J. M. Blondeau*, M. Suter, S. Borsos and the Canadian Antimicrobial Study Group

Journal of Antimicrobial Chemotherapy (1999) 43, Suppl. A, 25 30 JAC Determination of the antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis J. M. Blondeau*, M. Suter, S. Borsos and the Canadian Antimicrobial Study Group Division of Clinical Microbiology, Saskatoon District Health and St Paul s Hospital (Grey Nuns) and the Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada The susceptibility of Canadian isolates of three respiratory tract pathogens (Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae) to several antimicrobial agents were tested by two different methods. β-lactamase was produced by 68/211 (32.2%) of H. influenzae isolates and 64/75 (85.3%) of M. catarrhalis isolates. For S. pneumoniae, 19/156 (12.2%) isolates were resistant to penicillin (MIC 0.12 mg/l) and two isolates had MICs of 1.5 mg/l. For some combinations of agents and organisms, different methods gave different values for the proportion of isolates susceptible. Regardless of methodology, for H. influenzae, the most active antimicrobials based on proportion of strains susceptible were ciprofloxacin (100%) and cefpodoxime (98.5 100%). For M. catarrhalis, the most active agents were azithromycin, cefaclor, cefixime, cefpodoxime, cefuroxime, ciprofloxacin, clarithromycin and loracarbef (100% each); the least active was ampicillin. Against penicillin-sensitive and -resistant pneumococci, the activity was not significantly different for azithromycin and clarithromycin (93.4 100%) and ciprofloxacin (MIC 90 2.0 and 1.5 mg/l, respectively) but was different for cefuroxime (99.3% and 31.6%, respectively), cefaclor (MIC 90 0.75 and 256 mg/l, respectively), cefpodoxime (MIC 90 0.047 and 1.5 mg/l, respectively) and loracarbef (MIC 90 0.75 and 256 mg/l, respectively). This study indicates the increasing incidence, in Canada, of β-lactamase resistance in H. influenzae and M. catarrhalis and penicillin resistance in S. pneumoniae. Introduction Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are responsible for systemic as well as upper and lower respiratory tract infections including otitis media, sinusitis and community-acquired pneumonia. 1 3 In addition, H. influenzae and S. pneumoniae are frequently implicated as bacterial causes of meningitis, although the incidence of the former is decreasing. Treatment of these potentially serious infections has become problematic because of increasing global antimicrobial resistance. 4 6 Surveillance studies to detect and monitor antimicrobial resistance are now, therefore, needed. Recent publications suggest increasing resistance amongst H. influenzae, S. pneumoniae and M. catarrhalis to a variety of antimicrobial agents. 1,2,7 10 The methodology and interpretation used for susceptibility testing may influence the results and, therefore, resistance rates for some pathogens and antimicrobial agents. 4,11 In this study, we report the in-vitro susceptibilities of these commonly isolated respiratory pathogens, collected from Canadian laboratories, to several currently available antimicrobial agents using two different methods. Materials and methods Bacterial strains Isolates of H. influenzae, S. pneumoniae and M. catarrhalis from clinical specimens were collected from community and teaching hospitals during 1993 and 1994. 12 All isolates were sent to the study coordinator (J.M.B.) and tested at St *Corresponding address: Department of Clinical Microbiology, Royal University Hospital, 103 Hospital Drive, Saskatoon, Saskatchewan, Canada, S7N 0W8. Tel: 1-306-655-6943; Fax: 1-306-655-6947; E-mail: blondeauj@sdh.sk.ca See Acknowledgements. 1999 The British Society for Antimicrobial Chemotherapy 25

J. M. Blondeau et al. Paul s Hospital, Saskatchewan, Canada. Isolates were identified as H. influenzae, S. pneumoniae or M. catarrhalis by standard methods and frozen at 70 C in skimmed milk or on latex beads (Pro Lab Diagnostics, Toronto, Canada) until testing. Before susceptibility testing, isolates were subcultured on to chocolate agar plates (PML, Richmond, Canada) and incubated at 37 C in 5% CO 2 for 16 18 h. Duplicate isolates from the same patients were excluded. Susceptibility testing All isolates of H. influenzae and M. catarrhalis were tested for β-lactamase production with a nitrocefinase disc test (Cefinase; BBL Microbiology Systems, Cockeysville, MD, USA). MICs for all isolates were determined by two different methods: Etest (AB Biodisk, Solna, Sweden) and Kirby Bauer disc diffusion methodology. For both methods, a suspension of the test organism equivalent to a 0.5 McFarland standard was prepared and 150 mm diameter Mueller Hinton agar plates, supplemented with sheep blood were inoculated, using a non-toxic swab, to produce confluent growth. Etest strips or discs containing the antimicrobial agent were placed on the inoculated plates with sterile forceps. For isolates of H. influenzae and M. catarrhalis, susceptibility to ampicillin, azithromycin, cefaclor, cefixime, cefpodoxime, cefuroxime, clarithromycin, loracarbef and ciprofloxacin was determined. The susceptibility of S. pneumoniae to azithromycin, cefaclor, cefixime, cefpodoxime, cefuroxime, clarithromycin, loracarbef, oxacillin, penicillin and ciprofloxacin was determined. The plates were incubated for 18 24 h at 35 C in 5% CO 2. For the Etest, MICs were recorded at the point where growth of the micro-organism intersected the Etest strip. For the Kirby Bauer method, the diameter of the zone of inhibition was measured. Susceptibility was defined as the percentage of isolates that were susceptible at the established NCCLS breakpoint. 13 Isolates with intermediate susceptibility were included with resistant isolates. For S. pneumoniae, MICs of penicillin of 0.12 mg/l were categorized as resistant. The following strains were used for quality control and tested each time susceptibility testing was performed: S. pneumo - niae ATCC 49619, H. influenzae ATCC 49766 and 49247, and M. catarrhalis ATCC 25238. Results The susceptibilities of 211 H. influenzae, 156 S. pneumoniae and 75 M. catarrhalis isolates were determined for the selected antimicrobial agents by Etest or the Kirby Bauer method. Table I shows the antimicrobial susceptibilities of β-lactamase-positive and -negative isolates of H. influen - zae. Of the 211 isolates, 68 (32.2%) were β-lactamasepositive. Of the 143 β-lactamase-negative isolates, 11 (7.7%) were judged to be resistant to ampicillin by the Kirby Bauer method ( 18 mm diameter) and seven (4.9%) by the Etest (MIC 4 mg/l). Of the β-lactams Table I. Antimicrobial susceptibility of 211 Haemophilus influenzae isolates collected from across Canada Etest MIC (mg/l) Susceptibility (%) Antimicrobial β-lactamase agent production n 50% 90% range Etest Kirby Bauer Ampicillin positive 68 256 256 0.125 256 59 2.9 negative 143 0.38 0.75 0.019 256 95.1 92.3 Azithromycin positive 68 3.0 6.0 0.125 8.0 85.3 100 negative 143 3.0 4.0 0.38 12.0 93.0 100 Cefaclor positive 68 4.0 12.0 0.75 256 86.8 86.8 negative 143 3.0 8.0 0.19 256 93.0 90.9 Cefixime positive 68 ND ND ND ND 95.6 negative 143 ND ND ND ND 97.9 Cefpodoxime positive 68 0.064 0.125 0.023 256 100 98.5 negative 143 0.064 0.125 0.016 0.32 100 99.3 Cefuroxime positive 68 1.0 2.0 0.25 6.0 98.5 98.5 negative 143 1.0 2.0 0.25 12.0 99.3 97.9 Clarithromycin positive 68 8.0 24.0 0.25 96.0 73.5 70.6 negative 143 8.0 16.0 0.5 64.0 77.6 83.2 Loracarbef positive 68 2.0 8.0 0.5 256 98.5 94.1 negative 143 1.5 6.0 0.38 64.0 97.2 97.2 Ciprofloxacin positive 68 0.016 0.023 0.008 0.064 100 100 negative 143 0.016 0.023 0.006 0.094 100 100 ND, Not done. 26

Susceptibilities of Canadian isolates tested, cefuroxime and cefpodoxime were the most active agents against both enzyme-producing and non-enzymeproducing isolates of H. influenzae. Excluding ampicillin and cefaclor, all β-lactams had resistance rates of 3.4%. The presence of β-lactamase in H. influenzae isolates had little effect on the in-vitro activities of azithromycin and clarithromycin. However, there were several differences between the results of the Etest and those of the Kirby Bauer method in terms of susceptibility to azithromycin and clarithromycin. Against β-lactamase-positive isolates, azithromycin susceptibility rates were found to be 85.3% and 100% by the Etest and Kirby Bauer method respectively, while the corresponding values for clarithromycin were 73.5% and 70.6%, respectively. A similar trend was seen for β-lactamase-negative isolates: azithromycin susceptibility was 93.0% and 100% according to the Etest and the Kirby Bauer method, respectively, while the values for clarithromycin were 77.6% and 83.2%, respectively. All isolates were highly susceptible (100%) to ciprofloxacin by both methods. Ten antimicrobial agents were tested against the S. pneu - moniae isolates (Table II). Using the Kirby Bauer method, 12.2% had elevated MICs of oxacillin. In addition, 12.2% of isolates were determined to be penicillin-resistant by the Etest. High-level ( 2.0 mg/l) penicillin resistance was not detected, although two (10.5%) of 19 isolates had MICs of 1.5 mg/l. Similar discrepancies between methods were also noted for all other β-lactam antimicrobials tested. Susceptibility results were more consistent between the Etest and Kirby Bauer methodologies for clarithromycin (97.8% and 96.9%, respectively), and azithromycin (93.4% and 96.9%, respectively), when tested against penicillin-sensitive isolates. For penicillin-resistant isolates, susceptibility to both azithromycin and clarithromycin was 100% and 96.6% as determined by the Etest and the Kirby Bauer method, respectively. Of the penicillin-sensitive isolates, 99.3% were susceptible to cefuroxime as determined by the Etest. For penicillin-resistant isolates, cefpodoxime was the β- lactam agent that retained good activity (Etest); the others had MIC 90 s ranging from 4.0 to 256 mg/l. The Kirby Bauer method failed to detect differences in resistance rates between penicillin-sensitive and penicillin-resistant isolates and cross-resistance to other agents when compared with the Etest. There was no difference in the activity of ciprofloxacin against penicillin-sensitive or penicillin-resistant isolates when using the Etest. Nine antimicrobial agents were tested against 75 isolates of M. catarrhalis (Table III). Overall, 85% of the isolates were β-lactamase-positive. According to the Etest, all of the β-lactamase-positive isolates were susceptible to all of the agents except ampicillin. The Kirby Bauer test indicated 95.3% susceptibility to cefuroxime, and 100% susceptibility for the remaining agents. There was little Table II. Antimicrobial susceptibility of 156 Streptococcus pneumoniae isolates collected from across Canada Etest MIC (mg/l) Susceptibility (%) Antimicrobial Penicillin agent susceptibility n 50% 90% range Etest Kirby Bauer Azithromycin sensitive 137 0.38 0.75 0.019 32.0 93.4 96.9 resistant 19 0.5 0.75 0.19 0.75 100 96.6 Cefaclor sensitive 137 0.38 0.75 0.094 2.0 NA NA resistant 19 64 256 0.38 256 NA NA Cefixime sensitive 137 ND ND ND ND NA resistant 19 ND ND ND ND NA Cefpodoxime sensitive 137 0.023 0.047 0.016 1.0 NA NA resistant 19 1.0 1.5 0.032 2.0 NA NA Cefuroxime sensitive 137 0.023 0.25 0.016 2.0 99.3 NA resistant 19 2.0 4.0 0.125 4.0 31.6 NA Clarithromycin sensitive 137 0.064 0.094 0.016 64.0 97.8 96.9 resistant 19 0.047 0.047 0.023 0.094 100 96.6 Loracarbef sensitive 137 0.38 0.75 0.094 256 NA NA resistant 19 64.0 256 0.5 256 NA NA Oxacillin sensitive 137 0.094 0.19 0.008 8.0 NA 100 resistant 19 8.0 16.0 0.064 24.0 NA? Penicillin sensitive 137 0.016 0.032 0.004 0.094 100 100 resistant 19 0.75 1.0 0.125 1.5 0 0 Ciprofloxacin sensitive 137 1.0 2.0 0.012 32 NA NA resistant 19 1.5 1.5 0.75 4.0 NA NA ND, not done; NA, no approved NCCLS breakpoint. 27

J. M. Blondeau et al. Table III. Antimicrobial susceptibility of 75 Moraxella catarrhalis isolates collected from across Canada Etest MIC (mg/l) Susceptibility (%) Antimicrobial β -Lactamase Agent production n 50% 90% range Etest Kirby Bauer Ampicillin positive 64 0.75 2.0 0.094 8.0 37.5 NA negative 11 0.016 0.016 0.016 0.094 100 NA Azithromycin positive 64 0.125 0.125 0.016 0.25 100 100 negative 11 0.094 0.19 0.032 0.75 100 100 Cefaclor positive 64 1.0 1.5 0.75 6.0 100 100 negative 11 0.38 0.5 0.016 0.75 100 100 Cefixime positive 64 ND ND ND ND 100 negative 11 ND ND ND ND 100 Cefpodoxime positive 64 0.75 1.0 0.19 1.0 100 100 negative 11 0.125 0.125 0.016 0.125 100 100 Cefuroxime positive 64 1.5 2.0 0.5 4.0 100 95.3 negative 11 0.38 0.38 0.023 0.5 100 100 Clarithromycin positive 64 0.125 0.25 0.016 0.5 100 100 negative 11 0.094 0.25 0.016 0.38 100 100 Loracarbef positive 64 0.75 1.5 0.19 4.0 100 100 negative 11 0.19 0.25 0.016 0.25 100 100 Ciprofloxacin positive 64 0.047 0.064 0.023 0.25 100 100 negative 11 0.047 0.064 0.047 0.125 100 100 ND, not done; NA, no approved NCCLS breakpoint available. evidence of non-β-lactamase-mediated resistance since the susceptibilities of β-lactamase-negative isolates tested were similar to those of β-lactamase-positive isolates. All β-lactamase-positive and -negative isolates were susceptible to ciprofloxacin by both methods. Discussion Trembley et al. 14 previously reported on the incidence of H. influenzae resistance to antimicrobial agents across Canada. Of isolates collected in 1985 1987, 16.9% were β- lactamase-positive, varying from 12.8% to 19.6% in different regions of Canada. In total, 19.3% of H. influenzae isolates were resistant to ampicillin, 3.8% to erythromycin, 1.4% to cefaclor and 0.7% to cefuroxime. The newer macrolide/azalide agents were not tested in the aforementioned study. Scriver et al. 1 reported on the susceptibility of H. influenzae isolates to 11 antimicrobial agents collected in 1992 and 1993 from 23 Canadian medical centres. Of 1688 isolates tested, 28.4% were β-lactamase-positive, and five (0.4%) isolates that were β-lactamase-negative were resistant to ampicillin. In the same study, resistance to cephalosporins was 3.5%, while erythromycin resistance was 90%. Hoban et al. 15 determined antimicrobial susceptibility to 263 H. influenzae isolates from 18 Canadian medical centres. They reported that 29% of isolates were resistant to ampicillin, and 12% to cefaclor. Hoban et al. 16 tested 1000 H. influenzae isolates from five medical centres in the USA and Canada and found that 33% were resistant to ampicillin, 10% to cefaclor and 1% to cefixime. In our study, we found that 32.2% of isolates were β-lactamasepositive and that from seven to 11 of the β-lactamasenegative isolates were ampicillin-resistant, the actual number depending on the susceptibility testing method used. Resistance to cephalosporins (except cefaclor), was 3.4%, a finding identical to that of Scriver et al.; 1 resistance to loracarbef was similar to that of the cephalosporins. Additionally, the newer macrolides had resistance rates ranging from 0% to 29.4% depending on the agent tested and the methodology used for interpretation. The incidence of pneumococcal resistance to several antimicrobial agents has increased over the past few years. 8,10,17 21 Resistant S. pneumoniae had been infrequently identified in Canada with three previous Canadian surveys reporting resistance rates of 1.5% to 2.4%. 22 24 Recently, Simor et al. 10 tested 1089 S. pneumoniae isolates collected from 39 laboratories across Canada. They found that 11.7% of the isolates had reduced susceptibility to penicillin: 8.4% with intermediate resistance and 3.3% with high-level resistance. The frequency of resistance was not uniform across the country, with resistance rates increasing from East to West. In agreement with this present study, they concluded that there has been a recent significant increase in the prevalence of antibiotic-resistant S. pneu - moniae in Canada. Jacobs et al. 25 evaluated the Etest method for determining the MICs of four antimicrobial agents for S. pneumoniae and concluded that it was 28

Susceptibilities of Canadian isolates reliable. Similarly, the Etest has emerged as a reliable susceptibility testing method for the accurate detection of penicillin-resistant S. pneumoniae. Clark et al. 11 compared the Etest with five other methods and reported that it was reliable for detecting penicillin-resistant S. pneumoniae. Some (77) of the isolates used in this study were included in a comparative study of ciprofloxacin and 19 other antimicrobial agents. 12 The low susceptibility of S. pneumoniae to ciprofloxacin reported in that study may relate to the unsuitability of the Microscan panels for testing this pathogen, since recent results reported by Simor et al. 10 indicate that 97% of 1089 isolates were susceptible ( 1 mg/l) to ciprofloxacin when tested by a non-commercial method. We have recently reported that 91% of penicillin-sensitive S. pneumoniae isolates and 91% of penicillin-resistant S. pneumoniae isolates from Saskatchewan were susceptible to ciprofloxacin when tested by Etest and microbroth dilution. 26 M. catarrhalis has increasingly been cited in the literature as a pathogen associated with respiratory tract infections. 3 In our study, 85% of M. catarrhalis were β- lactamase-positive and resistant to penicillin. This finding is consistent with previously published results 15,27 and slightly higher than those reported by Hoban et al. 16 from five North American centres. Although none of the small number of β-lactamase-negative isolates were found to be resistant in this study, the low numbers make interpretation difficult. Other types of resistance mechanisms (non-β-lactamase-mediated), such as alterations in penicillin-binding proteins as in S. pneumoniae and H. influen - zae or changes in permeability, have been reported. 28,29 Our results indicate that while M. catarrhalis resistance to ampicillin is now extensive, susceptibilities to the broader spectrum agents reported here have not changed significantly. The current study measured the susceptibility of selected antimicrobial agents to three separate respiratory tract pathogens. This study adds information on the activity of some antimicrobial agents not tested before in the context of emerging antimicrobial resistance of respiratory pathogens isolated in Canada. Such data will continue to be important in helping clinicians to select the most appropriate microbial agent for the therapy of infected patients. Acknowledgements This work was presented, in part, at the Twentieth International Congress of Chemotherapy, Sydney, Australia, June 30 July 3, 1997. This work was funded, in part, by unrestricted grants from Bayer Canada Inc., Glaxo-Wellcome Inc., Lederle Canada Inc., Eli Lilly Canada Inc. and Pfizer Canada Inc. We thank Jonathan Harris and Teresa Tartaglione for editorial contributions. Canadian Respiratory Tract Pathogens Study Group: J. A. Smith, Vancouver General Hospital, Vancouver, British Columbia; M. A. Nobel, University Hospital UBC site, Vancouver, British Columbia; P. Kibsey, Grey Nuns Hospital, Edmonton, Alberta; J. M. Blondeau, St Paul s Hospital, Saskatoon, Saskatchewan; G.B. Horsman, H. E. Robertson Laboratory, Regina, Saskatchewan; D. J. Hoban, Health Sciences Center, Winnipeg, Manitoba; W. D. Colby, University Hospital, London, Ontario; P. Jessamine, Ottawa Civic Hospital, Ottawa, Ontario; A. E. Simor, Sunnybrook Medical Center, Toronto, Ontario; D. E. Low, Mount Sinai Hospital, Toronto, Ontario; A. M. Bourgault, Hôpital Saint-Luc, Montréal, Québec; M. Laverdiere, Hôpital Maisonneuve-Rosemont, Montréal, Québec; M. Kuhn, The Moncton Hospital, Moncton, New Brunswick; S. MacDonald, Halifax Infirmary/Camp Hill Complex, Halifax, Nova Scotia; W. Brown, Health Sciences Center, St John s, Newfoundland. References 1. 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 β-lactamases. Antimicrobial Agents and Chemotherapy 38, 1678 80. 2. Lonks, J. R. & Medeiros, A. A. (1995). The growing threat of antibiotic-resistant Streptococcus pneumoniae. Medical Clinics of North America 79, 523 35. 3. Catlin, B. W. (1990). Branhamella catarrhalis: an organism gaining respect as a pathogen. Clinical Microbiology Reviews 3, 293 320. 4. Felmingham, D. (1995). Antibiotic resistance. Do we need new therapeutic approaches? Chest 108, Suppl. 2, 70S 8S. 5. Gruneberg, R. N. & Felmingham, D. (1996). Results of the Alexander Project: a continuing, multicenter study of the antimicrobial susceptibility of community-acquired lower respiratory tract bacterial pathogens. Diagnostic Microbiology and Infectious Disease 25, 169 81. 6. Kunin, C. M. (1993). Resistance to antimicrobial drugs a worldwide calamity. Annals of Internal Medicine 118, 557 61. 7. Doern, G. V., Brueggemann, A. B., Pierce, G., Holley, H. P. & Rauch, A. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of β-lactamase-positive strains resistant to amoxicillin clavulanate: results of a national multicenter surveillance study. Antimicrobial Agents and Chemotherapy 41, 292 7. 8. Doern, G. V., Brueggemann, A., Holley, H. P. & Rauch, A. M. (1996). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-center national surveillance study. Antimicrobial Agents and Chemotherapy 40, 1208 13. 9. Doern, G. V., Brueggemann, A. B., Pierce, G., Hogan, T., Holley, H. P. & 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. 10. Simor, A. E., Louie, M., Low, D. E. & the Canadian Bacterial 29

J. M. Blondeau et al. Surveillance Network. (1996). Canadian national survey of prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 2190 3. 11. Clark, R. B., Giger, O. & Mortensen, J. E. (1993). Comparison of susceptibility test methods to detect penicillin- resistant Streptococcus pneumoniae. Diagnostic Microbiology and Infectious Disease 17, 213 7. 12. Blondeau, J. M., Yaschuk, Y. & the Canadian Ciprofloxacin Study Group. (1996). Canadian ciprofloxacin susceptibility study: comparative study from 15 medical centers. Antimicrobial Agents and Chemotherapy 40, 1729 32. 13. National Committee for Clinical Laboratory Standards. (1994). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A7. NCCLS, Villanova, PA. 14. Tremblay, L. D., L Ecuyer, J., Provencher, P., Bergeron, M. G. & the Canadian Study Group. (1990). Susceptibility of Haemophilus influenzae to antimicrobial agents used in Canada. Canadian Medical Association Journal 143, 895 901. 15. Hoban, D. J., Jones, R. N. & the Canadian Ofloxacin Study Group. (1995). Canadian ofloxacin susceptibility study: a comparative study from 18 medical centers. Chemotherapy 41, 34 8. 16. Hoban, D. J., Jones, R. N., Harrell, L. J., Knudson, M. & Sewell, D. (1993). The North American component (the United States and Canada) of an International Comparative MIC trial monitoring ofloxacin resistance. Diagnostic Microbiology and Infectious Disease 17, 157 61. 17. Baer, M., Vuento, R. & Vesikari, T. (1995). Increase in bacteraemic pneumococcal infections in children. Lancet 345, 661. 18. Foster, J. A. & McGowan, K. L. (1994). Rising rate of pneumococcal bacteremia at the Children s Hospital of Philadelphia. Pediatric Infectious Disease Journal 13, 1143 4. 19. Appelbaum, P. C. (1992). Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clinical Infectious Disease 15, 77 83. 20. Barry, A. L., Pfaller, M. A., Fuchs, P. C. & Packer, R. R. (1994). In vitro activities of 12 orally administered antimicrobial agents against four species of bacterial respiratory pathogens from US Medical Centers in 1992 and 1993. Antimicrobial Agents and Chemotherapy 38, 2419 25. 21. Breiman, R. F., Butler, J. C., Tenover, F. C., Elliott, J. A. & Facklam, R. R. (1994). Emergence of drug-resistant pneumococcal infections in the United States. Journal of the American Medical Association 271, 1831 35. 22. Dixon, J. M., Lipinski, A. E. & Graham, M. E. (1977). Detection and prevalence of pneumococci with increased resistance to penicillin. Canadian Medical Association Journal 117, 1159 61. 23. Jette, L. P. & Lamothe, F. (1989). Surveillance of invasive Streptococcus pneumoniae infection in Quebec, Canada, from 1984 to 1986: serotype distribution, antimicrobial susceptibility, and clinical characteristics. Journal of Clinical Microbiology 27, 1 5. 24. Mazzulli, T., Simor, A. E., Jaeger, R., Fuller, S. & Low, D. E. (1990). Comparative in vitro activities of several new fluoroquinolones and beta-lactam antimicrobial agents against community isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 34, 467 9. 25. Jacobs, M. R., Bajaksouzian, S., Appelbaum, P. C. & Bolmstrom, A. (1992). Evaluation of the Etest for susceptibility testing of pneumococci. Diagnostic Microbiology and Infectious Disease 15, 473 8. 26. Blondeau, J. M., Vaughan, D., Low, D., Graham, C. & Sutor, M. (1997). Regional resistance rates for penicillin resistant Streptococcus pneumoniae (PRSP) in Saskatchewan, Canada. In Proceedings of the Twentieth International Congress of Chemotherapy, Sydney, 1997. Abstract P032-3209, p. 80. Organizing Committee of the International Congress of Chemotherapy, Victoria, Australia. 27. Felmingham, D. & Gruneberg, R. N. (1996). A multicentre collaborative study of the antimicrobial susceptibility of communityacquired, lower respiratory tract pathogens 1992 1993: the Alexander Project. Journal of Antimicrobial Chemotherapy 38, Suppl. A, 1 57. 28. Quinn, J. P. (1989). Mechanism of resistance to beta-lactam antibiotics. Hospital Formulary 24, 204 5. 29. Hakenbeck, R., Tarpay, M. & Tomasz, A. (1980). Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 17, 364 71. 30