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Nonfluorinated quinolones, developed in the 1960s and 1970s, had a spectrum limited to aerobic gram-negative bacteria; they had a poor systemic distribution but reached high concentrations in urine; their use was therefore confined to the treatment of urinary tract infections. The introduction of fluoroquinolones (i.e., molecules fluorinated in the C-6 position) marked a dramatic improvement (2). The earlier drugs of this group, developed in the late 1970s and in the 1980s, were suitable for a far wider clinical use by virtue of a broader spectrum, encompassing gram-positive bacteria, and a good systemic distribution. Other fluoroquinolones, which had further-enhanced activity against gram-positive bacteria and were also variably active against anaerobes, were developed in the 1990s (10). However, several of these newer quinolones ended up being limited in their clinical use due to various, unexpected toxicity problems (1).

AF 3013, a fluoroquinolone formerly called NM394 (5, 8, 11), is the active compound derived from the transformation of the prodrug prulifloxacin (also called NM441 or AF 3012) after its oral administration and intestinal absorption. Prulifloxacin and AF 3013 were developed in the late 1980s in Japan. The in vitro activity of AF 3013 has so far been investigated only in that country, against strains mostly isolated in the 1980s (11). At present, prulifloxacin is being considered for marketing in Italy and other European countries. This prompted us to investigate the in vitro activity of its active form, AF 3013, against a variety of nosocomial and community bacterial strains recently isolated in Italy. AF 3013 was obtained from Angelini ACRAF, Pomezia, Italy. Of six additional fluoroquinolone molecules tested as comparators, two (ciprofloxacin and ofloxacin) were purchased from Sigma Chemical Co. (St. Louis, Mo.), and four (levofloxacin, sparfloxacin, trovafloxacin, and moxifloxacin) were from their respective manufacturers.

Bacteria. A total of 654 cultures of aerobic bacteria were examined, all freshly isolated (in 1998 to 2000) from clinical material in several Italian laboratories. Multiple isolates from the same patient were avoided. Most of these cultures (151 gram-positive strains and 245 gram-negative strains), isolated from inpatients >48 h after hospital admission, were regarded as being associated with nosocomial infections. The remaining (102 gram-positive and 156 gram-negative) strains, isolated from outpatients, were regarded as being associated with community infections. Most strains were initially identified using commercial and automated biochemical test systems, but the identification of several isolates was confirmed by evaluating additional distinguishing characters relating to the laboratory determination of genus and species (4). Based on MIC tests and breakpoints as recommended by the National Committee for Clinical Laboratory Standards (7), staphylococci were preliminarily characterized as oxacillin susceptible (MICs of 2 µg/ml for Staphylococcus aureus strains and 0.25 µg/ml for coagulase-negative staphylococci) or oxacillin resistant (MICs of 4 µg/ml for S. aureus strains and 0.5 µg/ml for coagulase-negative staphylococci).

MIC tests. MICs were determined by standard microdilution broth tests as recommended by the National Committee for Clinical Laboratory Standards (7). Antibiotics were tested at final concentrations, prepared from serial twofold dilutions, ranging from 0.015 to 128 µg/ml. Except for Haemophilus strains, the test medium was Mueller-Hinton II broth (BBL Microbiology Systems, Cockeysville, Md.), supplemented with 5% lysed horse blood when streptococci and listeriae were tested. The Haemophilus strains were tested in Haemophilus test medium (7). The inoculum was 5 × 10⁵ CFU/ml. The inoculated trays were incubated at 35°C for 18 to 24 h.

MBC tests. Minimal bactericidal concentrations (MBCs) were established by extending the MIC procedure to the evaluation of bactericidal activity (6). After the MIC was read, 0.01-ml volumes were drawn with an Eppendorf pipette from the wells showing no growth and spotted onto suitable agar plates. These plates were incubated at 35°C for 24 to 48 h. The MBC was read as the lowest antibiotic concentration which resulted in 0.1% survival in the subculture.

Time-kill assays. AF 3013 and ciprofloxacin were investigated for their killing kinetics against four test strains (A430, an oxacillin-susceptible S. aureus isolate; C330, a ciprofloxacin-susceptible Escherichia coli isolate; C1061, a ciprofloxacin-resistant E. coli isolate; and M578, a Proteus mirabilis isolate) (Fig. 1). Time-kill experiments were performed by standard procedures (6, 9) using tubes containing 10 ml of the test medium used in MIC assays. Three doubling concentrations of each antibiotic (1, 2, and 4 times the MIC) were tested. Drug-free control tubes were included in each run. To inoculate each tube of serially diluted antibiotic, 50 µl of diluted inoculum was delivered by pipette beneath the surface of the broth. The mixture was then vortexed and plated for viability counts (0 h) as reported below. Only tubes containing an initial inoculum within the range of 5 × 10⁵ to 1 × 10⁶ CFU/ml were considered acceptable. Cultures were incubated at 35°C with shaking. At 4-, 8-, and 24-h intervals, viable counts were performed in triplicate by spreading aliquots of 0.1 ml of the suitable dilutions onto plates of Trypticase soy agar (BBL). Plates were incubated for up to 48 h, and colony counts were performed on plates yielding 30 to 300 colonies (3). A threshold corresponding to the lower end of the range (30 colonies, i.e., 300 CFU/ml) was therefore used. Antibiotics were considered bactericidal when the original inoculum was reduced by 3 log₁₀ CFU/ml (99.9%).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Killing kinetics of AF 3013 and ciprofloxacin against four clinical isolates: S. aureus A430 (oxacillin susceptible), E. coli C330 (ciprofloxacin susceptible), E. coli C1061 (ciprofloxacin resistant), and P. mirabilis M578. , growth control; , 1× MIC; , 2× MIC; , 4× MIC. All experiments were done in triplicate. Standard deviations of the reported values were below 1 log.

Conclusion. On the whole, the in vitro activity of AF 3013 against gram-positive bacteria appeared to be greater than that of ofloxacin, similar to or greater than that of ciprofloxacin, similar to or at a lower level than those of levofloxacin and sparfloxacin, and at a lower level than those of trovafloxacin and moxifloxacin. Against gram-negative bacteria (with the exception of Stenotrophomonas maltophilia and Acinetobacter isolates), AF 3013 was more active than ciprofloxacin and generally even more active than the other compared drugs. With reference to the earlier Japanese study (11), the comparison with ofloxacin and sparfloxacin for gram-positive bacteria and with ciprofloxacin for both gram-positive and gram-negative bacteria appears to be more in favor of AF 3013 for recently collected Italian isolates. Time-kill experiments using selected isolates confirmed that the bactericidal activity of AF 3013 was at least similar to that of ciprofloxacin. Altogether, the excellent inhibitory and bactericidal activities exhibited by AF 3013 in vitro, in addition to the favorable characteristics shown by its prodrug (prulifloxacin) in preliminary in vivo studiesexperimental infections in mice (8), pharmacokinetic investigations with mice and dogs (8), and pharmacokinetic and safety studies with healthy human volunteers (5)warrant further in vitro and in vivo studies and appropriate clinical trials addressing in particular the treatment of urinary infections.