JAC Advance Access originally published online on March 23, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1240-1243; doi:10.1093/jac/dkl101
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Multicentre surveillance of the prevalence and molecular epidemiology of macrolide resistance among pharyngeal isolates of group A streptococci in the USA
1 Department of Pediatrics, Division of Infectious Diseases, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine Pittsburgh, PA, USA 2 Respiratory Diseases Branch, Centres for Disease Control and Prevention Atlanta, GA, USA 3 Children's Hospital of Columbus Columbus, OH, USA 4 Texas Children's Hospital Houston, TX, USA 5 Children's Hospital of San Diego San Diego, CA, USA 6 New Jersey Medical School—UMDNJ Newark, NJ, USA 7 Mott's Children's Hospital Ann Arbor, MI, USA 8 Arkansas Children's Hospital Littlerock, AR, USA 9 Vanderbilt University Medical Centre Nashville, TN, USA 10 Duke University Medical Centre Durham, NC, USA
*Corresponding author. Tel: +1-412-692-7438; Fax: +1-412-692-8499; E-mail: Michael.Green{at}chp.edu
Received 13 December 2005; returned 3 February 2006; revised 2 March 2006; accepted 3 March 2006
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Abstract |
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Objectives: Rates of macrolide resistance in group A streptococci (GAS) were reported to be low in the US in the 1990s. However, we documented an unexpectedly high rate of macrolide resistance among GAS in Pittsburgh, PA, in 2001 and 2002. In an effort to define the current prevalence of macrolide-resistant GAS in the US, a multicentre surveillance project was initiated.
Methods: Between October 2002 and May 2003, 50 pharyngeal GAS isolates per month were requested from each of the nine participating sites representing a wide geographical distribution. Standard susceptibility testing was performed and the macrolide resistance phenotype was assessed using double-disc diffusion testing. Monthly and annual rates of macrolide resistance were calculated for each site. An adjusted overall rate of macrolide resistance was determined to account for differences in the numbers of GAS isolates sent from each centre.
Results: Overall, 171 of the 2797 collected isolates of GAS (6.1%) were resistant to erythromycin. The adjusted overall resistance rate was 5.2%. Rates of macrolide resistance varied by site (range 3.0–8.7%) and also by month (<2% to >10%). The M phenotype of macrolide resistance accounted for >60% of all macrolide-resistant isolates recovered in this study.
Conclusions: These data suggest an increasing prevalence and broad geographical distribution of macrolide-resistant GAS in the US, indicating the need for ongoing local and national longitudinal surveillance to define the extent of this problem.
Keywords: antibiotic resistance , Streptococcus pyogenes , mef(A)
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Introduction |
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Although macrolide resistance among Streptococcus pyogenes has been recognized in Europe and Japan for many years,1 rates in group A streptococci (GAS) in the US have remained low.2 In the Spring of 2001, we documented the emergence of a high level of macrolide resistance among pharyngeal isolates of GAS acquired in Pittsburgh.3 During this time the monthly prevalence of macrolide resistance ranged from 0 to 41%, averaging 9.6%.4 The persistence of increased macrolide resistance in GAS at our centre over 2 years raises important questions regarding the current rate of resistance in the remainder of the US. The purpose of the current study was to define the prevalence of macrolide-resistant GAS in the US by performing longitudinal surveillance in multiple geographically separated centres.
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Materials and methods |
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Between October 2002 and May 2003, 50 pharyngeal isolates per month were requested from the clinical microbiology laboratory of each of the nine children's hospitals located in Ann Arbor, MI; Columbus, OH; Durham, NC; Houston, TX; Little Rock, AR; Nashville, TN; Newark, NJ; Pittsburgh, PA; and San Diego, CA. All GAS recovered from the Pittsburgh site were evaluated. Isolates were obtained from children presenting to emergency departments and ambulatory care centres with symptoms of acute pharyngitis. All isolates were sent to the clinical microbiology laboratory at the Children's Hospital of Pittsburgh. The Institutional Review Board of each participating centre approved the study.
In vitro susceptibility testing was performed against erythromycin and clindamycin using Kirby–Bauer discs (BBL Becton Dickinson, Sparks, MD, USA).5 MICs were determined using Etest (AB Biodisk, Piscataway, NJ, USA) for isolates demonstrating intermediate susceptibility or resistance to erythromycin or clindamycin. Breakpoints approved by the CLSI for GAS were used.5 Results of antimicrobial susceptibility testing have been reported in part in a separate manuscript.6
The monthly and annual rates of macrolide resistance in GAS were determined for each site by dividing the number of resistant isolates by the total number of GAS isolates received. The overall combined rate of macrolide resistance for the entire study was adjusted to account for differences in the actual number of isolates received from each centre by determining the corrected number of resistant isolates per site (multiplying the observed resistance rate for each site by 400, the number of isolates requested per site for the entire study period) and then dividing the sum of the corrected number of resistant isolates from all sites by 3600 (the total number of isolates requested from the nine sites over the study period).
The genetic relatedness of the erythromycin-resistant isolates was determined initially using field-inversion gel electrophoresis (FIGE) of ApaI-digested genomic DNA.6 emm typing and subtyping, as well as T-typing, were performed at the Streptococcal Laboratory of the Centers for Disease Control and Prevention (CDC) on representative isolates within each FIGE type, including those expressing different macrolide resistance phenotypes.7
Macrolide resistance isolates were categorized into those expressing an M phenotype, an inducible MLS (MLSi) phenotype or a constitutive MLS (MLSc) phenotype using the double-disc diffusion test (D-test).6 The presence of mef(A), erm(B) and erm(A) resistance genes was detected by PCR amplification.3
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Results |
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Overall, 171 of the 2797 collected isolates of GAS (6.1%) were resistant to erythromycin. The adjusted rate of macrolide resistance for all sites during this study period was 5.2%. The ‘projected’ annual rate of macrolide resistance by site varied from 3.0% to 8.7%. Although ‘projected’ annual rates of resistance were <5% at six of the nine sites during 2002–2003, the monthly rate of macrolide resistance exceeded 10% for at least 1 month during the study period at five sites. Remarkably, the rate of macrolide resistance exceeded 20% for at least 1 month at two sites. The ‘projected’ annual rate and range of monthly rates of GAS resistance for each site are shown in Table 1.
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The macrolide resistance phenotype was determined for 171 resistant isolates, of which 117 (68%) expressed the M resistance phenotype, 45 (27%) expressed the MLSi phenotype and 9 (5%) expressed the MLSc resistance phenotype. Although the M phenotype was expressed in more than two-thirds of resistant isolates overall, it accounted for
50% of the resistant isolates recovered from five of the participating centres (Table 1). Molecular epidemiological analysis was performed to determine the relative prevalence of clones of resistant GAS recovered during this study. Among the 171 resistant isolates, 12 emm types were identified. emm75 accounted for 47% of the resistant GAS and was recovered from seven of the nine participating centres. emm12 accounted for 26% of all resistant isolates and was recovered from eight of the nine sites. Table 2 shows the relative prevalence and geographical distribution of emm types within each macrolide resistance phenotype. Eight different emm types were identified among the 117 macrolide-resistant GAS isolates expressing the M phenotype. emm type 75 accounted for 62% of the isolates and was recovered from seven of the nine sites. Eight emm types were identified among the 45 MLSi phenotype isolates, while two emm types accounted for the 9 MLSc resistance phenotype isolates. emm type 12 was the most common type among both MLSi and MLSc phenotypes.
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PCR was performed to confirm the genetic basis of resistance for each of the macrolide resistance phenotypes. The mef(A) gene was detected in each of the eight emm types expressing the M phenotype; erm(A) was detected in seven of the eight emm types expressing the MLSi phenotype, and erm(B) was found in both isolates of the eighth emm type and also found in the single isolate of emm73.3 expressing the MLSc phenotype. No PCR products were obtained for eight MLSc isolates belonging to emm types 12, 12.7, and 12.25; an additional analysis has identified identical dual mutations (A2058G and U2166C) in domain V of 23S rRNA in these isolates.8
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Discussion |
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We recently documented the emergence and persistence of relatively high rates of macrolide resistance in pharyngeal isolates of GAS recovered from children in Pittsburgh.3,4 This multicentre study was initiated to determine the prevalence of macrolide resistance among GAS in the US. We found an overall adjusted rate of macrolide resistance in GAS of 5.2%. Although this rate is relatively low compared with much of the rest of the world, it represents a nearly twofold increase from rates reported in the US in the 1990s. Our results are consistent with three recently published studies evaluating GAS isolates from the US which reported rates of 6.2–6.8% for isolates obtained between 1998 and 20039–11 but differ from observations by Tanz et al.12 who reported a stable rate of macrolide resistance of <5% for 2000–2003.
We observed a high degree of month-to-month variability in the rate of macrolide-resistant GAS in many of our participating centres. Five of the nine centres had a rate of macrolide resistance in excess of 10% for at least 1 month during the surveillance period. This temporal variability is consistent with previous reports4,10 and may account for the differences in reported rates of macrolide resistance. Results reported by Tanz et al.12 were obtained during 2 week intervals from each site on three separate occasions. The narrow timeframe studied may have missed periods of increased prevalence, leading to an underestimation of the prevalence of macrolide resistance.
A fairly wide variation in rates of macrolide resistance between participating centres was observed during the current study (3.0–8.7%) and in reports by Barrozo et al.10 (<1–29%), Richter et al.11 (2.7–11%) and Tanz et al.12 (0–9.0%). In contrast to these observations, Critchley et al.9 found that the rate of resistance was fairly constant throughout the nine Census Regions of the US. The presence of geographical variability implies that overall rates of macrolide resistance in GAS cannot be extrapolated to individual geographical locations within the US. Accordingly, while national surveillance is necessary, results of regional analyses are needed to guide specific antibiotic choices for penicillin-allergic patients requiring treatment for GAS pharyngitis.
Twelve different emm types accounted for all macrolide-resistant GAS recovered during the study period; emm75 accounted for nearly half and emm12 accounted for one-quarter. These two emm types were recovered from the majority of the participating sites in our study and were also the most frequently recovered macrolide-resistant GAS types during the surveillance carried out by others.11,12 While emm12 and emm75 accounted for the majority of resistant isolates, macrolide resistance was found in numerous other emm types in all three studies. Of interest, we identified emm75 GAS among isolates expressing either the M or MLSi resistance phenotypes; emm12 GAS isolates were found to express M, MLSi or MLSc resistance phenotypes. Taken together, these results suggest that macrolide resistance in GAS in the US is being spread both by dissemination of specific clones and by spread of macrolide resistance genes [mef(A), erm(A) or erm(B)] from resistant isolates into previously susceptible strains of GAS. This latter phenomenon probably occurs by way of a transposon, as has been reported for both mef(A) and erm(B).13,14
In conclusion, these data suggest an increasing prevalence and broad geographical distribution of macrolide resistance in GAS in the US. Ongoing surveillance is needed to confirm these observations.
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Transparency declarations |
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Dr M. D. G. has previously served on an advisory board with Aventis-Pasteur, but does not currently have any active grant support or conflicts of interest. Drs C. H. A., K. M. E., E. R. W. and E. B. W. currently have grant support from Sanofi-Pasteur for work unrelated to the current study. Dr E. B. W. is currently on a Speaker's Bureau for Sanofi-Pasteur. Drs B. B., J. S. B., B. D., J. R. G., M. J. M., J. M. M., C. S. and G. E. S. as well as Ms K. A. B. do not currently have commercial or other associations that might pose a conflict of interest with this study beyond the unrestricted grant of support. There are no relevant issues to declare for any of the authors.
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Acknowledgements |
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This study was supported by an unrestricted grant from Aventis. Results were reported in part at the Forty-first Annual Meeting of the Infectious Diseases Society of America in San Diego, CA, October 2003 (Abstract 210), and the Forty-second Annual Meeting of the Infectious Diseases Society of America in Boston, MA, October 2004 (Abstract 343).
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Cresti S, Lattanzi M, Zanchi A, et al. (2002) Resistance determinants and clonal diversity in group A streptococci collected during a period of increasing macrolide resistance. Antimicrob Agents Chemother 46:1816–22.
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