Abstract
Against 33 Gram-positive and -negative bacteria, NXL 103 MICs were 0.03 to 1 μg/ml. NXL 103 was bactericidal by 12 h at 2 × MIC against all 5 pneumococci and at 2 × MIC after 24 h against all 5 group A and B β-hemolytic streptococci. NXL 103 was bactericidal against all 8 Haemophilus influenzae strains at 2× MIC and all 5 Moraxella catarrhalis strains at 4× MIC after 24 h but was mainly bacteriostatic against 5 methicillin-resistant Staphylococcus aureus strains. After the exposure of one strain of each species to NXL 103 for 10 daily subcultures, the MICs remained within ±1 dilution.
The resistotype of bacteria causing community-acquired respiratory tract infections is changing. Widespread use of the pediatric pneumococcal conjugate vaccine has selected multiresistant nonvaccine type 19A strains which cannot be treated in pediatric patients with any FDA-approved drug (17, 19), and the incidence of BLNAR (β-lactamase negative ampicillin resistant due to mutations in PBP3) Haemophilus influenzae strains is increasing. Although CLSI breakpoints are available, H. influenzae is macrolide and ketolide resistant in vitro due to efflux. Moraxella catarrhalis strains are uniformly β-lactamase positive (9, 10, 18).
Although Streptococcus pyogenes strains are penicillin G susceptible, macrolide resistance is increasing and quinolone resistance has appeared (13). Raised penicillin G MICs and macrolide resistance have appeared in group B streptococci (3). The problem of methicillin-resistant Staphylococcus aureus (MRSA) infections has been exacerbated by the emergence of glycopeptide-nonsusceptible strains (2, 5, 11). Most hospital-acquired MRSA strains are quinolone resistant (12, 15).
We need a new drug which covers all of these resistotypes. NXL 103 (XRP 2868) is an experimental broad-spectrum oral streptogramin comprising two components, RPR 202868 and RPR 132552 (7, 8, 14, 16). The clinically available intravenously administered streptogramin quinupristin-dalfopristin has rapid bactericidal activity against susceptible organisms. We did a time-kill study to test the activities of NXL 103, compared with those of other agents (see Table 2), against the bacterial groups listed above.
The strains we tested are listed in Table 1. MICs were determined by CLSI broth macrodilution (6). Viable counts were determined at 0, 3, 6, 12, and 24 h, with additional sampling at 1 h for Streptococcus pneumoniae. Previous studies (14, 16) have confirmed the stability of NXL 103, both of the components of NXL 103, and that of all of the other compounds tested, in broth through 24 h (4, 13). Recovery plates were incubated for up to 72 h, and colony counts were determined on plates yielding 30 to 300 colonies. Lack of drug carryover was verified by dilution (4, 13). One M. catarrhalis strain with regrowth at 2× MIC (Table 1) and all 5 bacteriostatically inhibited MRSA strains were retested for NXL 103 susceptibility after 24 h. One strain of each species (Table 1) was tested for resistance selection by multistep analysis (4, 13). No small-colony variants were observed.
TABLE 1.
Strains tested in this study
| Species and strain | Resistance phenotype | MIC (μg/ml) |
||||||
|---|---|---|---|---|---|---|---|---|
| NXL 103 | Amoxicillin- clavulanate | Cefuroxime | Azithromycin | Clarithromycin | Levofloxacin | Moxifloxacin | ||
| S. pneumoniae | ||||||||
| 7567 | Multiresistant serotype 19A | 0.25 | 8 | 8 | >64 | >64 | 1 | 0.25 |
| 7619 | Multiresistant serotype 19A | 0.5 | 8 | 8 | >64 | >64 | 1 | 0.25 |
| 1147d | Penicillin I and macrolide resistant (mef mutation) | 0.12 | 4 | 16 | 4 | 4 | 8 | 2 |
| 2874 | Penicillin R and macrolide resistant (L4 mutation) | 0.5 | 0.25 | 8 | >64 | >64 | 1 | 0.12 |
| 3548 | Penicillin R and macrolide resistant [erm(B) mutation] | 0.25 | 8 | 16 | >64 | >64 | 1 | 0.12 |
| H. influenzae | ||||||||
| 42 | β-Lactamase negative, quinolone nonsusceptible | 1 | 1 | 2 | 1 | 8 | 4 | 4 |
| 101 | β-Lactamase negative, macrolide hyperresistanta | 0.5 | 0.25 | 0.5 | >64 | >64 | 0.03 | 0.03 |
| 76 | β-Lactamase negative, macrolide hyperresistanta | 0.5 | 0.5 | 1 | >64 | >64 | 8 | 8 |
| 78d | BLNAR | 0.5 | 8 | 4 | 1 | 8 | 0.008 | 0.016 |
| 79 | BLNAR | 0.5 | 8 | 32 | 1 | 8 | 0.016 | 0.016 |
| 92 | β-Lactamase positive, amoxicillin-clavulanate resistant | 0.5 | 8 | 32 | 1 | 4 | 0.016 | 0.016 |
| 30 | β-Lactamase positive, macrolide hypersusceptibleb | 0.25 | 2 | 2 | 0.25 | 1 | 0.016 | 0.016 |
| 73 | β-Lactamase positive, macrolide hyperresistanta | 1 | 1 | 1 | >64 | >64 | 0.016 | 0.016 |
| M. catarrhalis | ||||||||
| 2 | β-Lactamase positive, macrolide susceptible | 0.03 | 0.25 | 1 | 0.03 | 0.12 | 0.03 | 0.06 |
| 6d | β-Lactamase positive, macrolide susceptible | 0.06 | 0.25 | 1 | 0.03 | 0.12 | 0.06 | 0.06 |
| 8 | β-Lactamase positive, macrolide susceptible | 0.06 | 0.5 | 4 | 0.03 | 0.12 | 0.06 | 0.06 |
| 11 | β-Lactamase positive, macrolide susceptible | 0.06 | 0.25 | 1 | 0.06 | 0.12 | 0.06 | 0.06 |
| 12 | β-Lactamase positive, macrolide susceptible | 0.06 | 0.5 | 1 | 0.06 | 0.06 | 0.06 | 0.06 |
| Group A streptococci | ||||||||
| 3139 | Macrolide susceptible | 0.06 | 0.016 | 0.016 | 0.12 | 0.06 | 0.5 | 0.25 |
| 3143 | Macrolide susceptible | 0.06 | 0.016 | 0.016 | 0.12 | 0.06 | 1 | 0.12 |
| 3145 | Macrolide susceptible | 0.06 | 0.016 | 0.016 | 0.12 | 0.03 | 1 | 0.25 |
| 3530d | Macrolide resistant [erm(B) mutation] | 0.06 | 0.016 | 0.016 | >64 | >64 | 1 | 0.12 |
| 3343 | Macrolide resistant [erm(B) mutation] | 0.06 | 0.03 | 0.016 | >64 | >64 | 1 | 0.12 |
| Group B streptococci | ||||||||
| 4 | Macrolide susceptible | 0.06 | 0.12 | 0.06 | 0.12 | 0.03 | 1 | 0.25 |
| 6 | Macrolide susceptible | 0.12 | 0.06 | 0.06 | 0.12 | 0.03 | 1 | 0.25 |
| 11 | Macrolide susceptible | 0.06 | 0.12 | 0.06 | 0.03 | 0.03 | 1 | 0.5 |
| 8 | Raised penicillin G MIC (0.12 μg/ml) | 0.12 | 0.25 | 0.25 | 0.12 | 0.06 | 1 | 0.25 |
| 7d | Macrolide resistant [erm(B) mutation] | 0.06 | 0.06 | 0.03 | >64 | >64 | 1 | 0.25 |
| MRSA | ||||||||
| 618 | hVISAe (hospital acquired; azithromycin MIC, >8 μg/ml) | 0.5 | 2 | 2 | 1 | 1 | 0.12 | 0.5 |
| 504d | VISAf (hospital acquired; azithromycin MIC, >8 μg/ml) | 0.25 | 8 | 4 | 2 | 1 | 0.5 | 0.5 |
| 555 | VISA (hospital acquired; azithromycin MIC, 1 μg/ml) | 0.06 | 8 | 16 | 8 | 2 | 0.12 | 0.12 |
| 510 | Hershey VRSAg (hospital acquired; azithromycin MIC, >8 μg/ml)c | 0.25 | 32 | 8 | 0.5 | 2 | 0.25 | 0.5 |
| 424 | MRSA (community acquired; azithromycin MIC, >8 μg/ml) | 0.12 | 1 | 0.5 | 0.5 | 4 | 0.25 | 0.25 |
Positive macrolide efflux plus ribosomal protein mutation(s) (18).
Negative macrolide efflux (18).
Reference 2.
Strains were tested by resistance selection. M. catarrhalis strain 6 and all 5 MRSA strains were tested for regrowth after 24 h.
hVISA, heterogeneous vancomycin-intermediate S. aureus.
VISA, vancomycin-intermediate S. aureus.
VRSA, vancomycin-resistant S. aureus.
S. pneumoniae MIC ranges (μg/ml) were as follows: NXL 103, 0.12 to 0.5; amoxicillin-clavulanate, 0.25 to 8; cefuroxime, 8 to 16; azithromycin, 4 to >64; clarithromycin, 4 to >64; levofloxacin, 1 to 8; moxifloxacin, 0.12 to 2. NXL 103, at 2× MIC, was bactericidal (99.9% killing) against all five strains tested after 12 h and gave 90% killing of all five strains at 2× MIC after 3 h. All of the β-lactams and quinolones tested gave kill kinetic patterns similar to those obtained with NXL 103 but relative to raised MICs, depending upon the resistotype.
H. influenzae MIC ranges (μg/ml) were as follows: NXL 103, 0.25 to 1; amoxicillin-clavulanate, 0.25 to 8; cefuroxime, 0.5 to 32; azithromycin, 0.25 to >64; clarithromycin, 1 to >64; levofloxacin, 0.016 to 8; moxifloxacin, 0.016 to 8. NXL 103 was bactericidal against all 8 strains tested at 2× MIC after 24 h and against all 8 strains at 4× MIC after 12 h; 90% inhibition of all strains was found at 4× MIC after 3 h. Amoxicillin-clavulanate, cefuroxime, levofloxacin, and moxifloxacin were bactericidal at 2 to 4× MIC after 24 h, and azithromycin and clarithromycin were bactericidal against all 5 strains at 2× MIC after 24 h. NXL 103 and quinolones showed the most rapid killing at earlier times.
S. pyogenes MIC ranges (μg/ml) were as follows: NXL 103, 0.06; amoxicillin-clavulanate, 0.016 to 0.03; cefuroxime, 0.016; azithromycin, 0.12 to >64; clarithromycin, 0.03 to >64; levofloxacin, 0.5 to 1; moxifloxacin, 0.12 to 0.25. NXL 103 was bactericidal against all 5 strains at 2× MIC after 12 h and against all 5 strains after 6 h at 4× MIC. Other quinolones and β-lactams had kinetics similar to those of NXL 103; macrolides showed slower killing.
S. agalactiae MIC ranges (μg/ml) were as follows: NXL 103, 0.06 to 0.12; amoxicillin-clavulanate, 0.06 to 0.25; cefuroxime, 0.03 to 0.25; azithromycin, 0.03 to >64; clarithromycin, 0.03 to >64; levofloxacin, 1; moxifloxacin, 0.25 to 0.5. NXL 103 was bactericidal against all 5 strains at 2× MIC at 24 h and killed 90% of the bacteria of all 5 strains at 2× MIC at 3 h.
M. catarrhalis MIC ranges (μg/ml) were as follows: NXL 103, 0.03 to 0.06; amoxicillin-clavulanate, 0.25 to 0.5; cefuroxime, 1 to 4; azithromycin, 0.03 to 0.06; clarithromycin, 0.06 to 0.12; levofloxacin, 0.03 to 0.06; moxifloxacin, 0.06. NXL 103 was bactericidal against all 5 strains at 4× MIC after 24 h, with 99% killing at 2× MIC, and showed 90% inhibition of all strains at 4× MIC after 3 h. Killing by amoxicillin-clavulanate, levofloxacin, and moxifloxacin was similar to that by NXL 103, with bactericidal activity against all 5 strains at 2× MIC after 24 h and significant killing at earlier times. Killing by azithromycin and clarithromycin was slower, with 5 and 2 strains showing bactericidal activity at 4× MIC after 24 h, respectively.
MRSA MIC ranges (μg/ml) were as follows: NXL 103, 0.06 to 0.5; vancomycin, 1 to 32; teicoplanin, 0.5 to 16; daptomycin, 0.5 to 8; linezolid, 1 to 4; tigecycline, 0.12 to 0.5; quinupristin-dalfopristin, 0.12 to 0.5. NXL 103, linezolid, tigecycline, and quinupristin-dalfopristin were mainly bacteriostatic. One of five MRSA strains was macrolide sensitive, and NXL 103 showed 99% killing of this strain at 1× MIC by 24 h. Vancomycin and teicoplanin were bactericidal against 3 and 4 strains, respectively, at 4× MIC after 24 h. Daptomycin showed the most rapid killing, being bactericidal against all 5 strains at 2× MIC after 12 h, with significant killing earlier.
The kill kinetics of all of the drugs tested at 12 and 24 h are summarized in Table 2 and graphically depicted for one strain each of S. pneumoniae, H. influenzae, and MRSA in Fig. 1. Time-kill data for each species tested are presented in the supplemental material. All 24-h subcultures yielded strains with the same MICs as the parents, and MICs after 10 daily subcultures were all within ±1 dilution of those of the parental strains.
TABLE 2.
Results of time-kill analyses after 24 h
| Drug | Total no. of strains | Species (no. of isolates tested) | Concn (fold MIC) | No. of strains showing indicated % killing at 24 h: |
||
|---|---|---|---|---|---|---|
| 90 | 99 | 99.9 | ||||
| NXL 103 | 33 | S. pneumoniae (5), H. influenzae (8), S. pyogenes (5), S. agalactiae (5), M. catarrhalis (5), MRSA (5) | 4 | 33 | 31 | 29 |
| 2 | 33 | 31 | 27 | |||
| 1 | 28 | 21 | 15 | |||
| Amoxicillin-clavulanate | 28 | S. pneumoniae (5), H. influenzae (8), S. pyogenes (5), S. agalactiae (5), M. catarrhalis (5) | 4 | 28 | 28 | 28 |
| 2 | 28 | 28 | 28 | |||
| 1 | 25 | 24 | 22 | |||
| Cefuroxime | 28 | S. pneumoniae (5), H. influenzae (8), S. pyogenes (5), S. agalactiae (5), M. catarrhalis (5) | 4 | 28 | 28 | 28 |
| 2 | 28 | 28 | 27 | |||
| 1 | 21 | 20 | 15 | |||
| Azithromycin | 18 | S. pneumoniae (1), H. influenzae (5), S. pyogenes (3), S. agalactiae (4), M. catarrhalis (5)a | 4 | 18 | 16 | 11 |
| 2 | 16 | 11 | 9 | |||
| 1 | 10 | 8 | 4 | |||
| Clarithromycin | 18 | S. pneumoniae (1), H. influenzae (5), S. pyogenes (3), S. agalactiae (4), M. catarrhalis (5)a | 4 | 17 | 14 | 10 |
| 2 | 14 | 9 | 7 | |||
| 1 | 9 | 7 | 5 | |||
| Levofloxacin | 28 | S. pneumoniae (5), H. influenzae (8), S. pyogenes (5), S. agalactiae (5), M. catarrhalis (5) | 4 | 28 | 28 | 28 |
| 2 | 28 | 28 | 27 | |||
| 1 | 24 | 23 | 18 | |||
| Moxifloxacin | 28 | S. pneumoniae (5), H. influenzae (8), S. pyogenes (5), S. agalactiae (5), M. catarrhalis (5) | 4 | 28 | 28 | 28 |
| 2 | 28 | 28 | 28 | |||
| 1 | 24 | 21 | 15 | |||
| Vancomycin | 5 | MRSA | 4 | 5 | 5 | 3 |
| 2 | 5 | 5 | 3 | |||
| 1 | 4 | 4 | 2 | |||
| Teicoplanin | 5 | MRSA | 4 | 5 | 5 | 4 |
| 2 | 5 | 5 | 3 | |||
| 1 | 3 | 2 | 0 | |||
| Daptomycin | 5 | MRSA | 4 | 5 | 5 | 5 |
| 2 | 5 | 5 | 5 | |||
| 1 | 4 | 2 | 2 | |||
| Tigecycline | 5 | MRSA | 4 | 5 | 3 | 0 |
| 2 | 5 | 2 | 0 | |||
| 1 | 3 | 0 | 0 | |||
| Quinupristin-dalfopristin | 5 | MRSA | 4 | 5 | 4 | 0 |
| 2 | 5 | 3 | 0 | |||
| 1 | 2 | 1 | 0 | |||
Ten strains with azithromycin and clarithromycin MICs of >64 μg/ml were not tested [S. pneumoniae (n = 4), H. influenzae (n = 3), S. pyogenes (n = 2), and S. agalactiae (n = 1)].
FIG. 1.
Twenty-four-hour kill kinetics of all of the drugs tested in this study for one strain each of S. pneumoniae, H. influenzae, and MRSA.
NXL 103 is an investigational streptogramin composed of a mixture of 70% RPR 132552 and 30% RPR 202868. Previous studies have shown that NXL 103 inhibits staphylococci, S. pneumoniae, nonpneumococcal streptococci, H. influenzae, and anaerobes at ≤1 μg/ml. NXL 103 is approximately 4 times as potent as quinupristin-dalfopristin against S. aureus, Enterococcus faecium, streptococci, and H. influenzae (7, 8, 14, 16). Our MIC results reflect those published previously by both us and others. Rapid kill kinetics against all of the strains tested, with the exception of MRSA, adds to the attractive properties of NXL 103. Quinupristin-dalfopristin has been described as often being bacteriostatic against MRSA, possibly mediated by constitutive erm(B) production by most MRSA strains (7, 8, 14, 16). This was reflected in the results obtained with NXL 103. The MRSA strains tested in this study are not representative of the usual strains seen in the hospital or the community.
We have previously found synergy between the two NXL 103 components against pneumococci; in contrast, only one component, RPR 132552, was active against all strains of H. influenzae, except BLNAR, where synergy occurred (16). We did not test the individual components against other species. Although both components were stable in broth through 24 h, other methods might yield maximal synergy in vitro and less in vivo, since drug concentrations may change over time.
Our results indicate a promising role for NXL 103 in the empirical treatment of community-acquired respiratory tract infections, subject to further pharmacodynamic studies (1), but future development of NXL 103 is in doubt.
Supplementary Material
Acknowledgments
This study was supported by a grant from Novexel Laboratories, Romainville, France.
We thank Ronald Jones for provision of drug-resistant strains of S. agalactiae.
Footnotes
Published ahead of print on 18 January 2011.
Supplemental material for this article may be found at http://aac.asm.org/.
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