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Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.

In a multicenter cooperative controlled study, individual test, accuracy, and precision control values were determined for 75-mug ticarcillin disks with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.

A regression line for correlating zones of inhibition of a 75-mug mezlocillin disk with minimal inhibitory concentrations of mezlocillin for Enterobacteriaceae and nonfermenters was calculated. Correlation was excellent (r = -0.96) for all species tested except Pseudomonas cepacia.

The minimal inhibitory concentrations of clindamycin and gentamicin alone and in combinations were determined by a microdilution method for 163 aerobic, facultative, and anaerobic clinical isolates. All 77 strains of Staphylococcus aureus, Diplococcus pneumoniae, Streptococcus pyogenes, and anaerobic bacteria (except for three strains of Clostridium) were inhibited by 1.6 mug or less of clindamycin per ml. Gentamicin did not interfere with the activity of clindamycin within the range of concentrations tested (0.1 to 100 mug/ml); for some strains combinations were synergistic. Sixty-two (94%) of 66 strains of Enterobacteriaceae and Pseudomonas aeruginosa were inhibited by 6.2 mug or less of gentamicin per ml. Combinations of clindamycin and gentamicin were indifferent for 29 strains and synergistic for 33 strains. All 20 strains of enterococcus, three strains of Clostridium, three strains of Escherichia coli, and one strain of Proteus rettgeri were resistant to both clindamycin (minimal inhibitory concentration greater than 3.1 mug/ml) and gentamicin (minimal inhibitory concentration greater than 6.2 mug/ml). Combinations of clindamycin and gentamicin were indifferent for 16 and synergistic for 11 of the resistant strains. Except for clindamycin-sensitive isolates, synergy was usually observed only at concentrations of one or both drugs which are not readily obtainable in vivo. Antagonism was never observed.

A simple, abbreviated broth dilution test (tube test) utilizing a commercially available medium and inexpensive disposable materials, and which could be performed entirely in room air, was developed and used to test the susceptibility of 100 strains of anaerobic bacteria to clindamycin, chloramphenicol, ampicillin, and tetracycline. Results are reported in categories of susceptibility: susceptible to concentrations surpassed in vivo with usual dosage, susceptible to concentrations surpassed in vivo with high dosage, and resistant to concentrations achievable in vivo. Results are compared to minimal inhibitory concentrations which were determined simultaneously by using a microdilution method in an anaerobic glove box. Twenty strains of Bacteroides fragilis, 10 strains of Fusobacterium, 20 strains of Clostridium, 10 strains of gram-positive non-sporeforming bacilli, and 30 strains of cocci grew to visible turbidity within 1 day of incubation. Of the 360 antibiotic-organism combinations tested, 98% were in a susceptibility category that corresponded (within one concentration) to the actual minimal inhibitory concentration as determined by the microdilution method. After 2 days of incubation, growth was more abundant, but results often indicated inappropriate degrees of resistance. Variation in inoculum size had little effect on results. Ten strains of B. melaninogenicus did not grow enough for susceptibility to be categorized accurately. The tube test could be used in any clinical microbiology laboratory for a limited number of susceptibility tests on anaerobic bacteria other than B. melaninogenicus without preparation of special media or purchase of special equipment.

The susceptibilities to spectinomycin of 303 recent urinary tract isolates were determined and compared to the susceptibilities of those strains to ampicillin, tetracycline, and gentamicin. Based on minimal inhibitory concentrations, 84% of Escherichia coli, Klebsiella, and Enterobacter, 31% of other Enterobacteriaceae, 7% of Staphylococcus aureus and Streptococcus (including enterococci), and 0% of Pseudomonas aeruginosa were susceptible to concentrations of spectinomycin that are easily surpassed in serum (

Endpoint determinations of endotoxin by the Limulus amoebocyte lysate assay have been facilitated by use of a microdilution procedure.

Pseudomonas aeruginosa was exposed to 0.1, 1.0, 10, 100, and 1,000 times the minimal inhibitory concentration of ticarcillin in vitro and subsequently examined with the scanning electron microscope. The morphological alterations observed were filamentation, mid-cell defects, and spheroplast formation, and these alterations were dependent upon the drug concentration.

The minimal inhibitory concentrations of gentamicin and minocycline alone and in combination were determined by a broth microdilution method for 100 aerobic, facultative, and anaerobic isolates representative of pathogens recovered from patients with intra-abdominal sepsis. Gentamicin inhibited all strains of Klebsiella, Enterobacter, and Pseudomonas aeruginosa in concentrations of 0.4 to 3.1 mug/ml and all strains of Escherichia coli and Proteus mirabilis in concentrations of 0.8 to 12.5 mug/ml. Whereas minocycline did not consistently inhibit these organisms in concentrations of 1.6 mug or less/ml, it did act synergistically with gentamicin against 43% of the Enterobacteriaceae tested in clinically achievable concentrations; significant synergy was most common with E. coli (60%). Minocycline inhibited 62% of Bacteroides fragilis, 71% of Clostridium, 40% of anaerobic cocci, and 40% of enterococci tested in concentrations of 1.6 mug or less/ml. Whereas gentamicin rarely inhibited these organisms in concentrations of 6.2 mug or less/ml, it did act synergistically with minocycline against 20% of B. fragilis, 67% of Clostridium, 22% of anaerobic cocci, and 22% of enterococci (which had minimal inhibitory concentrations of minocycline within the range tested) at clinically achievable concentrations. Although only four (13%) of the 30 isolates resistant to both gentamicin and minocycline alone were inhibited by clinically achievable concentrations of the combination, the observed synergy, particularly against strains of E. coli, was considered to be of potential clinical usefulness. Antagonism between gentamicin and minocycline was not observed at the concentrations tested.

To evaluate the potential usefulness of a single large oral dose of trimethoprim-sulfamethoxazole (TMP-SMZ) for the treatment of uncomplicated genitourinary gonorrhea, the pharmacokinetics of a 12-tablet dose containing 960 mg of TMP and 4,800 mg of SMZ were studied in 15 male volunteers, and the tolerance of this regimen was compared to that of a placebo in a double-blind crossover study. Both TMP and SMZ were rapidly absorbed. Peak mean serum concentrations (+/- standard deviation) of TMP, total SMZ, and free SMZ were 9.2 +/- 2.2, 259.4 +/- 40.9, and 233.7 +/- 33.6 mug/ml, respectively. Elimination half-lives were 16.7, 14.6, and 12.9 h, respectively. When results were compared to data from similar studies after smaller doses, peak mean serum concentrations were proportional to dose, but elimination half-lives were longer after larger doses. Urinary concentrations of TMP, total SMZ, and free SMZ were many-fold higher than serum concentrations. Percents recovery (+/- standard deviation) in urine were 60.6 +/- 10.6, 80.2 +/- 7.8, and 37.4 +/- 6.5%, respectively, during the 48 h after administration. The incidence of severe headache and of objective transient oliguria was significantly higher after TMP-SMZ than after placebo. Although the observed serum concentrations of TMP and SMZ surpassed concentrations necessary to inhibit clinical isolates of Neisseria gonorrhoeae in vitro for longer than 24 h, the adverse reactions associated with a 12-tablet dose of TMP-SMZ would preclude the clinical usefulness of such a therapeutic regimen.

The in vitro activities of ticarcillin (T) and carbenicillin (C) against 253 strains of Pseudomonas aeruginosa (108 randomly selected clinical isolates and 145 isolates determined to be C resistant by 10 referring hospitals) were evaluated using both broth microdilution and disk diffusion techniques. T activity was usually twofold or greater than C activity for individual strains in both sample populations. The minimal inhibitory concentration (MIC) values of T and C were 400 (median, 25) and 0.8 to >400 (median, 50) mug/ml, respectively, for the random sample and 1.6 to >400 (median, 50) and 12.5 to >400 (median, 200) mug/ml, respectively, for the select sample. For predicting susceptibility (MIC,

A microdilution technique using commercially available media and materials was developed and used to determine the minimal inhibitory concentrations (MICs) of clindamycin, chloramphenicol, tetracycline, minocycline, ampicillin, carbenicillin, cephalothin, and gentamicin for 101 anaerobic isolates. Representative strains of Bacteroides, Clostridium, Fusobacterium, Peptococcus, and Peptostreptococcus were tested. The use of Schaedler broth at pH 7.2, an inoculum of 10(5) to 10(7) colony-forming units per ml, and incubation at 35 C in an anaerobic glove box with an atmosphere of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide resulted in good growth and easily interpretable results. After 48 h of incubation, 97% of strains tested were inhibited by 3.1 mug or less of clindamycin per ml and 98% were inhibited by 12.5 mug or less of chloramphenicol per ml. Tetracycline and minocycline inhibited 81 and 88% of strains tested in concentrations of 1.6 mug or less per ml and 1.6 mug or less per ml, respectively. Ampicillin inhibited all strains other than B. fragilis in concentrations of 3.1 mug or less per ml. Excluding certain strains of Bacteroides and Clostridium, carbenicillin in concentrations of 12.5 mug or less per ml and cephalothin in concentrations of 6.2 mug or less per ml inhibited all strains tested. Gentamicin was inactive although some strains of anaerobic cocci and Bacteroides were inhibited by 3.1 mug or less per ml. After 18 to 24 h of incubation, eight of the 101 strains had not grown sufficiently for MICs to be determined; for the 93 strains which had grown sufficiently, 93% of 744 MICs were the same or one concentration lower than the 48-h MICs.

Chemical and cellular characteristics of fluid within subcutaneously implanted polyethylene chambers in rabbits were studied over a 3-month period. The fluid attained a relatively stable protein and cellular composition which was consistent with a mononuclear exudate. After a single dose of intramuscular penicillin G, the antibacterial activity of chamber fluid was found to be dynamic and similar to the serum antibacterial activity. This animal model may be useful for in vivo studies of the interaction of microorganisms with antimicrobial agents.

Antibiotic dynamics in the fluid obtained from subcutaneously implanted chambers in rats were similar to those observed in serum following single intramuscular injections of carbenicillin.