Pathological and Ultrastructural Characterization of an Edwardsiella ictaluri Triple hemR Mutant.

Enteric septicemia of catfish, which is caused by Edwardsiella ictaluri, is detrimental to farmed Channel Catfish Ictalurus punctatus. The hemin receptor HemR is involved in binding and uptake of heme into bacteria. Here, we explored pathological and ultrastructural changes in catfish fry that were immunized with a triple hemR mutant of E. ictaluri and challenged with wild-type E. ictaluri (EiWT) 28 d after immunization. Following immunization, pathological changes in the triple hemR-immunized fry were less severe compared to the EiWT-exposed control fry. Widely disseminated bacteria and severe necrosis in most organs, especially the kidney and spleen, were detected in both groups at days 4, 5, and 6. Multifocal granulomatous encephalitis with bacteria was seen in hemR-immunized fry at days 21 and 28 and in EiWT-exposed control fry at day 14. Phagocytic cells in the kidney and spleen of EiWT-exposed control fry contained more replicating bacteria compared to hemR-immunized fry. During the EiWT challenge of immunized fry, a robust immune response was observed in the triple hemR-immunized fry compared to the sham-vaccinated group. Many activated phagocytic cells were detected in the kidney and spleen with fragmented or no bacteria in the triple hemR-immunized fry. Our data suggested that virulence of triple hemR was lower and the onset of the lesions was delayed compared to EiWT. Additionally, triple hemR-immunized fry could mount an immune response and had milder lesions compared to the sham control after EiWT exposure.

Antibacterial Activities of Metabolites from Vitis rotundifolia (Muscadine) Roots against Fish Pathogenic Bacteria.

Enteric septicemia of catfish, columnaris disease and streptococcosis, caused by Edwardsiella ictaluri, Flavobacterium columnare and Streptococcus iniae, respectively, are the most common bacterial diseases of economic significance to the pond-raised channel catfish Ictalurus punctatus industry. Certain management practices are used by catfish farmers to prevent large financial losses from these diseases such as the use of commercial antibiotics. In order to discover environmentally benign alternatives, using a rapid bioassay, we evaluated a crude extract from the roots of muscadine Vitis rotundifolia against these fish pathogenic bacteria and determined that the extract was most active against F. columnare. Subsequently, several isolated compounds from the root extract were isolated. Among these isolated compounds, (+)-hopeaphenol (2) and (+)-vitisin A (3) were found to be the most active (bacteriostatic activity only) against F. columnare, with 24-h 50% inhibition concentrations of 4.0 ± 0.7 and 7.7 ± 0.6 mg/L, respectively, and minimum inhibitory concentrations of 9.1 ± 0 mg/L for each compound which were approximately 25X less active than the drug control florfenicol. Efficacy testing of 2 and 3 is necessary to further evaluate the potential for these compounds to be used as antibacterial agents for managing columnaris disease.

Increased Alternative Splicing as a Host Response to Edwardsiella ictaluri Infection in Catfish.

Alternative splicing is the process of generating multiple transcripts from a single pre-mRNA used by eukaryotes to regulate gene expression and increase proteomic complexity. Although alternative splicing profiles have been well studied in mammalian species, they have not been well studied in aquatic species, especially after biotic stresses. In the present study, genomic information and RNA-Seq datasets were utilized to characterize alternative splicing profiles and their induced changes after bacterial infection with Edwardsiella ictaluri in channel catfish (Ictalurus punctatus). A total of 27,476 alternative splicing events, derived from 9694 genes, were identified in channel catfish. Exon skipping was the most abundant while mutually exclusive exon was the least abundant type of alternative splicing. Alternative splicing was greatly induced by E. ictaluri infection with 21.9% increase in alternative splicing events. Interestingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced genes after infection. Sequence analyses of splice variants of a representative alternatively spliced gene, splicing factor srsf2, revealed that certain spliced transcripts may undergo nonsense-mediated decay (NMD), suggesting functional significance of the induced alternative splicing. Although statistical analysis was not possible with such large datasets, results from quantitative real-time PCR from representative differential alternative splicing events provided general validation of the bacterial infection-induced alternative splicing. This is the first comprehensive study of alternative splicing and its changes in response to bacterial infection in fish species, providing insights into the molecular mechanisms of host responses to biotic stresses.

The virulence and immune protection of Edwardsiella ictaluri HemR mutants in catfish.

Edwardsiella ictaluri is a Gram-negative facultative intracellular rod, causing enteric septicemia of catfish (ESC). Several heme uptake systems have been described in bacterial pathogens, most of which involve outer membrane proteins (OMPs). We have shown recently that heme/hemoglobin receptor family protein (HemR) is significantly up-regulated in E. ictaluri under iron-restricted conditions. In this work, our goal was to construct E. ictaluri HemR mutants and assess their virulence and immune protection potentials in catfish. To accomplish this, an in-frame deletion mutant (EiΔhemR) was constructed, and its virulence and immune protection were determined in catfish fingerlings and fry. The results indicated that the EiΔhemR was attenuated completely in catfish fingerlings, but it was virulent in 14 day-old catfish fry. To increase the attenuation of EiΔhemR in fry, we introduced frdA and sdhC gene deletions to the mutant, yielding two double (EiΔhemRΔfrdA and EiΔhemRΔsdhC) and one triple (EiΔhemRΔfrdAΔsdhC) mutants. Results indicated that two double HemR mutants did not exhibit increased attenuation, but the triple HemR mutant showed significantly less virulence and high protection in fry (p < 0.05). Histological examination of fry tissues vaccinated with the triple mutant displayed similar inflammation to that of wild-type infected fry, but much less necrosis and far fewer bacteria were observed. Immunohistochemistry (IHC) result indicated fewer numbers of bacteria around blood vessel and in the hematopoietic tissue in fry infected with triple mutant compared to control group infected with E. ictaluri wild-type. Our data indicated that EiΔhemR was safe and protective in catfish fingerlings, while EiΔhemRΔfrdAΔsdhC was much safer in catfish fry.

Improving safety of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish and evaluation of efficacy.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). Our recent work indicated that tricarboxylic acid cycle and one-carbon metabolism are critical pathways for E. ictaluri virulence. Although single and double gene deletions in these pathways resulted in safe and efficacious vaccines for use in catfish fingerlings, vaccine trials in catfish fry showed safety concerns. Therefore, we aimed to improve the safety of these mutants by constructing two triple mutant combinations. ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) was constructed by introducing an in-frame deletion of frdA in a gcvP-sdh mutant. ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) was constructed in a similar manner. ESC-NDKL1 strain was a better vaccine candidate compared to ESC-NDKL2, providing better safety and efficacy in catfish fry and catfish fingerlings. Field trials in earthen ponds under three vaccination conditions showed that survival was significantly higher in catfish vaccinated with ESC-NDKL1 by immersion at the fry stage, oral vaccination in ponds, and fry immersion-pond oral combination (86.74%, 81.67%, and 95.22%, respectively) compared to sham-vaccinated (42.75%), and Aquavac-ESC fry immersion vaccinated (61.51%) catfish. Our findings indicate that ESC-NDKL1 is a good candidate for further development as a vaccine for ESC.

Involvement of tolQ and tolR genes in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative intracellular facultative pathogen causing enteric septicemia of channel catfish (ESC). The Tol system, consisting of four envelope proteins TolQ, TolR, TolA, and TolB, are required for colicin import and contributes to bacterial virulence in several pathogenic bacteria. However, the Tol system and its importance in E. ictaluri virulence have not been investigated. Here we present construction and evaluation of the E. ictaluri TolQ, TolR and TolQR mutants (EiΔtolQ, EiΔtolR, and EiΔtolQR). The Tol mutants were developed using in-frame gene deletion and their attenuation and vaccine efficacy were determined in catfish fingerlings. The EiΔtolQ, EiΔtolR, and EiΔtolQR mutants showed reduced virulence in catfish (28.93%, 19.70%, and 39.82% mortality, respectively) compared to wild type (46.91% mortality). Further, vaccination with these mutants protected catfish against subsequent wild-type infection. This study suggests that the Tol system contributes to E. ictaluri virulence in catfish.

Influence of lipopolysaccharide outer-core in the intrinsic resistance to antimicrobial peptides and virulence in Edwardsiella ictaluri.

The genus Edwardsiella consists of bacteria with an intrinsic resistance to cyclic cationic antimicrobial peptides (CAMPs). Edwardsiella ictaluri, a pathogen of the catfish (Ictalurus punctatus) and the causative agent of a systemic infection, is highly resistant to CAMPs. Previously, we determined that the oligo-polysaccharide (O-PS) of the lipopolysaccharide (LPS) does not play a role in the E. ictaluri CAMP resistance and an intact core-lipid A structure is necessary for CAMPs resistance. Here, we evaluated the influence of the outer-core in the CAMPs resistance and fish virulence. E. ictaluri wabG, a gene that encodes for the UDP-glucuronic acid transferase that links the lipid A-inner-core to the outer-core-oligopolysaccharides, was deleted. Deletion of ΔwabG caused a pleiotropic effect, influencing LPS synthesis, CAMPs resistance, growth, and biofilm formation. E. ictaluri ΔwabG was attenuated in zebrafish indicating the important role of LPS during fish pathogenesis. Also, we evaluated the inflammatory effects of wabG LPS in catfish ligated loop model, showing a decreased inflammatory effect at the gut level respects to the E. ictaluri wild type. We conclude that E. ictaluri CAMPs resistance is related to the molecules present in the LPS outer-core and that fish gut inflammation triggered by E. ictaluri is LPS dependent, reinforcing the hypothesis that fish gut recognizes LPS in an O-PS dependent fashion.

Identification and expression analysis of 26 oncogenes of the receptor tyrosine kinase family in channel catfish after bacterial infection and hypoxic stress.

Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones. RTKs are not only key regulators of normal cellular processes, but are also involved in the progression of many types of tumors, and responses to various biotic and abiotic stresses. Catfish is a primary aquaculture species in the United States, while its industry is drastically hindered by several major diseases including enteric septicemia of catfish (ESC) that is caused by Edwardsiella ictaluri. Disease outbreaks are often accompanied by hypoxic stress, which affects the performance and survival of fish by reducing disease resistance. In this study, we identified 26 RTK oncogenes in the channel catfish genome, and determined their expression profiles after ESC infection and hypoxic stress. The 26 RTK genes were divided into four subfamilies according to phylogenetic analysis, including TIE (2 genes), ErbB (6 genes), EPH (14 genes), and INSR (4 genes). All identified RTKs possess a similar molecular architecture including ligand-binding domains, a single transmembrane helix and a cytoplasmic region, which suggests that these genes could play conserved biological roles. The expression analysis revealed that eight RTKs were significantly regulated after bacterial infection, with dramatic induction of insulin receptor genes including INSRb, IGF1Ra, and IGF1Rb. Upon hypoxic stress, EPHB3a, EGFR, ErbB4b, and IGF1Rb were expressed at higher levels in the tolerant catfish, while EPHA2a, EPHA2, TIE1 and INSRa were expressed at higher levels in the intolerant catfish. These results suggested the involvement of RTKs in immune responses and hypoxic tolerance.

Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in channel catfish macrophages.

Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H(+)-ATPases by use of the specific inhibitor bafilomycin A1 abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.

Effect of multiple mutations in tricarboxylic acid cycle and one-carbon metabolism pathways on Edwardsiella ictaluri pathogenesis.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC). We have shown recently that tricarboxylic acid cycle (TCA) and one-carbon (C1) metabolism are involved in E. ictaluri pathogenesis. However, the effect of multiple mutations in these pathways is unknown. Here, we report four novel E. ictaluri mutants carrying double gene mutations in TCA cycle (EiΔmdhΔsdhC, EiΔfrdAΔsdhC), C1 metabolism (EiΔglyAΔgcvP), and both TCA and C1 metabolism pathways (EiΔgcvPΔsdhC). In-frame gene deletions were constructed by allelic exchange and mutants' virulence and vaccine efficacy were evaluated using in vivo bioluminescence imaging (BLI) as well as end point mortality counts in catfish fingerlings. Results indicated that all the double gene mutants were attenuated compared to wild-type (wt) E. ictaluri. There was a 1.39-fold average reduction in bioluminescence, and hence bacterial numbers, from all the mutants except for EiΔfrdAΔsdhC at 144 h post-infection. Vaccination with mutants was very effective in protecting channel catfish against subsequent infection with virulent E. ictaluri 93-146 strain. In particular, immersion vaccination resulted in complete protection. Our results provide further evidence on the importance of TCA and C1 metabolism pathways in bacterial pathogenesis.

Phenotypic traits, virulence-associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea.

UNLABELLED: Edwardsiella tarda is the predominant bacterium in farm-cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I-CeuI-based pulsed-field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET-060 and ET-191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence-related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.

Mortality and pathology of hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), associated with Edwardsiella ictaluri infection in southern Thailand.

Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0- and 5.6-kb plasmids, compared with the 4.8- and 5.6-kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23-100% mortality within 14 days with a 168-h LD50 of 6.92 × 10(7)  CFU mL(-1) by immersion and a 96-h LD50 of 1.58 × 10(6)  CFU fish(-1) by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.

Comparison of Vietnamese and US isolates of Edwardsiella ictaluri.

We compared Edwardsiella ictaluri from striped catfish in Vietnam with US channel catfish isolates. Biochemical analyses and sequencing of the 16S rRNA gene confirmed that the Vietnamese isolates were E. ictaluri. Comparison using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into 4 plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the 4 profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only 1 contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type III secretion effector, EseI, and its chaperone, EscD, were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, 1 isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the lipopolysaccharide of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages.

Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri and preliminary investigation of its effects on virulence.

Edwardsiella ictaluri is the etiologic agent of enteric septicemia of catfish, which causes substantial losses in catfish aquaculture. To determine pathogen-host interactions, previous studies have used the green fluorescence protein (GFP) gene. Here, the pEI2 plasmid of E. ictaluri isolate I49 was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of open reading frame 2 and appeared to be at a neutral site in the plasmid. Purification of the pEI2::GFPKan plasmid and mobilization into E. coli resulted in GFP expression. The isolated pEI2::GFPkan plasmid was used to retransform the wild type I49 isolate (ensuring a single Tn10-GFP-kan insertion) and an independent E. ictaluri isolate, S97-73-3. The wild type and the green fluorescent-tagged strains were compared for modulation of pathogenicity in channel catfish Ictalurus punctatus by immersion challenge. A significant reduction in mortalities occurred for the I49GFPkan strain as compared to its isogenic parent, but no difference was observed between the S97-73-3GFPkan strain and the S97-73-3 wild type. This GFP-tagged plasmid will be useful for determining the effects that the pEI2::GFPkan plasmid has on virulence and host-pathogen interactions between E. ictaluri isolates.

Roles of plasmid-encoded proteins, EseH, EseI and EscD in invasion, replication and virulence of Edwardsiella ictaluri.

Native plasmids pEI1 and pEI2 were detected in Edwardsiella ictaluri HSN-1 isolated from diseased yellow catfish (Pelteobagrus fulvidraco). EseH encoded by pEI1 and other two proteins, EseI and EscD, encoded by pEI2, were found with homology to type III secretion system (T3SS) proteins. To investigate their roles in pathogenesis, the native plasmids were cured based on plasmid incompatibility by introducing a Kan positive and SacB negative selection marker into gene spacer of the native plasmids. Mutants with the deletion of the target genes were obtained by reverse PCR and self-ligation, and all mutants were examined for their virulence effect in yellow catfish. Compared with the HSN-1 strain, the two mutants ΔeseH and ΔeseI were attenuated, while mutant ΔescD had increased virulence with higher Competitive Index (CI) value. The adherence and invasion assays on fish EPC cells indicated that ΔeseH and ΔeseI had decreased ability in adherence. Using E. tarda as surrogate, EseH and EseI were detected in culture supernatants, but EscD was not, with the secretion of EseH depending on T3SS. In addition, EseH and EseI were found translocated into host cells, and by means of subcellular fractionation, EseH was localized in membrane fraction of ZF4 cells, and EseI in the cytosol fraction. Hence, the role of these three genes in adherence, invasion and cellular replication was revealed from the pathogenic bacterium E. ictaluri.

Tricarboxylic acid cycle and one-carbon metabolism pathways are important in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.

Mechanisms of intrinsic resistance to antimicrobial peptides of Edwardsiella ictaluri and its influence on fish gut inflammation and virulence.

The genus Edwardsiella comprises a genetically distinct taxon related to other members of the family Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cationic antimicrobial peptides (CAMPs) is a specific property of the genus Edwardsiella. In particular, Edwardsiella ictaluri, an important pathogen of the catfish (Ictalurus punctatus) aquaculture and the causative agent of a fatal systemic infection, is highly resistant to CAMPs. E. ictaluri mechanisms of resistance to CAMPs are unknown. We hypothesized that E. ictaluri lipopolysaccharide (LPS) plays a role in both virulence and resistance to CAMPs. The putative genes related to LPS oligo-polysaccharide (O-PS) synthesis were in-frame deleted. Individual deletions of wibT, gne and ugd eliminated synthesis of the O-PS, causing auto-agglutination, rough colonies, biofilm-like formation and motility defects. Deletion of ugd, the gene that encodes the UDP-glucose dehydrogenase enzyme responsible for synthesis of UDP-glucuronic acid, causes sensitivity to CAMPs, indicating that UDP-glucuronic acid and its derivatives are related to CAMP intrinsic resistance. E. ictaluri OP-S mutants showed different levels of attenuation, colonization of lymphoid tissues and immune protection in zebrafish (Danio rerio) and catfish. Orally inoculated catfish with O-PS mutant strains presented different degrees of gut inflammation and colonization of lymphoid tissues. Here we conclude that intrinsic resistance to CAMPs is mediated by Ugd enzyme, which has a pleiotropic effect in E. ictaluri influencing LPS synthesis, motility, agglutination, fish gut inflammation and virulence.

Use of bioluminescence mutant screening for identification of Edwardsiella ictaluri genes involved in channel catfish (Ictalurus punctatus) skin colonization.

Initial invasion of the host is the first and vital part of any infection process. We have demonstrated that Edwardsiella ictaluri is capable of colonizing and penetrating catfish skin. Therefore, a mutant library was constructed by random insertion of the Mar2xT7 transposon into the chromosome of E. ictaluri harboring the bioluminescence plasmid pAKgfplux1. This library was then screened through a series of three consecutive challenges for mutants showing a decreased ability to colonize the catfish epithelium. Eighteen mutants were identified that have decreased adhesion and virulence. Mutated genes encoded one sensor protein, two transport proteins, five enzymes, two regulatory proteins, and five hypothetical proteins. Among the mutated genes, the first one identified was a gene encoding for RstA/B, which is known to play a role in regulating the expression of invasion genes in Salmonella enterica Typhimurium. Another mutant was lacking a putative ribonuclease similar to a Shigella protein that regulates the expression of adhesin. A third mutant was defective in a protein similar to a Brucella protein that was initially identified as a transporter, but actually is a member of a newly discovered adhesin family. Results from this study could enable development of a new strategy for blocking E. ictaluri invasion at the initial adherence stage.

Construction and evaluation of an Edwardsiella ictaluri fhuC mutant.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in channel catfish. Iron is an essential micronutrient needed for bacterial virulence, and to acquire iron, many Gram-negative bacteria secrete ferric iron chelating siderophores. The ferric hydroxamate uptake (Fhu) system consists of four genes (fhuC, fhuD, fhuB, and fhuA), and is involved in the uptake of hydroxamate type siderophores across bacterial membranes. However, the Fhu system and its importance in E. ictaluri virulence have been uninvestigated. Here, we present construction and evaluation of an E. ictaluri ΔfhuC mutant. The E. ictaluri fhuC gene was deleted in-frame by allelic exchange, and the mutant's growth in media and virulence in catfish were determined. Our results indicated that deletion of the E. ictaluri fhuC gene did not affect the growth of E. ictaluri largely in both iron-replete and iron-depleted media. Addition of ferric iron sources into the iron-depleted medium improved the growth of both E. ictaluri ΔfhuC and wild type (WT). Catfish mortalities indicated that E. ictaluri ΔfhuC mutant was attenuated 2.05-fold compared with the parent strain. The catfish immunized with the E. ictaluri ΔfhuC mutant showed a high relative percent survival rate (97.50%) after re-challenge with the WT E. ictaluri strain. Taken together, our data indicates that the fhuC gene contributes to E. ictaluri virulence.

A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria.

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.