JAC
Journal of Antimicrobial Chemotherapy (1997) 40, 387–392
Increasing antimicrobial resistance in Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in Finland Raija Manninena,b*, Pentti Huovinena, Antti Nissinenc and The Finnish Study Group for Antimicrobial Resistance† a
Antimicrobial Research Laboratory, National Public Health Institute, Turku; bDepartment of Medical Microbiology, Turku University, Turku; cMicrobiology Laboratory, Central Hospital of Keski-Suomi, Jyväskylä, Finland Respiratory and otitis isolates of 807 Streptococcus pneumoniae, 816 Haemophilus influenzae and 446 Moraxella catarrhalis were collected from 21 clinical microbiology laboratories for antimicrobial susceptibility testing in 1995. After a period of relative stability in 1981 and 1987–1990, -lactamase production increased in H. influenzae. Among middle ear isolates from children under 6 years, -lactamase production increased from 8% to 24% in H. influenzae and from 81% to 96% in M. catarrhalis since the survey in 1987–1990. 1.2% of S. pneumoniae were penicillin-resistant and 4.2% intermediately resistant; 5 years earlier among otitis isolates of children only 1.7% intermediate resistance was found. Ampicillin resistance was seen among 1.9% of non- -lactamase-producing strains of H. influenzae. Resistance to trimethoprim–sulphamethoxazole occurred in 9.4% of S. pneumoniae, 7.4% of H. influenzae and 0.7% of M. catarrhalis. Frequencies of azithromycin resistance were 3.0% in S. pneumoniae and 1.6% in H. influenzae, and those of tetracycline resistance were 6.7% in S. pneumoniae and 1.2% in H. influenzae.
Introduction Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the predominant bacterial pathogens in respiratory infections.1 Antimicrobial resistance of these pathogens was assessed in 1987–1990 in a nationwide Finnish survey of a collection of middle ear isolates from children under six years of age. None of the S. pneumoniae strains were penicillin-resistant and 1.7
were intermediately resistant.2 -Lactamase production by M. catarrhalis increased in 1988–1990 from 69% to 81%, but other resistance was not encountered, except in one strain which was resistant to trimethoprim–sulphamethoxazole.3 The prevalence of -lactamase-producing H. influenzae and M. catarrhalis was comparable to the European average whereas resistance to other antimicrobial agents was much less common. As an increase in the resistance to all these pathogens was predicted, a new
*Corresponding author. National Public Health Institute, Kiinamyllynkatu 13, FIN-20520 Turku, Finland. Tel: +358-2-251-9255; Fax: +358-2-251-9254; E-mail:
[email protected] †The Finnish Study Group for Antimicrobial Resistance: Esa Ahonen, Central Hospital of Kainuu, Kajaani; Erkki Eerola, Turku University, Turku; Jussi Eskola, Aurora Hospital, Helsinki; Pirkko Hirvonen, Central Hospital of Keski-Suomi, Jyväskylä; Henrik Jägerroos, Central Hospital of Lappi, Rovaniemi; Marja-Leena Katila, Kuopio University Hospital, Kuopio; Maritta Kauppinen, Central Hospital of Etelä-Karjala, Lappeenranta; Marja-Liisa Klossner, Central Hospital of Satakunta, Pori; Sirkka Kontiainen, Aurora Hospital, Helsinki; Jukka Korpela, Central Hospital of Päijät-Häme, Lahti; Markku Koskela, Oulu University Hospital, Oulu; Anja Kostiala-Thompson, Jorvi Hospital, Espoo; Päivi Kärkkäinen, Central Hospital of Mikkeli, Mikkeli; Kaarina Lantto, Deaconess Institution of Oulu, Oulu; Ulla Larinkari, Central Hospital of Kymenlaakso, Kotka; Olli-Pekka Lehtonen, Turku University Central Hospital, Turku; Oili Liimatainen, Tampere University Hospital, Tampere; Antti Nissinen, Central Hospital of Keski-Suomi, Jyväskylä; Sinikka Oinonen, Hospital of Seinäjoki, Seinäjoki; Ilmo Pietarinen, Deaconess Institution of Oulu, Oulu; Olli-Veikko Renkonen, Helsinki University, Helsinki; Hannu Sarkkinen, Central Hospital of Pohjois-Karjala, Joensuu; Kristiina Schauman, Deaconess Institution of Helsinki, Helsinki; Aulikki Sivonen, Helsinki University, Helsinki; Martti Vaara, Helsinki University, Helsinki; Veli Vikberg, Central Hospital of Kanta-Häme, Hämeenlinna.
387 © 1997 The British Society for Antimicrobial Chemotherapy
R. Manninen et al. survey of all kinds of respiratory and otitis isolates of S. pneumoniae, H. influenzae and M. catarrhalis was performed in 1995. A subset, consisting of bacteria isolated from middle ear specimens of children under 6 years of age, was used for a comparison with the previous similarly structured material, to assess the increase of the resistance during the past 5 years.
Materials and methods Twenty-one clinical microbiology laboratories all over Finland were asked to collect 50 S. pneumoniae, 50 H. influenzae and 25 M. catarrhalis consecutive isolates from clinical respiratory or otitis specimens between January and June 1995, both from primary health care and hospitals. Identification and -lactamase production testing were performed in primary laboratories according to the standard methods used in each laboratory. The MICs were determined in the National Public Health Institute (NHPI). After repeated isolates from the same patient had been excluded, the total number of strains frozen in tryptone soya broth and glycerol tubes was 822 S. pneumoniae, 841 H. influenzae and 494 M. catarrhalis. Seven, 13 and 26 strains, respectively, of these three species repeatedly grew weakly in susceptibility testing and were thus omitted. In cases of unexpected resistance or atypical colony appearance, species identification was repeated in the NHPI. Pneumococci with reduced susceptibility to penicillin ( 0.06 mg/L) were identified with an optochin disc (Unipath Ltd, Basingstoke, UK). If the optochin results of the primary laboratory and the NHPI did not agree, the strain was identified by serotyping. Consequently, altogether 8/822 (1.0%) of the S. pneumoniae, 12/841 (1.4%) of the
H. influenzae and 22/494 (4.5%) of the M. catarrhalis strains were found to be other bacteria. The susceptibility results of 807 S. pneumoniae, 816 H. influenzae and 446 M. catarrhalis strains were included in the study. The middle ear isolates from children under 6 years formed a subset of strains, consisting of 378 S. pneumoniae, 297 H. influenzae and 197 M.catarrhalis. Forty-four percent of the S. pneumoniae, 33% of the H. influenzae and 52% of the M. catarrhalis strains originated from 0–1 year old infants. Thirty-one percent of S. pneumoniae, 39% of H. influenzae and 20% of M. catarrhalis originated from persons over 16 years old. Fifty-four percent of the S. pneumoniae, 41% of the H. influenzae and 46% of the M. catarrhalis were isolated from the middle ear. The nasopharynx and sinus isolates together represented a third of the material. The MICs of the following antimicrobial agents were determined with the agar dilution method outlined by the National Committee for Clinical Laboratory Standards (NCCLS):4,5 penicillin, MIC range 0.032–4 mg/L, ampicillin 0.06–64 mg/L, cefaclor 0.06–32 mg/L, cefuroxime sodium 0.06–32 mg/L, erythromycin 0.032–32 mg/L, tetracycline 0.06–32 mg/L, chloramphenicol 0.06–32 mg/L, trimethoprim 0.06–1024 mg/L, trimethoprim–sulphamethoxazole 0.06/1.19 mg/L to 16/304 mg/L, ciprofloxacin 0.016–8 mg/L (all above from Sigma, St Louis, MO, USA), loracarbef 0.06–32 mg/L (Lilly, Indianapolis, IN, USA), azithromycin 0.032–32 mg/L (Pfizer, Ringaskiddy, Ireland), cefixime 0.008–8 mg/L (Orion, Espoo, Finland), sparfloxacin 0.008–8 mg/L (Rhône–Poulenc Rorer, Vitry, France), and for S. pneumoniae ceftriaxone 0.016–8 mg/L (Sigma). For those M. catarrhalis strains which were not susceptible to erythromycin or trimethoprim–sulphameth-
Table I. Antimicrobial susceptibility of 807 S. pneumoniae in Finland in 1995 according to the MIC breakpoints of the NCCLS MIC (mg/L) MIC50
Antibiotic
% Resistant
% Intermediate
Penicillin Cefaclor Loracarbef Cefuroxime Ceftriaxone Cefixime Chloramphenicol Trimethoprim– sulphamethoxazole Tetracycline Erythromycin Azithromycin Sparfloxacin
1.2 2.0 2.0 2.4 0.3 2.7 1.9 9.4
4.2 0.4 0.3 0.1 1.8 1.0 0 6.9
0.032–4 0.25– 32 0.25– 32 0.06–16 0.016–2 0.06– 8 0.5–32 0.06/1.2–16/304
0.032 1 1 0.064 0.032 0.25 2 0.25/4.75
0.032 2 2 0.064 0.032 0.5 4 2/38
6.7 2.7 3.0 0.1
0.2 0.5 0.4 0
0.06– 0.032– 0.032– 0.032–
0.25 0.032 0.125 0.25
0.5 0.064 0.25 0.5
388
range
32 32 32 8
MIC90
S. pneumoniae, H. influenzae and M. catarrhalis oxazole according to the agar dilution, Etests (AB Biodisk, Solna, Sweden) were done for these antimicrobials. The test medium used for M. catarrhalis was Mueller– Hinton agar (Becton Dickinson, Cockeysville, MD, USA). For S. pneumoniae, Mueller–Hinton agar was supplemented with 5% sheep defibrinated blood and for trimethoprim and trimethoprim–sulphamethoxazole testing, 5% lysed horse blood. For H. influenzae Haemophilus Test Medium (HTM) (Unipath) was used. For trimethoprim and trimethoprim–sulphamethoxazole testing, 0.2 IU/mL thymidine phosphorylase was added to the HTM agar. Control strains were Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, M. catarrhalis ATCC 25238, H. influenzae ATCC 49247, H. influenzae PHLS 1141,6 a susceptible S. pneumoniae patient isolate IH 47186 and a multiresistant S. pneumo niae RH 43362. 2 Because the NCCLS did not give breakpoints for sparfloxacin, we used the breakpoints of the European Study Group on Antibiotic Breakpoints, namely 1 mg/L for susceptibility and 4 mg/L for resistance.7 The chi-square test was used for statistical analyses. The correlation of -lactamase-producing strains with patient age was compared with 95% confidence intervals (CI) for observed percentages.
Results S. pneumoniae Of the 807 pneumococci, ten (1.2%) were penicillinresistant (MIC 2 mg/L) and 34 (4.2%) were inter-
mediately resistant (MIC 0.125–1 mg/L) (Table I). All penicillin-resistant strains were also resistant to cefaclor, loracarbef, cefixime, trimethoprim and trimethoprim– sulphamethoxazole. Resistance to two or more unrelated drugs in addition to penicillins and second-generation cephalosporins was found among six penicillin-resistant strains, two of which were resistant to ceftriaxone, and among seven penicillin-intermediate strains. Two lactam-susceptible strains from the same laboratory were resistant to tetracycline, chloramphenicol, trimethoprim–sulphamethoxazole, erythromycin and clindamycin, the latter tested in the primary laboratory. Compared with the otitis-related strains studied in 1988–90,2 the paediatric middle ear isolates were significantly more often resistant to penicillin, cefaclor and erythromycin (P 0.001) and trimethoprim– sulphamethoxazole (P 0.014) (Table II).
H. influenzae The overall prevalence of -lactamase-producing strains was 19% (151/787) (95% CI 16–22%). Children under 4 years of age were more prone to harbour -lactamaseproducing strains than older ones: 26% (CI 21–30%) of their strains produced -lactamase compared with 13% (CI 7–19%) in the age group of 4–16 years (P 0.004) and 15% (CI 11–18%) in the age group over 16 years (P 0.0004). -Lactamase production varied between laboratories, from 0% to 40%, and there were no significant differences between geographical regions of Finland. Resistance to drugs other than ampicillin and cephalosporins was rare (Table III). Most noticeable was
Table II. Resistance of isolates from ear specimens of children under 6 years compared with the earlier study
Bacterium/antibiotic S. pneumoniae penicillin cefaclor erythromycin trimethoprim– sulphamethoxazole H. influenzae -lactamase production cefaclor trimethoprim– sulphamethoxazole M. catarrhalis -lactamase production cefaclor trimethoprim– sulphamethoxazole
Resistance rates (%) 1988–1990 1995 (n 0 0.3 0.6 4.5
639)
(n 8.0 0.5 3.6
578)
(n 401) 75.8 0 0.2
389
P
(n 378) 1.9 2.9 2.4 12.5
0.001 0.001 0.001 0.014
(n 297) 24.4 11.8 5.1
0.001 0.001 0.293
(n 197) 95.9 0 0.5
0.001 0.605
R. Manninen et al. the 7.4% resistance to trimethoprim–sulphamethoxazole. If the 608 specimens of the upper respiratory tract (including the sinus, nasopharynx and ear) are compared with the 102 lower respiratory tract specimens (sputum, bronchus and lung) the lower respiratory tract isolates were more resistant to trimethoprim–sulphamethoxazole than the upper respiratory isolates (14.1% versus 6.7%; P 0.01) and to azithromycin (7.0% versus 0.5%; P < 0.0001). One -lactamase-producing sinus isolate was resistant to both trimethoprim–sulphamethoxazole and tetracycline. Compared with the otitis-related strains studied previously,8 the paediatric middle ear isolates were significantly more often -lactamase-producing and resistant to cefaclor (Table II).
Moraxella catarrhalis -Lactamase was produced by 94% (95% CI 92–96%) of all M. catarrhalis strains. -Lactamase-producing M.
catarrhalis occurred in 97% (CI 95–99%) of isolates in the age group 0–3 years, in 94% (CI 88–100%) in the age group 4–16 years and in 86% (CI 79–93%) in the age group over 16 years. All 126 strains isolated from children under 1 year produced -lactamase. -Lactamase production was significantly (P 0.0001) more common among isolates of patients under 16 years than in those over 16 years. Three strains (0.7%) had an MIC of 4 mg/L of trimethoprim–sulphamethoxazole, which is just above the susceptibility/resistance breakpoint (Table IV). One isolate from a child’s middle ear was intermediately resistant to erythromycin. These four isolates were, however, susceptible according to the Etest with the MIC of erythromycin being 0.38 mg/L and those of trimethoprim–sulphamethoxazole being between 1 mg/L and 1.5 mg/L by this method. No resistance to cefuroxime, cefixime, chloramphenicol, tetracycline, azithromycin, ciprofloxacin or sparfloxacin was encountered. Compared with the otitis-related strains
Table III. Antimicrobial susceptibility of 816 H. influenzae isolates in Finland in 1995 according to the MIC breakpoints of the NCCLS, 151 strains were -lactamase producers and 636 non-producers
Antibiotic
% Resistant
Ampicillin 18.6 -lactamase-negative 1.9 Cefaclor 10.2 Loracarbef 1.2 Cefuroxime 0.6 Cefixime 0 Chloramphenicol 0 Trimethoprim– 7.4 sulphamethoxazole Tetracycline 1.2 Azithromycin 1.6 Ciprofloxacin 0 Sparfloxacin 0.4
% Intermediate
range
2.7 2.8 12.7 4.4 2.0 0 0.2 4.2 4.2 0 0 2.2
MIC (mg/L) MIC50
0.06– 64 0.06– 64 0.5– 32 0.5– 32 0.06– 32 0.008–0.5 0.25–4 0.06/1.2– 16/304
0.5 0.5 8 2 1 0.064 0.5 0.125/23.75
0.125–32 0.06–16 0.016–1 0.06– 8
2 2 0.016 0.25
MIC90 32 1 32 8 4 0.125 1 2/38 4 4 0.032 1
Table IV. Antimicrobial susceptibility of 446 M. catarrhalis isolates in Finland in 1995 according to the NCCLS MIC breakpoints
% Resistant
% Intermediate
Penicillin Cefaclor Loracarbef Trimethoprim– sulphamethoxazole Erythromycin
94.4a 0 0 0.7
0 0.9 0.2 0
0.032– 4 0.25–16 0.06–16 0.125/2.38–4/76
4 2 1 0.5/9.5
4 4 2 1/19
0
0.2
0.032–1
0.125
0.25
a
range
MIC (mg/L) MIC50
Antibiotic
All -lactamase producers were considered resistant.
390
MIC90
S. pneumoniae, H. influenzae and M. catarrhalis studied previously,3 the paediatric middle ear isolates produced -lactamase significantly more often (Table II).
Discussion Penicillin-resistant pneumococci have appeared in Finland and the rate of trimethoprim–sulphamethoxazole resistance has also increased. Penicillin resistance for all the S. pneumoniae strains was only 1.2% but half of the ten penicillin-resistant strains were multiresistant; resistance to ceftriaxone was also found. This pattern was seen all over the country. Cefaclor, loracarbef and cefixime appeared equally or less effective than penicillin against penicillin-resistant strains.9 After a stable period during the 1980s8,10 the prevalence of -lactamase-producing H. influenzae has increased from 8% to 24% in 1995 in middle ear isolates from children under 6 years. The overall rate, 19%, is almost at the same level as in western Europe and North America. Multicentre studies have shown a -lactamase production rate as high as 30% among respiratory isolates in USA in 1992–19939 and 25% in France in 1993.11 Ampicillin resistance among non- -lactamase-producing H. influenzae strains increased to 1.9%, and intermediate resistance to 2.8%, whereas it was only 0.2% in 1988– 1990 otitis, throat, blood and cerebrospinal fluid isolates.8 A high level (5.2%) of non- -lactamase-mediated ampicillin resistance was reported in sputum isolates in the UK in 1991, but this was based on a lower breakpoint for resistance, 1 mg/L.12 Ampicillin MICs in non- -lactamaseproducing ampicillin-resistant strains are lower than in -lactamase producers, often only causing intermediate resistance to ampicillin, but the MICs of the second generation cephalosporins are high enough to be interpreted as resistant.13 Cefaclor resistance in middle ear H. influenzae isolates from children have increased from 0.5% to 11.8% since the 1988–90 study.8 The increase of ampicillin-resistant non- lactamase-producing strains is one reason for this. The large unexpected difference between loracarbef (1.2%) and cefaclor resistance (10.2%) is partially explained by the fact that cefaclor is more labile in in-vitro testing than loracarbef14. Another reason is that, with H. influenzae, cessation of growth in the plate series was not distinct, possibly due to inoculum dependence. Thus the MIC distribution extends in the resistant direction, leading to intermediate or resistant interpretations, although the strains have no acquired resistance. Additionally, the incubation was performed in ambient air in the previous study, whereas it is now performed in a carbon dioxide atmosphere, which can reduce the potency of cefaclor and loracarbef.15 In middle ear isolates of M. catarrhalis from children aged less than 6 years the -lactamase production has increased from 81% in 1990 to 96% in 1995. This reflects
a steady increase in -lactamase-producing strains in Finland from the late 1980s3 and is consistent with trends elsewhere in Scandinavia,16 as well as in the USA9 and France.1 In contrast to the -lactamase-mediated resistance, resistance to other antimicrobials in M. catarrhalis has remained rare. Our 3% erythromycin resistance in pneumococci is good compared with the rates found in Spain (15.2%)17 and Belgium (21.5%).18 Erythromycin was also effective against M. catarrhalis but not against H. influenzae. Azithromycin was effective against H. influenzae. Respiratory infections are not a primary indication for ciprofloxacin use, since this antibiotic is effective against M. catarrhalis and H. influenzae, but less effective against S. pneumoniae. Sparfloxacin had better activity against all three bacteria. The rates of resistance of S. pneumoniae to tetracycline (6.7%) and trimethoprim–sulphamethoxazole were relatively high compared with the penicillin resistance, indicating that these agents are not always active against penicillin-susceptible pneumococci. The continuing clinical efficacy of ampicillin and penicillin in otitis media was supported by a meta-analysis of 5400 patients, which showed a high spontaneous rate of cure, and concluded that treatment with -lactamase-stable agents did not enhance the resolution of acute symptoms or middle ear effusion.19 These data indicate that in Finland the proportion of lactamase-producing H. influenzae and M. catarrhalis strains has increased during the early 1990s. Non- lactamase resistance in H. influenzae is less of a problem than in many other parts of Europe, and penicillinresistant pneumococci are not yet a serious problem in Finland. Penicillin and ampicillin/amoxycillin remain the primary choice for uncomplicated acute respiratory infections for the majority of patients.
Acknowledgements We thank the staff of the participating laboratories and Dr Maija Leinonen, the Oulu department of the NHPI, for identification of penicillin-resistant pneumococci. This work was supported by the Maud Kuistila Foundation, the Sigrid Juselius Foundation and Eli Lilly Finland Ltd, Vantaa, Finland.
References 1. Boulesteix, J., Bégué, P., Dubreuil, C., Mégraud, F., Dabernat, H., Geslin, P. et al . (1995). Acute otitis media in children: a study of nasopharyngeal carriage of potential pathogens and therapeutic efficacy of cefixime and amoxicillin–clavulanate. Infection 23, Suppl. 2, 79–81. 2. Nissinen, A., Leinonen, M., Huovinen, P., Herva, E., Katila, M. L., Kontiainen, S. et al. (1995). Antimicrobial resistance of Strepto coccus pneumoniae in Finland, 1987–1990. Clinical Infectious Diseases 20, 1275–80.
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R. Manninen et al. 3. Nissinen, A., Grönroos, P., Huovinen, P., Herva, E., Katila, M. L., Klaukka, T. et al. (1995). Development of -lactamasemediated resistance to penicillin in middle-ear isolates of Moraxella catarrhalis in Finnish children, 1978–1993. Clinical Infectious Diseases 21, 1193–6. 4. National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Second Edition: Approved Standard M7-A2. NCCLS, Villanova, PA. 5. National Committee for Clinical Laboratory Standards. (1994). Performance Standards for Antimicrobial Susceptibility Testing. Fifth Informational Supplement: NCCLS Document M100-S5. NCCLS, Villanova, PA. 6. Snell, J. J. S., Brown, D. F. J. & Phua, T. J. (1986). Antimicrobial susceptibility testing of Haemophilus influenzae: trial organized as part of United Kingdom national external quality assessment scheme for microbiology. Journal of Clinical Pathology 39, 1006–12. 7. European Study Group on Antibiotic Breakpoints. (1994). Breakpoint determination: sparfloxacin. European Journal of Clinical Microbiology and Infectious Diseases 13, 283–4. 8. Nissinen, A., Herva, E., Katila, M. L., Kontiainen, S., Liimatainen, O., Oinonen, S. et al. (1995). Antimicrobial resistance in Haemophilus influenzae isolated from blood, cerebrospinal fluid, middle ear fluid and throat samples of children. A nationwide study in Finland in 1988–1990. Scandinavian Journal of Infectious Diseases 27, 57–61. 9. 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 U.S. medical centers in 1992 and 1993. Antimicrobial Agents and Chemotherapy 38, 2419–25. 10. Karma, P., Luotonen, J., Pukander, J., Sipilä, M., Herva, E. & Grönroos, P. (1983). Haemophilus influenzae in acute otitis media. Acta Otolaryngologica (Stockholm) 95, 105–10. 11. Goldstein, F. W., Péan, Y., Gertner J. & The Vigil’roc Study Group (1995). Resistance to ceftriaxone and other -lactams in
bacteria isolated in the community. Antimicrobial Agents and Chemotherapy 39, 2516–9. 12. Powell, M., McVey, D., Kassim, M. H., Chen, H. Y. & Williams, J. D. (1991). Antimicrobial susceptibility of Streptococcus pneumo niae, Haemophilus influenzae and Moraxella (Branhamella) catarrhalis isolated in the UK from sputa. Journal of Antimicrobial Chemotherapy 28, 249–59. 13. Goldstein, F. W. & Acar, J. F. (1995). Epidemiology of antibiotic resistance in Haemophilus influenzae. Microbial Drug Resist ance 1, 131–5. 14. Knapp, C. C. & Washington, J. A. (1988). In vitro activities of LY163892, cefaclor, and cefuroxime. Antimicrobial Agents and Chemotherapy 32, 131–3. 15. Howard, A. J. & Dunkin, K. T. (1988). Comparative in-vitro activity of a new oral carbacephem LY163892. Journal of Anti microbial Chemotherapy 22, 445–56. 16. Forsgren, A. & Walder, M. (1994). Antimicrobial susceptibility of bacterial isolates in south Sweden including a 13-year follow-up study of some respiratory tract pathogens. APMIS 102, 227–35. 17. Moreno, S., Garcia-Leoni, M. E., Cercenado, E., Diaz, M. D., Bernaldo de Quiros, J. C. L. & Bouza, E. (1995). Infections caused by erythromycin-resistant Streptococcus pneumoniae: incidence, risk factors, and response to therapy in a prospective study. Clinical Infectious Diseases 20, 1195–200. 18. Verhaegen, J., Glupczynski, Y., Verbist, L., Blogie, M., Verbiest, N., Vandeven, J. et al. (1995). Capsular types and antibiotic susceptibility of pneumococci isolated from patients in Belgium with serious infections, 1980–1993. Clinical Infectious Diseases 20, 1339–45. 19. Rosenfeld, R. M., Vertrees, J. E., Carr, J., Cipolle, R. J., Uden, D. L., Giebink, G. S. et al. (1994). Clinical efficacy of antimicrobial drugs for acute otitis media: meta-analysis of 5400 children from thirty-three randomized trials. Journal of Pediatrics 124, 355–67. Received 26 November 1996; returned 4 February 1997; revised 18 March 1997; accepted 25 April 1997
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