Prevalence of macrolide and tetracycline resistance mechanisms in Streptococcus pyogenes isolates and in vitro susceptibility to telithromycin

Carmen Betriu*, Esther Culebras, Montserrat Redondo, Iciar Rodríguez-Avial, María Gómez, Ana Boloix and Juan J. Picazo

Department of Clinical Microbiology, Hospital Clínico San Carlos, Plaza Cristo Rey s/n, 28040 Madrid, Spain

Sir,

Among Streptococcus pyogenes, cross-resistance to macrolides, lincosamides and streptogramin B (MLSB) antimicrobials is encoded by two rRNA methylase genes [erm(B) and erm(A)]. In contrast, macrolide efflux, which is mediated by a membrane protein encoded by the mef(A) gene, produces resistance to 14- and 15-membered macrolides only. The purpose of this study was to determine the prevalence of erythromycin and tetracycline resistance genes among resistant isolates and their clonal relationships, and to evaluate the in vitro activity of the new ketolide telithromycin against erythromycin-resistant and -susceptible strains.

Seventy erythromycin-susceptible and 55 erythromycin-resistant S. pyogenes isolates consecutively collected from different patients in the Hospital Clínico San Carlos were tested. The sources of isolates and sites of infection were the upper respiratory tract (92), skin and soft tissues (16), blood (10), vagina (6) and urine (1). Erythromycin resistance phenotypes were determined by the double disc test. The presence of erythromycin resistance genes in the 55 resistant isolates was determined by PCR.1 Dot blot hybridization was used to confirm some results. The 26 tetracycline-resistant isolates were tested for the presence of tet(K), tet(L), tet(M) and tet(O) tetracycline resistance determinants by PCR. Randomly amplified polymorphic DNA (RAPD) analysis was carried out to determine the clonal characteristics of the erythromycin-resistant isolates, as described previously by Seppälä et al.,2 with two different primers (M13 and H2), and the same criteria were used to interpret and compare the patterns. The MICs of erythromycin, roxithromycin, azithromycin, miocamycin, clindamycin, tetracycline and telithromycin were determined by the agar dilution method in accordance with the NCCLS guidelines.3

According to the double disc test, three (5.5%) of the 55 erythromycin-resistant isolates showed the constitutive MLSB (cMLSB) resistance phenotype, five (9%) the inducible MLSB (iMLSB) phenotype, and the 47 (85.5%) remaining strains showed the M phenotype. Distribution of phenotypes and genotypes of erythromycin and tetracycline resistance are shown in Table 1. The most prevalent gene was mef(A) (in 85.4% of isolates) followed by erm(A) (32.7%). All 47 M phenotype isolates harboured the mef(A) gene, and 12 of them also had the erm(A) gene, the presence of which was confirmed by dot blot hybridization. The five iMLSB isolates harboured the erm(A) gene although three also contained erm(B). Other studies, such as that of Bemer-Melchior et al.,4 did not find iMLSB isolates possessing both erm(A) and erm(B) genes. Our results show that the tet(M) gene was the most prevalent (73.1%) in the tetracycline-resistant S. pyogenes isolates tested. Although tetracycline and erythromycin resistance genes are often found on the same mobile unit, in our S. pyogenes isolates, tet(M) was not associated with the erm(B) gene. The presence of the tet(M) gene was detected in the seven erythromycin-resistant isolates that were also resistant to tetracycline.


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Table 1.  Distribution of erythromycin and tetracycline resistance genes among 55 erythromycin-resistant S. pyogenes isolates with different resistance phenotypes and RAPD profiles
 
Amplification of genomic DNAs from the 47 M phenotype isolates using primer M13 resulted in 20 different patterns. Twenty-two RAPD patterns were produced by primer H2 (Table 1). The results of RAPD-M13 and RAPD-H2 analysis agreed for 64% of the isolates, as both methods identified the same 16 different strains among 30 isolates. Among the other four RAPD-M13 patterns, primer H2 differentiated three groups within one pattern and two groups in the other. The M13 primer was more discriminative in only one case and distinguished two groups in one RAPD-H2 pattern. RAPD-PCR typing of the strains with inducible and constitutive phenotype resulted in four and two fingerprint patterns, respectively, and agreed for both M13 and H2 primers. Combination of results with the two primers revealed that the strains studied had different clonal origins. Most of the patterns were observed in a small number (one, two or three) of isolates. There were two predominant fingerprint patterns, and both were shared by only eight isolates each.

Strains that were resistant to erythromycin were also resistant to the 14- and 15-membered macrolides tested. Clindamycin was active against strains with inducible erythromycin resistance (MIC90 0.2 mg/L) and, as Jalava et al.5 have described, it was more active than telithromycin against strains with an M phenotype (MIC range 0.01–0.06 mg/L). For the erythromycin-susceptible isolates, telithromycin was the most active drug tested; MICs of telithromycin were 0.008–0.01 mg/L, two- to four-fold lower than those of erythromycin. The five strains with the iMLSB phenotype, as well as the 47 M phenotype isolates, were inhibited by telithromycin at concentrations of 0.008–0.5 mg/L. However, the three cMLSB isolates had telithromycin MICs 2–16 mg/L. Our results agree with those described by Jalava et al.,5 who found that telithromycin MICs for S. pyogenes strains with the cMLSB phenotype of erythromycin resistance were also high. Interestingly, we found one iMLSB isolate that possessed both the erm(B) and erm(A) genes, and was susceptible to telithromycin (MIC 0.008 mg/L), unlike the iMLSB isolates with the erm(B) gene described by other groups,5,6 which were intermediately resistant to telithromycin (MICs 1–16 mg/L).

The findings of this study suggest that the high rate of erythromycin resistance in S. pyogenes in our hospital is not caused by the spread of a single clone. Furthermore, results of susceptibility testing indicate that the new ketolide telithromycin could be considered as an alternative for the treatment of infections caused by erythromycin-susceptible as well as erythromycin-resistant S. pyogenes isolates of the MLSB inducible or M phenotypes.

Acknowledgements

This work was supported by grant 99/0434 from the Fondo de Investigación Sanitaria, Madrid, Spain. This study was presented in part at the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 26–29 September 1999.

Footnotes

* Corresponding author. Tel: +34-913303486; Fax: +34-913303478; E-mail: cbetriu{at}efd.net Back

References

1 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

2 . Seppälä, H., He, Q., Osterblad, M. & Huovinen, P. (1994). Typing of group A streptococci by random amplified polymorphic DNA analysis. Journal of Clinical Microbiology 32, 1945–8.[Abstract]

3 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing. Ninth Informational Supplement M 100-S9. NCCLS, Wayne, PA, USA.

4 . Bemer-Melchior, P., Juvin, M. E., Tassin, S., Bryskier, A., Schito, G. C. & Drugeon, H. B. (2000). In vitro activity of the new ketolide telithromycin compared with those of macrolides against Streptococcus pyogenes: influences of resistance mechanisms and methodological factors. Antimicrobial Agents and Chemotherapy 44, 2999–3002.[Abstract/Free Full Text]

5 . Jalava, J., Kataja, J., Seppälä, H. & Huovinen, P. (2001). In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin-resistant Streptococcus species. Antimicrobial Agents and Chemotherapy 45, 789–93.[Abstract/Free Full Text]

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