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].

Multiresistant waterborne pathogens isolated from water reservoirs and cooling systems

Aims:  To determine the incidence of multiple antibiotic-resistant strains of the emergent human pathogens Legionella pneumophila, Pseudomonas aeruginosa and mesophilic Aeromonas species among those isolated from water reservoirs and industrial cooling systems. Methods and Results:  Water from four natural water reservoirs and four industrial cooling towers was sampled for 1 year period. The total heterotrophs, mesophilic Aeromonas, Pseudomonas spp. and Legionella spp. counts were performed as recommended by standard procedures, and the sensitivity of the isolates to 27 antibiotics was tested. A total of 117 Aeromonas, 60 P. aeruginosa and 15 L. pneumophila strains were isolated and identif…

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-dideoxy…

Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase)

C . E S T E V E A N D T . H . B I R K B E C K . 2004. Aims: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity. Methods and Results: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used. In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin. In contrast, proteases were mainly released during the stationary phase. Serine protease activity in EO63 culture s…

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 s…

Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels

Vibrio vulnificus biotype 2 is classically considered an obligate eel pathogen. However, it has recently been associated with one human septicemic case. In this paper, the opportunistic behavior of this pathogen is discussed. The bacterium can survive alone in brackish water or attached to eel surfaces for at least 14 days. It is able to spread through water and infect healthy eels by using skin as a portal of entry. These results suggest that water and infected eels may act as reservoirs of infection. A capsule seems to be essential for waterborne infectivity, which would explain why cells recovered from naturally diseased eels give rise to pure cultures of opaque colonies. The spread of t…

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10(4)-10(5) cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully de…

First record of Vibrio vulnificus biotype 2 from diseased European eel, Anguilla anguilla L.

Biochemical and toxigenic properties of Vibrio furnissii isolated from a European eel farm

Abstract The present study describes for the first time the isolation of Vibrio furnissii strains from a European eel culture system which are pathogenic for eels ( Anguilla anguilla ) (LD 50 dose, 10 6 cfu/fish). Biochemical characterization of the isolates was performed by API 20E system and by classical numerical taxonomy. Growth at 4 °C, and with 6–8% (w/v) NaCl in a broth medium were necessary to differentiate Vibrio furnissii from Aeromonas species when the API 20E system was used. Virulence for eels of V. furnissii isolates was demonstrated by intraperitoneal injection of living cells. The extracellular products (ECPs) produced by V. furnissii were lethal to elvers and induced some o…

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 …

Siderophore production in Aeromona spp. Isolated from European eel, Anguilla anfuilla L.

<i>Aeromonas hydrophila</i> subsp. <i>dhakensis</i> 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 t…

Occurrence of Drug-Resistant Bacteria in Two European Eel Farms

ABSTRACT 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.

Pathogenicity of live bacteria and extracellular products of motileAeromonasisolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD50 105·4‐107·6 cfu fish‐1) but no Aer. sobria, Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 108·4 cfu fish‐1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD50 1·0–3·2 μg (g fish)‐1) reproducing the symptoms associated with natur…

Presence of viruses in wild eels Anguilla anguilla L, from the Albufera Lake (Spain)

A virological analysis was conducted on wild eels from the Albufera Lake (Spain). A total of 179 individuals at different growth stages were collected in two different surveys (2004 and 2008). Presence of anguillid herpesvirus (AngHV-1), aquabirnavirus and betanodavirus was confirmed by PCR procedures in both surveys, although the number of detections was clearly higher in 2008 (83% of the eels analysed resulted positive for virus presence). AngHV-1 was the viral agent most frequently detected, followed by aquabirnaviruses. Betanodaviruses were detected by the first time in wild eels, and although the detections were only made by nested PCR, high percentage of positives were achieved. In ad…

Pathogenic Aeromonas hydrophila Serogroup O:14 and O:81 Strains with an S Layer

ABSTRACT 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-polysacchar…

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. …

Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas.

Aims:  This study was undertaken to cluster and identify a large collection of Aeromonas strains. Methods and Results:  Numerical taxonomy was used to analyse phenotypic data obtained on 54 new isolates taken from water, fish, snails, sputum and 99 type and reference strains. Each strain was tested for 121 characters but only the data for 71 were analysed using the `SSM' and `SJ' coefficients, and the UPGMA clustering algorithm. At SJ values of ≥ 81·6% the strains clustered into 22 phenons which were identified as Aer. jandaei, Aer. hydrophila, Aer. encheleia, Aer. veronii biogroup veronii, Aer. trota, Aer. caviae, Aer. eucrenophila, Aer. ichthiosmia, Aer. sobria, Aer. allosaccharophila, Ae…

DNA Relatedness among Aeromonas allosaccharophila Strains and DNA Hybridization Groups of the Genus Aeromonas

The genomic relatedness among three Aeromonas allosaccharophila strains, including the type strain, and other Aeromonas type and reference strains that were assigned to DNA hybridization groups was estimated by DNA-DNA hybridization (competition procedure using a membrane method). All A. allosaccharophila strains were highly related (70 to 100%) to strains 289T (= CECT 4199T) and ATCC 35942. Type strains of other validated Aeromonas species, reference strains of DNA groups 8 and 11, and the Aeromonas sp. strain ATCC 43946 (enteric group 501) were 0 to 41% related to A. allosaccharophila 289T and ATCC 35942. The G+Cs content of A. allosaccharophila strains were in the range 55.9 to 57.3 mol%…

Numerical Taxonomy of Aeromonadaceae and Vibrionaceae associated with Reared Fish and Surrounding Fresh and Brackish Water

Summary A numerical taxonomic study was performed on 51 isolates of gram-negative, oxidase-positive, rodshaped, heterotrophic, facultative anaerobic bacteria isolated from aquatic samples (fresh and brackish water, eels, turbot and rainbow trout). These bacteria were compared with 28 type and references strains which included most of Aeromonas phenospecies and DNA hybridization groups, as well as strains of some arginine-positive vibrios and Plesiomonas shigelloides . The strains were characterized by 137 morphological, biochemical, physiological and nutritional tests, and data were examined by simple matching (S SM ) and Jaccard (S J ) coefficients. Unweigthed pair group mathematical avera…

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 wer…

First description of nonmotileVibrio vulnificusstrains 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 re…

Plasmid-mediated QnrS2 determinant from a clinical Aeromonas veronii isolate.

The main objective of this study was to determine the prevalence of the Qnr determinants in clinical and environmental Aeromonas spp. A total of 52 Aeromonas sp. isolates identified by biochemical methods (5), 25 isolated from natural waters (1) and 27 isolated from clinical samples from hospitals in Valencia, Spain, were tested for quinolone resistance by the disk diffusion method (4) (nalidixic acid, 30 μg; oxolinic acid, 2 μg; flumequine, 30 μg; ciprofloxacin, 5 μg; and levofloxacin, 5 μg). Among the studied isolates, 27 showed resistance to nalidixic acid and susceptibility to ciprofloxacin, 24 isolates were susceptible to both nalidixic acid and ciprofloxacin, and only 1, the A. veroni…

Influence of diseases on the wild eel stock: The case of Albufera Lake

Abstract In recent years, eel stocks in the European Union have declined dramatically. This decline has been attributed to habitat loss, over fishing, and more recently, pathogens, but little data exist on bacterial diseases of wild eels. This study reports on the bacterial and parasitic diseases affecting the wild-eel population of the Albufera Lake. Over a 3-year period, 122 eels were analysed after having been randomly chosen from those caught in the lagoon via traditional fishing procedures. This collection of individuals comprised eels at different growth stages as well as some individuals showing pathological signs, such as haemorrhagic fins, petechiae on the belly and ulcers. Microbi…

An outbreak ofShewanella putrefaciensgroup in wild eelsAnguilla anguillaL. favoured by hypoxic aquatic environments

Microbiological analyses were conducted on wild eels from the L′Albufera Lake (Spain). A total of 174 individuals were collected in two surveys (i.e. year 2008 and autumn–winter 2014) among those caught by local fishermen into the lagoon. The prevalence of Shewanella putrefaciens group was 1.7% in 2008 and rose above 32% in 2014. It was due to an outbreak of shewanellosis that presented a morbidity rate of 64%. S. putrefaciens group strains were isolated as pure cultures from the sick eels that showed white ulcers surrounded by a reddish inflammation, damage of the mouth, extensive skin discoloration, exophthalmia, ascites and bad odour. The S. putrefaciens group was recovered from freshwat…

Seasonal recovery ofEdwardsiella piscicidafrom 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, Plesiomo…

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 ofShewanella putrefaciensand 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 …

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

Abstract 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 H2O–D2O mixture. The following structure of the polysaccharide was established by 1H and 13C NMR spectroscopy, including 2D ROESY and 1H,13C HMBC experiments: Download : Download full-size image where the degree of 6-O-acetylation of the later…

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 (…

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…

Vibrio Species in Seawater and Mussels: Abundance and Numerical Taxonomy

Summary Qualitative and quantitative studies were performed on Vibrio species from seawater and mussel samples at a hatchery in Valencia harbour, and from market mussels. Vibrio alginolyticus and V. harveyi were the most abundant species in all samples. Other species identified were V. pelagius, V. mediterranei, V. tubiashii, V. damsella, V. splendidus, V. fluvialis, V. parahaemolyticus, V. anguillarum, V. cholerae and Aeromonas spp . The abundance of V. harveyi and V. mediterranei was positively correlated with temperature whereas V. pelagius counts showed negative correlation with this parameter. The pathogenic species were recovered in very low numbers. A numerical taxonomic study was pe…

O-Serogrouping and surface components ofAeromonas hydrophilaandAeromonas jandaeipathogenic for eels

The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs] and outer membrane proteins [OMPs]) was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB+ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB- strains antigenic…

Evaluation of the API 20E system for identification and discrimination of Vibrio vulnificus biotypes 1 and 2

Aeromonas encheleia sp. nov., isolated from European Eels

Four strains isolated from European eels in Valencia, Spain, were found to constitute a DNA relatedness group which is 0 to 50% related to the 13 species and DNA group 11 of the genus Aeromonas. Phenotypically, these strains have all of the properties that define the genus Aeromonas. However, they differ from the previously described Aeromonas species by three or more properties. The strains are positive for motility, growth at 37 degrees C, indole production, and arginine dihydrolase activity. They exhibit negative reactions in tests for growth at 42 degrees C and in thiosulfate-citrate-bile salts-sucrose medium (Oxoid), Simmons citrate tests, and tests for lysine and ornithine decarboxyla…