The role of denitrification genes in anaerobic growth and virulence of Flavobacterium columnare.

AIMS: Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase) and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB and nosZ in anaerobic growth, nitrate reduction, biofilm formation and virulence. METHODS AND RESULTS: Four in-frame deletion mutants in virulent F. columnare strain 94-081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94-081, FcΔnasA,FcΔnirS and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS and FcΔnosZ compared to parent strain 94-081. Under anaerobic conditions, Fc∆nasA, Fc∆norB and Fc∆nosZ formed significantly less biofilm than the wild type strain at 24 and 96 h, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated in catfish, whereas FcΔnasA, FcΔnorB and FcΔnosZ had similar virulence to FcWT. CONCLUSIONS: These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate potential for F. columnare to grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that a Fc∆nirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease.

Differential susceptibility of white bass (Morone chrysops), striped bass (Morone saxatilis) and hybrid striped bass (M. chrysops × M. saxatilis) to Flavobacterium columnare and effects of mucus on bacterial growth and biofilm development.

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Antibiofilm activity in the culture supernatant of a marine Pseudomonas sp. bacterium.

In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using Flavobacterium sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit Flavobacterium sp. II2003 biofilm formation and the Pseudomonas sp. IV2006 strain was identified as producing a strong antibiofilm activity. The Pseudomonas sp. IV2006 culture supernatant (SNIV2006) inhibited Flavobacterium sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SNIV2006 had no effect on the Flavobacterium sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid-base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by Pseudomonas sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SNIV2006 and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SNIV2006 exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as Flavobacterium species that are pathogenic for fish, and human pathogens in both the medical environment, such as Staphylococcus aureus and Pseudomonas aeruginosa, and in the food industry, such as Yersinia enterocolitica. Thus, a wide range of applications could be envisaged for the SNIV2006 compounds, both in aquaculture and human health.

Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare.

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5-60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.

Transcriptome analysis of the fish pathogen Flavobacterium columnare in biofilm suggests calcium role in pathogenesis.

BACKGROUND: Flavobacterium columnare is the causative agent of columnaris disease that affects cultured freshwater fishes worldwide. F. columnare easily colonizes surfaces by forming biofilm, which helps the pathogen resist antibiotic and disinfectant treatments. Previously, we had shown that increasing concentrations of calcium (Ca2+) promoted biofilm formation by F. columnare. The objective of this study was to further characterize the role of Ca2+ on biofilm formation and to compare the transcriptome profiles of planktonic and biofilm cells. RESULTS: RNA-Seq analysis was conducted to identify genes that were differentially expressed between the following states: i) planktonic cells in control medium (P), ii) planktonic cells in calcium-enriched medium (P/Ca), and iii) biofilm cells in calcium-enriched medium (B/Ca). Overall, we identified 441 significant (FDR-adjusted p < 0.05, fold change > 2) differentially expressed genes (DEGs) between P and B/Ca samples; 112 significant DEGs between P/Ca and B/Ca samples, and 175 significant DEGs between P/Ca and P samples, corresponding to 15.87, 4.03 and 6.30% of the total protein-coding sequences, respectively. The significant DEGs fell into different functional categories including iron acquisition, oxidative stress response, extracellular protein secretion, and respiratory metabolism. CONCLUSIONS: Our results posit Ca2+ as a critical signal in regulating bacterial surface adhesion and biofilm formation in F. columnare. Living in biofilm elicited a shift in several metabolic pathways that allowed the cells to cope with oxidative stress and nutrient starvation. In addition, Ca2+ supplementation induced the expression of putative virulence factors in F. columnare, such as extracellular protein secretion and iron acquisition.

Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42.

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.

Bacterial Analogs of Plant Tetrahydropyridine Alkaloids Mediate Microbial Interactions in a Rhizosphere Model System.

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas species from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere.IMPORTANCE The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae, two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria.

Microbial basis of Fusarium wilt suppression by Allium cultivation.

Crop rotation and intercropping with Allium plants suppresses Fusarium wilt in various crops. However, the mechanisms underlying this phenomenon have not been fully elucidated. This study was designed to assess the role of microorganisms inhabiting Allium rhizospheres and antifungal compounds produced by Allium roots in Fusarium wilt suppression by Allium cultivation. Suppression of cucumber Fusarium wilt and the pathogen multiplication by Allium (Welsh onion and/or onion)-cultivated soils were eliminated by heat treatment at 60 °C, whereas those by Welsh onion-root extract were lost at 40 °C. The addition of antibacterial antibiotics eliminated the suppressive effect of Welsh onion-cultivated soil on pathogen multiplication, suggesting the contribution of antagonistic gram-negative bacteria to the soil suppressiveness. The Illumina MiSeq sequencing of 16S rRNA gene amplicons revealed that genus Flavobacterium was the predominant group that preferentially accumulated in Allium rhizospheres. Flavobacterium species recovered from the rhizosphere soils of these Allium plants suppressed Fusarium wilt on cucumber seedlings. Furthermore, confocal laser scanning microscopy revealed that Flavobacterium isolates inhibited the multiplication of the pathogen in soil. Taken together, we infer that the accumulation of antagonistic Flavobacterium species plays a key role in Fusarium wilt suppression by Allium cultivation.

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum).

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in freshwater-reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108  cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106 , 107 or 108  cfu/fish of F. psychrophilum FPG 101, the 108  cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2  = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety-seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108  cfu/fish.

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake.

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose-dependently inhibited by addition of D-glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose-mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N-acetyl-glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose-mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose-negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose-mediated inhibition of F. johnsoniae colony spreading.

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity.

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)-containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha-D-mannose/alpha-D-glucose >N-acetyl-beta-D-glucosamine >N-acetyl-beta-D-glucosamine/N-acetylneuraminic acid >N-acetyl-D-galactosamine >alpha-D-galactose/N-acetyl-alpha-D-galactosamine >beta-D-galactose = alpha-L-fucose. Virulence studies demonstrated isolate AL-02-36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%-100% versus 60% for isolate ALG-00-530 at equivalent doses (~3 × 106  CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2-3 logs) and survived (28 days) in formulated water-containing catfish mucus. Highly virulent isolate AL-02-36 possessed at least 2.5- to fivefold higher protease activity following growth in mucus than the less virulent ALG-00-530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.

Alkaline active cyanide dihydratase of Flavobacterium indicum MTCC 6936: Growth optimization, purification, characterization and in silico analysis.

The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. Km and Vmax for cyanide dihydratase of F. indicum was measured to be 4.76 mM and 45 U mg-1 with kcat calculated to be 27.3 s-1 and specificity constant (kcat/Km) to be around 5.67 mM-1 s-1. MALDI-TOF analysis of purified protein revealed that the amino acid sequence has 50% and 43% sequence identity with putative amino acid sequence of F. indicum and earlier reported cyanide dihydratase of Bacillus pumilus respectively. Homology modeling studies of cyanide dihydratase of F. indicum predicted the catalytic triad of the enzyme indicating Cys at 164, Glu at 46 and Lys at 130th position. The purified enzyme has potential applications in bioremediation and analytical sector.

Genetic manipulation of structural color in bacterial colonies.

Naturally occurring photonic structures are responsible for the bright and vivid coloration in a large variety of living organisms. Despite efforts to understand their biological functions, development, and complex optical response, little is known of the underlying genes involved in the development of these nanostructures in any domain of life. Here, we used Flavobacterium colonies as a model system to demonstrate that genes responsible for gliding motility, cell shape, the stringent response, and tRNA modification contribute to the optical appearance of the colony. By structural and optical analysis, we obtained a detailed correlation of how genetic modifications alter structural color in bacterial colonies. Understanding of genotype and phenotype relations in this system opens the way to genetic engineering of on-demand living optical materials, for use as paints and living sensors.

Ecosystem activation system (EAS) technology for remediation of eutrophic freshwater.

Ecosystem activation system (EAS) was developed to create beneficial conditions for microbiome recovery and then restore and maintain the ecological integrity (microbial community, phytoplankton, zooplankton) for eutrophic freshwater rehabilitation. A 30 day's filed test of EAS indicated that over 50% of contaminant was removed and the algae visibly disappeared. EAS treatment 2.5-fold increased the diversity of microbial community and changed the microbial community structure (e.g., two and three-fold decrease in the amount of Flavobacterium and Pseudomonas, typical abundant species of eutrophic freshwater, respectively). Further, the diversity of phytoplankton and zooplankton of treated water suggested that these species were diverse. Representative phytoplankton of eutrophic freshwater, Chlorella and Chlamydomonas were undetectable. The possible mechanism of EAS is restoring the trophic levels of the water body via bottom-up approach by microbial community.

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium.

Studies on species' responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to-head competition between mixed genotype co-cultures of a waterborne bacterial pathogen of fish, Flavobacterium columnare, at two different temperatures. We found that competition outcome in liquid cultures seemed to be well predicted by growth yield of isolated strains, but was mostly inconsistent with interference competition results measured in inhibition tests on solid agar, especially as no growth inhibition between strain pairs was detected at the higher temperature. These results suggest that, for a given temperature, the factors driving competition outcome differ between liquid and solid environments.

Different Phenotypes of Mature Biofilm in Flavobacterium psychrophilum Share a Potential for Virulence That Differs from Planktonic State.

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease and the rainbow trout fry syndrome in salmonid aquaculture worldwide. However, there have been few studies into the capacity of F. psychrophilum to form biofilms and how these cellular accretions differ from planktonic cells or how they affect potential virulence. We evaluated the biofilm formation by three Chilean isolates of F. psychrophilum (LM-02-Fp, LM-06-Fp, and LM-13-Fp) and two non-Chilean strains (JIP02/86 and NCMB1947T), and compared biofilm and planktonic states to obtain insights into expression differences of virulence- and biofilm-related genes (VBRGs). Our findings are based on scanning confocal laser microscopy (SCLM) and LIVE/DEAD staining, enzymatic reactions, reverse transcription-quantitative PCR (RT-qPCR) of genes encoding putative virulence factors, and transcriptomes (RNA-Seq). The LM-02-Fp and NCMB1947T strains were the strongest and weakest biofilm producers, respectively. The strong-biofilm producer showed different physiological cell states distributed in different layers of mature biofilms, whereas the NCMB1947T biofilms consisted of cells arranged in a monolayer. WGA-binding exopolysaccharides would be the main components of their corresponding extracellular matrices. Transcriptomes of F. psychrophilum NCMB1947T and LM-02-Fp were clustered by state (biofilm vs. planktonic) rather than by strain, indicating important state-dependent differences in gene expression. Analysis of differentially expressed genes between states identified putative VBRGs involved in polysaccharide biosynthesis, lateral gene transfer, membrane transport (e.g., for drugs and Fe3+), sensory mechanisms, and adhesion, and indicated that about 60-100% of VBRGs involved in these processes was significantly upregulated in the biofilm state. Conversely, upregulated motility-related genes in the biofilm state were not identified, whereas a lower fraction of proteolysis-related genes (33%) was upregulated in biofilms. In summary, F. psychrophilum strains that produce different biofilm phenotypes show global transcriptional activity in the mature biofilm state that differs significantly from their planktonic counterparts. Also, different biofilm phenotypes share a genetic potential for virulence that is transcriptionally enhanced with respect to free-living cells. Our results suggest that the F. psychrophilum biofilm lifestyle acts as a reservoir for a given set of putative virulence factors, and recommend a deeper understanding of which could help prevent recurring infections in salmonid farms.

Parasitism perturbs the mucosal microbiome of Atlantic Salmon.

Interactions between parasite, host and host-associated microbiota are increasingly understood as important determinants of disease progression and morbidity. Salmon lice, including the parasitic copepod Lepeophtheirus salmonis and related species, are perhaps the most important problem facing Atlantic Salmon aquaculture after feed sustainability. Salmon lice parasitize the surface of the fish, feeding off mucus, scales and underlying tissue. Secondary bacterial infections are a major source of associated morbidity. In this study we tracked the diversity and composition of Salmo salar skin surface microbiota throughout a complete L. salmonis infection cycle among 800 post-smolts as compared to healthy controls. Among infected fish we observed a significant reduction in microbial richness (Chao1, P = 0.0136), raised diversity (Shannon, P < 7.86e-06) as well as highly significant destabilisation of microbial community composition (Pairwise Unifrac, beta-diversity, P < 1.86e-05; P = 0.0132) by comparison to controls. While undetectable on an individual level, network analysis of microbial taxa on infected fish revealed the association of multiple pathogenic genera (Vibrio, Flavobacterium, Tenacibaculum, Pseudomonas) with high louse burdens. We discuss our findings in the context of ecological theory and colonisation resistance, in addition to the role microbiota in driving primary and secondary pathology in the host.

Cortisol directly impacts Flavobacterium columnare in vitro growth characteristics.

Teleost fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal axis to release glucocorticoids, in particular cortisol, into the blood. For the majority of bacterial fish pathogens, stress is considered a key factor in disease outbreaks. Based upon studies in mammals, there is considerable evidence to suggest that, besides impairing the immune system, cortisol can have a direct effect on bacterial cells. Hitherto, this intriguing field of microbial endocrinology has remained largely unexplored in aquatic diseases. The present study investigated in vitro the impact of cortisol on phenotypic traits of the fresh water fish pathogen Flavobacterium columnare. Colonies obtained from the highly virulent (HV) isolates resulted in significantly larger and more spreading colonies compared to those from the low virulent (LV) isolates. High cortisol doses added displayed a direct effect on the bacterial cells and induced a significant decrease in colony size. An additional intriguing finding was the inverse relationship between cortisol concentrations added to the broth and the spreading character of colonies retrieved, with higher cortisol doses resulting in less rhizoid to rough and even smooth colony formation (the latter only in the LV trout isolate), suggesting a dose-response effect. The loss of the rhizoid appearance of the F. columnare colonies upon administration of cortisol, and hence the loss of motility, might indicate a phenotypic change to the biofilm state. These findings form the basis for further research on the impact of glucocorticoids on other virulence factors and biofilm formation of F. columnare.

Comparative study of wastewater treatment and nutrient recycle via activated sludge, microalgae and combination systems.

Algal-bacterial synergistic cultivation could be an optional wastewater treatment technology in temperate areas. In this study, a locally screened vigorous Chlorella strain was characterized and then it was used in a comparative study of wastewater treatment and nutrient recycle assessment via activated sludge (AS), microalgae and their combination systems. Chlorella sp. cultured with AS in light showed the best performance, in which case the removal efficiencies of COD, NH3-N and TP were 87.3%, 99.2% and 83.9%, respectively, within a short period of 1day. Algal-bacterial combination in light had the best settleability. Chlorella sp. contained biomass, could be processed to feed, fertilizer or fuel due to the improved quality (higher C/H/N) compared with sludge. PCR-DGGE analysis shows that two types of rhizobacteria, namely, Pseudomonas putida and Flavobacterium hauense were enriched in sludge when cultured with algae in light, serving as the basics for artificial consortium construction for improved wastewater treatment.