Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.     

The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of d-serine

Human urine contains a relatively high concentration of d -serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d -serine deaminase gene ( dsd A) of UPEC. We found the gene in several clinical isolates of S. saprophyticus ; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii , phylogenetically close relatives of S. saprophyticus , and could also not be detected in isolates of Staphylococcus aureus , Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d -serine, whereas S. aureus , S. epidermidis and other staphylococcal species could not. The association of the dsd A gene with growth in media including d -serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d -serine utilization and detoxification may be a general property of uropathogenic bacteria.     

Micro determination of polyamines with snake venom oxidase.

An accurate micromethod suitable for the assay of polyamines in concentrations of 2 to 30 nmol is described. It is based on the oxidation of polyamines by purified fractions of crude l-amino acid oxidase from Russell's viper venom. By a combination of two enzyme fractions, one which oxidizes polyamines and amino acids (AAP) and another which oxidizes only amino acids (AA), the technique can accurately determine polyamine concentrations in extracts of sera which may not be free of amino acids. Experiments described show 90 to 100% recovery of added polyamines in the presence of varying amounts of amino acids. Polyamines added to serum also showed recoveries ranging from 96 to 98%. The enzymes do not oxidize histamine and epinephrine and are very stable. The method does not require sophisticated equipment and is suitable for screening of large number of clinical samples to assess the importance of polyamines as a diagnostic test or their prognostic value in diseases like cancer.     

Polyamines in representative taxa of coryneform and other bacteria

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.     

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.     

Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa

Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms.     

Mechanism of cGMP-gated channel block by intracellular polyamines

Polyamines block the retinal cyclic nucleotide-gated channel from both the intracellular and extracellular sides. The voltage-dependent mechanism by which intracellular polyamines inhibit the channel current is complex: as membrane voltage is increased in the presence of polyamines, current inhibition is not monotonic, but exhibits a pronounced damped undulation. To understand the blocking mechanism of intracellular polyamines, we systematically studied the endogenous polyamines as well as a series of derivatives. The complex channel-blocking behavior of polyamines can be accounted for by a minimal model whereby a given polyamine species (e.g., spermine) causes multiple blocked channel states. Each blocked state represents a channel occupied by a polyamine molecule with characteristic affinity and probability of traversing the pore, and exhibits a characteristic dependence on membrane voltage and cGMP concentration.     

Comparative evaluation of sequestration of polyamines by perfused rabbit and rat lungs

Differences were observed in the sequestration of polyamines putrescine, spermidine and spermine by isolated, ventilated, perfused rat and rabbit lungs, former being able to accumulate more polyamines compared to the latter. Steady state equilibrium was reached earlier for spermine in rat. Isolated ventilated lungs were perfused with harmaline and ouabain, inhibitors known to inhibit the sodium pump at a maximum concentration of 1 mM for rabbit lungs and 0.4 and 0.2 mM for rat lungs, respectively. They did not affect the uptake of polyamines by rat lung but decreased the uptake of putrescine by rabbit lung. Decreased sodium (50 meq/L) in the perfusate increased the uptake of spermine and spermidine by rabbit lung but again showed no effect with rat lung. However, the uptake of polyamines by isolated ventilated rat and rabbit lungs perfused for 60 min with these compounds was linear over the entire range of high concentrations studied. These results suggest that the major uptake process of polyamines by intact lungs of both animal species is primarily by simple diffusion. HPLC analysis of the perfusate and lungs from both animal species post-perfusion indicated no detectable metabolites of the polyamines.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.     

Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems

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.     

Distinct difference in the polyamine compositions of bryophyta and pteridophyta

Polyamine contents of various species of plants and fungi including Bryophyta, Pteridophyta, Gymnospermae, Ascomycota, Basidiomycota, and Lichenobionta were determined by the combination of six chromatographic techniques. Polyamines examined included putrescine, spermidine, spermine, 1,3-diaminopropane (diaminopropane), sym-norspermidine (norspermidine), sym-norspermine (norspermine), thermospermine, caldopentamine, homocaldopentamine, cadaverine, aminopropylcadaverine, sym-homospermidine (homospermidine), agmatine, and canavalmine. In addition to the widely occurring polyamines (putrescine, spermidine, and spermine), the "unusual" polyamines norspermidine and norspermine were found to be widely distributed in Bryophyta and Lichenobionta. These two polyamines were not detected in any species of Pteridophyta, Gymnospermae, and fungi even though their possible precursor, diaminopropane, was found in some species. Homospermidine was one of the major polyamines in Bryophyta and Lichenobionta, and was detected in most species of Pteridophyta and sporadically in higher plants. Agmatine was detected in most species of Bryophyta and in certain species of Gymnospermae. These data suggest that norspermidine, norspermine, and homospermidine can serve as chemical phylogenic and taxonomic markers in Plantae and Fungi.     

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo ( Raphidophyceae ), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua . These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.     

Role of polyamines in gibberellin-induced internode growth in peas

To determine the requirement for polyamines in gibberellin (GA) induced internode growth polyamine content was measured in internodes of peas of various internode phenotypes (slender, tall, dwarf, nana) with and without applied gibberellin (GA₃) and polyamine synthesis inhibitors. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. The amounts of polyamines in the different genetic lines of peas, which differed in internode lengths and extractable GA content, correlated with the extent of internode elongation. High polyamine concentrations were associated with young internodes and decreased with internode expansion. Extremely short internodes of nana plants without GA exhibited equal or higher amine concentrations relative to internodes of other lines of peas and GA-stimulated nana seedlings. The polyamine synthesis inhibitors, α-difluoromethylornithine and α-difluoromethylarginine, independently or in combination, inhibited polyamine accumulation and internode elongation of tall peas and GA-stimulated nana plants. Agmatine and putrescine restored growth and endogenous polyamine content to variable degrees. However, exogenous polyamines were not effective in promoting growth unless intracellular amines were partially depleted.

These results suggest that polyamines do not have a role in cell elongation, but may be required to support cell proliferation. Polyamines do not mediate the entire action of GA in internode growth of peas since GA induction of growth involves both cell division and cell elongation, whereas polyamines appear to affect cell division only.

     

Ethylene and polyamine interactions in morphogenesis of <Emphasis Type="Italic">Passiflora cincinnata</Emphasis>: effects of ethylene biosynthesis and action modulators,as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.     

Human DNA topoisomerase I inhibitory activities of synthetic polyamines: relation to DNA aggregation

DNA aggregation by polyamines has acquired importance as a prerequisite for the cellular uptake of DNA for gene therapy. Intracellular polyamines are constitutive components of mammalian cells and their availability is critical for cell proliferation. Interference of polyamine biosynthesis by synthetic polyamines leads to cytotoxicity. Optimization of the polyamine structural parameters is necessary to control their DNA aggregation, cytotoxic or enzyme inhibitory activities. We designed two series of tetra- and hexamines and compared their human DNA topoisomerase I (top1) inhibitory effects with the DNA aggregation properties. We show that hexamines are more efficient inhibitors of DNA relaxation by top1 than tetramines and that they suppress the top1-mediated DNA cleavage while tetramines do not. The DNA aggregation abilities within two series of polyamines correlate with the length of their central methylene chain. By contrast, the top1 inhibition within two series does not show the same correlation but demonstrates a threshold inhibitory effect on going from the (CH(2))(12) to the (CH(2))(14) central chain. We show further that the structures of DNA aggregates formed by polyamines with the (CH(2))(10-12) or with the (CH(2))(14-16) chains are very different. The first are a fluid cholesteric-type phases, whereas the second are well-structured aggregates similar to columnar liquid crystals with high packing density of DNA duplexes. The structures of polyamines-induced DNA aggregates are proposed to be crucial for top1 catalysis. The structure-function correlation described here may serve as a guide for rational design of polyamines with desired DNA-aggregation or anti-top1 activities.     

Antibiotic resistance in bacterial isolates from laboratory animal colonies naive to antibiotic treatment

Antiobiogrammes were made of a number of isolates of Staphylococcus aureus, Escherichia coli and Pasteurella pneumotropica derived from rodent, rabbit or minipig colonies never treated with antibiotics. For S. aureus no differences between rats and mice were found in the percentage of resistant isolates. Gentamicin and erythromycin were found to be the most efficient, while the highest percentages of resistance were found to be against penicillins and sulphonamides. In general, the results from antibiogrammes on E. coli were rather uniform, with only slight differences between isolates from different species, except that more vancomycin and tetracycline-resistant minipig isolates were found. In almost all isolates of E. coli, resistance was shown against penicillin, fucidin, macrolides, lincosamides and tiamulin. For a number of antibiotics, mouse isolates of P. pneumotropica were more frequently found to be sensitive than rat isolates. The resistance patterns of E. coli from the minipigs were quite similar to resistance patterns found in farm pigs, but apart from this, the resistance patterns of the bacterial species tested did not resemble human or farm animal patterns in any of the animal species, and, therefore, these studies do not support the theory that S. aureus and E. coli in laboratory animal colonies derive from the normal flora of the human caretakers. The fact that rodent species of E. coli, in contrast to human and farm animal species, are sensitive to ampicillin, tetracyclines, and the combination of sulphonamides and trimethoprim, might be due to the fact that these antibiotics are not used in rodent populations.