Molecular quantification of genes encoding for green-fluorescent proteins.

A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively coamplified DNA standard constructed by point mutation PCR. A single base difference was introduced to achieve a suitable migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant.

Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria.

A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA. The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively co-amplified DNA standard constructed by point mutation PCR. After computing the denaturation behavior of the target DNA stretch, a single base difference was introduced to achieve maximum migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to detect small sequence differences and to monitor recombinant DNA in effluxes of biotechnological plants.

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction.

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

Treatment of serious Pseudomonas infections with azlocillin.

Azlocillin is a semisynthetic acylureidopenicillin with increased activity against most strains of Pseudomonas aeruginosa. It was given as the sole antibacterial agent in the treatment of 21 patients with serious pulmonary, wound, bone or joint, or urinary tract infections, endocarditis, or malignant external otitis caused by Pseudomonas sp. In preliminary in vitro tests, azlocillin inhibited 90% of 36 clinical isolates, while carbenicillin and ticarcillin inhibited only 60% and 73%, respectively. Mean MIC of azlocillin against Ps. aeruginosa isolated from the 21 study patients was 9.8 mg/l; more than 50% of the strains were inhibited by a concentration of 6.25 mg/l. Intravenous administration of the antibiotic at a mean dosage of 17 g/day for 6 to 59 days resulted in an excellent or good clinical response in 90% (19) of the patients treated. Pseudomonas sp. was eliminated from the site of infection in 67% (14) of the patients. Azlocillin therapy was well tolerated; in only two patients, both of whom had penicillin-type rashes, was it necessary to discontinue therapy. Azlocillin was a safe and effective antimicrobial agent for the treatment of serious infections caused by strains of Pseudomonas sp., primarily Ps. aeruginosa.

A single injection of moxalactam for acute gonorrhea.

This study evaluated the efficacy of and tolerance to moxalactam in the treatment of uncomplicated gonorrhea, including two infections with penicillin-resistant strains. After appropriate cultures, 87 women and 64 men each received 1 gm of moxalactam intramuscularly as a single injection. Penicillinase-producing Neisseria gonorrhoeae was isolated from one man who had persistent urethritis after therapy with ampicillin. Another isolate showed high relative resistance to penicillin. At follow-up in three to seven days, moxalactam eradicated N gonorrhoeae from all 120 evaluable patients including the two with penicillin resistance and five women with rectal gonorrhea. Moxalactam administered intramuscularly was well tolerated and the few adverse effects were usually mild. In this study, 1 gm of moxalactam appeared to be virtually 100% effective and safe for therapy of adults with uncomplicated anogenital gonorrhea.

Moxalactam therapy of difficult infections in patients with serious underlying conditions.

Moxalactam was the single therapeutic agent used to treat a variety of infections in sixty-three patients, most of whom had serious concomitant illnesses. Fifty-three patient case reports qualified for evaluation, including those with pneumonia (8), urinary tract infections (18), superficial infections (6), orthopaedic infections (7), osteomyelitis (8), septicaemia (4), pansinusitis (1), and meningitis (1). Preliminary in vitro studies had indicated that most organisms, including those resistant to other antibacterial agents, would respond to moxalactam. Infecting bacteria from the fifty-three evaluable patients included a wide variety of Gram-positive and Gram-negative organisms. Doses of moxalactam ranged from 1 to 16 g/day administered intravenously or intramuscularly for 5 to 41 days. With few explainable exceptions, clinical and bacteriologic responses were adequate and satisfactory. Adverse effects were inconsequential. Allergic reactions were not observed, even in patients with a past history of reactions to penicillin.