Azithromycin 993

The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.

Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptibleStaphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 × 10−10 at 2 to 4× the MIC) than with levofloxacin or ciprofloxacin (3.0 × 10−7 to 3.0 × 10−8 at 2 to 4× the MIC). Mutants with a change in GrlA (Ser80→Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC50] and MIC90, 1 and 4 μg/ml, respectively); in comparison, MIC50s and MIC90s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 μg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 μg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80→Phe and Glu84→Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.

Bisulfite or sulfite was found to be inhibitory to Helicobacter pyloni growth. A modified version of Brucella broth (BB), bisulfite-less BB (BLBB), supported rapid, robust, and consistent growth of H. pylori. We suggest that BLBB simply be called "Pylori broth" for distinction from Brucella broth. Helicobacter pylori, a gram-negative, spiral bacterium, has been implicated as the etiological agent for gastritis and as a major contributing factor in the development of peptic gastroduodenal ulcers (2, 5, 8, 9, 12, 13). It has also been linked to an increased risk of gastric cancer (3). Since its first isolation in 1983 (9, 17), H. pylori has generally been described as an oxygen-susceptible, fastidious microaerophile (1, 10, 11, 14-16). Growth media and incubation conditions that provide an environment of reduced oxygen tension have commonly been used for culturing H. pylori. Brucella broth (BB) is one of the frequently used media (10, 11, 14-16). The medium consists of rich carbon and nitrogen sources, such as tryptone, peptamin, yeast extract, and glucose, and two salts, sodium chloride and sodium bisulfite. Bisulfite is thought to be useful in reducing oxygen tension in the medium. In our earlier experience with BB, growth of H. pylori was consistently more robust in older medium which had been stored in the refrigerator than in freshly prepared medium. Since bisulfite (HS03 1) and sulfite (S-3) can gradually be oxidized to sulfate (SO4 2) on

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