The bactericidal activities of HMR 3004, HMR 3647 and erythromycin against Gram-positive bacilli and development of resistance

Ricardo Fernández-Roblasa, Raquel Calvoa, Jaime Estebana, André Bryskierb and Francisco Sorianoa,*

a Department of Medical Microbiology. Fundación Jiménez Díaz, Madrid, Spain b Direction des Recherches, Anti-Infectieux, Hoechst Marion Roussel, Romainville, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The bactericidal activities of two new ketolides, HMR 3004 and HMR 3647, and the potential to develop resistance to these two antibiotics were studied in Gram-positive bacilli. As judged by time-kill kinetics both ketolides were mostly bacteriostatic, being bactericidal against only highly susceptible isolates of Corynebacterium striatum (two isolates) and Corynebacterium minutissimum (one isolate). Spontaneous resistant mutants were detected in seven of 30 strains tested, mainly in Rhodococcus equi, C. minutissimum and C. striatum, with a very low frequency of mutation (10-12– 10-15).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ketolides are a new class of semisynthetic macrolide antibiotics, with a 3-keto group instead of the L-cladinose moiety in the 14-membered ring. These new antibiotics are very active against Gram-positive bacteria, including erythromycin-resistant strains. 1,2,3,4,5 A recent study demonstrated that the ketolide HMR 3647 is predominantly bacteriostatic against bacteria implicated in respiratory tract infections, and against enterococci and Bacteroides fragilis. 6 An alarming rate of antibiotic resistance in Gram-positive bacteria, including coryneform species,4 is being detected. However, no data have been published on the bactericidal activity of ketolides and in-vitro selection of antibiotic-resistant mutants in coryneform organisms.

As a part of the evaluation of these antibiotics, we studied the bactericidal activity of two new ketolides, HMR 3004 and HMR 3647, and erythromycin, against Gram-positive bacilli, as well as the potential of some of these bacteria (principally members of the Corynebacterium genus), to develop resistance to HMR 3004, HMR 3647 and erythromycin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics and MIC determinations

HMR 3004 and HMR 3647 were obtained from Hoechst Marion Roussel (Romainville, Paris, France) and erythromycin from Pierrel (Milan, Italy). MICs were determined by a standard agar dilution method 7 in Mueller-Hinton agar (Difco Laboratories, Detroit, MI, USA) supplemented with 5% defibrinated sheep blood (MHA-EB). The plates were incubated aerobically at 35°C for 24 or 48 h, as required. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as controls.

Time-kill assay

The following 25 clinical isolates were studied: Corynebacterium minutissimum (3), Corynebacterium amycolatum (2), Corynebacterium urealyticum (2), Corynebacterium pseudodiphtheriticum (2), Corynebacterium striatum (2), Corynebacterium jeikeium (1), Erysipelothrix rhusiopathiae (2),Arcanobacterium haemolyticum (2), Rhodococcus equi (2), Oerskovia spp. (2), Listeria monocytogenes (2), Listeria murray (1), Listeria ivanovii (1) and Turicella otitidis (1). Cultures were grown at 35°C on MHA-EB. Approximately 15 min before the experiment, the colonies were harvested and a cell suspension with turbidity equal to that of a 1 McFarland standard, i.e. 3 x 108 cfu/mL, was prepared in deionized water. This suspension was diluted in cation adjusted Mueller-Hinton broth (Difco) (CAMHB) to achieve a cell concentration between 105 and 106 cfu/mL.

The time-kill reaction tubes were prepared to contain 10 mL of CAMHB, 0.1 mL of the antibacterial agent (diluted in broth to a concentration 100-fold the final test concentration) and 0.1 mL of inoculum. The final drug concentration was 0.5, 1, 2 and 4 x MIC for each test isolate. For highly susceptible isolates (MICs <= 0.015 mg/L) the MIC assumed for the time- kill assay was 0.015 mg/L. An identical reaction tube containing broth and inoculum, but without antibacterial agent, was used as a control. The tubes were incubated at 35°C, and samples were removed for viable counts at 0, 8 and 24 h. A 0.1 mL sample was removed from the reaction tube at each time point, and serial dilutions were prepared in CAMHB. The diluted samples (0.1 mL) were applied to the surface of duplicate agar plates spread over the surface with a sterile glass ` hockey stick' and, after 24-48 h incubation at 35°C, the colonies were counted and the viable count was calculated.8 Bactericidal activity was defined as a reduction in log10 cfu/mL of >=3.

Determination of spontaneous mutation frequency

Cultures of 30 ketolide and erythromycin-sensitive isolates were grown for 24-48 h at 35°C on MHA-EB. For each, the growth from two or three plates was harvested with sterile cotton swabs, and a dense cell suspension was prepared in 5 mL of Mueller-Hinton broth. The viable count of this suspension was taken as the average of three determinations. This cell suspension constituted the inoculum for the study. Serial dilutions of ketolide and erythromycin were prepared in 1 mL of sterile distilled water. To each 1 mL volume, 9 mL of molten MHA-EB was added. This agar was mixed, poured into 15 x 90 mm Petri dishes, and allowed to solidify and dry at room temperature before inoculation. Duplicate plates were prepared to contain the ketolide and erythromycin at 2, 4 and 8 x MIC. A 0.1 mL volume of the suspension was inoculated and spread over the agar surface with a sterile glass `hockey stick'. Once the inoculum had been absorbed into the agar, the plates were incubated at 35°C for 48-72 h. After incubation, the number of colonies growing on the drug-supplemented plates at each concentration was determined and the spontaneous mutation frequency was calculated. 9 The following 30 clinical isolates were studied: C. pseudodiphtheriticum (5), C. urealyticum (5), C. jeikeium (5), C. striatum (4), C. minutissimum (3), C. amycolatum (3), L. monocytogenes (3) and R. equi (2).

Induction of antibiotic-resistant mutants

To determine whether repeated exposure of organisms to subinhibitory concentrations of HMR 3004, HMR 3647 and erythromycin resulted in rapid development of resistance, we used a gradient method9 with plates with a continuous antibiotic concentration ranging from below to above the MIC. Molten MHA-EB was poured into a tilted dish (24.5 x 12 cm) and left to solidify. The dish was then put upright and another amount of molten MHA-EB with an antibiotic concentration close to the MIC of the organisms to be tested was poured and left to solidify. Such plates were inoculated with streaked lines of bacterial suspensions. Inocula were prepared in Mueller-Hinton broth to the turbidity of a 0.5 McFarland standard. After 24-48 h incubation the colonies present at the greatest distance from the lowest antibiotic concentrations were seeded in other plates prepared in the same way; a total of five serial transfers was performed. The colonies present at the greatest distance from the lowest concentrations were tested in parallel with the same organism unexposed to the antibiotic to determine the MIC. The following 25 clinical isolates were studied: C. jeikeium (5), C. pseudodiphtheriticum (4), C. striatum (4), C. minutissimum (3), C. urealyticum (3), R. equi (3) and L. monocytogenes (3).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the time-kill kinetic studies after 24 h for HMR-3004 and HMR-3647 are summarized in the Table. Only rates at concentrations of MIC and 4 x MIC (judged to be representative) are shown. Data for erythromycin are not shown. The data are presented in terms of the log10 cfu/mL change (positive or negative).


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Table. Bactericidal activity of HMR 3004 and HMR 3647 against 25 representative Gram-positive bacilli, studied by the time-kill method
 
Both ketolides demonstrated bacteriostatic activity against 22 of the 25 isolates tested. One isolate of C. striatum (CB-625) was killed at 24 h by both HMR 3004 and HMR 3647 at concentrations considered to be 1 and 4 x MIC, indicating extreme susceptibility to the two antibiotics (MICs <= 0.015 mg/L). Two additional isolates (C. striatum CB-807 and C. minutissimum CB-865) were killed only by HMR 3647 (MICs of 0.06 and <= 0.015 mg/L) after 24 h exposure at 4 x MIC. Erythromycin (data not shown) also demonstrated bacteriostatic activity against 23 of 25 isolates tested; it was only bactericidal against two isolates of C. striatum after 24 h exposure at 4 x MIC.

In this study no colonies resistant to ketolides or erythromycin were detected for 23 of the 30 isolates tested, indicating a spontaneous mutation frequency of <10-15. For the seven isolates (C. striatum (2), C. minutissimum (2), C. urealyticum (1) and R. equi (2)) spontaneous mutants were detected. For Corynebacteriumspp. the ketolides and erythromycin selected strains with MICs one to eight dilutions higher than the original strains, while in R. equi the ketolides and erythromycin selected strains with MICs one to six dilutions higher than the original strains. Mutation occurred in C. striatum, C. minutissimum, C. urealyticum and R. equi at frequencies of 1.1 x 10-12 to 6 x 10-15 for HMR 3004, 2.5 x 10-13 to 9 x 10-15 for HMR 3647 and 1.5 x 10-12 to 8 x10-15 for erythromycin. On the other hand, after five passages on plates containing antibiotic gradient no development of resistance to either ketolides or erythromycin was detected.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ketolides are a new class of antibiotics with good activity against Gram-positive organisms. 1,2,3 We have studied the activity of these antibiotics against Gram-positive bacilli, mainly members of the genus Corynebacterium, and have demonstrated that they are more active than erythromycin and other 14- 15- and 16-membered macrolides. 4,5

Time-kill curves are one of the most reliable methods of determining bactericidal activity of antibiotics. Previous studies have shown the bacteriostatic activity of HMR 3647 against selected Gram-positive and Gram-negative bacteria.6 Our study confirms that HMR 3004 and HMR 3647 are bacteriostatic drugs; HMR 3004 was only bactericidal against one highly susceptible isolate of C. striatum and HMR 3647 was only bactericidal against three highly susceptible isolates (two of C. striatum and one C. minutissimum).

In the study of development of resistance to antibiotics, it is of interest to know whether it is possible to select resistant mutants under laboratory conditions and how rapidly such mutants can arise.9 Antibiotic-resistant mutants can be selected by direct plating on agar plates containing a range of antibiotic concentrations or on gradient plates. Although the selection of mutants in gradient plates may be more efficient than direct plating, 9 our results suggest that when the frequency of resistance is very low (between 10-12 and 10-15) a very high inoculum is necessary to select resistant mutants. Such high inocula could only be used in the direct plating technique.

In summary, the two ketolides studied behaved mostly as bacteriostatic antibiotics and mutants resistant to both ketolides as well as to erythromycin occurred in only a few strains of coryneform organisms and only at very low frequencies. This fact, together with the lack of induction of the MLSB resistance phenotype by the ketolides,10 may be an advantage in the use of these new antibiotics for infections caused by susceptible organisms.


    Acknowledgments
 
This study was supported by a grant from Hoechst Marion Roussel, Romainville, France.


    Notes
 
* Correspondence addresss: Department of Medical Microbiology. Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. Tel: +34-91-550-49-00; Fax: +34-91-549-47-64; E-mail: fsoriano{at}microb.net Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ednie, L. M., Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Susceptibilities of 228 penicillin- and erythromycin-susceptible and -resistant pneumococci to RU 64004, a new ketolide, compared with susceptibilities to 16 other agents. Antimicrobial Agents and Chemotherapy 41, 1033–6.[Abstract]

2 . Jamjian, C., Biedenbach, D. J. & Jones, R. N. (1997). In-vitro evaluation of a novel ketolide antimicrobial agent, RU-64004.Antimicrobial Agents and Chemotherapy 41, 454–9.[Abstract]

3 . Schülin, T., Wennersten, C. B., Moellering, R. C. & Eliopoulos, G. M. (1997). In-vitro activity of RU 64004, a new ketolide antibiotic, against Gram-positive bacteria. Antimicrobial Agents and Chemotherapy 41, 1196–202.[Abstract]

4 . Soriano, F., Fernández-Roblas, R., Calvo, R. & García-Calvo, G. (1998). In-vitro susceptibilities of aerobic and facultative non- spore-forming Gram-positive bacilli to HMR 3647 (RU 66647) and 14 other antimicrobials. Antimicrobial Agents and Chemotherapy 42, 1028–33.[Abstract/Free Full Text]

5 . Soriano, F., Fernández-Roblas, R., Calvo, R., García-Calvo, G., Pardeiro, M. & Bryskier, A. (1998). In-vitro antimicrobial activity of HMR 3004 (RU 64004) against erythromycin A-sensitive and -resistant Corynebacterium spp. isolated from clinical specimens. Journal of Antimicrobial Chemotherapy 42, 647–9.[Abstract]

6 . Boswell, F. J., Andrews, J. M. & Wise, R. (1998). Pharmacodynamic properties of HMR 3647, a novel ketolide, on respiratory pathogens, enterococci and Bacteroides fragilis demonstrated by studies of time- kill kinetics and postantibiotic effect. Journal of Antimicrobial Chemotherapy 41, 149–53.[Abstract]

7 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically— Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

8 . Knapp, C. & Moody, J. A. (1995). Tests to assess bactericidal activity. In Clinical Microbiology Procedures Handbook, Vol. 1 (Isenberg, H. D., Ed.), pp. 5.16.1–5.16.33. American Society for Microbiology, Washington, DC.

9 . Murray, B. E. & Hodel-Christian, S. L. (1991). Bacterial resistance: theoretical and practical considerations, mutations to antibiotic resistance, characterization of R plasmids, and detection of plasmid-specified genes. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 556 –98. Williams & Wilkins, Baltimore, MD.

10 . Bonnefoy, A., Girard, A. M., Agouridas, C. & Chanot, J. F. (1997). Ketolides lack inducibility properties of MLSB resistance phenotype. Journal of Antimicrobial Chemotherapy 40, 85–9.[Abstract]

Received 11 May 1998; returned 2 July 1998; revised 22 July 1998; accepted 16 September 1998