Auris· Nasus· Larynx (Tokyo) 15,105-111 (1988)

THE ANTIBIOTIC SUSCEPTIBILITIES AND BETA­LACTAMASE PRODUCTION OF CLINICAL

ISOLATED Branhamella catarrhalis FROM ACUTE OTITIS MEDIA IN CHILDREN

Kenji SUZUKI, M. D., Shunkichi BABA, M. D., Mitsuaki INAGAKI, M. D., and Takehiro KOBAYASHI, M. D.

Department of Otorhinolaryngology, Nagoya City University, School of Medicine, Mizuho-ku, Nagoya

Branhamella catarrhalis has been misconsidered as a normal resident in human respiratory tract for a long time. However, many authors re­cently have reported its pathogenecity and isolated it from the otolaryn­gological region. In our study, this organism can be isolated from the ear and nasal discharge in the child with acute otitis media by the rate of 7.5 % and 21.4 % respectively. Out of this 107 isolated strains, 97 strains (90.7 %) were found to be beta-lactamase producing organisms. The MIC measurement of penicillins and cephems (except CEX) for inhibition of all these strains in our study is 6.25 ,ltg/ml or less and because of the unreli­ability of the ABPC's susceptibility test by disk method, it is necessary to check the beta-lactamase production in each strain. Becoming of the high emergence rate of beta-lactamase producing strains, B. catarrhalis should be considered to be as important pathogen as Streptococcus pneumoniae and Haemophilus injluenzae in upper respiratory tract in­fections in children.

Micrococcus (Branham ella) catarrhalis was first isolated by FROSCH and KOLLE (1896) and was termed as B. catarrhalis by CATLIN (1970). B. catarrrhalis has been thought to be the normal member of the flora in the respiratory tract, and little attention has been given to its pathogenic capacity or identification. How­ever, due to the increasing isolation of this organism as the pathogen from chronic respiratory tract infections in Japan as well as in overseas countries and, moreover, the increasing tendency toward the emergence of beta-lactamase producing strains, the significance of this organism has been recently pointed out (ELIASSON and KAMME, 1986). We have also devoted much attention to the role of this species in the field of otorhinolaryngology and found that B. catarrhalis is isolated from

Received for publication April 23, 1988

105

106 K. SUZUKI et al.

cultures of ear and nasal discharge at a relatively high frequency (INAGAKI, BABA, MORI, SUZUKI, SHIMADA, and SOYANO, 1987). In this paper, the sensitivity of B. catarrhalis to antibacterial drugs and its beta-lactamase production were studied.

MATERIALS AND METHODS

1. Subjects and identification. The ear or nasal swabs obtained from patients with otorhinolaryngological infection at their initial visit to our department were inoculated onto the sheep blood agar (BBL) or chocolate agar and incubated for 18-48 hr. Of the clinical isolates of Gram-negative diplococci, those which did not catabolize glucose and gave positive results for the nitrate reduction test, DNase test, and oxidase test were regarded as B. catarrhalis (MORELLO and BOHNHOFF, 1980).

2. Beta-lactamase production. The production of beta-Iactamases was determined in each strain by the use of disk nitrocefin (Difco).

3. Antibiotic susceptibility testing. The minimum inhibitory concentration (MIC) was determined according to the standard by the Japan Society of Chemo­therapy (FUJII, 1981). The organisms were incubated orver-night on the chocolate agar, and the colonies of the organisms were seeded onto Mueller-Hinton broth (BBL) and incubated for three hours. To this suspension, sterile physiological saline was added to adjust the density at 106 CFU/ml. The MICs were measured using the chocolate agar consisting of M ueller-Hinton agar (BBL) and 5 % horse defibrinated blood. A total of 14 antibiotics tested included three penicillins: ampicillin (ABPC), amoxycillin-clavulanic acid (AMPCjCV A), and ampicillin/ sulbactam (ABPCjSBT): four cephems: cephalexin (CEX), cefaclor (CCL), cefatri­zine (CFT), and cefixime (CFIX); three quinolones: ofloxacin (OFLX), enoxacin (ENX), and pipemidic acid (PPA), and other four drugs: erythromycin (EM), minocycline (MINO), amikacin (AMK), and chloramphenicol (CP).

RESULTS

The MICs of penicillins and cephems against beta-Iactamase non-producing strains were determined in 15 strains (Table 1). The MICs of ABPC, AMPCj CV A, and eCL against beta-Iactamase producing strains were determined in 75 strains (Table i). The MICs of other antibacterial agents against beta-lactamase producing strains were determined in 40 strains (Table 3). The MIC of CP, how­ever, was measured only in 12 strains (Table 4). The MICs of ABPC were 0.05-0.39 flg/ml against beta-Iactamase non-producing strains and 0.05-6.25 flg/ml against beta-lactamase producirrgstrains. The MIC 80% and MIC 90% of APBC against beta-Iactamase non-produCing strains were 0.10 and 0.39 flg/ml, respective­ly, and those against beta-lactamase producing strains were 1.56 and 3.13 flg/ml, respectively. The antibacterial activity of ABPC against beta-Iactamase producing

B. Catarrhalis IN OUR FIELD

Table 1. Cumulative percent of 15 strains of non-fi-lactamase-producing Branhamella catarrhalis inhibited at the indicated concentrations of antibiotics.

Antibiotics Minimum inhibitory concentration (f.tg/ml)

0.05 0. 10 0.20 0.39 0.78 1.56 3.13 6.25

Ampicillin 60 86.7 100 Amoxycillin

6.7 26.7 40.0 100 +c1avulanic acid Ampicillin

40 .0 73 .3 86.7 100 + sulbactam Cephalexin 20 .0 46 .7 86.7 Cefac10r 13.3 26.7 66.7 73.3 100 Cefatrizine 6.7 20.0 60.0 100 Cefixime 86.7 93.3 100

Inoculum size 106 CFU/rnl.

Table 2. Cumulative percent of 75 strains of fi-Iactamase-producing Bran-hamella catarrhalis inhibited at the indicated concentrations of antibiotics.

Minimum inhibitory concentration (f.tg/ml) Antibiotics

0.05 0.10 0.20 0 .39 0.78 1.56 3.13 6.25

Ampicillin 4.0 6.6 32.0 52.0 76.0 89.3 93.3 100 Amoxycillin

28.6 54 .7 78.7 93 .3 98.7 100 +clavulanic acid

Cefac10r 16.0 49.3 55.0 89.3 97.3 100

Inoculum size 106 CFU/ml.

Table 3. Cumulative percent of 40 strains of p-Iactamase-producing Bran-hamella catarrhalis inhibited at the indicated concentrations of an-tibiotics.

Minimum inhibitory concentration (f.tg/ml) Antibiotics

0.05 0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.5 25

Ampicillin/ 25.0 57.5 95 .0 97 .5 100 sulbactum

Cephalexin 2.5 7.5 17.5 32 .5 47.5 77.5 85.0 95.0 97 .5 Cefatrizine 32 .5 60.0 92 .5 97.5 100 Cefixime 50 .0 70.0 95 .0 100 Ofloxacin 27 .5 75.0 92.5 100 Enoxacin 5.0 45.0 95.0 97.5 100 Pipemidic

7.5 80.0 100 acid

Erythromycin 5.0 60 .0 92.5 100 Minocyc1ine 27.5 62.5 80.0 97.5 100 Amikacin 2.5 5.0 15.0 35.0 60.0 100

Inoculum size 106 CFU/ml.

107

12.5

100

12.5

50

100

108 K. SUZUKI et at.

Table 4. Cumulative percent of 12 strains of ;9-lactamase-producing Bran­hamella catarrhalis inhibited at the indicated concentration of chlor­amphenicol.

Minimum inhibitory concentration (,ugJml) Antibiotic

0.05 0.10 0.20 0.39 0.78 1.56

Chloramphenicol 66.7 100

Inoculum size 106 CFUJml

3.13

strains was considerably diminished. On the other hand, CFIX showed excellent antibacterial activities against these strains, and both of its MIC 80 % and MIC 90 % activities were 0.20 ,ug/ml. CEX showed the poorest antibacterial activity. Among other quinolone derivatives, the new quinolones such as OFLX and ENX showed excellent antibacterial activities, both of their MIC 80 % and MIC 90 % being 0.20 ,ug/ml. Meanwhile, the MIC 80% of PPA was 3.13 ,ug/ml. Both of the MIC 80% and MIC 90% of AMK were 3.13 ,ug/ml. The MIC 80% and MIC 90% of MINO were 1.56 and 3.13 ,ug/ml, respectively, and CP had a MIC range of 0.78 to 1.56 ,ug/ml.

DISCUSSION

In otorhinolaryngological field, partly because of the misperception that B. catarrhalis is the normal resident in the respiratory tract and has no pathogenicity, the reports on B. catarrhalis infections are insufficient. In otitis media, COFFY, BOOTH, and MARTIN (1966) first reported B. catarrhalis as the causative organism, and this was followed by the similar reports by KAMME (1970), KOVATCH, WALD, and MICHAELS (1983), SHURIN, MARCHANT, KIM, VAN HARE, JOHNSON, TUTIHASI,

Fig. 1. Pure culture of Branhamella catarrhalis from ear exudate (48 hr chocolate agar).

B. Catarrhalis IN OUR FIELD 109

Fig. 2. Branhamelfa catarrhalis phagocyted by neutrophil in nasal swab (Gram strain).

and KNAPP (1983), BLUESTONE (1986), and LUNDGREN and INGVARSSON (1986). With respect to sinusitis due to this organism, there are reports by BRORSON, AXELSSON, and HOLM (1976), KOFVATCH et al. (1983), WALD, REILLY, CASSELBRANT, LEDESMA-MEDINA, MILMOE, BLUESTONE, and CHIPONIS (1984), and BLUESTONE (1986). SCHALEN, CHRISTENSEN, KAMME, MIORNER, PETTERSSON, and SCHALEN (1980), BRORSON and MALMVALL (1981), and LUNDGREN and INGVARSSON (1986) reported nasopharyngeal infections caused by this organism. In Japan, the sole report on otitis media caused by this organism is the one by HIYOSHI, SEKIYA, KANESADA, NOGUCHI, OKINAKA, MATSUO, Kmo, MATSUDA, IKuMA, and OGATA

Table 5. Bacteriological findings in middle ear exudates and nasopharyngeal swabs from children with acute otitis media.

Middle ear exudate Nasopharyngeal swab Bacteria isolated

No. of strains % No. of strains % B. catarrhalis 13 7.5 59 21.3 S. pneumoniae 39 22.4 73 26.4 H. in/luenzae 37 21.4 61 22.0 S. aureus 28 16.2 11 4.3 C. N . S. 36 20 .8 7 2 .5 S. pyogenes 2 1.2 5 1.8 P. aeruginosa 4 2.3 1 0 .4 G. P.R. 11 6.4 36 13 .0 Moraxella sp. 0 0 18 6.5 Yeast like fungi 2 1.2 1 0.4 Others 0.6 4 1.4

Total 173 100 276 100

C. N. S., coagulase negative staphylococci; G. P. R., Gram positive rods.

110 K. SUZUKI et af.

(1986). However, this organism is sometimes isolated from ear and nasal swabs in a condition as if it were isolated from pure cultures (Fig. I), and, on micro­scopy of smears, phagocytosis by the neutrophils is not uncommon findings (Fig. 2). These suggest that B. catarrhalis is definitely pathogenic for humans. The relatively high isolation rates of this organism in our department from the ear (7.5 %) and nasal swabs (21.4 %) of children with acute otitis media clearly show that, as KOVATCH et af. (1983), SHURIN et af. (1983), and LUNDGREN and INGVARS­SON (1986) reported, this organism is an important pathogen in otorhinolaryn­gological infections, particularly in acute sinusitis and acute otitis media in children (Table 5). Since MALMVALL, BRORSON, and JOHNSON (1977) first reported that 3.8 % of the strains of B. catarrhalis were beta-Iactamase producers, KOVATCH et af. (1983) and SHURIN et af. (1983) reported that the percentage of those beta­lactamase producing strains increased up to 76.9 %, and LUNDGREN and INGVARS­SON (1986) and BLUESTONE (1986) reported that those accounted for 75 % of the isolates. MATSUMOTO, NAGATAKE, RIKITOMI, NISHIALA, and WATANABE (1985) reported that the percentage of beta-Iactamase producing strains is rapidly in­creasing every year. From our investigation, out of 107 strains isolated from nasal discharge, 97 strains (90.7 %) were beta-Iactamase producers (INAGAKI et af., 1987). In the present study, the MIC measurement showed that the antibacterial activities of not only penicillins but also cephems such as CCL and CFIX were slightly de­creased against beta-Iactamase producing strains as compared with those against non-producers. However, considering that both penicillins and cephems, with the exception of CEX, inhibited all strains at concentrations of 6.25 ,ugjml or less, the result of the ABPC susceptibility test by the disk method may be evaluated as ( + + ~ + + + ). Because of this unreliability of the disk technique, it appears reasonable, as has been pointed out with Haemophilus injiuenzae (NISHIOKA, 1983), to check the beta-Iactamase production of each strain. Our MIC measurement showed that the antibacterial activities decreased by three to four tubes at increased inoculum sizes. Such diminution in antibacterial activity is thought to be attri­butable to beta-Iactamase production by this organism. Isolation of B. catar­rhalis from upper respiratory tract infections in children is expected to increase to such an extent that the frequency almost parallels those of S. pneumoniae or H. injiuenzae. Considering this and such a high emergence rate of beta-Iactamase producing strains as described above, it is necessary to have adequate knowledge of this species.

REFERENCES

BLUESTONE, C. D.: Otitis media and sinusitis in children. Role of Branhamella catarrhalis. Drugs 31: 132-141, 1986.

BRORSON, J. E., AXELSSON, A., and HOLM, S. E.: Studies on Branhamella catarrhalis (Neisseria catarrhalis) with special reference to maxiIIary sinusitis. Scand. J. Infect. Dis. 8: 151-155, 1976.

B. Catarrhalis IN OUR FIELD 111

BRORSON, J. E., and MALMVALL, B. E.: Branhamella catarrhalis and other bacteria in the na­sopharynx of children with longstanding cough. Scand. J. Infect. Dis. 13: 111-113, 1981.

CATLIN, B. W.: Transfer of the organism named Neisseria catarrhalis to Branhamella gen. Nov. Int. J. Syst. Bacteriol. 20: 155-159, 1970.

COFFY, J. D., BOOTH, H. N., and MARTIN, A. D.: Otitis media in the practice of pediatrics. Bacteriological and clinical observations. Pediatrics 38: 25-32, 1966.

ELIASSON, I., and KAMME, c.: Upper respiratory tract infections ecological and therapeutic aspect of p-lactamase production with special reference to Branhamella catarrhalis. Drugs 31: 116-121,1986.

FROSCH, P., und KOLLE, W.: Die Mikrokokken. In Die Mikroorganismen, (Flugge, C., ed.), II. pp. 154-155, Verlag Von F. C. W. Vogel, Leipzing, 1896.

FUJII, Y.: The revision about the measurement of the minimum inhibitory concentration (MIC). Chemotherapy 29: 76-79,1981.

HIYOSHI, M., SEKIYA, T., KANESADA, K., NOGUCHI, T., OKINAKA, Y. MATSUO, T., KIDO, T., MATSUDA, Y., IKUMA, T., and OGATA, M.: A case report of otitis media caused by Bran­hamella catarrhalis. Clin. Otol. Jpn. 13: 408-409, 1986.

INAGAKI, M., BABA, S., MORI, Y., SUZUKI, K., SHIMADA, J., and SOYANO, K.: Chlinical signifi­cance of staphylococcus sp. in acute otitis media of children. J. Jpn. Soc. Infect. Dis. Otolaryngol. 5: 1-5, 1987.

KAMME, C.: Evaluation of the in vitro sensitivity of Neisseria catarrhalis to antibiotics with respect to acute otitis media. Scand. J. Infect. Dis. 2: 117-120, 1970.

KOVATCH, A. L., WALD, E. R., and MICHAELS, R. H.: p-Lactamase-producing Branhamella catarrhalis causing otitis media in children. J. Pediatr. 102: 261-·264, 1983.

LUNDGREN, K., and INGVARSSON, L.: Acute otitis media in Sweden. Role of Branhamella catarrhalis and the rationale for choice of antimicrobial therapy. Drugs 31: 125-131,1986.

MALMVALL, B. E., BRORSON, J. E., and JOHNSON, I.: In vitro sensitivity to penicillin V and 1'­lactamase production of Branhamella catarrhalis. J. Antimicrobiol. Chemother. 3: 374-375, 1977.

MATSUMOTO, K., NAGATAKE, T., RIKITOMI, N., NISHIALA, M., and WATANABE, K.: Infection with Branhamella catarrhalis. Infect. Inflam. Immun. 15: 147-158, 1985.

MORELLO, J. A., and BOHNHOFF, M.: Neisseria and Branhamella. In Manual of Clinical Micro­biology, IIIrd Ed. (Lennette, E. H., Balows, A., Hausler, W. J., and Truant, J. P., eds.), pp. 111-130, American Society for Microbiology, 1980.

NISHIOKA, K.: Method on testing of Haemophilus. Modern Med. Lab. 11: 1087-1092, 1983. SCHALEN, L., CHRISTENSEN, P., KAMME, C., MIORNER, H., PETTERSSON, K. I., and SCHALEN, C.:

High isolation rate of Branhamella catarrhalis from the nasopharynx in adults with acute laryngitis. Scand. J. Infect. Dis. 12: 227-280, 1980.

SHURIN, P. A., MARCHANT, C. D., KIM, C. H., VAN HARE, G. H., JOHNSON, C. E., TUTIHASI, M. A., and KNAPP, L. J.: Emergence of beta-lactamase-producing strains of Branhamella catarrhalis as important agents of acute otitis media. Pediatr. Infect. Dis. 2: 34-38. 1983.

WALD, E. R., REILLY, J. S., CASSELBRANT, M., LEDESMA-MEDINA, J., MILMOE, G. J., BLUESTONE, C. D., and CHIPONIS, D.: Treatment of acute maxillary sinusitis in childhood: A com­parative study of amoxicillin and cefaclor. J. Pediatr. 104: 297-302, 1984.

Request reprints to: K. Suzuki, M. D., Department of Otorhinolaryngology, Nagoya City University, School of Medicine, 1 Kawasumi Mizuho-cho, Mizuho­ku, Nagoya 467, Japan