Macrolide resistance in Belgian Streptococcus pneumoniae

K. Lagroua,*, W. E. Peetermansb, J. Verhaegena, S. Van Lierdec, L. Verbista and J. Van Elderea

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genetic basis of macrolide resistance was characterized in 59 Streptococcus pneumoniae isolates. All isolates were collected in 1995 and 1997 and were from invasive infections. The majority of the isolates (54 of 59 isolates) were erythromycin and clindamycin resistant (MLSB-phenotype) and carried the ermAM gene. Five isolates were erythromycin resistant but clindamycin susceptible (M-phenotype). Using PCR the mefE gene was detected in these five isolates. Contrary to the situation found in Canada and the USA, mefE-mediated erythromycin resistance in S. pneumoniae is uncommon in Belgium.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are three recognized mechanisms of resistance to macrolides; target modification, inactivation of the antibiotic and active efflux of the drug.1,2 The best known resistance mechanism is the production of an enzyme that methylates ribosomal binding sites. In pneumococci, this enzyme is encoded by the ermAM gene, which can be expressed constitutively or can be induced. Ribosomal methylation leads to resistance to macrolides, lincosamides and streptogramin B compounds, and is known as the MLSB-phenotype.1 However, recent epidemiological surveys in Canada and the USA have shown that 85 and 55.8%, respectively, of all macrolide-resistant isolates had an M-phenotype.2,3 M-phenotype strains are resistant to 14- and 15-membered macrolides but remain susceptible to 16-membered macrolides, lincosamides and streptogramin B antibiotics.4,5 Streptococcus pneumoniae strains with the M-phenotype carry the mefE gene which codes for an efflux mechanism.5

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

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 Mueller–Hinton 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 Mueller–Hinton 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fifty-four of 59 strains (91.5%) isolated in 1995 and 1997 had the MLSB-phenotype (Table IGo). All of these were high-level erythromycin resistant (MIC >= 16 mg/L). The MLSB-phenotype was inducibly expressed in only two strains. No strain was penicillin resistant (MIC >= 2 mg/L), ten strains showed intermediate resistance (0.12 mg/L <= MIC <= 1 mg/L). Five strains had the M-phenotype, four of these were low-level erythromycin resistant (MIC 1–16 mg/L). In these M-phenotype strains, the MICs of josamycin and miocamycin were <=2 mg/L and 0.5 mg/L, respectively. Comparison of the results for 1995 versus 1997 showed no increase of the M-phenotype.


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Table I. Characterization of 59 erythromycin-resistant Streptococcus pneumoniae isolated in 1995 and 1997
 
PCR detected ermAM in all 54 strains with the MLSB-phenotype (Table IGo). All strains with the M-phenotype contained the mefE gene. In three M-phenotype strains, the mefE gene was found together with the ermAM gene, but induction of resistance to clindamycin could not be achieved by either method we have described. The relationship between the MIC of erythromycin and the genetic characterization of the strains is given in Table IIGo.


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Table II. Relationship between the MIC of erythromycin and the genetic characterization of 59 Streptococcus pneumoniae isolated in 1995 and 1997
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Erythromycin resistance in Belgian pneumococcal isolates increased from 24% in 1995 to 28.5% in 1997. Our study shows that enzymatic modification of the MLSB antimicrobial binding site was the dominant mechanism of macrolide resistance both in 1995 and 1997. For 1995 and 1997 combined, 91.5% of the isolates expressed the MLSB-phenotype and this was confirmed at the genetic level by the presence of the ermAM gene.

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.


    Acknowledgments
 
We gratefully acknowledge the assistance of Joyce Sutcliffe (Groton, CT, USA) in supplying the control strains 02J1095 and 02J1175. This work was presented in poster form at the Ninth European Congress of Clinical Microbiology and Infectious Diseases, 21–24 March 1999, Berlin.


    Notes
 
1 Correspondence address. Rega Institute, Minderbroedersstraat 10, 3000 Leuven, Belgium. Tel: +32-16-33-73-72; Fax: +32-16-33-73-40; E-mail: katrien.lagrou{at}rega.kuleuven.ac.be Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Leclercq, R. & Courvalin, P. (1991). Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrobial Agents and Chemotherapy 35, 1267–72.[ISI][Medline]

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, 1817–24.[Abstract]

3 . 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, 2425–6.[Abstract/Free Full Text]

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, 73–8.[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, 2251–5.[Abstract]

6 . Ducoffre, G. (1998). Surveillance van Infectieuze Aandoeningen door een Netwerk van Laboratoria voor Microbiologie 1997 and Epidemiologische Trends 1983–1996. (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 Testing—Eight 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, 2562–6.[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, 786–9.[ISI][Medline]

Received 19 April 1999; returned 15 July 1999; revised 16 September 1999; accepted 20 September 1999