Enhanced activity of beta-lactam antibiotics with amdinocillin in vitro and and in vivo.

An important advantage of antibiotic combinations which can be determined is the increase in spectrum of activity. Ad hoc combinations of amdinocillin with ampicillin, ticarcillin, piperacillin, azlocillin, cefazolin, cefamandole, cefoxitin, and moxalactam in ratios similar to those in serum after parenteral administration of normal dosages of these agents were tested in vitro against over 200 strains of bacteria, including Staphylococcus aureus, Streptococcus faecalis, representative genera of the Enterobacteriaceae, and Pseudomonas aeruginosa. Independent of amdinocillin's activity, it was possible to show marked enhancement of the spectrum of all the beta-lactam agents tested, except moxalactam, against members of the Enterobacteriaceae. Little or no enhancement or antagonism of activity was seen against gram-positive bacteria or P. aeruginosa. The enhancing activity of amdinocillin on three beta-lactam antibiotics, ampicillin, cefazolin, and cefoxitin, was also demonstrated in experimental mouse infections with gram-negative bacteria.

Antimicrobial activity of ceftriaxone: a review.

Ceftriaxone possesses potent activity, both in vitro and in vivo, against a broad range of bacteria. MIC50 and MIC90 geometric means were calculated using the results of broth and agar dilution assays performed worldwide. The MIC90 for ceftriaxone overall was 8 micrograms/ml or less for Enterobacteriaceae and 0.024 microgram/ml or less for Neisseria and Hemophilus species. Moderate activity was noted against Pseudomonas and Acinetobacter species (MIC50 12 to 28 micrograms/ml). Ceftriaxone was extremely active against nonenterococcal streptococci (MIC90 0.07 microgram/ml or less) and quite active against methicillin-susceptible Staphylococcus aureus (MIC90 5 micrograms/ml or less). Ceftriaxone generally was inactive against enterococci and methicillin-resistant staphylococci. Activity against anaerobes was good, except for many strains of Bacteroides fragilis and B. thetaiotaomicron (MIC greater than 64 micrograms/ml). Ceftriaxone exhibited excellent stability to beta-lactamases. The effect of medium and inoculum on in vitro testing was minimal. Excellent activity was demonstrated in vivo. Against Enterobacteriaceae, nonenterococcal streptococci, and H. influenzae, the PD50 in mice generally was less than 1 mg/kg. S. aureus strains responded moderately (mean PD50 6.5 mg/kg), whereas against most P. aeruginosa strains, PD50s ranged from 5 to greater than 250 mg/kg. The superior pharmacokinetic profile of ceftriaxone compared with that of other new cephalosporins was demonstrated by use of a prophylactic treatment schedule. The ability of ceftriaxone to penetrate the cerebrospinal fluid and provide excellent therapeutic coverage was confirmed in experimental meningitis models.

Interpretation of susceptibility testing of ceftriaxone.

Susceptibility of a variety of bacterial isolates to ceftriaxone was determined by Kirby-Bauer assays using 30 micrograms ceftriaxone disks and by microdilution (MIC) assays using standard procedures. The relation between zones of inhibition and MICs was expressed by the following regression equation: zone diameter = 22.98-2.653 In (MIC). Using this regression line and the breakpoints estimated from ceftriaxone concentrations in plasma 12 to 24 hours after 1- and 2-g doses, the susceptibility of a pathogen to ceftriaxone was classified as follows: susceptible-zone 16 mm or greater, MIC 16 micrograms/ml or less; moderately susceptible-zone 13 to 15 mm, MIC 17 to 63 micrograms/ml; resistant-zone 12 mm or less, MIC 64 micrograms/ml or greater. These breakpoints were used to determine the susceptibility of organisms isolated during clinical studies in the United States. The correlation between the in vitro results and the bacteriologic outcomes achieved in the clinical cases was analyzed to assess the suitability of the chosen breakpoints. The results of the disk assays were correctly predictive of bacteriologic responses with 1,388 of 1,513 organisms (91.7 percent), whereas the results of dilution assays correctly predicted the response with 897 of 941 organisms (95.3 percent). The correlation between in vitro results and bacteriologic outcome in patients treated with ceftriaxone was equivalent or superior to that achieved in patients treated with the comparative agents cefamandole and cefazolin. Thus, the chosen cutoff points for indicating susceptibility and resistance to ceftriaxone appear to be suitable and highly predictive of clinical success.

Morphologic changes produced by amdinocillin alone and in combination with beta-lactam antibiotics: in vitro and in vivo.

Scanning electron microscopy was used to study the morphologic effects of amdinocillin (mecillinam) when combined with several beta-lactam antibiotics in vitro (Escherichia coli, three isolates; Klebsiella pneumoniae, one isolate) and also in vivo (E. coli, one isolate). Ovoid forms were found in the cultures of E. coli and K. pneumoniae following in vitro exposure to amdinocillin. This characteristic in vitro effect was also produced in the amdinocillin-treated E. coli-infected mouse. Varying degrees of filament formation were seen both in vitro and in vivo with the other beta-lactam antibiotics tested. The in vitro combination of amdinocillin with the beta-lactam antibiotics produced morphologic effects on E. coli and K. pneumoniae (enhanced cell distortion and lysis) not seen with the individual agents at the doses tested. Amdinocillin was synergistic with ampicillin, carbenicillin, and cephalothin in mice challenged with E. coli 736; scanning electron microscopy of bacteria from peritoneal lavages of mice treated with these synergistic combinations indicated that the organisms were more enlarged and distorted than those from animals receiving the individual agents. The enhanced morphologic effect observed in vivo was in agreement with the in vitro effect. Viable counts of bacteria recovered from mice treated with ampicillin plus amdinocillin were appreciably less than those from mice treated with each agent alone. The morphologic results from the scanning electron microscopy study point to a synergistic or enhanced effect of amdinocillin in combination with beta-lactam antibiotics and are in accord with prior reports of the synergistic effects of amdinocillin.

Therapeutic efficacy of fleroxacin for eliminating catheter-associated urinary tract infection in a rabbit model.

The efficacy of fleroxacin as therapy for experimentally induced catheter-associated urinary tract infection (CAUTI) was examined. A rabbit model of CAUTI using a closed urinary catheter drainage system and the mutant strain of Escherichia coli (WE 6933) were used to examine three dosage regimens (30 mg/kg q8h i.v.; 20 mg/kg q8h i.v.; and 10 mg/kg q8h++i.v.) of fleroxacin administered intravenously for 4 days. Quantitative bacterial counts, urinary concentrations of fleroxacin and desmethylferoxacin, histopathologic changes, and electron microscopic evaluation of catheter-associated biofilm and mucosal biofilm were performed. The results indicated that the bacterial biofilm on the urinary catheter could be eliminated by fleroxacin at 30 mg/kg q8h i.v. and 20 mg/kg q8h i.v. Fleroxacin concentrations in urine exceeded the levels necessary to destroy E. coli. Viable bacteria were eliminated with the third regimen (10 mg/kg q8h i.v.), but electron microscopy demonstrated remnants of bacterial biofilm. Histopathologic changes were significantly reduced in all fleroxacin-treated rabbits, and scanning electron microscopy showed deterioration of the bacterial biofilm on the surface of the Foley catheter in treated animals. These data suggest that fleroxacin may be useful for treating catheter-related infections because these therapeutic dosages limited ascending infections of the urethra and bladder, eliminated catheter-associated biofilms, and killed planktonic bacteria in urine.

Pharmacokinetics of fleroxacin as studied by positron emission tomography and [18F]fleroxacin.

A new method of tracing the disposition of fleroxacin was tested in infected and noninfected animals in an effort to develop a technique that might be applicable in humans. [18F]fleroxacin was synthesized and shown to be identical physically, chemically, and in its antimicrobial activity to the commercially produced product. Tracer amounts of [18F]fleroxacin were coinjected with a pharmacologic dose of unlabeled drug (10 mg/kg) into normal mice, rats with focal thigh infection due to Escherichia coli, and normal and infected rabbits. The rats and mice were killed at fixed time intervals after injection, and the concentration of drug was determined by radioactive counting in a well-type counter; the rabbits were studied both by this method and by positron emission tomographic (PET) imaging. These studies validated the reliability of the new approach and suggested that it could be applied safely to humans. In all three animal species studied, delivery of [18F]fleroxacin to most tissues was rapid, with the notable exception of the brain. Accumulation of drug in infected thigh muscle was similar to that in normal muscle. The concentrations of drug reached in various tissues suggest that fleroxacin will be particularly useful in the treatment of gastrointestinal, urinary tract, hepatobiliary, and skeletal infections and that it shows promise for the treatment of lung and soft tissue infection. The minimal concentrations of drug delivered to the brain should decrease the occurrence of central nervous system toxicity with this particular fluoroquinolone.

Cephalosporin 3'-quinolone esters with a dual mode of action.

According to the generally accepted mechanism by which bacterial enzymes react with cephalosporins, opening of the beta-lactam ring can lead to the expulsion of a 3'-substituent. A series of dual-action cephalosporins was prepared in which antibacterial quinolones were linked to the cephalosporin 3'-position through an ester bond in the expectation that, in addition to exerting their own beta-lactam activity, these cephalosporins would act as prodrugs for the second antibacterial agent. Compared to parent cephalosporins in which the 3'-substituent was acetoxy, the bifunctional cephalosporins exhibited a broadened antibacterial spectrum, suggesting that a dual mode of action may indeed be operative.

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.

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.

A simple, rapid 125I radioimmunoassay for the detection of barbiturates in biological fluids.

A simple, rapid radioimmunoassay employing 125-I-labeled secobarbital derivative has been developed and has been shown to be capable of detecting at the nanogram level a variety of barbiturates in urine as well as in plasma.

Bactericidal activity of daptomycin versus vancomycin in the presence of human albumin against vancomycin-susceptible but tolerant methicillin-resistant Staphylococcus aureus (MRSA) with daptomycin minimum inhibitory concentrations of 1-2microg/mL.

This study explored the influence of vancomycin tolerance and protein binding on the bactericidal activity of vancomycin versus daptomycin (protein binding 36.9% vs. 91.7%, respectively) against four vancomycin-tolerant methicillin-resistant Staphylococcus aureus (MRSA) [minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC)=0.5/16, 1/32, 2/32 and 1/32microg/mL for vancomycin and 1/1, 1/2, 2/2 and 2/4microg/mL for daptomycin]. Killing curves were performed with vancomycin/daptomycin concentrations equal to serum peak concentrations (C(max)) (65.70/98.60microg/mL) and trough concentrations (C(min)) (7.90/9.13microg/mL) in the presence and absence of a physiological human albumin concentration (4g/dL), controlled with curves with the theoretical free drug fraction of vancomycin/daptomycin C(max) (41.45/8.18microg/mL) and C(min) (4.98/0.76microg/mL). Vancomycin C(max) and C(min) concentrations, regardless of the media, showed a bacteriostatic profile not reaching a reduction of 99% or 99.9% of the initial inocula during the 24-h experimental time period. Daptomycin antibacterial profiles significantly differed when testing C(max) and C(min). C(max) was rapidly bactericidal (< or =4h) with >5 log(10) reduction in the initial inocula for all strains, regardless of the presence or not of albumin or the use of concentrations similar to free C(max). C(min) exhibited similar final colony counts at 0h and 24h in curves with albumin, but with >3 log colony-forming units (CFU)/mL reduction at < or =4h for strains with an MIC of 1microg/mL and ca. 2 logCFU/mL reduction at < or =6h for strains with an MIC of 2microg/mL. This activity was significantly higher than the activity of the free C(min) fraction. The results of this study reinforce the idea that pharmacodynamics using concentrations calculated using reported protein binding are unreliable. Daptomycin exhibited rapid antibacterial activity against vancomycin-tolerant MRSA isolates even against those with high daptomycin MICs in the presence of physiological albumin concentrations.

Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate.

Grunberg, E. (Hoffman-La Roche, Inc., Nutley, N.J.), and R. Cleeland. Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate. J. Bacteriol. 92:23-27. 1966.-Washed cell suspensions of Proteus mirabilis, under the proper conditions, stained well with fluorescein isothiocyanate with little or no loss of cell viability. The speed and intensity of the reaction was dependent on both the concentration of dye and pH. Within a range of pH 3.0 to 10.0, staining was most rapid at pH 5.0 to 6.0, with a slower and less intense reaction occurring at the other pH values. As the concentration of dye at either pH 5.0 or 9.0 was increased from 10 to 1,000 mug/ml, there was an increase in the rate of staining but a decrease in cell viability. After 24 hr of incubation at 4 C, pH 5.0, and a dye concentration of 10 mug/ml, all cells were stained, the majority exhibiting intense fluorescence with little or no loss of viability noted. In preliminary experiments with Staphylococcus aureus, similar results were obtained. Of various other fluorescent dyes tested, only rhodamine isothiocyanate was found to give satisfactory staining.

Automated technique for the detection of hemagglutinins to sheep erythrocytes.

The Technicon autoanalyzer system used for the detection of homologous human hemagglutinins has been modified for the detection of hemagglutinins to sheep red blood cells. Antibody quantitation was obtained by plotting the optical density (OD) readings of peak heights of a serially diluted reference serum versus the serum dilutions. When the OD of the peak height of an unknown sample fell within the linear portion of the plot, a direct determination of the antibody titer was made. If the OD of the sample fell outside the linear portion, dilutions of the sample were carried out until a direct reading could be made. Assay by this method of at least 140 samples was possible within a day. Titers obtained with the autoanalyzer agreed very closely with those obtained by manual titration.

Effect of mg and ethylenediaminetetraacetate on the in vitro activity of coumermycin a(1) and novobiocin against gram-negative bacteria.

The effect of Mg(2+) and ethylenediaminetetraacetic acid (EDTA) on the in vitro activity of coumermycin A(1) and novobiocin was determined against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis grown in Trypticase Soy Broth or defined synthetic medium. In the presence of EDTA, the activity of coumermycin A(1) was significantly enhanced (8- to 40-fold) against all four organisms in the synthetic medium lacking Mg(2+); novobiocin activity was increased to a lesser extent (4- to 16-fold) against three strains and remained unchanged against P. mirabilis. Addition of 200 mug of Mg(2+) per ml to the synthetic medium containing EDTA completely reversed the enhancement of activity of both antibiotics by EDTA only against P. aeruginosa. The addition of Mg(2+) to the synthetic medium appreciably inhibited the activity of novobiocin (sixfold) but not coumermycin A(1) against E. coli, and had little or no effect on the activity of either antibiotic against the remaining organisms. K. pneumoniae showed the greatest susceptibility to both antibiotics, and addition of EDTA enhanced this activity from 4-fold to 20-fold in all of the media tested. Subinhibitory concentrations of both antibiotics induced filament formation by all four organisms in all media. In addition, lysis of K. pneumoniae cells was observed.

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.

In vivo activity of amoxicillin and ampicillin against gram-positive bacteria: results of prophylactic studies.

In prophylactic treatment of intraperitoneal infections with Streptococcus pneumoniae (types 1 and 2) and Streptococcus pyogenes in mice, amoxicillin had a definite advantage over ampicillin in terms of protective effect. When the infecting agent was given to mice so as to produce an infection in the brain or lung (for example, S. pneumoniae given intracranially or intranasally), amoxicillin was also more effective prophylactically than ampicillin.

Activity of oral amoxicillin, ampicillin, and oxytetracycline against infection with chlamydia trachomatis in mice.

The effects in mice of oral treatment with amoxicillin, ampicillin, and oxytetracycline against an otherwise lethal intranasal infection with Chlamydia trachomatis (mouse pneumonitis) were studied. When treatment was started 30 min after infection and continued once daily thereafter for a total of seven treatments, the mean protective doses of amoxicillin, ampicillin, and oxytetracycline were 9.5, greater than 50, and 31.3 mg/kg, respectively. If 14 oral treatments were given, these values were 1.6 mg/kg for amoxicillin, 12.7 mg/kg for ampicillin, and 12.3 mg/kg for oxytetracycline.

Antiviral activity of 10-carboxymethyl-9-acridanone.

Intraperitoneal administration of 10-carboxymethyl-9-acridanone sodium salt (CMA) protected at least 50% of mice tested from otherwise lethal infections with Semliki forest, coxsackie B1, Columbia SK, Western equine encephalitis, herpes simplex, and pseudorabies viruses. The protective effect against influenza A2/Asian/J305 and coxsackie A21 viruses was less but was statistically significant. When administered either subcutaneously or orally, CMA protected at least 50% of mice against Semliki forest and pseudorabies viruses; the effect against coxsackie B1 and herpes simplex viruses was less but was statistically significant. Initiation of treatment could be delayed from 2 to 24 h after infection of mice with coxsackie B1, herpes simplex, Semliki forest, and Western equine encephalitis viruses without loss of an antiviral effect. CMA did not inactivate Semliki forest or coxsackie B1 viruses on contact and was without effect against any of the viruses tested in tissue culture by the tube dilution assay. The humoral antibody response in mice to both influenza virus and sheep erythrocytes was unaffected by CMA. After administration of CMA, an interferon-like substance was induced in mice or mouse cell culture but not in rabbits or rabbit cell culture.