a Servicio de Microbiología, Hospital Donostia and b Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad del País Vasco, San Sebastián, Spain
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Abstract |
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Introduction |
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Materials and methods |
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Identification
Based on the different colony morphology, two or three colonies from each selective culture were studied. Catalase-negative, non-ß-haemolytic and compatible colonies by Gram's stain were identified by rapid ID 32 STREP (bioMérieux, Marcy l'Étoile, France). The test strips were read after 4 h, and identification was obtained using the API LAB Plus Electronic Code Book (Version 3.3.3; bioMérieux).
Susceptibility studies and detection of clindamycin-inducible strains
All streptococci isolated from the selective plates were confirmed as erythromycin resistant by disc diffusion16 after 1824 h of incubation on MuellerHinton agar (Becton Dickinson) supplemented with 5% horse blood agar at 35°C in 5% CO2. At the same time clindamycin-inducible strains were detected. Two erythromycin discs were placed, one apart from and the other near to (at a distance of 15 20 mm) a clindamycin disc. After incubation the presence or absence of blunting was recorded in the zone of inhibition of the clindamycin disc.
MICs of erythromycin and clarithromycin (Abbott Laboratories), roxithromycin (Hoechst-Marion-Roussel, Romainville, France), azithromycin (Pfizer Inc., New York, NY, USA), josamycin (ICN Biomedicals, Costa Mesa, CA, USA), midekamycin (Meiji Pharmaceutical Co., Ltd, Tokyo, Japan), clindamycin (Upjohn, Kalamazoo, MI, USA), streptogramin A and streptogramin B (Rhone-Poulenc, Vitry-Alforville, France) were determined by agar dilution using MuellerHinton agar supplemented with 5% horse blood. The inoculum was 104 cfu/spot and plates were incubated for 1824 h in 5% CO2. S. pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were included as controls.
Presence of the mef and erm genes
Genomic DNA was prepared with Qiagen columns (Chatsworth, CA, USA) after initial incubation with lysozyme (Sigma Chemical Co., St Louis, MO, USA) (4 g/L). A polymerase chain reaction (PCR) was performed using the specific primers (Amersham Pharmacia, Amersham, UK) identified by Clancy et al.15 and Tait-Kamradt et al.17 in order to detect the genes originally described as mefA and mefE, now both known as mefA.18 The primers described by Sutcliffe et al.19 were used for ermA, ermB and ermC genes, whereas the primers identified by Kataja et al.20 were used to detect ermTR. When the PCR products yielded the expected size, microwell hybridization analysis21 was performed using specific biotinylated capture probes. Negative and positive controls were included in each run.
Sequencing
Following PCR, the amplicons obtained for the erythromycin-resistant strains were sequenced automatically with the primers described for the original mefE and ermB genes by ABI 300 Genetic Analyzer (Perkin Elmer Applied Biosystem, Foster City, CA, USA). These sequences were compared with those described for the original S. pneumoniae mefE gene (GenBank accession no. U83667), the original S. pyogenes mefA gene (GenBank accession no. U70055) and the S. pneumoniae ermB gene (GenBank accession no. X52632), respectively, using the software available at the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Mating experiments
An erythromycin-susceptible (MIC < 0.5 mg/L) S. pneumoniae R6 strain22 made rifampicin resistant (MIC 64 mg/L) by stepwise selection was used as the recipient. Conjugation was performed as described previously.7 Transconjugants were selected on blood agar supplemented with erythromycin 2 mg/L and rifampicin (Sigma Chemical Co.) 8 mg/L. DNase (Sigma Chemical Co.) at a final concentration of 1 g/L was added to some matings to rule out transformation.
Detection of the mefA gene and its sequencing in the commensal and transconjugant strains were performed as described previously.
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Results |
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Overall, 224 isolates belonged to the Streptococcus mitis group and 12 to the Streptococcus salivarius group. Seventeen isolates could not be attributed to any group (Table I). In vitro activities of nine antimicrobials in a randomly selected sample of 112 ErR strains were studied. MICs of macrolides, clindamycin and streptogramin B were higher for the strains with the MLSB phenotype than for the strains with the M phenotype (Table II
). Only five strains with the MLSB phenotype had an erythromycin MIC < 32 mg/L and those five were clindamycin inducible.
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The ermB gene was detected in 124 of the 127 ErR strains harbouring the MLSB resistance phenotype. In the other three strains no amplicons were detected using the primers described in Materials and methods. Sequencing the amplicon of the ermB gene of one S. oralis strain showed 96% identity when compared with the ermB gene described for S. pneumoniae.
One S. oralis strain bearing the MLSB phenotype carried both mefA and ermB genes. Sequencing the respective amplicons showed 98% identity with the original mefE gene described for S. pneumoniae and 98% identity with the ermB gene described for S. pneumoniae.
The mefA gene from clinical isolates of S. mitis and S. oralis was transferred by conjugation to an erythromycin-susceptible, rifampicin-resistant strain of S. pneumoniae. Both the donor and the transconjugant strains were resistant to erythromycin, clarithromycin and azithromycin but susceptible to clindamycin. The addition of DNase did not affect the gene transfer. The sequences obtained for the donor and transconjugant strains were >98% identical to the original mefA gene described for S. pyogenes.
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Discussion |
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With variations depending on which resistance phenotype is predominant in each country (M or MLSB), the macrolide resistance shown by S. pneumoniae or S. pyogenes is mediated mainly by two resistance genes: ermB and mefA. The ermB gene determines constitutive or inducible cross-resistance to macrolides, lincosamides and streptogramin B antibiotics. The mefA gene determines resistance only to 14- and 15-membered macrolides. In this study, 49.8% of the ErR strains harboured the mefA gene and 49% carried the ermB gene. With the exception of one strain that carried both resistance genes, each type of strain expressed the appropriate resistance phenotype: the M phenotype for those carrying the mefA gene and the MLSB phenotype for those harbouring the ermB gene. The strain that carried both the mefA and ermB genes showed the MLSB phenotype. This duplicity of genes and its expression as MLSB phenotype has already been identified in S. pneumoniae by other authors.7,29
The DNA of the original mefE13 and the original mefA15 genes is >90% identical. Nevertheless, this slight difference must be highlighted owing to the differences in prevalence depending on the Streptococcus species. Both genes have been identified in S. pneumoniae and S. pyogenes, although mefE is found more frequently in S. pneumoniae whereas mefA is predominant in S. pyogenes. Both variants of the new mefA gene have been detected in the ErR strains by sequencing. However, only the original mefA could be transferred by conjugation to the S. pneumoniae R6 strain. The experiment was not successful with the original mefE. Luna et al.7 transferred the mefA gene from a strain of -haemolytic streptococci to Enterococcus faecalis and S. pneumoniae recipients but they did not specify which type of mefA gene was transferred. Whether or not the large number of subjects with ErR strains in their pharynx influences the high percentage of clinical isolates of erythromycin-resistant S. pneumoniae30 and S. pyogenes14,31 found in Spain is presently unknown.
Taking into account the growing public health problem of antimicrobial resistance, more studies are needed to clarify the exact role played by commensal streptococci of the pharynx as recipients and donors of resistance genes.
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Notes |
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References |
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Received 25 January 2001; returned 29 March 2001; revised 30 April 2001; accepted 4 May 2001