Antibiotic resistance in respiratory tract isolates of Haemophilus influenzae and Moraxella catarrhalis collected from across Canada in 1997–1998

George G. Zhanela,b,c,d, James A. Karlowskya,b,d,*, Donald E. Lowe, The Canadian Respiratory Infection Study Group and Daryl J. Hobana,d

Department of Medical Microbiology, a Faculty of Medicine and b Faculty of Pharmacy, University of Manitoba and c Departments of Medicine and d Clinical Microbiology, Health Sciences Centre, Winnipeg, Man. R3A 1R9; e Department of Microbiology, Mt Sinai Hospital, Toronto, Ont. M5G 1XS, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Between September 1997 and November 1998 respiratory tract isolates of Haemophilus influenzae (n = 1352) and Moraxella catarrhalis (n = 428) were collected by 18 Canadian medical centres. ß-Lactamase was produced by 24.0 and 94.2% of H. influenzae and M. catarrhalis isolates, respectively. Resistance rates for H. influenzae were highest for ampicillin (24.0%), trimethoprim/sulphamethoxazole (13.7%), loracarbef (6.1%) and cefaclor (4.2%), and <= 1% for amoxycillin/clavulanate, cefotaxime, cefprozil, cefixime, imipenem, ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin and chloramphenicol. M. catarrhalis resistance rates, derived using NCCLS breakpoint criteria for Haemophilus spp., were <= 1% for all antibiotics tested except ampicillin (49.5%) and trimethoprim/sulphamethoxazole (1.6%).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are important bacterial pathogens in community-acquired respiratory tract infections.1 Effective empirical treatment of these infections is dependent upon continued regional and national surveillance studies and timely reporting. The most notable current trends among respiratory tract isolates in Canada and the USA are the increased isolation of penicillin- and multiple-antibiotic-resistant S. pneumoniae2 and a levelling off of ß-lactamase production rates among isolates of H. influenzae and M. catarrhalis.3

The most recent publication describing ß-lactamase production in Canadian respiratory tract isolates of H. influenzae reported on seven centres participating in the SENTRY antimicrobial surveillance programme.3 In that study 31.3% (75/240) of H. influenzae isolates collected in 1997 were ß-lactamase positive with rates among the seven centres ranging from 21.1 to 48.7%.3 Before that report three other Canadian national surveillance studies of antimicrobial resistance in H. influenzae have been published since 1990.46 Of the H. influenzae isolates collected from 23 centres across Canada in 1992–1993, 28.4% (474/1688) were ß-lactamase positive.4 The proportion of H. influenzae isolates producing ß-lactamase varied considerably by province in that study, ranging from 11.0 to 45.4%.4 A second study performed in 1991, involving eight Canadian centres, reported the prevalence of ß-lactamase production in H. influenzae to be 25.9% (182/702) with no regional variation.5 The third study determined that only 16.9% (424/2503) of H. influenzae isolates collected from 14 Canadian hospitals in 1985–1987 were ß-lactamase positive.6 ß-Lactamase-positive rates in that study varied modestly between hospitals and ranged from 12.8 to 19.6%.6 By comparison the three most recent North American studies, testing isolates collected in 1997, 1994–1995 and 1994–1995, reported ß-lactamase rates in H. influenzae of 34.2% (286/837),3 35.6% (571/1605)7 and 36.1% (822/2278).8 Doern and colleagues have suggested that the prevalence of ß-lactamase production among North American respiratory tract isolates of H. influenzae may have levelled off following the dramatic increases witnessed during the 1980s and early 1990s.3 Resistance to antibiotics other than cefaclor and loracarbef, including cephalosporins, ß-lactam/ß-lactamase inhibitor combinations, macrolides, tetracyclines, chloramphenicol and fluoroquinolones has remained relatively uncommon in H. influenzae in North America.3,7,8

Until recently3 Canadian data describing rates of ß- lactamase production and antibiotic susceptibilities for M. catarrhalis were not available. Recently published data from the SENTRY antimicrobial surveillance programme showed that 93.0% (120/129) of M. catarrhalis isolates collected by seven Canadian centres in 1997 were ß-lactamase positive.3 The two most recent North American studies reported similar rates of ß-lactamase production (92.0, 95.3%) in M. catarrhalis isolates.3,9 At present, resistance to antibiotics other than ampicillin has not emerged as a significant concern with M. catarrhalis.3 The purpose of the present study was to determine the prevalence of ß-lactamase production and antibiotic resistance in respiratory tract isolates of H. influenzae and M. catarrhalis recently collected in major population centres across Canada.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolate collection

Between September 1997 and November 1998, 18 Canadian hospital laboratories (Table IGo) collected lower respiratory tract isolates of H. influenzae (n = 100) and M. catarrhalis (n = 50). Isolates must have been deemed clinically significant by a site's current protocols to be accepted into the study. Only one specimen was considered per patient. All isolates were sent to the co-ordinating laboratory, the Health Sciences Centre (Winnipeg, Manitoba, Canada), on Amies charcoal swabs. The co-ordinating laboratory confirmed the identity of each isolate of H. influenzae by colonial morphology, Gram stain characteristics and X & V factor requirements. Similarly, colonial morphology and Gram stain, as well as oxidase and DNase production were used by the co-ordinating laboratory to confirm the identity of each isolate of M. catarrhalis. Production of ß-lactamase was assessed by use of the Cefinase disc test (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). In total, 1352 H. influenzae and 428 M. catarrhalis isolates were available for antibiotic susceptibility testing. Each isolate was stocked in skim milk and stored at –80°C in preparation for antibiotic susceptibility testing by the co-ordinating laboratory.


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Table I. Prevalence of ß-lactamase production in isolates of H. influenzae and M. catarrhalis collected across Canada from September 1997 to November 1998
 
Antibiotic susceptibility testing

The NCCLS M7-A4 microbroth dilution method10 was used to determine isolate susceptibilities to ampicillin, amoxycillin/clavulanate, cefotaxime, cefuroxime, cefaclor, cefprozil, loracarbef, cefixime, imipenem, erythromycin, azithromycin, clarithromycin, tetracycline, ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin, moxifloxacin, trimethoprim/sulphamethoxazole and chloramphenicol. Antibiotics were obtained from their respective manufacturers, purchased from the Sigma Chemical Company (St Louis, MO, USA) or supplied by Dade–MicroScan (Sacramento, CA, USA). Dehydrated microbroth 96-well panels were prepared commercially by Dade–MicroScan and contained doubling antibiotic dilutions encompassing NCCLS recommended interpretative breakpoints.10 Haemophilus test medium (PML Laboratories, Wilsonville, OR, USA) and cation-adjusted (Ca2+, 25 mg/L; Mg2+, 12.5 mg/L) Mueller–Hinton broth (PML Laboratories) were the susceptibility testing media used for H. influenzae and M. catarrhalis, respectively.10 Isolates were sub-cultured twice on to blood or chocolate agar from –80°C skim milk stocks before MIC testing. Panels were inoculated with bacteria to achieve a final concentration of approximately 5 x 105 cfu/mL in 100 µL and incubated at 35°C in ambient air for 24 h before reading. The MIC was defined as the lowest concentration of antibiotic inhibiting visible growth.10 MICs were interpreted using NCCLS recommended breakpoints.11 Colony counts were performed periodically to confirm initial inocula. Quality control organisms H. influenzae ATCC 49247 and ATCC 49766 were tested weekly.10


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 1352 isolates of H. influenzae tested 324 (24.0%) were ß-lactamase positive (Table IGo). The eastern provinces (Prince Edward Island, Nova Scotia, New Brunswick) generally demonstrated lower ß-lactamase-positive rates (range 14.4–20.0%) than the central (Quebec, Ontario) (range 25.1–30.4%) and western provinces (Manitoba, Saskatchewan, Alberta, British Columbia) (range 20.1–30.7%).

Antibiotic susceptibilities of the 1352 isolates of H. influenzae are presented in Table IIGo. Isolates were stratified by the presence or absence of ß-lactamase production and MICs are presented as antibiotic concentrations inhibiting 50 and 90% of isolates and MIC ranges (Table IIGo). NCCLS interpretative breakpoints11 were used to divide isolates into per cent susceptible and per cent resistant groups (Table IIGo). The ampicillin resistance rate (24.0%) in H. influenzae (Table IIGo) correlated with the ß-lactamase-positive rate (24.0%) (Table IGo). ß-Lactamase-positive ampicillin-susceptible isolates of H. influenzae were not identified (Table IIGo). One ß-lactamase-negative ampicillin-resistant (BLNAR) isolate of H. influenzae was detected. For this isolate, MICs of ampicillin, amoxycillin/clavulanate, cefotaxime, cefuroxime, cefaclor, cefprozil, loracarbef and cefixime of 8, 8/4, 0.06, 4, 16, 4, 8 and 0.25 mg/L, respectively, were required. A single ß-lactamase-positive amoxycillin/clavulanate-resistant (BLPACR) isolate of H. influenzae was also identified. For this isolate, MICs of ampicillin, amoxycillin/ clavulanate, cefotaxime, cefuroxime, cefaclor, cefprozil, loracarbef and cefixime of 8, 8/4, 0.06, 4, 16, 16, 16 and 0.25 mg/L, respectively, were required. The MICs for the BLNAR and BLPACR isolates were confirmed by repeat susceptibility testing.


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Table II. Antibiotic susceptibilities of 1352 isolates of H. influenzaea stratified by the presence or absence of ß-lactamase production
 
Among the cephalosporins tested cefaclor and loracarbef were the least active against H. influenzae with overall resistance rates of 4.2 and 6.1%, respectively (Table IIGo). Cefuroxime, cefixime, cefprozil and cefotaxime resistance rates for H. influenzae were lower than those for cefaclor and loracarbef, being 1.7, 1.0, 0.7 and 0.7%, respectively. For loracarbef, cefaclor and cefprozil 14.9, 8.8 and 5.9% of isolates were intermediately resistant.11 Cefaclor, loracarbef and cefprozil resistance rates were at least two-fold greater among ß-lactamase-positive H. influenzae compared with ß-lactamase-negative isolates (Table IIGo). The activities of cefotaxime, cefuroxime and cefixime appeared unencumbered by the presence of ß-lactamase. Imipenem was active against all isolates of H. influenzae tested (Table IIGo).

Of the H. influenzae isolates tested, 2.7 and 1.7% were resistant to azithromycin and clarithromycin, respectively (Table IIGo).11 As interpretative criteria are not available for erythromycin, only MIC50 and MIC90 information is presented in Table IIGo. Tetracycline and chloramphenicol resistance rates were 1.9 and 0.2%, respectively. Resistance rates for the fluoroquinolones were also low and ranged from 0 to 0.3% (Table IIGo). In total, four isolates of H. influenzae with ciprofloxacin MICs of 2 mg/L or greater were identified. Elevated MICs of the other fluoroquinolones tested were also demonstrated for all four isolates. One of the four ciprofloxacin-resistant isolates was also resistant to levofloxacin, grepafloxacin and trovafloxacin11 and demonstrated a moxifloxacin MIC of 2 mg/L. ß-Lactamase status did not appear to affect significantly the activity of macrolides, amoxycillin/clavulanate, cefuroxime, tetracycline, ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin and moxifloxacin against H. influenzae (Table IIGo). Trimethoprim/sulphamethoxazole resistance was identified in 13.7% of H. influenzae isolates, with approximately twice as many ß-lactamase-positive isolates (20.4%) demonstrating trimethoprim/sulphamethoxazole resistance as ß-lactamase-negative isolates (11.4%) (Table IIGo).

Of the 428 isolates of M. catarrhalis, 403 (94.2%) were ß-lactamase positive (Table IGo). ß-Lactamase rates ranged from 84.1% in Alberta to 100% in Saskatchewan (Table IGo). Regional differences in M. catarrhalis ß-lactamase production were not apparent (Table IGo). The in vitro activities of antibiotics against M. catarrhalis are given in Table IIIGo. The NCCLS currently does not provide MIC interpretative breakpoints for M. catarrhalis. The M. catarrhalis susceptibility testing data presented here were interpreted using Haemophilus spp. breakpoints.9 Other studies have interpreted M. catarrhalis MICs using NCCLS breakpoints for non-fastidious bacteria that grow well on unsupplemented Mueller–Hinton medium.3 The ampicillin resistance rate, using Haemophilus spp. breakpoints,11 was 49.5% with 30.6% of M. catarrhalis isolates susceptible to ampicillin (Table IIIGo). Isolates resistant to amoxycillin/ clavulanate, cefotaxime, cefprozil, azithromycin, clarithromycin, ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin and chloramphenicol were not identified (Table IIIGo). Resistance rates were less than 1% for cefuroxime, cefaclor, loracarbef, cefixime and tetracycline (Table IIIGo). Trimethoprim/sulphamethoxazole resistance was detected in 1.6% of M. catarrhalis isolates (Table IIIGo).


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Table III. Antibiotic susceptibilities of 428 isolates of M. catarrhalisa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Out-patient respiratory tract infections are commonly treated with empirical antibiotic therapy. The efficacy of this practice relies partially upon the access physicians have to current, accurate antibiotic surveillance data. The four most recent reports on the prevalence of ß-lactamase production among Canadian isolates of H. influenzae have reported rates of 31.3,3 28.4,4 26.05 and 16.9%6 based upon isolates collected during 1997, 1992–1993, 1991 and 1985–1987, respectively. In our study, 24.0% of H. influenzae isolates collected during 1997–1998 produced ß-lactamase, a result similar to those reported from isolates collected in 1992–1993 and 19914,5 but considerably lower than the rate reported by the SENTRY surveillance programme.3 Attributing significance to the differences between the ß-lactamase-positive rates determined regionally and nationally in the present study and those reported previously35 is difficult because of anomalies in sample size, collection periods, participating centres, restructuring of laboratory services within certain cities and provinces, and patient demographics. If the results of the present study are considered in the absence of the SENTRY surveillance programme,3 the data suggest that the prevalence of ß-lactamase production in Canada has levelled off between 1990 and 1997–1998 at approximately 24.0–28.4%. Doern et al. arrived at a similar conclusion concerning ß-lactamase rates in North America in general.3 The most notable difference between the present investigation (18 centres, n = 1352) and the recent SENTRY surveillance programme study (seven centres, n = 240)3 is sample size. Ampicillin-resistant ß-lactamase-negative isolates and amoxycillin/clavulanate-resistant ß-lactamase-positive isolates were uncommon, with rates of 0.1 and 0.3%, respectively, and considerably lower than previously reported in the USA.7

In the present study 13.7, 6.1 and 4.2% of H. influenzae isolates were resistant to trimethoprim/sulphamethoxazole, loracarbef and cefaclor, respectively (Table IIGo). The SENTRY surveillance programme also determined elevated trimethoprim/sulphamethoxazole and cefaclor resistance rates of 16.2 and 12.8%, respectively, for 240 isolates of H. influenzae from seven centres in Canada in 1997.3 Trimethoprim/sulphamethoxazole, loracarbef and cefaclor resistance rates in Canada have increased from levels in 1992–1993, when H. influenzae isolates were reported to be susceptible to these antibiotics at rates of 96.8–99.3, 97.9–100 and 93.7–94.7%, respectively.4 Resistance rates were higher to trimethoprim/sulphamethoxazole, loracarbef, cefaclor and cefprozil in ß-lactamase-positive than in ß-lactamase-negative isolates (Table IIGo). Of the ß-lactams tested, cefotaxime and cefixime were the most active ß- lactams against both ß-lactamase-positive and ß-lactamase-negative isolates of H. influenzae. However, the clinical implications of in vitro resistance to oral antibiotics, specifically cephalosporins, used to treat localized respiratory tract infections arising from H. influenzae has been questioned and remains to be fully elucidated.1214

The rate of ß-lactamase production among isolates of M. catarrhalis was similar to that reported previously by others.4,5 Ampicillin resistance, using Haemophilus spp. breakpoints11, was 49.5% with 30.6% of M. catarrhalis isolates susceptible to ampicillin (Table IIIGo). Applying the ampicillin-susceptible breakpoint for non-fastidious bacteria that grow well on unsupplemented Mueller–Hinton medium (<=8 mg/L),3,11 all isolates of M. catarrhalis would have been categorized as ampicillin susceptible. ß-Lactamase-positive M. catarrhalis harbour BRO ß-lactamases (BRO-1, BRO-2, BRO-3) and have been demonstrated to hydrolyse penicillin, ampicillin and amoxycillin.9 For 106 (26.3%) of the 403 ß-lactamase-positive isolates, the ampicillin MICs were <=1 mg/L. It is likely that these isolates produced the BRO-2 enzyme, which is known to be associated with low ampicillin MICs as a result of modest amounts of enzyme produced and low substrate affinity.15,16 A relationship between the production of BRO ß-lactamases by M. catarrhalis and clinical failures in patients treated with ß-lactams has not been determined.9

In conclusion, 24.0% of H. influenzae and 94.2% of M. catarrhalis isolates from across Canada in 1997–1998 produced ß-lactamase. ß-Lactamase production correlated with ampicillin resistance in isolates of H. influenzae but not M. catarrhalis principally because of the different ß- lactamase enzymes present in each species. H. influenzae demonstrated appreciable resistance to ampicillin (24.0% of isolates), trimethoprim/sulphamethoxazole (13.7% of isolates), loracarbef (6.1% of isolates) and cefaclor (4.2% of isolates). Isolates of M. catarrhalis were widely susceptible to all antibiotics tested other than ampicillin.


    Acknowledgments
 
The authors would like to thank each participating centre and their respective investigator for their support: Victoria General Hospital, Victoria, British Columbia, Dr P. Kibsey; Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, Dr D. Roscoe; Calgary Laboratory Services, Calgary, Alberta, Dr A. Gibb; University of Alberta Hospital, Edmonton, Alberta, Dr R. Rennie; Regina General Hospital, Regina, Saskatchewan, Dr E. Thomas; Royal University Hospital, Saskatoon, Saskatchewan, Dr J. Blondeau; St Boniface General Hospital, Winnipeg, Manitoba, Dr G. Harding; Health Sciences Centre, Winnipeg, Manitoba, Dr D. Hoban; St Joseph's Hospital, Hamilton, Ontario, Dr D. Groves; Hamilton Health Sciences Centre, Hamilton, Ontario, Dr F. Smaill; London Health Sciences Centre, London, Ontario, Dr Z. Hussain; Mount Sinai Hospital, Toronto, Ontario, Dr D. Low; Centre Universitaire de Santé de l'Estrie, Sherbrooke, Quebec, Dr J. Dubois; Maisonneuve–Rosemont, Montreal, Quebec, Dr M. Laveridière; Montreal General Hospital, Montreal, Quebec, Dr V. Loo; South East Health Care Corporation, Moncton, New Brunswick, Dr M. Kuhn; QEII Health Sciences Centre, Halifax, Nova Scotia, Dr K. Forward; and Queen Elizabeth Hospital, Charlottetown, Prince Edward Island, Dr L. Abbott. The authors would also like to gratefully acknowledge the financial support of Glaxo–Wellcome and Janssen–Ortho.


    Notes
 
* Correspondence address. Department of Clinical Microbiology, Health Sciences Centre, MS673, 820 Sherbrook Street, Winnipeg, Man. R3A 1R9, Canada. Tel: +1-204-787-4683; Fax: +1-204-787-4699; E-mail: jkarlowsky{at}hsc.mb.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Jorgensen, J. H., Doern, G. V., Maher, L. A., Howell, A. W. & Redding, J. S. (1990). Antimicrobial resistance among respiratory isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United States. Antimicrobial Agents and Chemotherapy 34, 2075–80.[ISI][Medline]

2 . Doern, G. V., Pfaller, M. A., Kugler, K., Freeman, J. & Jones, R. N. (1998). Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program. Clinical Infectious Diseases 27, 764–70.[ISI][Medline]

3 . Doern, G. V., Jones, R. N., Pfaller, M. A. & Kugler, K. (1999). Haemophilus influenzae and Moraxella catarrhalis from patients with community-acquired respiratory tract infections: antimicrobial susceptibility patterns from SENTRY antimicrobial surveillance program (United States and Canada, 1997). Antimicrobial Agents and Chemotherapy 43, 385–9.[Abstract/Free Full Text]

4 . Scriver, S. R., Walmsley, S. L., Kau, C. L., Hoban, D. J., Brunton, J., McGeer, A. et al. (1994). Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Canadian Haemophilus Study Group. Antimicrobial Agents and Chemotherapy 38, 1678–80.[Abstract]

5 . Scriver, S. R., Low, D. E., Simor, A. E., Toye, B., McGeer, A. & Jaeger, R. (1992). Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group. Journal of Clinical Microbiology 30, 2284–9.[Abstract]

6 . Tremblay, L. D., L'Ecuyer, J., Provencher, P. & Bergeron, M. G. (1990). Susceptibility of Haemophilus influenzae to antimicrobial agents used in Canada. Canadian Haemophilus Study Group. Canadian Medical Association Journal 143, 895–901.[Abstract]

7 . Doern, G. V., Brueggemann, A. B., Pierce, G., Hogan, T., Holley, H. P., Jr & Rauch, A. (1996). Prevalence of antimicrobial resistance among 723 outpatient clinical isolates of Moraxella catarrhalis in the United States in 1994 and 1995: results of a 30-center national surveillance study. Antimicrobial Agents and Chemotherapy 40, 2884–6.[Abstract]

8 . Jones, R. N., Jacobs, M. R., Washington, J. A. & Pfaller, M. A. (1997). A 1994–95 survey of Haemophilus influenzae susceptibility to ten orally administered agents. A 187 clinical laboratory center sample in the United States. Diagnostic Microbiology and Infectious Diseases 27, 75–83.[ISI][Medline]

9 . Doern, G. V., Brueggemann, A. B., Pierce, G., Holley, H. P., Jr & Rauch, A. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of ß-lactamase-positive strains resistant to amoxicillin– clavulanate: results of a national multicenter surveillance study. Antimicrobial Agents and Chemotherapy 41, 292–7.[Abstract]

10 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

11 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing—Ninth Informational Supplement M100-S9. NCCLS, Villanova, PA.

12 . Dagan, R., Abramson, O., Leibovitz, E., Greenberg, D., Lang, R., Goshen, S. et al. (1997). Bacteriologic response to oral cephalosporins: are established susceptibility breakpoints appropriate in the case of acute otitis media? Journal of Infectious Diseases 176, 1253–9.[ISI][Medline]

13 . Doern, G. V. (1992). In vitro activity of loracarbef and effects of susceptibility test methods. American Journal of Medicine 92, Suppl. 6A, 75–155.

14 . Doern, G. V. (1994). Does there exist a rational, objective in vitro basis for the management of selected infections of the respiratory tract? Infectious Diseases and Clinical Practice 3, 75–80.

15 . Farmer, T. & Reading, C. (1982). ß-Lactamases of Branhamella catarrhalis and their inhibition by clavulanic acid. Antimicrobial Agents and Chemotherapy 21, 506–8.[ISI][Medline]

16 . Stobberingh, E. E., van Eck, H. J., Houben, A. W. & van Boven, C. P. A. (1986). Analysis of the relationship between ampicillin resistance and ß-lactamase production in Branhamella catarrhalis. Drugs 31, Suppl. 3, 23–7.[ISI][Medline]

Received 8 October 1999; returned 25 November 1999; revised 16 December 1999; accepted 30 December 1999