Search

Aquaculture settings present a number of surfaces that can potentially act as a reservoir for Flavobacterium psychrophilum, resulting in a serious threat to the health of farmed salmonids. The aim of this study was to employ the CDC Biofilm Reactor to evaluate the capacity of F. psychrophilum to form biofilms on stainless steel, polystyrene, polyurethane, and polycarbonate. Scanning electron microscopy and confocal laser scanning microscopy were used to assess biofilm formation, while epifluorescence and qPCR methods were effectively used to determine cell contents. After a 72 h incubation period, F. psychrophilum had formed mature biofilms composed by dense, multilayered clusters of short bacilli on stainless steel, polystyrene, and polycarbonate. Nevertheless, biofilms formed on stainless steel presented a significantly higher cell density than those formed on polyurethane. Biofilm growth was poor on polyurethane surfaces. These findings will facilitate the design of appropriate guidelines for more efficiently managing F. psychrophilum and controlling the persistence of this pathogen in fish-farming environments.

Flavobacterium psyhrophilum affects farmed salmon and trout worldwide and is the etiological agent of bacterial cold water disease. The treatment of this disease is made with antibiotics. The emergence of resistant strains and its ability to form biofilms have increased the resistance to antibiotics. Considering the diverse bioactivity of Antarctic bacteria, we evaluated the inhibitory effect of compounds secreted by Antarctic bacterial strains on the biofilm formation of F. psychrophilum 19749. 26.9% of the Antarctic bacterial isolates inhibited strongly the biofilm, the greatest effect was shown by M19B. Proteins (48-56 kDa) secreted by M19B may be related to the inhibition of the biofilm formation of F. psychrophilum 19749.

The main goal of this study was to find bacterial isolates with the ability to inhibit the growth of the fish pathogens Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium psychrophilum and to inhibit the blockage of the quorum-sensing (QS) system. A total of 80 gram-negative strains isolated from various freshwater Chilean salmonid farms were studied. We determined that 10 strains belonging to the genus Pseudomonas inhibited at least one of the assayed fish pathogens. Of these, nine strains were able to produce siderophores and two strains were able to inhibit the growth of all assayed pathogenic species. When the 80 strains were examined for QS-blocking activity, only the strains Pseudomonas sp. FF16 and Raoultella planticola R5B1 were identified as QS blockers. When the QS-blocker strains were analyzed for their ability to produce homoserine lactone (HSL) molecules, thin-layer chromatography analysis showed that both strains were able to produce C6-HSL- and C8-HSL-type molecules. Strain R5B1 did not show growth inhibition properties, but strain FF16 also led to inhibition of growth in A. hydrophila and F. psychrophilum as well as to siderophore production. Pseudomonas sp. FF16 exhibited potentially useful antagonistic properties and could be a probiotic candidate for the salmon farming industry.

Industrial fishing effluents are characterized by high loads of protein and sulfate that stimulate the activity of proteolytic and sulfate reducing bacteria during anaerobic digestion. Their metabolic products (NH3 and H2S respectively) have a well-known detrimental effect on the activity of methanogens. Since methylamine is a carbon source used by methylaminotrophic methane producing archaea (mMPA) but not by sulfate reducing bacteria (SRB), enriched mMPA anaerobic biofilms have been developed on ceramics. We propose that methylated amines could be produced in the biofilm by using betaine, a known precursor of methylamine, as a carbon and energy source. We isolated an anaerobic betainotrophic methylaminogenic bacterial strain (bMB) from an anaerobic bioreactor, using betaine as the only carbon and energy source. This strain was identified by a standard biochemical test (API 20NE), cloning, and 16S rDNA sequencing. bMB biofilm structure and biofilm growth kinetic parameters were determined by means of scanning electron microscopy (SEM), and the Gompertz growth model, respectively. Monomethylamine production was determined by infrared spectroscopy and by high pressure liquid chromatography. The isolated bMB strain was determined as Stappia stellulata (Proteobacteria phylum). It was able to form biofilm on ceramics and its kinetic growth parameters resulted in: maximum biofilm bacterial count (A) of 6.25 x 10(8) UFC/cm(2) and maximum specific growth rate (mu(m)) of 0.0221/h. Production of monomethylamine was about 4.027 atogram/cell/day (at/cell/day) after 15 days of incubation in biofilms. This study confirms the adhesion capacity of this bMB strain on ceramic supports, assuring that monomethylamine production in biofilms could be enriched with mMPA that use monomethylamine.

Treatment of microbiological samples with viability dyes prior to extraction of DNA and PCR amplification for downstream analysis has evolved into a commonly applied method. The addition of this easy-to-perform step to the sample analysis procedure inhibits the amplification of DNA from dead cells with compromised cell membranes. The method is currently used both in combination with quantitative PCR (qPCR), end-point PCR, and isothermal amplification. We present here a detailed study of the effect of amplicon size on amplification signals from unstressed and heat-exposed cells after treatment with propidium monoazide (PMA). PMA treatment was shown to be more efficient in excluding dead cells from the analysis both in combination with qPCR (PMA-qPCR) and denaturing gradient gel electrophoresis (PMA-DGGE), when longer amplicons were used. When applied to pure cultures of the fish pathogens Vibrio anguillarum and Flavobacterium psychrophilum exposed to a heat gradient ranging from mild to lethal, qPCR product lengths did not influence PMA-qPCR results at low temperatures, whereas an increasingly strong impact was seen at higher temperatures. Membrane permeability as a result of heat exposure might however have to be considered a conservative parameter for cell death for these pathogens as culturability and redox activity were lost at lower stress intensities than membrane integrity. When applying PMA-DGGE to an environmental water sample which was either left untreated or was exposed to heat, differences to non-PMA treated samples tended to slightly increase when amplified fragments in the first round of the nested PCR were longer, whereas the impact of 1st-round cycle numbers remains unclear.

Reduced volatile sulphur compounds generate an impact on the environment, because of the bad smell and its low odour threshold. Compared with the existing physicochemical technologies for their elimination, biotrickling filters are an economically and environmentally sustainable alternative. Usually mixed cultures of microorganisms are used for inoculating biotrickling filters, in this case a pure culture of Thiobacillus thioparus is used for generating a biofilm, allowing to measure its capacity for the oxidation of four volatile reduced sulphur compounds: hydrogen sulphide, dimethyl sulphide, methyl mercaptan and dimethyl disulphide, using a residence time of 0.033 hrs. The viable cells of the biofilm were quantified by epifluorescence microscopy, staining the cells with ethidium bromide and acridine orange, polymerase chain reaction analysis in real time was used for testing the predominance of T. thiopharus in the biofilm. The microorganism was able to adhere and grow on the surface of rings made of polyethylene, with a viable population of 7·10 7 cell·ring -1 , a 74% of total cells. The real time PCR showed a persistence of the population of T. thioparus for more than 300 days of operation, without being displaced by other microbial species. The maximum elimination capacities for each compound were 34.4; 21.8; 30.8 and 25.6 gS·m -3 ·h -1 for H2S, dimethylsulphide, dimethyldisulphide and methyl mercaptan, respectively. We conclude that it is possible to implement a biotrickling filter with the bacteria T. thioparus, which can oxidize volatile reduced sulphur compounds efficiently.

The effect of pH and organic matter (total organic carbon, TOC) of effluents originating from fishing industries located in Talcahuano, Chile, on the ability to colonize different trophic group methanogenic bacteria (MB) over clay and polyethylene supports was evaluated. Both support materials enhanced all types of methanogenic bacterial growth compared to systems without support. The highest adherence was obtained at pH 8. At 8000 mg/l TOC, this had a significant effect on the growth rate of hydrogenotropic and methylaminotrophic methanogens on polyethylene and clay, respectively.

We herein report the diversity and relative abundance of chemolithotrophic nitrifying bacteria in a continuous‐flow bioreactor using 16S‐ribosomal RNA quantitative dot‐blot hybridizations. About 14.9% of the total bacterial population, determined by epifluorescence microscopy in the bioreactor suspended phase, was represented by nitrifying bacteria. Of this fraction, ammonia‐ and nitrite‐oxidizing bacteria accounted for 10% and 90%, respectively, the latter group being mostly N/frospira‐like. On the other hand, the nitrifiers adhesion/colonization capacity on polyethylene surfaces as evaluated by scanning electron microscopy and hybridizations analyses was 12.6% of the total bacterial community adhered. Finally, in spite of the relatively small contribution of nitrifiers to the total bacterial abundance in the bioreactor, we determined a mean ammonia removal rate of 170.48 ± 8.29 mg N 11 d1; thus, the low percentage of ammonia‐oxidizing bacteria, was not limiting the bioreactor performance.

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH 3 -N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37°C. The results showed that at 848.8 mg NH 3 -N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337±0.213 g COD methane/g VSS∗day when 48.8 mg NH 3 -N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH 3 -N/L, producing under 1.658±0.185 g COD methane/g VSS∗day. The lowest methanogenic activity reached was 0.639±0.162 g COD methane/g VSS∗day at the system added with 848.8 mg NH 3 -N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH 3 -N/L.

Sulphur Oxidizing Bacteria (SOB) is a group of microorganisms widely used for the biofiltration of Total Reduced Sulphur compounds (TRS). TRS are bad smelling compounds with neurotoxic activity which are produced by different industries (cellulose, petrochemical). Thiobacillus thioparus has the capability to oxidize organic TRS, and strains of this bacterium are commonly used for TRS biofiltration technology. In this study, two thiosulphate oxidizing strains were isolated from a petrochemical plant (ENAP BioBio, Chile). They were subjected to molecular analysis by real time PCR using specific primers for T. thioparus . rDNA16S were sequenced using universal primers and their corresponding thiosulphate activities were compared with the reference strain T. thioparus ATCC 10801 in batch standard conditions. Real time PCR and 16S rDNA sequencing showed that one of the isolated strains belonged to the Thiobacillus branch. This strain degrades thiosulphate with a similar activity profile to that shown by the ATCC 10801 strain, but with less growth, making it useful in biofiltration.

A cyanobacterial bloom occurring in 1998 in lake Tres Pascualas (Concepcion/Chile) was found to be dominated by Microcystis sp. The bloom contained both non-toxic (cyanopeptolin-type) and hepatotoxic (microcystin-type) peptides. Cyanopeptolin structure of the non-toxic peptides (called cyanopeptolin VW-1 and VW-2, respectively) was revealed by matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) of whole cells, showing dominant molecular ions at m/z = 975 and m/z 995, respectively. On post source decay (PSD), both cyanopeptolins showed fragments deriving from Ahp-Phe-MTyr (3-amino-6-hydroxy-2-piperidone), the characteristic partial structure of cyanopeptolins. The amounts of each of the two cyanopeptolins could only roughly be estimated to be >0.1% of bloom material dry weight. In addition the blooms contained microcystins (20 microg/g bloom dry weight as determined by RP-HPLC, 13 microg/g according to ELISA determination). MALDI-TOF-MS revealed several structural variants of microcystin: MCYST-RR (microcystin with Arg and Arg, indicated by m/z 1,038 and confirmed by PSD revealing a m/z = 135 fragment deriving from the Adda side chain, MCYST-FR (microcystin with Phe and Arg, indicated by m/z = 1,015). The presence of [Asp(3)]-MCYST-LR (microcystin with Leu and Arg, Asp non-methylated, indicated by m/z 981), and [Asp(3)]-MCYST-YR (microcystin with Tyr and Arg, Asp non-methylated, indicated by m/z 1,031) were likely. The relative amounts of the peptides varied between February, April, and May. Whole cell extracts from the bloom material revealed specific enzyme inhibitory activities. The serin-proteases trypsin, plasmin, elastase were inhibited, assumable due to the cyanopeptolins found. Elastase and the cysteine-protease papain were not inhibited, inhibitions of protein kinase and glutathione S-transferase (GST) were low. Strong inhibition was observed with protein-phosphatase-1, likely due to the microcystins present in the samples.

Cyanobacterial blooms dominated by Microcystis sp. occurred in lake Rocuant ("marisma", near Concepcion/Chile) in February 1995 and 1996. In the bloom samples collected in both years the hepatotoxin microcystin was detected by RP-HPLC in both samples and in the sample of 1995 also by a toxicity assay using primary rat hepatocytes. In the bloom of 1995, the microcystin content of the dry bloom biomass was determined to be 130 micrograms/g on the basis of the RP-HPLC peak area and 800 micrograms/g on the basis of the rat hepatotoxicity assay, respectively. In the bloom of 1996, RP-HPLC analysis revealed a microcystin content of 8.13 micrograms/g bloom material dry weight. In this year no hepatotoxicity was measured using a concentration range up to 0.8 mg (d. w.) of bloom material per ml in the rat hepatotoxicity assay. This is the first report on the detection of microcystins in Chilean water bodies.

Quorum sensing (QS) is a regulatory mechanism that enables bacteria to make collective decisions such as an increase in virulence factors and biofilm production. Inhibitors of QS are important research tools in the discovery of new potential anti-bacterial agents. Polygodial, drimenol and drimendiol are drimane sesquiterpenoids isolated from Drimys winteri, a Chilean native tree. Their QS activity, when tested on Chromobacterium violaceum ATCC 12472, showed that drimendiol is an inhibitor of QS, decreasing violaceine production in C. violaceum and decreasing biofilm formation of Pseudomonas syringae strains. Consequently it increased the biocide effects of CuSO4 on biofilms of P. syringae.

The most important bacterial pathology currently occurring in Chilean freshwater salmon farming is the cold-water disease produced by the psychrotrophic bacteria Flavobacterium psychrophilum. The main aim of this study was to characterize the inhibitory activity of an antagonist strain on the formation of biofilms of a F. psychrophilum strain. The antagonistic strain Pseudomonas fluorescens FF48 was isolated from the sediment beneath the salmon cages of a freshwater Chilean salmon farm and was identified by using the 16S rRNA gene sequence analysis. The production of siderophores, mainly during the stationary phase of growth of the antagonist strain was demonstrated using the Chrome Azurol S method and through F. psychrophilum inhibition under iron saturation conditions. Subsequently, the effect of the antagonist supernatant on the formation of F. psychrophilum biofilm was tested using the crystal violet staining method observing an inhibition of the growth of F. psychrophilum, but no effect was observed when iron saturation concentrations were used. Furthermore, when the antagonist strain was previously deposited on the support, it completely inhibited the formation of F. psychrophilum biofilms, but when both bacteria were inoculated simultaneously no inhibitory effect was detected. In conclusion, it was demonstrated that FF48 strain is able to inhibit the formation of F. psychrophilum biofilms in vitro probably mediated by the siderophore production, suggesting its potential use as a biocontrol biofilm in freshwater fish rearing systems to prevent the persistence of biofilms of the fish pathogenic species F. psychrophilum.