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Journal of Antimicrobial Chemotherapy (2007) 59, 1010– 1012 doi:10.1093/jac/dkm041 Emergence of a Streptococcus pneumoniae isolate resistant to streptogramins by mutation in ribosomal protein L22 during pristinamycin therapy of pneumococcal pneumonia Vincent Cattoir1*, Lilia Merabet1, Patrick Legrand1, Claude-James Soussy1 and Roland Leclercq2 1 Laboratoire de Bactériologie-Virologie-Hygiène, CHU Henri Mondor, Assistance Publique-Hôpitaux de Paris, Université Paris XII, Créteil, France; 2Service de Microbiologie, EA 2128 Relations Hôte et Microorganismes des Épithéliums, CHU Côte de Nacre, Université de Caen, 14033 Caen Cedex, France Received 5 December 2006; returned 5 January 2007; revised 17 January 2007; accepted 29 January 2007 Objectives: The aim of this study was to characterize the mechanism of resistance to macrolides and streptogramins of a Streptococcus pneumoniae strain isolated from blood cultures in an 80-year-old patient suffering from severe pneumonia unsuccessfully treated with pristinamycin. Methods: Resistance genes erm(B) and mef(A) were searched for by PCR. Portions of genes for domains V and II of the 23S rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22 (rplV) were amplified by PCR from total genomic DNA and sequenced. Results: Resistance genes erm(B) and mef(A) were not detected. Only mutation in the rplV gene encoding ribosomal protein L22 was detected. The strain contained a six amino acid insertion (107KRTAHI108) in the C-terminus of the ribosomal protein L22. Conclusions: This is the first report of emergence of a pneumococcus resistant to streptogramins by mutation in ribosomal protein L22 during treatment with pristinamycin. Keywords: macrolide resistance, pneumococcus, riboprotein, rplV Introduction Streptococcus pneumoniae is an important cause of communityacquired pneumonia. Penicillins are the treatment of choice of these infections and macrolides constitute an alternative in the case of intolerance to the drugs. However, increasing resistance to macrolides and related antibiotics [macrolides– lincosamides – streptogramins (MLS)] among S. pneumoniae clinical isolates constitutes a worldwide problem.1 In pneumococci, MLS resistance is mediated by two major mechanisms: target site modification and drug efflux.1 Ribosomal alteration is mediated by a methyltransferase, encoded by the erm(B) gene [or rarely the erm(A) gene], that methylates the A2058 residue (Escherichia coli numbering) in domain V of the 23S rRNA and confers the so-called MLSB phenotype of cross-resistance among macrolides, lincosamides and streptogramins B. Active efflux is mediated by the mef(A) gene [formerly mef(E)] that codes for an efflux pump and is responsible for the so-called M phenotype characterized by unique resistance to 14- and 15-membered-ring macrolides (azithromycin, erythromycin, clarithromycin and roxithromycin).1 In addition, mutations in 23S rRNA and the ribosomal proteins L4 and L22 (encoded by the rplD and rplV genes, respectively) have been infrequently described in pneumococci.2 Resistance to macrolides can reach percentages exceeding 50% in certain countries and reduced susceptibility to erythromycin has become more frequent than reduced susceptibility to penicillin in Europe overall.3 Telithromycin, a ketolide, and pristinamycin, an oral streptogramin, are molecules related to macrolides, which have demonstrated activity against pneumococci resistant to macrolides.4 Pristinamycin is a combination of streptogramin A-type ( pristinamycin IIA) and streptogramin B-type ( pristinamycin IA) antibiotics. Although Erm methylases confer cross-resistance to macrolides, lincosamides and streptogramins B, synergism between the two streptogramin components is maintained, explaining the activity of the streptogramin combination.2 Here, we report an S. pneumoniae isolate resistant to pristinamycin, an oral streptogramin, and mutated in ribosomal protein L22 isolated from blood cultures of a patient with lower respiratory tract infection unsuccessfully treated with pristinamycin. ..................................................................................................................................................................................................................................................................................................................................................................................................................................... *Corresponding author. Tel: þ33-1-49-81-68-16; Fax: þ33-1-49-81-28-39; E-mail: [email protected] ..................................................................................................................................................................................................................................................................................................................................................................................................................................... 1010 # The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] S. pneumoniae L22 ribosomal mutant Materials and methods Table 1. MICs of MLS antibiotics for S. pneumoniae HM8989 and for control strain, S. pneumoniae ATCC 49619 Bacterial strains and antimicrobial susceptibility testing The strain of S. pneumoniae HM8989 was isolated from two blood cultures taken from an 80-year-old male patient suffering from severe pneumonia. He had been treated empirically with pristinamycin (3 g day) for 1 week for bronchial infection and was hospitalized at Henri Mondor hospital (Créteil, France) for persistent fever and increasing dyspnoea. The S. pneumoniae isolate was phenotypically identified and antimicrobial susceptibility was tested by the disc diffusion method. MICs of MLS antibiotics were determined by the agar dilution method on Mueller –Hinton agar medium, according to the recommendations of Comité de l’Antibiogramme de la Société Française de Microbiologie.5 The reference strain S. pneumoniae ATCC 49619 was included as a control. MIC (mg/L) Antibiotics Erythromycin Clarithromycin Azithromycin Spiramycin Lincomycin Clindamycin Quinupristin Dalfopristin Quinupristin/ dalfopristin Pristinamycin IA Pristinamycin IIA Pristinamycin Telithromycin PCR and DNA sequence analysis Bacterial genomic DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France). Resistance genes erm(B) and mef(A) and portions of genes for domains V and II of the 23S rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22 (rplV) were amplified using PCR with specific primers, as previously described.6 PCR products were then sequenced in both directions using the ABI PRISM 3100 automatic sequencer (Applied Biosystems, Courtaboeuf, France). For identification of ribosomal mutations, sequences were compared with the genomic sequences of S. pneumoniae TIGR4 and R6 (GenBank accession numbers NC003098 and NC003028, respectively). Comparisons were done using the software available over the Internet at the National Center for Biotechnology Information web site (http:// www.ncbi.nlm.nih.gov/). Results Susceptibility to antimicrobial agents By the disc diffusion method, the pneumococcal strain HM8989 was fully susceptible to all b-lactams tested with MICs of penicillin G, ampicillin and cefotaxime equal to 0.016, 0.016 and 0.008 mg/L, respectively. It also showed an intrinsic low-level resistance to aminoglycosides (streptomycin, kanamycin and gentamicin) and was susceptible to tetracyclines, chloramphenicol, rifampicin, co-trimoxazole, levofloxacin and vancomycin. In contrast, it exhibited a very uncommon profile of resistance to macrolides and related antibiotics with an intermediate susceptibility to erythromycin and pristinamycin. The strain remained fully susceptible to lincomycin. No D-shaped zone was visible when the disc of erythromycin was placed close to the disc of clindamycin. MICs of MLS antibiotics are shown in Table 1. MICs of erythromycin, clarithromycin, azithromycin, telithromycin and spiramycin for the strain HM8989 were 32– 64-fold greater than those for the reference strain S. pneumoniae ATCC 49619. Despite the 32-fold increased MIC of telithromycin, the isolate remained categorized as susceptible to this antibiotic. MIC determination confirmed that the isolate was fully susceptible to lincosamides with MICs 2– 4-fold lower than those for the reference strain. In addition, the isolate was resistant to the streptogramin combination quinupristin/dalfopristin with an 8-fold increase in the MIC of quinupristin, whereas the MIC of dalfopristin was the same. As expected, this strain was classified as S. pneumoniae HM8989 S. pneumoniae ATCC 49619 1 1 2 8 0.12 0.03 8 32 16 0.03 0.016 0.06 0.12 0.5 0.06 1 32 0.25 16 4 2 0.25 4 2 0.12 0.008 intermediate to pristinamycin (MIC ¼ 2 mg/L), with MICs of pristinamycin IA and pristinamycin IIA equal to 16 and 4 mg/L, respectively. Mutation in the rplV gene No erm(B) methylase gene or mef(A) efflux gene could be detected by PCR with specific primers. No mutations were found in the rrl gene (domains II and V) or in the rplD gene for ribosomal protein L4. However, the strain displayed an 18 nucleotide tandem duplication at the 30 end of the rplV gene encoding ribosomal protein L22. This insertion corresponded to a six amino acid insertion (107KRTAHI108) tandemly duplicated (Figure 1). Discussion Macrolide resistance in S. pneumoniae is mostly mediated by erm(B), a 23S rRNA methylase, and by mef(A), a gene encoding an efflux pump.2 Since pristinamycin is still active against pneumococcal isolates displaying either mechanism,2 this oral antibiotic represents an attractive option for the empirical treatment of respiratory tract infections. This report shows that resistance to streptogramins may emerge in clinical isolates through mutation in ribosomal protein L22 and can be associated with clinical failure of pristinamycin therapy. No early pneumococcal isolate from the patient was available. Therefore, we do not know whether the patient was infected by a strain already intermediate resistant to pristinamycin or whether strain HM8989 was a mutant selected under pristinamycin therapy. A few cases of clinical failure of pristinamycin for the treatment of lower respiratory tract infections have been reported.7,8 However, in these episodes, the strains remained susceptible to pristinamycin. Only two pneumococcal isolates from blood cultures have been reported in 1989, resistant to 16-membered macrolides and intermediate to pristinamycin. This resistance was shown to be due to an A2062C mutation in domain V of the 23S rRNA.9 Since 1011 Cattoir et al. S. pneumoniae HM8989 10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK S. pneumoniae TIGR4 MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK S. pneumoniae R6 MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK S. pneumoniae HM8989 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....| ANLVVSEAFANEGPTMKRFRPRAKGSASPINKRTAHIKRTAHITVAVAEK S. pneumoniae TIGR4 ANLVVSEAFANEGPTMKRFRPRAKGSASPIN------KRTAHITVAVAEK S. pneumoniae R6 ANLVVSEAFANEGPTMKRFRPRAKGSASPIN------KRTAHITVAVAEK Figure 1. Alignment of portion of the deduced amino acid sequence of ribosomal protein L22 of S. pneumoniae HM8989 when compared with those of strains S. pneumoniae TIGR4 and R6. then, resistance to pristinamycin in clinical isolates of pneumococci from France has not been reported.10 Mutations in protein L22 seem to be uncommon in clinical isolates. In epidemiological surveys, mutations occurring in the 30 end of the rplV gene have recently been shown to confer unusual MLS-resistance patterns in clinical isolates [R22C, G95D, insertion 92VRPR93, A101P, tandem duplications (108RTAHI109 or 108 RTAHIT109), C117T].2 Emergence of an L22 mutant during treatment with azithromycin of a fatal pneumococcal pneumonia emphasizes the clinical importance of this mutation as a resistance mechanism.11 This isolate contained a tandem duplication 108 RTAHIT109 with resistance to erythromycin, azithromycin and quinupristin/dalfopristin (MICs 2 – 4 mg/L). The structure of the 50S ribosomal subunit containing a three-residue deletion mutant of L22 in Haloarcula marismortui showed a change in the L22 structure.11 L22 mutations at the C-terminus could change the conformation of the b-hairpin and enlarge the exit tunnel for polypeptides. This suggested that L22 mutants are resistant to macrolides, because the lumen of the tunnel has become so large that it can no longer be obstructed efficiently by macrolides. However, as pointed out by Tu et al.,12 the mechanism of L22 resistance is likely to be more complex, since macrolides bind to the tunnel entirely above the region affected by L22 mutations. The mechanism of resistance by mutation in L22 in S. pneumoniae has not been studied, but is probably similar. The tandem duplication that we found in the C-terminus of the L22 protein (107KRTAHI108) was not exactly the same as those that were previously described (108RTAHIT109) among clinical isolates of macrolide-resistant S. pneumoniae.2 However, these two close variations could be responsible for similar conformational changes in the riboprotein. In the literature, there is only one report of a macrolide-resistant pneumococcal mutant emerging during empirical treatment. Finally, L22 mutations did not affect the susceptibility to lincosamides but compromised the potency of the ketolides, with an increase in MIC of telithromycin from 0.008 to 0.25 mg/L. This report confirms the role of tandem duplications of ribosomal target protein L22 in MLS resistance in S. pneumoniae. This is the first report of emergence of a pneumococcal isolate resistant to streptogramins by mutation in ribosomal protein L22 during treatment with pristinamycin. Finally, it also highlights the risk of emergence of these mutants that present increased MICs of telithromycin. Transparency declarations None to declare. References 1. Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis 2002; 34: 482–92. 2. Leclercq R, Courvalin P. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: 2727–34. 3. Bruinsma N, Kristinsson KG, Bronzwaer S et al. Trends of penicillin and erythromycin resistance among invasive Streptococcus pneumoniae in Europe. J Antimicrob Chemother 2004; 54: 1045–50. 4. Pankuch GA, Visalli MA, Jacobs MR et al. Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents. Antimicrob Agents Chemother 1998; 42: 624 –30. 5. Comité de l’Antibiogramme de la Société Française de Microbiologie. Communiqué 2006. www.sfm.asso.fr (15 November 2006, date last accessed). 6. Canu A, Malbruny B, Coquemont M et al. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: 125–31. 7. Burucoa C, Pasdeloup T, Chapon C et al. Failure of pristinamycin treatment in a case of pneumococcal pneumonia. Eur J Clin Microbiol Infect Dis 1995; 14: 341–2. 8. Brevet-Coupe F, Guerin B, Watine J. Failure of pristinamycin treatment in a case of pneumonia caused by a streptogramins B-type resistant pneumococcus. Scand J Infect Dis 1998; 30: 419–20. 9. Depardieu F, Courvalin P. Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrob Agents Chemother 2001; 45: 319–23. 10. Weber P. Streptococcus pneumoniae: lack of emergence of pristinamycin resistance. Pathol Biol (Paris) 2001; 49: 840–5. 11. Musher DM, Dowell ME, Shortridge VD et al. Emergence of macrolide resistance during treatment of pneumococcal pneumonia. N Engl J Med 2002; 346: 630–1. 12. Tu D, Blaha G, Moore PB et al. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell 2005; 121: 257–70. 1012