Barbara La T. Prosser

Method of evaluating effects of antibiotics on bacterial biofilm.

Antibiotics are generally not effective against organisms in exopolysaccharide biofilms. A simple method of studying the effect of antibiotics on bacteria in established biofilms is reported. Escherichia coli ATCC 25922 cells grown overnight at 37 degrees C on Mueller-Hinton agar were suspended in buffer and dispensed on 0.5-cm2 catheter disks. The disks were incubated for 1 h at 37 degrees C, washed, transferred to petri dishes containing 20 ml of broth, and incubated at 37 degrees C for 20 to 22 h, at which time thick biofilms were established. Disks were washed, placed in broth or broth containing antibiotic, and incubated at 37 degrees C for 4 h. The disks were removed, and viable counts were determined. This process was repeated at other selected time intervals (e.g., 8 and 24 h). Viable bacterial counts decreased from 10(3) to 10(4) CFU/cm2 in 24 h with 400 micrograms of amdinocillin or cefamandole per ml. A combination containing 400 micrograms of each antibiotic per ml decreased the viable counts to an undetectable level (less than 100 CFU/cm2) in 24 h. Other antibiotics and organisms were also examined in this system. Images

Pharmacokinetics of [18F]fleroxacin in healthy human subjects studied by using positron emission tomography.

Positron emission tomography (PET) with [18F]fleroxacin was used to study the pharmacokinetics of fleroxacin, a new broad-spectrum fluoroquinolone, in 12 healthy volunteers (9 men and 3 women). The subjects were infused with a standard therapeutic dose of fleroxacin (400 mg) supplemented with approximately 20 mCi of [18F]fleroxacin. Serial PET images were made and blood samples were collected for 8 h, starting at the initiation of the infusion. The subjects were then treated with unlabeled drug for 3 days (400 mg/day). On the fifth day, infusion of radiolabeled drug, PET imaging, and blood collection were repeated. In most organs, there was rapid accumulation of radiolabeled drug, with stable levels achieved within 1 h after completion of the infusion. Especially high peak concentrations (in micrograms per gram) were achieved in the kidney (> 34), liver (> 25), lung (> 20), myocardium (> 19), and spleen (> 18). Peak concentrations of drug more than two times the MIC for 90% of Enterobacteriaceae strains tested (> 10-fold for most organisms) were achieved in all tissues except the brain and remained above this level for more than 6 to 8 h. The plateau concentrations in tissues (2 to 8 h, in micrograms per gram +/- standard error of the mean) of drug were as follows: brain, 0.83 +/- 0.032; myocardium, 4.53 +/- 0.24; lung, 5.80 +/- 0.48; liver, 7.31 +/- 0.33; spleen, 6.00 +/- 0.47; bowel, 3.53 +/- 0.74; kidney, 8.85 +/- 0.64; bone, 2.87 +/- 0.29; muscle, 4.60 +/- 0.33; prostate, 4.65 +/- 0.48; uterus, 3.87 +/- 0.39; breast, 2.68 +/- 0.11; and blood, 2.35 +/- 0.09. Concentrations of fleroxacin in tissue were similar in males and females, before and after pretreatment with unlabeled drug. Images

Multicenter in vitro comparative study of fluoroquinolones against 25,129 Gram-positive and Gram-negative clinical isolates☆

In vitro activities of fleroxacin, ciprofloxacin, ofloxacin, and lomefloxacin were evaluated against 25,129 fresh bacterial isolates from 51 US hospital or medical center laboratories, beginning in October of 1990. Susceptibility rates were > or = 85% against most species of Gram-negative bacteria. Notable exceptions were Pseudomonas, Acinetobacter, Xanthomonas, and Providencia. The study drugs displayed similar activity against most Gram-negative species. At least 90% of oxacillin-susceptible staphylococci were susceptible but, of oxacillin-resistant strains, only approximately 60% of Staphylococcus epidermidis and 25% of Staphylococcus aureus were susceptible to the quinolones tested. Staphylococcus saprophyticus strains were less susceptible to fleroxacin (42%) than to the other compounds (79%-97%). Ofloxacin and ciprofloxacin were more active against streptococci, and none of the compounds demonstrated appreciable activity against enterococci. Thus, the spectra of activity of fluoroquinolones illustrate that they remain effective agents for the treatment of many types of infections caused by Gram-negative pathogens.

Pharmacokinetics of 18F-labeled fleroxacin in rabbits with Escherichia coli infections, studied with positron emission tomography.

18F-labeled fleroxacin was used to measure the pharmacokinetics of fleroxacin in healthy and infected animals by positron emission tomography (PET) and tissue radioactivity measurements. In all experiments, a pharmacological dose of unlabeled drug (10 mg/kg) was coinjected with the tracer. The pharmacokinetics of [18F]fleroxacin was measured in groups of healthy mice (n = six per group) at 10, 30, 60, and 120 min after injection and in groups of rats with Escherichia coli thigh infections (n = six per group) at 60 and 120 min after injection by radioactivity measurements in excised tissues. In healthy rabbits (n = 4) and in rabbits with E. coli thigh infections (n = 4), tissue concentrations of drug were determined by serial PET imaging over 2 h; after the final image was acquired, animals were sacrificed and concentrations measured by PET were compared with the results of tissue radioactivity measurements. In all three species, there was rapid equilibration of [18F]fleroxacin to significant concentrations in most peripheral organs; low concentrations of drug were detected in the brain. Accumulations of radiolabeled drug in infected and healthy thigh muscles were similar. Peak concentrations of drug of more than three times the MIC for 90% of members of the family Enterobacteriaceae (greater than 100-fold for most organisms) were achieved in all tissues except brain and remained above this level for more than 2 h. Especially high peak concentrations were achieved in the kidney (greater than 75 micrograms/g), liver (greater than 50 micrograms/g), blood (greater than 25 micrograms/g), and bone and lung (greater than 10 micrograms/g).Since the MICs for 90% of all Enterobacteriaceae are <2 micrograms/ml, fleroxacin should be particularly useful in treating gram-negative infections affecting these tissues. In contrast, the low concentration of drug delivered to the brain should limit the toxicity of the drug for the central nervous system. Images

Comparative evaluation of fleroxacin, ampicillin, trimethoprimsulfamethoxazole, and gentamicin as treatments of catheter-associated urinary tract infection in a rabbit model

Fleroxacin, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin were comparatively evaluated for effectiveness in treating experimentally induced catheter-associated urinary tract infection and bacteriuria in a rabbit model with a closed drainage system. Fleroxacin, ampicillin and gentamicin effectively eliminated a lactose-negative, streptomycin-resistant uropathogenic strain of Escherichia coli (WE6933) from bag urine and catheter port urine, while trimethoprim-sulfamethoxazole only marginally reduced urine bacterial counts when compared to rabbits that received no antibiotic therapy. Fleroxacin eliminated E. coli from the catheter surfaces and from tissues adjacent to the catheter. Ampicillin or gentamicin therapy also eliminated biofilm bacteria from the catheter surfaces, but did not eliminate th residual bacteria from tissue adjacent to the septic catheters despite achieving urine levels of antibiotics substantially higher than minimum bactericidal concentrations for this pathogen. Trimethoprim-sulfamethoxazole was ineffective in eliminating E. coli from the catheter surfaces and the adjacent tissues. The ability of fleroxacin to effectively eliminate biofilm bacteria from catheter surfaces and tissues adjacent to such medical devices in the urinary tract may prove useful in the treatment of catheter-associated urinary tract infection and bacteriuria in mammals and humans.

Synthesis and biodistribution of 18F-labeled Fleroxacin

[18F]Fleroxacin (6,8-difluoro-1,4-dihydro-1-(2-[18F]fluoroethyl)-4- oxo-7-(4-methyl-1-piperazinyl)-3-quinolinecarboxylic acid) was synthesized from its methylsulfonyl ester precursor. 6,7,8-Trifluoro-4-hydroxyquinoline-3-carboxylic acid ethyl ester (Ro 19-7423) was alkylated with 2-bromoethanol to produce 6,7,8-trifluoro-1,4-dihydro-1-(2-hydroxyethyl)-4-oxo-3-quinolinecarboxyl ic acid ethyl ester in 76% yield which was then condensed with 1-methyl-piperazine to produce 6,8-difluoro-1,4-dihydro-1-(2-hydroxyethyl)-7-(4-methyl-1-piperazinyl)4- oxo-3- quinolinecarboxylic acid ethyl ester in 67% yield. This product was reacted with methanesulfonyl chloride to produce the mesylate precursor of fleroxacin in 66% yield. Nucleophilic substitution of the mesylate with 18F- in the presence of Kryptofix 2.2.2 followed by basic hydrolysis produced [18F]fleroxacin with a radiochemical yield of 5-8% [EOS] within 90 min. The pattern of biodistribution of [18F]fleroxacin was similar to the 14C-labeled drug.

Streptomyces longwoodensis sp. nov.

A new species of Streptomyces is described for which the name S. longwoodensis is proposed. The organism produces a polyether ionophorous antibiotic, lysocellin. It is characterized by its gray spore mass color, spiral spore chain with smooth spores, chromogenic reaction on ISP media 1 and 6, and carbon utilization characteristics. It differs from all streptomycetes described previously on the basis of its carbon utilization, protein hydrolysis, and pigment production. The type strain of S. longwoodensis is Roche X-14537 (=ATCC 29251).

Tissue pharmacokinetics of fleroxacin in humans as determined by positron emission tomography

The delivery of fleroxacin, a new broad-spectrum fluoroquinolone, to the major organs of the body was studied in 12 normal human volunteers (nine men and three women), utilizing positron emission tomography (PET). Following the infusion of 20 mCi of [(18)F]fleroxacin in conjuction with a standard therapeutic dose of 400 mg, images were acquired over 8 h. Beginning the next day, the subjects received unlabeled drug at a dose of 400 mg/day for 3 days, with a repeat PET study on the fifth day. Fleroxacin is distributed widely throughout the body, with the notable exception of the central nervous system, with stable levels achieved within 1 h after completion of the infusion. Especially high peak concentrations (18 mug/g) were achieved in the kidney, liver, lung myocardium, and spleen. The mean plateau concentrations (2-8 h post-infusion, mug/g) were: brain 0.83; myocardium, 4.53; lung, 5.80, liver, 7.31; spleen, 6.00; bowel, 3.53; kidney, 8.85; bone, 2.87; muscle, 4.60; prostate, 4.65; uterus, 3.87; breast, 2.68; and blood, 2.35. Repetitive dosing had no significant effect on the pharmacokinetics of the drug. Since the MIC(90)s of the family Enterobacterioaceae and Neisseria gonorrhoeae are <2 mug/ml, with the great majority of the individual species 1 mug/ml, these results suggest that a single daily dose of 400 mg of fleroxacin should be effective in the treatment of infections such as urinary tract infection and gonorrhea.

Nutritional Characterization of Some Selected Actinomycetes

Strains of several genetically important Streptomyces species and four strains of Nocardia mediterranei were screened for nutritional properties by using 150 different organic compounds. The methodology for an extensive nutritional analysis of actinomycetes is described, and the results are discussed on the basis of their taxonomic and ecological implications.

In vitro Activity of Ceftriaxone and Amikacin against Pseudomonas aeruginosa

Ceftriaxone and amikacin were combined at ratios of 1:1, 5:1, and 10:1 and tested in vitro against Pseudomonas aeruginosa. The activity was determined using the Steers-Foltz replicator and the agar dilution technique with Mueller-Hinton agar. Under all conditions tested, including those simulating severe infection (10(5) to 10(6) colony-forming units per spot), the organisms were more susceptible to the combination than to the single agents. With a conventional inoculum of 10(4) colony-forming units per spot, the combinations gave 97-100% coverage against P. aeruginosa. The increased activity of the combinations resulted in MIC90 values which were below the expected serum/plasma levels for significantly longer time periods than the MIC90 values observed with the individual agents.