Arctic Actinomycetes as Potential Inhibitors of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biofilm

The aim of this study was to identify novel biofilm inhibitors from actinomycetes isolated from the Arctic against Vibrio cholerae, the causative agent of cholera. The biofilm inhibitory activity of actinomycetes was assessed using biofilm assay and was confirmed using air–liquid interphase coverslip assay. The potential isolates were identified using 16S rRNA gene sequencing. Of all, three isolates showed significant biofilm inhibition against V. cholerae. The results showed that 20% of the actinomycetes culture supernatant could inhibit up to 80% of the biofilm formation. When different extracted fractions were assessed, significant biofilm inhibition activity was only seen in the diethyl ether fraction of A745. At 200 μg ml⁻¹ of diethyl ether fraction, 60% inhibition of V. cholerae biofilm was observed. The two potential isolates were found to be Streptomyces sp. and one isolate belonged to Nocardiopsis sp. This is the first report showing a Streptomyces sp. and Nocardiopsis sp. isolated from the Arctic having a biofilm inhibitory activity against V. cholerae. The spread of drug resistant V. cholerae strains is a major clinical problem and the ineffectiveness in antibiotic treatment necessitates finding new modes of prevention and containment of the disease, cholera. The formation of biofilms during the proliferation of V. cholerae is linked to its pathogenesis. Hence, the bioactive compound from the culture supernatant of the isolates identified in this study may be a promising source for the development of a potential quorum sensing inhibitors against V. cholerae.     

ClinicalPseudomonas aeruginosa: Potential factors of pathogenicity and resistance to antimicrobials

Resistance to 17 antimicrobials, surface hydrophobicity, motility, biofilm, production ofN-acylhomoserine lactone signal molecules (N-butyrylhomoserine lactone andN-3-oxolauroylhomoserine lactone) and response to oxidative stress were analyzed in 47 clinicalPseudomonas aeruginosa strains. In addition to natural resistance, the strains demonstrated the greatest level of resistance to cefotaxime (91.5 %). Isolates in the range of 44.7–57.4 % were resistant to aminoglycosides and ciprofloxacin, of 25.5–36.2 % to cephalosporins. On the other hand, 97.9 % remained susceptible to meropenem, 93.6 % to piperacillin + tazobactam and 87.2% to piperacillin. The majority of the strains (72.3 %) manifested their hydrophilic character. Higher zones of motility showed 12 isolates (in average 54.8 mm) as compared to the others (30.2 mm). Approximately 1/3 of the strains (29.8 %) produced a higher amount of biofilm quantified by measuring the absorbance of solubilized crystal violet (0.20–0.46) than the rest of isolates (0–0.19). All but two strains producedN-3-oxolauroylhomoserine lactone and in 48.9 % of samplesN-butyrylhomoserine lactone were detected. Only four isolates with higher biofilm production showed both types of homoserine lactone. Majority of the strains (70.2 %) manifested higher resistance to H₂O₂ than the rest of the strains. The group of strains resistant to aminoglycosides and ciprofloxacin revealed a significantly higher number of hydrophobic strains (compared with the sensitive ones). In contrast, higher number of strains sensitive to aminoglycosides and ciprofloxacin or only to ciprofloxacin producedN-butyrylhomoserine lactone and biofilm (compared to the resistant ones). Such association was not found among the rest of the tested parameters. The results indicate that the resistance to antimicrobials inP. aeruginosa isolates was not generally associated with changes in the production of the pathogenicity factors.     

Screening for marine nanoplanktic microalgae from Greek coastal lagoons (Ionian Sea) for use in mariculture

Mediterranean mariculture uses imported strains of marine phytoplankton, raising questions of ecological risk and ability to adapt to local conditions for mass culture outdoors. In this context, we report here on the mass-culture potential and chemical composition of six strains of Prasinophyceae (five strains of Tetraselmis sp. and one Pyramimonas sp.) isolated from a Greek coastal lagoon. Proximate composition had a pattern of 10–20% ash, 35–65% protein, 6–10% lipids, and 25–45% other organics including carbohydrates. The amino acid profiles were typical for the marine representatives of the class. All strains had a high PUFA content with dominant the ω3 fraction in four of them. The fatty acid profiles indicated a Tetraselmis strain with high EPA (14%) and a Pyramimonas strain with high DHA (6%). These strains might be a good alternative for the common commercial strains used in Mediterranean aquaculture.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

Production and characteristics of the exocellular polysaccharide of a mutantMycobacterium strain

Mutant strains ofMycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N′-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found thatMycobacterium sp. V649/15 produces a maximum of 15–19% polymer after a 5–6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.     

Formation and reversion of protoplasts in mutant strains ofBrevibacterium sp. M27

In mutant strains ofBrevibacterium sp. M27 requiring amino acids and resistant to antibiotics four methods were verified and a method inducing a high formation of protoplasts and their reversion was developed. Formation of protoplasts is 60–99% and their reversion 17–76%. In induced mutants a correlation between protoplast formation and growth rate could be demonstrated.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

Serratia ficaria sp. nov., a bacterial species associated with Smyrna figs and the fig waspBlastophaga psenes

Fourteen strains isolated from figs, caprifigs, and fig wasps collected in California and Tunisia, and from a small black ant in France, constitute a new DNA hybridization group that is 25–56% related toSerratia species, and 6–17% related to other species of Enterobacteriaceae. This homogeneous group (90% relatedness within the group) constitutes a new species that is namedSerratia ficaria sp. nov. (type strain, ICPB 4050, ATCC 33105). Strains of this species have a characteristic odor, similar to that ofS: odorifera andPseudomonas perolens. No strain ofS. ficaria has yet been recovered from clinical specimens.     

Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains ofTrichomonas vaginalis and five strains ofTritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point.Tritrichomonas foetus was more sensitive to the enzyme treatment thanT. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsintreated cells was inhibited 10–20% by addition of inhibitors of either protein synthesis (puromycin) orN-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM ofT. foetus andT. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40–50% and 17–30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge ofT. vaginalis than to that ofT. foetus.     

Antipathogenic potential of marine <Emphasis Type="Italic">Bacillus</Emphasis> sp. SS4 on <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine-lactone-mediated virulence factors production in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> (PAO1)

Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5–2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33–86%) and biofilm formation (33–88%), total protease (20–65%), LasA protease (59–68%), LasB elastase (36–68%), pyocyanin (17–86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751).     

Effect of tween 80 and dimethyl sulfoxide on biosynthesis ofl-lysine in regulatory mutants ofCorynebacterium glutamicum

By using dimethyl sulfoxide or Tween 80 (1 or 0.2 %), the production ofl-lysine was increased by 20–28 and 23–25%, respectively, in regulatory mutant strains ofCorynebacterium glutamicum. The stimulation observed is supposed to be caused by influencing cellular surface structures. Translated by Č. Novotny     

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 μmol photon m^-2 s^-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m^-2 d^-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m^-2 d^-1.     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Edwardsiella hoshinae,a new species of enterobacteriaceae

Eight strains isolated from birds, reptiles, and water constitute a new DNA hybridization group that is 37–58% related toEdwardsiella tarda and less than 10% related to other species of Enterobacteriaceae (SI nuclease method). This homogeneous group (78–100% relatedness within the group) constitutes a new species that is namedEdwardsiella hoshinae sp. nov. (type strain, CIP 78.56 ATCC 33379). Strains of this species produce acid fromd-mannitol, sucrose,d-trehalose, and salicin, and give a positive malonate test. Seven other strains that produced acid fromd-mannitol and sucrose (but not fromd-trehalose and salicin) and were malonate negative were found to belong to theEdwardsiella tarda DNA hybridization group. The base composition of the DNAs ofE. tarda andE. hoshinae is 55–58 mol% G+C.     

Field applications of a bio-trickling filter for the removal of nitrogen oxides from flue gas

A bio-trickling filter (BTF) packed with polyhedral spheres was used to remove nitrogen oxides (NOx) from the flue gas of a coal-fired power plant. The BTF system consistently removed 64–95% of the NOx after start-up and acclimation under dynamic conditions (e.g., 120–240 m³/h flue gas flow rate and inlet 300–900 mg NOx/m³). Scanning electron microscopy of the biofilms that were formed showed a shift in the predominating bacteria. Analyses by PCR-denaturing gradient gel electrophoresis showed that the naturally-selected mixed cultures in the biofilm under a flue gas environment were mainly Klebsiella sp. and Pseudomonas sp.     

Denitrifying and methanogenic bacteria in the biofilm of a fixed-film reactor operated with methanol/nitrate demonstrated by immunofluorescence and microscopy

 A denitrifying bacterial biofilm population established on a polypropylene substratum of a fixed-film reactor was characterized by microscopy, scanning electron microscopy and immunofluorescence after 120 days of operation. The reactor, operated at pH 7.0, 22°C, and −180 mV with synthetic wastewater containing methanol/nitrate, achieved a denitrification rate of 0.24 mol NO^- ₃ l^-1 day^-1 with a removal efficiency for nitrate of 95%–99% at an organic loading rate of 0.325 mol methanol l^-1 day^-1. The gas produced contained 2%–3% (v/v) methane and 3%–4% (v/v) carbon dioxide in addition to nitrogen. The biofilm contained mainly cells of Methanobrevibacter arboriphilus antigenically related to strain DC, short, flagellated, gram-negatively staining rods of Pseudomonas sp. antigenically related to Pseudomonas stutzeri strain AN11, non-identified pink-pigmented rods and small lemon-shaped cells with mono- and bipolar appendages resembling prosthecate Hyphomicrobium sp. The biofilm analysis provided evidence for a syntrophy between the denitrifying, methylotrophic, bacterial consortium and hydrogenotrophic methanogens, which were identified by antigenic fingerprinting with 17 antibody probes. Received: 11 July 1994/Received revision: 23 September 1994/Accepted: 28 September 1994     

Isolation and identification of a symbiotic bacterium fromSteinernema carpocapsae

Xenorhabdus nematophilus sp., an insect-pathogenic bacterium, was newly isolated from Korean entomopathogenic nematode ofSteinernema carpocapsae, which can be used as a useful bioinsecticide. Primary and secondary form variants ofXenorhabdus nematophilus were observed when culturedin vitro. Primary form variants adsorbed bromothymol blue, while secondary form did not. However, many other characters of two variants were very similar. The variants were all rod-shaped and cell size was highly variable ranging from 0.5 by 2.0 μm to 1.0 by 5.0 μm. Both produced highly toxic substances and killed the insect larva within 20–38 hr, indicating that insect pathogenicity ofXenorhabdus is not directly associated with its phase variation. In addition, cell-free culture supernatant ofXenorhabdus was sufficient to kill the insect larva by injecting it into insect hemolymph; however, cell-harboring culture broth was more effective for killing the insect. The use ofXenorhabdus nematophilus may provide a potential alternative toBacillus thuringiensis (Bt) toxins.     

Cyanobactericidal effect of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. isolated from eutrophic lake on <Emphasis Type="Italic">Microcystis</Emphasis> sp

A bacterium, which was observed in all cultivations of Microcystis sp., was isolated and designated as Rhodococcus sp. KWR2. The growth of bloom-forming cyanobacteria, including four strains of Microcystis aeruginosa and Anabaena variabilis, was suppressed by up to 75–88% by 2% (v/v) culture broth of KWR2 after 5 days. But KWR2 did not inhibit eukaryotic algae, Chlorella vulgaris and Scenedesmus sp. An extracellular algicidal substance produced by KWR2 showed a cyanobactericidal activity of 94% and was water-soluble with a molecular weight of lower than 8 kDa.     

Distribution of spore-positive and spore-negative nodules ofAlnus incana ssp.rugosa in Maine,USA

The distribution of spore-positive (sp+) and spore-negative (sp−) root nodules ofAlnus incana ssp.rugosa (DuRoi) Clausen (speckled alder) was examined at 29 sites with a wide range of environmental conditions in Maine, USA. These included: pH 3.4 to 7.0, soil texture ranging from coarse gravel to clay to organic soils, elevation from 3 to 591 m and latitude 43 to 47°N. Habitat types included disturbed areas, streamsides, swamps and old fields. Sp (−) nodules were substantially more common, making up 76% of all nodules, whereas only 24% were sp (+). Sp (−) nodules often occurred in pure stands and predominated at disturbed sites with mineral soils at the surface and in old fields and swamps with pH>4.0 Sp (+) nodules were nearly always found in mixture with sp (−) nodules. They occurred primarily at streamside and lakeshore sites where they made up 40% of the nodules and at sites with pH<4.0 regardless of habitat type. It is suggested that sp (−) strains ofFrankia may be maintained at a site by saprophytic growth in soil and thus nodulate newly established hosts, whereas sp (+) strains may be maintained primarily by spore production within nodules and thus depend on extended presence of the host.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.