Flavobacterium psychrophilum

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Higher order taxa Bacteria; Bacteroidetes; Flavobacteriia; Flavobacteriales; Flavobacteriaceae; Flavobacterium NCBI

Species Flavobacterium psychrophilum

NCBI: Taxonomy

Description and significance Flavobacterium psychrophilum is a yellow pigmented, psychrotophic, Gram-negative, rod-shaped, mobile bacterium approximately 0.4-0.5 x 1.0-5.0 µM that dwells in aquatic environments. The cells may appear flexible [4] and move over surfaces without pili or flagella by slow gliding motility [5]. Gliding motility promoted by low nutrition levels of the environment creates irregular colonies. On the other hand, a nutrient-rich environment produces circular, convex, translucent to opaque, and smooth and shiny cultures with defined edges. The optimal generation period of 2 hours occurs at strain dependent temperatures [3]. Growth is observed in temperatures between 3-23°C, but optimal growing temperature occurs between 15-20°C [3]. Once temperatures reach the 23-25°C range growth weakens [5]. Actively proteolytic, the proteases are considered to be virulence factors causing skin and muscular necrotic lesions on infected fish. Original isolation of F. psychrophilum derived from the external lesions and the internal organs of infected fish [3].

F. psychrophilum is of potential interest to research because the pathogen is the coldwater pathogenic agent of the severe fish diseases, bacterial cold-water disease and rainbow trout fry syndrome that are responsible for economic losses in salmonid and rainbow trout hatcheries. F. psychrophilum is common throughout the world, first reported in North America as bacterial cold-water disease and later identified as rainbow trout fry syndrome in Europe. However, the bacterium affects a wide range of fish, having been diagnosed in various types of salmon, rainbow trout, carp, eel, pal chub, roach, tench, and ayu. Infection is transmitted vertically and is difficult to control, causing high mortality rates and immunosuppression in fish producing difficulties in commercial and conservation aquaculture. A classical sign of infection is whitish material along the caudal fin of fish followed by necrosis lesions. Other infectious signs include ulcerations of the lower jaw, necrotic gills, and epidermal hyperplasmia [4].

At the moment, the most effective form of protection against F. psychrophilum is precautionary measure. There are effective antibiotics that can be emptied in the water system, however they are strenuous and hazardous to the environment [3,4]. Unfortunately, antibiotic resistance is common and there is currently no fully effective form of vaccination for the microbe [4].

Genome structure The F. psychrophilum JIP02/86 genome was recently determined and contains a circular chromosome of 2,861,988 base pairs and the pCP1 cryptic plasmid. The chromosome is double stranded with a G+C content of 32.54 mol% [3,5]. Fast generation time is incurred by the relatively large amount of 6 tRNA and 49 rRNA. The genome has 2432 predicted protein-coding sequences and 20 pseudogenes, of which 65% of genes have predicted functions and have an average gene length of 1003 base pairs [5]. The functions of 45% of the other genes were unknown and upon comparison of JIP02/86 to other published complete genomes, minimal conservation was observed [3]. Recent genome analysis has increased comprehension of F. psychrophilum metabolic pathways, pathogenic mechanisms, cell structure, psychrotrophic character, secretion systems, and stress-responses. The genome of F. psychrophilum provides a template of insight to the evolution and physiology of the Flavobacteriaceae family and the pathogen-host relationship that could help develop better disease control [5].

The F. psychrophilum genome encodes 13 putative secreted protease, of which Fpp1 and Fpp2 recently had their genes identified at locus tags FP0231 and FP0232, respectively. Fpp1 and Fpp2 display psychrophilic and thermolabile behavior allowing them to operate at low temperatures. Up to 27 genes involved in bacterial adhesion were identified based on their similar characteristics to genes of other organisms. Most of the genes encoded for the biosynthesis, export, modification, and polymerization of exopolysaccharides in F. psychrophilum were located at a 70-kilobase region of roughly 1,425,425 to 1,496,190 base pairs [5].

The pCP1 cryptic plasmid is 3407 base pairs long and was the source of cloning vectors pCP29 and pCP23, which are now used to manipulate Flavobacterium species with antibiotic resistance. The pCP1 plasmid has four reading frames: repA, ORF1, ORF2, and ORF3, of which only repA has a known function involving plasmid replication [7].

Cell structure and metabolism Many recent developments on metabolism and cell structure of F. psychrophilum JIP02/86 have appeared due to isolated homologous genes based on genomic comparisons to other published complete genomes [5].

Metabolism F. psychrophilum is a catalase positive, strict aerobic chemoorganotroph that undergoes respiration [3]. The recent sequencing of the genome exposed genes encoding for mechanisms of microaerobic conditions, indicating a shift in the electron transport chain in oxygen reduce conditions [3,5]. F. psychrophilum produces menaquinone 6 (MK-6) as the only major respiratory quinone in the respiratory electron transport chain [3]. The rest of the electron transport chain consists of 24 cytochrome oxidase family proteins and a cytochrome cbb3-type oxidase complex, which is commonly used in response to microaerobic conditions [5]. The combination of thermolabile enzymes and production of seven NAD(P)+-dependent, cold-active dehydrogenases allow F. psycrhrophilum to dwell in cold temperatures [3]. The microbe is missing sugar kinase and phosphotransferase system used for carbohydrate uptake, therefore F. psychrophilum is unable to metabolize carbohydrates and uses amino acids as a source of carbon and energy [3,5]. However, outside of glucose kinase, enzymes of the glycolytic pathway, pentose phosphate pathway, and tricarbocylic acid cycle are intact [5]. The tricarboxylic acid cycle is responsible for the degrading the products of proteins and lipids [3]. Proteins from the host fish cells are degraded into amino acids and oglionucleotides to make up the main source of carbon, nitrogen, and energy of the microbe [7]. In order to produce energy in nutrient-poor environments, two enzymes, cyanophycin synthetase and cyanophysinase, are produced. Cyanophycin synthetase produces the carbon and nitrogen storage compound, cyanophycin, which is then degraded by cyanophysinase for energy production [3]. Cyanophycin and amino acids can be stored in vacuoles until needed [4,5]. The enzyme putative phospholipase and three esterase-lipase-thioesterase family enzymes break down lipids and fatty acids. A long-chain fatty acid-CoA ligase, three fatty acid dehydrogenases, three thiolases, and a crotonase carry out β-oxidation [5].

Structure The microbe F. psychrophilum, is a Gram-negative rod that has a thin irregular capsule layer and phospholipid membrane that sometimes generate spheroplasts [3,4,5]. The phospholipid membrane undergoes constant modification by the addition of unsaturated fats in order to maintain membrane fluidity in the fluctuating cold temperatures [5]. The presence of MK-6 in the respiratory chain groups the Flavobacteriaceae into the Flavobacterium genus where all species have similar yellow non-diffusible pigments and similar fatty acid profiles [3].

Analogous to the rest of the Flavobacteriaceae class, F. psychrophilum has a unique composition of fatty acids dominated by branched-chained compounds, both non-hydroxylated and hydroxylated, plus both saturated and unsaturated straight-chain fatty acids and iso/anteiso-branched-chain fatty acids. The most prevalent are C15:0 iso, C15:0, C15:0 iso 3-OH, C15:0 anteiso, C15:0 ω6c, C15:0 iso G, C16:0 iso 3-OH, and C17:0 iso 3-OH. Due to the unique fatty acid composition in Flavobacteriaceae, researchers believe it can be used as an identification method for each bacterium [3].

When isolated in brothed culture, the bacterium produces long, tubular outgrowths from the cell surface that release membrane vesicles composed of a membrane bilayer, and various protease and antigenic proteins [3].

Ecology F. psychrophilum dwell in cold freshwater habitats and has a parasitic symbiosis with freshwater fish. The bacterium can survive for up to a few years in freshwater outside a fish host [4].

Optimal growth occurs on a Tryptone-yeast Extract-salts agar (TYES agar) with the addition of glucose [4,6]. It is not uncommon to see different types of colonies on the same plate. Growth is best in 0% NaCl, but the bacterium are able to withstand maximum concentrations between 0.5-1% NaCl [3].

Pathology Flavobacterium psychrophilum causes disease through the excretion degrading mechanisms that create skin and muscular necrotic lesions as well as swollen internal organs on fish. Entry points of the bacterium into fish are ambiguous but breaks in the skin of the fish are the most likely point of invasion [3,4]. Typical invaded regions are the lower jaw, fin, and caudal peduncle [4]. Virulence factors are poorly understood, however, theoretical methods are adhesive properties, agglutination, lysis of fish red blood cells, the ability to escape the bactericidal mechanisms of phagocytes, and production of extracellular proteases and chondroitin AC lyase [3]. Invasion of the microbe generates immunosuppression decreasing the presence of the host’s phagocyctes and developing possibilities for other infections to occur [4]. Evidence of the immunosuppression onset derives from the spread of F. psychrophilum into the spleen of the host fish. F. psychrophilum is also transmitted through reproduction from mother to egg via vertical transmission seeing that the presence of the bacterium on the exterior of fish eggs has been documented [4].

Thermolabile proteases are secreted by F. psychrophilum upon entry of the fish [3]. Long tubular brebs develop from the cell surface to obtain calcium to extend protease activity [3,4]. Once enough tissue is degraded by protease, the microbe spreads through the host fish’s collagenous connective tissue. The sequencing of the genome revealed mechanisms to colonize and degrade fish tissues via gliding mobility, adhesion, biofilm formation, iron acquisition, production of proteases that hydolyze cell tissue, production of bacteria-host cell interaction proteins, and toxin secretion systems to the bacterial surface [3,5,7]. The bacterium creates open ulcers and lesions on internal organs that ultimately lead to death [3,4,5,7].

Symptoms of bacterial cold-water disease and rainbow trout fry syndrome include erosive tissue usually along the lower jaw, fin, and caudal peduncle of the host fish. Whitish material develops along these regions and progressive symptoms involve tissue necrosis, ulcerations, mucus production, epidermal and internal organ hyperplasia, increased pigmentation, nervous disorders, and other virulent ailments. One distinguishable symptom referred to as “black tail” is the increased pigmentation of the posterior region of the body. Neurological symptoms of spiral swimming or spinal compressions may occur over an extended period of time. Estimated mortality rates around the world fluctuate between 10-90% [4].

F. psychrophilum has intrinsic antibiotic resistance to tobramycin, neomycin, polymyxin B, oxytetracycline, oxolonic acid, and amoxicillin. Bath or oral antibiotic treatments offer the highest efficiency against F. psychrophilum, however problematic environmental issues arise with their use [3].

Application to Biotechnology Various biotechnology mechanisms are used to detect and identify F. psychrophilum. First method of identification was an enzyme-linked immuno-sorbant assay test but an ELISA is the preferred method [6]. Genetic tools including selectable markers, plasmid cloning vectors, a reporter system, and a transposon have been developed for F. psychrophilum [4].

Current Research Antimicrobial Susceptibility of F. pschryophilum Antimicrobial resistance of F. psychrophilum is a particular concern of aquacultural farmers because a resistance to the antibiotics used could lead to drastic economical and ecological losses. Using a cation-adjusted Mueller-Hinton broth in custom Trek Sensitive susceptibility plates for aquaculture, researchers collected 72 F. psychrophilum isolates and tested them for resistance against 10 antimicrobial agents. Broth microdilution antimicrobial susceptibility testing practices were used to conclude the minimum inhibitory concentrations (MIC) of the microbial strains. High MIC indicates resistance and the results showed many of the F. psychrophilum strains to be resistant to ampicillin, oxolinic acid, gentamicin, florenicol, oxytetracycline, and erythromycin. Low MIC results demonstrated F. psychrophilum was susceptible to trimethoprim-sulfamethoxazole and ormetoprim-sulfadimethoxine [9].

Diversity of F. psychrophilum and Use of Host Phagocytes The inability to properly identify strains of F. psychrophilum averts researchers from producing a proper means of protection against it. Researchers explored the use of F. psychrophilum bacteriophages as a protection assay to see if it could increase the defense of salmonids against the microbe in aquaculture. The researchers isolated and characterized 12 strains of F. psychrophilum and 15 of their bacteriophages. Minimal DNA diversity was found between the F. psychrophilum strains even though each one was isolated from a different location. Methods of infection resulted in rare infection of a bacteriophage on a F. psychrophilum different from the original strain used for enrichment and isolation. Intraperitoneal injection of bacteriophages with host strain did decrease mortality when added in a ratio of 10 plaque-forming units per colony forming unit. The researchers recognize that the results are rather artificial but it exhibits the protection assay could protect fish from cold water disease and rainbow trout fry syndrome [8].

Outer-membrane Subproteome and Identification of Antigenic Targets of F. psychrophilum The outer-membrane proteins in the subproteome of F. psychrophilum are significant to the interaction between a cell and its habitat. Researchers examined the outer-membrane region of F. psychrophilum to identify antigens targeted by the rainbow trout. A cell-envelope suspension made from differential sodium lauryl sacrosinate solubility prepared the outer-membranes. Further examination of the suspension via two-dimensional electrophoresis and LC-MS/MS resulted in the isolation and identification of 36 outer-membrane proteins. Creating an immunoproteomic assay from antibodies of CWD-convalescent rainbow trout, 25 of the 36 proteins were identified as immunoreactive F. psychrophilum antigens. Each of these 25 antigens may offer potential candidacy for developing a vaccine against F. psychrophilum [10].

References 1) “Flavobacterium.” NCBI. U.S. National Library of Medicine. Web. 9 May 2012. <http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=237>.

2) “Flabobacterium psychrophilum.” NCBI. U.S. National Library of Medicine. Web. 9 May 2012. <http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree>.

3) Bergey, D. H., William B. Whitman, Noel R. Krieg, and James T. Staley. Bergey's Manual of Systematic Bacteriology: The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydia, and Planctomycetes. 2nd ed. Vol. 4. New York: Springer, 2011. Print.

4) Barnes, Michael E., and Michael L. Brown. “A Review of Flavobacterium psychrophilum Biology, Clinical Signs, and Bacterial Cold Water Disease Prevention and Treatment.” The Open Fish Science Journal. Vol. 4. 2011. www.benthamscience.com/open/tofishsj/articles/.../40TOFISHSJ.pdf

5) Duchaud, Eric, Mekki Boussaha, et al. "Complete Genome Sequence of the Fish Pathogen Flavobacterium Psychrophilum." Nature Biotechnology 25.7 (2007): 763-69. Nature Biotechnology. 24 June 2007. Web. 9 May 2012. <http://www.nature.com/nbt/journal/v25/n7/abs/nbt1313.html>.

6) Antaya, Claire. "Current Eco-Economical Impacts of Flavobacterium Psychrophilum." MMG 445 Basic Biotechnology EJournal 4 (2008): 16-21. Microbiology and Molecular Genetics. Michigan State University. Web. 9 May 2012. <ejournal.vudat.msu.edu/index.php/mmg445/article/.../289/329>.

7) Alvarez, B., P. Secades, M. J. McBride, and J. A. Guijarro. "Development of Genetic Techniques for the Psychrotrophic Fish Pathogen Flavobacterium Psychrophilum." Applied and Environmental Microbiology 70.1 (2004): 581-87. NCBI. Web. 9 May 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321288/>.

8) Castillo, D., G. Higuera, et al. “Diversity of Flavobacterium psychrophilum and the Potential Use of Its Phages for Protection Against Bacterial Cold Water Disease in Salmonids.” Journal of Fish Disease. 35(3). (2012): 193-201. Web. 9 May 2012. http://www.ncbi.nlm.nih.gov/pubmed/22324343

9) Hesami, Shohreh, Julia Parkman, Janet I. MacInnes, Jeffrey T. Gray, Carlton L. Gyles, and John S. Lumsden. "Antimicrobial Susceptibility of Flavobacterium Psychrophilum Isolates from Ontario." Journal of Aquatic Animal Health 22.1 (2010): 39-49. Taylor and Francis Online. 9 Jan. 2011. Web. 9 May 2012. <http://www.tandfonline.com/doi/abs/10.1577/H09-008.1#preview>.

10) Dumetz, Fabien, and Eric Duchuad. "Analysis of the Flavobacterium Psychrophilum Outer-membrane Subproteome and Identification of New Antigenic Targets for Vaccine by Immunomics." Microbiology 154.6 (2008): 1793-801. Microbiology. 6 Mar. 2008. Web. 9 May 2012. <http://mic.sgmjournals.org/content/154/6/1793.full>.

11) Delmas, B. "Flavobacterium Psychrophilum Genome Server." I.N.R.A. VIM & MIG Laboratories, 18 May 2011. Web. 9 May 2012. <http://migale.jouy.inra.fr/psychrophilum/?q=en>.

Edited by student of Dr. Lynn M Bedard, DePauw University http://www.depauw.edu