Elena Alcaide

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.

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.

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.

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, 104.61 to 107.11), 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.

Susceptibility of Nile tilapia (Oreochromis niloticus) to vibriosis due to Vibrio vulnificus biotype 2 (serovar E)

The present study documents the susceptibility of Nile tilapia to the experimental vibriosis caused by Vibrio vulnificus biotype 2 (serovar E) using a reference strain (Spanish Collection of Type Cultures, CECT 4604) selected for its high degree of virulence for eels. The biotype 1 of this species is one of the usual organisms involved in epizootics occurred in tilapia. After intraperitoneal injection, the selected strain developed a haemorrhagic septicaemia similar to eel vibriosis with a LD50 four log units lower than that exhibited by the type strain of the species, which belongs to the biotype 1. The results obtained in waterborne and intubation challenges indicated that water and feed could act as a vehicle for vibriosis transmission to healthy tilapia. Moreover, live cells and the extracellular products derived from the strain CECT 4604 showed remarkable activity against tilapia erythrocytes, which correlated with the in vivo production of extensive haemorrhagic areas. Our results suggest that this bacterium could constitute a serious health hazard for tilapia, especially if it is cocultured with eels. Thus, vaccination of tilapia with a vaccine against V. vulnificus biotype 2 could be the best strategy to prevent any cross transmission of the disease from eels to tilapia under intensive rearing conditions.

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 (LD50 of 3.3×106 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.

Numerical Taxonomy of Vibrionaceae Isolated from Cultured Amberjack (Seriola dumerili) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with SSM and SJ coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at SSM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish.

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 addition, in 2008, seven aquabirnaviruses were isolated. Phylogenetic analysis performed using partial sequences of both genomic segments of aquabirnaviruses indicated that the seven isolates could be typed as WB (genogroup I) on the basis of segment A sequences, but when segment B was used six of them clustered with C1 strain (genogroup V) and one was typed as Ab (genogroup II). These results indicate natural reassortment between different strains of aquabirnaviruses in the eels. Although betanodaviruses were not isolated in cell culture, the analysis of the sequence of the nested PCR product indicated that they clustered with SJNNV genotype. The diversity of viral agents and the high level of viral detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought.

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.

Numerical Taxonomy of Pseudomonads Isolated from Water, Sediment and Eels

Summary A numerical taxonomic study was performed on 129 pseudomonad isolates from aquatic environments. 67 out of 98 field strains were isolated from water and sediment samples from Albufera lake, a hypereutrophic lake near Valencia, and 31 strains came from an eel hatchery. 10 clusters were formed at 84% similarity level when the Sokal-Michener similarity coefficient was used. Five clusters were designated as Pseudomonas putida (16 strains), P. fluorescens biotype I (9 strains), P. pseudoalcaligenes (11 strains), P. stutzen (4 strains) and Shewanella putrefaciens (3 strains). A subcluster of 7 strains (out of 15) was ascribed to the species Xanthomonas maltophilia , while the other strains remained unidentified. Two clusters comprise members of the new family Comamonadaceae , as they share the same pattern of substrate utilization as sole carbon source, but only one could be ascribed to the genus Acidovorax .