1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Carretera. de Colmenar, Km 9.1, 28034-Madrid, Spain; 2 Dipartimento di Patologia, Sezione di Microbiologia, Strada Le Grazie 8, 37134-Verona, Italy
Received 14 October 2004; returned 12 November 2004; revised 21 December 2004; accepted 22 December 2004
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Abstract |
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Methods: The distribution of telithromycin MICs was analysed for two distinct collections of Italian (n=486) and Spanish (n=210) S. pyogenes strains. The effect of an efflux mechanism was investigated using [3H]telithromycin.
Results: Telithromycin MIC ranges were 0.0040.06 mg/L (MIC50 and MIC90, 0.01 mg/L) in erythromycin-susceptible strains (lacking both mef and erm genes) and 0.011 mg/L (MIC50 and MIC90, 0.5 mg/L) in strains endowed with the M phenotype and expressing the mef(A) gene. A distinct telithromycin efflux was detected in the strains expressing the mef(A) gene, but not in those expressing the erm(B) gene, nor in the susceptible strains lacking mef(A) or erm genes. Efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. An msr-like sequence was also found in all strains effluxing telithromycin, but not in the others.
Conclusions: This study shows that telithromycin can be removed from S. pyogenes by efflux. That the efflux is related to the presence of the mef(A) gene is demonstrated, butowing to the increasingly evident complexity of S. pyogenes efflux systemsthe possibility that other genes may contribute to the efflux cannot be excluded.
Keywords: ketolides , macrolides , antimicrobial resistance
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Introduction |
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Telithromycin is a ketolide, i.e. a 14-membered derived macrolide designed to overcome erythromycin resistance in Gram-positive cocci. This compound has been shown to have low MIC values for both erythromycin-susceptible and erythromycin-resistant S. pyogenes isolates, irrespective of the macrolide resistance mechanism.5,13 Telithromycin is also commonly regarded as being capable of overcoming the efflux mechanism of resistance because of its excellent activity against macrolide-resistant isolates with the M phenotype.11 Nevertheless, telithromycin MIC values in many MLSB erythromycin-resistant strainsmostly those endowed with the erm(B) geneand in the M-type erythromycin-resistant strains appear to be distinctly higher than in the erythromycin-susceptible ones.5 But, as far as we know, the possibility of telithromycin acting as an actual substrate for an efflux pump has not been previously investigated in S. pyogenes.
In this study, clinical isolates of S. pyogenes from both Italy and Spain were tested for susceptibility to erythromycin, clindamycin, telithromycin and quinupristin/dalfopristin. MIC distributions of strains with different macrolide resistance phenotypes and genotypes conferring resistance to these antibiotics were analysed. The possible effect of a proton-dependent efflux pump on telithromycin extrusion was also investigated using a radiolabelled telithromycin compound in macrolide-resistant strains of different genotypes. Given the recent finding that Streptococcus pneumoniae isolates endowed with a mef gene also carry an open reading frame homologous to msr(A)14 encoding for a putative efflux system in Staphylococcus epidermidis15 , the presence of such a gene in S. pyogenes was also investigated.
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Materials and methods |
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A total of 696 S. pyogenes isolates recovered over the period 19992002 in Italy (486 isolates) and Spain (210 isolates) were included. All strains were isolated from clinical specimens (mostly throat swabs) obtained in different geographical regions from patients of different ages (mostly paediatric).
Susceptibility testing
The MICs for the S. pyogenes isolates were determined by the standard agar dilution test recommended by the NCCLS.16
MuellerHinton agar (Oxoid Ltd, Basingstoke, UK) supplemented with 5% sheep blood was used, and the plates were incubated overnight in ambient air at 35 °C. Standard quality control strains S. pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were included in each run. Interpretation of the results was based on the NCCLS guidelines for most antibiotics.16
For telithromycin, the breakpoints that have been validated by two committees in Europe, including MENSURA (Mesa Española de Normalización de las Sensibilidad y Resistancia a los Antimicrobianos) and CA-SFM (Comité de l'Antibiogramme de la Société Française de Microbiologie)17
were followed. The categories used were susceptible ( 0.5 mg/L), intermediate (12 mg/L) and resistant ( >2 mg/L).
Antibiotics were supplied as powders of known potency by their respective manufacturers or purchased from Sigma Chemical Co., St Louis, MO, USA.
Phenotype detection of resistance mechanisms
The resistance phenotype was determined by the double disc diffusion test with erythromycin (15 µg) and clindamycin discs (2 µg) using MuellerHinton agar supplemented with 5% sheep blood. Erythromycin and clindamycin discs were placed on the plates 1520 mm apart. Blunting of the clindamycin inhibition zone in the vicinity of the erythromycin disc was taken as indicating inducible MLSB resistance (iMLSB). Resistance to clindamycin with no blunting of the clindamycin inhibition zone indicated constitutive MLSB resistance (cMLSB). The M phenotype was characterized by susceptibility to clindamycin alone but with no blunting of the inhibition zone around the clindamycin disc.18
Discs were purchased from Oxoid Ltd (Basingstoke, UK).
PCR-based detection of resistance genes
Erythromycin-resistant strains with M phenotype were investigated to detect the mef(A) gene and the msr-like sequence. The presence of both erm(B) and erm(A) was also determined. Total DNA was obtained using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). PCR procedures were as previously described for mef, erm5 or msr.19
Erythromycin and telithromycin efflux by actively growing cells
The presence of macrolide efflux was assessed indirectly by measuring the accumulation of radioactive [N-Me-14C]erythromycin or [3H]telithromycin in selected clinical strains, including erythromycin- and telithromycin-susceptible strains (n=1), isolates expressing the M phenotype and positive for mef(A) gene (n=3), isolates expressing an inducible (iMLSB) (n=1) and constitutive (cMLSB) (n=1) resistance phenotype. This assay was performed following the protocol previously described by Sutcliffe et al.18 Freshly grown colonies from blood agar plates were grown in BrainHeart Infusion broth in 5% CO2 at 37 °C without shaking until an OD650 of 0.3 was reached. At this point, erythromycin (0.02 mg/mL) was added to ensure full induction of the efflux determinant and the culture was incubated for another hour. Radiolabelled antibiotics were added at a final concentration of 0.2 mg/mL, and uptake was assessed by removing aliquots of the culture in duplicate, filtering each sample onto pre-wet GF/C filters, and washing the filters twice with 0.9% NaCl containing 1 mg of either antibiotic per mL. The amount of cell-associated radiolabelled antibiotic was determined after scintillation counting of the dried filters. Samples receiving 10 mM sodium arsenate, an agent disrupting proton motive force, were exposed to this agent for 10 min prior to the addition of radiolabelled antibiotic.
Experiments were performed in triplicate for all strains.
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Results |
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The MIC range, MIC50 and MIC90 values and percentage of overall susceptibilities to antibiotics for all S. pyogenes isolates from both countries are shown in Table 1. Of the 486 Italian isolates of S. pyogenes, 334 were erythromycin-susceptible (68.7%). Of the erythromycin-non-susceptible isolates, 28.3% (43 out of 152 erythromycin resistant) showed an M phenotype, whereas 24.3% (37/152) and 47.2% (72/152) exhibited the constitutive and inducible MLSB phenotype, respectively. Overall telithromycin susceptibility among Italian isolates was 94.0%. Fully resistant telithromycin ( > 2 mg/L) and intermediate (12 mg/L) isolates were within the population expressing the MLSB phenotype. All telithromycin-resistant isolates (n=26) carried an erm(B) gene; 80.8% of them (21 isolates) had a constitutive MLSB phenotype and 19.25% (5 isolates) an inducible MLSB phenotype. Neither resistant nor intermediate isolates to telithromycin were found among isolates expressing an M phenotype. Nevertheless, telithromycin MIC values were displaced to the right in the MIC distribution within the population displaying this phenotype (erythromycin-susceptible isolates: range, 0.0040.06 mg/L; MIC50 and MIC90=0.01 mg/L; isolates endowed with the M phenotype: 0.011 mg/L; MIC50 and MIC90=0.5 mg/L) (Figure 1).
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Telithromycin MIC values in the Spanish isolates were in the range 0.0040.06 mg/L (MIC50=0.008 mg/L and MIC90=0.01 mg/L) within the erythromycin-susceptible population. As in the Italian isolates, telithromycin MIC values were higher in the isolates expressing the M phenotype and possessing the mef(A) (29 isolates) gene (range, 0.121 mg/L; MIC50 and MIC90=0.5 mg/L) when compared with susceptible isolates without mef(A) (Figure 1). The concomitant presence of the mef(A) gene with the erm(A) gene corresponded to lower susceptibility in only one strain (intermediate to telithromycin, MIC = 1 mg/L and expressing an M phenotype) as opposed to when the simultaneous presence of mef(A) and erm(B) genes (two isolates) was detected. As in the Italian isolates, all telithromycin-resistant isolates expressed a constitutive MLSB resistance phenotype. As expected, both in Italy and Spain, all isolates were fully susceptible to penicillin (MIC values
0.01 mg/L).
Efflux assay
The putative effect of a proton-dependent pump was investigated in actively growing cells using radiolabelled erythromycin and telithromycin in selected strains with and without the presence of the mef(A) gene. As expected, the efflux effect of either erythromycin or telithromycin was not observed in susceptible strains in which no mef(A), erm(B) or erm(A) genes were found (Figure 2). On the contrary, a clear telithromycin efflux was demonstrated in the strains expressing the M phenotype linked to the presence of the mef(A) gene. This effect was lower than that affecting erythromycin (Figure 2). Reversibility of both erythromycin and telithromycin efflux by addition of the inhibitor compound sodium arsenate was also demonstrated in isolates expressing the mef(A) gene. These results confirmed the effect of a proton-dependent pump on telithromycin, as well as on erythromycin. No substantial retention of the radiolabelled antibiotics could be observed in the isolates expressing the MLSB resistance phenotype, either constitutive or inducible, a previously observed phenomenon that has been explained by the lack of macrolide binding to methylated ribosomes.18 Among the strains considered, all those with the mef gene, but none of the others, were also found to carry an msr-like sequence. It is of note that a small amount of radiolabelled compounds were accumulated after arsenate pre-treatment both in susceptible and erm(B)-positive strains. This could suggest the possible presence of an additional naturally occurring efflux pump in S. pyogenes that is also inhibited by this compound.
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Discussion |
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Although no telithromycin-resistant isolates were observed within the population expressing the M phenotype, the MIC values were distinctly higher than those observed in the erythromycin-susceptible isolates (at least a 32-fold difference in both the MIC50 and MIC90 values). This finding has also been clearly observed in streptococci from the viridans group27 and to a lesser extent in S. pneumoniae isolates28 and strongly suggests the possibility of telithromycin being actively effluxed from S. pyogenes. By means of radiolabelled compounds, efflux has been shown to affect 14- and 15-membered macrolides, such as erythromycin, clarithromycin and azithromycin, but not 16-membered macrolides such as josamycin.18 In our experiments using radiolabelled erythromycin and telithromycin, we demonstrated that both compounds were pumped out in S. pyogenes isolates endowed with a mef(A) gene, but not in susceptible isolates or in the MLSB-resistant ones. This effect proved to be reversible by the addition of the pump inhibitor sodium arsenate, whose addition did not have any effect on the small amount of erythromycin or telithromycin accumulated in other erythromycin-resistant strains (MLSB).
The demonstration that telithromycin can be pumped out by an efflux system has been provided recently in Enterobacter aerogenes and Escherichia coli isolates.29 Although macrolide resistance is mainly associated with the AcrAB-TolC system in Gram-negative organisms, telithromycin resistance was only weakly mediated by this pump, suggesting the participation of other putative efflux pumps. In Haemophilus influenzae, macrolide resistance has also been related to the presence of an acrAB gene cluster.30
Other efflux pumps linked to the presence of the mre(A) gene in staphylococci and Streptococcus agalactiae or of the msr(A) gene in staphylococci and S. pneumoniae have recently been reported to affect macrolides and/or ketolides.14,3132 In the present study, we have shown that an msr-like gene coexists in S. pyogenes strains harbouring mef (but not in those with erm or devoid of either major resistance determinant). The results of our study may also suggest the possible presence in S. pyogenes of a slow-acting efflux pump affecting macrolides and ketolides, sinceeven in strains lacking the mef(A) determinantthe addition of arsenate produced a slight accumulation of erythromycin and telithromycin.
A new gene, mat(A), which is always adjacent to mef(A) and is homologous to the ATP-binding transporter msr(SA) of S. aureus, has also been described recently,33 and the presence of a novel efflux systemnot related to the mef(A) genehas been inferred in inducible erythromycin-resistant strains of S. pyogenes.34
Despite being affected by efflux, the activity of telithromycin in S. pyogenes with the M phenotype remains remarkable, possibly resultingas a net effectfrom its high intrinsic potency due to its ribosome binding properties.35 None of the isolates from our collection with this phenotype was classified as fully resistant to telithromycin according to the common interpretive categories, unless the concomitant presence of erm(B) was observed. However, the possibility of telithromycin being affected by even a low-level resistance mechanism is a disturbing finding for at least three reasons, namely: (i) its possible additive effect to another mechanism of reduced susceptibility: the two coexisting mechanisms would raise the MIC values above the clinical resistance threshold even though each of them alone would be unable to do so. It should be recalled that, as in our study, mef plus erm resistant strains have actually been reported, though at highly variable rates.5,36 This possibility has been recognized as an emerging problem in S. pneumoniae;37 (ii) the possibility that this reduced susceptibility (or low-level intrinsic resistance) may represent a first step towards acquiring higher resistance levels (i.e. via a different resistance mechanism, independent of the efflux contribution); and (iii) the uncertain in vivo behaviour of isolates which, though susceptible in vitro, show reduced MIC values and harbour a definite mechanism of resistance. Thus, the use of telithromycin should be carefully considered not only when erythromycin resistance is related to the erm(B) gene but also when it depends on the presence of efflux.
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Acknowledgements |
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