Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into ‘columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

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Cholinesterases characterization of three tropical fish species, and their sensitivity towards specific contaminants

Ecotoxicology and Environmental Safety ◽

10.1016/j.ecoenv.2019.01.105 ◽

2019 ◽

Vol 173 ◽

pp. 482-493 ◽

Cited By ~ 6

Author(s):

Beatriz V.R. Pereira ◽

Elaine C.M. Silva-Zacarin ◽

Monica Jones Costa ◽

André Cordeiro Alves Dos Santos ◽

Janaina Braga do Carmo ◽

...

Keyword(s):

Fish Species ◽

Tropical Fish

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The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare

Applied and Environmental Microbiology ◽

10.1128/aem.01769-17 ◽

2017 ◽

Vol 83 (23) ◽

Cited By ~ 25

Author(s):

Nan Li ◽

Yongtao Zhu ◽

Benjamin R. LaFrentz ◽

Jason P. Evenhuis ◽

David W. Hunnicutt ◽

...

Keyword(s):

Secretion System ◽

Gliding Motility ◽

Control Measures ◽

Flavobacterium Columnare ◽

Wild Type ◽

Secretion Systems ◽

Content Type ◽

Freshwater Aquaculture ◽

Columnaris Disease ◽

Spent Media

ABSTRACT Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare. The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS. IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.

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Isolation and characterization of polymorphic microsatellite loci from elongate loach (Leptobotia elongate), a threatened fish species endemic to the Yangtze River

Conservation Genetics Resources ◽

10.1007/s12686-011-9491-7 ◽

2011 ◽

Vol 4 (1) ◽

pp. 129-131 ◽

Cited By ~ 3

Author(s):

Hong Yan Liu ◽

Fei Xiong ◽

Xin Bin Duan ◽

Da Qing Chen

Keyword(s):

Fish Species ◽

Microsatellite Loci ◽

Yangtze River ◽

The Yangtze River ◽

Threatened Fish ◽

Isolation And Characterization ◽

Polymorphic Microsatellite

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Molecular characterization of Flavobacterium columnare isolated from a natural outbreak of columnaris disease in farmed fish, Catla catla from India

The Journal of General and Applied Microbiology ◽

10.2323/jgam.59.417 ◽

2013 ◽

Vol 59 (6) ◽

pp. 417-424 ◽

Cited By ~ 14

Author(s):

Dev Kumar Verma ◽

Gaurav Rathore

Keyword(s):

Molecular Characterization ◽

Catla Catla ◽

Flavobacterium Columnare ◽

Farmed Fish ◽

Columnaris Disease ◽

Natural Outbreak

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Isolation and Characterization of Aeromonas veronii from Ornamental Fish Species in China

Israeli Journal of Aquaculture - Bamidgeh ◽

10.46989/001c.20844 ◽

2017 ◽

Author(s):

L Li ◽

C. Zhang ◽

C. Pei ◽

Y. Yiksung ◽

J. Sun ◽

...

Keyword(s):

Fish Species ◽

Ornamental Fish ◽

Aeromonas Veronii ◽

Isolation And Characterization

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Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease

Vaccines ◽

10.3390/vaccines9111370 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1370

Author(s):

Wenlong Cai ◽

Covadonga R. Arias

Keyword(s):

Vaccine Strain ◽

Molecular Basis ◽

Live Vaccine ◽

Gliding Motility ◽

Genome Comparison ◽

Comparative Genomic ◽

Flavobacterium Columnare ◽

Secretion Systems ◽

Columnaris Disease ◽

Modified Live Vaccine

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

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Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and Type IX Secretion System

10.1101/2020.10.02.323337 ◽

2020 ◽

Author(s):

Heidi M. T. Kunttu ◽

Anniina Runtuvuori-Salmela ◽

Krister Sundell ◽

Tom Wiklund ◽

Mathias Middelboe ◽

...

Keyword(s):

Phage Therapy ◽

Secretion System ◽

Gliding Motility ◽

Genetic Mutations ◽

Phage Resistance ◽

Flavobacterium Columnare ◽

Aquaculture Industry ◽

Columnaris Disease ◽

Phage Sensitivity ◽

Type Ix Secretion System

AbstractIncreasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

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