a Laboratoire de Biologie Médicale BIO VSM, 10 rue de la Gare, F-77360 Vaires-sur-Marne; b Laboratoires Abbott, Rungis; c Service de Microbiologie, Centre Hospitalo-Universitaire Robert Debré, Paris; d Service des Maladies Infectieuses, Centre Hospitalo-Universitaire du Bocage, Dijon, France
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Abstract |
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Introduction |
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Three distinctive phenotypes of resistance to the macrolide, lincosamide and streptogramin B antibiotics (MLS) are now recognized in GAS:7 (i) constitutive resistant phenotype (cMLS) and (ii) inducible resistant phenotype (iMLS), both related to ribosome methylation; and (iii) M phenotype, a new resistance pattern related to an efflux mechanism and characterized by susceptibility to lincosamides. Recently, Giovanetti et al.3 described three subtypes of iMLS strains based on their susceptibility profile to 16-membered macrolides, the phenotypic characteristics of these subtypes correlated relatively well with genotypes.
The objectives of this study were: (i) to assess the current prevalence of erythromycin and clarithromycin resistance in GAS isolated in French adults; and (ii) to determine the phenotypes of macrolidelincosamide resistance and the genetic mechanisms employed in the resistant strains.
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Materials and methods |
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A total of 303 consecutive strains were collected during 19981999 from patients (1240 years of age) enrolled in the clinical and microbiological evaluation of short course therapy with clarithromycin-modified release. Throat swabs were obtained by general practitioners at the enrolment visit. The majority of samples were collected from four different regions of France (281 of 303 strains): Pays de la Loire, RhônesAlpesBourgogne, ProvenceAlpesCôte d'Azur and AlsaceLorraine. Twenty-two additional swabs were obtained in two different towns outside these four regions. Specimens were mailed the same day to the central microbiology laboratory (Laboratoire BIO VSM) using Amies transport medium (Bio-Rad, Marnes-La-Coquette, France). On the day of arrival, the swabs were plated on Columbia agar supplemented with 5% sheep blood, colistin and nalidixic acid (bioMérieux, Marcy l'Étoile, France), and then incubated for a total of 48 h in anaerobiosis. Colonies of GAS were identified on the basis of ß-haemolysis, Gram's stain and agglutination with a commercial latex reagent (bioMérieux). Susceptibility testing was performed and strains were stored at 70°C for further MIC determination and the search for macrolide resistance genes after all isolates were collected.
Susceptibility testing
All strains were tested for susceptibility to erythromycin by a standard diffusion method using MuellerHinton agar supplemented with 5% sheep blood (Bio-Rad) and erythromycin discs (15 µg, Bio-Rad). MICs of clarithromycin (Abbott, Rungis, France) were determined by broth microdilution in accordance with the NCCLS8 method using MuellerHinton broth (Bio-Rad) supplemented with a final concentration of 2.5% lysed horse blood (bioMérieux) and incubation in ambient air for 2024 h. A quality control strain of Streptococcus pneumoniae ATCC 49619 was introduced into each batch of experiments. An additional strain of Streptococcus pyogenes ATCC 19615 was used for the control of in-laboratory reproducibility.
Phenotypes of MLS resistance were identified further by a triple disc diffusion test derived from Giovanetti et al.,3 using MuellerHinton agar supplemented with 5% sheep blood and erythromycin (15 µg), clindamycin (2 µg) and spiramycin (100 µg), a 16-membered macrolide, discs (Bio-Rad). After 1824 h of incubation in ambient air, the absence of inhibition around the three discs indicated constitutive resistance (cMLS phenotype), and susceptibility to clindamycin and spiramycin with no blunting indicated the M phenotype of resistance. Blunting of the clindamycin zone of inhibition indicated inducible resistance (iMLS phenotype). The three subtypes of inducible resistance were searched for using the 16-membered macrolide disc: (i) the iMLS-A subtype was characterized by the absence of any zone of inhibition around the spiramycin and erythromycin discs; (ii) the iMLS-B subtype was characterized by blunting of the spiramycin zone of inhibition with no zone of inhibition around the erythromycin disc; and (iii) the iMLS-C subtype was characterized by blunting of the spiramycin zone of inhibition with moderate reduction of the inhibition zone around the erythromycin disc.
Determination of erythromycin resistance genes
Erythromycin-resistant isolates were screened for the usual genes of macrolide resistance in GAS. The ermB, ermTR and mefA genes were detected by PCR amplification as described previously.6 The following primers were used: 5'-CGA GTG AAA AAG TAC TCA ACC-3' and 5'-GGC GTG TTT CAT TGC TTG ATG-3' to detect ermB; 5'-GCA TGA CAT AAA CCT TCA-3' and 5'-AGG TTA TAA TGA AAC AGA-3' to detect ermTR; and 5'-AGT ATC ATT AAT CAC TAG TGC-3' and 5'-TTC TTC TGG TAC TAA AAG TGG-3' to detect mefA (Genset, Paris, France). Amplification was performed in a DNA thermal cycler (no. 9600; Perkin-Elmer Cetus, Norwalk, CT, USA) programmed for one cycle of denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 1 min, primer annealing at 55°C for 1 min and extension at 72°C for 1 min. Amplification products were run through 2% agarose gels. Gels were stained with ethidium bromide and the DNA bands were visualized with a UV transilluminator.
S. pyogenes 02C 1061, S. pyogenes 02C 1110 and S. pyogenes 02C 1064 (kindly provided by J. Sutcliffe) were used as positive PCR controls for the ermB, ermTR and mefA genes, respectively. Amplification of DNA from the positive controls with the corresponding primers yielded PCR products of the expected sizes (616, 348 and 206 bp for ermB, ermTR and mefA, respectively).
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Results |
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The Table summarizes MICs of clarithromycin in relation to the susceptibility of isolates to erythromycin and the phenotypes of resistance. MICs of the iMLS isolates were lower than those of the M phenotype isolates, and cMLS isolates were of high-level resistance.
PCR amplification for the 29 resistant strains (Table) showed that all cMLS, iMLS and M phenotype isolates harboured ermB, ermTR and mefA genes, respectively. No strain harboured more than one gene simultaneously.
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Discussion |
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Phenotypic characteristics of these iMLS isolates together with low MICs of clarithromycin are consistent with the description of the iMLS-C phenotype recently made by Giovanetti et al.3 However, the ermTR gene alone was detected in our iMLS isolates, while Giovanetti et al. detected both the ermTR and mefA genes in 11 of their 12 strains. Moreover, none of the cMLS isolates in the present study harboured both the ermB and mefA genes, contrary to the Italian study.3 Similar genotypes in cMLS strains were also observed in recent French6 and Finnish9 isolates. The M phenotype is predominant amongst strains isolated in European countries such as Spain,4 some areas of Italy3 and Finland9, which have high rates of erythromycin resistance. This phenotype accounted for only one-third of our resistant strains.
The type of macrolide prescribed predominantly in each country, e.g. short- versus long-acting macrolides, or C-14 versus C-15 and C-16 macrolides, could impact on the prevalence of resistance and the distribution of genotypes. Such a hypothesis warrants further investigation.
The present study confirms that the macrolide resistance rate in France seems stabilized at 10% amongst GAS isolates. With this relatively low resistance rate compared with those of other European countries, erythromycin and clarithromycin still remain an alternative to penicillin G for the treatment of streptococcal pharyngo-tonsillitis in France. However, continued surveillance of macrolide resistance in GAS is advisable.
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Acknowledgments |
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Notes |
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References |
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2
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Savoia, D., Avanzini, C., Bosio, K., Volpe, G., Carpi, D., Dotti, G. et al. (2000). Macrolide resistance in group A streptococci. Journal of Antimicrobial Chemotherapy 45, 417.
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Giovanetti, E., Montanari, M. P., Mingoia, M. & Varaldo, P. E. (1999). Phenotypes and genotypes of erythromycin resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains. Antimicrobial Agents and Chemotherapy 43, 193540.
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Baquero, F., Garcia-Rodriguez, J. A., de Lomas, J. G., Aguilar, L. & the Spanish Surveillance Group for Respiratory Pathogens. (1999). Antimicrobial resistance of 914 beta-hemolytic streptococci isolated from pharyngeal swabs in Spain: results of a 1-year (19961997) multicenter surveillance study. Antimicrobial Agents and Chemotherapy 43, 17880.
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Arpin, C., Canron, M. H., Noury, P. & Quentin, C. (2000). Emergence of mefA and mefE genes in beta-haemolytic streptococci and pneumococci in France. Journal of Antimicrobial Chemotherapy 44, 1338.
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Bingen, E., Fitoussi, F., Doit, C., Cohen, R., Tanna, A., George, R. et al. (2000). Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients. Antimicrobial Agents and Chemotherapy 44, 14537.
7 . Seppälä, H., Nissinen, A., Yu, Q. & Huovinen, P. (1993). Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland. Journal of Antimicrobial Chemotherapy 32, 88591.[ISI][Medline]
8 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.
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Kataja, J., Huovinen, P., Skurnik, M., the Finnish Study Group for Antimicrobial Resistance & Seppälä, H. (1999). Erythromycin resistance genes in Group A streptococci in Finland. Antimicrobial Agents and Chemotherapy 43, 4852.
Received 16 January 2001; returned 29 March 2001; revised 26 April 2001; accepted 4 May 2001