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In Vitro Biofilm Formation of Flavobacterium psychrophilum

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Abstract

The fish pathogen Flavobacterium psychrophilum has been shown to occur in two colony phenotypes, rough and smooth. The aim of this study was to examine the effect of environmental and nutritional factors on the biofilm formation of both phenotypes under in vitro static conditions. The present study revealed that smooth cells displayed higher abilities of biofilm formation compared to rough cells in nutrient-rich media (TYES broth and fish feed) under static conditions, while biofilms formed by cells of both phenotypes were mainly Ca²⁺ dependent than Mg²⁺. Neither of the colony phenotypes was able to form biofilms in the low-nutrient medium (lake water) within 5 days.

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... In polluted water, both circulatory and structural changes to the gills occur, which may facilitate bacterial adhesion [26,40]. Lack of Ca 2+ or acid/alkaline pH inhibits adhesion [41], as has been described for other bacterial species [42][43][44][45]. Differences in adherence activity on various surfaces have also been reported in previous studies; F. psychrophilum adheres more readily to fin tissue, caudal peduncle, and lower jaw than to other fish body surfaces [36,46]. ...
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Flavobacterium psychrophilum is currently one of the most important pathogens in aquaculture worldwide, causing high losses to farmed salmonids particularly during early growth stages with significant economic impact. Despite previous attempts, no effective vaccine has been developed, and protection against introduction into farms is difficult due to the ubiquitous occurrence of the pathogen. A better understanding of the mechanism of disease development is essential for targeted therapeutic and preventive measures in farms. Unfortunately, the pathogenesis of diseases caused by F. psychrophilum has not been elucidated yet. Previously, several putative virulence factors have been identified. Some appear to be essential for disease development, while others are probably dispensable. The importance of some factors has not yet been explored. This review focuses on the supposed virulence factors of F. psychrophilum and the current knowledge about their importance in the pathogenesis of the disease.
... As it has been demonstrated that some sturgeon species are slow to fully develop their immune systems [11,12], larval lake sturgeon may rely on innate immune responses during early developmental stages, which could leave them vulnerable to pathogenic infection and may in part explain high mortalities often observed during early rearing in this species. While free-flowing riverine environments typically have low levels of bacterial endotoxins (usually < 1 μg ml − 1 ; [13,14]), hatchery environments may compound the effects of pathogenic bacteria with high densities of vulnerable eggs and larvae, accumulation of waste by-products, lower flow rates than may be expected in natural water ways, and the accumulation of biofilms on hatchery surfaces or sturgeon eggs themselves, which could collectively lead to increased endotoxin concentrations [15,16]. Under hatchery conditions, increases in the presence of bacterial pathogens such as Flavobacterium sp., have been associated with higher rates of lake sturgeon embryonic mortality [17]. ...
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Larval lake sturgeon, Acipenser fulvescens, reared in hatcheries for stock enhancement of wild populations may be susceptible to early opportunistic bacterial infection. Thus, we examined survival and whole-body mRNA expression of both stress- and immune-related genes (MyD88, IL-1β, StAR, GR1, and HSP70) in 30 days post fertilization larval lake sturgeon following immune challenge with lipopolysaccharides (LPS). Larval sturgeon were exposed to 0, 25, 50, 100, 150, and 200 μg.ml⁻¹ LPS and sampled after 30 min, 4 h, and 48 h. Mortality was zero in 0 and 25 μg.ml⁻¹ LPS; 37.5% in 50 μg.ml⁻¹ LPS and 100% in the higher concentrations. Expression of MyD88 and StAR mRNA were positively correlated and increased with time in the 50 μg.ml⁻¹ LPS treatment. There was an influence of both treatment and time on IL-1β mRNA, with expression 10-fold higher than controls after 4 h. Expression of HSP70 mRNA was suppressed within 30 min of 50 μg.ml⁻¹ LPS exposure and remained so throughout the time course. Correlated mRNA expression of GR1 with MyD88, StAR and IL-1β suggests a potential relationship between the innate immune and glucocorticoid responses of larval Lake Sturgeon during this early developmental stage. Data presented suggest that larval Lake sturgeon largely responded with predicted changes in gene expression of immune related and stress response genes following an LPS challenge. This study provides a foundation for future research examining the effects of hatchery and naturally occurring stressors on the immune responses of larval lake sturgeon.
... To estimate the optimal incubation time for F. psychrophilum biofilm maturation before the detachment phase, the bacterial biomass of isolate FPS-S6 attached to polystyrene wells (NunclonÔ Delta surface) was quantified using a previously described crystal violet (CV) staining method after 3, 5, and 7 days of incubation at 15°C (Ref. 12 ). In brief, 20 lL of the prepared bacterial test suspension (*10 5 CFUs) was added to wells containing 180 lL of FALW and incubated for 1 h at 15°C. ...
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Background: Bacteriophages (phages) have been proposed as an alternative to antibiotics and surface disinfectants for treatment of Flavobacterium psychrophilum biofilms and fish infections in aquaculture settings. The aim of the study was to estimate the minimal phage:host ratio (PHR) required for the control of in vitro biofilm formation and mortalities caused by F. psychrophilum in experimentally infected fish. Materials and Methods: F. psychrophilum cells in different stages of biofilm formation were exposed to the lytic phage FPSV-D22 at different PHRs. Results: Our results show that an initial PHR of 0.01 is sufficient for more than an 80% inhibition of attachment and colonization, and disruption of maturated F. psychrophilum biofilms, whereas greater ratios resulted in almost complete interruption of the different biofilm stages. Interestingly, a similar response was observed in a phage therapy trial with live rainbow trout (Oncorhynchus mykiss), where treatment of F. psychrophilum-infected fish by injection of serial bacteriophage doses resulted in significantly (***p ≤ 0.001) higher survival already at a PHR of 0.02. Conclusions: These results indicate that phages have the potential to be effective for control and treatment of F. psychrophilum infections in fish farms even when applied in concentrations lower than previously expected.
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Bacterial coldwater disease and other infections caused by Flavobacterium psychrophilum are a worldwide concern, particularly for freshwater salmonid hatcheries. F. psychrophilum infections can be difficult to control; antibiotic resistance is common and no effective vaccines are currently available. This review summarizes the biology and charac-teristics of this important pathogen, as well as the techniques required for isolation and identification. In addition, the epi-demiology, clinical signs, treatment, and possible preventative measures of bacterial coldwater disease are discussed.
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Flavobacterium psychrophilum, a bacterium known for its adhesion ability to surfaces, has recently been shown to express phenotypic variation, as smooth and rough colony types in vitro. The aim of this study was to assess the effect of different compounds on adhesion of both phenotypes of F. psychrophilum to polystyrene surfaces of 96-well microtiter plates. Cells of F. psychrophilum of both phenotypes (10(8) CFU ml(-1)) were treated with different compounds for one hour at 15°C and were subsequently allowed to adhere to polystyrene surfaces. The adhered cells were stained with crystal violet and optical density measured at 595 nm. The compounds were classified as non, weak, moderate or strong inhibitors of the F. psychrophilum adhesion. The results showed that a combination of selected carbohydrates, D- and L-amino acids, phytochemicals, an ion chelating agent (EDTA) and proteinase K strongly inhibited the adhesion of mainly smooth cells. We suggest that the compounds inhibit the cell adhesion by presumably disrupting the protein-protein interactions that hold smooth cells together and by negatively affecting the surface hydrophobicity of smooth cells. In contrast, rough cells exhibit resistance to most inhibitor compounds.
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Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome and cold water disease in salmonids, causes serious disease outbreaks in fish farms worldwide. The aim of the present study was to examine the survival capacity of F. psychrophilum in laboratory microcosms containing sterilised water under different environmental conditions and to examine the virulence of starving F. psychrophilum cells. The results showed that F. psychrophilum survived for very long time in sterilised fresh water at 15 degrees C and the cells were still culturable after starvation for 300 days. A high salinity of the water (30 per thousand) drastically reduced the number of culturable cells below detection limit after incubation for 1 day. A water salinity of approximately 6 per thousand initially reduced the number of culturable cells below the detection limit, but cells were again recovered on agar plates at the end of the experiment. The presence of sediment containing nutrients in the experimental water microcosms increased the survival of F. psychrophilum. The challenge experiments indicated that the virulence of starving F. psychrophilum is maintained for at least seven days after the transfer of the bacterial cells to fresh water.
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