Gluconimycin,a new antibiotic

Summary A new antibiotic, gluconimycin, was isolated from Streptomyces AS 9. The systematic position of the organism is discussed. Gluconimycin has a polypeptide nature. It contains iron and gluconic acid in its molecule. Thus it has been classified as a member of sideromycins. Gluconimycin is considered from the fast moving type when chromatographed by butanolacetic acid-water. The antibiotic is active against Gram+ve, Gram-ve bacteria and some fungi. The antibiotic exerts high toxicity when injected in mice.     

Long-term starvation-induced loss of antibiotic resistance in bacteria

Escherichia coli, Pseudomonas fluorescens, and aPseudomonas sp. strain 133B containing the pSa plasmid were starved in well water for up to 523 days. There were two patterns of apparent antibiotic resistance loss observed. InPseudomonas sp. strain 133B, there was no apparent loss of antibiotic resistance even after starvation for 340 days. InE. coli, by day 49 there was a ten-fold difference between the number of cells that would grow on antibiotic- and nonantibiotic-containing plates. However, over 76% of the cells that apparently lost their antibiotic resistance were able to express antibiotic resistance after first being resuscitated on non-selective media. By day 523, only 12% of these cells were able to express their antibiotic resistance after being resuscitated. After starvation for 49 days, cells that could not grow on antibiotic medium even after resuscitation, showed a permanent loss of chloramphenicol (Cm) resistance but retained resistance to kanamycin (Km) and streptomycin (Sm). Restriction enzyme digests show that a 2.5 to 3.0 Kb region from map location 12.5 to 15.5 Kb was deleted. This coincides with the 2.5 Kb reduction in plasmid size observed in 3 isolates that had lost antibiotic resistance after starvation for 49 days.Published as Technical Paper #9224, Oregon Agricultural Experiment Station.     

Studies on Fungal Tannase

The possibility that selective inhibition of phage by antibiotic may be achieved by using bacterial mutant resistant to the antibiotic was investigated in the system of HM-phages of Clostridium saccharoperbutylacetonicum, a butanol-producing bacterium.

Consequently, it was found that Oxytetracycline, using the antibiotic-resistant mutant as host, inhibited selectively the growth of HM-phages. The bacterial mutant termed type A (one-step mutant resistant to 30 μg/ml of Oxytetracycline) did not permit the growth of HM-phages (HM 2 and HM 3) in the presence of the antibiotic (ca. 10 μg/ml), though it permitted the growth of the phages in the absence of the antibiotic.

An analysis of the mode of action of Oxytetracycline in HM 2-phage system revealed the following, (i) The antibiotic had a slight phagicidal action, (ii) It did not prevent the phage adsorption, (iii) It inhibited the protein synthesis in phage-infected cells, (iv) It inhibited the lysis of infected cells. Active phages were, however, not detected when the lysis-inhibited cells were artificially lysed.

Another type of bacterial mutant was also encountered. In this mutant termed type B the development of resistance to Oxytetracycline (30 μg/ml) was associated with a simultaneous loss of sensitivity to particular phages (HM 2 group).     

Telithromycin Inhibition of Protein Synthesis and 50S Ribosomal Subunit Formation in <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Cells

The new ketolide antibiotic telithromycin (HMR3647) has been examined for inhibitory effects in cells of Streptococcus pneumoniae. The antibiotic caused a proportional decline in cell growth rate and viability with an IC₅₀ of 15 ng/ml. At a concentration of 7.5 ng/ml, protein synthesis in these cells was reduced by 50%. As seen in other organisms, this compound was also a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays defined the reduced rate of 50S synthesis in antibiotic treated cells. At 7.5 ng/ml the rate was reduced to 50% of the control synthesis rate. An IC₅₀ of 15 ng/ml was found for the effect on this process. 30S ribosomal subunit formation was unaffected by the antibiotic. Inhibition of translation and 50S particle formation are equivalent targets for this antibiotic. The effects of telithromycin in S. pneumoniae are compared with those found in Staphylococcus aureus cells. Received: 29 October 2001 / Accepted: 1 February 2002     

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild‐type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild‐types attained similar population densities. Given the importance of the methyl‐directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild‐type phenotype.     

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.     

Mechanism of Antimycin A Resistance in Bacillus subtilis

The site of action of antimycin A is known to lie between cytochrome b and c in the respiratory chain of mammalian cells. But in general, bacteria, even those which have cytochromes similar to those of mammalian cells such as Bacillus subtilis, are naturally resistant to this antibiotic.

The mechanism of this natural resistance is studied using a strain of B. subtilis. Succinoxidase activity of the intact cells of this bacterium showed very low sensitivity to the antibiotic, but on disruption of the cells, the sensitivity increased 7.5 times. Moreover, the activity of the intact cells could be sensitized by treatment with cationic detergent. In addition to the permeability barrier suggested by the above results, it was found that the electron transport system of this bacterium contained antimycin A insensitive by-path.     

Accumulation and turnover of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutant strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by ³H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.     

Liberation of sisomicin from cells by sodium chloride

Summary Most sisomicin produced during fermentation by Micromonospora inyoensis remained bound inside the cells. When cells were suspended in buffer solutions containing sodium chloride, the bound antibiotic was increasingly liberated by increasing salt concentration. These results were applied to fermentation cultures and, as a result, up to 46% increase in final product titre was achieved.     

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg²⁺ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

The ketolide antibiotic ABT-773 is a specific inhibitor of translation and 50S ribosomal subunit formation in Streptococcus pneumoniae cells

ABT-773 is a new 3-keto macrolide antibiotic that has been shown to be very effective against infections by Gram-positive microorganisms. This work examines its inhibitory effects in cells of Streptococcus pneumoniae. ABT-773 caused a proportional decline in cell growth rates and viability with an IC₅₀ of 5 ng/ml. Protein synthesis in these cells was reduced by 50% at an antibiotic concentration of 2.5 ng/ml. This compound was also found to be a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays revealed a reduced rate of 50S synthesis in antibiotic-treated cells. At 2 ng/ml, the rate was reduced to 33% of the control synthesis rate. An IC₅₀ of 5 ng/ml was found for the effect on this process, indicating an equal effect of the drug on translation and assembly. Synthesis of the 30S ribosomal subunit was unaffected by this antibiotic. The effects of ABT-773 in S. pneumoniae are compared with those of the related ketolide antibiotic telithromycin in S. pneumoniae and in Staphylococcus aureus. Received: 6 November 2001 / Accepted: 14 December 2001     

Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Penicillin production and its mode of inheritance inAspergillus nidulans

Previous workers have shown that some strains ofAspergillus nidulans produce penicillin-like substances. In the present studies, shake-flask cultures of 101 wild-type strains ofA. nidulans, representatives of 18 different heterokaryon-compatible groups, were examined and filtrates of most found to inhibit the growth of a strain ofBacillus subtilis sensitive to penicillin, although members of two of these groups had no detectable antibiotic activity. Five strains with antibacterial properties were chosen for detailed investigation as well as two genetically labelled derivatives obtained from one of these after ultraviolet light treatments; one derivative had a similar antibiotic yield to its original wild-type parent but the other was selected as having increased antibiotic yield. The antibiotic produced by these seven strains was by all tested criteria, including chromatographic and electrophoretic behaviour, indistinguishable from penicillin. A heterokaryon test between the two mutants indicated that antibiotic productivity was under nuclear control.     

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces

Aim: We compared the MBEC?‐HTP assay plates made of polystyrene with metal discs composed of TMZF^® and CrCo as substrates for biofilm formation. Methods and Results: Staphylococcus aureus was grown on polystyrene and on metal discs made of titanium and chrome–cobalt. Antibiotic susceptibility was assessed by examining the recovery of cells after antibiotic exposure and by measuring the biofilm inhibitory concentration (BIC). The minimal inhibitory concentration (MIC) was assessed with planktonic cells. Bacterial growth was examined by scanning electron microscopy. The antibiotic concentration for biofilm inhibition (BIC) was higher than the MIC for all antibiotics. Microscopic images showed the biofilm structure characterized by groups of cells covered by a film. Conclusions: All models allowed biofilm formation and testing with several antibiotics in vitro. Gentamicin and rifampicin are the most effective inhibitors of Staph. aureus biofilm‐related infections. We recommend MBEC?‐HTP assay for rapid testing of multiple substances and TMZF^® and CrCo discs for low‐throughput testing of antibiotic susceptibility and for microscopic analysis. Significance and Impact of the Study: In vitro assays can improve the understanding of biofilms and help developing methods to eliminate biofilms from implant surfaces. One advantage of the TMZF^® and CrCo discs as biofilm in vitro assay is that these metals are commonly used for orthopaedic implants. These models are usable for future periprosthetic joint infection studies.     

Plant selection principle based on xylose isomerase

Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.     

Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7–57.6%) and sterols (48.5–96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.     

Action of FR901228, a Novel Antitumor Bicyclic Depsipeptide Produced by Chromo-bacterium violaceum No. 968, on Ha-ras Transformed NIH3T3 Cells

FR901228, a novel antitumor antibiotic, reversed the transformed morphology of the Ha-ras transformants, Ras-1 cells, and inhibited their growth. The reduction of c-myc expression was observed in FR901228-treated Ras-1 cells by RNA dot-blot hybridization. This reduction of c-myc expression and morphological reversion of the transformed cells to normal were correlated with growth inhibition (G₀/G₁ arrest in cell cycle).     

Susceptibility of Pseudomonas aeruginosa Urinary Tract Isolates and Influence of Urinary Tract Conditions on Antibiotic Tolerance

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe urinary tract infections (UTIs). Because of the high intrinsic antibiotic resistance of P. aeruginosa and its ability to develop new resistances during antibiotic treatment, these infections are difficult to eradicate. The antibiotic susceptibility of 32 P. aeruginosa isolates from acute and chronic UTIs were analysed under standardized conditions showing 19% multi-drug resistant strains. Furthermore, the antibiotic tolerance of two P. aeruginosa strains to ciprofloxacin and tobramycin was analysed under urinary tract-relevant conditions which considered nutrient composition, biofilm growth, growth phase, and oxygen concentration. These conditions significantly enhance the antibiotic tolerance of P. aeruginosa up to 6000-fold indicating an adaptation of the bacterium to the specific conditions present in the urinary tract. This reversible phenomenon is possibly due to the increased formation of persister cells and is based on iron limitation in artificial urine. The results suggest that the general high antibiotic resistance of P. aeruginosa urinary tract isolates together with the increasing tolerance of P. aeruginosa grown under urinary tract conditions decrease the efficiency of antibiotic treatment of UTIs.     

Permanent mycoplasma removal from tissue culture cells: A genetic approach

Mycoplasma contamination of tissue culture cells easily evades detection and, thus, represents a continous threat to cell biologists. In cases where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplasma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains; however, cell associated mycoplasmas are often protected from antibiotics at concentrations shown to be effectivein vitro. Antibiotic concentrations high enough to be lethal to cell asso|ciated mycoplasmas frequently are also detrimental to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistant even to high concentrations of the antibiotic applied. Here, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes these limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent,Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycoplasma while leaving the host cells unharmed. Upon successful mycoplasma eradication, cultivation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibility to the toxic agent. Cessation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chromosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.