Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

Killer systems of Saccharomyces cerevisiae yeasts

The killer systems of Saccharomyces cerevisiae are a peculiar group of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. Their basic properties are linearity of genome, its fragmentation, resulting in two separately replicating major and minor segments, and the ability to control the synthesis of secretory proteins--mycocins which can kill the taxonomically related strains. Secretion of mycocins also confers immunity to their action. The strains containing killer symbionts are toxigenic and resistant to their own toxins, while those with no killer double-stranded RNA are sensitive to mycocins. The killer systems of Saccharomyces cerevisiae possess some properties relevant to viruses and evidently are evolved during the evolution of infectious viruses. Occurrence of such systems in monocellular eucaryotic organisms is an example of genome complication in the course of putting together the virus-like components. The peculiarities of replication and expression of killer systems and their utilization for the construction of vector molecules are discussed.     

Selection of plant growth-promoting Pseudomonas spp. that enhanced productivity of soybean-wheat cropping system in Central India

The aim of this investigation was to select effective Pseudomonas sp. strains that can enhance the productivity of soybean-wheat cropping systems in Vertisols of Central India. Out of 13 strains of Pseudomonas species tested in vitro, only five strains displayed plant growth-promoting (PGP) properties. All the strains significantly increased soil enzyme activities, except acid phosphatase, total system productivity, and nutrient uptake in field evaluation; soil nutrient status was not significantly influenced. Available data indicated that six strains were better than the others. Principal component analysis (PCA) coupled cluster analysis of yield and nutrient data separated these strains into five distinct clusters with only two effective strains, GRP3 and HHRE81 in cluster IV. In spite of single cluster formation by strains GRP3 and HHRE81, they were diverse owing to greater intracluster distance (4.42) between each other. These results suggest that the GRP3 and HHRE81 strains may be used to increase the productivity efficiency of soybean-wheat cropping systems in Vertisols of Central India. Moreover, the PCA coupled cluster analysis tool may help in the selection of other such strains.     

Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems

The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to possess different excretion systems. Export in strain KK 25 is much slower than in the two mutants. The differences between the two lysine-producing strains are more subtle. K(m) and V(max) are similar, but pH dependence and membrane potential dependence reveal differences in the intrinsic properties of the carrier system.     

Differences and dynamic changes in the cell surface properties of three Pseudomonas aeruginosa strains isolated from petroleum-polluted soil as a response to various carbon sources and the external addition of rhamnolipids

Three Pseudomonas aeruginosa strains isolated from petroleum-polluted soil were the subject of studies concerning changes in cell surface properties. Fundamentally different reactions could be observed for each of the studied strains after a cultivation on various carbon sources. The experiments carried out during the logarithmic growth phase showed, that the changes in the cell surface hydrophobocity values were dynamic and substrate dependant. An external addition of rhamnolipids to the tested systems resulted in further shifts in the CSH values. All of the strains displayed miscellaneous phenotypic properties during MATH, sedimentation profile, Zeta potential and surface tension measurements. The obtained results lead to a conclusion, that the presence of rhamnolipids seems to be the key factor to this phenomenon, as all of the studied strains exhibited the ability to produce this biosurfactant in a different degree.     

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems.

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.     

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.     

Fermentation technologies for the production of probiotics with high viability and functionality

There is growing scientific evidence supported by mechanistic and clinical studies that probiotics can provide health benefits. As probiotics are highly sensitive to many environmental factors, and because the propagation of many strains of intestinal origin is not straightforward, most commercial strains are selected on the basis of their technological properties - ruling out some strains with promising health properties. To date, probiotic production has almost exclusively been carried out using conventional batch fermentation and suspended cultures, in some cases combined with the use of sublethal stresses to enhance cell viability, the addition of protectants or microencapsulation to provide cell protection. However, other less conventional fermentation technologies, such as continuous culture and immobilized cell systems, could have potential for enhancing the performance of these fastidious organisms. These technologies might be employed to develop strains with improved physiology and functionality in the gut and to enlarge the range of commercially available probiotics, as well as expanding product applications.     

The lack of mutagenic properties of patulin and patulin adducts formed with cysteine in Salmonella test systems.

The mutagenic properties of patulin and the patulin adducts formed with cysteine were tested with histidine auxotroph Salmonella typhimurium strains as indicator organisms. The tests were performed by microsomal activation and host-mediated assay. Neither patulin nor patulin--cysteine reaction mixture was mutagenic in these test systems.     

Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems

The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.     

Fusion and hybridization of marsupial and eutherian cells. V. Development of selective systems

The availability of systems which permit the selective elimination of marsupial cells from fused cultures is an essential requirement for the production of marsupial X eutherian somatic cell hybrids. Such hybrids have particular advantages for genetic studies of mammalian cells. We describe the isolation and characterization of several drug-resistant marsupial cell strains. We have selected strains resistant to concentrations of 10 micrograms/ml of the purine analogues 8-azaguanine and 6-thioguanine. Several of these strains were found to be deficient in the enzyme hypoxanthine phosphoribosyltransferase and consequently sensitive to hypoxanthine-aminopterin-thymidine (HAT) selective medium. We have also isolated marsupial cell strains resistant to concentrations of 22 micrograms/ml of the thymidine analogue 5-bromodeoxyuridine. These strains were thymidine kinase deficient and HAT sensitive. Drug resistance was a stable characteristic maintained for many generations in the absence of the drug. However, inhibition of growth of these drug-resistant strains was strongly density dependent, a factor that caused difficulties in the selection of hybrids. We have also developed selective systems which exploit differences between marsupial and eutherian cells in sensitivity to growth in ouabain, and in adhesiveness and other growth properties. Marsupial cells were found to be naturally much more sensitive to ouabain than rodent cells, a phenomenon that should be useful in the selection of marsupial X rodent cellular hybrids. We discuss a number of difficulties associated with the derivation and use of variant marsupial cell strains.     

Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems

The occurrence of mucoid Pseudomonas aeruginosa strains was investigated in water samples and surface material from non-clinical aquatic environments. Ten of 81 environmental isolates displayed a mucoid colony type after incubation at 36°C for 24 h on Pseudomonas Isolation Agar. The mucoid strains obtained exclusively from surfaces of technical water systems were characterized in terms of medium-dependent expression of mucoid colonial phenotype, exoenzyme profile, pigment production and O-antigen type. The mucoid strains secreted substantially higher quantities of carbohydrate and uronic acid-containing material compared to non-mucoid environmental isolates. Major slime components of the mucoid strains were identified as O-acetylated alginates that contained higher proportions of mannuronate than guluronate monomer residues and were composed of blocks of poly-mannuronate and poly-mannuronate/guluronate, whereas blocks of poly-guluronate were absent. The results suggest that surfaces in aquatic environments may represent a natural habitat for mucoid (i.e. alginate-overproducing) strains of Ps. aeruginosa with properties similar to clinical mucoid strains.     

Use of Analogues and the Substrate-Sensitivity of Mutants in Analysis of Purine Uptake and Breakdown in Aspergillus nidulans

Aspergillus mutants resistant to various purine analogues (purine, 8-azaguanine, 2-thioxanthine, and 2-thiouric acid) are defective in at least one step of purine uptake or breakdown. The properties of these mutants show that there are two uptake systems for purines, one which mediates the uptake of hypoxanthine, guanine, and adenine, and the other, xanthine and uric acid. Allantoinase-less strains are sensitive to the toxic effects of allantoin accumulation. They are severely inhibited when grown in the presence of naturally occurring purines. Mutant strains derived from these, resistant to naturally occurring purines, may be isolated. These are either wild-type revertants, or carry a second metabolic block in the uptake or breakdown of purines. The properties of these double mutants confirm the interpretation of the nature of the analogue-resistant mutants.     

Using the topology of metabolic networks to predict viability of mutant strains

Understanding the relationships between the structure (topology) and function of biological networks is a central question of systems biology. The idea that topology is a major determinant of systems function has become an attractive and highly disputed hypothesis. Although structural analysis of interaction networks demonstrates a correlation between the topological properties of a node (protein, gene) in the network and its functional essentiality, the analysis of metabolic networks fails to find such correlations. In contrast, approaches utilizing both the topology and biochemical parameters of metabolic networks, e.g., flux balance analysis, are more successful in predicting phenotypes of knockout strains. We reconcile these seemingly conflicting results by showing that the topology of the metabolic networks of both Escherichia coli and Saccharomyces cerevisiae are, in fact, sufficient to predict the viability of knockout strains with accuracy comparable to flux balance analysis on large, unbiased mutant data sets. This surprising result is obtained by introducing a novel topology-based measure of network transport: synthetic accessibility. We also show that other popular topology-based characteristics such as node degree, graph diameter, and node usage (betweenness) fail to predict the viability of E. coli mutant strains. The success of synthetic accessibility demonstrates its ability to capture the essential properties of the metabolic network, such as the branching of chemical reactions and the directed transport of material from inputs to outputs. Our results strongly support a link between the topology and function of biological networks and, in agreement with recent genetic studies, emphasize the minimal role of flux rerouting in providing robustness of mutant strains.     

Suppression of development of vancomycin-resistant <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> by low-molecular-weight cationic peptides of the lantibiotic family

Low-molecular-weight cationic peptides warnerin and hominin activate the autolytic systems and cause cell death of Staphylococcus epidermidis 33 GISK, as well as its vancomycin-resistant variant. Minimal bactericidal concentrations of warnerin for both strains studied were determined. Efficiency of antibacterial action of the peptide was found to depend directly on its concentration. Comparative investigation of adhesive properties and biofilm-forming ability of two strains was carried out. The cationic peptide warnerin was found to suppress biofilm formation by both vancomycin-sensitive and resistant strains of S. epidermidis 33 GISK and to have a pronounced destructuring effect on formed biofilms.     

Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin

Virulence properties of pathogenic bacteria, as well as resistance to antibiotics, are thought to arise through a specialization process favoured by the strong selection pressure imposed in clinical treatments. Nevertheless, in the case of opportunistic pathogens, it is unclear whether strains can be classified into virulent and non-virulent isolates. Clones of the opportunistic pathogen Pseudomonas aeruginosa do not seem to be associated to a particular biovar or pathovar, which suggests that virulence characteristics in opportunistic pathogens may already be present in environmental (non-clinical) isolates. We have explored this possibility, studying environmental isolates (mainly from oil-contaminated soils) and clinical isolates (from bacteraemia and cystic fibrosis patients) of P. aeruginosa . All environmental strains were found to actively efflux quinolones, which are synthetic antibiotics not expected to be present in the environment. These strains contained multidrug resistance determinants, were capable of invading epithelial cells and presented genes from the quorum-sensing and type III secretion systems. Some of them expressed either haemolytic or proteolytic activities or both, characteristics considered to be typical of virulent strains. All the strains tested, of clinical or environmental origin, could use alkanes (oil hydrocarbons) as a carbon source. Our results suggest that clinical and non-clinical P. aeruginosa strains might be functionally equivalent in several traits relevant for their virulence or environmental properties. Selection of clinically relevant traits, such as antibiotic resistance or cellular invasiveness, in opportunistic pathogens present in soil ecosystems is discussed.     

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

The antimicrobial properties of thermophilic bacilli

Lytic activity and antagonistic properties of 11 thermophilic strains in different species of the genus Bacillus have been studied. It is shown that the spectra of lytic and antagonistic effect of the studied thermophiles do not coincide: filtrates of cultural liquids of these strains implement the degradation of living cells of Gram-negative bacteria and yeast but inhibit the growth of Gram-positive bacteria. Both the intensities of lytic and antagonistic actions of the extracellular products of the studied strains of bacilli do not correlate. An analysis of the obtained data shows that the lysis of test cultures takes place as a result of degrading action of extracellular hydrolases of thermophiles but not as a result of activation of autolytic systems of test cultures by the substances of antibiotic nature synthesized by the studied sporeforming bacteria.