Department of Microbiology, Bristol-Myers Squibb Company, Wallingford, CT 06492, USA
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Abstract |
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Introduction |
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In this study, we determined the activity of gatifloxacin in more than 60 bacterial species and compared it with those of ciprofloxacin and ofloxacin.
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Materials and methods |
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Gatifloxacin was obtained from Kyroin Pharmaceutical Co. Ltd, Tochigi, Japan. Ciprofloxacin was prepared at BristolMyers Squibb France, Montpellier, France. Ofloxacin was purchased from Sigma Chemical Co., St Louis, MO, USA.
Bacterial strains
All bacterial strains used in this study were clinical isolates obtained from numerous sources of broad geographical distribution. Isolates were maintained frozen in liquid nitrogen.
Growth inhibitory activity
MIC values were determined by an agar dilution method in accordance with the procedures outlined by the NCCLS.5 Inocula were adjusted to yield approximately 5 x 104 cfu/spot. MuellerHinton II agar (MHA; BBL Microbiology Systems, Cockeysville, MD, USA) was used for all but the following: streptococci, Listeria monocytogenes, Corynebacterium spp., Leuconostoc spp., Pediococcus spp., Lactobacillus spp., Gardnerella vaginalis and Campylobacter jejuni (all MHA plus 5% defibrinated sheep blood); Haemophilus spp. (Haemophilus test medium); Legionella spp. (buffered starch yeast extract agar); Neisseria gonorrhoeae [GC medium base (BBL) supplemented with 1% Supplement C]; Helicobacter pylori (MHA plus 1% glycerol and 10% sheep blood), Bartonella spp. (TSA II agar plus 5% rabbit blood and 1% yeast extract) and anaerobes [WilkinsChalgren agar (Oxoid) supplemented with 5% defibrinated sheep blood]. Culture plates were incubated aerobically at 35°C for 18 h (G. vaginalis and Haemophilus, Neisseria, Legionella, Pediococcus, Lactobacillus and Leuconostoc spp. in 5% CO2 for 24 h; H. pylori in 10% CO2 for 72 h; Bordetella spp. in ambient air for 72 h; Bartonella spp., in 5% CO2 for 5 days), or in a microaerophilic atmosphere with the Campy-Pak system (BBL Microbiology Systems) for C. jejuni, or for 48 h in an anaerobic atmosphere for anaerobes. The MIC was defined as the lowest concentration of drug that prevented visible growth.
Susceptibility tests with Mycobacterium spp. were performed by a macrobroth dilution method in 7H9 broth (Difco) and 2 x 107 cfu/ml final bacterial inocula. The tubes were incubated at 35°C in 5% CO2. Inhibition of growth was monitored for 3 weeks although growth controls almost always demonstrated very heavy growth after 7 days' incubation. The MIC was defined as the lowest concentration of drug that inhibited visible growth after 3 weeks' incubation. All work involving mycobacteria was done under a biological safety cabinet in a high-containment facility.
The MICs for Chlamydia trachomatis and Chlamydia pneumoniae were determined using, respectively, McCoy (ATCC) and HL6 cells (Washington Research Foundation, Seattle, WA, USA) in a microtitre format. Chlamydial suspensions prepared in a maintenance medium [Eagle's minimal essential medium (Gibco) supplemented with fetal bovine serum for C. trachomatis and Iscove medium for C. pneumoniae] contained approximately 1001000 inclusion-forming units per mL, with 0.05 mL of the suspension added per well. The plates were centrifuged at 1000g for 60 min. Two-fold serial dilution of the quinolones in maintenance medium was added (100 µL). Plates were incubated for 48 h (for C. trachomatis) or 72 h (C. pneumoniae) in 5% CO2 and then fixed with methanol and stained with 0.02 mL of fluorescein-conjugated monoclonal antibody specific to the chlamydial lipopolysaccharide. The presence of chlamydial inclusion bodies was detected via an inverted fluorescent microscope at x100 magnification. The MIC was defined as the lowest drug concentration with no observable inclusion bodies.
MICs for Mycoplasma pneumoniae, Mycoplasma hominis and Ureaplasma urealyticum were performed by a microbroth dilution method. The test medium was PPLO medium (Difco) supplemented with yeast extract, horse serum and either dextrose (for M. pneumoniae), arginine (for M. hominis) or urea (for U. urealyticum). Two-fold serial dilutions of the quinolones were prepared in the test medium. The inoculum contained 104105 colour-changing units (CCU) per mL, and 100 µL of the adjusted inoculum was added to the 100 µL of the diluted drug in each well. A CCU is the minimum inoculum required for growth as indicated by a colour change in phenol indicator. MIC was defined as the lowest dilution of antibiotics that inhibited growth as indicated by the lack of a colour change, relative to the growth control with colour change.
Susceptibility testing of Borrelia burgdorferi was done in 48-well microtitre plates using BSK II medium supplemented with 6% haemolysed rabbit serum. Quinolones were two-fold serially diluted in growth medium. Two- to 3 day growth of B. burgdorferi was added to a final inoculum of approximately 1 x 105 cfu/mL. The plates were incubated for 4 days at 34°C in a microaerophilic atmosphere (Campy-Pak) and then 10 µL samples were removed and examined under dark-field microscopy at x200 magnification. The MIC was defined as the lowest drug concentration that inhibited all growth per 10 fields examined.
The interpretative MIC breakpoints used to analyse the data in this study were those recommended by the NCCLS.5 The MIC breakpoints for ciprofloxacin are 1 mg/L, susceptible; 2 mg/L, intermediate; and
4 mg/L, resistant. For ofloxacin, the MIC breakpoints are:
2 mg/L, susceptible; 4 mg/L, intermediate; and
8 mg/L, resistant.5 The proposed gatifloxacin interpretative criteria are
2 mg/L, susceptible; 4 mg/L, intermediate; and
8 mg/L, resistant.
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Results and discussion |
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Gatifloxacin was about two- to four-fold more potent than ciprofloxacin and ofloxacin against methicillin-susceptible (MS) strains of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus saprophyticus, with modal MIC90s of 0.12 mg/L for gatifloxacin and 0.250.5 mg/L for ciprofloxacin and ofloxacin (Table I). These quinolone MIC90 values increased >16-fold against methicillin-resistant (MR) strains of staphylococci. Based on their interpretative criteria for susceptibility, all of the MS S. aureus strains were susceptible to the three quinolones. In contrast, 51%, 55% and 78% of the 58 MR S. aureus strains had MICs at or below the susceptible breakpoint for ciprofloxacin, ofloxacin and gatifloxacin, respectively. Nevertheless, since clinical resistance to ciprofloxacin developed quickly among MR S. aureus,7,8 the utility of gatifloxacin against this group of organisms may depend also on the rate of resistance development to gatifloxacin. We have reported that the frequency of selecting for less susceptible variants of MR S. aureus by gatifloxacin was 100-fold less than by ciprofloxacin.9 It remains to be determined whether these in vitro observations on gatifloxacin will translate to its clinical usefulness in the treatment of MR S. aureus infections.
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Gatifloxacin was active against Enterococcus faecalis (MIC50 and MIC90 of 0.5 and 1 mg/L), and against some strains of Enterococcus faecium (MIC50 and MIC90 of 1 and 4 mg/L). Ciprofloxacin and ofloxacin had lower potency against enterococci.
Gatifloxacin was the most potent agent against L. monocytogenes, G. vaginalis and Gram-positive bacterial species that are intrinsically resistant to vancomycin (i.e. Leuconostoc, Lactobacillus and Pediococcus spp.) (Table II). All three quinolones were less potent against Pediococcus spp. For the most part, gatifloxacin was active against many imipenem-resistant strains of Corynebacterium spp., including strains of Corynebacterium jeikeium and Corynebacterium urealyticum (formerly CDC group D2).
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The quinolones were very active against members of the family Enterobacteriaceae (Escherichia coli, Klebsiella spp., Citrobacter spp., Enterobacter/Pantoea spp., Serratia marcescens, Salmonella spp., Shigella spp. and Yersinia spp.), with lesser activity against Providencia spp. (Table I). Gatifloxacin (MIC90s, 0.060.5 mg/L) was often two-fold less potent than ciprofloxacin, and the same as or two-fold more potent than ofloxacin against the Enterobacteriaceae. The quinolones were less potent against Providencia rettgeri (MIC90, 1, 2 and 4 mg/L of gatifloxacin, ofloxacin and ciprofloxacin, respectively), and Providencia stuartii (MIC90, 4 mg/L for gatifloxacin compared with >16 mg/L for ciprofloxacin and ofloxacin, respectively). Gatifloxacin and ofloxacin were two- to four-fold less potent than ciprofloxacin against Pseudomonas aeruginosa. Against Pseudomonas fluorescens and Pseudomonas stutzeri, the corresponding gatifloxacin MIC90s were 8 and 0.25 mg/L, the same as ofloxacin MIC90s, while ciprofloxacin MIC90s were four- to eight-fold lower. Gatifloxacin and ciprofloxacin had comparable, though poor, potencies against Burkholderia cepacia (MIC50s and MIC90s, 4 and 8 mg/L), but gatifloxacin was more potent (MIC50 and MIC90, 1 and 4 mg/L) than either ciprofloxacin or ofloxacin against Stenotrophomonas maltophilia.
Gatifloxacin had good potency against Acinetobacter spp. (MIC90s, 0.51 mg/L), Alcaligenes and Flavobacterium spp. All three quinolones were very potent (MIC90s 0.25 mg/L) against Vibrionaceae and Campylobacteraceae.
Fastidious microorganisms
Gatifloxacin was exquisitely potent (MIC90s, 0.016 0.25 mg/L) against N. gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Haemophilus parainfluenzae, H. pylori, Bordetella spp. and Legionella spp. (Table I
). While gatifloxacin and ciprofloxacin had comparable potencies (MICs
1 mg/L) against B. burgdorferi and Bartonella spp., they were about two- to eight-fold more potent than ofloxacin against these species (Table II
).
Of the three quinolones, gatifloxacin (MICs, 0.13 mg/L) was the most potent against C. trachomatis and C. pneumoniae, being eight- to 16-fold more potent than ofloxacin and ciprofloxacin (Table I). Thus, given its higher intracellular accumulation in eukaryotic cells,10,11 gatifloxacin should be very effective against these pathogenic organisms.
Based on MIC50 values, gatifloxacin was four- to eight-fold more potent than ciprofloxacin and ofloxacin against ureaplasma, 10-fold more potent against M. pneumoniae and eight- to 16-fold more potent against M. hominis (Table I). The MICs of gatifloxacin for mycoplasma were
0.25 mg/L, while they were much higher (MIC range, 0.54 mg/L) for ureaplasma. While ureaplasma might be intrinsically less susceptible than mycoplasma to quinolones, one primary contributing factor for the higher MICs could be the use of urea as the indicator in ureaplasma testing. Urea supplementation results in an acidic pH, a condition known to affect adversely the activity of quinolones.12 Thus, it is likely that quinolones are intrinsically more active against ureaplasma than these MICs suggest.
Mycobacterium spp.
Gatifloxacin was active against Mycobacterium tuberculosis, being eight- to 16-fold more potent than ciprofloxacin and ofloxacin (Table I). Though gatifloxacin was less potent against Mycobacterium aviumintracellulare (MAI) strains (Table II
), it was again four-fold more active than comparator quinolones. Interestingly, although complete inhibition of growth was achieved at 2 mg/L of gatifloxacin in three MAI strains, partial inhibition of two additional MAI strains was noted.
Anaerobic bacteria
Unlike ciprofloxacin and ofloxacin, which were active only against Clostridium perfringens and Propionibacterium acnes (MIC90s, 1 mg/L), gatifloxacin was active (MICs, 0.52 mg/L) against Bacteroides fragilis, Clostridium difficile, Peptostreptococcus spp. and other Bacteroides spp. (Tables I and II). Ciprofloxacin and ofloxacin MIC90s for B. fragilis and C. difficile were > 8 mg/L. With the exception of the five Fusobacterium varium (gatifloxacin MICs, 24 mg/L), gatifloxacin MICs were
0.5 mg/L for the other Fusobacterium spp.
In summary, gatifloxacin has a broad antibacterial spectrum. Compared with ciprofloxacin and ofloxacin, gatifloxacin has notably enhanced potency against contemporary Gram-positive aerobic bacteria, S. maltophilia, anaerobic bacteria, chlamydia, mycoplasma/ureaplasma and mycobacteria.
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Notes |
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References |
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2
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Received 28 July 1999; returned 18 October 1999; revised 19 October 1999; accepted 28 October 1999