Abstract
MICs, time-kills, and postantibiotic effects (PAEs) of ABT-773 (a new ketolide) and 10 other agents were determined against 226 pneumococci. Against 78 ermB- and 44 mefE-containing strains, ABT-773 MICs at which 50% of the isolates tested were inhibited (MIC50s) and MIC90s were 0.016 to 0.03 and 0.125 μg/ml, respectively. Clindamycin was active only against macrolide-resistant strains containing mefE (MIC50, 0.06 μg/ml; MIC90, 0.125 μg/ml). Activities of pristinamycin (MIC90, 0.5 μg/ml) and vancomycin (MIC90, 0.25 μg/ml) were unaffected by macrolide or penicillin resistance, while β-lactam MICs rose with those of penicillin G. Against 19 strains with L4 ribosomal protein mutations and two strains with mutations in domain V of 23S rRNA, ABT-773 MICs were 0.03 to 0.25 μg/ml, while macrolide and azalide MICs were all ≥16.0 μg/ml. ABT-773 was bactericidal at twice the MIC after 24 h for 8 of 12 strains (including three strains with erythromycin MICs greater than or equal to 64.0 μg/ml). Kill kinetics of erythromycin, azithromycin, clarithromycin, and roxithromycin against macrolide-susceptible strains were slower than those of ABT-773. ABT-773 had longer PAEs than macrolides, azithromycin, clindamycin, or β-lactams, including against ermB-containing strains. ABT-773, therefore, shows promising in vitro activity against macrolide-susceptible as well as -resistant pneumococci.
The incidence of pneumococci resistance to penicillin G and other β-lactam and non-β-lactam compounds has increased worldwide at an alarming rate, including in the United States. Major foci of infections currently include South Africa, Spain, Central and Eastern Europe, and parts of Asia (1, 8, 9, 13). The problem is exacerbated by the tendency of these strains to spread from country to country and from continent to continent (18, 19). In the United States, a recent survey has shown an increase in resistance to penicillin from <5% before 1989 (including <0.02% of isolates for which the MIC of penicillin is ≥2.0 μg/ml) to 6.6% in 1991 to 1992 (with penicillin MICs ≥2.0 μg/ml for 1.3% of isolates) (3). In another, more recent, survey, 23.6% (360) of 1,527 clinically significant pneumococcal isolates were not susceptible to penicillin (6). It is also important to note the high rates of isolation of penicillin-intermediate and -resistant pneumococci (approximately 30%) in middle ear fluids from patients with refractory otitis media, compared to pneumococci isolated from other sites (2).
Pneumococcal strains with intermediate and, especially, full resistance to penicillin G are often resistant to erythromycin (7, 12, 15). In the United States, Breiman and coworkers in 1991 to 1992 demonstrated erythromycin resistance rates of 3.7 and 2.2% in patients 1 to 2 and ≥4 years of age, respectively (3). A recent study by Doern and coworkers (6) has documented erythromycin resistance rates of 19 to 20% and 49% in penicillin-intermediate and -resistant strains, respectively (6). In Europe, erythromycin resistance rates are generally higher. For example, 27.5% of all pneumococci studied in France in 1992 (63% of penicillin-resistant strains) were erythromycin resistant (11).
Macrolide resistance in pneumococci is predominantly mediated by two mechanisms: (i) strains containing the ermB gene, coding for a ribosomal methylase, are resistant to 14-membered macrolides such as erythromycin, clarithromycin, and roxithromycin; 15-membered azalides such as azithromycin; and 16-membered macrolides such as josamycin and spiramycin, as well as the lincosamide clindamycin; (ii) strains containing the mefE gene, coding for an efflux pump, are resistant to 14-membered macrolides and azalides but are susceptible to 16-membered macrolides as well as clindamycin. Although clarithromycin generally yields MICs for pneumococci which are one or two dilutions lower than those of other macrolides, erythromycin-resistant pneumococci are resistant in vitro to all other existing macrolides (7, 31). However, recent work indicates that achievable levels of clarithromycin may be achievable in mefE-containing strains, in which macrolide MICs are lower (usually 1.0 to 16.0 μg/ml) than is the case with ermB-containing strains (≥64.0 μg/ml) (D. Shortridge, G. Doern, J. Beyer, A. Brueggemann, and R. K. Flamm, Abstr. 36th Infect. Dis. Soc. Am. Annu. Meet., abstr. 225F, 1998).
Up to now, the only members of the macrolide lincosamide streptogramin group which is consistently active against all pneumococci, irrespective of their penicillin- or erythromycin-susceptibility status, have been pristinamycin and quinupristin/dalfopristin, two parenteral streptogramins, and the ketolide group. HMR 3647 (telithromycin), the first ketolide to be developed, has been shown to be very active against pneumococci irrespective of their macrolide or penicillin susceptibility status (22, 25–27).
ABT-773 is a recently developed ketolide (4) (A. M. Nilius, M. Bui, L. Almer, D. Hensey, J. Beyer, Z. Ma, Y. S. Orr, and R. Flamm, Abstr. 9th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P-177, 1999; Z. Ma, R. F. Clark, and Y. Or, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2133, 1999; Z. Cao, R. Hammond, S. Pratt, A. Saiki, C. Lerner, and P. Zhong, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2135, 1999).
The present study examined (i) the susceptibility by agar dilution of 226 penicillin- and erythromycin-susceptible and -resistant pneumococci to ABT-773, compared to erythromycin, azithromycin, clarithromycin, roxithromycin, clindamycin, pristinamycin, amoxicillin, ceftriaxone, imipenem, and vancomycin; (ii) the activity of the above compounds against six erythromycin-susceptible and six erythromycin-resistant pneumococci by time-kill methodology; and (iii) the postantibiotic effect of the above drugs against eight pneumococcal strains, including three ermB-containing strains.
MATERIALS AND METHODS
Bacteria.
For agar dilution MICs, pneumococci comprised 83 macrolide-susceptible (MIC, ≤0.25 μg/ml) and 143 macrolide-resistant (MIC, ≥0.5 μg/ml) strains. Of these, 55 were penicillin susceptible, 85 were penicillin intermediate, and 86 were penicillin resistant. Of the macrolide-resistant strains (which included intermediate strains by NCCLS classification), 75 carried the ermB and 44 carried the mefE gene; three strains carried both erm and mef in spite of repeated subcultures in an attempt to separate the two genes from a possible heterogenous population (these were included in the erm-containing group for data analysis, because they exhibited the Erm phenotype). In addition, 19 strains with the L4 ribosomal protein mutation and two with mutations in domain V of the 23S rRNA (A. Tait-Kamradt, T. Davies, M. Jacobs, P. Appelbaum, and J. Sutcliffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 842, 1999) were tested to determine the MIC of macrolides. For time-kill studies, six macrolide-susceptible and six macrolide-resistant strains (three carrying erm, three carrying mef) were tested, while for postantibiotic effect (PAE) studies, five macrolide-susceptible and three macrolide-resistant strains were studied.
Antimicrobials and MIC testing.
ABT-773 powder was obtained from Abbott Laboratories (Abbot Park, Ill.); other antimicrobials were obtained from their respective manufacturers. Agar dilution methodology was performed on 226 strains (12, 13) by using Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% sheep blood. Broth MICs for eight strains tested by PAE were performed according to NCCLS recommendations by using cation-adjusted Mueller-Hinton broth with 5% lysed defibrinated horse blood. Standard quality control strains, including Streptococcus pneumoniae ATCC 49619, were included in each run of agar and broth dilution MICs. All incubation took place in air (20).
Time-kill testing.
For time-kill studies, glass tubes containing 5 ml of cation-adjusted Mueller-Hinton broth (Difco) plus 5% lysed horse blood with doubling antibiotic concentrations were inoculated with 5 × 105 to 5 × 106 CFU of bacteria per ml and were incubated at 35°C in a shaking water bath. Antibiotic concentrations were chosen to comprise two doubling concentrations above and one concentration below the agar dilution MIC. Growth controls with inoculum but no antibiotic were included with each experiment (22, 25).
Lysed horse blood was prepared as described previously (22, 25). The initial bacterial inoculum was prepared by making a 0.5-McFarland-standard suspension in Mueller-Hinton broth containing 5% lysed horse blood from an 18-h culture on a sheep blood agar plate. Dilutions required to obtain the correct final inoculum (5 × 105 to 5 × 106 CFU/ml) were determined by prior viability studies using each strain (22, 25).
To inoculate each tube of serially diluted antibiotic, 50 μl of diluted inoculum was delivered by pipette beneath the surface of the broth. Tubes were then vortexed and plated for viability counts within 10 min (approximately 0.2 h). The original inoculum was determined by using the untreated growth control. Only tubes containing an initial inoculum within the range of 5 × 105 to 5 × 106 CFU/ml were acceptable.
Viability counts of antibiotic-containing suspensions and controls without drug were performed by plating 1:10 dilutions of 0.1-ml aliquots from each tube in sterile Mueller-Hinton broth onto 5% sheep blood agar plates (BBL). The number of dilutions required varied with the initial inoculum on plates containing undiluted inoculum and depended upon the original MIC of the antibiotic for the strain. Recovery plates were incubated for up to 72 h. Colony counts were performed on plates yielding 30 to 300 colonies. The lower limit of sensitivity of colony counts was 300 CFU/ml (22–25).
Time-kill assays were analyzed by determining the number of strains which yielded a Δlog10 CFU per ml of 1, 2, and 3, at 0, 3, 6, 12, and 24 h, compared to counts at 0 h. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by ≥3 log10 CFU/ml (99.9%) at each of the time periods and were considered bacteriostatic if the inoculum was reduced by 0 to 3 log10 CFU/ml. With the sensitivity threshold and inocula used in these studies, no problems were encountered in delineating 99.9% killing, when present. The problem of bacterial carryover was addressed as described previously (22, 25). For macrolide time-kill testing, only strains with MICs ≤4.0 μg/ml were tested.
PAE testing.
The PAE was determined by the viable plate count method (5, 29, 30) using Mueller-Hinton broth (MHB) supplemented with 5% lysed horse blood. The PAE was induced by exposure to 10 times the MIC for 1 h except in the case of pristinamycin where, owing to rapid bactericidal activity, 1 times the MIC was used. Additionally, the one quinolone-resistant strain was exposed at quinolone concentrations 5 times the MIC. Tubes containing 5 ml of broth with antibiotic were inoculated with approximately 5 × 106 CFU/ml. Growth controls with inoculum but no antibiotic were included with each experiment. Tubes were placed in a shaking water bath at 35°C for 1 h. At the end of the exposure period, cultures were diluted 1:1,000 to remove antibiotic. A control containing bacteria preexposed to antibiotic at a concentration of 0.01 times the MIC was also prepared (29, 30).
Viability counts were determined before exposure and immediately after dilution (0 h) and then every 2 h until tube turbidity reached 1 McFarland standard. Inocula were prepared by suspending growth from an overnight blood agar plate in broth. The broth was incubated at 35°C for 2 to 4 h in a shaking water bath until turbidity matched 1 McFarland standard and was checked for viability by plate counts (29, 30).
The PAE was defined as PAE = T−C (where T is time required for viability counts of an antibiotic-exposed culture to increase by 1 log10 above counts immediately after dilution and C is corresponding time for growth control [5]). For each experiment, viability counts (log10 CFU per milliliter) were plotted against time, and the results were expressed as the mean of two separate assays ± standard deviation.
Characterization of macrolide-resistant strains.
Macrolide-resistant strains were screened by the erythromycin-clindamycin double disk method (7). PCR primers were purchased from Sigma/Genosys Biotechnologies (The Woodlands, Tex.). Primers and PCR conditions for ermB and mefE have been described previously (31). Primers and PCR conditions for L4 ribosomal protein in S. pneumoniae have been described (A. Tait-Kamradt et al., 39th ICAAC, abstr. 842; A. Tait-Kamradt, T. Davies, M. Cronan, M. Jacobs, P. Appelbaum, and J. Sutcliffe, submitted for publication). To locate mutants in 23S rRNA, the genes were initially amplified from total genomic DNA as three overlapping contigs by using primers and PCR conditions described previously (A. Tait-Kamradt et al., 39th ICAAC, abstr. 842). The peptidyl transferase region from each allele of 23S rRNA was amplified by using unique primers beyond the 3′ end of each 23S rRNA as described previously (A. Tait-Kamradt et al., 39th ICAAC, abstr. 842). PCR products were purified by using QIAquick PCR Purification Kit (QIAGEN, Valencia, Calif.) and were sequenced by using an Applied Biosystems model 373 DNA sequencer. Sequence comparisons were performed by using Vector NTI sequence analysis software (InforMax, Inc., North Bethesda, Md.).
RESULTS
Results of agar dilution testing with 205 strains (excluding those with the L4 and 23S rRNA mutations) classified by penicillin susceptibility are presented in Table 1. The ketolide ABT-773 had the lowest MICs of all macrolides and azalides tested, irrespective of penicillin MIC. For other macrolides and azalides, MICs rose with those of penicillin; however, owing to the inclusion of several macrolide-resistant but penicillin-susceptible strains from our collection, macrolide MICs were lower for penicillin-intermediate than for penicillin-susceptible organisms. This is currently not the case in the United States, where less than 5% of penicillin-susceptible pneumococci are macrolide resistant (6). All strains were susceptible to pristinamycin and vancomycin, while β-lactam MICs rose with those of penicillin G.
TABLE 1.
Agar dilution MICs of antimicrobial agents against 205 strains classified by penicillin susceptibility
| Drug | Penicillin sensitivity of straina | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Penicillin | S | ≤0.008–0.06 | 0.03 | 0.06 |
| I | 0.125–1.0 | 0.25 | 1.0 | |
| R | 2.0–>8.0 | 2.0 | 4.0 | |
| ABT-773 | S | ≤0.008–0.25 | 0.016 | 0.06 |
| I | ≤0.008–4.0 | 0.016 | 0.03 | |
| R | ≤0.08–2.0 | 0.03 | 0.125 | |
| Erythromycin | S | 0.016–>64.0 | 2.0 | >64.0 |
| I | ≤0.008–>64.0 | 0.06 | >64.0 | |
| R | ≤0.008–>64.0 | 8.0 | >64.0 | |
| Azithromycin | S | 0.03–>64.0 | 4.0 | >64.0 |
| I | 0.016–>64.0 | 0.125 | >64.0 | |
| R | 0.03–>64.0 | 8.0 | >64.0 | |
| Clarithromycin | S | 0.016–>64.0 | 1.0 | >64.0 |
| I | ≤0.008–>64.0 | 0.03 | >64.0 | |
| R | 0.016–>64.0 | 8.0 | >64.0 | |
| Roxithromycin | S | 0.125–>64.0 | 8.0 | >64.0 |
| I | 0.03–>64.0 | 0.25 | >64.0 | |
| R | 0.125–>64.0 | 32.0 | >64.0 | |
| Clindamycin | S | 0.016–>64.0 | 0.06 | >64.0 |
| I | 0.016–>64.0 | 0.06 | >64.0 | |
| R | 0.03–>64.0 | 0.06 | >64.0 | |
| Pristinamycin | S | 0.125–0.5 | 0.25 | 0.5 |
| I | 0.125–1.0 | 0.25 | 0.5 | |
| R | 0.125–1.0 | 0.25 | 0.5 | |
| Amoxicillin | S | ≤0.008–0.25 | 0.016 | 0.06 |
| I | ≤0.008–2.0 | 0.125 | 1.0 | |
| R | 0.25–8.0 | 1.0 | 4.0 | |
| Ceftriaxone | S | ≤0.008–0.25 | 0.016 | 0.06 |
| I | 0.016–2.0 | 0.25 | 1.0 | |
| R | 0.5–8.0 | 1.0 | 2.0 | |
| Imipenem | S | ≤0.008–0.125 | ≤0.008 | 0.016 |
| I | 0.016–0.25 | 0.03 | 0.25 | |
| R | 0.3–1.0 | 0.25 | 0.5 | |
| Vancomycin | S | 0.125–0.25 | 0.25 | 0.25 |
| I | 0.125–0.25 | 0.25 | 0.25 | |
| R | 0.125–0.25 | 0.25 | 0.25 | |
S, sensitive; I, intermediate; R, resistant.
Results of agar dilution testing with the above 205 strains classified by erythromycin susceptibility are presented in Table 2. ABT-773 was very active irrespective of erythromycin susceptibility. ABT-773 MICs for erm- and mef-containing strains did not differ significantly. Against 83 erythromycin-susceptible and 122 erythromycin-resistant pneumococci (carrying erm and mef), the agar dilution MIC at which 50% of the isolates tested were inhibited (MIC50)/MIC90s in micrograms per milliliter) were as follows: ABT-773, ≤0.008/0.016, 0.03/0.125 (erm and mef); erythromycin, 0.03/0.06, >64.0/>64.0 (erm), 2.0/8.0 (mef); azithromycin, 0.06/0.125, >64.0/>64.0 (erm), 2.0/8.0 (mef); clarithromycin, 0.03/0.06, >64.0/>64.0 (erm), 1.0/8.0 (mef); roxithromycin, 0.25/0.25, >64.0/>64.0 (erm), 8.0/32.0 (mef); and clindamycin, 0.06/0.06, >64.0/>64.0 (erm), 0.06/0.125 (mef). Pristinamycin and vancomycin MICs remained unchanged irrespective of macrolide susceptibility, and amoxicillin, ceftriaxone, and imipenem MICs rose with those of penicillin G.
TABLE 2.
Agar dilution MICs of antimicrobial agents against 205 strains classified by macrolide susceptibility
| Drug | Macrolide sensitivity of straina | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| ABT-773 | S | ≤0.008–0.03 | ≤0.008 | 0.016 |
| erm | ≤0.008–4.0 | 0.03 | 0.125 | |
| mef | 0.016–0.125 | 0.016 | 0.125 | |
| Erythromycin | S | ≤0.008–0.125 | 0.03 | 0.06 |
| erm | 2.0–>64.0 | >64.0 | >64.0 | |
| mef | 0.5–8.0 | 2.0 | 8.0 | |
| Azithromycin | S | 0.016–0.25 | 0.06 | 0.125 |
| erm | 8.0–>64.0 | >64.0 | >64.0 | |
| mef | 0.5–16.0 | 2.0 | 8.0 | |
| Clarithromycin | S | ≤0.008–0.06 | 0.03 | 0.06 |
| erm | 1.0–>64.0 | >64.0 | >64.0 | |
| mef | 0.25–8.0 | 1.0 | 8.0 | |
| Roxithromycin | S | 0.03–0.25 | 0.25 | 0.25 |
| erm | 8.0–>64.0 | >64.0 | >64.0 | |
| mef | 1.0–>64.0 | 8.0 | 32.0 | |
| Clindamycin | S | 0.016–0.06 | 0.06 | 0.06 |
| erm | 0.25–>64.0 | >64.0 | >64.0 | |
| mef | 0.03–0.125 | 0.06 | 0.125 | |
| Pristinamycin | S | 0.125–0.5 | 0.25 | 0.5 |
| erm | 0.125–1.0 | 0.5 | 1.0 | |
| mef | 0.125–1.0 | 0.25 | 0.5 | |
| Amoxicillin | S | ≤0.008–2.0 | 0.125 | 1.0 |
| erm | ≤0.008–8.0 | 0.25 | 2.0 | |
| mef | ≤0.008–8.0 | 1.0 | 2.0 | |
| Ceftriaxone | S | ≤0.008–2.0 | 0.125 | 1.0 |
| erm | ≤0.008–8.0 | 0.25 | 1.0 | |
| mef | ≤0.008–2.0 | 0.5 | 2.0 | |
| Imipenem | S | ≤0.008–0.5 | 0.03 | 0.25 |
| erm | ≤0.008–0.5 | 0.125 | 0.25 | |
| mef | ≤0.008–1.0 | 0.25 | 0.5 | |
| Vancomycin | S | 0.125–0.25 | 0.25 | 0.25 |
| erm | 0.125–0.25 | 0.25 | 0.25 | |
| mef | 0.125–0.25 | 0.125 | 0.25 | |
S, sensitive; erm, strain carrying erm, including three strains carrying erm and mef (see Materials and Methods); mef, strain carrying only mef.
MICs of ABT-773 were compared to those of macrolides and azalides against 21 ermB- and mefE-negative macrolide-resistant strains, comprising 19 strains with L4 ribosomal protein mutations (69GTG71→TPS) and 2 with a 23S rRNA mutation (A2059→G). ABT-773 MICs ranged between 0.03 and 0.25 μg/ml, compared to 16.0 to >64.0 μg/ml for erythromycin, azithromycin, clarithromycin, and roxithromycin. Clindamycin MICs ranged between 0.06 and 0.125 μg/ml for strains containing the L4 mutation but were higher (0.5 to 2.0 μg/ml) for 23S rRNA strains.
Time-kills for six macrolide-susceptible, three ermB- and three mefE-carrying strains, for which MICs were similar to those presented in Tables 1 and 2, are shown in Table 3. MICs were based upon agar dilution and macrodilution, which were all within 1 dilution of each other. The ketolide ABT-773 was bactericidal at 2 times the MIC for 8 of 12 strains (including those highly resistant to macrolides [MICs greater than or equal to 64 μg/ml]). After 12 h, ABT-773 gave 90% killing of 10 of 12 strains at 2 times the MIC. Kill kinetics of the macrolides erythromycin, clarithromycin, and roxithromycin, and the azalide azithromycin, against macrolide-susceptible strains were slower than those of ABT-773. ABT-773 was bacteriostatic against all four strains which did not give 99.9% killing after 24 h (data not shown). Clindamycin, for all strains except those for which the MIC of the antibiotic was greater than or equal to 64 μg/ml, was bactericidal for eight of nine strains tested after 24 h at 2 times the MIC. Pristinamycin was rapidly bactericidal, showing significant killing after 3 h, with bactericidal activity against all 12 strains after 24 h at 2 times the MIC. β-Lactams (amoxicillin, ceftriaxone, and imipenem) started to show significant killing after 12 h; after 24 h at 2 times the MIC, bactericidal activity was seen against 11 strains. Vancomycin gave rapid kill kinetics at early time periods, with bactericidal activity against all 12 strains tested at 2 times the MIC.
TABLE 3.
Time-kill results of 12 pneumococcal strains
| Drug and concn | MIC range (μg/ml) | No. of strains showing bactericidal activity at the specified time period (h)
|
|||
|---|---|---|---|---|---|
| 3 | 6 | 12 | 24 | ||
| ABT-773 | |||||
| 4 times MIC | 0.004–0.06 | 0 | 0 | 4 | 8 |
| 2 times MIC | 0 | 0 | 4 | 8 | |
| MIC | 0 | 0 | 4 | 5 | |
| Erythromycina | |||||
| 4 times | 0.03–>64.0 | 0 | 0 | 2 | 5 |
| 2 times | 0 | 0 | 2 | 4 | |
| MIC | 0 | 0 | 0 | 0 | |
| Azithromycina | |||||
| 4 times MIC | 0.03–>64.0 | 0 | 0 | 2 | 5 |
| 2 times MIC | 0 | 0 | 1 | 5 | |
| MIC | 0 | 0 | 0 | 2 | |
| Clarithromycina | |||||
| 4 times MIC | 0.016–>64.0 | 0 | 0 | 2 | 5 |
| 2 times MIC | 0 | 0 | 2 | 4 | |
| MIC | 0 | 0 | 1 | 2 | |
| Roxithromycina | |||||
| 4 times MIC | 0.06–>64.0 | 0 | 0 | 2 | 5 |
| 2 times MIC | 0 | 0 | 1 | 5 | |
| MIC | 0 | 0 | 1 | 4 | |
| Clindamycinb | |||||
| 4 times MIC | 0.03–>64.0 | 0 | 0 | 3 | 8 |
| 2 times MIC | 0 | 0 | 3 | 8 | |
| MIC | 0 | 0 | 2 | 6 | |
| Pristinamycin | |||||
| 4 times MIC | 0.25–1.0 | 2 | 4 | 11 | 12 |
| 2 times MIC | 2 | 4 | 10 | 12 | |
| MIC | 2 | 4 | 8 | 8 | |
| Amoxicillin | |||||
| 4 times | 0.016–2.0 | 0 | 2 | 9 | 12 |
| 2 times MIC | 0 | 2 | 9 | 11 | |
| MIC | 0 | 1 | 8 | 9 | |
| Ceftriaxone | |||||
| 4 times MIC | 0.03–4.0 | 0 | 1 | 6 | 12 |
| 2 times MIC | 0 | 1 | 6 | 11 | |
| MIC | 0 | 2 | 5 | 10 | |
| Imipenem | |||||
| 4 times MIC | 0.008–0.5 | 2 | 6 | 10 | 11 |
| 2 times MIC | 1 | 5 | 10 | 11 | |
| MIC | 1 | 5 | 9 | 7 | |
| Vancomycin | |||||
| 4 times MIC | 0.25–0.5 | 0 | 3 | 10 | 12 |
| 2 times MIC | 0 | 2 | 10 | 12 | |
| MIC | 0 | 0 | 9 | 5 | |
Only six strains for which MICs were ≤0.125 μg/ml were tested.
Only nine strains for which MICs were ≤0.125 μg/ml were tested.
Microdilution MICs as well as PAEs for the eight strains tested are presented in Table 4. Pristinamycin PAEs (MICs, 0.125 to 0.5 μg/ml) are not given due to rapid bactericidal activity (25). PAEs of macrolides and clindamycin were only tested against five strains with MICs less than or equal to 0.25 μg/ml. Macrolide PAEs were longer than those of β-lactams. ABT-773 had longer PAEs strain for strain (individual data not shown) than macrolides, azalides, or clindamycin. Additionally, the three strains for which macrolide, azalide, and clindamycin MICs were greater than 64.0 μg/ml yielded mean ABT-773 PAEs of 5.6 h (range, 3.7 to 7.5 h), 1.7 h (range, 1.4 to 2.0 h), and 2.9 h (range 2.4 to 3.4 h) (Table 4).
TABLE 4.
MICs and PAEs (h) of eight pneumococcal strains
| Drug | MIC range (h) | Mean PAE (range) (h)a |
|---|---|---|
| ABT-773 | ≤0.008–0.06 | ≥5.6 (1.7–≥7.7) |
| Erythromycinb | 0.03–>64.0 | 3.8 (1.9–7.7) |
| Azithromycinb | 0.06–>64.0 | 2.9 (0.8–5.8) |
| Clarithromycinb | 0.03–>64.0 | 3.9 (2.2–7.7) |
| Roxithromycinb | 0.125–>64.0 | 3.0 (1.9–5.2) |
| Clindamycinb | 0.016–>64.0 | 2.7 (1.6–4.7) |
| Imipenem | ≤0.008–0.5 | 1.5 (1.0–2.4) |
| Ceftriaxone | ≤0.008–2.0 | 1.2 (0–3.0) |
| Amoxicillin | 0.016–2.0 | 1.7 (0.5–2.7) |
| Vancomycin | 0.125–0.25 | 2.2 (1.3–4.1) |
Exposure at 10 times MIC for 1 h. Pristinamycin not included because of rapid bactericidal activity (25).
Three strains not tested because antibiotic MICs were greater than 64.0 μg/ml.
DISCUSSION
ABT-773 is a new ketolide (4) (A. M. Nilius et al., Abstr. 9th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P-177; Z. Ma et al., 39th ICAAC, abstr. 2133; Z. Cao et al., 39th ICAAC, abstr. 2135) which, in preliminary studies, has been reported to be more potent in vitro than the macrolides against Haemophilus influenzae, Moraxella catarrhalis, Legionella spp., Neisseria gonorrhoeae, and Listeria monocytogenes. ABT-773 was also more potent against macrolide-susceptible strains of S. pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, enterococci, Helicobacter pylori, and Mycobacterium avium complex, and also against Corynebacterium spp., Mycoplasma pneumoniae, Chlamydia trachomatis, Borrelia burgdorferi, and Toxoplasma gondii. ABT-773 had potent activity against macrolide-resistant streptococci and enterococci irrespective of their macrolide resistance mechanism but had little detectable activity against constitutively macrolide-resistant staphylococci and macrolide-resistant H. pylori and M. avium complexes (4) (D. Shortridge, N. C. Ramer, J. Beyer, Z. Ma, Y. Or, and R. K. Flamm, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2136, 1999; M. M. Neuhauser, J. L. Prause, R. Jung, N. Boyea, J. M. Hackleman, L. H. Danziger, and S. L. Pendland, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2139, 1999; F. Goldstein, M. D. Kitzis, M. Miegi, and J. F. Acar, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2142, 1999; A. L. Barry, P. C. Fuchs, and S. D. Brown, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2144, 1999; S. L. Pendland, J. L. Prause, M. M. Neuhauser, N. Boyea, J. M. Hackleman, and L. H. Danziger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2145, 1999; R. Jung, D. H. Li, S. L. Pendland, and L. H. Danziger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2146, 1999; A. A. Khan, F. G. Araujo, J. C. Craft, and J. S. Remington, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2147, 1999). ABT-773 has been shown to be effective against S. pneumoniae in an experimental rat lung model (J. Meulbroek, M. Mitten, K. W. Mollison, P. Ewing, J. Alder, A. M. Nilius, R. K. Flamm, Z. Ma, and Y. Or, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2151, 1999).
Results of this study indicate that ABT-773 was very active against macrolide-susceptible and -resistant pneumococci. MICs against macrolide-resistant pneumococci carrying erm did not differ significantly from those seen in mef-carrying strains. Similar results have been reported by others for ABT-773 and by us as well as other workers for telithromycin (4, 26). Additionally, ABT-773 gave low MICs against strains with the L4 and 23S rRNA mutations. The latter mechanism has been recognized only recently, and is present in strains isolated from Central and Eastern Europe (P. C. Appelbaum, unpublished data). A few strains with the 23S rRNA mutation have also been found in clinical specimens (J. A. Sutcliffe, personal communication). The significance of these new resistance mechanisms is currently unknown.
Results for other macrolides, azalides, and other compounds are similar to those reported previously (7, 15–17, 21, 23, 24, 27, 28, 32), with lower macrolide MICs against strains carrying mef than against those carrying erm. In the United States, both mechanisms of macrolide resistance are commonly encountered in clinical specimens (D. Shortridge et al., Abstr. 36th Infect. Dis. Soc. Am. Annu. Meet., abstr. 225F).
Long macrolide PAEs have previously been compared to those of other compounds (10, 14, 29, 30). In the present study, ABT-773 gave the longest PAEs of all drugs tested, including macrolide and nonmacrolide compounds.
Results of the present study document low ABT-773 MICs against macrolide-susceptible and -resistant pneumococci. Although MICs were slightly higher against strains containing erm and mef, MICs were significantly lower than those of other macrolides and azalides. If results of pharmacokinetic, pharmacodynamic (area under the concentration-time curve and MIC), and animal studies are promising, this compound is of potential use for therapy of macrolide-susceptible and -resistant pneumococci.
ACKNOWLEDGMENTS
This study was supported by a grant from Abbott Laboratories, Chicago, Ill.
REFERENCES
- 1.Appelbaum P C. Antimicrobial resistance in Streptococcus pneumoniae—an overview. Clin Infect Dis. 1992;15:77–83. doi: 10.1093/clinids/15.1.77. [DOI] [PubMed] [Google Scholar]
- 2.Block S, Harrison C J, Hedrick J A, Tyler R D, Smith R A, Keegan E, Chartrand S A. Penicillin-resistant Streptococcus pneumoniae in acute otitis media: risk factors, susceptibility patterns and antimicrobial management. Pediatr Infect Dis J. 1995;14:751–759. doi: 10.1097/00006454-199509000-00005. [DOI] [PubMed] [Google Scholar]
- 3.Breiman R F, Butler J C, Tenover F C, Elliott J A, Facklam R R. Emergence of drug-resistant pneumococcal infections in the United States. JAMA. 1994;271:1831–1835. [PubMed] [Google Scholar]
- 4.Brueggemann A B, Doern G V, Huynh H K, Wingert E M, Rhomberg P R. In vitro activity of ABT-773, a new ketolide, against recent clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Antimicrob Agents Chemother. 2000;44:447–449. doi: 10.1128/aac.44.2.447-449.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Craig W A, Gudmundsson S. Postantibiotic effect. In: Lorian V, editor. Antibiotics in laboratory medicine. Baltimore, Md: Williams and Wilkins; 1996. pp. 296–329. [Google Scholar]
- 6.Doern G V, Brueggemann A, Holley H P, Rauch A M. Antimicrobial resistance of Streptococcus pneumoniae isolated from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-center national surveillance study. Antimicrob Agents Chemother. 1996;40:1208–1213. doi: 10.1128/aac.40.5.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Fasola E L, Bajaksouzian S, Appelbaum P C, Jacobs M R. Variation in erythromycin and clindamycin susceptibilities of Streptococcus pneumoniae by four test methods. Antimicrob Agents Chemother. 1997;41:129–134. doi: 10.1128/aac.41.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Friedland I R, Istre G S. Management of penicillin-resistant pneumococcal infections. Pediatr Infect Dis J. 1992;11:433–435. doi: 10.1097/00006454-199206000-00002. [DOI] [PubMed] [Google Scholar]
- 9.Friedland I R, McCracken G H., Jr Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. N Engl J Med. 1994;331:377–382. doi: 10.1056/NEJM199408113310607. [DOI] [PubMed] [Google Scholar]
- 10.Fuursted K, Knudsen J D, Petersen M B, Poulsen R K, Rehm D. Comparative study of bactericidal activities, postantibiotic effects, and effects on bacterial virulence of penicillin G and six macrolides against Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:781–784. doi: 10.1128/aac.41.4.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Geslin P, Fremaux A, Sissia G, Spicq C, Aberrane A. Epidémiologie de la résistance aux antibiotiques de Streptococcus pneumoniae en France. Réseau national de surveillance (1984–1993) Méd Mal Infect. 1994;24:948–961. [Google Scholar]
- 12.Jacobs M R. Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clin Infect Dis. 1992;15:119–127. doi: 10.1093/clinids/15.1.119. [DOI] [PubMed] [Google Scholar]
- 13.Jacobs M R, Appelbaum P C. Antibiotic-resistant pneumococci. Rev Med Microbiol. 1995;6:77–93. [Google Scholar]
- 14.Licata L, Smith C E, Goldschmidt R M, Barrett J F, Frosco M. Comparison of the postantibiotic and postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:950–955. doi: 10.1128/aac.41.5.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Liñares J, Alonso T, Pérez J L, Ayats J, Domínguez M A, Pallarés R, Martín R. Decreased susceptibility of penicillin-resistant pneumococci to twenty-four β-lactam antibiotics. J Antimicrob Chemother. 1992;30:279–288. doi: 10.1093/jac/30.3.279. [DOI] [PubMed] [Google Scholar]
- 16.Liñares J, Pallares R, Alonso T, Perez J L, Ayats J, Gudiol F, Viladrich P F, Martin R. Trends in antimicrobial resistance of clinical isolates of Streptococcus pneumoniae in Bellvitge hospital, Barcelona, Spain (1979–1990) Clin Infect Dis. 1992;15:99–105. doi: 10.1093/clinids/15.1.99. [DOI] [PubMed] [Google Scholar]
- 17.Mason E O, Jr, Kaplan S L, Lamberth L B, Tillman J. Increased rate of isolation of penicillin-resistant Streptococcus pneumoniae in a children's hospital and in vitro susceptibilities to antibiotics of potential therapeutic use. Antimicrob Agents Chemother. 1992;36:1703–1707. doi: 10.1128/aac.36.8.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.McDougal L K, Facklam R, Reeves M, Hunter S, Swenson J M, Hill B C, Tenover F C. Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States. Antimicrob Agents Chemother. 1992;36:2176–2184. doi: 10.1128/aac.36.10.2176. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Munoz R, Musser J M, Crain M, Briles D E, Marton A, Parkinson A J, Sorensen U, Tomasz A. Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis. Clin Infect Dis. 1992;15:112–118. doi: 10.1093/clinids/15.1.112. [DOI] [PubMed] [Google Scholar]
- 20.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 3rd ed. 1997. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Villanova, Pa. [Google Scholar]
- 21.Nelson C T, Mason E O, Jr, Kaplan S L. Activity of oral antibiotics in middle ear and sinus infections caused by penicillin-resistant Streptococcus pneumoniae: implications for treatment. Pediatr Infect Dis J. 1994;13:585–589. doi: 10.1097/00006454-199407000-00001. [DOI] [PubMed] [Google Scholar]
- 22.Pankuch G A, Jacobs M R, Appelbaum P C. Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, ciprofloxacin, and vancomycin by using time-kill methodology. Antimicrob Agents Chemother. 1994;38:2065–2072. doi: 10.1128/aac.38.9.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Pankuch G A, Jacobs M R, Appelbaum P C. Activity of CP99,219 compared with DU-6859a, ciprofloxacin, ofloxacin, levofloxacin, lomefloxacin, tosufloxacin, sparfloxacin and grepafloxacin against penicillin-susceptible and -resistant pneumococci. J Antimicrob Chemother. 1995;35:230–232. doi: 10.1093/jac/35.1.230. [DOI] [PubMed] [Google Scholar]
- 24.Pankuch G A, Jacobs M R, Appelbaum P C. Comparative activity of ampicillin, amoxycillin, amoxycillin/clavulanate and cefotaxime against 189 penicillin-susceptible and -resistant pneumococci. J Antimicrob Chemother. 1995;35:883–888. doi: 10.1093/jac/35.6.883. [DOI] [PubMed] [Google Scholar]
- 25.Pankuch G A, Lichtenberger C, Jacobs M R, Appelbaum P C. Antipneumococcal activities of RP 59500 (quinupristin/dalfopristin), penicillin G, erythromycin, and sparfloxacin determined by MIC and rapid time-kill methodologies. Antimicrob Agents Chemother. 1996;40:1653–1656. doi: 10.1128/aac.40.7.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Pankuch G A, Visalli M A, Jacobs M R, Appelbaum P C. Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents. Antimicrob Agents Chemother. 1998;42:624–630. doi: 10.1128/aac.42.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Spangler S K, Jacobs M R, Appelbaum P C. Susceptibilities of penicillin-susceptible and -resistant strains of Streptococcus pneumoniae to RP 59500, vancomycin, erythromycin, PD 131628, sparfloxacin, temafloxacin, Win 57273, ofloxacin, and ciprofloxacin. Antimicrob Agents Chemother. 1992;36:856–859. doi: 10.1128/aac.36.4.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Spangler S K, Jacobs M R, Pankuch G A, Appelbaum P C. Susceptibility of 170 penicillin-susceptible and -resistant pneumococci to six oral cephalosporins, four quinolones, desacetylcefotaxime, Ro 23-9424 and RP 67829. J Antimicrob Chemother. 1993;31:273–280. doi: 10.1093/jac/31.2.273. [DOI] [PubMed] [Google Scholar]
- 29.Spangler S K, Lin G, Jacobs M R, Appelbaum P C. Postantibiotic effect of sanfetrinem compared with those of six other agents against 12 penicillin-susceptible and -resistant pneumococci. Antimicrob Agents Chemother. 1997;41:2173–2176. doi: 10.1128/aac.41.10.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Spangler S K, Lin G, Jacobs M R, Appelbaum P C. Postantibiotic effect and postantibiotic sub-MIC effect of levofloxacin compared to those of ofloxacin, ciprofloxacin, erythromycin, azithromycin and clarithromycin against 20 pneumococci. Antimicrob Agents Chemother. 1998;42:1253–1255. doi: 10.1128/aac.42.5.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother. 1996;40:2562–2566. doi: 10.1128/aac.40.11.2562. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Tweardy D J, Jacobs M R, Speck W T. Susceptibility of penicillin-resistant pneumococci to eighteen antimicrobials: implications for treatment of meningitis. J Antimicrob Chemother. 1983;12:133–139. doi: 10.1093/jac/12.2.133. [DOI] [PubMed] [Google Scholar]