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...

Resistance of Escherichia coli to trimethoprim (TMP)-sulfamethoxazole remains at 3%-8% at many medical centers within the United States. In this study a 44% resistance rate was observed among E. coli isolated at a pediatric hospital in Santiago, Chile, and a 40% resistance rate at a general teaching hospital in Bangkok, Thailand. Most isolates were from urinary tract infections and showed high-level resistance (minimal inhibitory concentration of TMP greater than 1,000 micrograms/ml). Nineteen of 35 isolates tested transferred resistance to TMP; most cotransferred resistance to streptomycin and sulfonamides. Dihydrofolate reductase type I was detected by gene probing in 14 of 35 strains. Subsequent investigations in Brazil, Honduras, and Costa Rica revealed that this high rate of resistance was not an isolated phenomenon.

Several 8-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae were examined for R plasmids. Penicillin-resistant strains isolated from men returning from the Far East and their contacts contained a 4.4 x 106-dalton plasmid in common. Transformation studies and the isolation of a spontaneous penicillin-susceptible segregant showed that the structural gene for f8-lactamase was part of the 4.4 x 106-dalton plasmid. An additional penicillinresistant gonococcal strain isolated in London was found to harbor a 3.2 x 106-dalton R plasmid. Deoxyribonucleic acid (DNA)-DNA duplex studies revealed that the penicillin-resistant gonococcal isolates contained a significant portion (about 40%) of the transposable DNA sequence, TnA, which includes the f8-lactamase gene commonly found on R plasmids of the Enterobacteriaceae and Haemophilus influenzae.It has been known for some time that the R plasmids of enteric species enjoy a very broad host range (1,14). The recent finding (reviewed in 4, 6, 28) that certain pIa'smid-mediated antibiotic resistance genes reside upon discrete sequences of deoxyribonucleic acid (DNA) that have the capacity to transpose from plasmid to plasmid has further demonstrated that the range ofplasmid-mediated antibiotic resistance genes might be even broader than that of the whole plasmid genome. Therefore, plausible mechanisms exist by which pathogenic microorganisms, previously antibiotic susceptible, may be converted to stable antibiotic resistance by the direct extension of an entire R plasmid or by the simple addition of only a segment of DNA to a preexisting indigenous plasmid gene pool (6,15,19).Previous studies from this lAboratory (21) and others (3, 24, 30) have established that most strains of Neisseria gonorrhoeae contain plasmid species that are cryptic in the phenotypic sense. For example, our examination of30 antibiotic-susceptible gonococcal isolates showed that 28 strains harbored a multicopy (2.6 + 6.2) x 106--dalton (2.6 MDal) plasmid, and 1 strain contained a 24.5-MDal plasmid species, whereas 1 strain was plasmid free (13; L. P. Elwell and S. Falkow, in R. Roberts, ed., The Gonococcus, in press). The demonstration of a widespread plasmid gene pod in gonococci together with the emergence, epidemiology, and molecular nature of R plasmids mediating ampicillin resistance in Haemophilus influenzae (11, 12) led us to speculate (15) that gonococci were likely candidates to acquire plasmidmediated penicillin resistance specified by a (8-lactamase. Consequently, the recent isolation of f8-lactamase-producing strains of N. gonbrrhoeae (2, 25, 26) prompted us to examine them for the presence of a novel DNA species that might represent an R plasmid.In this study we report the detection of plasmid species from three penicillin-resistant clinical isolates of N. gonorrhoeae. We furher demonstrate that these plasmids probably represent a direct or indirect extension of the plasmid gene pool of enteric species to gonococci.

Four ampicillin-resistant, f,-lactamase-producing strains of Haemophilus influenzae type b were examined for the presence of plasmid deoxyribonucleic acid (DNA). Three resistant strains contained a 30 x 106-dalton (30 Mdal) plasmid and one resistant strain contained a 3-Mdal plasmid. The ampicillin-sensitive Haemophilus strains examined did not contain plasmid DNA. Transformation of a sensitive H. influenzae strain to ampicillin resistance with isolated plasmid DNA preparations revealed that the structural gene for ,B-lactamase resided on both plasmid species. DNA-DNA hybridization studies showed that the 30-Mdal Haemophilus plasmid contained the ampicillin translocation DNA segment (TnA) found on some R-factors of enteric origin. The significance of this finding is discussed in relation to the possible origin of the H. influenzae plasmids. Disease due to ampicillin-resistant strains of Haemophilus influenzae type b has become an increasingly serious and widespread problem (1, 3, 25). Ampicillin-resistant strains have been isolated from cases of epiglottitis, pneumonia, otitis media, and meningitis, including three fatal cases of infant meningitis (15, 17). The primary cause of ampicillin resistance appears to be the production of a d-lactamase shown in one strain to possess a broad-spectrum penicillinase-cephalosporinase activity (9). Thorne and Farrar (J. Infect. Dis., in press) recently have demonstrated transfer of the ampicillin resistance from one strain to an ampicillin-sensitive strain during mixed incubation, presumably by conjugation. The sudden emergence of ampicillin resistance in H. influenzae due to a transferable #-lactamase gene suggested to us, and others (9,15), that an R-factor might be involved. In this study we report the isolation of plasmid deoxyribonucleic acid (DNA) from four ampicillin-resistant strains of H. influenzae. Three strains contained a 30 x 108-dalton (30 Mdal) plasmid and one strain contained a 3.0-Mdal plasmid, and evidence is presented indicating that the ,3-lactamase gene is located on those plasmid species. Resistance to ampicillin mediated by the TEM-type ,B-lactamase in enteric bacteria is found on a segment of DNA that can be translocated from replicon to replicon (13). This same segment is found on the plasmid DNA isolated from resistant H. influenzae.