Genotypic characterization of macrolide-resistant strains of Streptococcus pneumoniae isolated in Quebec, Canada, and in vitro activity of ABT-773 and telithromycin

K. Weiss1,*, C. Guilbault1, L. Cortes1, C. Restieri1, D. E. Low and the EQUERE project (Etude QUEbecoise des pathogènes REspiratoires)2,§

1 Hôpital Maisonneuve-Rosemont, 5415 L’Assomption, 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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Increasing resistance of Streptococcus pneumoniae to macrolides represents a challenge for clinicians. New ketolides have an enhanced activity against macrolide-resistant strains. Four hundred and seventy-four strains of S. pneumoniae were collected during the 2000–2001 season in Quebec through a surveillance network. Macrolide resistance was 20.2%, and significantly higher in non-invasive strains versus invasive ones (22.4% versus 14.8%), and in children (30%) versus adults (14.8%). For susceptible strains, MIC90s of ABT-773 and telithromycin were 0.008 and 0.015 mg/L. Among the 96 macrolide-resistant strains, 56 (58%) were erm(B), 35 (37%) carried the mef(A) gene, four were carrying both genes and one none. ABT-773 and telithromycin were very active against all these resistant strains irrespective of the resistance mechanism, with MIC90s of 0.25 and 0.5 mg/L, respectively.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Increasing resistance of Streptococcus pneumoniae to macrolides has been reported in several countries.1,2 In Canada, newer macrolides such as azithromycin and clarithromycin are increasingly used for the treatment of respiratory tract infections. Macrolide consumption went from 14.3 prescriptions per 100 population in 1995 to 15.3 in 2000 (+7%), whereas the Canadian antibiotic market shrank by 14% in the same period (data provided by IMS Canada, Pointe Claire, Quebec, Canada). They still represent excellent treatment options for patients with community-acquired pneumonia, otitis media, sinusitis and acute bacterial exacerbation of chronic bronchitis.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Strains

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: Gram’s 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 Mueller–Hinton 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


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Overall resistance to macrolides was 96/474 (20.2%). This represents a definite increase compared with the last decade where resistance in Quebec and Canada as a whole was <10%.7 There was also a significant difference in terms of level of resistance to macrolides between invasive strains (14.6%) and non-invasive strains (22.4%) (P < 0.05); this was also true for other classes: penicillin, cephalosporins. This raises the issue of the energy cost associated with the acquisition of resistance for bacteria, which may render them less virulent. Macrolide resistance was also associated with more paediatric isolates (50/164, 30%) compared with adults (46/310, 14.8%) (P < 0.01). There was no significant statistical relationship with regard to gender; the percentage of resistance was 19% and 22% in males and females, respectively. Molecular analysis of the 96 macrolide-resistant strains showed that 56 (58%) carried the erm(B) genotype, 35 (37%) were mef(A), four were both erm(B) and mef(A) and one strain was neither. This finding, specific to the province of Quebec, is very different from a previous Canadian study that included Quebec, and showed a much higher proportion of mef(A)-mediated resistance overall.7 There seems to be no obvious notable explanation in terms of consumption of antibiotics, including macrolides, for explaining the higher proportion of erm(B)-mediated resistance in Quebec compared with the rest of the country. In North America in general, mef(A) resistance is the most prevalent type of macrolide resistance, in contrast to many European countries where erm-type resistance is more common.79

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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Table 1.  Genotypic and phenotypic results for S. pneumoniae
 

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Table 2.  MIC distribution (mg/L) and cumulative percentage for ABT-773 and telithromycin among the 96 macrolide-resistant strains
 
Seventy-one per cent of macrolide-resistant strains were not susceptible to penicillin (46% intermediate, 25% resistant) and 54% were also resistant to cefuroxime. Only nine strains (1.8%) had an MIC of >=1 mg/L of ABT-773 and 11 strains (2%) had an MIC of >=1 mg/L of telithromycin. All of these strains originated from two centres in Montreal, were mainly paediatric isolates and may thus represent the same clone. No further molecular analysis was performed to assess this possibility.

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


    Acknowledgements
 
The EQUERE project is supported in part by grants from Abbott Canada, Aventis Pharma Canada, Bayer Inc. and Bristol-Myers Squibb Pharmaceutical group.

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 d’Or; J. F. Paradis, Chicoutimi; H. Senay, Québec; C. Tremblay, Québec; P. Vigeant, Valleyfield.


    Footnotes
 
* Corresponding author. Tel: +1-514-252-3817; Fax: +1-514-252-3898; E-mail: weisscan{at}aol.com Back

§ The EQUERE project participants are listed in the Acknowledgements. Back


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 Introduction
 Materials and methods
 Results and discussion
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