Argarose gel electrophoresis may be employed effectively for the detection and preliminary characterization of plasmid deoxyribonucleic acid (DNA) present in clinical isolates and laboratory strains of gram-negative microorganisms. The method is sensitive and does not require radioisotopes or ultracentrifugation. The estimation of plasmid mass from the extent of DNA migration in gels compares favorably with results obtained by electron microscopy of plasmid DNA purified by equilibrium density centrifugation. The method has proved to be a useful tool for survey work and the epidemiological investigation of plasmid dissemination, as well as an important adjunct to the genetic analysis of plasmids.

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or,most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: (i) AZT was a substrate for E. coli thymidine kinase; (ii) spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and (iii) intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.Nucleoside antibiotics have been under investigation for many years (27). Some of the most clinically effective antiviral agents currently in use are purine or pyrimidine nucleoside analogs (24). For example, ribavirin, a synthetic nucleoside similar in structure to guanosine and inosine, has potent in vitro activity against a broad spectrum of viruses, including the epidemic respiratory viruses (3,25). Two effective inhibitors of bacteria are 9-,B-D-arabinofuranosyladenine and 2',3'-dideoxyadenosine. reported the lethality of the former to a purinerequiring strain of Escherichia coli B. In this organism, 9-4-D-arabinofuranosyladenine markedly inhibited DNA synthesis and had virtually no effect upon RNA synthesis. In addition, 2',3'-dideoxyadenosine was shown to be lethal to selected strains of E. coli by irreversibly inhibiting DNA synthesis in susceptible microorganisms (5, 28).As a result of screening synthetic compounds for potential antimicrobial activity, we have observed that compound BW A509U (3'-azido-3'-deoxythymidine, referred to as AZT in this paper; Fig. 1) has potent, bactericidal in vitro activity against various members of the family Enterobacteriaceae. This report describes the extent of the in vitro growthinhibiting activity of AZT and proposes a mechanism to explain its lethal properties. In addition, the antibacterial activity of AZT is discussed in light of the recent finding that this compound inhibits human T-cell lymphotropic virus type III/lymphadenopathy-assoc...

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The molecular nature of two distinct gonococcal R plasmids, 4.4 X 10(6) and 3.2 X 10(6) daltons, encoding beta-lactamase activity were examined. Both plasmids contained about 40% of the transposable ampicillin resistance sequence Tn2. Deoxyribonucleic acid-deoxyribonucleic acid polynucleotide sequence studies have shown that the two gonococcal plasmids share about 70% of their sequences and are closely related to RSF0885, a 4.1 X 10(6)-dalton plasmid found in a beta-lactamase-producing strain of Haemophilus influenzae. All three of these R plasmids possess a guanine-plus-cytosine content of 0.40 to 0.41 mol fraction and are present as multicopy gene pools in their bacterial hosts.

Type I and II topoisomerase activities were partially purified from Pneumocystis carinii. The catalytic (strand-passing) activities of both enzymes were selectively inhibited by members of a series of dicationicsubstituted bis-benzimidazoles compared with those of topoisomerases of mammalian (calf thymus) origin.The most active inhibitors of the parasite enzymes were also highly effective in an in vivo animal model of P.carinii pneumonia. Selected dicationic-substituted bis-benzimidazoles also strongly inhibited the induction of the topoisomerase I-and 11-mediated cleavable complex, suggesting that the biologically active DNA minor groove-binding molecules inhibit the enzyme-DNA binding step of the topoisomerase reaction sequence. The apparent selectivities for the parasite enzymes and the low levels of toxicity to mammalian cells for the biologically active bis-benzimidazoles suggest that these compounds hold promise as effective therapeutic agents in the treatment of a life-threatening AIDS-related disease, P. carinii pneumonia.Dicationic-substituted aromatic molecules related to pentamidine have long been known to be effective antiparasitic agents (35). Recent studies have found that a number of direct analogs of pentamidine have activity against Pneumocystis carinii (19), Giardia lamblia (3), Toxoplasma gondii (25), Cryptosporidium parvum (7), Leishmania amazonensis subsp. mexicana (5), and Plasmodium falciparum (5). A strong correlation was observed between compound activity against G. lamblia in vitro and DNA-binding ability (3). On the basis of those initial results, studies of antiparasitic compounds were extended to a series of compounds with much stronger DNA-binding affinities, the dicationic bis-benzimidazoles. As observed with the pentamidine analogs, a strong correlation was found between DNA binding strength and antigiardial activity for the bisbenzimidazole series [r2 = 0.96 versus calf thymus DNA and i2 = 0.97 versus poly(dA) * (dT)](4). Other studies confirmed that the effective antigiardial compounds were strong DNA minor groove-binding agents with an AT base pair preference (14). Molecular modeling calculations in that study showed that the DNA-binding strength for this class of compound depended on the radius of curvature on the basis of four defined moieties within the molecules, the distance between cationic moieties, the electronic effects from cationic substituents, and hydrogen bonding. There was no evidence of either an intercalative or a covalent interaction of these compounds with nucleic acids. The antigiardiasis study also revealed a convincing correlation between antitopoisomerase II activity and in vitro activity against G. lamblia for the dicationic bis-benzimidazoles (r2 = 0.91) (4). It is unclear whether these compounds manifest their antiparasitic activity primarily by binding to DNA, topoisomerase, or the enzyme-DNA binary complex.As part of a continuing investigation to determine the mechanism of the anti-P. carinii action of these dicationic molecules and to develop new ag...