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Antimicrobial Agents and Chemotherapy, April 2005, p. 1646-1648, Vol. 49, No. 4
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.4.1646-1648.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
and
Claire Poyart2,
Service de Microbiologie, CHU Côte de Nacre, Caen,1 Laboratoire Mixte Pasteur Necker de Recherche sur les Streptocoques, Faculté de Médecine Necker, Paris, France2
Received 19 October 2004/ Returned for modification 18 November 2004/ Accepted 28 November 2004
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A collection of 135 streptococci identified as S. bovis biotype I or II.2 and obtained between 1994 and 2003 from clinical samples was studied. Seventy-two were isolated from blood cultures or cardiac vegetations and were responsible for endocarditis. Of these endocarditis isolates, 46 were from various French hospitals (including 23 kindly provided by J. Etienne and F. Vandenesch), and 26 came from Belgium and The Netherlands, kindly provided by W. H. F. Goessens. Fifty-six other isolates were from blood cultures obtained in French hospitals, four were from other clinical samples, and three were isolated in one cat and two dogs. All strains were stored at –80°C in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) containing 20% glycerol until being tested.
All isolates were identified as S. gallolyticus using the API 20Strep and rapid-identification 32Strep systems (bio-Mérieux). Species identification was also performed by amplification and sequencing of the sodA gene as previously described (7). Among the strains responsible for endocarditis, 71 (98.4%) belonged to the species S. gallolyticus, whereas the remaining strain was identified as Streptococcus lutetiensis. Furthermore, for the 56 strains isolated from blood cultures, 50 (89.3%), 3 (5.3%), 2 (3.6%), and 1 (1.8%) were identified as S. gallolyticus, Streptococcus pasteurianus, Streptococcus infantarius, and S. lutetiensis, respectively. Only the 128 S. gallolyticus strains were retained for the study. Screening for antibiotic resistance was first carried out by the disk diffusion method with Mueller-Hinton plates containing 5% sheep blood (Bio-Rad, Marnes-la-Coquette, France) in accordance with the guidelines of the Comité de l'Antibiogramme de la Société Française de Microbiologie (2). The resistance phenotypes of erythromycin-resistant S. gallolyticus were further characterized by the agar diffusion method using erythromycin and clindamycin disks. Blunting of the clindamycin inhibition zone proximal to the erythromycin disk indicated an inducible type of macrolide-lincosamide-streptogramin B (MLSB) resistance. Resistance to both erythromycin and clindamycin indicated MLSB cross-resistance. Susceptibility to clindamycin with no blunting defined the M phenotype (efflux mechanism).
By the disk diffusion technique, no strain was resistant to penicillin, ampicillin, rifampin, vancomycin, teicoplanin, and high levels of gentamicin. Acquired resistances were detected for macrolides, tetracyclines, streptomycin, and kanamycin. MICs of erythromycin, kanamycin, clindamycin, spiramycin, streptomycin, telithromycin, and tetracyclines were determined by the agar dilution method in accordance with the guidelines of the Comité de l'Antibiogramme de la Société Française de Microbiologie (2). Categorization as nonsusceptible (intermediate or resistant) was done on the basis of the following Comité de l'Antibiogramme de la Société Française de Microbiologie breakpoints: erythromycin, >1 µg/ml; clindamycin, >2 µg/ml; kanamycin and streptomycin, >1,000 µg/ml; telithromycin, >1 µg/ml; tetracycline, >8 µg/ml (2).
Seventy-six isolates (59.4%) were intermediate or resistant to erythromycin. Among these, 64 (84.2%) were cross-resistant to erythromycin and clindamycin (MIC at which 90% of the isolates tested are inhibited [MIC90], >128 µg/ml) whereas 11 (14.5%) were susceptible to clindamycin with blunting, indicating an MLSB inducible phenotype (erythromycin MIC90, >4 µg/ml; clindamycin MIC90, 0.125 µg/ml) (Table 1). One isolate was intermediate to erythromycin but susceptible to clindamycin (MIC < 0.12 µg/ml) without blunting, displaying an M phenotype. The percentages of strains resistant to tetracyclines and to high levels of kanamycin and streptomycin were 77.7, 34.7, and 37.3, respectively. All strains resistant to high levels of kanamycin and nearly all strains highly resistant to streptomycin were coresistant to erythromycin and tetracyclines. Resistances to erythromycin and tetracyclines were also often combined.
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Two known mechanisms of resistance to tetracyclines have been reported in streptococci and enterococci: efflux by proton antiporters [Tet(L) and Tet(K)] and ribosome protection [Tet(M), Tet(O), Tet(S), and Tet(T)]. A total of 77.7% of isolates were resistant to tetracyclines, and tet(M) was the most prevalent determinant of resistance to tetracyclines, accounting for 97% of tetracycline-resistant isolates. This determinant is usually highly prevalent in gram-positive bacteria resistant to tetracyclines and is often associated with conjugative elements of the Tn916 family (1). Consistently, the intTn gene, encoding the integrase of Tn916, was found in all strains harboring tet(M)-like genes. These results show that tetracycline resistance in S. gallolyticus is due to the acquisition of Tn916-related transposons. A tet(O)-like determinant was detected alone in one isolate and combined with tet(M)-like genes in 13 isolates. tet(L)-like determinants were found alone in three isolates and combined with tet(M) in five isolates, whereas six isolates contained a combination of tet(M)-, tet(L)-, and tet(O)-like determinants. Combination of tetracycline resistance determinants has been reported previously in various gram-positive bacteria (9, 10). The tet(K), tet(S), and tet(T) determinants were not detected.
All isolates highly resistant to kanamycin contained an aphA-3-like gene responsible for phosphorylation of kanamycin and all isolates, but four isolates highly resistant to streptomycin contained an aad-6-like gene responsible for adenylylation of streptomycin. The streptomycin resistance in the four PCR-negative strains might be due to a chromosomal mutation, as reported previously for enterococci (6). The results reported in this study are similar to those reported previously in two studies on strains isolated in Taiwan (12) and in humans and animals in Belgium (4).
In conclusion, our results show that tetracycline resistance is widespread in S. gallolyticus, due to the presence of tet(M) probably borne by conjugative transposons such as Tn916, which was detected in 77.7% of the isolates studied. Prevalence of erythromycin resistance was also high, which might be explained by the notion that tetracycline and erythromycin resistance determinants are often colocated on large conjugative transposons (1). These results emphasize the need to monitor the epidemiology and the genetic basis of antibiotic resistance in streptococci.
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ACKNOWLEDGMENTS |
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This work was supported by the Institut Pasteur, INSERM, and the Universities of Paris V and Caen.
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FOOTNOTES |
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Present address: Unité de Biologie des Bactéries Pathogènes à Gram Positif, Institut Pasteur, Paris Cedex 15, France.
Present address: Service de Bactériologie, CHU Cochin, Paris Cedex 14, France.
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