Infectious Diseases Research Group, a Departments of Microbiology and Immunology, b Internal Medicine and c Paediatrics, Rega Institute for Medical Research, K.U. Leuven, Belgium
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Abstract |
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Introduction |
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In Belgium, erythromycin resistance in pneumococci has increased from 11.5% in 1988 to 31.0% in 1998.6 From 1995 to 1997, erythromycin resistance increased from 24 to 28.5%. In this study, strains from the Belgian National Reference Collection from 1995 and 1997 were examined in order to define the molecular mechanism of macrolide resistance and to investigate whether there was a significant increase in the prevalence of macrolide resistance owing to efflux.
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Materials and methods |
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Fifty-nine randomly chosen erythromycin-resistant S. pneumoniae isolates from the Belgian National Reference Laboratory for pneumococci (University Hospital Gasthuisberg, Leuven, Belgium) were included: 29 isolates were from 1995; 30 isolates from 1997. The total number of erythromycin-resistant pneumococci sent to the reference laboratory was 239 in 1995 and 355 in 1997. Accordingly, 12.1 and 8.4% of the erythromycin-resistant strains from 1995 and 1997, respectively, were included in this study. According to NCCLS criteria,7 strains were considered erythromycin resistant if an inhibition zone <15 mm was found using a 15 µg erythromycin disc (bioMérieux, Marcy l'Étoile, France).
Determination of the antibiotic resistance phenotype
MICs of erythromycin, clindamycin, azithromycin, clarithro-mycin and penicillin were assessed by Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions. Plates were incubated overnight at 36°C in 5% CO2. Susceptibility testing for josamycin and miocamycin was performed following NCCLS recommendations using agar dilution on MuellerHinton agar containing 5% sheep blood and incubation at 36°C in 5% CO2 for 18 h.7 Josamycin was from Rhône-Poulenc-Rorer (Paris, France) and miocamycin from Menarini (Firenze, Italy).
Clindamycin-susceptible strains were tested for inducible resistance by placing 78 µg erythromycin discs (neoSensitabs, ROSCO, Taastrup, Denmark) and 25 µg clindamycin discs (neo-Sensitabs) 15 mm apart on a blood agar plate. Induction was considered present when the inhibition zone around the clindamycin disc was blunted on the side opposite to the erythromycin disc. A second method for the determination of induction was used in strains carrying the ermAM gene but in which resistance to clindamycin could not be induced by the disc method. For these strains, the MIC of clindamycin was determined on MuellerHinton blood agar with and without the addition of 0.1 mg/L erythromycin.4 When the MIC of clindamycin was higher in the presence of erythromycin, induction was considered to be present.
DNA isolation and PCR reaction
S. pneumoniae isolates were grown overnight on blood agar in 5% CO2 at 36°C. Colonies were taken from the agar and grown in 5 mL BHI broth at 37°C for 4.5 h. Total genomic DNA was isolated with InstaGene Matrix (BIORAD, CA, USA). The PCR reaction mixture was as recommended by Perkin-Elmer, with the concentration of magnesium optimized for each primer set (ermAM: 2 mM, mefE: 4 mM). Primer sets for ermAM and mefE were as described previously.4,8 The PCR conditions were as described by Sutcliffe et al.2 An erythromycin-susceptible strain was used as negative control. S. pneumoniae strains 02J1095 (ermAM) and 02J1175 (mefE) were used as positive-control strains (Dr J. A. Sutcliffe, Pfizer Central Research, Groton, CT, USA).
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Results |
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Discussion |
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Since efflux-based erythromycin resistance is uncommon in Belgian isolates, clindamycin and 16-membered macrolides cannot be considered as therapeutic alternatives for the treatment of infections with erythromycin-resistant pneumococci. Our results are in contrast to reports from Canada and the USA, where the M-phenotype is the most common resistance phenotype.2,3 The geographical difference in the prevalence of the M-phenotype in pneumococci is interesting. There are as yet no data suggesting that this may be due to clonal spread of erythromycin-resistant S. pneumoniae. In one study of M-phenotype S. pneumoniae in Canada,3 all isolates were genetically different. Macrolide resistance in Canada is, however, low (2.9%). On the other hand, a single clone predominates among erythromycin-resistant Streptococcus pyogenes in Finland.9 The ermAM gene is located on a transposon (Tn1545) which facilitates spread amongst different strains.1
Interestingly, both ermAM and mefE were detected in three low-level erythromycin-resistant M-phenotype isolates. Although the ermAM gene was present, clindamycin resistance was not expressed constitutively and could not be induced. The amplicon from the ermAM PCR had the correct size but sequencing is needed to exclude the presence of a mutation preventing its expression.
We conclude that efflux-mediated erythromycin resistance in S. pneumoniae is still uncommon in Belgium. Neither does it appear that macrolide resistance owing to the mefE gene is increasing significantly in Belgium.
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Acknowledgments |
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Notes |
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References |
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2 . Sutcliffe, J., Tait-Kamradt, A. & Wondrack, L. (1996). Strepto-coccus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrobial Agents and Chemotherapy 40, 181724.[Abstract]
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Johnston, N. J., De, A. J., Kellner, J. D. & Low, D. E. (1998). Prevalence and characterization of the mechanisms of macrolide, lincosamide, and streptogramin resistance in isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 24256.
4 . Shortridge, V. D., Flamm, R. K., Ramer, N., Beyer, J. & Tanaka, S. K. (1996). Novel mechanism of macrolide resistance in Streptococcus pneumoniae. Diagnostic Microbiology and Infectious Disease 26, 738.[ISI][Medline]
5 . Tait-Kamradt, A., Clancy, J., Cronan, M., Dib-Hajj, F., Wondrack, L., Yuan, W. et al. (1997). mefE is necessary for the erythromycin-resistant M-phenotype in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 22515.[Abstract]
6 . Ducoffre, G. (1998). Surveillance van Infectieuze Aandoeningen door een Netwerk van Laboratoria voor Microbiologie 1997 and Epidemiologische Trends 19831996. (Institute of Public Health Louis Pasteur), Ministry of Social Services, Public Health and Environment, Brussels.
7 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility TestingEight Informational Supplement: Approved Standard M100-S8. NCCLS, Wayne, PA.
8 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 25626.[Abstract]
9 . Kataja, J., Huovinen, P., Muotiala, A., Vuopio-Varkila, J., Efstratiou, A., Hallas, G. et al. (1998). Clonal spread of group A streptococcus with the new type of erythromycin resistance. Journal of Infectious Diseases 177, 7869.[ISI][Medline]
Received 19 April 1999; returned 15 July 1999; revised 16 September 1999; accepted 20 September 1999