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Antimicrobial Agents and Chemotherapy, July 2005, p. 3031-3033, Vol. 49, No. 7
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.7.3031-3033.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Johnson & Johnson Pharmaceutical Research & Development, L.L.C.,1 Ortho-McNeil Pharmaceutical, Inc., Raritan, New Jersey,2 Focus Bio-Inova, Herndon, Virginia3
Received 12 January 2005/ Returned for modification 31 January 2005/ Accepted 6 March 2005
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In order to determine the specific ribosomal mutations (i.e., ribosomal protein or 23S rRNA) that exist in macrolide-resistant clinical isolates in the United States, isolates that were mefA and ermB negative were checked for mutations in the genes coding for ribosomal proteins L4 and L22 and 23S rRNA. Isolates were also checked for the presence of ermA. Specifically, from previous studies in our lab, we had in our possession 70 macrolide-resistant S. pneumoniae isolates collected in 1999 and 252 macrolide-resistant isolates collected in 2000, representing a partial collection of all the macrolide-resistant isolates from TRUST 3 and TRUST 4 surveillance studies, respectively. Most of the TRUST 3 isolates (53/70) were highly resistant to azithromycin (MIC 
16 µg/ml; range, 4 to >128 µg/ml), while the 252 TRUST 4 isolates included strains with lower macrolide MICs (azithromycin MIC range, 1 to >128 µg/ml).
MICs were determined by broth microdilution using panels manufactured by Trek Diagnostic Systems (Westlake, OH) and using NCCLS methods (10). The ermB and mefA genes were detected by PCR as described by Sutcliffe et al. (14), and mutations in 23S rRNA, L4, and L22 genes were detected as described by Tait-Kamradt et al. (17). Detection of ermA by PCR was done as described by Syrogiannopoulos et al. (15). Genetic relatedness among macrolide-resistant isolates containing ribosomal mutations was determined by serotyping and pulsed-field gel electrophoresis (PFGE) as described previously (2).
The genotypes of the 70 TRUST 3 isolates and 252 TRUST 4 isolates are presented in Table 1. For the TRUST 3 isolates, ermB was the most prevalent resistance mechanism (Table 1), which can be attributed to the bias for very high levels of macrolide resistance in this isolate collection: 76% of isolates had azithromycin MICs of 16 µg/ml. However, mefA was the most common resistance mechanism in the TRUST 4 isolates (Table 1); these isolates had a broader range of azithromycin MICs and, hence, were more representative of the natural distribution of macrolide-resistant isolates (7). Mutations in the 23S rRNA genes were much more common than L4 mutations. Two isolates (5459 and 5486) had mefA but exhibited uncharacteristically high macrolide MICs (i.e., azithromycin MICs of >128 µg/ml) and resistance to clindamycin (Table 2). Upon further characterization, these two isolates were also found to contain 23S rRNA mutations (Table 2). In addition, one of these isolates (5486) had a mutation leading to a Glu77-to-Gly substitution in L22. While mutations in the gene encoding L22 have been shown to cause macrolide resistance in clinical isolates (5, 6, 8), this particular substitution has not been previously reported, and its role in resistance is uncertain.
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0.5 µg/ml) (Table 2). While the isolate with ermA and an L4 mutation (5430) was susceptible to clindamycin by broth microdilution, a double-disk test with erythromycin and clindamycin showed this isolate to be inducibly clindamycin resistant.
A majority of isolates (14/21) with ribosomal mutations had low telithromycin MICs (
0.06 µg/ml); however, four isolates with mutations at A2058/A2059 had elevated telithromycin MICs (0.25 to 1.0 µg/ml) (Table 2). A recent publication by Doern and Brown showed that in the PROTEKT 2000-2001 surveillance study, the telithromycin MIC90 among all isolates of S. pneumoniae was 0.5 µg/ml (4). However, among penicillin-susceptible pneumococci, the telithromycin MIC90 was 0.03 µg/ml. Three isolates with elevated telithromycin MICs in our study were penicillin-susceptible; thus, their telithromycin MICs were 8- to 32-fold higher than the reported MIC90 from the PROTEKT study (Table 2). This indicates that isolates with elevated telithromycin MICs were present before the introduction of telithromycin in the United States.
Other ribosomal-acting agents (tetracycline, linezolid, and quinupristin-dalfopristin) were tested and all isolates were susceptible except for isolate 5459, which was resistant to tetracycline (Table 2). Nine (43%) isolates were penicillin resistant (Table 2).
PFGE analyses showed that three isolates (5719, 5892, and 5948) with serotype 23F had almost identical SmaI patterns, but they did not all have the same resistance mechanisms (Table 2). Two isolates (5430 and 5501) with serotype 29 had the same PFGE pattern but different resistance mechanisms (Table 2). All other isolates had unique PFGE patterns.
The macrolide-resistant isolates with ribosomal mutations that were identified in this study had mutations primarily in domain V of 23S rRNA, and most were not clonally related. Non-Erm, non-Mef macrolide-resistant S. pneumoniae isolates from Finland were recently reported to contain mutations primarily in 23S rRNA. Some of these isolates were clonally related (12). In contrast, non-Erm, non-Mef macrolide-resistant S. pneumoniae isolates from Eastern Europe were reported to primarily have mutations in the ribosomal protein L4, and a majority of these isolates were clonally related (9, 16).
Macrolide/azalide usage continues to increase (7). Macrolide prescriptions increased 13% from 1993 to 1999, despite total antibiotic prescriptions decreasing 15% during the same time frame. The greatest increase in macrolide prescriptions was reported for children less than 5 years of age, for whom the number of prescriptions increased 320% from 1993 to 1999 (7). Twenty-nine percent (6/21) of the isolates containing ribosomal mutations in this study were isolated from children less than 5 years of age.
In summary, 21 of 322 macrolide-resistant S. pneumoniae isolates contained ribosomal mutations (23S rRNA or L4), with mutations in 23S rRNA being predominant. Two novel combinations of A2059G/mefA and L4 (69GTG71-TPS)/ermA were identified. The appearance of these mechanisms may in part be due to the continued widespread use of macrolide and azalide antibiotics. Recent TRUST 7 (2003) surveillance data showed that 27.5% of S. pneumoniae in the United States are macrolide resistant (13). How the introduction of telithromycin in the United States will affect macrolide resistance rates and the types of resistance mechanisms observed among pneumococci remains to be determined. This study provides a framework of the types of ribosomal mutations that existed among macrolide-resistant pneumococci prior to the use of telithromycin in the clinic.
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ACKNOWLEDGMENTS |
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Financial support for this work was provided by Johnson & Johnson Pharmaceutical Research and Development, L.L.C., and Ortho-McNeil Pharmaceutical, Inc.
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FOOTNOTES |
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