1 Hôpital Maisonneuve-Rosemont, 5415 LAssomption, Montreal, Quebec H1T 2M4; 2 Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
Received 2 January 2002; returned 21 March 2002; revised 30 May 2002; accepted 13 June 2002
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Abstract |
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Introduction |
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Macrolide resistance in S. pneumoniae can occur by target modification through erm genes [mostly erm(B)], which encode 23S rRNA methylases and which are associated with high-level resistance and cross resistance to clindamycin. Macrolides bind weakly to the domain II and strongly to the domain V of the 23S rRNA. The methylase activity of Erm(B) decreases the affinity of all macrolides to the domain V, thus leaving only the very weak link to domain II as the primary target.3 The other type of resistance mechanism is encoded by the mef(A) gene, which acts as an efflux pump for 14- and 15-membered macrolides. This type of resistance has been associated with much lower MICs and susceptibility to clindamycin.
Ketolides are a newer generation of antibiotics with enhanced activity against S. pneumoniae. They are a new class of agents within the macrolide family. They have a 14-membered ring where the cladinose at the C-3 position has been replaced by a keto group, and an 11,12-carbamate side chain in telithromycin.4 The main purpose of this study was to evaluate and compare the activity of ABT-773 and telithromycin against macrolide-resistant strains of S. pneumoniae in the province of Quebec.
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Materials and methods |
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Four hundred and seventy-four non-duplicate S. pneumoniae isolates (one strain per patient) were collected through a surveillance network in Quebec (13 centres: four teaching hospitals, nine regional hospitals) from October 2000 to March 2001; 346 strains (73%) were isolated from non-sterile sites (276 sputum, 57 ear and 13 eye) and 128 (27%) were isolated from either blood or CSF. Patients comprised 200 females and 274 males; 310 (66%) isolates were from adults and 164 (34%) isolates were paediatric isolates (patients aged 16 years and under).
All collected strains were sent to a central laboratory for susceptibility testing and molecular analysis. Strains were identified using standard procedures: Grams stain, optochin susceptibility test and bile solubility test.
Antibiotics and susceptibility testing
MICs were determined using a broth microdilution method according to NCCLS recommendations.5 Bacterial strains were thawed and incubated overnight on sheep blood agar (Quélab, Montreal, Quebec, Canada) at 35°C in a CO2 environment. From each of these cultures 0.5 McFarland equivalent suspensions were prepared, which were then diluted at 1:100 in a cation-adjusted MuellerHinton broth supplemented with 5% lysed horse blood. Each inoculum was added to the prepared dilutions of each antibiotic in 96-well plates (final concentration was 5 x 104 cfu/mL) and incubated overnight at 35°C in ambient air. The MIC was then determined as the lowest antibiotic concentration able to inhibit S. pneumoniae growth. The following antibiotics were tested and provided by their respective manufacturers: ABT-773 and clarithromycin (Abbott, Abbott Park, IL, USA), penicillin, clindamycin, cefuroxime, levofloxacin (Nucrotechnics, Scarborough, Ontario, Canada), azithromycin (Pfizer Canada, Montreal, Quebec, Canada) and telithromycin (Aventis Pharma, Romainville, France). Other antibiotics were also tested in this study [ciprofloxacin and moxifloxacin (Bayer Inc., Toronto, Ontario, Canada); gatifloxacin and cefprozil (Bristol-Myers Squibb, Montreal, Quebec, Canada)]. Quality control was assessed by testing S. pneumoniae ATCC 49619.
Determination of macrolide resistance mechanisms
The 96 strains were screened for the presence of erm(B) and mef(A) genes.
The 96 macrolide-resistant strains were thawed and incubated with CO2 overnight at 37°C on blood agar (Quélab). Four colonies of each strain were suspended in 25 µL of distilled water and lysed by incubating for 10 min at 95°C. For the direct multiplex PCR, the 25 µL of treated specimen was added to 25 µL of a master mix containing 0.5 µM of mef(A) primers (Gibco), 0.5 µM of erm(B) primers (Gibco), 3 mM of Mg2+, 10 mM of each dNTP, 5 µL of 10x PCR buffer and 0.5 µL of Taq DNA polymerase, all provided by the Qiagen Taq PCR kit, as described previously.6
S. pneumoniae ATCC 49619 and three strains characterized previously [one mef(A), one erm(B) and one susceptible] were used as controls; these control strains were characterized in an earlier study.7
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Results and discussion |
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The in vitro activity of ABT-773 and telithromycin against susceptible S. pneumoniae strains (378 strains) was excellent, with MIC90s of <0.008 and 0.015 mg/L, respectively. Table 1 states the activity of both ketolides to macrolides depending on the type of resistance. The MIC distribution and cumulative percentage for ABT-773 and telithromycin among the macrolide-resistant strains are described in Table 2. Apart from a few exceptions, MICs of both ketolides were lower for strains with erm-mediated resistance compared with strains harbouring the mef(A) gene.
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Both ketolides were very active against all macrolide-resistant S. pneumoniae. The MIC50 and MIC90 of ABT-773 were one to two dilutions lower compared with telithromycin, and this was consistent irrespective of resistance status. Very few studies have directly compared both ketolides so far. Although in vitro resistance for newer macrolides (clarithromycin, azithromycin) does not seem to translate very often into clinical failure, especially in the case of respiratory tract infections, the arrival of newer agents adds new options to our therapeutic arsenal.10 ABT-773 and telithromycin had the lowest MIC90s (0.03 and 0.06 mg/L, respectively) of all the 13 antibiotics tested in this study including the fluoroquinolones (MIC90s of 0.125 mg/L of moxifloxacin and 0.25 mg/L of gatifloxacin, and 2 mg/L of ciprofloxacin and levofloxacin). As ketolides are also known to have good in vitro activity against other respiratory tract pathogens, such as Haemophilus influenzae, Moraxella catarrhalis, Legionella spp. and Chlamydia pneumoniae, the additive activity against macrolide-resistant S. pneumoniae is a definite advantage for these new antibiotics.11,12
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Acknowledgements |
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The EQUERE Group: M. Cayouette, Joliette; P. Dolce, Rimouski; L. A. Galarneau, Trois-Rivières, Arthabaska, Shawinigan and Drummonville; P. Harvey, Rivière du Loup; C. Laférriere, Montreal; I. Lecorre, Longueuil; M. Libman, Montreal; A. Marcoux, Val dOr; J. F. Paradis, Chicoutimi; H. Senay, Québec; C. Tremblay, Québec; P. Vigeant, Valleyfield.
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Footnotes |
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The EQUERE project participants are listed in the Acknowledgements.
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References |
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