Abstract
The in vitro activities of GAR-936, the 9-t-butylglycylamido derivative of minocycline, were compared with those of doxycycline, minocycline, and tetracycline against 527 gram-positive clinical isolates. GAR-936 inhibited all strains, including those resistant to other tetracyclines, at concentrations of ≤2 μg/ml, except two strains of JK diphtheroids for which the MIC was 4 μg/ml.
Although tetracyclines remain valuable therapeutic agents for a variety of infections, resistance to this class limits their use against many important gram-positive bacterial pathogens. For example, only 33% of enterococci recovered at our hospital during 1997 to 1999 were susceptible to tetracycline. The glycylcyclines are novel tetracycline analogs that have activity against organisms resistant to older compounds of this class (5, 14). They inhibit protein synthesis on wild-type ribosomes and on TetM-protected, tetracycline-resistant ribosomes (15). These compounds also inhibit organisms with tetracycline efflux determinants (14). GAR-936, the 9-t-butylglycylamido derivative of minocycline, appears to be both better tolerated by hosts and more active against tetracycline-resistant strains than earlier glycylcyclines (14). The present study examined the in vitro activities of GAR-936 against gram-positive bacteria, including strains resistant to β-lactams, glycopeptides, and other tetracyclines.
Routine clinical isolates collected at the Beth Israel Deaconess Medical Center were included regardless of tetracycline resistance patterns. Staphylococci and most pneumococci were recovered in 1998. Strains with unusual resistance traits, including glycopeptide-resistant or β-lactamase-producing enterococci and β-lactam-resistant streptococci, had been referred to our laboratory from various sources over several years (2, 4, 16). Wyeth-Ayerst Laboratories, Pearl River, N.Y., kindly provided GAR-936. Tetracycline, minocycline, and doxycycline were purchased from Sigma Chemical Company, St. Louis, Mo.
Activities of the antimicrobials were determined by agar dilution methods on Mueller-Hinton II agar (12). Media were supplemented with 5% sheep blood for testing streptococci and corynebacteria. Inocula of approximately 104 CFU were applied to the surfaces of plates and were incubated for 16 to 20 h at 35°C in air or in 5% CO2 (for Lactobacillus spp., Leuconostoc spp., Streptococcus spp., Pediococcus spp., and diphtheroids). Pneumococci were also tested by broth microdilution (12).
The results of susceptibility studies are shown in Table 1. GAR-936 was four times less active than minocycline against oxacillin-susceptible and -resistant strains of Staphylococcus aureus, according to a comparison of MICs at which 90% of the isolates tested were inhibited (MIC90s). However, isolates intermediately susceptible or resistant to the other compounds were inhibited by GAR-936 at ≤1 μg/ml. GAR-936 inhibited two strains of vancomycin-intermediate S. aureus (resistant to tetracycline and minocycline) at concentrations of 1 and 2 μg/ml.
TABLE 1.
Comparative in vitro activity of GAR-936
| Organism(s) and relevant characteristic (no. of isolates) | Antimicrobial agent | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Enterococcus faecalis (42) | GAR-936 | 0.06–0.5 | 0.12 | 0.25 |
| Minocycline | 0.06–32 | 16 | 32 | |
| Doxycycline | 0.12–32 | 8 | 16 | |
| Tetracycline | 0.12–>128 | 32 | 128 | |
| Enterococcus faecalis, β-lactamase producing (10) | GAR-936 | 0.12–0.25 | 0.25 | 0.25 |
| Minocycline | 8–16 | 16 | 16 | |
| Doxycycline | 8–16 | 8 | 16 | |
| Tetracycline | 32–>128 | 32 | 64 | |
| Enterococcus faecalis, vancomycin resistant (VanA) (10) | GAR-936 | 0.12–1 | 0.25 | 0.5 |
| Minocycline | 0.12–32 | 0.12 | 16 | |
| Doxycycline | 0.25–16 | 0.25 | 16 | |
| Tetracycline | 0.5–>128 | 0.5 | 64 | |
| Enterococcus faecalis, vancomycin resistant (VanB) (20) | GAR-936 | 0.12–0.25 | 0.25 | 0.25 |
| Minocycline | 0.12–32 | 0.25 | 16 | |
| Doxycycline | 0.12–32 | 0.5 | 32 | |
| Tetracycline | 0.03–>128 | 1 | >128 | |
| Enterococcus faecium (40) | GAR-936 | 0.06–0.25 | 0.12 | 0.25 |
| Minocycline | 0.06–64 | 0.06 | 16 | |
| Doxycycline | 0.12–64 | 0.12 | 16 | |
| Tetracycline | 0.12–>128 | 0.25 | 64 | |
| Enterococcus faecium, vancomycin resistant (VanA) (24) | GAR-936 | 0.06–0.25 | 0.12 | 0.25 |
| Minocycline | 0.06–32 | 0.12 | 16 | |
| Doxycycline | 0.12–32 | 0.12 | 16 | |
| Tetracycline | 0.03–128 | 0.25 | 64 | |
| Enterococcus faecium, vancomycin resistant (VanB) (20) | GAR-936 | 0.12–0.5 | 0.25 | 0.25 |
| Minocycline | 0.06–32 | 16 | 32 | |
| Doxycycline | 0.12–32 | 8 | 32 | |
| Tetracycline | 0.12–128 | 32 | 64 | |
| Enterococcus faecium, vancomycin resistant (VanD) (4) | GAR-936 | 0.06 | ||
| Minocycline | 0.06–2 | |||
| Doxycycline | 0.12–4 | |||
| Tetracycline | 0.25–16 | |||
| Enterococcus avium (10) | GAR-936 | 0.06–0.12 | 0.06 | 0.06 |
| Minocycline | 0.06–16 | 8 | 16 | |
| Doxycycline | 0.25–32 | 8 | 32 | |
| Tetracycline | 0.5–>128 | 32 | 64 | |
| Enterococcus raffinosus (10) | GAR-936 | 0.06–0.5 | 0.06 | 0.12 |
| Minocycline | 0.06–32 | 0.06 | 16 | |
| Doxycycline | 0.12–16 | 0.25 | 16 | |
| Tetracycline | 0.25–128 | 0.5 | 64 | |
| Enterococcus casseliflavus (14) | GAR-936 | 0.12–0.5 | 0.25 | 0.25 |
| Minocycline | 0.06–0.12 | 0.06 | 0.12 | |
| Doxycycline | 0.12–0.25 | 0.25 | 0.25 | |
| Tetracycline | 1–2 | 2 | 2 | |
| Enterococcus gallinarum (11) | GAR-936 | 0.06–2 | 0.12 | 0.25 |
| Minocycline | 0.06–32 | 0.12 | 16 | |
| Doxycycline | 0.12–16 | 0.25 | 16 | |
| Tetracycline | 0.25–128 | 1 | 128 | |
| Staphylococcus aureus, oxacillin susceptible (30) | GAR-936 | 0.25–0.5 | 0.5 | 0.5 |
| Minocycline | 0.06–8 | 0.12 | 0.12 | |
| Doxycycline | 0.25–8 | 0.25 | 0.5 | |
| Tetracycline | 0.12–64 | 0.5 | 1 | |
| Staphylococcus aureus, oxacillin resistant (28) | GAR-936 | 0.25–1 | 0.5 | 1 |
| Minocycline | 0.06–8 | 0.25 | 0.25 | |
| Doxycycline | 0.12–16 | 0.25 | 2 | |
| Tetracycline | 0.25–64 | 0.5 | 1 | |
| Staphylococcus aureus, vancomycin intermediate (2) | GAR-936 | 1–2 | ||
| Minocycline | 16 | |||
| Doxycycline | 8 | |||
| Tetracycline | 64 | |||
| Coagulase-negative staphylococci, oxacillin susceptible (24) | GAR-936 | 0.12–1 | 0.25 | 1 |
| Minocycline | 0.12–1 | 0.12 | 0.5 | |
| Doxycycline | 0.12–64 | 0.25 | 2 | |
| Tetracycline | 0.25–>128 | 0.5 | 4 | |
| Coagulase-negative staphylococci, oxacillin resistant (30) | GAR-936 | 0.12–2 | 0.5 | 1 |
| Minocycline | 0.06–2 | 0.25 | 0.5 | |
| Doxycycline | 0.12–32 | 1 | 8 | |
| Tetracycline | 0.25–>128 | 2 | 64 | |
| Streptococcus pyogenes (30) | GAR-936 | 0.06–0.25 | 0.12 | 0.12 |
| Minocycline | 0.12–16 | 0.12 | 0.25 | |
| Doxycycline | 0.25–16 | 0.25 | 0.5 | |
| Tetracycline | 0.25–32 | 0.5 | 4 | |
| Streptococcus agalactiae (10) | GAR-936 | 0.06–0.25 | 0.12 | 0.12 |
| Minocycline | 0.12–32 | 16 | 32 | |
| Doxycycline | 0.25–16 | 16 | 16 | |
| Tetracycline | 0.5–64 | 64 | 64 | |
| Viridans group streptococci, penicillin susceptible (15) | GAR-936 | 0.03–0.25 | 0.06 | 0.25 |
| Minocycline | 0.03–4 | 0.12 | 2 | |
| Doxycycline | 0.06–8 | 0.25 | 4 | |
| Tetracycline | 0.03–8 | 0.25 | 8 | |
| Viridans group streptococci, penicillin resistant (15) | GAR-936 | 0.02–0.12 | 0.03 | 0.06 |
| Minocycline | 0.06–32 | 0.12 | 16 | |
| Doxycycline | 0.02–32 | 0.25 | 16 | |
| Tetracycline | 0.02–64 | 0.25 | 64 | |
| Streptococcus pneumoniae, penicillin susceptible (28) | GAR-936 | 0.03–0.25 | 0.06 | 0.12 |
| Minocycline | 0.06–16 | 0.06 | 8 | |
| Doxycycline | 0.12–16 | 0.25 | 16 | |
| Tetracycline | 0.12–64 | 0.25 | 32 | |
| Streptococcus pneumoniae, penicillin resistant (30) | GAR-936 | 0.03–0.25 | 0.06 | 0.12 |
| Minocycline | 0.06–16 | 0.12 | 16 | |
| Doxycycline | 0.06–32 | 0.25 | 16 | |
| Tetracycline | 0.12–64 | 0.25 | 32 | |
| Listeria monocytogenes (20) | GAR-936 | 0.25–0.5 | 0.25 | 0.5 |
| Minocycline | 0.12–0.25 | 0.25 | 0.25 | |
| Doxycycline | 0.12–0.25 | 0.25 | 0.25 | |
| Tetracycline | 0.5–4 | 4 | 4 | |
| JK diphtheroids (20) | GAR-936 | 0.12–4 | 0.5 | 2 |
| Minocycline | 0.12–4 | 0.25 | 1 | |
| Doxycycline | 0.25–8 | 0.25 | 4 | |
| Tetracycline | 0.25–64 | 0.5 | 32 | |
| Lactobacillus spp. (12) | GAR-936 | 0.03–0.12 | 0.06 | 0.12 |
| Minocycline | 0.03–2 | 0.12 | 0.5 | |
| Doxycycline | 0.12–8 | 0.25 | 2 | |
| Tetracycline | 0.12–8 | 0.5 | 8 | |
| Leuconostoc spp. (10) | GAR-936 | 0.12 | 0.12 | 0.12 |
| Minocycline | 0.25–0.5 | 0.25 | 0.5 | |
| Doxycycline | 1–4 | 1 | 2 | |
| Tetracycline | 1–8 | 1 | 4 | |
| Pediococcus spp. (8) | GAR-936 | 0.03–1 | ||
| Minocycline | 0.5–2 | |||
| Doxycycline | 2–8 | |||
| Tetracycline | 4–128 | |||
Against coagulase-negative staphylococci, GAR-936 was consistently 1 dilution less active than minocycline, based on the MIC50s and MIC90s. Again, however, the new compound inhibited tetracycline- and doxycycline-resistant strains at ≤2 μg/ml. The MICs of GAR-936 for staphylococci resistant to both tetracycline and minocycline were higher than those for strains susceptible to minocycline (Fig. 1).
FIG. 1.
MICs (geometric means) of GAR-936 for gram-positive cocci based on resistance (R) or susceptibility (S) to minocycline (Min) or tetracycline (Tet). Among enterococci and staphylococci, susceptible isolates were those for which MICs were ≤4 μg/ml and resistant isolates were those for which MICs were ≥16 μg/ml. Streptococci were grouped according to susceptibility criteria for pneumococci: MICs for susceptible isolates were ≤2 μg/ml, and MICs for resistant isolates were ≥8 μg/ml. Isolates intermediately susceptible to Tet and Min (i.e., enterococci or staphylococci for which MICs were 8 μg/ml and streptococci for which MICs were 4 μg/ml) were included with resistant groups.
Against streptococci other than pneumococci, the intrinsic potency of GAR-936, based on a comparison of MIC50s, was equivalent or slightly superior to that of minocycline. Median (modal) MICs for GAR-936 and minocycline were 0.06 (0.06) and 0.12 (0.12) μg/ml, respectively. However, all streptococcal isolates, including strains resistant to doxycycline or minocycline, were inhibited by GAR-936 at ≤0.25 μg/ml.
For pneumococci, the results shown in Table 1 were obtained by agar dilution. The activity of GAR-936 against 60 strains of Streptococcus pneumoniae, half of which were not susceptible to penicillin, was also evaluated by microdilution. The MIC50 and MIC90 were 0.03 and 0.06 μg/ml. These were within 1 dilution of agar dilution results. Individually, microdilution results were 2 dilutions lower than the agar dilution results for 11 strains, 1 dilution lower for 27, equivalent for 13, and 1 dilution greater for 4. For S. pneumoniae ATCC 49619, the broth microdilution result was 1 dilution lower than the agar dilution result.
Approximately one-third of enterococci were resistant to doxycycline or minocycline and 44% were resistant to tetracycline, whereas all were inhibited by GAR-936 at ≤2 μg/ml. Against 20 strains of Listeria monocytogenes, GAR-936 was equal in activity to minocycline and doxycycline, based on a comparison of MIC50s, but eight times more active than tetracycline. GAR-936 was the most active agent tested against Lactobacillus, Pediococcus, and Leuconostoc. Against Pediococcus spp., GAR-936 was up to 4 times more active than minocycline and 8 to 128 times more active against doxycycline and tetracycline, based on a comparison of MIC90s. The isolates least susceptible to the new drug were Corynebacterium jeikeium. Two isolates of this group were inhibited only at 4 μg/ml.
Figure 1 shows the mean MICs of GAR-936 for isolates classified by susceptibility to tetracycline and minocycline. The geometric mean MIC of GAR-936 for tetracycline-resistant, minocycline-resistant staphylococci (1 μg/ml) was 2.4 times higher than that against tetracycline-susceptible strains. However, only four staphylococcal isolates were represented in the former group. For streptococci and enterococci, the resistance phenotype did not consistently influence GAR-936's geometric mean MICs.
The MICs (in micrograms per milliliter) of GAR-936 for control organisms were as follows (subscript indicates number of times that the value was observed): for S. aureus ATCC 29213 (n = 18), 0.121, 0.2510, 0.55, and 1.02; for Escherichia coli ATCC 25922 (n = 18), 0.251, 0.58, and 1.09; for S. pneumoniae ATCC 49619 by agar dilution (n = 2), 0.062; and for S. pneumoniae ATCC 49619 by broth microdilution (n = 2), 0.032. All results were within quality control (QC) limits for tetracycline against S. pneumoniae ATCC 49619 and for tetracycline and minocycline against S. aureus ATCC 29213 (12). NCCLS-approved QC ranges are not available for minocycline against the pneumococcus or for doxycycline against either control strain. For E. coli ATCC 25922, 17 of 18 observations were within limits for tetracycline and within the previously utilized QC range (0.5 to 2 μg/ml) for minocycline, with the remaining data point being 1 dilution above that range. However, the QC range for this antibiotic and organism was recently revised to 0.25 to 1 μg/ml, based on new microdilution study results; 8 of 18 values which we obtained by agar dilution fell above this range.
The present study confirmed the findings of Petersen et al. (14) (who demonstrated MIC90s for staphylococci, streptococci, and enterococci that were generally ≤0.5 μg/ml) and extended the results to include more vancomycin-resistant enterococci, penicillin-resistant S. pneumoniae, and oxacillin-resistant staphylococci. Resistance to antibiotics has become an increasingly difficult problem in the management of infections with gram-positive bacteria (11). Methicillin-resistant S. aureus, coagulase-negative staphylococci, penicillin-resistant S. pneumoniae, and vancomycin-resistant enterococci all cause significant morbidity or mortality or both in U.S. hospitals (1, 3, 6, 7–11, 13). Such isolates are frequently resistant to multiple classes of antibiotics, including tetracyclines, erythromycin, chloramphenicol, β-lactams, and trimethoprim-sulfamethoxazole (8). If studies in vivo confirm the efficacy and tolerability of GAR-936, the glycylcycline would appear to have the potential for broad therapeutic application against infections with gram-positive bacteria.
Acknowledgments
This study was supported by a grant from Wyeth-Ayerst Research, Pearl River, N.Y.
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