overnight. From this culture 30 ~1 were transferred to a tube containing 3 ml fresh TY-medium. The tube was incubated at 37°C until the bacterial concentration showed an optical density of 0.3 at 500 nm. Ten ~1 from this bacterial suspension were transferred to tubes containing 100 ~1 of penicillin V and to tubes containing 100 ~1 of ampicillin in concentrations from 32 pg/ml to 0.0075 pg/ml (Zstep dilutions). The tubes were incubated at 37°C for 24 h. MIC for the bacteria was defined as the lowest antibiotic concentration that completely inhibited visible growth of the bacteria in the tube. To define MBC, 100 pl from the tubes with no visible growth were cultivated on blood agar or McLeod agar, depending on the species of bacteria tested. The plates were incubated at 37°C for 24 h and the number of colonies counted. MBC was determined as the lowest concentration of antibiotics that killed 99.9% of the bacteria. (II) Two to three colonies of the bacteria were suspended in 0.9% NaCl. This suspension was poured on an ASM-agar plate [19], an ASM-agar plate with human blood, or a McLeod agar plate, depending on the bacteria tested. Discs containing penicillin V and ampicillin were placed on each plate. The plates were incubated at 37°C for 24 h and the zone around each disc was measured. Then the disc was replaced, on the same spot, by a new disc containing penicillinase. The plate was
158
reincubated at 37°C for 24 h and were bacteria growing in the zone tion of bacterial growth outside tolerant to the antibiotic. Bacteria-producing P-lactamase
the zone of inhibition measured again. If there in amounts of more than 0.1% of the concentrathe zone the bacterial strain was regarded as was not tested for tolerance.
Statistics
For statistic analyses x2-test and Yates corrections for small numbers were used.
Results
The number of bacteria in NPH ranged from 5 x lo6 to 3 x lo* cfu/ml. No differences were found in the number of bacteria classified as pathogenic compared to the number of Moruxella and staphylococci. No significant differences in the number of bacteria could either be found between the sick patients and the healthy carriers in the control group. In the patients with more than one pathogenic bacterium in NPH none of the pathogenic bacteria were growing in a significantly higher number than the others. None of the non-/?-lactamase-producing strains showed tolerance to penicillin V or ampicillin. In only two of the 38 (5%) patients in the group of children with AOM no pathogens (Pn, H.i., B.C., or GAS) were found in NPH, while the corresponding figure for the healthy children in the control group was 7/25 (28%). About half of the children in the two groups had more than one bacteria in the NPH culture. B.C. was the most common finding in NPH in the children with AOM (60%) and in the control group (48%). One of the patients with AOM harboured GAS as a single pathogen. One patient had a non+lactamase-producing staphylococcus in combination with a H.i.-strain. Eleven out of 23 (48%) of B.C. strains produced /3-lactamase in the infected children while the corresponding figures for the control group were 2/12 (17%). Of the H.i. strains 5/18 (28%) were p-lactamase producers in the children with AOM
TABLE I Results from the bacteriological
cultures before treatment. Patients n = 38
Haemophilw influenrae Branhamella catarrhalis
18 (5) 23 (11)
Pneumococci Group A streptococci
17 1
Beta-lactamase-producing
strains in brackets
Controls n = 25 11 (0)
12 (2) 9 0
Moraxella
3 (2)
0
Staphylococci
1 (0)
1 (0)
159
and O/11 in the control group (Table I). However, these differences were not significant. Out of the 21 patients with treatment failure or a relapse 11 patients (52%) harboured aerobic &lactamase-producing bacteria in NPH. The corresponding figure for the 17 children with a new AOM more than 1 month after termination of therapy was 4 (24%) and 2 (6%) in the control group. There was a significant difference at the 1% level between the patients with treatment failure and the control group. Out of the 11 patients with treatment failure or relapse 9 had been treated with penicillin V or ampicillin. Three of the 10 patients lacking /I-lactamase-producing bacteria had been treated with one of these two antibiotics.
Clinical outcome After treatment of the 38 patients the clinical outcome was as follows: 2 patients experienced a recurrent otitis media within one month. They had both been treated with cefaclor. B.C. was isolated in one patient and B.C. plus H.i. in the other from NPH. In the 6 children in whom bilateral paracentesis was performed due to a new treatment failure, 5 had been treated with cefaclor and one with trimethoprim. The paracentesis was made within 5 days, X = 3 when the patient still was treated with antibiotic. Seven of the 12 middle-ear secretions harboured pathogenic bacteria and in 4 children the same pathogenic bacteria were found both in the middle-ear secretion and in the NPH (Table II). Initially all healed from the infection, but 4 later required transmyringeal drainage due to long-standing secretory otitis media (> 3 months). The remaining 30 children had a new appointment 4 weeks after the acute infection. Of these 30 children, 13 were healed and 17 had SOM. The secretion in the middle ears of 9 of these 17 children disappeared after 4 further weeks of expectation, while 8 had persistent SOM and were later treated with paracentesis and transmyringeal drainage.
TABLE
II
Cube results from 6 children with another treatmenf failure in whom paracentesis was performed wltthrnj (X = 3) days from start of treatment Patient No. 1 2 3 4 5 6
Right ear
Lefr ear
Lutest treatment
Initial NPH culture
Cefaclor Cefaclor Cefaclor Cefaclor Cefaclor Trimethoprim
H.i. Pn. Pn. H.i. Hi. 0
H.i.
H.i.
H.i.
0
Pn. 0 0 0
Pn. H.i. * 0 H. i.
* Beta-lactamase-producing
strain.
B.C. * Pn. H.i.
B.C. *
B.C. *
*
Preop NPH culture Hi H.i. Pn.
Pn. B.C. * H.I. * 0 0
160
Discussion This is a clinical study, based on 38 children with different histories of earlier episodes of AOM which we have followed bacteriologically during their last AOM. The wide variation in the number of the bacteria cultured from the NPH makes it impossible to judge which bacteria can cause AOM if more than one bacterium is present. Neither can this quantification differentiate between a patient with AOM and a healthy carrier, which has been shown in e.g. streptococcal tonsillitis by Roos
WI. The high presence of B.C. and the low numbers of Pn isolates seen in this study are not in accordance with the results reported by others [8]. The initial therapy might explain the high incidence of B.C. found in the children with AOM in our study but does not account for the high incidence in the control group. It is tempting to assume a special epidemiological situation or an increased frequency of B.C. in children. Forty-eight percent of the B.C. strains from the infected patients were /3-lactamase producers, which is in accordance with other studies [18]. However, as much as 28% of H.i strains produced P-lactamase in the infected children, in contrast to none in the control group. The high incidence of fi-lactamase-producing H.i. in the group of children with AOM contradicts the results of others [10,18], but could be a reflection of a selection process due to earlier antibiotic treatment of this patient group. Relapses are commonly looked upon as a new infection caused by bacteria from the NPH, the latter being a reservoir for reinfections. Accordingly, not only potentially pathogenic bacteria with the capacity to produce P-lactamase, but also other P-lactamase-producing bacteria in the NPH should be considered. Brook [3] has pointed out the possibility that P-lactamase-producing bacteria in NPH, by releasing /3-lactamase to its surroundings, might protect non-/?-lactamase-producing strains from the action of /3-lactam antibiotics. The control group had fewer P-lactamase-producing strains than the children with treatment failure. Every other child with treatment failure or relapse of AOM had some aerobic /3-lactamase-producing bacteria in NPH. As mentioned earlier, it is likely that this is a reflection of the selection of P-lactamase-producing bacteria in the nasopharyngeal flora due to earlier treatment. Nevertheless it indicates a greater role of fi-lactamase-producing strains in children with recurrent AOM than has previously been suggested [lo]. The existence of bacteria in 7 out of 12 of the middle-ear secretions (6 children) at the time of paracentesis in patients with treatment failure of AOM after receiving antibiotics for less than 5 days, stresses the importance of treating AOM with a full lo-day course of antibiotics in these children. Penicillin V tolerance has been shown in streptococci group A [9], C [15] and G [13] as well as in staphylococci [17], pneumococci [ll] and Huemophilus influenzae [2]. This has also been suggested in treatment failure of streptococcal tonsillitis [5]. We could, with the methods described here, not find any strains tolerant to penicillin V or ampicillin. Thus, tolerance to these antibiotics does not seem to be
161
the reason for treatment Thore and Lid& [18].
failure
or relapse
of AOM
and
confirms
the results
of
Acknowledgement The study was supported
by Gijteborg’s
Medical
Association
(L253/86).
References 1 Andersson, A.W. and Cruikshank, J.G.. Endocarditis due to viridans type streptococci tolerant to /3-lactam antibiotics; therapeutic problems, Br. Med. J., 285 (1982) 854-856. 2 Bergeron, M.G. and Lavoie, G.Y., Tolerance of Haemophilur influenzue to /%lactam antibiotic. Antimicrob. Agents Chemother., 28 (1985) 320-325. 3 Brook, I.. Aerobic and anaerobic bacteriology of adenoids in children; a comparision between patients with chronic adenotonsillitis and adenoid hypertrophy, Laryngoscope, 91 (1981) 377-382. 4 Eliasson, J. and Kamme, C., Characterization of the plasmid-mediated /I-lactamase in Brunhamellu catarrhahs, with special reference to substrate affinity, J. Antimicrob. Chemother.. 15 (1985) 1399149. 5 Grahn, E., Holm, S.E. and Roos, K., Penicillin tolerance in fi-streptococci isolated from patients with tonsillitis, Stand. J. Inf. Dis., 19 (1987) 421-426. 6 Holm, S.-E. and Falsen, E., An antigen-free medium for cultivation of /I-hemolytic streptococci. Acta Pathol. Microbial. Stand., 69 (1967) 264-276. 7 Kallings, J., Bengtsson, S.. Christensen, P., Holm, S.E., Lind. L. and Kahn, M.. Antibiotic sensitivity of Huemophilus influenzue, Streptococcus pneumoniue, Streptococcus pyogenes and Branhamellu cuturrhulis isolated from upper respiratory tract infection in Sweden, Stand. J. Infect. Dis., Suppl. 39 (1983) 100-105. 8 Kamme, C., Lundgren, K. and Mardh. P.-A., The aetiology of acute otitis media in children. Stand. J. Inf. Dis.. 3 (1971) 217-223. 9 Kim, K.S. and Kaplan, E.L., Association of penicillin tolerance with failure to eradicate group A streptococci from patients with pharyngitis, J. Paediatr., 107 (1985) 681-684. 10 Laurin, L.. Prellner, K. and Kamme, C., Phenoxymethylpenicillin and therapeutic failure in acute otitis media, Stand. J. Inf. Dis., 17 (1985) 367-370. 11 Lin. H. and Tomaz, A., Penicillin tolerance in multiply drug-resistant natural isolates of Streprococcus pneumomae, J. Infect. Dis., 152 (1985) 365-372. 12 Malmvall. B.-E.. Brorson, J.-E. and Johnsson, J.. In oitro sensitivity to penicillin V and P-lactamase production of Branhumellu euturrhulis, J. Antimicrob. Chemother.. 3 (1977) 374-375. 13 Noble, IT., Tyburski, M.B., Berman, M.. Greenspan, J. and Tenenbaum. M.J., Antimicrobial tolerance in Group G streptococci, Lancet, 2 (1980) 982. 14 O’Callaghan, C.H., Morris, A., Kirby, SM. and Shingler, A.H., Novel method for detection of beta-lactamase by using a chromogenic cephalosporin substrate, Antimicrob. Agents Chemother., 1 (1972) 283-288. 15 Portnoy, D., Prentis, J. and Richards, G.-K., Penicillin tolerance of human isolates of group C streptococci, Antimicrob. Agents Chemother., 20 (1981) 235-238. 16 Roos. K., The diagnostic value of symptoms and signs in acute tonsillitis in children over the age of ten and in adults, Stand. J. Infect. Dis., 17 (1985) 259-267. 17 Sabath, L.D., Wheeler, N., Laverdiere, M., Blazeric, D. and Wilkinson, B.J., A new type of penicillin resistance of Staphylococcus aureus, Lancet, 1 (1977) 443-447. 18 Thore, M. and Lid&, M., Relapse of acute purulent otitis media: antibiotic sensitivities of nasopharyngeal pathogens, Stand. J. Infect. Dis., 19 (1987) 3155323. 19 Wretlind, B., Nord, C.E. and Wadstrom, T., In uitro sensitivity of isolates of Pseudomonas aerugmosa to carbenicillin, gentamicin. tobramycin and some other antibiotics. Stand. J. Infect. Dis., 6 (1974) 49-52.
PEDOT
155
00702
Beta-lactamase production and bacterial tolerance in recurrent acute otitis media Kristian
Roos
i, Lena
Lind
* and Stig E. Holm
3
’ ENT Department, Lundby Hosprtal, Giiteborg (Sweden), ’ Department of Clinical Bactenolom, Gijteborg (Sweden) and -’ Department of Clinrcal Bacteriology, Limed (Sweden)
Key words: Treatment
failure;
Otitis media;
/3-Lactamase;
Tolerance
Abstract Different reasons for treatment failure or relapse of acute otitis media (AOM) have been suggested. In this study 38 children (8 treatment failures, 13 relapses of AOM within one month and 17 with a new AOM) were compared to 25 matching healthy children, regarding fi-lactamase producing bacteria and tolerance to penicillin V and ampicillin. Branhamella catarrhalis was the most common bacteria found in the nasopharynx and was isolated in 60% of children with AOM and in 48% of the control group. Fifty-two percent of the children classified as treatment failure or relapse of AOM had aerobic /?-lactamase producing bacteria in NPH. No bacteria tolerant to penicillin or ampicillin were found. Thus, P-lactamase-producing bacteria seem to play a decisive role in treatment failure and relapse of AOM. On the other hand, tolerance to penicillin V or ampicillin does not seem to have any impact on healing of AOM.
Introduction Treatment failure and relapse of acute otitis media (AOM) are common problems especially in young children. Treatment failure is often defined as continued infection in spite of antibiotic treatment or a recurrence within a few days after treatment is finished. Relapse is often regarded as a new infection from bacteria persisting in the nasopharynx (NPH). Pneumococci (Pn) account for about 30-50% of the episodes of AOM, while Haemophilus influenzae (H.i.) is suggested as the causative agent in 20%. In Sweden, about 8% of H.i. isolates are fi-lactamase
Correspondence: K. Roos, ENT Department,
Sweden.
Lundby
Hospital,
Wieselgrensplatsen
2A, S-417 17 Giiteborg.
156
producers [7]. Branhamella catarrhalis (B.C.) is believed to cause about 10% of AOM episodes in children. The number of P-lactamase-producing strains of this species have increased from 4% in 1977 [12] to 40-45% in 1984 [4] in Sweden. Group A streptococci (GAS) are only found in about 5% of children with AOM [8]. The overall risk for infection with a j?-lactamase-producing strain has been estimated to be about 4-5% among consecutive cases of AOM [lo]. Tolerance to penicillin V, defined as the MBC/MIC ratio 2 16 or 2 32 has been suggested as one reason for treatment failure in various types of infections such as streptococcal tonsillitis [5] and endocarditis [l]. In a study from Canada, tolerance was reported in 5% of H.i. isolates [2]. Penicillin V tolerance has occasionally been demonstrated in isolates of pneumococci in e.g. South Africa [ll]. The aim of the present study was to investigate whether p-lactamase production or tolerance were correlated to treatment failure or relapse of AOM.
Materials and methods Patients Thirty-eight children, 17 girls and 21 boys, with a mean age of 2.9 years, sought medical help in an open care unit due to acute purulent otitis media. Twenty-two of the patients were less than 2 years of age. The study went on during January 1986-April 1987. Eight children had completed treatment with antibiotics due to acute otitis media less than one week earlier. These patients were regarded as treatment failures. Thirteen children had had an infection between one week and one month earlier and were regarded as relapses of AOM. Another 17 children had had otitis media more than one month earlier. All children were looked upon by an ENT-specialist with otomicroscopy. The patients had, prior to entering the study, been treated with penicillin V (17 patients) ampicillin (2), trimethoprim (3), cefaclor (10) or erythromycin (4). Two patients did not know what antibiotic they had previously been given. Twenty-five children, without clinical signs of upper respiratory tract infection and who had not been treated with antibiotics during the preceding month, served as controls. They matched in age and sex and entered the study during the same time of the year as the patients studied. Treatment Patients with treatment failure or relapse of AOM were treated with cefaclor or trimethoprim. Patients with a primary AOM were given penicillin V or, if allergic to penicillin, erythromycin. The 38 children, who got antibiotic treatment, had a follow-up at 4-week intervals. Prior to the present AOM 6 of the 8 children classified as treatment failure had recently had another AOM before this last failure. These 6 children were treated with paracentesis during the following days under general anesthesia and were simultaneously given antibiotics.
157
Sampling Nasopharyngeal (NPH) samples were taken with a swab, via the nasal route, both at the initial visit and at the first control visit after 1 month. The swab was stored in 1 ml TY-medium [6] and immediately sent for bacteriological examination. The middle-ear secretions from the 6 children with a new treatment failure were aspirated with a 2 ml syringe and the secretion was immediately transported to the bacteriological laboratory. Cultivation The NPH sample in TY-medium was stirred in a Vortex mixer for 20 s after arrival at the bacteriological laboratory. An amount of 0.003 ml was spread on Colombia blood agar, streptoagar (containing human blood, Gentiana violet and inhibitors of gram-negative rods) and McLeod agar plates. The TY-medium was then diluted to 10-l in fresh TY-medium. From this dilution 0.1 ml was spread on the same plates as mentioned above. The plates were incubated in CO, atmosphere for 24 h and 48 h. The bacterial growth on the plates was counted and the bacteria typed by conventional techniques. The secretions from the middle ears were cultivated on Colombia blood agar. streptoagar and McLeod agar. The plates were incubated in CO, atmosphere for 24 h and 48 h. The bacterial colonies were counted and typed by conventional techniques. P-Lactamase production was tested by the nitrocefin method [14]. Tolerance Two different methods were used to test if the bacterial strain was tolerant (MBC/MIC 2 16) to penicillin V or ampicillin. (1) The bacteria were cultivated in TY-medium at 37°C overnight. From this culture 30 ~1 were transferred to a tube containing 3 ml fresh TY-medium. The tube was incubated at 37°C until the bacterial concentration showed an optical density of 0.3 at 500 nm. Ten ~1 from this bacterial suspension were transferred to tubes containing 100 ~1 of penicillin V and to tubes containing 100 ~1 of ampicillin in concentrations from 32 pg/ml to 0.0075 pg/ml (Zstep dilutions). The tubes were incubated at 37°C for 24 h. MIC for the bacteria was defined as the lowest antibiotic concentration that completely inhibited visible growth of the bacteria in the tube. To define MBC, 100 pl from the tubes with no visible growth were cultivated on blood agar or McLeod agar, depending on the species of bacteria tested. The plates were incubated at 37°C for 24 h and the number of colonies counted. MBC was determined as the lowest concentration of antibiotics that killed 99.9% of the bacteria. (II) Two to three colonies of the bacteria were suspended in 0.9% NaCl. This suspension was poured on an ASM-agar plate [19], an ASM-agar plate with human blood, or a McLeod agar plate, depending on the bacteria tested. Discs containing penicillin V and ampicillin were placed on each plate. The plates were incubated at 37°C for 24 h and the zone around each disc was measured. Then the disc was replaced, on the same spot, by a new disc containing penicillinase. The plate was
158
reincubated at 37°C for 24 h and were bacteria growing in the zone tion of bacterial growth outside tolerant to the antibiotic. Bacteria-producing P-lactamase
the zone of inhibition measured again. If there in amounts of more than 0.1% of the concentrathe zone the bacterial strain was regarded as was not tested for tolerance.
Statistics
For statistic analyses x2-test and Yates corrections for small numbers were used.
Results
The number of bacteria in NPH ranged from 5 x lo6 to 3 x lo* cfu/ml. No differences were found in the number of bacteria classified as pathogenic compared to the number of Moruxella and staphylococci. No significant differences in the number of bacteria could either be found between the sick patients and the healthy carriers in the control group. In the patients with more than one pathogenic bacterium in NPH none of the pathogenic bacteria were growing in a significantly higher number than the others. None of the non-/?-lactamase-producing strains showed tolerance to penicillin V or ampicillin. In only two of the 38 (5%) patients in the group of children with AOM no pathogens (Pn, H.i., B.C., or GAS) were found in NPH, while the corresponding figure for the healthy children in the control group was 7/25 (28%). About half of the children in the two groups had more than one bacteria in the NPH culture. B.C. was the most common finding in NPH in the children with AOM (60%) and in the control group (48%). One of the patients with AOM harboured GAS as a single pathogen. One patient had a non+lactamase-producing staphylococcus in combination with a H.i.-strain. Eleven out of 23 (48%) of B.C. strains produced /3-lactamase in the infected children while the corresponding figures for the control group were 2/12 (17%). Of the H.i. strains 5/18 (28%) were p-lactamase producers in the children with AOM
TABLE I Results from the bacteriological
cultures before treatment. Patients n = 38
Haemophilw influenrae Branhamella catarrhalis
18 (5) 23 (11)
Pneumococci Group A streptococci
17 1
Beta-lactamase-producing
strains in brackets
Controls n = 25 11 (0)
12 (2) 9 0
Moraxella
3 (2)
0
Staphylococci
1 (0)
1 (0)
159
and O/11 in the control group (Table I). However, these differences were not significant. Out of the 21 patients with treatment failure or a relapse 11 patients (52%) harboured aerobic &lactamase-producing bacteria in NPH. The corresponding figure for the 17 children with a new AOM more than 1 month after termination of therapy was 4 (24%) and 2 (6%) in the control group. There was a significant difference at the 1% level between the patients with treatment failure and the control group. Out of the 11 patients with treatment failure or relapse 9 had been treated with penicillin V or ampicillin. Three of the 10 patients lacking /I-lactamase-producing bacteria had been treated with one of these two antibiotics.
Clinical outcome After treatment of the 38 patients the clinical outcome was as follows: 2 patients experienced a recurrent otitis media within one month. They had both been treated with cefaclor. B.C. was isolated in one patient and B.C. plus H.i. in the other from NPH. In the 6 children in whom bilateral paracentesis was performed due to a new treatment failure, 5 had been treated with cefaclor and one with trimethoprim. The paracentesis was made within 5 days, X = 3 when the patient still was treated with antibiotic. Seven of the 12 middle-ear secretions harboured pathogenic bacteria and in 4 children the same pathogenic bacteria were found both in the middle-ear secretion and in the NPH (Table II). Initially all healed from the infection, but 4 later required transmyringeal drainage due to long-standing secretory otitis media (> 3 months). The remaining 30 children had a new appointment 4 weeks after the acute infection. Of these 30 children, 13 were healed and 17 had SOM. The secretion in the middle ears of 9 of these 17 children disappeared after 4 further weeks of expectation, while 8 had persistent SOM and were later treated with paracentesis and transmyringeal drainage.
TABLE
II
Cube results from 6 children with another treatmenf failure in whom paracentesis was performed wltthrnj (X = 3) days from start of treatment Patient No. 1 2 3 4 5 6
Right ear
Lefr ear
Lutest treatment
Initial NPH culture
Cefaclor Cefaclor Cefaclor Cefaclor Cefaclor Trimethoprim
H.i. Pn. Pn. H.i. Hi. 0
H.i.
H.i.
H.i.
0
Pn. 0 0 0
Pn. H.i. * 0 H. i.
* Beta-lactamase-producing
strain.
B.C. * Pn. H.i.
B.C. *
B.C. *
*
Preop NPH culture Hi H.i. Pn.
Pn. B.C. * H.I. * 0 0
160
Discussion This is a clinical study, based on 38 children with different histories of earlier episodes of AOM which we have followed bacteriologically during their last AOM. The wide variation in the number of the bacteria cultured from the NPH makes it impossible to judge which bacteria can cause AOM if more than one bacterium is present. Neither can this quantification differentiate between a patient with AOM and a healthy carrier, which has been shown in e.g. streptococcal tonsillitis by Roos
WI. The high presence of B.C. and the low numbers of Pn isolates seen in this study are not in accordance with the results reported by others [8]. The initial therapy might explain the high incidence of B.C. found in the children with AOM in our study but does not account for the high incidence in the control group. It is tempting to assume a special epidemiological situation or an increased frequency of B.C. in children. Forty-eight percent of the B.C. strains from the infected patients were /3-lactamase producers, which is in accordance with other studies [18]. However, as much as 28% of H.i strains produced P-lactamase in the infected children, in contrast to none in the control group. The high incidence of fi-lactamase-producing H.i. in the group of children with AOM contradicts the results of others [10,18], but could be a reflection of a selection process due to earlier antibiotic treatment of this patient group. Relapses are commonly looked upon as a new infection caused by bacteria from the NPH, the latter being a reservoir for reinfections. Accordingly, not only potentially pathogenic bacteria with the capacity to produce P-lactamase, but also other P-lactamase-producing bacteria in the NPH should be considered. Brook [3] has pointed out the possibility that P-lactamase-producing bacteria in NPH, by releasing /3-lactamase to its surroundings, might protect non-/?-lactamase-producing strains from the action of /3-lactam antibiotics. The control group had fewer P-lactamase-producing strains than the children with treatment failure. Every other child with treatment failure or relapse of AOM had some aerobic /3-lactamase-producing bacteria in NPH. As mentioned earlier, it is likely that this is a reflection of the selection of P-lactamase-producing bacteria in the nasopharyngeal flora due to earlier treatment. Nevertheless it indicates a greater role of fi-lactamase-producing strains in children with recurrent AOM than has previously been suggested [lo]. The existence of bacteria in 7 out of 12 of the middle-ear secretions (6 children) at the time of paracentesis in patients with treatment failure of AOM after receiving antibiotics for less than 5 days, stresses the importance of treating AOM with a full lo-day course of antibiotics in these children. Penicillin V tolerance has been shown in streptococci group A [9], C [15] and G [13] as well as in staphylococci [17], pneumococci [ll] and Huemophilus influenzae [2]. This has also been suggested in treatment failure of streptococcal tonsillitis [5]. We could, with the methods described here, not find any strains tolerant to penicillin V or ampicillin. Thus, tolerance to these antibiotics does not seem to be
161
the reason for treatment Thore and Lid& [18].
failure
or relapse
of AOM
and
confirms
the results
of
Acknowledgement The study was supported
by Gijteborg’s
Medical
Association
(L253/86).
References 1 Andersson, A.W. and Cruikshank, J.G.. Endocarditis due to viridans type streptococci tolerant to /3-lactam antibiotics; therapeutic problems, Br. Med. J., 285 (1982) 854-856. 2 Bergeron, M.G. and Lavoie, G.Y., Tolerance of Haemophilur influenzue to /%lactam antibiotic. Antimicrob. Agents Chemother., 28 (1985) 320-325. 3 Brook, I.. Aerobic and anaerobic bacteriology of adenoids in children; a comparision between patients with chronic adenotonsillitis and adenoid hypertrophy, Laryngoscope, 91 (1981) 377-382. 4 Eliasson, J. and Kamme, C., Characterization of the plasmid-mediated /I-lactamase in Brunhamellu catarrhahs, with special reference to substrate affinity, J. Antimicrob. Chemother.. 15 (1985) 1399149. 5 Grahn, E., Holm, S.E. and Roos, K., Penicillin tolerance in fi-streptococci isolated from patients with tonsillitis, Stand. J. Inf. Dis., 19 (1987) 421-426. 6 Holm, S.-E. and Falsen, E., An antigen-free medium for cultivation of /I-hemolytic streptococci. Acta Pathol. Microbial. Stand., 69 (1967) 264-276. 7 Kallings, J., Bengtsson, S.. Christensen, P., Holm, S.E., Lind. L. and Kahn, M.. Antibiotic sensitivity of Huemophilus influenzue, Streptococcus pneumoniue, Streptococcus pyogenes and Branhamellu cuturrhulis isolated from upper respiratory tract infection in Sweden, Stand. J. Infect. Dis., Suppl. 39 (1983) 100-105. 8 Kamme, C., Lundgren, K. and Mardh. P.-A., The aetiology of acute otitis media in children. Stand. J. Inf. Dis.. 3 (1971) 217-223. 9 Kim, K.S. and Kaplan, E.L., Association of penicillin tolerance with failure to eradicate group A streptococci from patients with pharyngitis, J. Paediatr., 107 (1985) 681-684. 10 Laurin, L.. Prellner, K. and Kamme, C., Phenoxymethylpenicillin and therapeutic failure in acute otitis media, Stand. J. Inf. Dis., 17 (1985) 367-370. 11 Lin. H. and Tomaz, A., Penicillin tolerance in multiply drug-resistant natural isolates of Streprococcus pneumomae, J. Infect. Dis., 152 (1985) 365-372. 12 Malmvall. B.-E.. Brorson, J.-E. and Johnsson, J.. In oitro sensitivity to penicillin V and P-lactamase production of Branhumellu euturrhulis, J. Antimicrob. Chemother.. 3 (1977) 374-375. 13 Noble, IT., Tyburski, M.B., Berman, M.. Greenspan, J. and Tenenbaum. M.J., Antimicrobial tolerance in Group G streptococci, Lancet, 2 (1980) 982. 14 O’Callaghan, C.H., Morris, A., Kirby, SM. and Shingler, A.H., Novel method for detection of beta-lactamase by using a chromogenic cephalosporin substrate, Antimicrob. Agents Chemother., 1 (1972) 283-288. 15 Portnoy, D., Prentis, J. and Richards, G.-K., Penicillin tolerance of human isolates of group C streptococci, Antimicrob. Agents Chemother., 20 (1981) 235-238. 16 Roos. K., The diagnostic value of symptoms and signs in acute tonsillitis in children over the age of ten and in adults, Stand. J. Infect. Dis., 17 (1985) 259-267. 17 Sabath, L.D., Wheeler, N., Laverdiere, M., Blazeric, D. and Wilkinson, B.J., A new type of penicillin resistance of Staphylococcus aureus, Lancet, 1 (1977) 443-447. 18 Thore, M. and Lid&, M., Relapse of acute purulent otitis media: antibiotic sensitivities of nasopharyngeal pathogens, Stand. J. Infect. Dis., 19 (1987) 3155323. 19 Wretlind, B., Nord, C.E. and Wadstrom, T., In uitro sensitivity of isolates of Pseudomonas aerugmosa to carbenicillin, gentamicin. tobramycin and some other antibiotics. Stand. J. Infect. Dis., 6 (1974) 49-52.
