In vitro selection of mutants of Streptococcus pneumoniae resistant to macrolides and linezolid: relationship with susceptibility to penicillin G or macrolides

H. Carsenti-Dellamonica1,*, M. Galimand2, F. Vandenbos1, C. Pradier1, P. M. Roger1, B. Dunais1, M. Sabah1, G. Mancini1 and P. Dellamonica1

1 Infectious Disease Department, Archet Hospital, Route Saint Antoine de Ginestière, Nice 06202, France; 2 Institut Pasteur, 25 Rue du Docteur Roux, 75724, Cedex 15 Paris, France


* Corresponding author. Tel: +33-492035626; Fax: +33-493965454; E-mail: carsenti.h{at}chu-nice.fr

Received 1 March 2005; returned 27 April 2005; revised 21 June 2005; accepted 25 July 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To evaluate the rate of acquisition of resistance to linezolid and macrolides in Streptococcus pneumoniae isolates with different levels of susceptibility to penicillin and erythromycin.

Materials and methods: Thirty strains of S. pneumoniae were tested by serial passages in subinhibitory concentrations of each antibiotic by the spiral method. The four copies of the 23S rRNA rrl gene of parent strains and linezolid-resistant mutants were amplified and sequenced.

Results: The rate of acquisition of macrolide resistance did not differ when C-14 and C-16 macrolides were tested. Resistance to linezolid in strains susceptible to penicillin and erythromycin was difficult to produce. For mutants with low-level resistance to linezolid the cut-off value of the MIC was between 6 and 8 mg/L depending on the strain. All linezolid-resistant mutants displayed a mutation in 2–4 copies of the 23S rRNA rrl gene, mainly the G2576U mutation (27/30) with an additional C2610U mutation observed in certain mutants. Two new mutations were also noted, namely C2612A and C2571G. In three linezolid-resistant mutants no mutation was identified within the studied domain, suggesting another mechanism of resistance.

Conclusions: Linezolid resistance in pneumococcal strains susceptible to penicillin and macrolides was more difficult to obtain than with macrolides. Increased resistance to these agents may therefore influence the clinical use of linezolid.

Keywords: pneumococcus , oxazolidinones , resistance


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to macrolides is increasingly reported in clinical isolates of Streptococcus pneumoniae.1,2 The predominant mechanisms of resistance involve modification of the ribosomal target by methylation of a specific adenine residue (A2058) in 23S rRNA conveyed by a family of methyltransferase enzymes encoded in pneumococci by genes belonging to the erm(B) or erm(A) class. This leads to cross-resistance to macrolides, lincosamides and group B streptogramins, resulting in what is known as the MLSB phenotype.3 The second mechanism of resistance, macrolide-specific efflux, is mediated by a membrane protein encoded by the mef gene and confers resistance only to the 14- and 15-member macrolides.4 Ribosomal mutation has also been reported recently in a few clinical isolates of S. pneumoniae (1.5%).57 The target modification can be achieved by mutations in domains II and V of 23S rRNA (in one to four of the four copies of this gene present in S. pneumoniae) and in the genes encoding riboproteins L4 and L22.6,7 Mutations in domain V of the 23S rRNA were the most frequent, in particular substitutions of A2058G/U, A2059G, A2062C, C2610A and C2611A/G. These mutations can confer resistance to MLSB antibiotics and, in some cases, to ketolides.

Two other mechanisms associated with unusual resistance phenotypes to MLSB antibiotics have been identified in clinical isolates of S. pneumoniae.5,6 Macrolide–streptogramin resistance (MS phenotype) is due to a 3 amino acid substitution, whereas further resistance to ketolides is due to a 6 amino acid insertion in a highly conserved region of the ribosomal protein L4.6 The macrolide–lincosamide resistance phenotype (ML) observed in clinical isolates or laboratory mutants is due to an A2059G (Escherichia coli numbering) change in two, three or four copies of 23S rRNA.6 Recent case reports describing invasive macrolide-resistant pneumococcal disease during or shortly after initiation of macrolide treatment raise concerns about the effectiveness of these agents against suspected pneumococcal infections.8

Oxazolidinones, of which linezolid is the first registered for clinical use, are new compounds.9,10 One of their main advantages is their ability to retain activity against most resistant pneumococcal isolates, methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, and vancomycin-resistant isolates of Enterococcus spp.

Early studies have demonstrated that linezolid exerts a bacteriostatic effect by inhibiting formation of the initiation complex. Linezolid appears to have a binding site in domain V of the 23S rRNA that overlaps at least partially with the binding sites for chloramphenicol and lincosamides, although, in contrast to those agents, oxazolidinones do not inhibit peptidyl transferase activity.

Laboratory-derived linezolid-resistant mutants display mutations in the central loop of domain V, mainly G2447U in S. aureus,11 G2528U in Enterococcus faecalis and Enterococcus faecium12, and G2032A/U/C in E. coli.13 Identical mutations and others were found in E. faecium and E. faecalis clinical isolates resistant to linezolid (G2576U, G2505A),12,14,15 which have emerged during linezolid treatment16,17 or following prior exposure to linezolid.14 Clinical isolates of S. aureus were found with mutations G2576T and T2500A.18,19

The present study was performed to investigate the rate of appearance of resistance to linezolid and macrolides (erythromycin and spiramycin) in S. pneumoniae with different levels of resistance to penicillin G and to erythromycin.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Twenty-seven isolates of S. pneumoniae were selected from nasopharyngeal aspirates in children aged 6 months to 3 years and were named according to their susceptibility to penicillin. Reference strains from the collection of the Institut Pasteur (S. pneumoniae 102911, 104485 and 104471) displaying various levels of resistance to penicillin (S, I and R) were included (Table 1). These organisms included five strains susceptible to penicillin (MIC ≤ 0.064 mg/L) and erythromycin (MIC ≤ 0.25 mg/L), five strains susceptible to penicillin and resistant to erythromycin (MIC ≥ 1 mg/L), five strains intermediate to penicillin (MIC 0.125–1 mg/L) and susceptible to erythromycin, five strains intermediate to penicillin and resistant to erythromycin, five strains resistant to penicillin (MIC > 1 mg/L) and susceptible to erythromycin, and five strains resistant to penicillin and erythromycin.


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Table 1. Rate of appearance of resistance

 
Organisms were identified by optochin susceptibility, latex agglutination and API Strept (BioMérieux, Marcy l'Etoile, France). Serotyping of isolates was performed by the standard quellung method using sera from the Statens Serum Institut (Copenhagen, Denmark). Isolates were stored at –80°C in brain heart broth plus 10% ethylene glycol (Merck, Darmstadt, Germany).

Antimicrobials

Antimicrobials were obtained as laboratory-grade powders from their respective manufacturers; erythromycin and spiramycin from Aventis-Sanofi (Paris, France), and linezolid from Pfizer (Pharmacia, Paris, France).

Susceptibility testing

MICs were determined by using the Etest method (AB Biodisk, Solna, Sweden) on Mueller–Hinton (MH) agar supplemented with 5% horse blood. Cut-off points for resistance were those published by the NCCLS.20 MICs were also determined by antibiotic-containing gradient plates. At the beginning of the study and after each series of seven passages, MICs were determined by Etest to correlate MICs on gradient plates with the Etest method. Reference strains were used as internal controls for antibiotic solutions and Etest.

Susceptibility of each parental strain and respective resistant mutants was tested by disc diffusion method (Kirby Bauer method) to chloramphenicol, erythromycin, clindamycin, spiramycin and tetracycline. Using the triple disc method as described by Giovanetti et al.,21 blunting of the clindamycin and spiramycin inhibition zone towards the erythromycin disc was interpreted as inducible resistance. Resistance to clindamycin with no blunting of the clindamycin inhibition zone and erythromycin resistance indicated constitutive resistance. Resistance mediated by efflux was characterized by susceptibility to clindamycin and resistance to erythromycin.

Selection of resistant mutants

Mutants resistant to linezolid, erythromycin and spiramycin were obtained from parental isolates by daily passages in subinhibitory concentrations of each antibiotic.22 A continuous concentration gradient of each antibiotic was plated on to blood agar using a spiral inoculator. Plates were left at room temperature for at least 6 h to ensure diffusion of the antibiotic. Bacterial inoculum size was 106 cfu/mL. MICs were determined after overnight incubation at 37°C in a 5% CO2 atmosphere. Colonies nearest to the inhibition zone (cultured with sub-MICs) were selected and diluted to the inoculum size. This procedure was done daily until a significant increase in the MIC (at least 4-fold) was obtained. Resistant mutants were subcultured in antibiotic-free medium for 10 serial passages. Mutants were determined to be of the same clonal type as that of the parental strains by PFGE and by serotyping.

Determination of ribosomal mutations in linezolid mutants

Bacterial DNA was extracted using GFX Genomic Blood DNA Purification Kit (Amersham Pharmacia Biotech, Piscataway, USA). The four copies of the 23S rRNA rrl gene were amplified separately using primers as described by Depardieu and Courvalin.23 PCR products were purified using QIAquick PCR purification kit (QIAGEN, Courtaboeuf, France) and visualized by 1% agarose electrophoresis and ethidium bromide staining. PCR amplification of a fragment of 553 bp representing domain V of each rrl gene between coordinates 2167 and 2729 (E. coli numbering) was carried out with primers labelled using cyanine fluorophore Cy 5.5: 5'-CTTTCAGCGTTTATCCCTTCC-3'-Cy5 and 5'-AGGAGACGCTGTTGGGATACT-3'-Cy 5.0. Amplification with these primers was carried out in a total volume of 7.8 µL containing 4.8 µL of diluted amplification product added to each of the nucleotide wells (3 µL). The amplification product contained 2.5 µL of sequencing buffer, 3 µM of each labelled primer, 3.5 µL of DMSO, 6.5 µL of DNAse-free water, 2 µL of template DNA and 3 µL of Thermosequenase. The cycling program started with initial denaturation at 94°C for 1 min. A total of 35 amplification cycles of denaturation at 94°C for 40 s, annealing at 56°C for 20 s, and primer extension at 72°C for 60 s were carried out followed by a final extension primer step at 72°C for 2 min. Control DNA was a single standard M13 template. Sequencing was performed on a Visible Genetics sequencer (Bayer, Paris, France). Sequences were compared with that of wild-type S. pneumoniae from the Institute for Genomic Research (http//www.tigr.org). All parental isolates, all linezolid-resistant and certain intermediate mutants at different MIC levels were sequenced.

Statistics

Rates of acquisition of resistance in each susceptibility group were compared by the Mann–Whitney test with an alpha risk of 5%.


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 Materials and methods
 Results
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 References
 
Selection of erythromycin-resistant mutants

Resistant mutants were always obtained when erythromycin was used as the selecting agent. The mean numbers of passages in subinhibitory concentrations of erythromycin were 21.2 ± 15.2 for isolates susceptible to penicillin, 16.6 ± 13.2 for isolates intermediate to penicillin and 6.8 ± 1.3 for isolates resistant to penicillin. The rate of acquisition of erythromycin resistance was comparable regardless of penicillin susceptibility (Table 1). Out of 15 erythromycin-resistant mutants, 10 were resistant to clindamycin and spiramycin without any blunting of the zone (phenotype MLSB constitutive) and remained susceptible to linezolid. The MIC of linezolid was increased 2- to 3-fold. For one mutant (R1), the linezolid MIC increased from 0.25 to 1.5 mg/L (6-fold), but the mutant remained susceptible to linezolid (Table 1). Five mutants (S3, S4, I1, I2 and R5) remained clindamycin-susceptible with no blunting of the inhibition zone around the clindamycin disc. They were intermediate or resistant to spiramycin and cannot be interpreted as phenotype M strains. All the erythromycin-resistant mutants were susceptible to linezolid and no change in chloramphenicol or tetracycline susceptibility was noted.

Selection of spiramycin-resistant mutants

Resistant mutants were always obtained with spiramycin as the selecting agent. The mean numbers of passages were 10 ± 3.7 for isolates susceptible to penicillin, 9 ± 3.7 for those intermediate to penicillin and 5.2 ± 1.8 for those resistant to penicillin. Rate of acquisition of resistance was significantly more rapid when isolates were penicillin resistant (P = 0.03) compared with penicillin-susceptible parental isolates. Spiramycin-resistant mutants selected from penicillin-susceptible (S2), penicillin-intermediate (I1, I2, I3 and I4 mutants) and penicillin-resistant isolates (R1, R8, R9) were susceptible to erythromycin and clindamycin at the end of passage (Table 1). This particular phenotype, observed in 8 out of 15 spiramycin-resistant mutants, was verified twice by the disc diffusion method and by Etest. The seven other spiramycin-resistant isolates were resistant to erythromycin and clindamycin. No change in susceptibility to chloramphenicol or tetracycline was observed and all remained susceptible to linezolid.

Selection of linezolid-resistant mutants

All isolates yielded mutants resistant to linezolid with MICs ≥ 12 mg/L (Table 1). When serial passages were performed with linezolid using isolates susceptible to penicillin and erythromycin, resistant mutants were obtained after an average of 56.2 ± 14.2 passages. The rate of selection (16.6 ± 7.6) was significantly different when isolates were susceptible to penicillin but erythromycin resistant (P = 0.0005). The rate of acquisition of resistance between isolates intermediate to penicillin also differed significantly according to erythromycin susceptibility (P = 0.008).

When serial passages were performed with linezolid using isolates resistant to penicillin and susceptible to erythromycin, the number of passages required to generate resistance was 19.2 ± 11.6, which was not significantly different from that required for isolates resistant to penicillin and erythromycin (18 ± 6.5; P = 0.86).

With erythromycin-susceptible isolates, the difference in rate of acquisition of resistance to linezolid was not different between isolates susceptible or intermediate to penicillin but a significant difference was observed with isolates resistant to penicillin compared with those susceptible to penicillin (P = 0.0019), and also between isolates intermediate or resistant to penicillin (P = 0.035) (Figure 1).



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Figure 1. Number of passages required for S. pneumoniae to acquire linezolid resistance according to penicillin and erythromycin susceptibility.

 
Cross-resistance

In 22 cases, the antibiotic-resistance profile was similar between the parental isolates and the corresponding linezolid-resistant mutants. No acquisition of resistance to erythromycin, clindamycin or spiramycin was noted in any of the mutants. Two of 15 pneumococci that were initially resistant to macrolides and clindamycin (S8 and R2) became susceptible to these antibiotics during acquisition of resistance to linezolid.24 Loss of tetracycline resistance was also observed for S8. MICs of erythromycin decreased from >256 to 0.5 mg/L for S8 and 0.19 mg/L for R2.

Six linezolid-resistant mutants derived from erythromycin-resistant parental strains (four susceptible and two intermediate to penicillin) and initially susceptible to chloramphenicol became resistant to this antibiotic at the end of serial passage.

Mutations in domain V of 23S rRNA

Parental strains did not display any mutation in the four copies of the gene rrl compared with the S. pneumoniae R6 sequence. With an MIC for linezolid of 4 mg/L no mutation was observed. One mutation appeared when the linezolid MIC reached 6 mg/L in five out of the seven mutants tested at this level (Table 2). This result is concordant with the breakpoint for resistance (>4 mg/L).


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Table 2. Rate of appearance of mutations in linezolid mutants according to increase in MIC

 
Linezolid-resistant mutants had one mutation on at least three of the four copies in 27 cases (Table 2). In the three other cases (S8, I1 and R9), no mutation was found in the studied domain and these results were verified on mutants obtained in fewer passages that had different levels of resistance to linezolid.

Twenty-five mutants out of 30 had the G2576U mutation. In five mutants (S10, I7, I8, R6 and R10) an additional mutation, C2610U, was observed. This mutation appeared when the MIC of linezolid was ≥12 mg/L (Table 2). Two linezolid-resistant mutants displayed a different mutation, C2612A for S2 and C2571G for I3. An additional mutation (A2503G) was observed when the MIC reached 24 mg/L for the latter mutant (Table 2).


    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
Oxazolidinones are a new antibiotic class with activity against multiresistant strains. To avoid emergence of resistance to this agent, it is important to understand the mechanisms of resistance and to compare selection rates for linezolid and macrolides.

Erythromycin-resistant mutants were easily obtained, the majority showing the constitutive MLSB phenotype. For five mutants a particular phenotype was observed, with resistance to erythromycin and spiramycin and susceptibility to clindamycin with no blunting zone. This is not consistent with the M phenotype because resistance to a C-16 membered macrolide was obtained and may be compared with mutations observed by Tait-Kamradt et al.6 All mutants remained susceptible to linezolid.

The rate of acquisition of resistance to spiramycin was significantly higher when parental strains had decreased susceptibility to penicillin. Eight mutants remained erythromycin- and clindamycin-susceptible, which was consistent with the A2062C mutation described by Depardieu23 in a clinical isolate with resistance to spiramycin alone. These strains may be missed, as routine laboratory tests do not commonly include a C-16 membered macrolide. All mutants remained susceptible to linezolid and no modification of susceptibility to tetracycline and chloramphenicol was observed.

Tait-Kamradt et al.6 showed that macrolide resistance in mutants selected from susceptible strains was not related to any clinically documented resistance determinant but to mutations in 23S rRNA and ribosomal protein L4.

An important result of our study is the relationship between penicillin and erythromycin susceptibility and rate of acquisition of resistance to linezolid. Resistance was very difficult to obtain with isolates susceptible to penicillin and macrolides. Linezolid resistance was reached more rapidly when strains susceptible or intermediate to penicillin were resistant to erythromycin (Figure 1). Macrolides and linezolid bind near each other on the 50S ribosomal subunit, inhibiting protein synthesis.25 Macrolides bind to the 50S ribosomal subunit and more particularly to domains II and V of rRNA.26 The main resistance mechanism to macrolides is the modification of the target site by methylation of adenine at position 2058. The main mutation G2576U obtained in mutants resistant to linezolid is far from this adenine position. The hypothesis that methylation in adenine 2058 may modify the affinity of linezolid and facilitate the emergence of linezolid resistance merits further investigations. Ribosomal mutations in domain V of 23S rRNA in macrolide-resistant S. pneumoniae mutants have been described as A2059G, A2062C and A2058G, and C2611A and found respectively in clinical and in vitro isolates.3,6 A C2610U mutation conferred a small increase in the MICs of macrolides and clindamycin.7 In our study, this second mutation (C2610U) appeared only in 5 out of 30 linezolid-resistant mutants and only when the linezolid MIC was ≥12 mg/L.

Mechanisms of resistance to linezolid are independent of those involved in penicillin resistance, so the MIC values for linezolid did not vary with penicillin susceptibility of the isolates. Linezolid resistance, difficult to obtain with penicillin- and erythromycin-susceptible strains, was significantly easier to obtain when pneumococci had associated resistances. Pneumococci appeared more prone to acquire further resistance and one hypothesis is that these strains may be more hypermutable.

Our in vitro results showing that resistance to linezolid was most likely to emerge in isolates that were already resistant to macrolides are concordant with the first two such clinical strains of S. pneumoniae recently described.5

Oxazolidinones possess a distinctive mechanism of action and this can explain the absence of cross resistance with other antibiotics. However a decrease in susceptibility to chloramphenicol has been reported in linezolid-resistant S. aureus.27

Similar results were noted in six linezolid-resistant mutants obtained from 19 chloramphenicol-susceptible parental isolates. The site of action of chloramphenicol is located on the central loop of domain V of 23S rRNA, as for linezolid. The ribosomal mutation involved in linezolid resistance may confer a structural modification of the ribosome and decrease the affinity for chloramphenicol, or the two drugs may compete for closely neighbouring sites of action.28

Four copies of the rrl gene are present in S. pneumoniae. For E. faecium the increase in linezolid MIC is related to the number of mutated copies (1–5 out of 6 copies) when the MIC increased from 8 to 64 mg/L.12,28 This appears to be different in S. pneumoniae since the number of mutated copies is generally 3 or 4 when resistance becomes apparent.5 It is intriguing to note that the same mutation can give completely different MICs for linezolid in different isolates. Our results are consistent with those of Tait-Kamradt et al.6 who showed that acquisition of resistance to macrolides in S. pneumoniae required the mutation of at least two copies. The G2576U mutation found in 27 mutants of linezolid-resistant S. pneumoniae has been identified in all clinical strains of linezolid-resistant staphylococci and enterococci.14,29 Mutation A2160G has been described in an animal model of selection of linezolid resistance in pneumococci but was not found in our study.11 Mutations G2576U and G2032A, and T2500A were respectively described in E. coli13 and S. aureus.18,19 In two cases, the mutations observed (C2571G and C2612A) were original and their implication in the development of resistance had to be confirmed.

For three mutants, no mutation was found on any of the four copies of the rrl gene. This absence of mutation was confirmed by the study of three mutants with intermediate levels of resistance. These results suggest a mutation in another site such as ribosomal protein L22 or L4. Recently, Farrell et al.5 described two clinical isolates of S. pneumoniae with macrolide resistance due to ribosomal mutations and with resistance to linezolid (MIC 4 mg/L). This resistance was associated with two separate novel mutations, namely 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.

An additional mutation, C2610U, was observed when the MIC increased to >12 mg/L, associated with G2576U in five cases, while A2503G was added to C2571G in one mutant. The C2610U mutation was previously reported in one strain of E. faecalis in association with G2576U15 and a C2611A mutation was also found associated with G2576U in a strain of S. pneumoniae resistant in vitro to linezolid.30

These data associated with our results suggest that the C2610U, C2611A/G/U and C2612A mutations may be involved in the resistance to linezolid.

The use of antimicrobial agents in areas where there is a high prevalence of penicillin-resistant and macrolide-resistant S. pneumoniae isolates is likely to induce mutations in vivo and lead to therapeutic failure. Recommendations for antibiotic prescriptions should therefore take local resistance rates into account. Study of the capacity to generate resistance should be one of the points to consider when deciding on large-scale use in respiratory infections, and comparative studies should be carried out to determine the clinical repercussions of in vitro results. The initial acquisition of a resistance mechanism, even unrelated, may influence the rate of acquisition of resistance to other agents. This hypothesis is particularly important for verification as penicillin and macrolide resistance is increasing worldwide and may influence the future of linezolid in clinical use.


    Acknowledgements
 
Molecular biology investigations in this work received financial support from Pfizer Inc.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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