Showing papers in "Antimicrobial Agents and Chemotherapy in 1985"

Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material.

Abstract: When disks of urinary catheter material were exposed to the flow of artificial urine containing cells of Pseudomonas aeruginosa, a thick adherent biofilm, composed of these bacteria and of their exopolysaccharide products, developed on the latex surface within 8 h. After this colonization, sterile artificial urine containing 1,000 micrograms of tobramycin per ml was flowed past this established biofilm, and a significant proportion of the bacterial cells within the biofilm were found to be still viable after 12 h of exposure to this very high concentration of aminoglycoside antibiotic. Planktonic (floating) cells taken from the test system just before the exposure of the biofilm to the antibiotic were completely killed by 50 micrograms of tobramycin per ml. The MIC of tobramycin for cells taken from the seeding cultures before colonization of the catheter material, and for surviving cells recovered directly from the tobramycin-treated biofilm, was found to be 0.4 micrograms/ml when dispersed cells were assayed by standard methods. These data indicate that growth within thick adherent biofilms confers a measure of tobramycin resistance on cells of P. aeruginosa.

The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro.

Abstract: INTRODUCTION During the past 5 to 7 years, much attention has been given to the synthesis of new 4-quinolone-3-carboxylates and to the evaluation of these newly prepared agents for antibacterial activity, a revival of interest generated by the discovery of nalidixic and oxolinic acids mtny years ago. This renewed effort uncovered a. number of 6-fluoro-7piperazino-4-quinolones noteworthy for both the breadth and intensity of their activities against gram-negative bacilli and cocci in vitro and for the capacity to control experimentally induced systemic infections with selected bacteria when administered orally in well-tolerated doses (21). The most active representatives of this compound class, designated fluoroquinolones, include norfloxacin (AM-715, MK-0366), ofloxacin (DL-8280), ciprofloxacin (Bay o 9867), amifloxacin (WIN 49375), enoxacin (AT-2266, CI-919), and pefloxacin (1589-RB). The prim4ary target of nalidixic acid and probably also all the fluoroquinolones is DNA gyrase, an essential bacterial enzyme that maintains superhelical twists in DNA (13, 18, 24). The promise of the fluoroquinoloies led to their prompt evaluation for pharmacokinetics in human volunteers and cautious assessments of therapeutic potential, including tolerability in selected patients. The encouraging results of these early trials, with the burgeoning interest in further studies, have made it desirable to review the status of these agents as revealed in the published English literature. We have attempted this task in a two-part minireview. The first part here considers the structures, mechanisms of action and resistance, and spectra of activity of the six aforementioned fluoroquinolones with reference to nalidixic and oxolonic acids for comparison. The second part will consider pharmacology, clinical activity, and toxicities in humans (D. C. Hooper and J. S. Wolfson, Antimicrob. Agents Chemother., in press). Other quinolones, such as cinoxacin, pipemidic acid, rosoxacin, and flumequine, will not be discussed directly. Several reviews of cinoxacin have recently appeared (28, 63).

Evolution of plasmid-coded resistance to broad-spectrum cephalosporins

Abstract: A clinical isolate of Klebsiella ozaenae with transferable resistance to broad-spectrum cephalosporins produces a beta-lactamase determined by plasmid pBP60. The beta-lactamase had the same isoelectric point as SHV-1 (7.6). From heteroduplex analysis, an extensive homology between the two bla genes could be deduced; therefore, the new beta-lactamase was designated SHV-2. Enzymatic studies revealed that SHV-2 was able to hydrolyze broad-spectrum cephalosporins due to an increased affinity of these compounds for the enzyme. The assumption that SHV-2 is a natural mutant of SHV-1 was strongly supported by the isolation of a laboratory mutant of SHV-1 that showed activities similar to those of SHV-2.

The fluoroquinolones: pharmacology, clinical uses, and toxicities in humans.

Abstract: The structures, mechanisms of action and resistance, and spectra of activity of the fluoroquinolones in vitro were considered in the first part of our two-part minireview (48). To place this foregoing information in clinical perspective, we now discuss in this the second part of the minireview the published English literature on the pharmacology, proved and potential clinical uses, and toxicities of the fluoroquinolones in humans.

Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles

Abstract: The peptide antibiotic nisin was shown to cause a rapid efflux of amino acids and Rb+ from the cytoplasm of gram-positive bacteria (Staphylococcus cohnii 22, Bacillus subtilis W 23, Micrococcus luteus ATCC 4698, and Streptococcus zymogenes 24). It strongly decreased the membrane potential of cells as judged by the distribution of the lipophilic tetraphenylphosphonium cation. Ascorbate-phenazine methosulfate-driven transport of L-proline by cytoplasmic membrane vesicles was blocked after addition of nisin, and accumulated amino acids were released from the vesicles. Soybean phospholipid (asolectin) vesicles were not affected by nisin. The data suggest that the cytoplasmic membrane is the primary target and that membrane disruption accounts for the bactericidal action of nisin.

Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus.

Abstract: About 80% of methicillin- and cefazolin-resistant strains of Staphylococcus aureus isolated clinically in Japan in 1982 retained their resistance even after elimination of penicillinase-encoding plasmids. The penicillin-binding proteins (PBPs) of the penicillinase-free, methicillin- and cephem-resistant subclones of Staphylococcus aureus (MRSA) were compared with those of spontaneous susceptible revertants which had been obtained by the replica method after 10 subcultures in drug-free media. A new PBP fraction (PBP2') having a molecular weight of 78,000 and low binding affinities for various beta-lactam antibiotics was found in MRSA exclusively. The levels of resistance of MRSA strains were reduced markedly by culturing them at 43 degrees C or at pH 5.2 or both. We found that the binding capacity of PBP2' for 14C-labeled penicillin G was decreased by preincubation of the membrane fractions of MRSA strains at 43 degrees C for 60 min and that the amount of PBP2' in MRSA strains grown at pH 5.2 was less than that the amount of PBP2' in MRSA strains grown at pH 7.0. Temperature- and pH-dependent expression of resistance in MRSA is likely to reflect the temperature sensitivity and neutral pH-dependent production of the specific PBP fraction (PBP2'). We suggest that MRSA strains can grow in the presence of beta-lactam antibiotics because of the low affinities of the specific PBP2' fraction for various beta-lactam antibiotics.

Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in animals and humans.

Abstract: The pharmacokinetic profile of UK-49,858 (fluconazole), a novel triazole antifungal agent which is being developed for oral and intravenous use, was determined in mice, rats, dogs, and humans. Comparative data following oral and intravenous administration showed that bioavailability was essentially complete in all four species. Peak concentrations in plasma of drug normalized to a 1-mg/kg dose level following oral administration, were relatively high: 0.7, 0.6, 1.1, and 1.4 micrograms/ml in mice, rats, dogs, and humans, respectively. The volumes of distribution ranged between 1.1 liter/kg in mice and 0.7 liter/kg in humans, which are approximate to the values for total body water. Whole body autoradiography studies in mice following intravenous administration of [14C]UK-49,858 demonstrated that the drug was evenly distributed throughout the tissues, including the central nervous system and the gastrointestinal tract. Plasma protein binding was low (11 to 12%) in all species. Marked species differences were observed in elimination half-lives, with mean values of 4.8, 4.0, 14, and 22 h in mice, rats, dogs, and humans, respectively. The major route of elimination of the drug was renal clearance, with about 70% of the dose being excreted unchanged in the urine in each species. Studies with [14C]UK-49,858 on metabolism and excretion (intravenous and oral) in mice and dogs showed that about 90% of the dose was recovered as unchanged drug in urine and feces, confirming the metabolic stability of the drug. This pharmacokinetic profile is markedly different from that of imidazole antifungal drugs and undoubtedly contributes to the excellent efficacy of UK-49,858 in vivo.

Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12.

Abstract: Diffusion rates of various beta-lactam antibiotics through the OmpF and OmpC porin channels of Escherichia coli K-12 were measured by the use of reconstituted proteoliposomes. The results can be interpreted on the basis of the gross physicochemical properties of the antibiotics along the following lines. (i) As noted previously (Nikaido et al., J. Bacteriol., 153:232-240, 1983), there was a monotonous dependence of the penetration rate on the hydrophobicity of the molecule among the classical monoanionic beta-lactams, and a 10-fold increase in the octanol-water partition coefficient of the uncharged molecule decreased the penetration rate by a factor of 5 to 6. (ii) Compounds with exceptionally bulky side chains, such as mezlocillin, piperacillin, and cefoperazone, showed much slower penetration rates than expected from their hydrophobicity. (iii) The substituted oxime side chain on the alpha-carbon of the substituent group at position 7 of the cephem nucleus decreased the penetration rate almost by an order of magnitude; this appears to be largely due to the steric effect. (iv) The presence of a methoxy group at position 7 of the cephalosporins also reduced the penetration rate by 20%, probably also due to the steric hindrance. (v) Zwitterionic compounds penetrated very rapidly, and the correlation between the rate and hydrophobicity appeared to be much weaker than with the monoanionic compounds. Imipenem showed the highest permeability among the compounds tested, presumably due, at least in part, to its compact molecular structure. (vi) Compounds with two negative charges penetrated more slowly than did analogs with only one negatively charged group. Among them, only moxalactam, ceftriaxone, and azthreonam showed penetration rates corresponding to, or higher than, 10% of that of imipenem.

Antibacterial activity of mupirocin (pseudomonic acid), a new antibiotic for topical use.

Abstract: Mupirocin (pseudomonic acid A), an antibiotic produced by Pseudomonas fluorescens, showed a high level of activity against staphylococci and streptococci and against certain gram-negative bacteria, including Haemophilus influenzae and Neisseria gonorrhoeae, but was much less active against most gram-negative bacilli an anaerobes. Nearly all clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis, including multiply resistant strains, were susceptible (mupirocin MIC, less than or equal to 0.5 microgram/ml). There was no cross-resistance between mupirocin and clinically available antibiotics, and the selection of resistant variants in vitro occurred at a low frequency. Mupirocin was highly bound (95% bound) to the protein of human serum, and activity was reduced 10- to 20-fold in the presence of human serum. The activity of mupirocin was not greatly influenced by inoculum size but was significantly enhanced in acid medium. In tests of bactericidal activity, MBCs were 8- to 32-fold higher than MICs and the antibiotic demonstrated a slow bactericidal action in time-kill tests, resulting in 90 to 99% killing after 24 h at 37 degrees C.

Pharmacokinetics of ciprofloxacin after oral and parenteral administration.

Abstract: In 12 fasting volunteers, the pharmacokinetics of ciprofloxacin (Bay o 9867; 1-cyclopropyl-6-fluor-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carbonic acid) were determined after the administration of 50, 100, and 750 mg orally as well as 50 and 100 mg intravenously over 15 min. Serum and urine concentrations were detected with a bioassay. In addition, urine concentrations after a 50-mg dosing were measured by high-pressure liquid chromatography. The serum course of ciprofloxacin could best be described by an open three-compartment model. High volumes of distribution (exceeding 200 liters/100 kg) suggested effective diffusions in the extravascular space. The terminal half-life of ciprofloxacin ranged between 3 and 4 h. High total and renal clearances suggested additional elimination pathways, such as tubular secretion, metabolism, or biliary excretion. After oral administration, absorption was sufficient, and the absolute bioavailability varied between 0.77 and 0.63. Maximal serum concentrations were attained 0.5 to 1 h after dosing; the higher dosage tended towards a delay in absorption. The proportion of the relative amount of metabolites to the total amount of drug excreted in urine increased from 29.7% after intravenous administration to 42.7% after oral dosing, indicating a first-pass effect of the liver. Ciprofloxacin concentrations with a bioassay were 3 to 27% higher than with high-pressure liquid chromatography, which may indicate the presence of biologically active metabolites. No side effects were recorded.

Occurrence of a beta-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci.

Abstract: The mechanism of methicillin resistance was investigated in methicillin-resistant staphylococci (MRS) and in variants which had lost methicillin resistance. Phase-contrast microscopy showed that cells swelled at low concentrations of beta-lactam antibiotics in both MRS and variants which had lost methicillin resistance. Cells of variants which had lost methicillin resistance were lysed easily when higher concentrations of antibiotic were used. In contrast, MRS cells remained swollen at even higher concentrations of antibiotics. Furthermore, bacterial growth was inhibited at antibiotic concentrations much lower than MICs for MRS. Examination of the penicillin-binding proteins (PBPs) in MRS revealed that a new PBP-2' (molecular weight, 74,000) was induced in large quantity by exposure to beta-lactams. PBP-2' was produced constitutively in variants of MRS which had lost a penicillinase plasmid. The induction of PBP-2' by beta-lactams was not detected in variants which had lost methicillin resistance. High concentrations of beta-lactam were required for saturation of PBP-2'. The optimum antibiotic concentration for the induction of PBP-2' varied with the beta-lactam used as the inducer, and PBP-2' was produced in a larger amount at 32 degrees C than at 37 degrees C. From these results, we suggest that the mechanism of methicillin resistance depends on the induction of PBP-2', which may function as a detour enzyme for PBP-2 or PBP-3 or may be a particular enzyme involved in peptidoglycan synthesis.

Antimicrobial susceptibility of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae.

Abstract: Broth microdilution MICs were determined for 258 clinical isolates of Mycobacterium fortuitum (3 biovariants) and M. chelonae (2 subspecies) with amikacin, tobramycin, cefoxitin, doxycycline, erythromycin, and sulfamethoxazole-trimethoprim and with several new beta-lactams and aminoglycosides and ciprofloxacin. Variations in susceptibility by and within species subgroups confirm the need for susceptibility testing against clinically important strains.

Antiviral and antimetabolic activities of neplanocins.

Abstract: Of a series of carbocyclic analogs of adenosine, in which the ribose moiety was replaced by a cyclopentenyl ring, neplanocin A, or (-)-9-[trans-2, trans-3-dihydroxy-4-(hydroxymethyl)cyclopent-4-enyl]adenine proved particularly effective in inhibiting the multiplication of DNA viruses (i.e., vaccinia), (-)RNA viruses (i.e., parainfluenza, measles, and vesicular stomatitis), and double-stranded RNA viruses (i.e., reo) in vitro in cell culture. Depending on the cells used, the MIC of neplanocin A for these viruses ranged from 0.01 to 4 micrograms/ml, and depending on the parameter used to assess toxicity for the host cell, the specificity index of neplanocin A ranged from 50 to 4,000. As postulated before for other adenosine analogs, neplanocin A may owe its antiviral action to inhibition of S-adenosylhomocysteine hydrolase, hence perturbation of transmethylation reactions. In vivo, neplanocin A afforded only marginal protection against a lethal infection of mice with vesicular stomatitis virus.

In vitro activities of and mechanisms of resistance to antifol antimalarial drugs.

Abstract: Certain drugs that interfere with folate metabolism (sulfones, sulfonamides, and inhibitors of dihydrofolate reductase) play an important role in the chemotherapy and prophylaxis of malaria. The activities and mechanisms of action of these drugs are regarded as similar in most respects to their activities against procaryotic microorganisms. Believed incapable of utilizing intact exogenous folates, plasmodia have been regarded as dependent on de novo synthesis of required folate cofactors. The present investigation, conducted in pursuit of a method for testing the in vitro susceptibility of Plasmodium falciparum to antifol antimalarial drugs, produced evidence that earlier assumptions about the folate metabolism of this organism are not correct. Three of four isolates of P. falciparum were successfully maintained in a culture medium depleted of folic acid and p-aminobenzoic acid. The antimalarial activities of sulfonamides and dihydrofolate reductase inhibitors were, furthermore, variably antagonized by the presence of folic acid and p-aminobenzoic acid in the culture medium. Optimum conditions for assessment of antifol antimalarial activity in vitro therefore require precise control of these factors in the culture medium. Our results suggest that resistance to antifol antimalarial drugs involves a complex of factors related to both the de novo synthesis of active folate cofactors and the ability to utilize exogenous intact folates in various forms.

Susceptibility of clinical isolates of Campylobacter pyloridis to 11 antimicrobial agents.

Abstract: The activities of 11 antimicrobial agents, including two bismuth salts, against 70 strains of Campylobacter pyloridis isolated from gastric biopsy specimens were tested. The isolates were very susceptible to penicillin (the MIC for 90% of the strains tested [MIC90] was 0.03 microgram/ml), erythromycin, cefoxitin (MIC90, 0.12 microgram/ml), gentamicin, and ciprofloxacin (MIC90, 0.25 microgram/ml). The bismuth salts and nalidixic acid had moderate activity (MIC90, 16 to 64 micrograms/ml). Twenty percent of the isolates were resistant to metronidazole (MIC, greater than 1 micrograms/ml), and all were resistant to sulfamethoxazole and trimethoprim (MIC90, greater than 256 micrograms/ml).

Role of beta-lactam hydrolysis in the mechanism of resistance of a beta-lactamase-constitutive Enterobacter cloacae strain to expanded-spectrum beta-lactams.

Abstract: Enterobacter cloacae strains producing chromosomally mediated beta-lactamase constitutively show high degrees of resistance to most of the third-generation beta-lactams. It has been proposed that this resistance is due to the nonhydrolytic binding or trapping of beta-lactams by the enzyme. We found that the outer membrane of E. cloacae strain 55M indeed had permeability to cefazolin about 14-fold lower than that of Escherichia coli, and that the number of beta-lactamase molecules produced by this constitutive mutant was exceptionally large (2 X 10(5) per cell). These conditions are expected to produce a low degree of resistance, but could not explain the high resistance level of the mutant. We showed that the beta-lactamase of this strain hydrolyzed third-generation beta-lactams at measurable rates. Although the V max for these compounds was less than 0.01% of that for cefazolin, the enzyme could hydrolyze them at rates comparable to the rate for cefazolin when the substrate concentration was near 0.1 microM, a concentration thought to be physiologically relevant for the inhibition of cell growth, because of the exceptionally high affinity of the enzyme to many third-generation compounds. Calculations based on kinetic parameters of the enzyme, outer membrane permeability, and affinity toward penicillin-binding proteins succeeded in predicting the MICs for several third-generation beta-lactams. The data suggest that hydrolysis may be more important than nonhydrolytic binding for the expression of the resistant phenotype, and that studies on the susceptibility of beta-lactams to beta-lactamases should be carried out at physiologically relevant, very low concentrations of the drug, rather than the customary very high concentrations, such as 100 microM.

Comparison of a new cephalosporin, BMY 28142, with other broad-spectrum beta-lactam antibiotics.

Abstract: BMY 28142, a new broad-spectrum semisynthetic cephalosporin, was evaluated in vitro and in vivo in comparison with ceftazidime, cefotaxime, moxalactam, and cefoperazone. The activity of BMY 28142 compared favorably with the activities of the other compounds against both Pseudomonas aeruginosa and Staphylococcus aureus and was somewhat greater against members of the family Enterobacteriaceae. The influence of inoculum size on MICs of BMY 28142 was small for most of the isolates tested, except Enterobacter species. With Enterobacter strains, a marked inoculum effect was found with all of the compounds, and the effect was more pronounced in broth than agar. Still, MICs of BMY 28142 in broth did not exceed 16 micrograms/ml. Of 37 Enterobacteriaceae strains resistant to one or more of the comparison beta-lactams, none were resistant, at a low inoculum size (10(4) CFU), to BMY 28142, compared with 3 for moxalactam, 18 for ceftazidime, 23 for cefotaxime, and 34 for cefoperazone; at an inoculum size of 10(6) CFU, the number of resistant strains was 12, 17, 25, 34, and 37, respectively. Binding to human serum proteins approximated 19%. Recovery of 73% of the drug in mouse urine indicated good bioavailability. The in vitro profile was sustained in vivo by the results obtained with experimental infections in mice. BMY 28142 was as effective as ceftazidime against systemic infection with P. aeruginosa and as effective as cefotaxime against systemic infection with S. aureus. Overall, infections with 18 of 20 strains representing nine genera were responsive to BMY 28142 at doses equivalent to or lower than those of the most effective of the comparison compounds.

Activity of UK-49,858, a bis-triazole derivative, against experimental infections with Candida albicans and Trichophyton mentagrophytes.

Abstract: The therapeutic potential of UK-49,858, a difluorophenyl bis-triazole derivative, has been assessed by evaluating its activity against systemic infections with Candida albicans in normal mice and rats and in mice with impaired defence mechanisms, against vaginal infections with C. albicans in mice, and against dermal infections with Trichophyton mentagrophytes in guinea pigs. Orally administered ketoconazole was used as a comparative agent throughout, and parenterally administered amphotericin B was included in the study of C. albicans systemic infection in normal mice. The activity of UK-49,858 given orally to mice or rats infected systemically with C. albicans was far superior to that of ketoconazole. In addition, UK-49,858 showed activity comparable to that of amphotericin B when given parenterally, although the latter gave more prolonged protection. UK-49,858 was also effective orally in curing experimental candidal vaginitis in mice and trichophytosis in guinea pigs, against which it was approximately 10 times more active than ketoconazole. These data suggest that UK-49,858 may be of value in the treatment of both C. albicans and dermatophyte fungal infections in man.

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent.

Abstract: SF 86-327 is a new antimycotic agent of the allylamine type. Its primary action appears to be the inhibition of ergosterol biosynthesis at the point of squalene epoxidation, as was previously found with the related compound naftifine. Biosynthesis was measured by incorporation of [14C]acetate into sterols in cells of Candida albicans, Candida parapsilosis, Torulopsis glabrata, and the dermatophyte Trichophyton mentagrophytes. There was a positive correlation between the SF 86-327 concentrations needed for inhibition of growth and of sterol synthesis in these four fungi. The greater antifungal efficacy of SF 86-327 in comparison with naftifine was also reflected in the relative activities of the two compounds as sterol synthesis inhibitors. Inhibition was maximal at neutral pH. A similar degree of inhibition was found in cell-free extracts when [14C]mevalonate was used as substrate. In all cases, inhibition of sterol synthesis was accompanied by a parallel accumulation of labeled squalene. SF 86-327 and naftifine had no significant effect on initial enzymes of the ergosterol pathway, measured by incorporation of [14C]acetyl coenzyme A, or on steps distal to squalene epoxidation, measured by conversion of labeled squalene 2,3-epoxide or lanosterol. Both allylamines were highly selective for fungal, as opposed to mammalian, sterol biosynthesis. SF 86-327 caused slight inhibition of squalene epoxidation in a rat liver cell-free system, but at concentrations three to four orders of magnitude greater than those required for inhibition of the fungal pathway.

Tetracycline-resistant Mycoplasma hominis strains contain streptococcal tetM sequences.

Abstract: Clinical isolates of Mycoplasma hominis resistant to high levels of tetracycline contained DNA sequences homologous to the streptococcal tetracycline determinant, tetM. In contrast, none of the susceptible M. hominis isolates tested carried this determinant. This is the first description of tetM in an unrelated genus and suggests the spread of tetM from Streptococcus spp. to Mycoplasma spp.

Antimicrobial activity of ciprofloxacin against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus determined by the killing curve method: antibiotic comparisons and synergistic interactions.

Abstract: A derivative of quinolinecarboxylic acid, ciprofloxacin (BAY o 9867) was found to be an effective bactericidal agent against Pseudomonas aeruginosa and Escherichia coli. A bactericidal effect was achieved immediately after the addition of ciprofloxacin. At a concentration of 0.5 micrograms/ml, culture viability was reduced from 5 X 10(5) to about 5 X 10(3) CFU/ml within 15 min, and at 0.1 micrograms/ml, a greater than 10-fold reduction in viability resulted during the first hour after exposure. This bactericidal activity observed during the lag phase in Mueller-Hinton broth was also demonstrated in a nongrowing system. The antibiotics used in comparative studies, i.e., tobramycin, aztreonam, cefotaxime, and azlocillin, did not show this initial bactericidal activity, and ciprofloxacin prevented culture regrowth at lower concentrations. Staphylococcus aureus was not as susceptible to ciprofloxacin; killing occurred at a concentration of 0.5 micrograms/ml only after the onset of exponential growth in the control culture. Synergistic interactions were observed with ciprofloxacin in combination with tobramycin and azlocillin against P. aeruginosa and with cefotaxime and tobramycin against E. coli.

Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

Abstract: The tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicillium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2+ and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25 degrees C and 7.8, respectively. The reaction required O2 and was stimulated by increasing the dissolved oxygen concentration of the reaction mixture. Purification of the enzyme to a single major band in polyacrylamide gel electrophoresis was achieved by protamine sulfate precipitation, ammonium sulfate fractionation (50 to 80% of saturation), DEAE-Sephacel chromatography, and gel filtration on Sephacryl S-200. The estimated molecular weight was 39,000 +/- 1,000. The apparent Km of isopenicillin N synthetase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.13 mM. The enzyme activity was strongly inhibited by glutathione, which acts as a competitive inhibitor. A good correlation was observed between the isopenicillin N synthetase activity in extracts of four different strains of P. chrysogenum (with widely different penicillin-producing capability) and the amount of penicillin production by these strains.

Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture.

Abstract: Several antibiotics which inhibit protein synthesis on 70S ribosomes, including clindamycin, pirlimycin, 4'-pentyl-N-demethyl clindamycin, four tetracyclines, chloramphenicol, thiamphenicol, and erythromycin, had antimalarial effects against Plasmodium falciparum in culture which were greatly influenced by the duration of drug exposure and by oxygen tension. In 96-h incubations, potency was increased by a factor of up to 10(6) over the first 48-h period and by a factor of up to 10(4) in 15% O2 versus 1% O2. Two aminoglycosides, kanamycin and tobramycin, had no antimalarial activity. Rifampin and nalidixic acid, which inhibit nucleic acid synthesis, were not similar to the 70S inhibitors. The mitochondrial inhibitors Janus Green, rhodamine 123, antimycin A1, and 8-methylamino-8-desmethyl riboflavin had activities which were influenced by the duration of exposure and oxygen tension. Quinoline-containing antimalarial agents, ionophores, and other antimalarial drugs were influenced to a minor extent by the duration of exposure but were not affected by oxygen tension. These data can best be explained by the hypothesis that antimalarial 70S ribosome-specific protein synthesis inhibitors are toxic to the parasites by acting on the mitochondrion.

Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses.

Abstract: The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality.

In vitro activity of WIN 51711, a new broad-spectrum antipicornavirus drug.

Abstract: WIN 51711 (5-[7-[4-(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3-methylisoxazole), a new antipicornavirus drug, is a potent inhibitor of human entero- and rhinoviruses at concentrations not inhibitory to HeLa cell growth. In plaque reduction assays, WIN 51711 reduced plaque formation by 9 enteroviruses and 33 rhinoviruses, with MICs of 0.004 to 0.17 and 0.004 to 6.2 micrograms/ml, respectively. Addition of WIN 51711 to infected cells at concentrations of 0.02 to 5.0 micrograms/ml reduced the yield of picornaviruses by 90%. Other RNA viruses (nonpicornaviruses) and DNA viruses were unaffected by the compound.

Comparison of aminoglycoside resistance patterns in Japan, Formosa, and Korea, Chile, and the United States.

Abstract: The resistance mechanisms of more than 2,000 aminoglycoside-resistant gram-negative aerobic bacteria were estimated by a method that assigned a biochemical mechanism based on susceptibility to selected aminoglycosides. Strains from hospitals in Japan, Formosa, and Korea (the Far East) were compared with strains from Chile and the United States. Of the strains from Chile, 90% had an aminoglycoside resistance pattern indicative of the 3-N-acetyltransferase [AAC(3)-V] enzyme. Of the strains from the Far East, 78% had susceptibility patterns suggesting the presence of AAC(6') enzymes. In contrast, strains from the United States had a wider variety of resistance mechanisms including 2''-O-adenylyltidyltransferase [ANT(2'')], AAC(3), AAC(6'), and AAC(2'). Reflecting these differences in resistance patterns, the frequencies of resistance to gentamicin, tobramycin, dibekacin, and amikacin in strains from the United States were different from those in strains from the Far East. These differences seem to be correlated with different aminoglycoside usage in the two regions. In the United States, where gentamicin was the most widely used aminoglycoside, 92% of the strains were resistant to gentamicin, 81% were resistant to dibekacin, and 8.8% were resistant to amikacin. In the Far East, dibekacin and kanamycin were widely used in the past and more recently amikacin has been frequently used. Of the strains from this region, 99% were resistant to dibekacin, 85% were resistant to gentamicin, and 35% were resistant to amikacin.

Cloning and expression of Staphylococcus aureus plasmid-mediated quaternary ammonium resistance in Escherichia coli.

Abstract: The Staphylococcus aureus plasmid pSK1 carries Tn4001, a 4.7-kilobase (kb) transposon which specifies resistance to gentamicin, tobramycin, and kanamycin. In addition, pSK1 mediates resistance to trimethoprim and linked resistance to ethidium bromide (Ebr) and to quaternary ammonium compounds (Qar). Restriction endonuclease analysis of pSK1 and a deleted derivative of pSK1 revealed that the gene(s) responsible for Ebr Qar lies within a 5.2-kb HindIII fragment. This fragment has been cloned into the Escherichia coli plasmid vector pBR322, and transformants of an E. coli K-12 strain exhibited Ebr Qar. Subcloning of the 5.2-kb insert, combined with data from electron microscopic analysis of deleted derivatives of pSK1, located the Ebr Qar determinant(s) on a 2.3-kb segment of pSK1 DNA.

Comparative in vitro activity of sanguinarine against oral microbial isolates.

Abstract: MICs of sanguinarine were determined for 52 oral reference strains and 129 fresh isolates from human dental plaque. Sanguinarine was found to completely inhibit the growth of 98% of the isolates at a concentration of 16 micrograms/ml.

Penetration of oral doxycycline into the cerebrospinal fluid of patients with latent or neurosyphilis.

Abstract: Five patients with laboratory evidence of latent or neurosyphilis were treated orally with doxycycline (200 mg) twice a day for 21 days. After the seventh dose, the mean level of doxycycline in serum was 5.8 micrograms/ml, with a mean drug level in cerebrospinal fluid of 1.3 micrograms/ml. The mean penetration into cerebrospinal fluid was 26%. These preliminary findings suggest that doxycycline, administered orally at a dose of 200 mg twice a day, reaches a sufficient concentration in cerebrospinal fluid to be worthy of further evaluation as an alternative regimen to penicillin therapy for latent or neurosyphilis.

Resistance caused by decreased penetration of beta-lactam antibiotics into Enterobacter cloacae.

Abstract: Strains of Enterobacter cloacae were selected on the basis of resistance to aztreonam, ceftazidime, moxalactam, or imipenem. All strains produced the same E2 beta-lactamase, with an isoelectric point greater than 9.5 and with high hydrolytic activity in the presence of cephaloridine. Resistance to beta-lactams could not be correlated with the amount of beta-lactamase present in the various strains. beta-Lactamase activity was induced strongly by moxalactam and imipenem in the wild-type and moxalactam-resistant strains, with beta-lactamase representing as much as 4% of the total cellular protein after induction (2 X 10(5) molecules per cell). Ceftazidime and aztreonam were poor inducers. None of the antibiotics studied was readily hydrolyzed by the E2 beta-lactamase; aztreonam and moxalactam inhibited the enzyme with apparent Ki values of 1.2 and 100 nM, respectively. Aztreonam, which bound covalently to the E2 beta-lactamase with a half-life of 2.3 h at 25 degrees C, was used to measure penetrability of beta-lactam into the periplasmic space of the resistant E. cloacae strains. In all of the E2-producing organisms studied, a significant permeability barrier existed. A maximum concentration of 0.02 microgram of aztreonam per ml should have saturated the periplasmic beta-lactamase in the highest enzyme producers studied. However, fully active beta-lactamase was observed in the periplasm of cells exposed to aztreonam at concentrations at least 1,000-fold higher than that theoretically necessary to inhibit the total enzyme within the cell. Thus, the major cause for resistance to beta-lactam antibiotics in these E. cloacae strains was lack of penetration across the outer membrane.