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
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Abstract |
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Introduction |
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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 19921993, 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 19851987 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, 19941995 and 19941995, 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.
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Materials and methods |
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Between September 1997 and November 1998, 18 Canadian hospital laboratories (Table I) 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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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 DadeMicroScan (Sacramento, CA, USA). Dehydrated microbroth 96-well panels were prepared commercially by DadeMicroScan 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) MuellerHinton 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
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Results |
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Antibiotic susceptibilities of the 1352 isolates of H. influenzae are presented in Table II. 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 II
). NCCLS interpretative breakpoints11 were used to divide isolates into per cent susceptible and per cent resistant groups (Table II
). The ampicillin resistance rate (24.0%) in H. influenzae (Table II
) correlated with the ß-lactamase-positive rate (24.0%) (Table I
). ß-Lactamase-positive ampicillin-susceptible isolates of H. influenzae were not identified (Table II
). 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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Of the H. influenzae isolates tested, 2.7 and 1.7% were resistant to azithromycin and clarithromycin, respectively (Table II).11 As interpretative criteria are not available for erythromycin, only MIC50 and MIC90 information is presented in Table II
. 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 II
). 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 II
). 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 II
).
Of the 428 isolates of M. catarrhalis, 403 (94.2%) were ß-lactamase positive (Table I). ß-Lactamase rates ranged from 84.1% in Alberta to 100% in Saskatchewan (Table I
). Regional differences in M. catarrhalis ß-lactamase production were not apparent (Table I
). The in vitro activities of antibiotics against M. catarrhalis are given in Table III
. 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 MuellerHinton 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 III
). Isolates resistant to amoxycillin/ clavulanate, cefotaxime, cefprozil, azithromycin, clarithromycin, ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin and chloramphenicol were not identified (Table III
). Resistance rates were less than 1% for cefuroxime, cefaclor, loracarbef, cefixime and tetracycline (Table III
). Trimethoprim/sulphamethoxazole resistance was detected in 1.6% of M. catarrhalis isolates (Table III
).
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Discussion |
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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 II). 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 19921993, when H. influenzae isolates were reported to be susceptible to these antibiotics at rates of 96.899.3, 97.9100 and 93.794.7%, respectively.4 Resistance rates were higher to trimethoprim/sulphamethoxazole, loracarbef, cefaclor and cefprozil in ß-lactamase-positive than in ß-lactamase-negative isolates (Table II
). 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 III). Applying the ampicillin-susceptible breakpoint for non-fastidious bacteria that grow well on unsupplemented MuellerHinton 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 19971998 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.
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Acknowledgments |
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Notes |
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References |
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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, 167880.[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, 22849.[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, 895901.[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, 28846.[Abstract]
8 . Jones, R. N., Jacobs, M. R., Washington, J. A. & Pfaller, M. A. (1997). A 199495 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, 7583.[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, 2927.[Abstract]
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Received 8 October 1999; returned 25 November 1999; revised 16 December 1999; accepted 30 December 1999