1 Division of Infectious Diseases and 5 Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Il-won dong, Kangnam-ku, Seoul 135710; 2 Department of Medicine, Kyunghee University Medical Center, Seoul; 3 Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul; 4 Chonnam National University Hospital, Kwangju, Korea; 6 Beijing Childrens Hospital, Beijing, China; 7 Chulalongkorn University, Bangkok; 8 Siriraj Hospital, Bangkok, Thailand; 9 Chang Gung Childrens Hospital, Taipei, Taiwan; 10 Christian Medical College, Vellore, India; 11 University of Colombo, Colombo, Sri Lanka; 12 National University of Singapore, Singapore; 13 Universiti Putra Malaysia, Kuala Lumpur; 14 University Malaya, Kuala Lumpur, Malaysia; 15 University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam; 16 Princess Margaret Hospital, Hong Kong
Received 15 August 2003; returned 29 October 2003; revised 17 December 2003; accepted 17 December 2003
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Abstract |
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Methods: Phenotypic and genotypic characterization of the isolates and their resistance mechanisms.
Results: Of 555 isolates studied, 216 (38.9%) were susceptible, 10 (1.8%) were intermediate and 329 (59.3%) were resistant to erythromycin. Vietnam had the highest prevalence of erythromycin resistance (88.3%), followed by Taiwan (87.2%), Korea (85.1%), Hong Kong (76.5%) and China (75.6%). Ribosomal methylation encoded by erm(B) was the most common mechanism of erythromycin resistance in China, Taiwan, Sri Lanka and Korea. In Hong Kong, Singapore, Thailand and Malaysia, efflux encoded by mef(A) was the more common in erythromycin-resistant isolates. In most Asian countries except Hong Kong, Malaysia and Singapore, erm(B) was found in >50% of pneumococcal isolates either alone or in combination with mef(A). The level of erythromycin resistance among pneumococcal isolates in most Asian countries except Thailand and India was very high with MIC90s of >128 mg/L. Molecular epidemiological studies suggest the horizontal transfer of the erm(B) gene and clonal dissemination of resistant strains in the Asian region.
Conclusion: Data confirm that macrolide resistance in pneumococci is a serious problem in many Asian countries.
Keywords: erythromycin, erm(B), mef(A), pneumococci
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Introduction |
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The three main mechanisms of macrolide resistance in S. pneumoniae are ribosomal methylation encoded by the erm(B) gene, macrolide efflux encoded by the mef(A) gene and mutations within rRNA and ribosomal protein.79 Macrolide resistance in pneumococci from Europe and South Africa is predominantly mediated by the erm(B) gene, which is usually associated with high-level resistance, whereas efflux, encoded by the mef(A) gene, shows low-level resistance and accounts for 61%85% of macrolide resistance in North America.10,11 However, the distribution of macrolide resistance genes in pneumococci has not been investigated in many Asian countries despite the high prevalence of resistance. The current study was performed to characterize pneumococcal isolates from Asian countries with regard to the prevalence and level of macrolide resistance, the genetic mechanisms of resistance and the genetic relatedness of resistant isolates.
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Materials and methods |
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Isolates of S. pneumoniae from a range of infections were collected from 10 centres during January 1998March 2001. The 10 centres were each in different Asian countries belonging to the ANSORP Study Group and included Korea (Samsung Medical Center, Seoul), China (Beijing Childrens Hospital, Beijing), Hong Kong (Princess Margaret Hospital, Hong Kong), Thailand (Chulalongkorn University, Bangkok), Taiwan (Chang Gung Childrens Hospital, Taipei), India (Christian Medical College, Vellore), Sri Lanka (University of Colombo, Colombo), Singapore (National University of Singapore, Singapore), Malaysia (University Malaya, Kuala Lumpur) and Vietnam (University of Medicine and Pharmacy, Ho Chi Minh City).
Antimicrobial susceptibility tests
Pneumococcal isolates were screened for susceptibility to penicillin with 1 µg oxacillin discs (BBL Microbiology systems, Cockeysville, MD, USA) in accordance with the NCCLS performance standards.12 Isolates with a zone of inhibition of 20 mm were considered susceptible to penicillin. MICs of penicillin, erythromycin, clarithromycin, azithromycin, clindamycin and trimethoprimsulfamethoxazole were determined by the agar dilution method, in accordance with NCCLS guidelines.12 S. pneumoniae ATCC 49619 was used as a control. An isolate for which the MIC of erythromycin was
1 mg/L was interpreted as resistant to erythromycin.
Detection of the erm(B) and mef(A) genes
erm(B) and mef(A) genes were detected by the duplex PCR technique, as described previously.11,13 Amplified fragments of the erm(B) (639 bp) and mef(A) (348 bp) genes were obtained by using the following primer pairs: erm(B) (upstream, 5'- GAA AAG GTA CTC AAC CAA ATA-3'; downstream, 5'-GTA ACG GTA CTT AAA TTG TTT AC-3') and mef(A) (upstream, 5'-AGT ATC ATT AAT CAC TAG TGC-3'; downstream, 5'-TTC TTC TGG TAC TAA AAG TGG-3'). The PCR mixture was amplified for 35 cycles. Each cycle consisted of 30 s at 94°C for denaturation, 30 s at 50°C for annealing and 1 min 30 s at 72°C for extension, and this was followed by a final extension at 72°C for 10 min.
Serotyping and pulsed-field gel electrophoresis (PFGE)
Serotype was determined by the capsular Quellung method using commercial antisera (Statens Seruminstitut, Copenhagen, Denmark) as recommended by the manufacturer. PFGE was performed as described previously14 for 82 erythromycin-resistant isolates, which were randomly selected among those that were highly resistant (MIC 128 mg/L) with prevalent serotypes 19F, 19A, 14, 23F, 6B or 6A. These 82 isolates possess the erm(B) gene either alone or together with the mef(A) gene. For restriction analysis, each plug was treated with 20 units of SmaI (Roche Molecular Biochemicals, Milan, Italy) in accordance with the manufacturers recommendations. PFGE patterns of Asian isolates were compared with those of the Spain23F clone (SP-264) and an Iceland6B reference strain (IC-2). The PFGE patterns were analysed visually and interpreted according to the criteria described by Tenover et al.15 A divergence in more than three bands was interpreted as indicative of different clones.
Multilocus sequence typing (MLST)
MLST was carried out as described previously.16 PCR fragments of the aroE, gdh, gki, recP, spi, xpt and ddl genes were obtained from chromosomal DNA and directly sequenced using the primers described by Enright & Spratt.16 The sequences of the seven loci were compared with those of corresponding genes at the MLST website (http://www.mlst.net) and were then assigned an allele number. Allele profiles are shown as a series of seven integers corresponding to the alleles at each of the loci, in the order aroE, gdh, gki, recP, spi, xpt and ddl.
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Results |
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A total of 555 isolates of S. pneumoniae were obtained (Table 1). The most common specimen source was sputum (217 isolates), followed by blood (157 isolates), middle ear fluid (54 isolates) and CSF (40 isolates). Among the 555 isolates, 216 (38.9%) were susceptible, 10 (1.8%) intermediate and 329 (59.3%) resistant to erythromycin (Table 1). Vietnam showed the highest prevalence rate of erythromycin resistance (88.3%), followed by Taiwan (87.2%), Korea (85.1%), Hong Kong (76.5%) and China (75.6%). This is in contrast to India (1.5%) and Sri Lanka (10.3%), which showed the lowest prevalence rates. In most countries, MIC90s of erythromycin among pneumococcal isolates were 128 mg/L or higher, except among isolates from Thailand and India (4 and 0.12 mg/L, respectively). In these Asian countries, there were significant increases in the prevalence rates of erythromycin resistance compared with those in 19961997, particularly in Vietnam, Malaysia, Singapore, China and Korea (Figure 1). The prevalence of erythromycin resistance in Taiwan has been persistently high since 1996.
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Among the 329 erythromycin-resistant pneumococci, erm(B) and mef(A) were detected in 157 (47.7%) and 101 (30.7%) isolates, respectively. In 71 (21.6%) isolates, both erm(B) and mef(A) were detected (Table 1). The erm(B) gene was predominant among strains from China (76.9%), Sri Lanka (75%) and Taiwan (70.7%), whereas mef(A) prevailed among strains from Hong Kong (66.7%) and Malaysia (64.3%). The presence of both erm(B) and mef(A) was observed among isolates from Vietnam (49.1%), Korea (38.6%), Malaysia (21.4%), China (20%) and Hong Kong (8.9%). All isolates carrying the erm(B) gene or both the erm(B) and mef(A) genes showed high-level erythromycin resistance (MICs 128 mg/L), and MICs of erythromycin for isolates carrying only the mef(A) gene were 164 mg/L. Eighty-six percent of the isolates with an M phenotype (resistant to macrolides but susceptible to clindamycin) had mef(A), whereas all the isolates with the MLSB phenotype had erm(B) alone, or both erm(B) and mef(A).
Correlation between erythromycin and other drug resistance
Pneumococcal isolates that were resistant to erythromycin were commonly resistant to other antimicrobial agents (Table 2). Erythromycin-resistant isolates were resistant to azithromycin (97%), clarithromycin (96.4%), trimethoprimsulfamethoxazole (77.2%), clindamycin (64.1%) and penicillin (59.9%). Most penicillin-resistant isolates (94.2%) were resistant to erythromycin, whereas 26.7% of the penicillin-susceptible isolates were resistant to erythromycin.
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The most prevalent serotypes among erythromycin-resistant pneumococcal isolates from Asian countries were 19F, 23F, 14, 6B and 6A, which accounted for 74.8% of total isolates (Table 4). The erm(B) gene was more common than the mef(A) gene among isolates with serotypes 6A, 6B, 14 and 23F, whereas the mef(A) gene was predominant in isolates with serotype 19F. Interestingly, 69% of pneumococcal isolates with both the erm(B) gene and the mef(A) gene belong to serotype 19F. Serotype distribution of penicillin-resistant S. pneumoniae was similar to that of erythromycin-resistant isolates; serotypes 19F and 23F accounted for 41.6% and 30.1% in 209 penicillin-resistant isolates, respectively.
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Eighty-two high-level erythromycin-resistant isolates demonstrated 18 different PFGE patterns (Table 5). The type A and B patterns on PFGE were the most common, being found in 50 isolates from five countries (Korea, China, Hong Kong, Vietnam and Malaysia). These isolates belong to serotypes 19F or 19A and (apart from one isolate) possessed both erm(B) and mef(A) genes. However, erythromycin-resistant isolates with the erm(B) gene showed multiple PFGE patterns.
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Nine isolates (two from Korea, two from Hong Kong, two from Vietnam, two from China and one from Malaysia) that showed identical fragment patterns (A or B) on PFGE and that contained both erm(B) and mef(A) genes were subjected to MLST. Allele profiles of all of these isolates were very similar to that of the Taiwan19F-14 clone (1516191562026) with a maximum of one or two different loci (Table 6).
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Discussion |
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Generally, erythromycin resistance in pneumococci results from either modification of the drug-binding site [encoded by erm(B)] or active efflux of the drug [encoded by mef(A)].21 The efflux mechanism is predominant in macrolide-resistant pneumococci in North America,22 whereas ribosomal methylation has been found in >80% of erythromycin-resistant S. pneumoniae isolates in most European countries,11,2326 except Germany.27 However, despite the high prevalence, there have been only a few reports on the mechanism of macrolide resistance in pneumococci in the Asian region. In Hong Kong, 73% of isolates displayed the M phenotype encoded by the mef(A) gene,19 whereas 65% of Taiwanese isolates had the MLSB phenotype encoded by the erm(B) gene.20 Our data provide further information on the distribution of resistance determinants in erythromycin-resistant pneumococcal isolates in the Asian region. Ribosomal methylation by the erm(B) gene was the most common mechanism of erythromycin resistance in China, Taiwan, Sri Lanka and Korea, whereas efflux was more common in erythromycin-resistant isolates from Hong Kong, Singapore, Thailand and Malaysia. In most Asian countries except Hong Kong, Malaysia and Singapore, the erm(B) gene was found in >50% of pneumococcal isolates either singly or dually with the mef(A) gene. Consequently, the level of erythromycin resistance among pneumococci in most Asian countries except Thailand and India was very high, with MIC90s of >128 mg/L. Moreover, most of these erythromycin-resistant pneumococcal isolates from Asian countries showed multiple resistance to other antimicrobial agents. Multidrug resistance in pneumococci may further restrict the selection of effective antimicrobial agents in the treatment of pneumococcal infections. The relationship between antibiotic usage and prevalence of macrolide resistance in Asian countries could not be evaluated in this study because we had no data on antibiotic usage from each country.
Recently, clinical failure of macrolide treatment in pneumococcal infections caused by macrolide-resistant strains has been reported in various parts of the world.2830 Such failure was not confined to pneumococcal diseases caused by highly resistant strains but was evident in those caused by low-level resistant strains. Given the widespread emergence of high-level resistance to erythromycin in pneumococci in the Asian region, as documented in this study, single use of macrolides in the treatment of pneumococcal diseases may result in clinical failure of antimicrobial therapy.
An interesting finding concerning the distribution of resistance determinants was the high rate of the dual presence of the erm(B) and the mef(A) genes among isolates from Vietnam (49.1%) and Korea (38.6%). Most of these isolates belonged to the serotype 19F and had an identical PFGE pattern (A or B). Furthermore, nine of these isolates had an MLST profile similar to the reference strain of the Taiwan19F-14 clone.31,32 Based on the data from PFGE and MLST in this study, the serotype 19F isolates with the erm(B) and mef(A) genes could be variants of the Taiwan19F-14 clone. Since the Taiwan19F-14 clone was originally known to have only an mef(A) gene,31 horizontal transfer of the erm(B) gene into this clone, and subsequent dissemination of this variant, could be responsible for the high rate of the dual presence of the erm(B) and mef(A) genes among isolates from some Asian countries. Recently, novel serotype 23F and 19F clones, which were resistant to penicillin, cephalosporins and macrolide, were found in Taiwan, which could partly explain the high prevalence of erythromycin resistance in Taiwan.33
In summary, surveillance has shown increases in both the prevalence of erythromycin resistance and the distribution of major determinants of macrolide resistance in pneumococci from Asian countries. In addition, molecular studies suggest the clonal spread of resistant strains in the Asian region, which could be one of the major reasons for the rapid increase in macrolide resistance. Given the clinical relevance of macrolide resistance in the treatment of pneumococcal infections, continuous surveillance of resistance and the documentation of clinical outcomes of macrolide treatment in pneumococcal infections caused by macrolide-resistant strains in the Asian region are warranted.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work.
¶ These authors contributed equally to this work.
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