In Vitro Bactericidal Activities of ABT-773 against ermB Strains of Streptococcus pneumoniae

Abstract

The bactericidal activities of ABT-773, a new ketolide, were compared to those of cefuroxime and amoxicillinclavulanate against 10 strains of Streptococcus pneumoniae containing the ermB gene. MICs and time-kill curves were determined in duplicate per NCCLS guidelines with cation-adjusted Mueller-Hinton broth with 3% lysed horse blood. Viable counts were done at 0, 2, 6, and 24 h. Antibiotic concentrations tested were two and eight times the MIC. ABT-773 MICs ranged from 0.008 to 1.0 g/ml. Bactericidal activity was observed with ABT-773 at eight times the MIC against 4 of 10 strains at 24 h compared to 10 of 10 strains with the beta-lactam antibiotics.The ketolides, semisynthetic 14-membered ring macrolides, represent a new subclass of agents in the macrolide-lincosamide-streptogramin group. The substitution of the L-cladinose sugar with a 3-keto group on the erythronolide A ring is the major differing structural component of the ketolides (2). Despite structural similarity, the ketolides maintain in vitro activity against macrolide-resistant strains of Streptococcus pneumoniae. One of the newest agents, ABT-773, has demonstrated higher rates of accumulation as well as stronger ribosome binding than erythromycin with macrolide-sensitive strains of S. pneumoniae (2). ABT-773 has also demonstrated the ability to accumulate in S. pneumoniae isolates containing the mef gene. Even in the presence of the erm gene, ABT-773 was shown to bind to methylated ribosomes, although binding was less than that to the wild type. It is these properties that allow the ketolide to remain active against macrolide-resistant organisms (2). The MIC of ABT-773 at which 90% of the isolates tested are inhibited has been reported previously to be 0.125 g/ml for 78 ermB-and 44 mefE-containing strains (3).With in vitro activity against macrolide-resistant S. pneumoniae (1), the ketolides are prime candidates for the treatment of community-acquired respiratory tract infections. The purpose of this study was to compare the in vitro bactericidal activities of ABT-773, amoxicillin-clavulanate, and cefuroxime against ermB-containing strains of S. pneumoniae. MIC and time-kill assays were performed on 10 clinical isolates of S. pneumoniae containing the ermB gene. PCR was used to determine the genes present in the macrolide-resistant strains of S. pneumoniae. Bacterial DNA was prepared with the Wizard genomic DNA purification kit (Promega, Madison, Wis.). Primers (mefE and ermB) were prepared at the University of Illinois at Chicago Protein Research Laboratory as previously described (8, 9).The isolates were stored at Ϫ70°C and underwent three subcultures prior to MIC and time-kill studies. The control strain, S. pneumoniae ATCC 49619, was used for validation of MIC results (5). For each experiment, the organisms were freshly subcultured on blood agar plates (Remel, Lenexa, Kans.) and incubated overnight at 35°C with 5% CO 2 . Each inoculum was prepared by direct suspension and adjusted with sterile saline until the turbidity matched a 0.5 McFarlan...

Interaction of the New Ketolide ABT-773 (Cethromycin) with Human Polymorphonuclear Neutrophils and the Phagocytic Cell Line PLB-985 In Vitro

“…Uptake was more rapid at 40°C than at 37°C (C/E, 253,218 [P Ͻ 0.05]), but there were no significant differences at further incubation times. The activation energy (⌬G; in calories per mole) was calculated as described previously (14,23,24) after incubation of PMNs and ketolides at 0, 20, 37, and 40°C for 5 min by using the Arrhenius equation: ⌬G ϭ ϪRT ln K eq , where R is a constant (1.98 cal/mol), T is temperature (in degrees Kelvin), and K eq is the C/E at 5 min. ⌬G values for ABT-773 were 50 kJ/mol for PMNs, 40 kJ/mol for D-PLB, and 37 kJ/mol for ND-PLB.…”

“…ABT-773 is active in vitro (9,19,24,27) and in vivo (12,21) against penicillin-resistant and inducibly erythromycin-resistant staphylococci, streptococci, and pneumococci. No data are yet available, however, on its uptake by phagocytes.…”

“…Various experimental conditions were used to study the mechanism of uptake, as follows: cell viability (pretreatment of PMNs with 10% formaldehyde, followed by two washes in HBSS); pH, 7 to 9; temperature, 0, 20, 37, and 40°C; extracellular concentrations, 2.5 to 50 g/ml; and pretreatment for 15 min with various metabolic inhibitors (NaF, KCN, or 2,4-dinitrophenol at 1 mM each) or with various activators or inhibitors of PMN functions which have been reported to interfere with macrolide uptake (17,23,24), namely, Ni 2ϩ , a blocker of the Na ϩ -Ca 2ϩ exchanger (0.625 to 5 mM); PMA, a protein kinase C (PKC) activator (100 and 10 ng/ml); H89, a protein kinase A (PKA) inhibitor (50 M); and verapamil, a Ca 2ϩ channel blocker (62.5 to 250 M). We also studied the inhibitory effects of various macrolides and ketolides, and the quinolone levofloxacin (100 g/ml) was used as a control of passive accumulation (28).…”

“…A total of 2.5 ϫ 10 6 cells were loaded with the drugs at 10 g/ml (30 min at 37°C) and centrifuged through the oil cushion. The cell pellet was sonicated in the presence of 0.5% Triton X-100 or 0.73 M sucrose to protect the granules (17,23,24). After centrifugation, the amounts of lysozyme (a granule marker) and radiolabeled drugs in the pellet and the supernatant were determined as described previously (23).…”

Efficacy of Cethromycin, a New Ketolide, against Streptococcus pneumoniae Susceptible or Resistant to Erythromycin in a Murine Pneumonia Model

“…Moreover, ketolides showed a second ribosomal binding site at domain V, as well as at domain II of the 23S RNA subunit (13). Cethromycin (ABT-773), one of the newest agents, has a high in vitro activity against a variety of resistant phenotypes, including activity against penicillin-and macrolide-resistant gram-positive bacteria (15,18,24).…”