Abstract
The in vitro activities of ABT-773 were evaluated against 324 strains of gram-positive bacteria, including multidrug-resistant Staphylococcus spp. and Enterococcus spp. ABT-773 had lower MIC ranges, MICs at which 50% of isolates are inhibited (MIC50s), and MIC90s than erythromycin or clindamycin for almost all isolates tested. The MICs of ABT-773 were also lower than those of quinupristin-dalfopristin (Q-D) for methicillin-susceptible Staphylococcus aureus, Rhodococcus spp., and Streptococcus spp., while the MICs of Q-D were lower than those of ABT-773 for methicillin-resistant S. aureus and Enterococcus faecium, including vancomycin-resistant isolates.
The emergence of resistance in gram-positive bacteria and other bacteria to currently available antimicrobials such as vancomycin (VAN), macrolides, beta-lactam antibiotics, and quinolones (5, 9, 10) and the reported isolation of VAN-intermediate (VANi) Staphylococcus aureus strains (3, 6) have resulted in a clear need to discover new antibiotics. Ketolides have been reported to be active against resistant gram-positive bacteria including erythromycin-resistant (ERYr) and penicillin-resistant Streptococcus pneumoniae strains (1, 4). ABT-773 is a new ketolide which has been reported to have excellent activity against S. pneumoniae, staphylococci, and other respiratory pathogens (2; M. H. Bui, L. S. Almer, D. M. Hensey, Z. Ma, Y. S. Or, A. M. Nilius, and R. K. Flamm, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2138, 1999; D. Shortridge, N. C. Ramer, J. Beyer, Z. Ma, Y. S. Or, and R. K. Flamm, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2136, 1999). However, its activity against multidrug-resistant enterococci has not been well represented in those studies. In the present study, we determined the in vitro activities of ABT-773 by the agar dilution and the broth microdilution methods and compared these to the activities of ERY, clindamycin (CLI), and quinupristin-dalfopristin (Q-D) against 324 gram-positive organisms (including 156 enterococci) that display various degrees of resistance to antimicrobial agents.
(This work was presented in part at the 5th International Conference on the Macrolides, Azalides, Streptogramins, Ketolides and Oxazolidinones, Seville, Spain, 2000, abstr. 2.14.)
The organisms tested in the present study included isolates of Enterococcus spp. (n = 156; β-lactamase producers, high-level gentamicin and VAN resistant), Staphylococcus spp. (n = 34; methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), Streptococcus spp. (n = 102), Rhodococcus spp. (n = 14), Pediococcus spp. (n = 6), Leuconostoc spp. (n = 7), Lactobacillus spp. (n = 3), and Corynebacterium spp. (n = 2) which were collected between 1980 and 1999 from the United States, Argentina, Belgium, Chile, and Thailand. These isolates were identified to the species level by defined biochemical tests, with commercially available Analytab Products strips (bioMérieux Vitek, Inc. Hazelwood, Mo.), and/or by using species-specific DNA gene probes (15). The ketolide compound ABT-773 was obtained from Abbott Laboratories, North Chicago, Ill.; ERY and CLI were obtained from Sigma, St. Louis, Mo.; and Q-D was obtained from Rhône-Poulene Rorer, Vitry sur Seine, France. The MIC of each antimicrobial agent was determined by following the National Committee for Clinical Laboratory Standards (NCCLS) guidelines for antimicrobial susceptibility testing by the agar dilution and broth microdilution methods (11). Mueller-Hinton medium (Becton Dickinson and Company, Cockeysville, Md.) with serial twofold dilutions of antibiotics was used. Incubation was carried out at 35 ± 1°C. The standard reference strains used were Enterococcus faecalis ATCC 29212, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and Escherichia coli ATCC 25922. The susceptibilities to ERY, CLI, and Q-D were interpreted according to the recommendations of NCCLS (12). The susceptibility breakpoints of ERY (12) for Streptococcus spp. including S. pneumoniae are ≤0.25 μg/ml for susceptibility (ERYs), 0.5 μg/ml for intermediate (ERYi), and ≥1 μg/ml for resistance (ERYr), while for Enterococcus spp. and Staphylococcus spp., these breakpoints are ≤0.5 μg/ml for ERYs, 1 to 4 μg/ml for ERYi, and ≥8 μg/ml for ERYr (12). The presence of ERY resistance genes erm(B) and mef(A/E) (13, 16) was determined by hybridization of DNA probes with colony lysates under high-stringency conditions (15).
The results of susceptibility testing of 324 organisms with ABT-773 and the other antibiotics tested are summarized in Tables 1 and 2. Among the 19 MSSA isolates, 2 isolates were ERYr (MIC of ERY, >512 μg/ml; MIC of CLI, 0.125 μg/ml) and were classified by the double-disk diffusion method (8) as having an inducible macrolide-lincosamide-streptogramin B (iMLS) phenotype. Both of these isolates with the iMLS phenotype were inhibited by ABT-773 at 0.031 to 0.125 μg/ml. For the remaining 17 MSSA isolates, ABT-773 showed 8 times lower MICs at which 50% of isolates are inhibited (MIC50s) and MIC90s than ERY and 16 to 32 times lower MIC50s and MIC90s than CLI and Q-D. All MRSA isolates used in the present study were resistant to ERY and CLI (ERY and CLI MICs, >512 and >256 μg/ml, respectively), and ABT-773 at concentrations ≤64 μg/ml was unable to inhibit these strains.
TABLE 1.
MIC ranges, MIC50s, and MIC90s of ABT-773 compared with those of other antibiotics, determined by agar dilution method
| Bacterial strains (no. isolates tested) and drug | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| S. aureus | |||
| MSSA, total (19) | |||
| ABT-773 | 0.015–0.125 | 0.031 | 0.031 |
| ERY | 0.25–>512 | 0.25 | >512 |
| CLI | 0.125–>256 | 0.125 | 1 |
| Q-D | 0.25–1 | 0.5 | 1 |
| MSSA, ERYs (17) | |||
| ABT-773 | 0.015–0.031 | 0.031 | 0.03 |
| ERY | 0.25 | 0.25 | 0.25 |
| CLI | 0.062–1 | 0.125 | 1 |
| Q-D | 0.25–1 | 0.5 | 1 |
| MSSA, ERYr (2) | |||
| ABT-773 | 0.031, 0.125 | ||
| ERY | >512 | ||
| CLI | 0.12 | ||
| Q-D | 0.25, 0.5 | ||
| MRSA, all ERYr (15) | |||
| ABT-773 | >64 | >64 | >64 |
| ERY | >512 | >512 | >512 |
| CLI | >256 | >256 | >256 |
| Q-D | 0.5–1 | 1 | 1 |
| E. faecalis | |||
| Total (92) | |||
| ABT-773 | <0.007–>64 | 0.25 | 64 |
| ERY | 0.062–>512 | >512 | >512 |
| CLI | 16–>256 | >256 | >256 |
| ERYs (13) | |||
| ABT-773 | ≤0.007–0.015 | ≤0.007 | 0.015 |
| ERY | 0.062–0.5 | 0.5 | 0.5 |
| CLI | 16–32 | 32 | 32 |
| ERYi or ERYr (79) | |||
| ABT-773 | ≤0.007–>64 | 1 | 64 |
| ERY | 1–>512 | >512 | >512 |
| CLI | 32–>256 | >256 | >256 |
| VANs (80) | |||
| ABT-773 | ≤0.007–>64 | 0.25 | 16 |
| ERY | 0.062–>512 | >512 | >512 |
| CLI | 16–>256 | >256 | >256 |
| VANs, ERYs (13) | |||
| ABT-773 | ≤0.007–0.015 | ≤0.007 | 0.015 |
| ERY | 0.062–0.5 | 0.5 | 0.5 |
| CLI | 16–32 | 32 | 32 |
| VANs, ERYi or ERYr (67) | |||
| ABT-773 | ≤0.007–>64 | 0.5 | 32 |
| ERY | 1–>512 | >512 | >512 |
| CLI | 16–>256 | >256 | >256 |
| VANr (12) | |||
| ABT-773 | 0.015–>64 | 32 | >64 |
| ERY | 2–>512 | >512 | >512 |
| CLI | 16–>256 | >256 | >256 |
| E. faecium | |||
| Total (64) | |||
| ABT-773 | ≤0.007–>64 | 8 | 32 |
| ERY | 2–>512 | >512 | >512 |
| CLI | 0.062–>256 | >256 | >256 |
| Q-D | 0.5–8 | 1 | 4 |
| VANs, ERYi or ERYr (22) | |||
| ABT-773 | 0.015–>64 | 4 | 64 |
| ERY | 2–>512 | >512 | >512 |
| CLI | 0.125–>256 | >256 | >256 |
| Q-D | 0.5–16 | 1 | 8 |
| VANr, ERYi or ERYr (42) | |||
| ABT-773 | ≤0.007–64 | 16 | 32 |
| ERY | 2–>512 | >512 | >512 |
| CLI | 0.062–>256 | >256 | >256 |
| Q-D | 0.5–32 | 1 | 4 |
| Rhodococcus spp. (14) | |||
| ABT-773 | 0.015–1 | 1 | 1 |
| ERY | 0.062–16 | 2 | 8 |
| CLI | 0.25–>256 | 128 | >256 |
| Q-D | 2–128 | 64 | 128 |
| Group A Streptococcia (29) | |||
| ABT-773 | ≤0.007–0.062 | 0.031 | 0.031 |
| ERY | 0.031–16 | 0.125 | 0.125 |
| CLI | 0.062–0.25 | 0.062 | 0.125 |
| Q-D | 0.125–2 | 0.5 | 1 |
| Group B streptococci (10) | |||
| ABT-773 | 0.015–0.031 | 0.031 | 0.031 |
| ERY | 0.125 | 0.125 | 0.125 |
| CLI | 0.125 | 0.125 | 0.125 |
| Q-D | 1–4 | 2 | 4 |
| S. pneumoniae | |||
| Total (42) | |||
| ABT-773 | ≤0.007–2 | 0.031 | 0.25 |
| ERY | 0.015–>128 | 0.125 | >128 |
| CLI | 0.015–>16 | 0.125 | >16 |
| Q-D | 0.25–8 | 0.5 | 2 |
| ERYs (17) | |||
| ABT-773 | ≤0.007–0.031 | ≤0.007 | 0.031 |
| ERY | 0.015–0.125 | 0.062 | 0.125 |
| CLI | 0.015–0.125 | 0.125 | 0.125 |
| Q-D | 0.25–8 | 0.5 | 2 |
| ERYr (25) | |||
| ABT-773 | ≤0.007–2 | 0.031 | 0.25 |
| ERY | 1–>128 | 64 | >128 |
| CLI | 0.015–>16 | 0.25 | >16 |
| Q-D | 0.25–2 | 0.5 | 2 |
| Alpha-hemolytic streptococci | |||
| Total (21) | |||
| ABT-773 | ≤0.007–0.062 | 0.015 | 0.062 |
| ERY | 0.031–>512 | 0.5 | 8 |
| CLI | ≤0.007–256 | 0.062 | 0.25 |
| Q-D | 0.25–4 | 1 | 4 |
| ERYs (13) | |||
| ABT-773 | ≤0.007–0.062 | ≤0.007 | 0.031 |
| ERY | 0.031–0.5 | 0.125 | 0.5 |
| CLI | ≤0.007–0.25 | 0.031 | 0.125 |
| Q-D | 0.25–4 | 0.5 | 2 |
| ERYr (8) | |||
| ABT-773 | ≤0.007–0.062 | ||
| ERY | 1–>512 | ||
| CLI | 0.031–256 | ||
| Q-D | 1–4 | ||
One isolate was ERYr and two isolates were ERYi.
TABLE 2.
Susceptibilities of the HLER, erm(B)-positive E. faecalis and E. faecium isolates tested
| Organism and no. of isolates | MIC (μg/ml)
|
||
|---|---|---|---|
| ABT-773 | ERY | CLI | |
| E. faecalis(n = 60) | |||
| 20 | 0.031–0.5 | >512 | 32–>256a |
| 16 | 1–4 | >512 | >256 |
| 13 | 8–32 | >512 | >256 |
| 11 | ≥64 | >512 | >256 |
| E. faecium(n = 54) | |||
| 4 | 0.25 | >512 | 4–>256b |
| 10 | 1–4 | >512 | ≥256 |
| 37 | 8–32 | 256–>512 | 0.125–>256c |
| 3 | >64 | >512 | >256 |
The MICs were <256 μg/ml for only two isolates (MICs for both isolates, 32 μg/ml).
The MIC was <256 μg/ml for only one isolate (MIC, 4 μg/ml).
The MIC was <256 μg/ml for only one isolate (MIC, 0.125 μg/ml).
Of 92 E. faecalis isolates tested, 13 were VAN susceptible (VANs) and ERYs, 67 were VANs and ERYi or ERYr, and 12 were VANr and ERYi or ERYr. In comparison to ERY MIC50s and MIC90s for E. faecalis isolates, ABT-773 MIC50s and MIC90s were 64 and 32 times lower for VANs and ERYs isolates, respectively; 1,024 and 16 times lower for VANs and ERYi or ERYr isolates, respectively, and >16 and >16 times lower for VANr and ERYi or ERYr isolates, respectively. All ERYs and 13 of 79 ERYi or ERYr E. faecalis isolates were inhibited by ABT-773 at ≤0.062 μg/ml. For all E. faecalis isolates the ABT-773 MIC was >0.062 μg/ml and the ERY MIC was >512 μg/ml. A total of 64 Enterococcus faecium isolates (22 VANs isolates and 42 VANr isolates, all of which were ERYi or ERYr) were tested, and the MIC50s and MIC90s of ABT-773 were found to be 8 to 128 times and 16 to 32 times lower than the ERY MIC50s and MIC90s for both the VANs and the VANr and ERYi or ERYr groups, respectively. All E. faecalis isolates and 54 E. faecium isolates which had high-level ERY resistance (HLER) (ERY MIC, 256 to >512 μg/ml) tested positive for erm(B) (13). Of the 60 HLER isolates of E. faecalis, 20 were inhibited by ABT-773 at 0.015 to 0.5 μg/ml, 16 were inhibited by ABT-773 at 1 to 4 μg/ml, 13 were inhibited by ABT-773 at 8 to 32 μg/ml, and 11 were inhibited by ABT-773 at ≥64 μg/ml (Table 2). Similarly, of 54 HLER isolates of E. faecium, 4 were inhibited by ABT-773 at 0.25 μg/ml, 10 were inhibited by ABT-773 at 1 to 4 μg/ml, 37 were inhibited by ABT-773 at 8 to 32 μg/ml, and 3 were inhibited by ABT-773 at ≥64 μg/ml (Table 2). For all HLER E. faecalis isolates tested, CLI MICs were 32 to ≥256 μg/ml, while for 52 of 54 HLER E. faecium isolates CLI MICs were ≥256 μg/ml.
Of 29 group A streptococci and 10 group B streptococci tested, only one isolate, a group A streptococcus was ERYr (MIC, 16 μg/ml); the MIC of ABT-773 for that isolate was 0.062 μg/ml. ABT-773 was found to be more active against both group A and group B streptococcal strains, with MIC50s and MIC90s being four or more times lower than those of ERY, CLI, and Q-D. ABT-773 had MIC90s 128, 4, and 64 times lower than those of ERY, CLI, and Q-D, respectively, for 21 alpha-hemolytic streptococci, which included 13 ERYs isolates and 8 ERYr isolates.
All 17 ERYs S. pneumoniae isolates were inhibited by ≤0.031 μg of ABT-773 per ml, while 90% of these isolates were inhibited by 0.125 μg of ERY and CLI per ml and 2 μg of Q-D per ml. Of 25 ERYr S. pneumoniae isolates tested, 5 isolates contained the mef(A) gene (13) and 18 contained the erm(B) gene (13). These 18 isolates with the erm(B) gene consisted of 9 isolates for which ERY MICs were 1 to >128 μg/ml and CLI MICs were 0.031 to 0.25 μg/ml and 9 isolates for which ERY MICs were <128 μg/ml and CLI MICs were >16 μg/ml. Twenty-four of the 25 ERYr S. pneumoniae isolates tested were inhibited by ABT-773 at ≤0.25 μg/ml, while the MIC90s of ERY, CLI, and Q-D for these isolates were >128, >16, and 2 μg/ml, respectively; the remaining one erm(B) isolate was inhibited by ABT-773 at 2 μg/ml, which was severalfold higher than the MICs for other erm(B)-containing S. pneumoniae isolates.
Of 14 Rhodococcus spp. tested, 4 isolates were inhibited by 0.06 to 0.5 μg of ERY per ml and 10 were inhibited by 2 to 16 μg of ERY per ml; the MIC90s of ABT-773 were 8 to 256 times lower than the MIC90s of ERY, CLI, or Q-D for these isolates. Leuconostoc spp., Pediococcus spp., Corynebacterium spp., and Lactobacillus spp. were inhibited by ABT-773 at concentrations in the range of ≤0.007 to 0.062 μg/ml, while these isolates were inhibited by ERY at concentrations in the range of 0.062 to 16 μg/ml.
Broth microdilution MIC results for ABT-773 for all bacteria tested were either the same as or within 1 dilution of the agar dilution MICs (data not shown). The results of the present study show that ABT-773 is more potent than ERY against almost all isolates. However, the MICs of ABT-773 tended to vary with the MIC of ERY. The enterococcal isolates which were susceptible or intermediate in susceptibility to ERY were highly susceptible to ABT-773, while for isolates which were highly resistant to ERY, the MICs of ABT-773 were usually higher. The MIC50s and MIC90s of ABT-773 for ERYr isolates were 0.031 and 0.25 μg/ml, except one erm(B) isolate, suggesting a possibility that other mechanisms are respectively, for all involved in the higher MIC for this strain.
A macrolide efflux pump encoded by mef(A) and ribosomal alterations encoded by erm(B) have been described as the major mechanisms of ERY resistance in S. pneumoniae isolates in the literature (7, 14). In a study with MLS-resistant S. pneumoniae isolates, ABT-773 was shown to still have binding affinity for methylated ribosomes (J. O. Capobianco, V. Shortridge, Z. Ma, L. Phan, Y. S. Or, and P. Zhong, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2134, 1999). It has also been suggested that the effectiveness of ABT-773 in mef(A)-containing macrolide-resistant S. pneumoniae isolates is due to either the lack of drug recognition by the efflux pump or the fact that the influx rate exceeded the capacity of the pump to remove the ketolide from the cell (Capobianco et al., 39th ICAAC).
On the basis of the potent activity of ABT-773 against the Streptococcus spp. tested and the lower MICs of ABT-773 for most of the Enterococcus spp., Rhodococcus spp., and MSSA strains tested, this new ketolide might prove useful for the treatment of infections caused by gram-positive bacteria which are susceptible to this compound and merits further study.
Acknowledgments
This study was funded by a grant from Abbott Laboratories.
We thank Audrey Wanger, University of Texas Health Science Center, and Kenneth V. I. Rolston, M. D. Anderson Cancer Center, Houston, Tex., for providing some of the isolates.
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