Antimicrobial Agents and Chemotherapy, August 2004, p . 3169-3171, Vol . 48, No . 8

PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) is a longitudinal, multicenter surveillance study of antibacterial resistance among respiratory tract pathogens from numerous countries worldwide . PROTEKT US is a sister study involving the United States only and began in 2000 . All macrolide-resistant S . pneumoniae strains isolated during year 1 of the PROTEKT study (1999 to 2000) were screened for the common efflux and methylase genes associated with macrolide resistance to determine the global distributions of these mechanisms . Of 1,043 macrolide-resistant isolates screened, 16 (1.5%) isolates repeatedly tested negative for the erm and mef genes, while the remaining isolates were resistant to macrolides (4, 5) . All isolates were found to harbor ribosomal mutations, most commonly A2059G (12 isolates) (4) .

(Preliminary data were presented at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 2003 [D . J . Farrell, I . Morrissey, S . Bakker, S . Buckridge, J . Borger, and D . Felmingham, Abstr . 43rd Intersci . Conf . Antimicrob . Agents Chemother., abstr . C2-69, 2003].)

Of the 23,543 isolates of S . pneumoniae collected from 1999 to 2002 in the PROTEKT and PROTEKT US studies, 7,746 (32.9%) were macrolide resistant; 86 (0.37%) macrolide-resistant isolates (including the 16 isolates reported previously) were negative for methylase and efflux mechanisms . The ribosomal mutations associated with macrolide resistance in these isolates were determined in this study . Before the isolates were sequenced for ribosomal mutations, macrolide resistance was confirmed by repeating the MIC determinations and comparing the MIC data with the initial results . Detection of L4 and L22 riboprotein and 23S rRNA gene mutations was performed as described previously (3) . Briefly, the complete L4 and L22 operons and all four 23S rRNA operons were sequenced .

In total, 23 different mutational combinations were found in the 86 isolates, with A2059G in all four copies of the 23S rRNA gene being the most prevalent mutation (29 of 86 isolates [33.7%]) (Table 1) . Eight isolates had two mutations, and two isolates had three mutations . Sixty isolates had mutations that have been described previously (13 mutational combinations), while 13 isolates had mutations not described previously (10 mutational combinations) (2, 9, 10) . A mutational event was not found in nine isolates, and hence, the mechanism(s) of resistance remains unknown .

 
Ribosomal mutations were well distributed globally, being found in the following countries: Australia (n = 1 mutation), Belgium (n = 1), Canada (n = 17), Germany (n = 6), Hong Kong (n = 1), Hungary (n = 1), Japan (n = 7), Poland (n = 1), Switzerland (n = 1), Turkey (n = 1), and the United States (n = 49) . Care should be taken in interpreting the prevalence of mutations by country, as the majority (47 of 49 [95.9%]) of strains from the United States were obtained from the PROTEKT US (2000 to 2001) surveillance study .

All 86 isolates were susceptible to telithromycin at the published NCCLS tentative susceptibility breakpoint of 1 µg/ml (8) . If the European Union susceptibility breakpoint of 0.5 µg/ml is applied, three isolates would be classified as having intermediate susceptibility to telithromycin (MIC = 1 µg/ml) .

Six isolates were found to be resistant to quinupristin-dalfopristin; for five isolates the quinupristin-dalfopristin MIC was 4 µg/ml and for one isolate the quinupristin-dalfopristin MIC was 8 µg/ml . Resistance to quinupristin-dalfopristin in clinical isolates of S . pneumoniae is very uncommon, with only three reports thus far in the literature (3, 6, 7) . Five separate mutations in the 23S rRNA, L4, and L22 genes were associated with this resistance, with three of these described previously (a 109RTAHIT114 tandem duplication in L22, 23S rRNA C2611G, and G95D combined with A2059G in L22) (3, 7) . Two of 8,837 (0.02%) clinical isolates collected in the SENTRY study (2001 and 2002) were found to be resistant to quinupristin-dalfopristin, with the MICs for both isolates being 4 µg/ml and with both isolates having a similar L22 mutation, 109RTAHI113 (6) .

Two isolates were found to be resistant to linezolid (MICs = 4 µg/ml) . This resistance was associated with two separate novel mutations (A2059G in three copies of the 23S rRNA gene in combination with G2057A in four copies of the 23S rRNA gene and a 6-bp deletion in the L4 riboprotein gene, ₆₄PWRQ₆₇ to ₆₄P_Q₆₇) . This is the first report of linezolid resistance in clinical isolates of S . pneumoniae .

It was previously suggested (3) that resistance might be dependent upon the number of 23S rRNA genes mutated in a dose-response manner . The data from this larger population of isolates argues against this, as the MIC ranges overlap for isolates with A2059G mutations in one to four alleles (Table 1) . Differential expression of mutated and nonmutated copies of the gene in response to environmental pressure, particularly antimicrobial exposure, is a possibility that requires investigation .

In summary, telithromycin retained good activity against all 86 isolates, demonstrating that so far this antibacterial is refractory to resistance associated with a wide variety of ribosomal mutations and ribosomal mutation combinations, including mutations associated with linezolid and quinupristin-dalfopristin resistance; however, causality needs to be confirmed by mutational analysis with isogenic strains . A mutational event was not found in 9 of the 86 isolates, and hence, the mechanism(s) of macrolide resistance remains to be elucidated .

Aventis is acknowledged for its financial support of the PROTEKT study .

1. Canu, A., B . Malbruny, M . Coquemont, T . A . Davies, P . C . Appelbaum, and R . Leclercq. 2002 . Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae . Antimicrob . Agents Chemother . 46:125-131.
2. Depardieu, F., and P . Courvalin. 2001 . Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae . Antimicrob . Agents Chemother . 45:319-323.
3. Farrell, D . J., S . Douthwaite, I . Morrissey, S . Bakker, J . Poehlsgaard, L . Jakobsen, and D . Felmingham. 2003 . Macrolide resistance by ribosomal mutation in clinical isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 study . Antimicrob . Agents Chemother . 47:1777-1783.
4. Farrell, D . J., I . Morrissey, S . Bakker, and D . Felmingham. 2001 . Detection of macrolide resistance mechanisms in Streptococcus pneumoniae and Streptococcus pyogenes using a multiplex rapid cycle PCR with microwell-format probe hybridization . J . Antimicrob . Chemother . 48:541-544.
5. Farrell, D . J., I . Morrissey, S . Bakker, and D . Felmingham. 2002 . Molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae and Streptococcus pyogenes isolated from the PROTEKT 1999-2000 study . J . Antimicrob . Chemother . 50(Suppl . 1):39-47.
6. Jones, R . N., D . J . Farrell, and I . Morrissey. 2003 . Quinupristin-dalfopristin resistance in Streptococcus pneumoniae: novel L22 ribosomal protein mutation in two clinical isolates from the SENTRY antimicrobial surveillance program . Antimicrob . Agents Chemother . 47:2696-2698.
7. Musher, D . M., M . E . Dowell, V . D . Shortridge, R . K . Flamm, J . H . Jorgensen, P . Le Magueres, and K . L . Krause. 2002 . Emergence of macrolide resistance during treatment of pneumococcal pneumonia . N . Engl . J . Med . 346:630-631.
8. NCCLS. 2004 . Performance standards for antimicrobial susceptibility testing; twelfth information supplement . NCCLS document M100-S14 . NCCLS, Wayne, Pa.
9. Pihlajamaki, M., J . Kataja, H . Seppala, J . Elliot, M . Leinonen, P . Huovinen, and J . Jalava. 2002 . Ribosomal mutations in Streptococcus pneumoniae clinical isolates . Antimicrob . Agents Chemother . 46:654-658.
10. Tait-Kamradt, A., T . Davies, P . C . Appelbaum, F . Depardieu, P . Courvalin, J . Petitpas, L . Wondrack, A . Walker, M . R . Jacobs, and J . Sutcliffe. 2000 . Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America . Antimicrob . Agents Chemother . 44:3395-3401.
11. Tait-Kamradt, A., T . Davies, M . Cronan, M . R . Jacobs, P . C . Appelbaum, and J . Sutcliffe. 2000 . Mutations in 23S rRNA and ribosomal Protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage . Antimicrob . Agents Chemother . 44:2118-2125.

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