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JAC Advance Access originally published online on April 3, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1171-1176; doi:10.1093/jac/dkm089
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Activities of 16-membered ring macrolides and telithromycin against different genotypes of erythromycin-susceptible and erythromycin-resistant Streptococcus pyogenes and Streptococcus pneumoniae

Annarita Mazzariol1, Raffaella Koncan1, Luca Agostino Vitali2 and Giuseppe Cornaglia1,*

1 Dipartimento di Patologia, Sezione di Microbiologia, Università degli Studi di Verona, Strada Le Grazie 8, 37134 Verona, Italy 2 Dipartimento di Biologia Molecolare, Cellulare e Animale, Università degli Studi di Camerino, Via F. Camerini 5, 62032 Camerino, Italy

* Corresponding author. Fax: +39-045-58-46-06; E-mail: giuseppe.cornaglia{at}univr.it

Received 4 August 2006; returned 19 September 2006; revised 24 January 2007; accepted 5 March 2007


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Objectives: To test four 16-membered macrolides (josamycin, spiramycin, midecamycin and rokitamycin) along with other compounds in the same class (erythromycin, clarithromycin, roxithromycin and azithromycin) plus clindamycin and telithromycin, against Streptococcus pyogenes and Streptococcus pneumoniae isolates with well-characterized resistance genotypes.

Methods: Four hundred and eighty-six isolates of S. pyogenes and 375 isolates of S. pneumoniae were assayed for their macrolide susceptibilities and investigated by PCR to detect their different erythromycin resistance genes. All strains had been isolated over the period 2002–2003 from specimens of different human origin obtained in 14 different Italian centres.

Results: All 16-membered macrolides showed very low MICs (MIC50s and MIC90s, ≤0.06–0.5 mg/L) for the erythromycin-susceptible isolates and for those with the M phenotype, but the telithromycin MICs for the M-type isolates were at least four times higher (MIC90s, 0.5 mg/L). In S. pyogenes, the MIC50s of 16-membered macrolides for the cMLSB isolates were ≥ 256 mg/L, whereas that for telithromycin was 4 mg/L; the MIC50s of 16-membered macrolides and telithromycin ranged from ≤ 0.06 to 0.5 mg/L for the iMLSB isolates with erm(A) and from 0.12 to ≥ 256 mg/L for those with erm(B). In S. pneumoniae, the MIC50s of the 16-membered macrolides for the cMLSB isolates ranged from 0.5 to 128 mg/L, whereas for the iMLSB isolates their values ranged from ≤ 0.06 to 4 mg/L; the MIC50s and MIC90s of telithromycin for both the cMLSB and the iMLSB isolates ranged from ≤ 0.06 to 0.12 mg/L.

Conclusions: MICs ranged for all the drugs, except telithromycin, from ≤ 0.06 to ≥ 256 mg/L, with 15% to 30% resistant S. pyogenes for all drugs tested except clindamycin (8%) and telithromycin (5.4%) and 10% to 40% resistant S. pneumoniae for all drugs tested except telithromycin (0.3%). In both S. pyogenes and S. pneumoniae, erythromycin resistance related to a mef gene meant that telithromycin MICs were definitely higher than in erythromycin-susceptible isolates, although telithromycin susceptibility was preserved in all cases. In S. pyogenes, the activity of both 16-membered macrolides and telithromycin against the iMLSB strains proved to be dependent on the erm gene involved, being greater against isolates with erm(A).

Keywords: antimicrobial resistance surveillance , macrolides , azalides , group A streptococci , GAS


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Since the early 1990s, a dramatic increase in the isolation of erythromycin-resistant streptococci has been observed worldwide, some of the highest prevalence values being reported in Italy.1–3 The precise implications for clinical outcomes of the different resistance phenotypes need to be fully elucidated, also with a view to the possibility of both testing and using alternative compounds in the same class.

Data regarding susceptibility to 16-membered macrolides are few and far between, often involving a limited number of compounds and suggesting heterogeneous susceptibility patterns. This prompted us to test four 16-membered macrolides, along with other compounds in the same class and clindamycin and telithromycin, against a substantial number of Streptococcus pyogenes and Streptococcus pneumoniae isolates representative of different Italian geographical areas and well-characterized with regard to their resistance genotype.


   Materials and methods
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Four hundred and eighty-six isolates of S. pyogenes and 375 isolates of S. pneumoniae were tested for their susceptibility to four 16-membered macrolides (josamycin, spiramycin, midecamycin and rokitamycin), three 14-membered macrolides (erythromycin, clarithromycin and roxithromycin), the 15-membered azalide azithromycin, the lincosamide clindamycin and the ketolide telithromycin.

All strains had been isolated over the period 2002–2003 from specimens of different human origin (92% throat swabs and the others being from either blood or wound exudates) obtained in different geographical areas and from patients of different ages.

MICs were determined by agar dilution on Mueller–Hinton plates supplemented with 5% sheep blood and inoculated with 104–105 cfu/mL using a Steer inoculator. The plates were incubated overnight at 35°C with 5% CO2. S. pneumoniae ATCC 49619 was used as a control strain. Interpretation of the results was basically as outlined in the latest CLSI (formerly NCCLS) guidelines.4 When CLSI breakpoints were not available, the results were interpreted according to either the breakpoints proposed by the French Society for Microbiology (telithromycin, roxithromycin, josamycin, spiramycin and midecamycin)5 or those published by Ono et al.6 (rokitamycin).

The resistance phenotype was determined by the double disc test with erythromycin and clindamycin as described previously.7

The presence of resistance genes was determined by PCR amplification, as described by Daly et al.8 PCR primers for mef(E), mef(A), erm(A) and erm(B) were designed to provide specific PCR products of 363, 553, 590 and 764 bp, respectively. The mef(E) primers were as described by Daly et al.8 (5'-GGG AGA TGA AAA GAA GGA GT-3' and 5'-TAA AAT GGC ACC GAA AG-3'). The mef(A) primers were as described by Daly et al.8 (5'-TGG TTC GGT GCT TAC TAT TGT-3' and 5'-CCC CTA TCA ACA TTC CAG A-3'). The erm(A) primers were 5'-CCC GAA AAA TAC GCA AAA TTT CAT-3' and 5'-CCC TGT TTA CCC ATT TAT AAA CG-3', and the erm(B) primers were 5'-CAC TTC AGG AGT GAT TAC ATG AA-3' and 5'-CTC ATA GAA TTA TTT CCT CCC GT-3'. Briefly, 1 µL of each lysate was used in a 25 µL reaction mixture at an annealing temperature of 52°C (mef), 48°C [erm(A)] or 56°C [erm(B)]. Products were run on a 1.5% agarose gel and visualized with ethidium bromide staining. Data were validated by sequencing selected PCR products.


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Table 1 lists the MICs and interpretative categories for S. pyogenes and S. pneumoniae. One hundred and fifty-two S. pyogenes isolates (31.3% of the total) were erythromycin-resistant. All of the erythromycin-resistant strains were also resistant to clarithromycin, azithromycin and roxithromycin. Forty-eight isolates (9.9% of the total) proved non-susceptible to clindamycin. The number of isolates non-susceptible to 16-membered macrolides ranged from 78 (rokitamycin, 16% of the total) to 95 (midecamycin, 19.5% of the total). Four hundred and fifty-seven isolates (94% of the total) were susceptible to telithromycin, with MIC50 and MIC90 values of ≤ 0.06 and 0.5 mg/L, respectively.


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  		Table 1.. MICs (mg/L) and interpretative categories for S. pyogenes (486 isolates) and S. pneumoniae (375 isolates)

 
Table 1 also shows that 145 S. pneumoniae isolates (38.7% of the total) were erythromycin-resistant, virtually all of them also proving resistant to clarithromycin and azithromycin. The results for roxithromycin were very similar, too. The number of isolates non-susceptible to 16-membered macrolides ranged from 48 (rokitamycin, 12.8% of the total) to 97 (spiramycin, 25.8% of the total). Only a single isolate (0.3% of the total) was non-susceptible to telithromycin (MIC, 8 mg/L).

In terms of genotype distribution, 40 S. pyogenes isolates (26.3% of all erythromycin-resistant isolates) exhibited constitutive MLSB resistance (cMLSB), 69 (45.4%) inducible MLSB resistance (iMLSB) and 43 (28.3%) M-type resistance. At PCR analysis, all cMLSB isolates showed the erm(B) gene, all M isolates showed the mef(A) gene and no resistance genes were found in the erythromycin-susceptible isolates. Fifty-one of the iMLSB isolates had the erm(A) gene and 18 had the erm(B) gene. Concerning S. pneumoniae, 87 isolates (60% of all erythromycin-resistant isolates) were cMLSB, 32 (22.1%) iMLSB and 26 (17.9%) M-type. In all the MLSB isolates, whether constitutive or inducible, PCR analysis showed an erm(B) gene. All the M-type isolates had a mef gene, no phenotypic difference being observed between strains harbouring mef(A) (69.2%) and mef(E) (30.8%), respectively (data not shown).

Table 2 breaks down the activities of 16-membered macrolides and telithromycin by different erythromycin resistance phenotypes. In S. pyogenes, all 16-membered macrolides showed indistinctly low MICs both for the erythromycin-susceptible strains and for those with the M phenotype, whereas telithromycin MICs for the M-type isolates were at least eight times higher than for the erythromycin-susceptible isolates. All the 16-membered macrolides showed MIC50s equal to or above 256 mg/L for all the cMLSB isolates. Among the iMLSB isolates, those with erm(B) turned out to be the ones with the higher level of resistance (MIC50 = 32 mg/L and MIC90 = 256 mg/L in the case of rokitamycin; MIC50 and MIC90 ≥ 256 mg/L in all other cases), whereas those with erm(A) were much more susceptible, ranging in activity from rokitamycin (MIC50 and MIC90 ≤ 0.06 mg/L) to spiramycin (MIC50 = 0.5 mg/L and MIC90 = 8 mg/L). Telithromycin proved more active than the 16-membered macrolides against both the cMLSB and the iMLSB isolates, a further distinction being observed between the iMLSB isolates with erm(B) (MIC50 = 0.12 mg/L and MIC90 = 8 mg/L) and those with erm(A) (MIC50 and MIC90 ≤ 0.06 mg/L). All telithromycin-resistant strains carried an erm(B) gene; 80.7% of them (21 isolates) had a cMLSB phenotype and 19.3% (5 isolates) had an iMLSB phenotype.


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  		Table 2.. MICs for S. pyogenes and S. pneumoniae strains classified by erythromycin susceptibility

 
Table 2 also shows that all of the 16-membered macrolides were very active against both the erythromycin-susceptible and the M-type isolates of S. pneumoniae (MIC90s, ≤0.06 to 0.12 mg/L). As with S. pyogenes, telithromycin MICs were higher (at least 4-fold) for the M-type isolates than for the erythromycin-susceptible isolates. All of the 16-membered macrolides showed a wide range of activities against the cMLSB isolates (≤0.06 to > 256 mg/L), although the MIC90 for these isolates was always ≥ 128 mg/L. The iMLSB isolates showed a greater susceptibility, rokitamycin being the most active compound (MIC50 ≤ 0.06 mg/L and MIC90 = 0.5 mg/L). Telithromycin MICs were consistently low both for the cMLSB and for the iMLSB isolates (MIC90, 0.12 and ≤0.06 mg/L, respectively); only one cMLSB (MIC90, 8 mg/L) and no iMLSB strains proved resistant.


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Resistance to 14- and 15-membered macrolides commonly used in clinical practice could be theoretically overcome by employing ‘diverse’ macrolides, such as 16-membered compounds and telithromycin.

Although telithromycin has been clearly shown to be active against most streptococcal strains, irrespective of their erythromycin susceptibility, data regarding 16-membered compounds are few and far between, often collected on the occasion of specific outbreaks only and limited to the compounds used in those specific countries.

All of the M isolates and 65% of the iMLSB isolates (but none of the cMLSB isolates) were susceptible to the 16-membered compound miokamycin in the Finnish outbreak,7 and all of the M isolates and roughly 50% of the iMLSB isolates (but again none of the cMLSB isolates) were susceptible to another 16-membered compound, namely josamycin, in a survey carried out during the Italian outbreak.9 In another Italian survey, only the iMLSB isolates with erm(A) presented a definite zone of inhibition around the josamycin or spiramycin discs in the agar diffusion test.10

Consistent with those partial reports, our results showed that all four 16-membered macrolides we tested were generally active against M-type isolates of S. pyogenes (MIC50s and MIC90s, ≤0.06 to 0.5 mg/L), but not against the cMLSB isolates (MIC50s, ≥ 256 mg/L). As regards the iMLSB isolates, the susceptibility values were distinctly lower in the isolates with erm(A) (ranging from ≤ 0.06 to 0.5 mg/L) than in those with erm(B) (ranging from 0.12 to ≥ 256 mg/L). Against pneumococcal isolates too, all 16-membered macrolides tested showed very good activity against the M-type erythromycin-resistant isolates, with a wide activity range (≤0.06 to 4 mg/L) against the iMLSB isolates even though only erm(B) isolates were represented.

Most S. pyogenes and S. pneumoniae isolates examined in the present study (94.0% and 99.7%, respectively) were susceptible to telithromycin, but the susceptibility values for the M-type erythromycin-resistant isolates (MIC90s, 0.5 mg/L) were at least four times higher than for the erythromycin-susceptible ones (and for most of the erythromycin-resistant MLSB isolates, too), which has been shown to be related to telithromycin acting as a substrate for an efflux pump.11,12

Despite the fair in vitro activity of some compounds, most notably rokitamycin, any clinical use against iMLSB streptococci should be regarded with the utmost caution, also on the basis of previous experience with ‘dissociated’ erythromycin resistance in staphylococcal strains, whose in vitro susceptibility to non-inducing 16-membered macrolides proved to be illusory in the clinical setting because of the ease with which mutants constitutively resistant to all MLSB antibiotics arose as a consequence of nucleotide sequence alterations.13

Moreover, the virtual absence of widely accepted and internationally validated breakpoints for 16-membered macrolides often makes it difficult to translate their in vitro activity into precise clinical recommendations and to evaluate the correlation between these susceptibility results and those obtained for erythromycin and the other macrolides commonly used in clinical practice.


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G. C. has received funds for speaking at symposia organized on behalf of Pfizer and GSK.


   Acknowledgements
 
No specific financial support has been received for this work beyond the research funding from the University of Verona to A. M. and G. C.


   References
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1 Cornaglia G, Bryskier A. Macrolide resistance of Streptococcus pyogenes. In: Optimal Management of Streptococcal Pharyngitis: Epidemiology, Diagnosis, Treatment and Public Health Considerations—Pechère JC, Kaplan EL, eds. (2003) Basel: Karger AG. 150–65.

2 Kaplan EL, Cornaglia G. Persistent macrolide resistance among group A streptococci: the lack of accomplishment after 4 decades. Clin Infect Dis (2005) 41:609–11.[CrossRef][ISI][Medline]

3 Cornaglia G, Ligozzi M, Mazzariol A, et al. Rapid increase of resistance to erythromycin and clindamycin in Streptococcus pyogenes in Italy, 1993–1995. Emerg Infect Dis (1996) 2:339–42.[ISI][Medline]

4 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16 (2005) Villanova, PA, USA: CLSI.

5 Comité de l'Antibiogramme de la Société Française de Microbiologie. Communiqué 2006. http://www.sfm.asso.fr/nouv/general.php?pa=2.

6 Ono T, Shiota S, Hirota K, et al. Susceptibilities of oral and nasal isolates of Streptococcus mitis and Streptococcus oralis to macrolides and PCR detection of resistance genes. Antimicrob Agents Chemother (2000) 44:1078–80.[Abstract/Free Full Text]

7 Seppälä H, Nissinen A, Yu Q, et al. Three different phenotypes of erythromycin-resistant S. pyogenes in Finland. J Antimicrob Chemother (1993) 32:885–91.[Free Full Text]

8 Daly MM, Doktor S, Flamm R, et al. Characterization and prevalence of MefA, MefE, and the associated msr(D) gene in Streptococcus pneumoniae clinical isolates. J Clin Microbiol (2004) 42:3570–4.[Abstract/Free Full Text]

9 Cocuzza C, Mattina R, Mazzariol A, et al. High incidence of erythromycin-resistant Streptococcus pyogenes in Monza (North Italy) in untreated children with symptoms of acute pharyngo-tonsillitis: an epidemiologic and molecular study. Microb Drug Res (1997) 3:371–8.

10 Giovanetti E, Montanari MP, Marchetti F, et al. In vitro activity of ketolides telithromycin and HMR 3004 against Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility. J Antimicrob Chemother (2000) 46:905–8.[Abstract/Free Full Text]

11 Cantón R, Mazzariol A, Morosini M-I, et al. Telithromycin activity is reduced by efflux in Streptococcus pyogenes. J Antimicrob Chemother (2005) 55:489–95.[Abstract/Free Full Text]

12 Benvenuti C, Koncan R, Bahar G, et al. Telithromycin activity is reduced by efflux in Streptococcus pneumoniae. In: Abstracts of the Sixteenth European Congress of Clinical Microbiology and Infectious Diseases, Nice, France, 2006. Basel, Switzerland: European Society of Clinical Microbiology and Infectious Diseases. Abstract P1591.

13 Weisblum B. Insights into erythromycin action from studies of its activity as inducer of resistance. Antimicrob Agents Chemother (1995) 39:797–805.[ISI][Medline]


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