1 Laboratoire du Centre National de Greffe de Moelle Osseuse, 1006 Tunis, Tunisia; 2 Service de Microbiologie, CHU Côte de Nacre, 14033 Caen Cedex, France
Received 19 February 2004; returned 31 March 2004; revised 2 May 2004; accepted 8 May 2004
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Abstract |
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Methods: MICs of erythromycin, spiramycin, lincomycin and pristinamycin were determined for S. mitis isolates. Macrolide-resistance genes were characterized by PCR and ribosomal mutations by sequencing.
Results: A total of 169 S. mitis isolates were recovered from 66 patients at the Tunisian Bone Marrow Transplant Centre. Of these, 120 (70%) were non-susceptible to erythromycin and one was resistant to pristinamycin; 48.5% of isolates had an MLSB phenotype with cross-resistance between erythromycin, spiramycin and lincomycin, 4% had a dissociated MLSB phenotype with resistance to erythromycin and spiramycin but apparent susceptibility to lincomycin and 47.5% displayed the M phenotype. Resistance determinants were characterized in 33 isolates. Ten of 14 isolates with the cross MLSB resistance contained an erm(B)-like gene and four a combination of erm(B)- and mef(A)-like genes. Four of the five isolates with a dissociated MLSB phenotype contained erm(B)-like and one a combination of erm(B)- and mef(A)-like genes. All the 14 isolates with an M phenotype contained mef(A)-like genes. The pristinamycin-resistant strain had G105 and A108 substitutions in the conserved C terminus of the L22 ribosomal protein.
Conclusions: The prevalence of macrolide resistance is high in S. mitis from neutropenic patients and is due to the spread of erm(B)- or mef(A)-like genes alone or combined. Resistance to streptogramins is rare and in this case associated with ribosomal mutation.
Keywords: oral streptococci , ribosome , mutation , drug resistance
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Introduction |
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In streptococci, resistance to macrolides is conferred either by methylases, encoded by erm genes that modify the ribosomal target of macrolides or by pumps, encoded by mef(A) genes, that efflux these antibiotics. Ribosomal methylation results in cross-resistance to macrolide, lincosamide and streptogramin B antibiotics (MLSB phenotype) and can be expressed either constitutively or inducibly. In streptococci, MLSB resistance is mostly mediated by determinants belonging to the erm(B) class. Another methylase, first described in Streptococcus pyogenes, and then found to be extensively present in this species but rare in S. pneumoniae, is mediated by a gene originally called ermTR and belonging to the erm(A) class.3,4 Recently, mutations in 23S rRNA or ribosomal proteins leading to macrolide resistance in clinical isolates of S. pneumoniae have also been described.4
Macrolide efflux is mediated by a membrane protein encoded by the mef(A) gene and is characterized by resistance to 14- and 15-membered ring macrolides, whereas 16-membered ring macrolides, lincosamides and streptogramins, remain active even after induction with erythromycin (M phenotype).5
Gram-negative bacteria and some Gram-positive organisms, such as Enterococcus faecalis are intrinsically resistant to streptogramins. Acquired resistance to streptogramins is mostly found in staphylococci and Enterococcus faecium where it is usually due to the acquisition of extrinsic genes responsible for inactivation (vat and vgb genes) or efflux of streptogramin components (vga genes).6 In S. pneumoniae, resistance to streptogramins is rare and has been related to ribosomal mutations such as an A2062C mutation in domain V of 23S rRNA, or to L22 and L4 protein mutations.4,7,8
Few studies have investigated the macrolide resistance mechanisms in S. mitis, in particular in immunocompromised patients who are exposed to multiple courses of antimicrobial treatments. The purpose of this study is to determine the susceptibility to antibiotics of S. mitis isolated in the Tunisian Bone Marrow Transplant Centre in 2002 and to characterize the mechanisms of macrolide and streptogramin resistance in erythromycin- and pristinamycin-resistant strains.
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Materials and methods |
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In 2002, 169 S. mitis isolates were obtained from 66 patients hospitalized at the Tunisian Bone Marrow Transplant Centre. Of these, 85% of strains were isolated from systematic nasopharyngeal specimens, 5% from the upper respiratory tract and 10% from other sites including blood cultures. In 2002, S. mitis represented 9.5% of the total and 2% of the blood culture isolates in our centre. Isolates were identified by API Rapid ID 32 Strep system (bioMérieux, La-Balme-Les-Grottes, France). Only isolates for which identification results were rated as typical were retained for the study. When multiple isolates were obtained for a single patient, duplicates were eliminated on the basis of identical patterns of antimicrobial susceptibility. The following strains were used as controls in PCR: six S. mitis strains susceptible to erythromycin, S. pneumoniae HM28 containing the erm(B) gene, S. pneumoniae O2J1175 containing the mef(A) gene and S. aureus BM3002 containing vga(A), vgb(A) and vat(A) genes.9,10
Antibiotic susceptibility testing
Susceptibility of S. mitis to penicillins, gentamicin, erythromycin, spiramycin, lincomycin and pristinamycin was tested by the agar diffusion technique using MuellerHinton medium (bioMérieux) supplemented with 5% horse blood and incubated at 37°C for 24 h in an aerobic atmosphere under 5% CO2 as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM).11 Resistance to erythromycin, spiramycin and lincomycin indicated a cross MLSB resistance. Blunting of the lincomycin inhibition zone proximal to the erythromycin disc indicated an inducible dissociated MLSB resistance. Resistance to erythromycin and susceptibility to lincomycin with no blunting zone defined the M phenotype (efflux mechanism).
The susceptibility of 120 erythromycin-resistant S. mitis to the following eight antibiotics was tested by the agar dilution technique: penicillin G (Biochemie GmbH, Vienna, Austria), ampicillin (Bristol-Myers Squibb S.P.A., Italy), erythromycin (Abbott, Rungis, France), lincomycin (Pfizer France, Paris, France), pristinamycin (Aventis, Vitry-sur-Seine, France), gentamicin (A. Menarini, Florence, Italy), vancomycin (Eli-Lilly France, S. A., Suresnes, France) and teicoplanin (Aventis).11 MICs were determined on MuellerHinton agar supplemented with 5% horse blood and incubated at 37°C for 24 h in an aerobic atmosphere under 5% CO2. An inoculum of 104 cfu per spot was used. S. pneumoniae CIP 104485 and E. faecalis ATCC 29212 were used as controls. Results were interpreted according to the recommendations of the CA-SFM.11
Polymerase chain reaction and sequencing
We used a multiplex PCR technique previously described which allowed amplification of erm(B), mef(A) and ermTR[erm(A)] genes.9 For the strain resistant to pristinamycin, known streptogramin resistance genes belonging to the families of vat, vga and vgb genes were searched for by PCR as described.12 In addition, for this strain, portions of rrl genes encoding 23S ribosomal RNA, and rplD and rplV genes encoding ribosomal proteins L4 and L22, respectively, were amplified. Analysis of the genomic sequence of S. mitis NCTC 12261, obtained at The Institute for Genomic Research website at http://www.tigr.org, allowed the design of 12 pairs of primers to amplify rrl, rplD and rplV genes. PCR products were sequenced on both strands. Oligonucleotide primers used are shown in Table 1. The amino acid sequences of proteins L22 of the strain resistant to pristinamycin and of S. mitis NCTC 12261 were deduced from the sequence of the rplV gene and compared. Conserved regions in the protein L22 were identified by comparison with that of other Gram-positive cocci, in particular S. pneumoniae.7
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Results |
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Of the 169 non-repetitive strains of S. mitis isolated in 2002, 70% (120 strains) were not susceptible to erythromycin and 83% to penicillin G whereas only 0.6% (one strain) was resistant to pristinamycin by the disc-diffusion technique. No strain was resistant to high levels of gentamicin or to vancomycin or teicoplanin. Three phenotypes were observed among the 120 erythromycin-resistant isolates. Fifty-eight strains (48.5%) displayed the cross MLSB resistance, five (4%) a dissociated MLSB resistance phenotype and 57 (47.5%) the M phenotype.
A summary of MIC range, MIC50 and MIC90 of seven antibiotics for erythromycin-resistant strains is listed in Table 2. Teicoplanin was the most active antibiotic against erythromycin-resistant strains with MIC90 <0.125 mg/L.
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Characterization of macrolide resistance in selected strains
Thirty-three erythromycin-resistant strains were studied further. Fourteen displayed the cross-MLSB resistance, five the dissociated MLSB phenotype and 14 the M phenotype. erm(B)-like sequences were amplified from the 19 strains with the MLSB phenotype. Five of these strains contained in addition mef(A)-like genes. The 14 strains with the M phenotype contained mef(A)-like genes. No DNA fragment could be amplified from the six erythromycin-susceptible S. mitis isolates with any of the primers tested. Distribution of erythromycin resistance genes according to erythromycin resistance phenotypes is reported in Table 3.
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Characterization of resistance to pristinamycin
The strain resistant to pristinamycin contained an erm(B)-like gene. Neither vat, vga nor vgb genes conferring streptogramin resistance could be amplified by PCR. The analysis of the sequence of genes encoding the ribosomal structures which participate in the binding of streptogramins to their target did not reveal any mutation in the sequences of rrl and rplD genes. In contrast, comparison of the deduced amino acid sequences of the rplV genes of the pristinamycin-resistant strain and of S. mitis NCTC 12261 and S. pneumoniae R6 showed substitution of two amino acids in the L22 ribosomal protein, A105G and T108A, located at the conserved C terminus region of the protein 91PRAKGSASPINKRTGHIAVAVAEKG115 (substitutions underlined).
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Discussion |
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In S. mitis, MLSB and M phenotypes were present at a similar frequency in our strains. This was also the case in a Spanish study.19 However, the relative proportion of the two resistance phenotypes may vary according to the country or even within a given country. For instance, the M phenotype was found to be highly prevalent in another Spanish study and in Finland (80.6%).18,20
Literature describing molecular mechanisms conferring resistance to macrolides in S. mitis is limited to a few reports. In our study, the molecular characterization of S. mitis clinical isolates revealed that the erm(B) and mef(A) genes are found with similar frequencies. Similar findings were reported in Spanish and Japanese studies with viridans streptococci.19,21 However, erm(B) was found more prevalent than mef(A) in a Canadian study whereas mef(A) genes were prevalent in a Spanish study.20,22
In our study, the MLSB and M phenotypes correlated with the presence of erm(B)-like and mef(A)-like genes, respectively. The strains resistant to erythromycin but susceptible to lincomycin which contained an erm(B)-like gene could be phenotypically distinguished from those with the M phenotype on the basis of resistance to spiramycin and antagonism between erythromycin and lincomycin. Strains carrying a mef(A)-like gene generally displayed lower MICs of erythromycin (464 mg/L) than those with an erm(B)-like gene (MICs from 4 to >1024 mg/L) with some exceptions. Surprisingly, in five of 19 strains with the MLSB phenotype, the erm(B)-like gene was combined with a mef(A)-like gene, the M phenotype conferred by the latter gene being masked by the MLSB phenotype. Combination of both genes has already been detected in erythromycin-resistant S. pneumoniae and S. agalactiae,9,23,24 but has not been previously found in S. mitis and has been reported in one study in only 0.8% of S. oralis.19,20,22 Viridans streptococci have been suspected of being a possible reservoir of resistance genes and this accumulation of resistance genes in a non-negligible proportion of strains may contribute to rapid spread of resistance determinants to pathogenic species of streptococci.2
We confirmed that streptogramin resistance is very uncommon in viridans streptococci.16 The only strain resistant to pristinamycin contained an erm(B)-like gene which alone could not explain the streptogramin resistance. Indeed, the synergy is usually maintained between the factors A and B of the streptogramin combination despite the resistance to factor B conferred by the gene.4 In contrast, the mutation that we found in the conserved C terminus of the L22 ribosomal protein might explain the streptogramin resistance. Mutations in the same conserved region and conferring resistance to streptogramins have been reported in clinical isolates and laboratory mutants of S. aureus25 and S. pneumoniae.7,8 This is the only protein to interact with RNA sequences belonging to all six domains of 23S rRNA and is important for the folding of the 23S rRNA. It is also a partner of L4 in the probable formation of a gated opening for the tunnel exit.7 This is the first report of L22 mutation in S. mitis.
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Footnotes |
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References |
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