Natural antibiotic susceptibilities of Edwardsiella tarda, E. ictaluri, and E. hoshinae. (1/29)

The natural antibiotic susceptibilities to 71 antibiotics of 102 Edwardsiella strains belonging to E. tarda (n = 42), E. ictaluri (n = 41), and E. hoshinae (n = 19) were investigated. MICs were determined using a microdilution procedure according to NCCLS criteria and German standards. All edwardsiellae were naturally sensitive to tetracyclines, aminoglycosides, most beta-lactams, quinolones, antifolates, chloramphenicol, nitrofurantoin, and fosfomycin. Edwardsiella species were naturally resistant to macrolides, lincosamides, streptogramins, glycopeptides, rifampin, fusidic acid, and oxacillin. Although slight species-dependent differences in natural susceptibilities to some antibiotics (e.g., macrolides and cefaclor) were seen, differences in natural susceptibility affecting clinical assessment criteria were only seen with benzylpenicillin. Whereas E. tarda was naturally resistant to benzylpenicillin, E. hoshinae was naturally sensitive. Natural sensitivity and resistance to this penicillin were found among the strains of E. ictaluri. The observed oxacillin sensitivity of E. ictaluri was attributed to the failure of the species to grow at higher salt concentrations found in oxacillin-containing microtiter plates. The present study describes a database concerning the natural susceptibility of Edwardsiella species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these microorganisms.  (+info)

Edwardsiella ictaluri invasion of IEC-6, Henle 407, fathead minnow and channel catfish enteric epithelial cells. (2/29)

Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested. Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells. All isolates were invasive, but at differing efficiencies. Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells. Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E. ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency. This is possibly due to loss of outer membrane receptors during enzymatic cell harvest. This study provides the first documentation of the invasion of cultured mammalian and fish cells by E. ictaluri, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I. punctatus).  (+info)

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection. (3/29)

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.  (+info)

The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils. (4/29)

Edwardsiella ictaluri, the causative agent of enteric septicaemia of catfish (ESC), expresses long O polysaccharide (OPS) chains on its surface. The authors previously reported the construction of an isogenic Ed. ictaluri OPS mutant strain and demonstrated that this strain is avirulent in channel catfish. This paper reports the cloning of the Ed. ictaluri OPS biosynthesis gene cluster and identification of the mutated gene in the OPS-negative strain. The sequenced region contains eight complete ORFs and one incomplete ORF encoding LPS biosynthesis enzymes. The mutated gene (designated wbiT) was similar to other bacterial galactose-4-epimerases. Glycosyl composition analysis indicated that wild-type Ed. ictaluri OPS contains higher amounts of galactose and N-acetylgalactosamine than the OPS mutant strain, which correlated well with predicted functions of the genes identified in the OPS biosynthesis cluster. The OPS mutant had a relatively small, but significant, decrease in its ability to survive in normal catfish serum compared to wild-type Ed. ictaluri, but it retained the ability to resist killing by catfish neutrophils.  (+info)

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol. (5/29)

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.  (+info)

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. (6/29)

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.  (+info)

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare. (7/29)

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.  (+info)

Mortality and pathology in brown bullheads Amieurus nebulosus associated with a spontaneous Edwardsiella ictaluri outbreak under tank culture conditions. (8/29)

Brown bullheads Amieurus nebulosus (family Ictaluridae) are commonly used as a sentinel of environmental contamination. These fish are not generally cultured under laboratory conditions and little is known about their disease susceptibility. Here we report an outbreak of disease due to Edwardsiella ictaluri in a laboratory population of tank-reared, wild-caught brown bullheads. The isolate was positively identified as E. ictaluri using standard bacteriological substrate utilization tests and a monoclonal antibody specific for this bacterium. This pathogen causes a significant disease in channel catfish Ictalurus punctatus and is associated with disease in other ictalurid and non-ictalurid fishes. It appears that E. ictaluri is also a significant pathogen in brown bullheads and produces clinical signs and lesions similar but not identical to those observed in channel catfish. Since commercial sources of bullheads for laboratory tank studies are not available, precautions should be taken to prevent potential E. ictaluri disease outbreaks from wild-caught bullheads intended for laboratory research.  (+info)

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