43rd ICAAC
A meeting of the American Society for Microbiology

September 14-17, 2003, Chicago, IL


For more information on any presentation at the 43rd ICAAC contact Jim Sliwa, ASM Office of Communications at jsliwa@asmusa.org



EMBARGOED UNTIL:  Wednesday, September 17, 10:00 a.m.

(Session 221, Paper C1-2124)

Helene Carsenti
Archet hospital
Nice, France
Phone: 33663712865


Results and conclusions: Antimicrobial resistance is a growing problem worldwide. Streptococcus pneumoniae is of major clinical importance for pneumonia, bacterial meningitis , and otitis media. Many countries have reported that its resistance to the penicillins, betalactams, macrolides and, recently, fluoroquinolones is increasing. Improved strategies for antimicrobial usage and development of new agents that select resistance at lowest rate are essential to safeguard antimicrobial efficacy.

Linezolid is the first member of a new synthetic class of antimicrobials, the oxazolidinones, to enter clinical practice. It is characterized by a broad spectrum of activity against Gram-positive organisms and acts by inhibition of an early stage in the protein synthesis pathway. This mode of action, although not fully characterized, is distinct from those of other antibacterials and cross-resistance does not occur with linezolid. As a result, linezolid retains activity against clinically important methicillin-resistant Staphylococcus aureus, glycopeptide-intermediate S.aureus, glycopeptide-resistant enterococci and penicillin-resistant S. pneumoniae.

Our work was aimed at verifying that resistance to linezolid is slow to develop and that mutants resistant to linezolid are not cross resistant to other antibiotics. In vitro selection of resistance to linezolid is difficult when the S. pneumoniae are susceptible to penicillin G and erythromycin. Although the mechanisms of resistance are unrelated, resistance to penicillin G and to erythromycin are involved and the rate of acquisition of resistance to linezolid is faster in strains resistant to erythromycin and, or to penicillin G (Poster ICAAC 03-A-2497-ASM).

The other surprising results were that an inverse balance occurred between development of resistance to linezolid and resistance to macrolides or tetracycline. In mutants resistant to linezolid, we observed restoration of susceptibility to macrolides for 2 out of 15 parental strains resistant to erythromycin, and a decrease in MIC from > 256 mg/L to 12 mg/L for two others

Resistance to erythromycin in the parental strains was associated with resistance to lincomycin as a result of ribosomal methylation encoded by erm(B) gene. In the two strains which became susceptible to erythromycin, loss of the erm(B) gene was visualized in agarose gel electrophoresis of PCR products and confirmed by quantification by real time PCR. (figures 1,2,3)

Restoration of susceptibility to tetracycline in one out of 9 parental strains resistant to tetracycline was observed when resistance to linezolid developed ( linezolid MIC 24 mg/l) but tet (M) gene was still present. This strain also lost its resistance to trimethoprim-sulfamethoxazole.

Inverse correlation between development of resistance to linezolid and to erythromycin was first observed in S. aureus and our work confirm it also exists for S. pneumoniae. Investigations are needed to understand the mechanisms of resistance to linezolid since, in one strain, no mutations in the 23S rRNA domain V were observed. We need to understand why some strains became susceptible to erythromycin and to reproduce this phenomenon, may be the use of linezolid could restore susceptibility to macrolides, a future wish

This work was done in the Antibiology laboratory in Nice-Sophia Antipolis University (P. Dellamonica) by H. Carsenti and F. Vandenbos

-Primer sequences of domain V of 23S rRNA domain V were designed by M. Galimand at the Institut Pasteur ( Paris), then adapted in Nice and sequences performed in our laboratory.

-PCR and real time PCR were performed in our laboratory .

-R. Leclercq ( Caen) verified the results of erm(B) PCR and added the genes tet (M) and int-Tn1545

Part of the work had a financial support from Pharmacia

Scientific summary

Linezolid ( LIN) belongs to the oxazolidinone class of antibiotics. It inhibits protein synthesis and binds to the 50S ribosomal subunit in a similar way to chloramphenicol, macrolides, lincosamides and streptogramins. Its binding site is close to that of lincomycin and chloramphenicol, however it has a different mechanism of action : it has no effect on the peptidyltransferase activity but inhibits the formation of the initiation complex in bacterial translation system. It is not cross-resistant with other antimicrobial classes, including inhibitors of protein synthesis that bind to the 50S ribosomal subunit. Studies have shown that resistance to linezolid can appear by point mutation in the 23S rRNA domain V in S. aureus and Enterococcus faecalis ,probably at the binding site.

LIN resistant mutants are difficult to obtain in vitro but in patients during linezolid therapy emergence of resistant strains of E. faecium, fa ecalis or S. aureus was described.

We demonstrated that , for S. pneumoniae, it was related to resistance to the penicillins or to the macrolides (Poster ICAAC 03-A-2497-ASM)

From 28 parental strains of S. pneumoniae with various levels of susceptibility to penicillin G ( PEN) or erythromycin (ERY) we increased MICs of LIN by serial daily passages in subMICs of LIN by a gradient plate method

Daily passages in subMICs of LIN were done each day until R to LIN

10 PEN S ( 5 ERY S, 5 ERY R)

10 PEN I ( 5 ERY S, 5 ERY R)

8 PENI R ( 3 ERY S, 5 ERY R)

Among the 15 ERY R strains, we were surprised to see that :

2 strains ( S8 and S22) lost R to ERY when MICs to LIN increased

For 2 other strains (S16, S17), MICs of ERY dropped from > 256 mg/L to 12mg/L but erm(B) gene was still present. Studies of erm(B PCR products on agarose gels showed disappearence of the band for S22 and S8 mutants . For S8 loss of erm(B was associated with loss of tet (M) and int of transposon Tn1545 while only erm(B) gene was lost for S22.

Real time PCR showed a significant decrease of erm(B) gene for the two strains .

Sequences of domain V of the 23SrRNA genes of parental S8 and S22 strains and of mutants at intermediate and resistant level to linezolid were done on the 4 copies of the rrl S.pneumoniae gene .

For S8 mutants at MIC of LIN 6mg/L, 12mg/l and 24mg/L no mutations were observed while point mutation G2576U ( E. coli numbering) was observed in the four copies for S22, in three copies for S16. In S17 mutants, mutation G2576U was present on the four copies with an additional mutation C2610U on two copies.

Restoration of susceptibility to tetracycline in one out of 9 parental strains (S12) resistant to tetracycline was observed when resistance to linezolid developed ( linezolid MIC 24 mg/l) but tet (M) gene was still present. This strain lost also its resistance to trimethoprim-sulfamethoxazole.