Journal of Antimicrobial Chemotherapy (2001) 47, 27-31
© 2001 The British Society for Antimicrobial Chemotherapy
In vitro activity of ketolides HMR 3004 and HMR 3647 and seven other antimicrobial agents against Corynebacterium diphtheriae
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
The in vitro activities of two ketolides, HMR 3004 and HMR 3647 (telithromycin), and the comparator agents erythromycin A, azithromycin, clarithromycin, roxithromycin, levofloxacin, ofloxacin and penicillin G were determined by an agar dilution method against 410 isolates of Corynebacterium diphtheriae. Test isolates originated from diverse geographical locations, including the former USSR, where epidemic diphtheria has re-emerged during the 1990s. All isolates tested were susceptible to penicillin G, ofloxacin and levofloxacin. The two ketolides and four macrolides were highly active against 405 of the 410 isolates. HMR 3004 was the most active of the drugs, followed by HMR 3647, clarithromycin, erythromycin A, roxithromycin and azithromycin. Five isolates showed reduced susceptibility to all macrolides and ketolides tested; three were non-toxigenic isolates from Australia and the remaining two were from cases of diphtheria in Vietnam. Inducible (MLSB) resistance was detected in the isolates from Vietnam, but not in the isolates originating from Australia. Significant antimicrobial resistance remains rare amongst C. diphtheriae; nevertheless, new ketolide antimicrobials may have a role to play in the treatment and control of this re-emergent pathogen.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Diphtheria is an acute infectious disease caused by toxigenic Corynebacterium diphtheriae, manifested by local infection of the upper respiratory tract and occasionally the skin, together with toxic systemic effects, particularly affecting the heart, kidneys and peripheral nerves. The introduction of routine immunization with diphtheria toxoid in the 1940s greatly reduced the incidence of diphtheria and led to its virtual elimination in many countries of the developed world. In the early 1990s however, diphtheria re-emerged in countries of the former Soviet Union and over 170000 cases and 4000 deaths were reported to the European Region of the WHO between 1990 and 1998.1 In addition, diphtheria has remained endemic in many other areas of the world, including parts of southeast Asia, Africa and South America. Although immunization is the most important long-term factor for the prevention of diphtheria, antibiotics play a significant role in the treatment and control of the disease. Antibiotics are used to prevent dissemination and toxin production in infected patients, to eradicate the organism in asymptomatic carriers and to prevent infection of contacts.2 C. diphtheriae has been shown to be susceptible to a wide range of antibiotics3 and benzylpenicillin and erythromycin remain the recommended drugs for the antimicrobial treatment and control of diphtheria.4 No resistance to penicillin has been documented;3,5,6 however, inducible resistance to erythromycin has been reported in the USA7,8 and Vietnam.9
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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Isolates
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
.
|
Antimicrobial agents
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 104–105 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 18–20 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).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The results of the susceptibility testing of 410 isolates of C. diphtheriae against nine antimicrobial agents are shown in Table II
. The susceptibilities of the control strains tested were within reference ranges. Variations in biotype and toxigenicity of the isolates had no effect on the susceptibility of the strains to any of the antimicrobial agents tested. Levofloxacin, ofloxacin and penicillin G were active against all isolates (MIC90 of 0.215, 0.43 and 0.43 mg/L, respectively). The ketolides and macrolides were highly active against the majority of isolates tested (405 out of 410, 99%). HMR 3004 was the most active (MIC90 0.004 mg/L) and HMR 3647 was active at the same concentrations as HMR 3004 or within one to two doubling dilutions. Of the four macrolides tested, clarithromycin was the most active (MIC90 0.008 mg/L), followed by erythromycin A (MIC90 0.026 mg/L), roxithromycin (MIC90 0.03 mg/L) and azithromycin (MIC90 0.058 mg/L).
|
Clinically relevant breakpoints for C. diphtheriae have not been defined; however, five isolates showed reduced susceptibilities to the two ketolides and the four macrolides (Table III
). Two of the five isolates were from Vietnam from cases of diphtheria and the remaining three were non-toxigenic strains isolated in Australia. The isolates from Australia showed a lower level of resistance to erythromycin A (agar dilution MIC of 2 mg/L and Etest MIC of 3 mg/L) and the other macrolides (Table III). The MICs of the macrolides for these isolates were six two-fold dilutions higher than the relevant MIC90s. In comparison, the MICs of the two ketolides were only two two-fold dilutions higher than the relevant MIC90s and inducible resistance was not detected in these isolates using either the double disc diffusion assay or the Etest method. The isolates from Vietnam showed a higher level of resistance to the erythromycin A (agar dilution MIC 4 mg/L and Etest MIC 128–192 mg/L) and the other macrolides than those from Australia (Table III). The MICs of the macrolides for these isolates were at least 12 two-fold dilutions higher than the respective MIC90s, whereas those of the ketolides were only four or five two-fold dilutions higher than the relevant MIC90s. Inducible MLSB resistance was detected in both these isolates using the double disc diffusion assay and the Etest method. These findings suggest that the mechanism of resistance varies between the two geographical locations. Ribotyping has proven useful in the epidemiological investigation of C. diphtheriae and was performed on these isolates, as described previously.14 The three isolates from Australia all produced a single, identical ribotype pattern (D9), which was distinct from the single ribotype pattern (D61) produced by both isolates from Vietnam (A. De Zoysa, personal communication).
|
Current recommendations for the antimicrobial treatment of diphtheria require parenteral administration of procaine benzylpenicillin or erythromycin A, followed by oral therapy.4 No resistance to penicillin has been reported,3,5,6 and this finding was confirmed in this study. Five of 410 isolates showed reduced sensitivity to erythromycin A and the other macrolides tested. Erythromycin-resistant C. diphtheriae have been documented previously in Vietnam,9 France15 and the USA7 and resistance in the latter was found to be plasmid mediated.8 In the study reported from Vietnam, 27% (4/15) of isolates were reported to be resistant to erythromycin (MIC
64 mg/L) and benzylpenicillin is recommended as the first-line antimicrobial treatment for diphtheria in this country.9 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.
|
Acknowledgments |
|---|
We thank Dr A. Bryskier, Hoechst-Marion-Roussel, for advice and critical review of the manuscript. We also thank all the persons who kindly provided the strains examined in this study, in particular Dr C. Parry, Wellcome Trust Clinical Research Unit, Centre for Tropical Diseases, Cho Quan Hospital, Ho Chi Minh City, Vietnam and Professor G. L. Gilbert, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Australia. This study was supported by a grant from Hoechst-Marion-Roussel, Romainville, France.
|
Notes |
|---|
* Correspondence address. WHO Collaborating Centre for Diphtheria and Streptococcal Infections, Respiratory and Systemic Infection Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK. Tel: +44-20-8200-4400; Fax: +44-20-8205-6528; E-mail: kengler{at}phls.nhs.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
1 . WHO, Department of Vaccines and Other Biologicals. (1999). WHO Vaccine Preventable Diseases Monitoring System: 1999 Global Summary. WHO/V&B/99.17. World Health Organization, Geneva.
2 . Farizo, K. M., Strebel, P. M., Chen, R. T., Kimbler, A., Cleary, T. J. & Cochi, A. L. (1993). Fatal respiratory disease due to Corynebacterium diphtheriae: case report and review of guidelines for management, investigation and control. Clinical Infectious Diseases 16, 59–68[Web of Science][Medline]
3
.
Maple, P. A., Efstratiou, A., Tseneva, G., Rikushin, Y., Deshevoi, S., Jahola, M. et al. (1994). The in-vitro susceptibilities of toxigenic strains of Corynebacterium diphtheriae isolated in North-western Russia and surrounding areas to ten antibiotics. Journal of Antimicrobial Chemotherapy 34, 1037–40.
4 . Begg, N. (1994). Manual for the Management and Control of Diphtheria in the European Region. WHO Regional Office for Europe, Copenhagen. ICP/EPI 038(B).
5 . Gordon, R. C., Yow, M. D., Clark, D. J. & Stephenson, W. B. (1971). In vitro susceptbility of Corynebacterium diphtheriae to thirteen antibiotics. Applied Microbiology 21, 548–9.[Web of Science][Medline]
6
.
Zamiri, I. & McEntegart, M. G. (1972). The sensitivity of diphtheria bacilli to eight antibiotics. Journal of Clinical Pathology 25, 716–7.
7 . Jellard, C. H. and Lipinski, A. E. (1973). Corynebacterium diphtheriae resistant to erythromycin and lincomycin. Lancet i, 156.
8
.
Coyle, M. B. Minshew, B. H., Bland, J. A. & Hsu, P. C. (1979) Erythromycin and clindamycin resistance in C. diphtheriae from skin lesions. Antimicrobial Agents and Chemotherapy 16, 525–7.
9 . Kneen, R., Pham, N. G., Solomon, T., Tran, T. M., Nguyen, T. T., Tran, B. L. et al. (1998). Penicillin vs. erythromycin in the treatment of diphtheria. Clinical Infectious Diseases 27, 845–50.[Web of Science][Medline]
10 . Bryskier A., Agouridas, C. & Chantot, J. F. (1997). Ketolides: new semi-synthetic 14-membered ring macrolides. In Expanding Indications for the New Macrolides, Azalides and Streptogramins, (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 39–47. Marcel Dekker, New York.
11
.
Malathum, K., Coque, T. M., Singh, K. V. & Murray, B. (1999). In vitro activities of two ketolides, HMR 3647 and HMR 3004 against gram-positive bacteria. Antimicrobial Agents and Chemotherapy 43, 930–6.
12 . Efstratiou, A. & Maple, P. A. C. (1994). Manual for the Laboratory Diagnosis of Diphtheria. WHO Regional Office for Europe, Copenhagen. ICP/EPI 038(C).
13
.
Hamilton-Miller, J. M. (1991). Calculating MIC50. Journal of Antimicrobial Chemotherapy 27, 863–4.
14 . De Zoysa, A., Efstratiou, A., George, R. C., Jahkola, M., Vuopio-Varkila, J., Deshevoi, S. et al. (1995). Molecular epidemiology of Corynebacterium diphtheriae from north-western Russia and surrounding countries studied by using ribotyping and pulsed-field gel electrophoresis. Journal of Clinical Microbiology 33, 1080–3.[Abstract]
15
.
Patey, O., Bimet, F., Emond, J. P., Estrangin, E., Riegel, P., Halioua, B. et al. (1995). Antibiotic susceptibilities of 38 nontoxigenic strains of Corynebacterium diphtheriae. Journal of Antimicrobial Chemotherapy 36, 1108–10.
Received 14 February 2000; returned 26 May 2000; revised 18 July 2000; accepted 25 September 2000
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Ackermann and A. C. Rodloff Drugs of the 21st century: telithromycin (HMR 3647)--the first ketolide J. Antimicrob. Chemother., March 1, 2003; 51(3): 497 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. von Hunolstein, F. Scopetti, A. Efstratiou, and K. Engler Penicillin tolerance amongst non-toxigenic Corynebacterium diphtheriae isolated from cases of pharyngitis J. Antimicrob. Chemother., July 1, 2002; 50(1): 125 - 128. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||