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PubMed1997

Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase.

The vast majority of AIDS-related deaths are associated with opportunistic infections. For fungal infections, there are few effective antifungals, particularly for systemic use. The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls. This report describes one of the genes cloned by complementation of the blm1-1 mutation of S. cerevisiae using a YCp50-based yeast genomic library. Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases. The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle. Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.

1-Phosphatidylinositol 4-Kinase↗

PubMed1975

Survival of human enteric and other sewage microorganisms under simulated deep-sea conditions.

The survival of pure cultures of Escherichia coli, Streptococcus faecalis, Clostridium perfringens, and Vibrio parahaemolyticus under simulated deep-sea conditions of low temperature (4 C), seawater, and hydrostatic pressures ranging from 1 to 1,000 atm was determined over a period exceeding 300 h. The viability of E. coli and total aerobic bacteria in seawater-diluted raw sewage subjected to these deep-sea conditions was also measured. There was a greater survival of both E. coli and S. faecalis at 250 and 500 atm than at 1 atm at 4 C. S. faecalis was quite insensitive to 1,000 atm, whereas with E. coli there was a 10-fold die-off per 50-h exposure to 1,000 atm. In contrast, V. parahaemolyticus and C. perfringens were quite sensitive to pressures exceeding 250 atm, and with both of these species there was a total loss of viability of approximately 10(8) cells per ml within 100 h at 1,000 arm and within 200 h at 500 atm. The viability of the naturally occurring fecal coliforms in sewage exposed to moderate pressures at 4 C was found to be similar to the survival patterns demonstrated with pure cultures of E. coli. The total numbers of aerobic bacteria in these sewage samples, however, stabilized at 500 and 1,000 atm after 100 h, and at 1 and 250 atm there was significant growth of sewage-associated bacteria, which apparently utilized the organic compounds in the seawater-diluted sewage samples. A preliminary classification of some of these bacteria indicated that approximately 90% (160 isolates) of the organisms that survived over a 400-h exposure to 500 and 1,000 atm were Arthrobacter/Corynebacterium species, and the representative organisms capable of growing at 1 and 250 atm in seawater at 4 C were gram-positive cellulose digesters and an unidentified gram-negative coccus. The significance of these results with respect to the contamination of the deep ocean with human pathogens and the possibility of sewage-associated microorganisms growing and competing with indigenous marine microbial flora in situ is discussed.

Arthrobacter↗

PubMed1992

Mode of photocatalytic bactericidal action of powdered semiconductor TiO2 on mutans streptococci.

Powdered semiconductor TiO2 has a photocatalytic bactericidal capacity on some kinds of bacteria, but its mechanism still remains unclear. The mode of its photocatalytic bactericidal action on the mutans group of streptococci was investigated. Powdered TiO2 had a bactericidal capacity on all serotypes of mutans streptococci. Streptococcus sobrinus AHT was mainly used for these experiments. The most effective concentration of TiO2 was about 1 mg ml-1 and, at this concentration, 10(5) colony-forming units of S. sobrinus AHT per millilitre were completely killed within 1 min. In order to search for the mechanism of this effect, a high bacterial cell density (10(9) colony-forming units ml-1) was used in the following studies. "Rapid" leakage of potassium ions from the bacteria occurred parallel to the decrease in cell viability. Protein and RNA were "slowly" released from bacterial cells for a reaction time up to 120 min. The pH of the reaction mixture decreased continuously to 4.5 after 120 min. Co-aggregation of S. sobrinus AHT and powdered TiO2 occurred at high bacterial densities (above 10(8) colony-forming units ml-1). Aggregates gradually decomposed with light irradiation. Transmission electron microscopy of S. sobrinus AHT after photocatalytic action for 60-120 min indicated complete destruction of bacterial cells. From these results, bacterial death appears to be caused by a significant disorder in cell membranes and finally the cell walls were decomposed.

Hydrogen-Ion Concentration↗

PubMed2006

Rapid test for distinguishing membrane-active antibacterial agents.

In the search for antibacterial agents with a novel mode-of-action (MOA) many targeted cellular and cell-free assays are developed and used to screen chemical and natural product libraries. Frequently, hits identified by the primary screens include compounds with nonspecific activities that can affect the integrity and function of bacterial membrane. For a rapid dereplication of membrane-active compounds, a simple method was established using a commercially available Live/Dead(R) Bacterial Viability Kit. This method utilized two fluorescent nucleic acid stains, SYTO9 (stains all cells green) and propidium iodide (stains cells with damaged membrane red) for the drug-treated bacterial cells. The cells were then either examined visually by fluorescence microscopy or their fluorescence emissions were recorded using a multi-label plate reader set to measure emissions at two different wavelengths. The ratio of green versus red was compared to a standard curve indicating the percentage of live versus dead bacteria. Nine known antibiotics and 14 lead compounds from various antibacterial screens were tested with results consistent with their MOA.

Anti-Bacterial Agents↗

PubMed2003

Inhibition of HSV-1 replication and reactivation by the mutation-insensitive transcription inhibitor tetra-O-glycyl-nordihydroguaiaretic acid.

Methylated derivatives of nordihydroguaiaretic acid (NDGA)were previously shown to be potent mutation-resistant inhibitors of herpes simplex virus type 1 (HSV-1) which target Sp1 protein binding to critical viral promoters. The hydrophobic nature of these agents, however, renders them relatively water-insoluble and, therefore, limits their applicability. We report here on the anti-HSV-1 properties of a related but water-soluble glycylated derivative of NDGA, tetra-O-glycyl-NDGA (G(4)N). In yield reduction assays, G(4)N inhibited replication of laboratory and clinical strains of wild type HSV-1 and ACV-resistant (HSV-1(R)) strains of HSV-1 in a dose-dependent manner, with average IC(50) values of 4.7 and 3.2 microM against wild-type and HSV-1(R) strains, respectively. An MTT-based cytotoxicity assay revealed a TC(50) value of 73.2 microM for G(4)N on Vero cells, with no reduction in viability detected at concentrations below 30 microM. Similar to its methylated counterparts, G(4)N was found to inhibit transcription of the HSV-1 ICP4 gene, a major immediate early viral regulator, and gel mobility shift assays showed it can block Sp1 protein binding to cognate sites on the ICP4 promoter. In anticipation of its potential use as a systemic anti-HSV-1 agent, we tested G(4)N in a murine trigeminal ganglia (TG) explant model system, and found G(4)N was able to prevent HSV-1 reactivation from explanted and cultured latently infected TG.

Animals↗

PubMed2002

Impact of lysozyme and lactoferrin on oral Candida isolates exposed to polyene antimycotics and fluconazole.

OBJECTIVE: To assess the antifungal activity of lysozyme and lactoferrin on 10 oral isolates each of Candida albicans and Candida tropicalis following their brief exposure (I h) to subtherapeutic concentrations of two polyene drugs - nystatin, amphotericin B, and an azole -fluconazole. METHODS: Yeasts were sequentially exposed to subtherapeutic concentrations of antifungals and then to either lysozyme or lactoferrin and the viability evaluated by quantifying colony-forming units. RESULTS: The exposure of both C. albicans and C. tropicalis isolates to all three antifungal agents significantly increased their susceptibility to lysozyme (P < 0.05) but not to lactoferrin. Exposure to the two polyene drugs had a lesser impact on the lysozyme susceptibility of both Candida species compared with the azole drug. Both interspecies and intraspecies sensitivity to lysozyme was noted and C. albicans was less susceptible than C. tropicalis. CONCLUSIONS: Lysozyme, in addition to being a potent natural antifungal agent, may act synergistically with the studied antimycotics.

Amphotericin B↗

PubMed2000

Changes in viability and macromolecular content of long-term batch cultures of Salmonella typhimurium measured by flow cytometry.

Exposure of many Gram-negative bacteria to prolonged starvation induces alternative programmes of gene expression, along with a transition into a dormant condition sometimes referred to as a viable non-culturable (VBNC) state. Knowledge of how pathogenic species respond to nutrient limitation is therefore important for their detection and dissemination. This study used flow cytometry, coupled with fluorescent dyes for viability and macromolecular content, to study the responses of the pathogen Salmonella typhimurium to prolonged batch culture. Statistical analysis of the flow cytometric data, together with total and culturable cell counts, failed to demonstrate a VBNC state in this pathogen, contrary to reports from other workers. Analysis of rRNA and protein content identified a small proportion of cells in 110 day-old cultures that represented an active sub-population. This observation may provide an explanation for the long-term survival properties of this organism during prolonged exposure to nutrient limitation, as well as the high degree of heterogeneity observed in labelled cells.

Bacterial Proteins↗

PubMed2003

In vitro effect on Cryptosporidium parvum of short-term exposure to cathelicidin peptides.

Two laboratory methods, a cell culture system and double fluorogenic staining, were used to study the viability and infective ability of Cryptosporidium parvum sporozoites and oocysts after short-term exposure to four cathelicidin peptides. The compounds, SMAP-29, BMAP-28, PG-1 and Bac7(1-35), exerted a strong cytotoxic effect on sporozoites, but did not affect the viability and function of oocysts consistently. Overall, in the sporozoite series, a percentage of the viable population decreased rapidly to less than detectable levels after 15 and 60 min exposure to the peptides at concentrations of 100 and 10 micro g/mL, respectively. In the oocyst series, no compound produced complete inhibition of parasite growth: 60-85% of the oocyst population was viable after 180 min exposure at 100 micro g/mL. SMAP-29 exerted the highest activity against both sporozoites and oocysts.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Inhibition of adherence and killing of Candida albicans with a 23-Mer peptide (Fn/23) with dual antifungal properties.

Candida albicans adheres to host tissue and then proliferates in order to establish a commensal as well as a pathogenic state. Specific adherence to proteins is provided by several surface adhesins of Candida. Two well-studied proteins, Als1p and Als5p, do not require energy for adherence to occur (dead as well as living cells adhere) and have a multiplier effect of cell-cell aggregation that mediates the formation of microcolonies of Candida cells. The entire process is spontaneous, reversible, and stable for physiologically relevant chemical and physical forces. This adherence process is inhibited by the addition of free peptide ligands, including a 23-mer derived from fibronectin (Fn/23) that binds to the adhesins through H bond formation. Adherence was measured by determining the number of yeast cells that adhered to 90-microm-diameter polyethylene glycol (PEG) beads with a 7-mer peptide (KLRIPSV) synthesized on the surfaces of the beads. The concentration of the Fn/23 peptide that inhibited the adherence of cells to the peptide-coated beads by 50% was 4 to 5 microM, and the magnitudes of adherence were similar regardless of the presence or absence of physiologic salt concentrations. The minimum fungicidal concentration of Fn/23 was 2 to 4 microM in water, but there was no killing in physiologic salt concentrations. Peptides from the C and N termini or the center sequence of Fn/23 had no effect on inhibition of adherence and little effect on fungal viability. The fungicidal effect was similar to that seen with 23-, 19-, and 18-mer peptides derived from porcine myeloid cells, a Helicobacter pylori ribosomal protein, and a hybrid of cecropin and magainin, respectively. However, these fungicidal peptides did not inhibit C. albicans adherence to the peptide-coated PEG beads. This dual property of Fn/23, i.e., inhibition of adherence and killing of C. albicans, may provide important adjuvant effects in the treatment of disease caused by this fungus.

Amino Acid Sequence↗

PubMed1985

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

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.

Azlocillin↗

PubMed1971

Glucose and fructose metabolism in a phosphoglucoisomeraseless mutant of Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae deficient in phosphoglucoisomerase (EC 5.3.1.9) is described. It does not grow on glucose or sucrose but does grow on galactose or maltose. Addition of glucose to cultures growing on fructose, mannose, or acetate arrests further growth without altering viability; removal of glucose permits resumption of growth. Glucose causes accumulation of nearly 30 mumoles of glucose-6-phosphate per g (wet weight) of cells and suppresses synthesis of ribonucleic acid. Inhibition of growth by glucose does not appear to be due to a loss of adenosine triphosphate or inorganic orthophosphate. The mutant, however, utilizes glucose-6-phosphate produced intracellularly. Release of carbon dioxide from specifically labeled glucose suggests a C-l preferential cleavage. The kinetics of glucose-6-phosphate accumulation during glucose utilization in the mutant is not consistent with the notion that the utilization of glucose is controlled by glucose-6-phosphate.

Adenine Nucleotides↗

PubMed1977

Recent advances in microbiology in leprosy.

The recent advances in microbiology of leprosy are reviewed. Till now the leprosy bacillus had not been cultivated in laboratory media; the recent claims of success have not been confirmed. There has been a breakthrough in the experimental transmission of leprosy to experimental animals--the white mice, the immune depressed white mice, and the nine-banded armadillo. Apart from providing definite proof for the causative relationship of the bacillus discovered by Hansen and the disease leprosy, the experimental transmission to animals have considerably advances our knowledge about the disease. The mouse has provided a suitable model for screening of antileprosy drugs, detecting development of drug resistance, ascertaining viability of the bacilli and determining the genuineness of a supposed culture of the leprosy bacillus. The armadillo has provided a model for making basic studies of the disease as it occurs in man. Further, the generalised infection in armadillo has provided large amounts of leprosy bacilli for preparing standardised lepromin, for preparing a specific skin-testing antigen containing the active protein fraction of the bacillus, and a step towards the production of a vaccine against leprosy. It is interesting to note that the fact that the protein fraction of the bacillus was responsible for the positive lepromin reaction was discovered by Dharmendra far back in 1941.

Animals↗

PubMed1993

Actinobacillus actinomycetemcomitans and localized juvenile periodontitis. Clinical, microbiologic and histologic studies.

The present studies examined Actinobacillus actinomycetemcomitans and its role in localized juvenile periodontitis (LJP). The distribution of the bacteria was studied in healthy normals, patients with adult periodontitis, diabetics, and those with LJP. Over 95% of the LJP patients harbored A. actinomycetemcomitans, whereas only 17% of healthy subjects, 21% of adult periodontitis patients, and 5% of diabetics were positive. All members of a LJP family harboring the organism yielded isolates of the same biotype and serotype. The transmission of the bacteria was studied after transfer of the bacteria, with periodontal probes from infected to healthy gingival sites, within the oral cavity of LJP patients. Newly colonized gingival sites, 50% of those involved, became free of A. actinomycetemcomitans after only 3 weeks. A purposely forceful inoculation contributed to a more predictable colonization (89%), but only prolonged the colonization with one week. Treatment of LJP lesions with scaling and root planing resulted in minimal clinical and microbiological changes during a 16 week follow-up period. However, gingival curettage and modified Widman flap surgery suppressed A. actinomycetemcomitans in 75% and 89% of the sites, and resulted in resolution of periodontal pocket depth and gain in attachment level. Gingival tissue specimens, from 35 LJP sites, 3 control sites, and one monkey biopsy, were studied to verify the hypothesis of gingival infiltration of A. actinomycetemcomitans. Bacteria were identified immunohistologically with rabbit antisera serospecific to the three A. actinomycetemcomitans serotypes. Positive staining was observed in the tissue from all but one LJP patient. Twenty-eight (80%) lesions were positive for A. actinomycetemcomitans antigens in the gingival connective tissue, often with antigens located both between and within cells. The specimen from a culture positive control demonstrated no signs of invasion, similar to the monkey specimen. Transmission electron microscopic examination verified gram-negative bacteria that appeared as single microbes or small clusters in the connective tissue. The viability of the invading bacteria was studied by selective culture of minced tissue specimens and a series of washing solutions, after a rigorous surface disinfection. The washings successfully removed the bacteria in most cases, and after mincing 73% of the biopsies showed to be culture positive. The culture recovery of A. actinomycetemcomitans from minced tissues correlated exceedingly well with the presence of specific antigens in the gingival tissues from corresponding gingival biopsies. To further demonstrate the importance of A. actinomycetemcomitans in LJP, six culture positive LJP patients were treated by systemic tetracycline alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1982

Fate in model ecosystems of microbial species of potential use in genetic engineering.

The changes in populations of Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Klebsiella pneumoniae, Agrobacterium tumefaciens, Rhizobium meliloti, and Saccharomyces cerevisiae were measured after their introduction into samples of sewage, lake water, and soil. Enumeration of small populations was possible because the strains used were resistant to antibiotics in concentrations and combinations such that few species native to these ecosystems were able to grow on agar containing the inhibitors. Fewer than 2 cells per ml of sewage or lake water and 25 cells per g of soil could be detected. A. tumefaciens and R. meliloti persisted in significant numbers with little decline, but S. aureus, K. pneumoniae, S. typhimurium, S. cerevisiae, and vegetative cells of B. subtilis failed to survive in samples of sewage and lake water. In sterile sewage, however, K. pneumoniae, B. subtilis, S. typhimurium, A. tumefaciens, and R. meliloti grew; S. cerevisiae populations were maintained at the levels used for inoculation; and S. aureus died rapidly. In sterile lake water, the population of S. aureus and K. pneumoniae and the number of vegetative cells of B. subtilis declined rapidly, R. meliloti grew, and the other species maintained significant numbers with little or a slow decline. The populations of S. aureus, K. pneumoniae, A. tumefaciens, B. subtilis, and S. typhimurium declined in soil, but the first four species grew in sterile soil. It is suggested that some species persist in environments in which they are not indigenous because they tolerate abiotic stresses, do not lose viability readily when starved, and coexist with antagonists. The species that fails to survive need only be affected by one of these factors.

Bacillus subtilis↗

PubMed2002

CAY-I, a fungicidal saponin from Capsicum sp. fruit.

Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal saponin, CAY-1, with a molecular weight of 1243.35 Da, was isolated and purified to homogeneity from commercially available dry, ground fruit of Capsicum frutescens. CAY-1 was shown to be a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus and A. niger with species-dependent LD90 values between 3 and 20 microM. Activity against some Aspergillus species was affected by the test medium used. In vitro assays, CAY-1 was effective against Pneumocystis carinii (IC50): 9.5 microM) and Candida albicans (IC90: 6.2 microM). CAY-1 had no effect on the viability of the nongerminating conidia of the two filamentous fungi, P. carinii and C. albicans, nor on the conidial type of Fusarium oxysporum. It was ineffective against the bacteria Enterobacter agglomerans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. CAY-1 was not cytotoxic to A 549 lung carcinoma cells or HeLa cells at effective fungicidal concentrations. The results indicate that CAY-1 is an effective fungicide for Aspergillus species, C. albicans and P. carinii at concentrations below the threshold for mammalian cell toxicity.

Antifungal Agents↗

PubMed2000

Ursolic acid triggers calcium-dependent apoptosis in human Daudi cells.

Ursolic acid (UA) is a pentacyclic triterpenoid compound which occurs naturally in a large variety of vegetarian foods, medicinal herbs and plants. In the present study, ursolic acid was found to decrease cell viability in human lymphoma Daudi cells in a dose-dependent manner. UA also induced morphological changes in cells as well as loss of membrane asymmetry, DNA fragmentation and nuclei condensation, indicating that the mechanism by which UA induced cell death was through apoptosis. Treatment with UA increased intracellular Ca2+ levels. Use of Ca2+ channel inhibitors like verapamil blocked this Ca2+ influx and also the triggering of apoptosis. We hypothesized that the binding of UA to glucocorticoid receptors and the Ca2+ currents induced constituted the first steps of apoptosis.

Antineoplastic Agents, Phytogenic↗

PubMed1981

Antibiotic action of pyocyanin.

Biologically produced pyocyanin was purified, and the nature of its antibacterial action was determined for several bacteria. The pigment was shown to be bactericidal for all susceptible organisms. The bactericidal effect was dependent upon pyocyanin concentration and resulted in decreases in viability ranging from 1 to 8 log viable cells ml-1. The gram-positive bacteria were more susceptible as a group to the antibiotic action than were the gram-negative bacteria. All apyocyanogenic pseudomonads tested were totally resistant to the pigment, suggesting that resistance may be a characteristic of the genus. Pseudomonas aeruginosa, the producer organism, was also essentially unaffected by high concentrations of pyocyanin. Facultative anaerobes were twofold or more times resistant to the action of the pigment under fermentative conditions; however, the antibiotic action did not require oxygen since denitrifying bacteria were more susceptible during anaerobic respiration than during aerobic respiration.

Anti-Bacterial Agents↗

PubMed2000

The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum.

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.

Anti-Bacterial Agents↗