a Respiratory and Systemic Infection Laboratory and b Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK
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Abstract |
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Introduction |
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Ketolides are a new class of macrolide-like antimicrobial agents, characterized by a 3-keto group instead of an l-cladinose at position 3 on the erythronolide A ring. Their mechanism of action is similar to that of the macrolides: they bind to the 50S ribosomal subunit and inhibit bacterial protein synthesis.10 Ketolides are active against a variety of Gram-positive organisms including those resistant to erythromycin and other macrolides.11
We have determined the susceptibility of 410 isolates of C. diphtheriae to two ketolides, HMR 3647 and HMR 3004, in comparison with four macrolides (erythromycin A, azithromycin, clarithromycin and roxithromycin), two fluoroquinolones (levofloxacin and ofloxacin) and penicillin G.
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Materials and methods |
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The 410 isolates used in this study were selected from the culture collection at the PHLS Streptococcus and Diphtheria Reference Unit, Central Public Health Laboratory, London, UK, referred between 1988 and 1998 from the UK and 15 other countries. Isolates were selected to be representative of geographical origin, biotype and toxigenicity. Biotyping was performed using conventional biochemical methods and toxigenicity testing was performed using the Elek test, as described previously.12 The origin, biotype and toxigenicity of the isolates are summarized in Table I.
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Nine antimicrobial agents were tested against C. diphtheriae, including HMR 3004, HMR 3647, erythromycin A, clarithromycin, azithromycin, roxithromycin, levofloxacin, ofloxacin and penicillin G. All were supplied by Hoechst-Marion-Roussel, Romainville, France.
Determination of MICs
MICs were determined by the agar dilution method on Diagnostic Sensitivity Test (DST) agar (Oxoid, Basingstoke, UK) supplemented with 5% saponin-lysed horse blood, as described previously.3 Inocula comprised 104105 cfu/spot and were delivered using a multipoint inoculator (Mast Laboratories Ltd, Merseyside, UK). Non-toxigenic C. diphtheriae biotype mitis (NCTC 11397), Staphylococcus aureus (NCTC 6571, ATCC 29213 and 25923) and Enterococcus faecalis (ATCC 29212) were used as controls. Plates were incubated for 1820 h at 37°C in air and the MIC was defined as the lowest concentration required to completely inhibit visible growth. MIC50s and MIC90s were calculated using cumulation and interpolation.13 Isolates showing reduced susceptibility to erythromycin by agar dilution (erythromycin MIC 1 mg/L) were re-tested against the four macrolides using Etest strips (AB Biodisk, Solna, Sweden), in accordance with the manufacturer's instructions.
Induction of MLS resistance
Inducible MLSB resistance to erythromycin A was determined using a double disc diffusion test as described previously3 and by a novel method using Etest strips. For both methods, a semi-confluent lawn of each isolate was prepared by swabbing a suspension (corresponding to a McFarland no. 0.5) on to DST agar containing saponin-lysed horse blood (5%). For the disc diffusion assay, clindamycin (2 µg) and erythromycin A (15 µg) discs (Oxoid) were placed 25 mm apart. For the Etest method, an erythromycin Etest strip was placed on a plate for 1 h at room temperature, then removed. A clindamycin Etest strip was then placed in exactly the same position and plates were incubated for 24 h at 37°C in air. As a control, Etests for clindamycin only were also performed for each strain, in accordance with the manufacturer's instructions. Inducible resistance was detected by a D-shaped zone of inhibition around the clindamycin disc (disc diffusion assay) and a significant increase in the MIC of clindamycin in the presence of erythromycin (Etest strips).
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Results and discussion |
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Etest strips may be used for the detection of MLSB resistance and showed good correlation with the conventional double disc diffusion test for the five isolates tested in this study. Although the Etest method is more expensive than the disc diffusion assay, it eliminates the need to determine the appropriate distance between the discs.
Currently, the recommended treatment regimes (penicillin and erythromycin) for diphtheria have some drawbacks in terms of tolerance and compliance and these factors may compromise the treatment and prophylaxis of the disease. The newer agents studied here have more favourable pharmacological properties, are less likely to cause adverse reactions (thus promoting compliance with therapy) and demonstrate equivalent or improved in vitro activity. The ketolides and quinolones may therefore be useful in the treatment of infection with C. diphtheriae and in the clearance of colonization and carriage of the organism. However, the cost of these newer antimicrobial agents is likely to preclude their use in regions where diphtheria remains endemic and/or epidemic. Clinical trials are necessary in order to comment on the efficacies of the agents and on their ability to eradicate colonization and infection by C. diphtheriae.
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Acknowledgments |
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Notes |
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References |
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Received 14 February 2000; returned 26 May 2000; revised 18 July 2000; accepted 25 September 2000