The in vitro activity of ABT773, a new ketolide antimicrobial agent

J. M. Andrews, T. M. A. Weller*, J. P. Ashby, R. M. Walker and R. Wise

Department of Medical Microbiology, City Hospital NHS Trust, Dudley Road, Birmingham B18 7QH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in vitro activity of ABT773, a ketolide antimicrobial agent, was investigated and compared with those of seven other antibiotics. Type strains and 733 Gram-positive, Gram-negative and anaerobic isolates of clinical origin and four Chlamydia isolates were used. The activity of ABT773 was very similar to that of telithromycin, the other ketolide tested. The MIC90 was <= 0.5 mg/L for all bacteria examined except methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae and Bacteroides spp. The antichlamydial activity of ABT773 was greater than that of telithromycin, erythromycin and ciprofloxacin. Neither an increase in the size of the inoculm nor the addition of human serum had any marked affect on the in vitro activity of ABT773.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Ketolide antibiotics are a group of new semi-synthetic agents, derived from the macrolide class, designed to combat the rise in resistance amongst respiratory pathogens. Telithromycin (formerly HMR 3647), the first to be developed, has a broad antibacterial spectrum, including streptococci resistant to erythromycin A by both the efflux mechanism and by the inducible MLSB mechanism.1,2 These properties are conferred by the characteristic 3-keto group on the erythonylide A ring in contrast to the usual {alpha}-l-cladinose moiety. Another ketolide, ABT773, has a cyclic carbamate group at the 11,12-position in addition to the 3-keto group.3 Studies in vitro have indicated that ABT773 has activity against Streptococcus pneumoniae, Haemophilus influenzae, group A streptococci, Moraxella catarrhalis, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella pneumophila,3 Helicobacter pylori4 and Toxoplasma gondii.5 In these investigations we compared the in vitro activity of ABT773 with those of telithromycin and six other antimicrobial agents. They were tested against a range of Gram-positive, Gram-negative and anaerobic bacteria and Chlamydia spp.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial agents

The following agents were used: ABT773 and clarithromycin (Abbott Laboratories, Abbott Park, IL, USA); telithromycin (Aventis, Romaineville, France); erythromycin (Sigma–Aldrich Company Ltd, Poole, UK); amoxycillin and clavulanic acid (SmithKline Beecham, Worthing, UK); ciprofloxacin and moxifloxacin (Bayer AG, Wuppertal, Germany). All agents were stored and prepared in accordance with the manufacturer's instructions.

Susceptibility testing

The MICs for appropriate type strains and 733 clinical isolates, collected over the previous 3 years from many centres, were determined using a standard agar plate dilution method.6 A final inoculum of 104 cfu/spot was employed for all bacteria using a multipoint inoculator (Mast, Bootle, UK) on the following media: Iso-sensitest agar (ISTA) (Oxoid, Basingstoke, UK) for staphylococci and enterococci; ISTA supplemented with 5% defibrinated horse blood (DHB) (TCS Microbiology, Buckingham, UK) for streptococci and M. catarrhalis; ISTA, 5% DHB and 20 mg/L ß-nicotinamide adenine dinucleotide (NAD) (Sigma) for Neisseria spp. and H. influenzae; Wilkins–Chalgren agar (Oxoid) with 5% DHB for anaerobic bacteria. Ten isolates each of M. catarrhalis, S. aureus and Enterococcus faecalis and nine isolates of Staphylococcus epidermidis were also tested at an inoculum of 106 cfu/spot. The plates were incubated at 35–37°C for 18–24 h in air for the majority of organisms, supplemented with 4–6% CO2 for S. pneumoniae, H. influenzae and Neisseria spp., or in an anaerobic cabinet (Don Whitley Scientific Ltd, Skipton, UK). H. influenzae was incubated in both air and 4–6% CO2. In all tests the MIC was defined as the lowest concentration of antimicrobial to inhibit bacterial growth. Amoxycillin and clavulanic acid were combined in a ratio of 2:1 and the results were reported in terms of the amoxycillin MIC.

Chlamydial susceptibility testing

The in vitro activity of ABT-773, telithromycin, erythromycin and ciprofloxacin was tested against one reference strain of Chlamydia pneumoniae and three clinical isolates of Chlamydia trachomatis. The technique of Webberley et al.7 was employed. McCoy cell coverslip cultures were infected with c. 1000 inclusion-forming units of chlamydia then exposed to doubling dilutions of the above antimicrobials. Following incubation at 35–37°C in 4–6% CO2 for 48 h, the coverslips were stained using a fluorescein-labelled immuno-antibody kit (Imagen Chlamydia Kit, Organon Teknika Ltd, Cambridge, UK). The MIC was defined as the lowest concentration of antimicrobial to inhibit the development of inclusions. The minimum lethal concentration (MLC) was taken as the lowest concentration of antimicrobial to inhibit further inclusion development after incubation in drug-free media for a further 48 h.

Effect of human serum

The effects of human serum on the MIC and minimum bactericidal concentration (MBC) of ABT773 and telithromycin were determined for two isolates each of group A ß-haemolytic streptococci, S. pneumoniae, methicillin-sensitive S. aureus (MSSA), M. catarrhalis, E. faecalis and Enterococcus faecium. A micro-dilution method was employed using Iso-sensitest broth (Oxoid) containing 0%, 20% or 70% human serum (TCS Microbiology) and supplemented with 5% laked horse blood (Oxoid) in a final volume of 200 µL. Doubling dilutions of the antimicrobials were inoculated with 105 cfu/mL of the test bacteria and incubated at 35–37°C for 18–24 h in the appropriate atmosphere. The MIC was defined as above. The MBC was defined as the lowest concentration of antimicrobial to prevent growth when broth from each well containing no visible growth was subcultured on to appropriate antibiotic-free media and examined for growth after a further 18–24 h incubation.


    Results and discussion
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 Materials and methods
 Results and discussion
 References
 
The in vitro antimicrobial activities of all agents tested are shown in Table IGo along with the breakpoint, where available. The MICs of the reference strains were all within the acceptable range, no more than one dilutional step away from the expected value (data not shown).


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Table I. The comparative in vitro antimicrobial activity of ABT773
 
The ketolides were developed primarily to be used against respiratory pathogens resistant to other agents. In this respect these data are very promising. Both ketolides were highly active against S. pneumoniae, including isolates resistant to macrolides. In view of this dissociation, ketolides may prove to be useful oral agents for the treatment of penicillin- and erythromycin-resistant pneumococci. The five isolates of S. pneumoniae not susceptible to ABT773 were also resistant to telithromycin, macrolides and quinolones.

ABT773 and telithromycin were also tested against other respiratory pathogens. Neither ketolide was as active as the quinolones and co-amoxiclav against H. influenzae, but they compared favourably with the other antibiotics for M. catarrhalis. The MIC of ABT773 for some isolates of H. influenzae was higher when incubated in air rather than CO2, although it is not known which conditions provide the most clinically relevant result. ABT773 showed very good activity against two species of Chlamydia, although the number of isolates examined was small (Table IIGo). The ketolides were more active than erythromycin, which in turn had lower MICs than ciprofloxacin. ABT773 was two to four times more active than telithromycin against the isolates examined. This activity, and that previously reported for L. pneumophila,3 indicate that ketolides have the potential for use as a single agent in community-acquired pneumonia when the causative pathogen is undetermined.


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Table II. The comparative in vitro anti-chlamydial activity (MIC and MLC, in mg/L) of ABT773
 
ABT773 showed greater activity against MSSA, Staphylococcus saprophyticus and S. epidermidis than all other antimicrobials tested, although macrolide–ketolide cross-resistance did occur in three isolates of the latter. The MIC90 of >128 mg/L for methicillin-resistant S. aureus (MRSA) reflects the fact that nine of these isolates were resistant to both ABT773 and telithromycin, as well as to quinolones and macrolides. However, resistance to macrolides, or ciprofloxacin, did not necessarily predict a lack of sensitivity to ketolides. In contrast, a previous study found no ciprofloxacin-resistant, ketolide-sensitive MRSA.8 This inconsistency is probably explained by a difference in the strain of endemic MRSA in the two testing centres. A knowledge of the local susceptibility pattern will be necessary if ketolides are to be used as empirical therapy for infections thought to be caused by MRSA.

The activity of ABT773 against group A streptococci, group B streptococci and ‘Streptococcus milleri' was comparable to that of telithromycin, the macrolides and the ß-lactams but greater than that of the quinolones. Amoxycillin, co-amoxiclav and telithromycin all proved to have lower MIC90s than ABT773 against E. faecalis, but only the last of these was as active against E. faecium. Against Neisseria gonorrhoeae and Neisseria meningitidis, the ketolides and the quinolones were more active than the other agents.

Moxifloxacin was the most active agent when Bacteroides spp. were examined. ABT773 had similar activity to co-amoxiclav and clarithromycin in most cases. Against both Clostridium spp. and Peptostreptococcus spp., ABT773 was the most active compound tested with an activity four times that of telithromycin.

Results were largely unaffected by changes in the inoculum and by the presence of human serum. When an inoculum of 106 cfu/spot was used the only isolates affected were two of 10 E. faecalis where the MIC increased eight-fold. There was an eight-fold rise in the MBC of ABT773 for one isolate of E. faecalis with 70% human serum and for one isolate of S. pneumoniae at both serum concentrations. The MIC of ABT773 for one isolate of E. faecium also rose eight-fold in the presence of 20% human serum. The MIC of telithromycin did not change in this study, but previous work has shown that it may vary for some Gram-positive cocci in the presence of human serum.2 It is not known whether this finding will have any significant effect on the use of ketolides in vivo.

In summary, the results presented here are consistent with those collected in earlier susceptibility studies in vitro.3,9 The activity of ABT773 is very similar to that of telithromycin, although there were some differences, such as greater activity of the former against all Bacteroides spp., Clostridium spp. and Peptostreptococcus spp. ABT773, and other ketolides, are potentially useful oral antibiotics for the community. Further clinical research is needed to determine the value of ABT773 in practice and its side-effect profile.


    Acknowledgments
 
Financial support was provided by Abbott Laboratories.


    Notes
 
* Corresponding author. Tel: +44-121-507-5742; Fax: +44-121-551-7763; E-mail: T.M.A.Weller{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Bryskier, A. (1998). Novelties in the field of anti-infectives in 1997. Clinical Infectious Diseases 27, 865–83.[ISI][Medline]

2 . Boswell, F. J., Andrews, J. M., Ashby, J. P., Fogarty, C., Brenwald, N. P. & Wise, R. (1998). The in vitro activity of HMR 3647, a new ketolide antimicrobial agent. Journal of Antimicrobial Chemotherapy 42, 703–9.[Abstract]

3 . Ma, Z., Clark, R. F. & Or, Y. (1999). Design, synthesis, and characterization of ABT-773: a novel ketolide highly active against multidrug-resistant pathogens. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract F2133, p. 345. American Society for Microbiology, Washington, DC.

4 . Pendland, S. L., Prause, J. L., Neuhauser, M. M., Boyea, N., Hackleman, J. M. & Danziger, L. H. (1999). In vitro activity of ABT-773, a new ketolide antibiotic, alone and in combination with metronidazole, amoxicillin, or tetracycline against Helicobacter pylori. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract F2145, p. 349. American Society for Microbiology, Washington, DC.

5 . Khan, A. A., Araujo, F. G., Craft, J. C. & Remington, J. S. (1999). The ketolide ABT-773 is active against Toxoplasma gondii. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract F2147, p. 349. American Society for Microbiology, Washington, DC.

6 . Working Party of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

7 . Webberly, J. M., Matthews, R. S., Andrews, J. M. & Wise, R. (1987). Commercially available fluorescein-conjugated monoclonal antibody for determining the in vitro activity of antimicrobial agents against Chlamydia trachomatis. European Journal of Microbiology and Infectious Diseases 6, 587–9.

8 . Barry, A. L., Fuchs, P. C. & Brown, S. D. (1999). Comparative in vitro antimicrobial activity of ABT-773 and tentative disk test interpretive criteria. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract F2144, p. 348. American Society for Microbiology, Washington, DC.

9 . Bui, M. H., Almer, L. S., Hensey, D. M., Ma, Z., Or, Y., Nilius, A. M. et al. (1999). Antibacterial effects of ABT-773 against respiratory tract pathogens. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract F2138, p. 347. American Society for Microbiology, Washington, DC.

Received 26 May 2000; returned 2 August 2000; revised 21 August 2000; accepted 18 September 2000