Pathogenic strains of Shewanella putrefaciens contain plasmids that are absent in the probiotic strain Pdp11.

Shewanella putrefaciens Pdp11 is a strain described as a probiotic for use in aquaculture. However, S. putrefaciens includes strains reported to be pathogenic or saprophytic to fish. Although the probiotic trait has been related to the presence of a group of genes in its genome, the existence of plasmids that could determine the probiotic or pathogenic character of this bacterium is unknown. In the present work, we searched for plasmids in several strains of S. putrefaciens that differ in their pathogenic and probiotic character. Under the different conditions tested, plasmids were only found in two of the five pathogenic strains, but not in the probiotic strain nor in the two saprophytic strains tested. Using a workflow integrating Sanger and Illumina reads, the complete consensus sequences of the plasmids were obtained. Plasmids differed in one ORF and encoded a putative replication initiator protein of the repB family, as well as proteins related to plasmid stability and a toxin-antitoxin system. Phylogenetic analysis showed some similarity to functional repB proteins of other Shewanella species. The implication of these plasmids in the probiotic or pathogenic nature of S. putrefaciens is discussed.

Monitoring the Starvation-Survival Response of Edwardsiella piscicida and E. tarda in Freshwater Microcosms, at Various Temperatures.

Edwardsiella piscicida is an important fish pathogen responsible for economic losses in global aquaculture, and E. tarda is also a human zoonotic pathogen. In this study, the survival of E. piscicida and E. tarda strains kept in filtered and sterilized lake water microcosms was investigated during a 20-week period at 7 °C, 15 °C and 25 °C, as well as its pathogenicity retention during a starvation period. E. tarda V43.2 stayed culturable for 6 weeks at 7 °C, 9 weeks at 25 °C and 12 weeks at 15 °C. Both E. piscicida strains (V12.1 and V57.2) stayed culturable even longer, for at least 12 weeks at 7 °C, 15 °C and 25 °C under the same starvation conditions. After Edwardsiella cells entered into the VBNC state, some became shorter and "rounded up," but others aggregated and retained a short rod shape. Aggregates of Edwardsiella cells were common throughout the VBNC period, and a well-formed biofilm was observed for all tested strains at the end of the experiment. The growth capacity of VBNC cells was restored by cultivating microcosm water samples in LB broth at 28 °C. Resuscitated E. piscicida cells were as virulent for the European eel as the controls. Natural waters can be a reservoir for Edwardsiella, and its underestimation in environmental samples poses a risk to public health and aquaculture.

TarSynFlow, a workflow for bacterial genome comparisons that revealed genes putatively involved in the probiotic character of Shewanella putrefaciens strain Pdp11.

Probiotic microorganisms are of great interest in clinical, livestock and aquaculture. Knowledge of the genomic basis of probiotic characteristics can be a useful tool to understand why some strains can be pathogenic while others are probiotic in the same species. An automatized workflow called TarSynFlow (Targeted Synteny Workflow) has been then developed to compare finished or draft bacterial genomes based on a set of proteins. When used to analyze the finished genome of the probiotic strain Pdp11 of Shewanella putrefaciens and genome drafts from seven known non-probiotic strains of the same species obtained in this work, 15 genes were found exclusive of Pdp11. Their presence was confirmed by PCR using Pdp11-specific primers. Functional inspection of the 15 genes allowed us to hypothesize that Pdp11 underwent genome rearrangements spurred by plasmids and mobile elements. As a result, Pdp11 presents specific proteins for gut colonization, bile salt resistance and gut pathogen adhesion inhibition, which can explain some probiotic features of Pdp11.

Seasonal recovery of Edwardsiella piscicida from wild European eels and natural waters: Isolation methods, virulence and reservoirs.

A total of 127 wild eels caught in the L'Albufera Lake (Spain) and 24 samples of lagoon freshwater were analysed for 1-year period. Edwardsiella strains were isolated from liver/kidney on TSA-1 plates in 31.9% of total diseased specimens, and the edwardsiellosis prevalence in the fishery was of 11.8%. The use of double-strength Salmonella-Shigella (DSSS) broth and SS agar yielded Edwardsiella isolation from intestine in 100% of those edwardsiellosis-diseased eels, but also in 40.4% of other sick fish with vibriosis or aeromonosis and in 28.8% of healthy eels, as well as from freshwater in 8.3% of samples. Pure cultures were isolated on SS agar from the former, but motile Aeromonas, Plesiomonas shigelloides and Hafnia alvei were recovered along with Edwardsiella in the other samples. Edwardsiella isolates identification at species level revealed that E. piscicida was distributed between wild eels and freshwater but E. tarda only did in freshwater. All E. piscicida strains were virulent for eels (LD  < 1.0 × 106 CFU/fish) but that of E. tarda was not. This is the first report of E. piscicida in wild eel intestines and natural freshwater, highlighting its role as potential reservoirs for the bacterium. A seasonal recovery was found for E. piscicida at water temperature above 20°C.

Aeromonas hydrophila subsp. dhakensis isolated from feces, water and fish in Mediterranean Spain.

Eight Aeromonas hydrophila-like arabinose-negative isolates from diverse sources (i.e., river freshwater, cooling-system water pond, diseased wild European eels, and human stools) sampled in Valencia (Spain) during 2004-2005, were characterized by 16S rRNA gene sequencing and extensive biochemical testing along with reference strains of most Aeromonas species. These isolates and all reference strains of A. hydrophila subsp. dhakensis and A. aquariorum showed a 16S rRNA sequence similarity of 99.8-100%, and they all shared an identical phenotype. This matched exactly with that of A. hydrophila subsp. dhakensis since all strains displayed positive responses to the Voges-Prokauer test and to the use of dl-lactate. This is the first report of A. hydrophila subsp. dhakensis recovered from environmental samples, and further, from its original isolation in India during 1993-1994. This was accurately identified and segregated from other clinical aeromonads (A. hydrophila subsp. hydrophila, A. caviae, A. veronii biovars veronii and sobria, A. trota, A. schubertii and A. jandaei) by using biochemical key tests. The API 20 E profile for all strains included in A. hydrophila subsp. dhakensis was 7047125. The prevalence of this species in Spanish sources was higher for water (9.4%) than for feces (6%) or eels (1.3%). Isolates recovered as pure cultures from diseased eels were moderately virulent (LD(50) of 3.3×10(6) CFU fish(-1)) to challenged eels in experimental trials. They were all resistant to ticarcillin, amoxicillin-clavuranic acid, cefoxitin, and imipenem, regardless of its source. Our data point to A. hydrophila subsp. dhakensis as an emerging pathogen for humans and fish in temperate countries.

The effect of metals on condition and pathologies of European eel (Anguilla anguilla): in situ and laboratory experiments.

Forty-nine wild eels (Anguilla anguilla) caught in the Albufera Lake (Spain), measuring 24.0-75.0 cm in length and 25.0-637.7 g in weight, were examined for metals (Cd, Co, Cr, Cu, Fe, Hg, Mn, Pb, Se and Zn), condition (CI and HSI indices), as well as for diseases (Anguillicola infestation; bacterial infections). Total metal load significantly increased in eel liver tissue parallel to total length and body weight (log), while silvering females (W(B) > 200 g; L ≥ 500 mm) exhibited the highest amounts of Co, Cu, Hg, Se and Zn. Diverse effects may be expected in these big eels due to long-term metal exposure. In fact, IMBI (individual mean (multi-metal) bioaccumulation index) and copper load (Ln) in particular, were significantly related with a decrease in the HSI, reflecting lower eel fitness. In addition, most silvering females (75%) showed a CI below 0.2, and this size group presented the highest prevalence of chronic diseases, at significant levels, that are non-lethal in the short term, but degenerative in the long term. Amounts of hepatic iron were not correlated with eel size; however, a significant, strong negative correlation between this metal (Ln) and HSI and CI was found for wild eels suffering from diseases of any aetiology. This also included small eels (W(B) <67 g; L < 350 mm), as this size group presented a significant prevalence of acute diseases caused by single virulent bacterial pathogens (i.e. Edwardsiella tarda and Vibrio vulnificus biotype 2). To assess the effect of metals on susceptibility to disease, yellow eels were maintained and exposed to iron, copper, and pathogens, in captivity under laboratory conditions. Co-exposure of eels to iron (9 µg of Fe/g of fish) and bacterial pathogens by intraperitoneal injection (IP), yielded a hundred-fold reduction in the LD50 of all bacteria assayed (i.e. E. tarda, V. vulnificus, and motile Aeromonas), and also the time taken to cause eel death. Short-term aqueous exposure of eels to 0.4, 0.7, 1.7 and 3.9 µM of copper, yielded increasing mortality among eels IP challenged by a single dose of 1.90 × 10(6) E. tarda cells, and this effect was significant for 1.7 µM of copper. These results suggest a synergistic interaction among copper and iron, and bacterial disease agents, with respect to their effect on eel health, considering sublethal levels of metals that are currently found in natural waters.

Mechanisms of quinolone resistance in Aeromonas species isolated from humans, water and eels.

Mechanisms of resistance were determined in 33 quinolone-resistant isolates of the species Aeromonas hydrophila, Aeromonas caviae, Aeromonas media, Aeromonas salmonicida, Aeromonas popoffii and Aeromonas veronii, recovered from humans, freshwater and eels. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were sequenced in these resistant strains, as well as in 8 quinolone-sensitive Aeromonas used as controls. All quinolone-resistant Aeromonas carried point mutations in the gyrA QRDR at codon 83, respectively giving rise to substitutions Ser(83)-->Ile (32 strains) or Ser(83)-->Val (1 strain). Almost half of these isolates (48%) carried additional point mutations in the gyrA QRDR at codon 92 and/or in the parC QRDR at codon 80 corresponding to substitutions Leu(92)-->Met and Ser(80)-->Ile. In all cases, MICs of quinolones were determined in the presence and absence of the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAbetaN). Addition of PAbetaN had no effect on the levels of resistance observed in these isolates. In conclusion, the mechanism of quinolone resistance in the Aeromonas isolates studied was related to mutations in QRDR regions of gyrA and parC genes, with little obvious involvement of pumps inhibited by PAbetaN.

Structure of a polysaccharide from the lipopolysaccharides of Vibrio vulnificus strains CECT 5198 and S3-I2-36, which is remarkably similar to the O-polysaccharide of Pseudoalteromonas rubra ATCC 29570.

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with (1)H and (13)C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure: -->4)-beta-d-GlcpNAc3NAcylAN-(1-->4)-alpha-l-GalpNAmA-(1-->3)-alpha-d-QuipNAc-(1--> where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4-d-malyl ( approximately 30%) or 2-O-acetyl-4-d-malyl ( approximately 70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid (l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.

Structure of a polysaccharide from the lipopolysaccharide of Vibrio vulnificus clinical isolate YJ016 containing 2-acetimidoylamino-2-deoxy-L-galacturonic acid.

A polysaccharide isolated after mild acid degradation of the lipopolysaccharide of Vibrio vulnificus clinical isolate YJ016 was found to contain L-Fuc, D-GlcpNAc, 2,4-diacetamido-2,4,6-trideoxy-D-glucose (di-N-acetylbacillosamine, D-QuiNAc4NAc), and 2-acetimidoylamino-2-deoxy-L-galacturonic acid (L-GalNAmA). The last sugar derivative was confirmed by correlations for nitrogen-linked protons in 2D TOCSY and ROESY spectra measured in a H(2)O-D(2)O mixture. The following structure of the polysaccharide was established by (1)H and (13)C NMR spectroscopy, including 2D ROESY and (1)H,(13)C HMBC experiments: [structure: see text], where the degree of 6-O-acetylation of the lateral beta-GlcNAc residue is approximately 70%.

Structure of a polysaccharide from the lipopolysaccharide of Vibrio vulnificus CECT4602 containing 2-acetamido-2,3,6-trideoxy-3-[(S)- and (R)-3-hydroxybutanoylamino]-L-mannose.

A polysaccharide was isolated by GPC after mild acid treatment of the lipopolysaccharide of Vibrio vulnificus CECT4602 and found to contain L-Rha, D-GlcpNAc and 2-acetamido-2,3,6-trideoxy-3-(3-hydroxybutanoylamino)-L-mannose (L-RhaNAc3NHb). GLC analysis of the trifluoroacetylated (S)-2-octyl esters derived by full acid hydrolysis of the polysaccharide showed that approximately 80% of the 3-hydroxybutanoic acid has the S configuration and approximately 20% the R configuration. The following structure of the polysaccharide was established by (1)H and (13)C NMR spectroscopies, including 2D ROESY and (1)H/(13)C HMBC experiments: [carbohydrate sequence see in text].

First description of nonmotile Vibrio vulnificus strains virulent for eels.

Nonmotile Vibrio vulnificus strains were isolated as pure cultures from body ulcers and internal organs of wild diseased European eels caught in a Mediterranean freshwater coastal lagoon. All 54 V. vulnificus isolates were nonmotile, indole-, ornithine decarboxilase-, mannitol- and cellobiose-positive, developed the opaque variant in culture, belonged to the O-antigenic serovar A and were highly virulent for eels by both intraperitoneal injection and immersion challenges. The nonmotile phenotype found in our V. vulnificus isolates was stable: nonmotile cells were always recovered from experimentally infected eels; no variation in the immobility of the V. vulnificus cells was observed for repeated subculture by daily passages on solid media, at different temperatures or incubation times and with or without magnesium sulfate. Many of the fla genes of Vibrio were present in the genome of the nonmotile strains (flaCDE and flaFBA for flagellins and flaH for the distal capping protein), although we observed by transmission electron microscopy that these V. vulnificus strains always lacked the polar flagellum. This is the first report on the existence of nonmotile wild-type V. vulnificus strains.

Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain.

Pure cultures of Edwarsiella tarda were isolated from body ulcers and internal organs of wild European eels caught in a Mediterranean freshwater coastal lagoon (Albufera Lake, Valencia, Spain) over a 1 yr period. Overall, the E. tarda isolation rate from wild eels was 9%, but this increased to 22.8% in diseased individuals. All 22 E. tarda isolates belonged to the 'wild-type' biogroup of the species and were virulent for eels (lethal dose that kills 50% of exposed individuals [LD50 dose]: 10(4.85) to 10(6.83) CFU ind.(-1)), and therefore represented the aetiological agent of the haemorrhagic disease observed in wild European eels. The E. tarda isolates and E. tarda CECT 894T type strain were biochemically and serologically related and resistant to macrolides, antifolates, and glycopeptides, but only the isolates from wild eels were resistant to clindamicyn. This study is the first description of edwardsiellosis in a wild European eel population, and alerts us to the presence of E. tarda in natural wetland environments in Mediterranean Europe.

Occurrence of drug-resistant bacteria in two European eel farms.

The occurrence of strains that are resistant to oxolinic acid, oxytetracycline, sulfamethoxazole-trimethoprim, and nitrofurantoin among heterotrophic bacteria, including human and fish pathogens, in two freshwater eel farms was investigated. High levels of individual- and multiple-drug-resistant bacteria were detected, although sampling events were not correlated with clinical outbreaks and drug therapy.

Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer.

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10(4.61) to 10(7.11)), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.

Taxonomic study of sucrose-positive Aeromonas jandaei-like isolates from faeces, water and eels: emendation of A. jandaei Carnahan et al. 1992.

Fourteen sucrose-positive Aeromonas jandaei-like isolates from fresh water and reared European eels were subjected to a polyphasic study to determine their taxonomic position. Numerical taxonomy was used to analyse phenotypic data obtained for these isolates and 43 type and reference strains representative of recognized Aeromonas species. The A. jandaei cluster (phenon 1) was defined at 81.6 % similarity (S(J)); this included the A. jandaei-like isolates, the sucrose-positive strain Aeromonas veronii biogroup sobria CECT 4910 and nearly all A. jandaei reference strains used in the study. Four other reference strains of A. veronii biogroup sobria and the type strain of Aeromonas ichthiosmia were peripheral to the A. jandaei cluster. The supra-group 'A. jandaei-A.veronii biogroup sobria-A. ichthiosmia' was linked at 80.7 % similarity (S(J)) and was clearly segregated from the phenotypic core of the A. veronii biogroup sobria species, which was related to the reference strain Popoff 224 (CECT 4835). DNA relatedness between strains grouped in the A. jandaei cluster (phenon 1) and A. jandaei CECT 4228(T) ranged from 70 to 100 %, but was below 50 % when DNAs from A. veronii biogroup sobria CECT 4835, A. veronii biogroup veronii CECT 4257(T) and A. ichthiosmia CECT 4486(T) were used. In addition, DNA relatedness between peripheral A. veronii biogroup sobria strains and the species A. jandaei (CECT 4228(T)), A. veronii (CECT 4257(T), CECT 4835) and A. ichthiosmia (CECT 4486(T)) was always below 54 %, as it was between the species A. ichthiosmia (CECT 4486(T)) and A. veronii (CECT 4257(T), CECT 4835). Emendation of A. jandaei is proposed; this taxon now includes sucrose-positive clinical and environmental strains as well as environmental isolates that are pathogenic for fish and humans. Other new traits for this species are the ability to grow at 4-42 degrees C, acid production from glycerol but not from lactose, D-melibiose or D-raffinose, the use of D-gluconate, L-glutamate or L-proline but not L-lactate, L-alanine, L-arabinose or L-arginine, hydrolytic activity against casein, elastin, starch and lecithin and the inability to lyse arbutin. The DNA G+C content of A. jandaei is also reported for the first time; it ranges from 58.1 to 61.1 mol%. On the other hand, the DNA relatedness data support the classification of peripheral reference strains of A. veronii biogroup sobria outside this taxon, indicating that biogroup sobria requires further revision.