Synthesis and antimicrobial evaluation of TAN-1057A/B analogs.

TAN-1057A-D, dipeptides isolated from bacteria Flexibacter sp. PK-74 and PK-176, are new antibiotics with potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We describe, in detail, the synthesis of several TAN-1057A/B analogs by a convergent route featuring a new method to construct the cyclic amidinourea functional group. The biological activity of these substances against methicillin-resistant Staphylococcus aureus (MRSA) is reported.

Discovery of RWJ-54428 (MC-02,479), a new cephalosporin active against resistant gram-positive bacteria.

The discovery of RWJ-54428 (MC-02,479), a new cephalosporin displaying promising activity against sensitive and resistant Gram-positive bacteria, is described. Progressive structural modification from the previously reported 3-phenylthiocephem MC-02,331 afforded an overall increase in potency against MRSA while retaining other key properties such as acceptable solubility and serum binding. Evaluation of the in vitro potency and in vivo efficacy of a series of closely related compounds resulted in selection of RWJ-54428 (MC-02,479) for further studies.

SAR studies of anti-MRSA non-zwitterionic 3-heteroarylthiocephems.

SAR studies in a series of 3-heteroarylthio substituted cephalosporins established that high activity against methicillin-resistant Staphylococcus aureus (MRSA) can be achieved with various heteroaryl substituents. Early results showed that highly lipophilic 3-heteroarylthio substituents, which were necessary for anti-MRSA activity, caused high affinity of such cephems toward serum proteins. Our earlier published efforts described discovery of zwitterionic cephems MC-02,331 and RWJ-54428 (MC-02,479), where serum binding was reduced by employing basic, positively charged functionalities attached to the 3-heteroarylthio substituent. In order to avoid low solubility problems associated with most such zwitterionic cephalosporins a wide variety of non-basic heteroaryl substituents was tested (non-zwitterionic cephems are more easily formulated as water soluble sodium salts for intravenous administration). Considerable reduction in serum binding was obtained in some analogs while maintaining high anti-MRSA potency.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.

Microbial fermentation-derived inhibitors of efflux-pump-mediated drug resistance.

A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa and Candida albicans. New compounds EA-371alpha and EA-371delta were isolated and demonstrated to be potent and specific inhibitors of the MexAB-OprM pump in P. aeruginosa. Two series of fungal metabolites, enniatins and beauvericins, were found to be ubiquitous and potent inhibitors of ABC transporters. Milbemycins were rediscovered as potent inhibitors of the CDRI pump in C. albicans, and demonstrated to potentiate effectively the antifungal activity of fluconazole and SCH-56592 against a wide variety of Candida clinical isolates.

In vitro evaluation of the activities of azithromycin alone and combined with pyrimethamine against Toxoplasma gondii.

By using an in vitro microassay to assess drug interaction, azithromycin combined to pyrimethamine was found more active than pyrimethamine alone against Toxoplasma gondii, and additivity between those drugs was demonstrated. Our results show that the combination of azithromycin and pyrimethamine may lead to a more rapid control of T. gondii.

Azithromycin uptake and intracellular accumulation by Toxoplasma gondii-infected macrophages.

The uptake of azithromycin and erythromycin was measured in RAW 264.7 mouse macrophages infected with Toxoplasma gondii to determine whether the activity of macrolides could be correlated with their degree of host cell penetration. Uptake was expressed as the ratio of the intracellular (I) to the extracellular (E) concentrations. After infection, the intracellular accumulation of macrolides was equivalent to that measured in uninfected cells and azithromycin reached an I/E ratio of 105.8 +/- 8.0 in infected macrophages incubated with 20 mg/L of drug. The release of azithromycin from macrophages previously exposed to the drug was enhanced by exposure to Micrococcus luteus and phorbol myristate acetate but not after infection with T. gondii. Azithromycin accumulates readily and remains inside T. gondii-infected macrophages thereby interfering with the growth of the parasite which was confirmed by growth-inhibition experiments and by electron microscopy.

Inhibition of Toxoplasma gondii protein synthesis by azithromycin.

Azithromycin was shown to specifically inhibit the protein synthesis of Toxoplasma gondii in experimental systems by using free tachyzoites and T. gondii-infected mouse macrophages. RNA synthesis of the parasite was not affected by azithromycin. Inhibition of protein synthesis was also proportional to the relative anti-Toxoplasma activity of three macrolides.

Use of mouse macrophage cell lines for in vitro propagation of Toxoplasma gondii RH tachyzoites.

In most laboratories, Toxoplasma gondii is maintained in mice and is studied in vitro using nonlymphoid cell lines or primary mouse macrophages. In this study, three rapidly dividing mouse macrophage cell lines (J774 A.1, P388D1, RAW264.7) were evaluated for their suitability for studying the RH strain of T. gondii. For comparison, tachyzoites were also grown in two slowly dividing epithelial cell types: a rat lung cell line (L2) and a bovine turbinate cell line (BT). Various inocula of T. gondii were added to the above cells and tachyzoites were harvested from the culture supernatants after 2-8 days of infection. The mouse macrophage cell lines supported rapid growth of T. gondii RH allowing up to a 300-fold increase of the inoculum in 2-4 days. L2 and BT supported slower growth of T. gondii (10- to 90-fold increase of inoculum in 5 to 8 days) and, thus, may be more suitable for assessment of host cell-parasite interactions and drug activity. Toxoplasma gondii RH isolated from each of the cell cultures described were able to multiply in all cell types used. Protein profiles of whole tachyzoite isolated from mice or cell cultures and protein profiles of the corresponding soluble and membrane fractions of the intraphagosomal membrane network were similar as seen after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In mice, intraperitoneal injection of 10(6), 10(5), and 10(3) tachyzoites isolated from the cell cultures or from infected mice caused death after 4, 5, and 8 days, respectively, indicating that parasites grown in vitro retained virulence.

The legumin boxes and the 3' part of a soybean beta-conglycinin promoter are involved in seed gene expression in transgenic tobacco plants.

beta-conglycinin is one of the major seed storage proteins in soybean. It is composed of three subunits, namely alpha, alpha' and beta. The expression of beta-conglycinin is highly regulated, being restricted to the embryo during the mid-maturation phase of embryogeny. Two series of constructs were made with the alpha' subunit promoter and the GUS reporter gene to investigate the cis-acting elements involved in the regulated expression of this promoter. The activity of each construct was tested in transgenic tobacco plants. In the first series of constructs, we checked if the 'legumin box', a sequence found in most legume seed storage protein genes as well as in other seed-specific genes, is involved in the regulated expression of the alpha' subunit of the beta-conglycinin gene in tobacco. To this end, both copies of the alpha' subunit promoter legumin boxes were mutagenized in vitro. The transcriptional activity of the single mutants and the double mutant were compared with that of the wild-type promoter. Our results show that the legumin boxes act together to increase transcription of the beta-conglycinin alpha' subunit gene by about a factor of ten. This is the first demonstration of a function for the legumin box in transcriptional regulation. In the second series of experiments, we wished to determine if the 3' part of the promoter (the CCAAT and TATAA region) contains important regulatory elements. We found that this small fragment (-82 to +13 bp) can confer by itself a low level of seed-specific gene expression. Chimaeric promoters constructed from parts of the alpha' subunit promoter and of the constitutive CaMV 35S promoter were also analysed. These constructs also revealed the importance of the CCAAT and TATAA region of the alpha' subunit promoter in seed-specific gene expression.

Effect of clindamycin on intracellular replication, protein synthesis, and infectivity of Toxoplasma gondii.

We studied the effects of clindamycin and a combination of clindamycin and pyrimethamine on the proliferation of Toxoplasma gondii in cultured mammalian cells and the effect of clindamycin on the parasite's RNA and protein syntheses. Infected macrophages were treated for 48 h with clindamycin or a combination of clindamycin and pyrimethamine, and the 50% inhibitory concentrations for parasite growth were 32.50 +/- 1.30 and 10.78 +/- 0.56 micrograms/ml, respectively. A modified susceptibility assay was also used to measure the effect of low concentrations of clindamycin on T. gondii. Macrophages and bovine turbinate cells were infected with low numbers of tachyzoites and were exposed to low concentrations of clindamycin for 5 days. In these systems, a concentration of 10 ng of clindamycin per ml inhibited 50% of the growth of the parasite in macrophages, while it completely prohibited the growth of the parasite in epithelial cells. When free tachyzoites were preexposed to clindamycin for 4 h, the reduction of parasite infectivity was proportional to the amount of drug; 100 ng of clindamycin per ml reduced the infectivity of T. gondii to 46.5% +/- 8.5% of that of the untreated control. A concentration of 40 micrograms of clindamycin per ml reduced protein synthesis by 56.2% +/- 6.0% but had no effect on RNA synthesis after a 4-h exposure of free tachyzoites of T. gondii to the drug. Our results show that long-term exposure to low concentrations of clindamycin reduces the level of replication of T. gondii, that clindamycin affects the protein synthesis of free parasites, and that clindamycin impairs the ability of tachyzoites to infect host cells.

Characterization of multiresistant strains of Neisseria gonorrhoeae isolated in Nicaragua.

The extensive use of antibiotics in Nicaragua raises concerns about the resulting levels of susceptibility of pathogenic bacteria. This is the first study that characterizes 18 strains of N. gonorrhoeae isolated in Nicaragua (1989), for their antibiotic susceptibility. Strains were predominantly of the auxotype/serotype Proto/PIB. There was no difference in lipopolysaccharides profiles obtained after SDS-PAGE for all strains. Variable expression of the PII outer membrane protein was not associated to antimicrobial resistance. All strains were susceptible to ceftriaxone, spectinomycin, rifampin and cefoxitin. The strains were classified in five groups based on plasmid profiles. A total of 78% of the isolates were penicillinase-producing (PPNG) and 22% were tetracycline-resistant N. gonorrhoeae (TRNG). One PPNG strain showed a concomitant decreased of penicillin binding to penicillin-binding protein 2. These randomly chosen isolates of N. gonorrhoeae from Nicaragua possess high levels of resistance to multiple families of drugs.

PIP: In Nicaragua, in 1989, health workers obtained urethral or cervical samples from 18 people with gonorrhea attending public health clinics in Managua and sent them to the National Laboratory of Public Health in Managua for characterization of their antibiotic susceptibility. Of the 18 strains, 15 (83.3%) were of the auxotype/serotype Proto/PIB. Electrophoresis of lipopolysaccharides on SDS-polyacrylamide gels (15%) with 4 M urea revealed no difference in lipopolysaccharide profiles for all strains. The variable expression of the 31-kDa opacity outer membrane protein was not related to antimicrobial resistance. All isolates exhibited susceptibility to ceftriaxone, spectinomycin, cefazolin, cefoxitin, and rifampin. 78% of the strains produced beta-lactamase. 89% of the strains were resistant to penicillin and ampicillin, 44% were resistant to tetracycline, 28% were resistant to cefamandol, 22% were resistant to chloramphenicol, and 11% were resistant to erythromycin. There were 5 distinct groups of Neisseria gonorrhoeae isolated according to their plasmid profiles. The largest was plasmid profile group 1 (55.6%), defined as carrying the 24.5, 3.2, and 2.6 MDa plasmids. It produced beta-lactamase. Penicillinase-producing N. gonorrhoeae (PPNG) comprised 78% of the isolates, 22% of whom were tetracycline-resistant N. gonorrhoea. One PPNG strain exhibited a parallel decrease of penicillin binding to penicillin-binding protein 2. These findings confirmed the presence of multiresistant N. gonorrhoeae strains in Managua, Nicaragua. Based on these findings, the researchers recommended that penicillin and tetracycline not be used to treat gonorrhea in Nicaragua; they recommended ceftriaxone and spectinomycin.

Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay.

The utility of spiramycin for preventing transplacental transmission of toxoplasmosis and the efficacy of conventional macrolides against Toxoplasma gondii are subjects of active debate. An in vitro microassay was developed to determine the relative inhibitory activity against T. gondii of 24 conventional macrolides derived from erythromycin and tylosin (14- and 16-membered macrolides, respectively). Macrolides and T. gondii RH tachyzoites were added to monolayers of BT cells grown in 96-well plates. Plates were incubated for 20 h at 37 degrees C, and the growth of T. gondii was then measured by the selective incorporation of [3H]uracil in trichloroacetic acid-precipitable material during an additional incubation of 20 h. Dose-response curves and 50 and 90% inhibitory concentrations (IC50 and IC90, respectively) were determined for each drug. Microscopic examination was performed on stained replicates of the infected monolayers, and the relative toxicities of the drugs for host cells were determined. Spiramycin and tylosin showed only limited activity against T. gondii (IC50 of 20.16 and 20.00 micrograms/ml, respectively). Erythromycin and azithromycin had a better anti-Toxoplasma activity with IC50 of 14.38 and 8.61 micrograms/ml, respectively, whereas drugs like desmycosin, dirithromycin, and roxithromycin had no detectable activity. Although many macrolides inhibited intracellular proliferation of T. gondii, azithromycin was the only macrolide demonstrating prolonged inhibitory activity on the replication of intracellular tachyzoites. We conclude that conventional 14- and 16-membered macrolides often interfere with the growth of, but may not kill, T. gondii RH tachyzoites in vitro.

Characterization of lithium-induced enzyme release from human polymorphonuclear leukocytes.

Lysozyme release from purified human polymorphonuclear leukocytes was found to be uniquely enhanced by 2.5-20 mM LiCl. This effect was dose dependent and was not detected when the media was supplemented with NaCl, KCl, MgCl2, or CaCl2. The purified isotopes of Li+, 6Li, and 7Li were equally effective in enhancing lysozyme release from the cells at 10 and 20 mM, but 6Li was more effective than 7Li at 5 mM. The enhancement of enzyme release in the presence of Li+ was comparable to the enhancement observed in the presence of N-formylmethionylleucylphenylalanine (fMLP). Addition of LiCl plus fMLP did not result in lysozyme release in excess of each stimulant alone, except when the cells were incubated with 20 mM 6Li + 10(-5) M fMLP. In addition, enzyme release induced by these two agents could be further enhanced to the same degree by addition of cytochalasin D to the incubation mixtures. While similarities between enzyme release induced by LiCl and fMLP were detected, optimal stimulation of enzyme release by Li+ was much more sensitive to inhibition by pertussis toxin than was maximal fMLP stimulation. Therefore, the intracellular events altered by Li+ and the peptide may share some metabolic steps, but they differ in their sensitivity to alterations in cAMP metabolism.

Quinolone antimicrobial agents: mechanism of action and resistance development.

The primary target of fluoroquinolone antimicrobial agents is the A subunit of DNA gyrase. In several cases a close relationship to ID50 (inhibitory dose-50%) and minimum inhibitory concentration (MIC) has been shown for gram-negative bacteria, although this has not been regularly observed for gram-positive bacteria to date. Cellular entry is by means of diffusion and involves, at least in part, the porin pathway in the outer membrane of gram-negative bacteria. An energy dependent efflux occurs which is more active in brain heart infusion broth than nutrient broth and which, to date, has not been shown to contribute to inhibition of growth or bacterial lethality. The extent of uptake by different gram-negative bacteria varies and may contribute in some cases to determination of the MIC. Resistance to fluoroquinolones is by means of mutations affecting the gyrase gene coding for the A subunit and mutations which affect cell permeability particularly involving porin proteins of the outer membrane. Combined target and permeability resistance has been reported but involves two or more mutational steps. Resistance during clinical treatment has been observed, but is most likely to be of low magnitude and to be detected in patients with significantly compromised host defenses.

Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility.

Cell composition and surface properties of Escherichia coli were modified by using various growth media to investigate the role of yet uncharacterized components in ceftazidime susceptibility. An eightfold dilution of Luria broth was used as the basic growth medium and was supplemented with up to 4% phosphate, 5% glucose, or 12% L-glutamate. Decreases in cephaloridine and ceftazidime susceptibility, of two- and eightfold, respectively, were observed only in the glucose-enriched medium. The outer membrane permeability to ceftazidime and cephaloridine was evaluated by crypticity indices. Indices were unchanged under all growth conditions. Fluorometry of whole cells with 1-N-phenylnaphthylamine showed that glucose does not affect the interaction of this hydrophobic probe with the membranes but showed that elevated concentrations of phosphate or glutamate cause a marked increase in cell hydrophobicity, which, in turn, correlates with an increase in the susceptibility of E. coli to nalidixic acid. Growth in phosphate- or glutamate-enriched media caused an augmentation in major phospholipid species and may explain the increased hydrophobicity and susceptibility of E. coli to nalidixic acid. These data showed that E. coli susceptibility to ceftazidime is not influenced by cell surface hydrophobicity and suggested that the contribution of a nonspecific lipophilic diffusion route for entry of ceftazidime into cells is not likely to occur or is distinct from that of more hydrophobic molecules such as nalidixic acid. Finally, the penicillin-binding proteins of the E. coli cells were also investigated. Penicillin-binding protein 8 was only markedly labeled with 125I-penicillin V in inner membranes extracted from cells grown with glucose. Results of this study suggest that the unexpected change in penicillin-binding protein 8 observed in the presence of glucose may be responsible for the increase in MICs of cephaloridine and ceftazidime.

Contribution of permeability and sensitivity to inhibition of DNA synthesis in determining susceptibilities of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis to ciprofloxacin.

To examine the correlation between bacterial cell susceptibility to ciprofloxacin and the magnitude of uptake and cell target sensitivity, the relative contribution of ciprofloxacin accumulation in intact cells and its ability to inhibit DNA synthesis were investigated among strains of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis. Uptake studies of [14C]ciprofloxacin demonstrated diffusion kinetics for P. aeruginosa and E. coli. Ciprofloxacin was more readily removed from E. coli J53 and A. faecalis ATCC 19018 by washing than from P. aeruginosa PAO503. These results indicate that the process of cell accumulation is different for P. aeruginosa in that the drug is firmly bound at an extracellular site. Whatever the washing conditions, A. faecalis accumulated less drug than either of the other two bacteria. Magnesium chloride (10 mM) caused a substantial decrease of ciprofloxacin accumulated and an increase in the MIC, depending upon the nature of the medium. The addition of carbonyl cyanide m-chlorophenylhydrazone caused a variable increase in drug accumulated, depending on the medium and the bacterial strain. The concentration of ciprofloxacin required to obtain 50% inhibition (ID50) of DNA synthesis for P. aeruginosa PAO503 and A. faecalis ATCC 19018 did not correlate with their corresponding MICs but did for E. coli J53. Treatment with EDTA decreased the ID50 of ciprofloxacin for P. aeruginosa PAO503 and its gyrA derivative by 5- and 2-fold, respectively, and decreased the ID50 for E. coli JB5R, a strain with a known decrease in OmpF, by 1.4-fold but did not decrease the ID50 for the normally susceptible E. coli J53. The ID(50) for P. aeruginosa obtained after EDTA treatment or in ether-permeabilized cells was higher than that obtained for the other two strains. The protonophore carbonyl cyanide m-chlorophenylhydrazone prevented killing by low ciprofloxacin concentrtaions, but sodium azide did not. The latter compound did not enhance killing in association with inhibition of a previously described energy-dependent efflux of ciprofloxacin susceptibility being the susceptibility to inhibition of DNA synthesis in E. coli, poor premeability associated with the small pore size of A. faecalis, and a combination of low permeability and reduced susceptibility of DNA synthesis to inhibition for P. aeruginosa.

Antibiotic susceptibility profiles of 941 gram-negative bacteria isolated from septicemic patients throughout Canada. The Canadian Study Group.

The choice of antimicrobial therapy for the treatment of bacteremia is often empirical and based on the knowledge of antibiotic susceptibility profiles of the most common bacteria causing such infections. It therefore is crucial to survey the susceptibility of bacteria causing sepsis. This study examines the susceptibility profiles of 941 gram-negative bacteria, isolated from septic patients in 10 Canadian hospitals, to 28 antimicrobial agents. Among the isolates, 30 different species were represented; Escherichia coli dominated, representing 52.5% of isolates. More than 50% of all bacteria were resistant to ampicillin. Only 67% of the E. coli isolates were susceptible to ampicillin, while 30% of all strains were resistant to ticarcillin. Of the cephalosporins, ceftazidime and cefoperazone/sulbactam were the agents to which isolates were the most susceptible (90%). Only 51% of the E. coli strains were susceptible to cephalothin, while 91% were still susceptible to cefazolin. A total of 93% and 98% of the strains were susceptible to aztreonam and imipenem, respectively. Aminoglycosides were highly active against most isolates, in general in the following order: netilmicin greater than tobramycin greater than gentamicin greater than amikacin. Tobramycin was the most active against Pseudomonas aeruginosa. Nearly all isolates were susceptible to the quinolones. Tolerance (MBC/MIC ratio, greater than or equal to 32) was rarely observed. This survey of the susceptibility of gram-negative bacteria causing sepsis provides valuable information for implementing the chemotherapy for gram-negative septicemia and demonstrates that several older and newer agents, alone or in combination, can be used as adequate initial therapy for gram-negative sepsis in Canada.