a Servicio de Microbiología, Hospital de Móstoles, 28935 Móstoles, Madrid; b Area de Bioquímica y Biología Molecular, Universidad de la Rioja, Logroño, Spain
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Abstract |
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Introduction |
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Therefore, the emergence and spread of resistance to macrolides in S. pyogenes constitute an important problem in the management of streptococcal infections. In recent years a variety of studies in different areas and countries have been published, showing great diversity in rates of resistance to macrolides.711 Recent data from various parts of Spain indicate increasing prevalence of resistance to macrolides over the years.1215 The mechanisms of macrolide resistance have been elucidated and involve target modification mediated by a methylase (encoded by erm genes), which modifies an adenine in 23S rRNA,16 and an efflux mechanism encoded by the mef gene.17,18 The gene is a novel one with sequence homology to membraneassociated pump proteins.18 Target site modification due to methylase activity confers resistance to macrolides, lincosamides and streptogramin B (MLSB) antibiotics, and is expressed constitutively or inducibly. The efflux mechanism selectively pumps 14- and 15-membered macrolides out of the cell but not 16-membered macrolides or lincosamides. The efflux determinant in streptococci seems to be distinct from the multicomponent macrolide efflux system in coagulase-negative staphylococci.17
Previous studies have used different methods (agar diffusion, agar dilution, broth microdilution), different interpretations of the results and different populations. Moreover, in several of these studies the genes responsible for antibiotic resistance were not determined.
The aim of the present study was to investigate, by the standard agar dilution method, the prevalence of susceptibility to 14-, 15- and 16-membered macrolides and clindamycin in S. pyogenes isolated in Spain in 1998. The numbers of strains collected were proportional to the numbers of inhabitants in each geographical area. The different susceptibility phenotypes and the genetic basis for the antibiotic resistance were also determined.
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Materials and methods |
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A total of 486 unique isolates of S. pyogenes collected in 21 laboratories in Spain from February 1998 to September 1998 were used. The country was arbitrarily divided into 21 geographical areas. The sample size was stratified in proportion to the number of inhabitants of each area, with a ratio of approximately one strain per 80,000 inhabitants. Throat swab samples provided 359 isolates (73.9%), and the remaining 127 isolates were from other sources, including pus from cutaneous lesions (n = 42), otorrhoea (n = 32), vaginal swabs (n = 19) and others (n = 34). Three hundred and eighty-five (79.2%) were isolated from children and 101 (20.8%) from adults. Identification of strains was by standard criteria.19 Strains were kept frozen in skim milk at 30°C.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards.20 Antibiotics were obtained as standard reference powders of known potency from Sigma Chemical Co. (St Louis, MO, USA; penicillin G, erythromycin and clindamycin), Pfizer Inc. (New York, NY, USA; azithromycin) and Menarini (Barcelona, Spain; diacetil-midekamycin = miocamycin). The antimicrobials were incorporated into the medium in a log2 dilution series from 0.008 to 2 mg/L for penicillin G, from 0.06 to 64 mg/L for erythromycin, azithromycin and clindamycin and from 0.12 to 64 mg/L for miocamycin. MuellerHinton agar medium with 5% sheep blood was used. Inocula were prepared by diluting bacterial suspensions equivalent in turbidity to a McFarland 0.5 standard, resulting in c. 104 cfu/spot when applied by a Steer's replicator (Craft Machine Inc., Chester, PA, USA). The plates were incubated overnight at 35°C in an atmosphere containing 5% carbon dioxide. The interpretative categories for each antibiotic were those recommended by the NCCLS.21 The MIC breakpoint for miocamycin resistance was 4 mg/L, as defined by the Comité de lAntibiogramme de la Societé Française de Microbiologie.22 Staphylococcus aureus ATCC 29213 and Streptococcus pneumoniae ATCC 49619 were used as quality control strains. All susceptibility tests were performed in the same laboratory to avoid interlaboratory variation in the results.
Discs containing erythromycin (15 µg) or clindamycin (2 µg) were used to identify antibiotic resistance phenotypes; different phenotypes of MLS resistance were identified according to the description by Seppälä et al.23 After 24 h incubation at 35°C, blunting of the clindamycin zone of inhibition proximal to the erythromycin disc was taken to indicate inducible resistance. Resistance to clindamycin (confirmed by the agar dilution method) with no blunting of the clindamycin zone of inhibition indicated constitutive resistance. The novel resistance phenotype, designated the M phenotype, was characterized by susceptibility to clindamycin with no blunting of the inhibition zone around the clindamycin disc.
Detection of macrolide resistance genes
Twenty-five erythromycin-resistant strains were selected, 20 with the M phenotype, one per laboratory, and all those with the MLSB phenotype. The MLS resistance mechanism was determined by PCR with amplification of erm genes, using degenerate erm primers (E1 5'-GARATIGGIIIIGGIAAGAGGICA-3'; E2 5'-AAYTGRTTITTIGTRAA-3'),24 and specific primers for ermA (A1 5'-TCTAAAAAGCATGTAAAAGAA-3'; A2 5'-CTTCGATAGTTTATTAATATTAGT-3'), ermB (B1 5'-GAAAAGRTACTCAACCAAATA-3'; B2 5'-AGTAACGGTACTTAAATTGTTTAC-3'), ermC (C1 5'-TCAAAACATAATATAGATAAA-3'; C2 5'-GCTA- ATATTGTTTAAATCGTCAAT-3')25 and ermTR genes (TR1 5'-ATAGAAATTGGGTCAGGAAAAGG-3'; TR2 5'-TTGATTTTTAGTAAAAAG-3').26 The conditions used in each case were as previously recommended.18,24,26 The efflux pump mechanism was determined by PCR using primers and specific conditions for amplification of mefA/E genes (A/E1 5'-AGTATCATTAATCACTAGTGC-3'; A/E2 5'-TTCTTCTGGTACTAAAAGTGG-3')18,25 and the msrA gene (msrA1 5'-GCAAATGGTGTAGGTAAGACAACT-3'; msrA2 5'-ATCATGTGATGTAAACAAAAT-3').27 Positive and negative controls of our collection were used in all cases. Genomic DNA for PCR reactions was obtained with the Instagene matrix system (BioRad, Hecules, CA, USA) according to the manufacturer's instructions.
Statistical analysis
The chi-squared test was used.
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Results |
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One hundred and fourteen (23.5%) of the isolates were resistant to erythromycin (MIC breakpoint 1 mg/L). The resistance to both the 14- and 15-membered ring macrolides tested was 23.5%, whereas the resistance to miocamycin, a 16-membered ring macrolide, was 1.0% and the resistance to clindamycin was 0.8%.
The phenotypes of susceptibility to macrolides and lincosamides are shown in Table II. One hundred and nine (95.6%) of the 114 erythromycin-resistant strains were susceptible to clindamycin (MICs
0.060.25 mg/L) and miocamycin (MICs
0.121 mg/L), and induction with erythromycin did not modify susceptibility to the latter antibiotics; these strains were designated as having the M phenotype. Four isolates (3.5%) were resistant to erythromycin (MICs > 64 mg/L), azithromycin (MICs > 64 mg/L), miocamycin (MICs > 64 mg/L) and clindamycin (MICs > 64 mg/L), which indicates a constitutive type of resistance. The remaining erythromycin-resistant strain (MIC 4 mg/L) was susceptible to clindamycin (MIC 0.12 mg/L) and had intermediate resistance to miocamycin (MIC 2 mg/L), but showed an inducible type of resistance.
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There were marked variations according to geographical area. As an example, 31/74 strains (41.9%) isolated in the three laboratories in the south of Spain were erythromycin resistant, versus 83/412 strains (20.1%) isolated in the rest of the country (P = 0.00005). There were no significant differences in the number of erythromycin-resistant strains recovered from children (93/385 or 24.1%) compared with adults (21/101 or 20.8%).
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Discussion |
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The design of our surveillance study, based on the collection of strains per unit population, avoids any bias resulting from some laboratories contributing a greater number of strains with respect to the size of population assigned to them. In addition, we tested all the strains in the same laboratory using standard agar dilution methodology, determined the susceptibility phenotype for each strain and identified the genes responsible for macrolide resistance in a selected sample.
In spite of the extensive use of penicillins and other ß-lactam antibiotics, all our strains were susceptible to penicillin G, as in other parts of the world.29
Until the early 1990s there was a low rate of erythromycin resistance in S. pyogenes isolated in some parts of Spain.30,31 However, the frequency of erythromycin resistance has increased in recent years.1215 In this national study we observed a high prevalence of resistance to both 14- and 15-membered macrolides, although regional variations were found. If we compare data obtained by the same methods in 199613 and 1998 from the same five laboratories, a dramatic increase in resistance is observed in the two-year period. In 1996, 15/103 (14.6%) isolates were erythromycin resistant, but in 1998 resistance had increased to 29.4% (32/109 isolates) (P = 0.009).
The prevalence of resistance to erythromycin but susceptibility to clindamycin and miocamycin in S. pyogenes is one of the highest reported for any country. The great majority of resistant strains (95.6%) have the M phenotype, conferred by the mef gene, as reported in some studies,1315,18,3234 although not in other studies where greater variety in the percentages of various phenotypes was found.79 Miocamycin, the 16-membered ring macrolide tested, and the lincosamides retained full activity against strains with the M phenotype, and could be an alternative for treatment, although more studies are needed to confirm its clinical efficacy. In our study, MICs of erythromycin and azithromycin for strains with the M phenotype are homogeneous. There appears to be a homogeneous population without subphenotypes as in the strains studied recently in Sweden.32
In Spain, with approximately 40000000 inhabitants, outpatient consumption of macrolides was 9.3 x 106 units in 1987 and 14.9 x 106 units in 1996, an increase of >60% (International Marketing Service, Madrid, Spain). In Finland an increase in erythromycin resistance was linked to increased use of erythromycin.11 Therefore in Spain selection pressure by macrolides has probably played a significant role in the spread of resistance. In Finland, after a significant reduction of the use of macrolides in outpatients, there was a significant decrease in the rate of resistance to erythromycin in S. pyogenes isolates.35 Judicious use of macrolides would probably help contain the spread of resistance, as has been seen in other countries.35
Among studies covering entire countries, our study demonstrates one of the highest rates of S. pyogenes erythromycin resistance with clindamycin and miocamycin susceptibility in the world. Strains with the M phenotype account for the great majority of erythromycin-resistant isolates. The high prevalence observed and the geographical variations suggest a need for all laboratories to test for at least a 14-membered macrolide and a 16-membered macrolide or clindamycin in their S. pyogenes isolates as routine practice, informing physicians of the results. Long-term monitoring of the antibiotic resistance to observe its evolution is also advisable, as is study of the prevalence of macrolide resistance in S. pyogenes in other countries, since bacteria do not recognize national boundaries.
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Acknowledgments |
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Notes |
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References |
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Received 15 July 1999; returned 20 October 1999; revised 12 November 1999; accepted 26 November 1999