1 Service de Microbiologie, CHU Côte de Nacre, 14033 Caen, France; 2 Microbiology Unit, Canterbury Health Laboratories, Christchurch; 3 Department of Pathology, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand
Received 29 June 2004; returned 10 August 2004; revised 1 October 2004; accepted 5 October 2004
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Abstract |
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Methods: Nineteen erythromycin-susceptible, clindamycin-resistant S. agalactiae isolates from New Zealand were studied. MICs of macrolide, lincosamide and streptogramin antibiotics were determined. Clindamycin and streptogramin resistance genes were searched for by PCR. Isolates were compared by serotyping and by DNA macrorestriction patterns determined by PFGE. Conjugative transfer of resistance traits to recipient strains of S. agalactiae and Enterococcus faecium was assayed.
Results: The 19 S. agalactiae isolates were intermediate or resistant to clindamycin (MIC range: 0.52 mg/L) and lincomycin (MIC range: 18 mg/L) and had high MICs of dalfopristin (432 mg/L), a streptogramin A-type antibiotic, compared with controls. By contrast, the strains were susceptible to macrolides and quinupristin, a streptogramin B-type antibiotic. This new phenotype was called LSA (lincosamidestreptogramin A). Clindamycin resistance could not be transferred to recipient strains. Thirteen isolates belonged to serotype III and to a single PFGE genotype A, and five isolates belonged to serotype I and to genotype B. One isolate was non-typeable and belonged to a distinct genotype C.
Conclusions: We have characterized a new LSA phenotype in S. agalactiae. Analysis of restriction patterns of S. agalactiae chromosomal DNA showed that the resistance was spread in a minimum of three bacterial clones. The genetic and biochemical basis for the resistance remains unknown.
Keywords: streptococci , resistance mechanism , inhibition of protein synthesis , drug resistance , group B streptococcus
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Introduction |
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Materials and methods |
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Nineteen S. agalactiae susceptible to erythromycin but resistant to clindamycin were obtained from vaginal swabs (n=9), urine (n=2) and various other sites (n=8). Eighteen were isolated from women (age: 1980 years) and one from a newborn. The isolates were identified by conventional methods and by latex agglutination assay (Murex Diagnostics, Dartford, UK). Serotyping of isolates was determined by using a latex agglutination assay (Bio-Rad, Marnes-la-Coquette, France).
Antibiotic susceptibility
The MICs of ampicillin, clindamycin, dalfopristin, erythromycin, lincomycin, quinupristin, quinupristindalfopristin and doxycycline were determined by the microbroth dilution method with MuellerHinton broth (bioMérieux) supplemented with 5% defibrinated sheep blood, as recommended by the NCCLS (tested range: 0.0132 mg/L).8 All antibiotics were provided by their manufacturers. The plates were incubated overnight at 35°C in ambient air. MICs of clindamycin were also determined by the Etest technique, as recommended by the manufacturer (AB Biodisk, Uppsala, Sweden). MICs were also determined in the presence and absence of 10 mg/L of reserpine (Sigma Chemicals, St Louis, MO, USA), as described previously.9 An efflux mechanism could be suspected when there was at least a four-fold lower MIC in the presence of reserpine. A double disc-diffusion test with discs of erythromycin and clindamycin was used to test inducibility of resistance.10 Blunting of the clindamycin zone of inhibition proximal to the erythromycin disc was taken to indicate an inducible type of resistance.
Inactivation of antimicrobials
Inactivation of clindamycin, lincomycin or dalfopristin was checked using a previously described technique.11 Briefly, strains were streaked on brain heart infusion agar plates containing a heavy inoculum of Micrococcus luteus ATCC 9341 as an indicator organism, and 0.5 mg/L of dalfopristin or 0.2 mg/L of clindamycin, a concentration slightly higher than the MIC for M. luteus. Inactivation of the antibiotic in the culture medium would allow growth of the indicator in the surrounding medium. Staphylococcus aureus RN4220/pVMM26 inactivating lincomycin and clindamycin and S. aureus BM3002 inactivating dalfopristin were used as controls.11,12
Molecular techniques
PFGE was used to compare the isolates, as previously described.13 Genomic DNA was isolated from an overnight-grown culture and the agarose-embedded DNA was digested with the enzyme SmaI (New England BioLabs Inc., Beverly, MA, USA). PFGE was performed using the CHEF-DR-III system (Bio-Rad). Gels were run at 6 V/cm, 14°C, on a 1.2% agarose gel with pulse times of 535 s for 24 h. The bacteriophage DNA ladder (New England BioLabs Inc.) was used as a size standard. The PFGE banding patterns were compared visually and interpretation of gels was performed using the criteria of Tenover et al.14 Strains were considered genetically distinct if their restriction patterns differed by three or more bands.
The lincomycin-resistant isolates were screened for lincosamide and streptogramin A-type resistance genes. The erm(A), erm(TR), erm(B) and erm(C) genes encoding ribosomal methylases and the lnu(A) and lnu(B) genes (previously called linA and linB) encoding lincosamide nucleotidyltransferases were detected by PCR amplification, as described previously.12,15 The primers used to detect dalfopristin resistance genes were those described previously: vat(A),16 vat(B),17 vat(C),18 vat(E),19 vga(A),20 vga(B)21 and vga(Av).22 S. aureus strain BM3002 for vga(A) and vat(A),11 BM3318 for vga(Av),22 HM290 for erm(A), HM1054R for erm(C), BM4611 for lnu(A'),23 RN4220/pVMM26 for lnu(B),12 Staphylococcus haemolyticus BM4610 for lnu(A),23 E. faecium HM1032 for erm(B) and vat(D), and E. faecium UW 1965 for vat(E)19 were used as controls.
To detect mutations in the ribosomal target of lincosamides, we amplified a portion of the rrl gene for domain II from nt 452835 (Escherichia coli numbering), four fragments of domain V of 23S rRNA (nt 1601 to 2900) and the entire rplV gene (for L22 ribosomal protein) using primers previously described for Streptococcus pyogenes and Streptococcus pneumoniae.24,25 For amplification of the entire rplD gene (L4 ribosomal protein), primers 5'-ggtaacgtaccaggtgctaag (direct) and 5'-gatttcaacgcaaggcgacg (reverse), and primers 5'-ggtggtggtgttgtctttgg (direct) and 5'-gcacgtgtgtcaagttcaaatg (reverse) were used. The amplicons were analysed by single-strand conformation polymorphism (SSCP) analysis, as described previously.25
Plasmids were extracted from streptococcal strains, as described previously by Ehrenfeld & Clewell.26 Enterococcus faecalis JH2-2 containing plasmid pAD1 was used as a control.26
Filter mating
Transfer of lincomycin and dalfopristin resistance from strains of S. agalactiae to the recipient strains of E. faecium HM1070 and S. agalactiae 132 (both susceptible to lincosamides and streptogramins A-type and resistant to rifampicin and fusidic acid) was attempted by filter mating, as previously described.12 Transconjugants were selected on brain heart infusion containing rifampicin (20 mg/L), fusidic acid (10 mg/L) and lincomycin (2 mg/L) or dalfopristin (10 mg/L). Experiments were repeated independently three times.
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Results |
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MICs of macrolide, lincosamide and streptogramin antibiotics for the 19 strains and for controls are shown in Table 1 and Figures 1 and 2. All strains were susceptible to ampicillin and resistant to doxycycline (not shown). Compared with susceptible controls, MICs of clindamycin, lincomycin and dalfopristin were increased by a two- to four-fold dilution factor. This shift in MICs of lincosamides and dalfopristin contrasted with maintained susceptibility to 14-, 15- and 16-membered macrolides, telithromycin and quinupristin. The synergism between quinupristin (a streptogramin B) and dalfopristin (a streptogramin A) was retained with no significant increase in MICs. The double-disc diffusion test did not demonstrate inducible resistance to erythromycin. The MICs of lincosamides and dalfopristin were unchanged in the presence of the efflux pump inhibitor, reserpine. To the best of our knowledge, this phenotype is new in S. agalactiae and is called LSA (lincosamidestreptogramin A). Thirteen LSA isolates belonged to serotype III, five to serotype I and one was non-typeable. The SmaI macrorestriction of genomic DNA generated about 1520 fragments of different sizes. Analysis of macrorestriction patterns led to the identification of three different genotypes. All serotype III isolates had DNA profiles that were indistinguishable (five isolates) or differing by one or two bands (eight isolates) and were therefore clustered in a single genotype A (Figure 3). The five serotype I isolates formed a single genotypic cluster with indistinguishable profiles, whereas the non-typeable isolate displayed a distinct genotype C. Therefore, the spread of resistance appeared to be multiclonal.
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To determine the resistance genotype of strains, we used primers specific for genes encoding resistance to lincosamides or to streptogramin A-type antibiotics in clinical isolates. Although these genes confer resistance to lincosamides or streptogramins A alone, and have not been previously reported to confer an LSA phenotype, a combination was possible. All PCR controls gave the expected results. However, no PCR product was obtained for the study isolates. Genes that encode ribosomal structures composing the target of lincosamides, rrl, rplD and rplV genes, from two strains with serotype I and III were analysed by PCR-SSCP. Compared with the sequences of S. agalactiae 2603 V/R and S. agalactiae NEM316 obtained from The Institute for Genomic Research website at http://www.tigr.org and from The Institut Pasteur de Paris at http://www.pasteur.fr/recherche/unites/gmp/, respectively,27,28 no mutation was found.
Four strains of S. agalactiae of serotype I and III were tested for the transfer of clindamycin- or dalfopristin-resistance trait. No transfer to S. agalactiae 132 or to E. faecium HM1070 could be detected. No plasmid could be visualized after DNA extraction from the cells.
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Discussion |
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Clindamycin resistance may be misidentified in strains with the LSA phenotype if only erythromycin is tested. This may be of clinical importance in countries where clindamycin is recommended as an ampicillin substitute for the treatment or prevention of S. agalactiae infections in case of allergy. The emergence of LSA resistance justifies the in vitro test of clindamycin, in particular in countries where this phenotype is prevalent.
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Acknowledgements |
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Footnotes |
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References |
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