Isolates Of Edwardsiella Tarda Research Articles

Molecular characterization, virulence and antimicrobial and biocidal susceptibility of selected bacteria isolated from the cloaca of nestling ospreys (Pandion haliaetus) from Mono Lake, California, USA.

In the present study, the presence of the Enterobacterales, Staphylococcus spp., Mammaliicoccus spp., and Enterococcus spp. in cloacal samples of nestling ospreys (Pandion haliaetus), a fish-eating specialist, from Mono Lake, California, USA was examined by a multiphasic approach, including antimicrobial and biocide susceptibility testing, genotyping, and whole genome sequencing of selected isolates. The most commonly detected species was Escherichia coli, followed by Mammaliicoccus sciuri, Staphylococcus delphini, Enterococcus faecalis, Enterococcus faecium, Hafnia alvei, Klebsiella pneumoniae, Citrobacter braakii and single isolates of Edwardsiella tarda, Edwardsiella albertii, Klebsiella aerogenes, Plesiomonas shigelloides and Staphylococcus pseudintermedius. Multi-drug resistance (MDR) was observed in two E. coli isolates and in an Enterococcus faecium isolate. The MDR blaCTX-M-55-positive E. coli belonged to the pandemic clone ST58. The results of the present study suggest that nestling ospreys are exposed to MDR bacteria, possibly through the ingestion of contaminated fish. Ospreys may be good biosentinels for the presence of these microorganisms and antibiotic resistance in the local environment and the risk for other wildlife, livestock and humans.

Biofilm formation Edwardsiella tarda isolated from fresh water fishes

A B S T R A C T Edwardsiella tarda is one of the serious pathogenic bacteria, infected both cultured and wild fishes species. and has many virulence factors that enhance pathogenesis of bacterial in fishes and cause severe economic losses in aquaculture industry so This study was aimed to isolated Edwardsiella tarda from 150 diseased fish samples ,(60 Nile tilapia (Oreochromis niloticus) ,60 cat fish (Claris gariepinus) , 30 (Mullet (Mugil cephalus) (bayad). from internal organs after clinical and postmortem examination The results revealed that, about 24 isolates of Edwardsiella tarda 12(20%);7 (11.67%) and 5 (16.66%) were isolated from C. gariepinus ,O. niloticus, and M. cephalus fishes respectively. 8 strains formed strong formation biofilm (black colonies ) on cong red agar , 11 moderate(pink ) and 6 negative (white colonies ) 8 out 24 showed resistance to five antibacterial sensitive (oxacillin , ampicillin , sulphamethoprime , gentamycin and Norofloxacin and. Positive results in the amplification of virulence-related genes associated with the pathogenicity of the Edwardsiella tarda in the 8 resistance isolate are positive for production of chondroitinase enzyme gene (cds1), Nacylhomoserine lactones (AHLs)( edwI )gene, vibrioferringene (pvsA) gene and sensor protein implicated gene (qseC) The sequences obtained for ) (qseC) and ( edwI ) genes had accession number MW362141 and MW362142at GenBankand were identical to the corresponding GenBank sequences.which isolated from different sources and countries with no significant differences depending strains host or its area of isolation

Typhlocolitis by Edwardsiella tarda in a Cow

Typhlocolitis by Edwardsiella tarda in a Cow

Antibiotics Resistance Pattern and Plasmid Profiling of <i>Edwardsiella tarda</i> Isolated from <i>Heterobranchus longifilis</i>

A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.

Edwardsiella tarda Tunes Tricarboxylic Acid Cycle to Evade Complement-Mediated Killing.

Evasion of complement-mediated killing is a common phenotype for many different types of pathogens, but the mechanism is still poorly understood. Most of the clinic isolates of Edwardsiella tarda, an important pathogen infecting both of human and fish, are commonly found serum-resistant. To explore the potential mechanisms, we applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics approaches to profile the metabolomes of E. tarda EIB202 in the presence or absence of serum stress. We found that tricarboxylic acid (TCA) cycle was greatly enhanced in the presence of serum. The quantitative real-time PCR (qRT-PCR) and enzyme activity assays validated this result. Furthermore, exogenous succinate that promotes the TCA cycle increased serum resistance, while TCA cycle inhibitors (bromopyruvate and propanedioic acid) that inhibit TCA cycle, attenuated serum resistance. Moreover, the enhanced TCA cycle increased membrane potential, thus decreased the formation of membrane attack complex at cell surface, resulting serum resistance. These evidences suggested a previously unknown membrane potential-dependent mechanism of serum resistance. Therefore, our findings reveal that pathogen mounts a metabolic trick to cope with the serum complement-mediated killing.

Studies on Edwardsiellosis in Some Marine Fishes Using Molecular Diagnosis at Suez bay

A total of 240 marine fishes Mugil cephalus, Rastrelliger kanagurta and Nemipterus japonicas were randomly collected from Attaka fishing port, Suez-bay, Suez Governorate (60 in each winter and fall and 120 in spring).The infected fishes showed hemorrhages all over the body surface including gills cover and fins, scales detachment accompanied by slight swelling of abdomen. Isolation was done from gills, liver, kidneys and intestines using Tryptone Soya Agar medium and X.L.D medium. Morphological and biochemical identification of bacterial isolates revealed three different isolates of Edwardsiella tarda (A, B and C). Their diagnosis was confirmed using Microbact 24E and 16S RNA gene for PCR. Their virulence was differentiated by H2S, Indole, Catalase test and haemolysis on blood agar (α, β and γ). The total prevalence of E. tarda isolates was (9.6%) among all examined marine fishes while seasonal prevalence was (15%) in spring, (3.3%) in winter and (5%) in fall.

Epidemiological cut-off values for disc diffusion data generated by standard test protocols from Edwardsiella tarda and Vibrio harveyi

The susceptibility of 52 isolates of Edwardsiella tarda and 54 isolates of Vibrio harveyi to 12 antibiotics was established using internationally standardised disc diffusion protocols. Normalised resistance interpretation was employed to generate statistically valid epidemiological cut-off values for these susceptibility data. For three of these 24 species/antibiotic data sets, there were insufficient fully sensitive strains to allow analysis and for an additional two there were sufficient fully susceptible isolates to allow only a provisional estimate to be made. The data sets for a further two species/antibiotic combinations were considered too diverse to allow the calculation of a valid cut-off value. Valid epidemiological cut-off values were, therefore, calculated for 17 species/antibiotic combinations. It is argued that these cut-off values will greatly facilitate the performance of studies designed to monitor the consequence of antibiotic use in aquaculture. They will also facilitate the prudent and rational choice of antibiotics to be applied therapeutically in commercial farming of aquatic animals.

Pharmacokinetics, pharmacokinetic–pharmacodynamic relationship, and withdrawal period of amoxicillin sodium in olive flounder (Paralichthys olivaceus)

1. The pharmacokinetics (PK) and withdrawal period of amoxicillin sodium in olive flounder and its activity against pathogenic bacteria of olive flounder were investigated.2. Intramuscular administration (12.5 or 125 mg/kg, n = 160) and HPLC analysis of sera were used.3. Rapid absorption (Tmax 2.6 and 2.2 h), prolonged action (terminal half-life, 15.52 and 10.42 h; MRT, 18.79 and 14.44 h), and dose-proportional exposure (AUC0–∞, 273.69 and 2755.37 h. μg/ml) were observed after 12.5 and 125 mg/kg doses.4. The withdrawal period of amoxicillin sodium from muscle plus skin of olive flounder (n =40, water temperature, 23 °C) was 12 d (276 degree days).5. Amoxicillin sodium had small MICs against Streptococcus iniae (0.008–0.06 μg/ml) and Streptococcus parauberis (0.03–1.0 μg/ml), whereas higher concentrations were required to inhibit Edwardsiella tarda isolates (0.06–16 μg/ml).6. While large AUC0–24 h/MIC90 and Cmax/MIC90 ratios were obtained for S. iniae and S. parauberis, with drug concentrations in serum greater than MICs for the entire dosing interval (T > MIC90 of 100%), the lower dose (12.5 mg/kg) could not achieve target values of the PK–pharmacodynamic (PD) indices for E. tarda isolates, suggesting the need for higher doses to combat pathogenic bacteria with large MICs.

Antimicrobial susceptibility and genetic characterisation of oxytetracycline-resistant Edwardsiella tarda isolated from diseased eels

Edwardsiellosis is one of the most important bacterial diseases in eels. Edwardsiella tarda (E. tarda) isolates (n=94) from diseased eels were screened against the eight most commonly used antimicrobial agents...

Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species.Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardseilla. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity-related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR-based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus-PCR and BOX-PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain-specific or origin-specific clades. Biochemical characterization was sensitive for interspecific typing, while PCR-based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. PCR-based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.

Phenotypic diversity of Edwardsiella tarda isolated from different origins

To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1-7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra-species level. Furthermore, evaluation of virulence-associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.

Pathogenic Potential of Edwardsiella tarda Strains Isolated from Turbot

In this study, pathogenicity of Edwardsiella tarda isolates from turbot Scophthalmus maximus as well as other strains from different hosts were tested against turbot, sole Solea senegalensis and sea bass Dicentrarchus labrax. In addition, the influence of challenge route and temperature in the pathogenic potential of E. tarda strains from turbot were also examined. The results obtained showed that E. tarda did not have host specificity in virulence and that the turbot isolates were highly virulent regardless of the inoculation route and temperature. Moreover, inoculation experiments performed in mice suggest that these isolates are virulent for mammals. All these findings suggest that this emergent turbot pathogen constitutes an important risk factor for the marine aquaculture.

Antimicrobial Property of 2-Hydroxypropane-1,2,3-Tricarboxylic Acid Isolated from Citrus microcarpa Extract

This article described antimicrobial property and structure analysis of 2-hydroxypropane-1,2,3-tricarboxylic acid isolated from the crude extract of Citrus microcarpa. Presently, there was no report on compound from C. microcarpa that possessed antimicrobial property against fish pathogenic bacteria. Therefore, in this study, the bioactive principle in C. microcarpa extract was isolated using thin layer chromatography. It's structure was elucidated based on nuclear magnetic resonance (NMR) spectroscopic data, such as proton NMR (1HNMR), correlation spectroscopy, carbon 13 NMR, and heteronuclear multiple bond correlation data. This study showed that the bioactive compound isolated from C. microcarpa was 2-hydroxypropane-1,2,3-tricarboxylic acid monohydrate. The minimum inhibitory concentration (MIC) values of crude C. microcarpa extract and its bioactive component, 2-hydroxypropane-1,2,3-tricarboxylic acid as well as commercially available synthetic 2-hydroxypropane-1,2,3-tricarboxylic acid, were determined against 18 isolates of Edwardsiella tarda and 7 bacterial reference strains, namely, Escherichia coli (ATCC 25922), Citrobacter freundii (ATCC 8090), Aeromonas hydrophila (ATCC 49140), Pseudomonas aeruginosa (ATCC 35032), Streptococcus agalatiae (ATCC 13813), E. tarda (ATCC 15947), and Yersinia enterocolitica (ATCC 23715), using two-fold microdilution method. The MIC values for both the natural 2-hydroxypropane-1,2,3-tricarboxylic acid and the synthetic one were ranging from 15.6 to 62.5 mg mL−1, whereas that of the crude extract was ranging from 7.8 to 31.3 mg mL−1. These findings showed that both the crude extract and its bioactive component might have potential as antimicrobial agent for aquaculture use.

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.

Combination effects of cephalexin and gentamicin on Edwardsiella tarda and Streptococcus iniae

The objective of this study was to determine the in vitro activity of cephalexin–gentamicin combination by a microbroth chequerboard technique against clinical isolates of Edwardsiella tarda and Streptococcus iniae. Gentamicin was shown more susceptible than cephalexin against both bacteria. The effect of cephalexin–gentamicin combination against both bacteria represented additive interaction. The combination even showed synergic interaction (22%) against E. tarda, with a FIC index of <0.5 as a borderline. No antagonism for cephalexin–gentamicin combination was observed for any bacterial strain.

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1 h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.

Effect of Incubation Temperature and Salinity on Expression of the Outer Membrane Protein Profile ofEdwardsiella tarda

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.

Edwardsiella tarda induces plasma membrane ruffles on infection of HEp-2 cells

Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated. By electron microscopy we observed at 1 h post infection that E. tarda induced formation of extensive plasma membrane projections resembling membrane ruffles. The ruffles did not coincide with adhering bacteria. Only few invading bacteria were seen. Vacuolated nuclear membrane was occasionally observed. Three hours post infection, E. tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus. Only one of the E. tarda strains was seen residing within the host cell remains. The results indicate that E. tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis.

Edwardsiella tarda induces plasma membrane ruffles on infection of HEp-2 cells.

Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated. By electron microscopy we observed at 1 h post infection that E. tarda induced formation of extensive plasma membrane projections resembling membrane ruffles. The ruffles did not coincide with adhering bacteria. Only few invading bacteria were seen. Vacuolated nuclear membrane was occasionally observed. Three hours post infection, E. tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus. Only one of the E. tarda strains was seen residing within the host cell remains. The results indicate that E. tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis.