Alicia E. Toranzo

Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds

A survey of antibiotic-producing bacteria from the microbial flora attached to seaweeds and the study of their antibiotic capacities were carried out. From 5 species of green and brown marine algae, 224 bacterial strains were isolated and tested for antibiotic production. A total of 38 strains displayed antibiotic activity, withEnteromorpha intestinalis being the source of the highest number of producer strains. All epiphytic bacteria with antibiotic activity were assigned to thePseudomonas-Alteromonas group. Antagonism assays among the isolates demonstrated that each producer strain inhibits the growth of the other producers, as well as of some nonproducer strains also isolated from seaweeds. Likewise, an autoinhibitory effect was observed in all antibiotic-producing strains. Antibacterial spectra of all the strains include activity againstStaphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, andAchromobacter. A preliminary characterization of the antibiotic substances produced by these epiphytic bacteria demonstrated that they are low molecular weight compounds, thermolabile, and anionic and are not affected by proteolytic enzymes. The role that these inhibitory substances can play in the natural environment is discussed.

Pasteurellosis in cultured gilthead seabream (Sparus aurata): first report in Spain

Abstract The first documented epizootic of pasteurellosis in juvenile gilthead seabream ( Sparus aurata ) cultured in Spain is described. The affected fish showed no apparent surface lesions and only some of them displayed slight haemorrhagic areas in the head and gills. However, fish often exhibited enlarged spleen with typical whitish tubercles. Losses of approximately 26 000 fingerlings (40% of the stock) occurred over a 4-week period (August–September 1990). Microbiological analysis of moribund fish revealed the presence in pure culture in all the organs examined of a bacterium which was characterized biochemically and serologically as Pasteurella piscicida . The isolate was sensitive to most antimicrobial agents tested, and the epizootic was effectively controlled by oral administration of chloramphenicol and oxytetracycline. The virulence assays revealed that the P. piscicida isolate was pathogenic for gilthead seabream with a LD 50 ranging from 1.6 × 10 5 to 9.5 × 10 5 (depending on the fish size), and also for turbot ( Scophthalmus maximus ) with LD 50 ≤ 1.6 × 10 4 . The failure to recover the inoculated strain from the survivor gilthead seabream indicates that the carrier state of P. piscicida in the infected population cannot be demonstrated. The histopathological changes observed in the internal organs of diseased fish are typical of a bacterial septicaemia showing extensive, acute multifocal necrosis in spleen and kidney with large masses of bacterial cells.

Phenotypic and pathobiological characteristics of Pasteurella piscicida

Abstract Pasteurellosis, caused by Pasteurella piscicida , is one of the most threatening diseases of wild and cultured marine fish, and has been reported from many geographical areas including the USA, Japan and the Mediterranean countries. The objective of this article is to construct a picture of the current state of knowledge about this bacterial pathogen and the pathogenesis of the disease it causes. We review some important questions such as the controversial taxonomic position of the bacterium, and its main virulence mechanisms. The epidemiology of the disease, the routes of transmission and the putative reservoirs of P. piscicida in the environment are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, including new diagnostic procedures, chemotherapy, employment of immunostimulants, and improvements in immunization programs.

Comparison between the bacterial flora associated with fingerling rainbow trout cultured in two different hatcheries in the North-West of Spain

Abstract A comparative study was conducted of the microflora associated with gills, intestine, liver and kidney of healthy rainbow trout (Salmo gairdneri) taken at two different hatcheries (O and C) located in the North-West of Spain. The main bacterial groups isolated from fish in both rearing facilities were Aeromonas, Pseudomonas-Xanthomonas, Enterobacteria and Gram (+) cocci. Members of Vibrio, Corynebacteria, Flavobacterium-Cytophaga and Moraxella-Acinetobacter were also detected. The distribution in the various fish organs of these eight bacterial groups revealed the greatest divesity in the gills and intestinal tract. Pseudomonas-Xanthomonas and Aeromonas were the predominant genera isolated from gills in the two hatcheries; however, Vibrio and Corynebacteria were not detected in this organ in hatchery O. Whereas in hatchery C all Flavobacterium-Cytophaga strains were present only on the gills of fish, in hatchery O this group was distributed among gills, intestine and liver. In both rearing units, Enterobacteria and Aeromonas comprised 50% of the total microflora present in the intestinal tract. The lowest diversity was found in the liver and kidney, emphasizing the high levels of Aeromonas in the kidney and Gram (+) cocci in the liver of fish from hatchery C. Examination of the bacteria present in water samples from each hatchery indicated that, in general, the bacterial population of apparently healthy rainbow trout was a reflection of their respective aqueous environments. Whereas Pseudomonas and Aeromonas were the more characteristic bacteria of the freshwater fish microflora regardless of the sampling location, other groups, such as Enterobacteria, appeared to be significantly influenced by the water quality. Pathogenicity assays revealed that the fish carried a diversity of potential pathogens, since strains belonging to Aeromonas, Vibrio and Pseudomonas displayed some degree of virulence for rainbow trout.

Specificity of slide agglutination test for detecting bacterial fish pathogens

Abstract The usefulness of the slide agglutination assay for a rapid diagnosis of fish diseases was evaluated using a total of 80 pathogenic bacteria and environmental isolates belonging to the genera Vibrio (28), Pasteurella (5), Aeromonas (26), Yersinia (6), Edwardsiella (8), Pseudomonas (6) and Lactobacillus (1). Selected strains from each bacterial group were used as antigens for rabbit immunization. The specificity of the reactions between whole-cell antigens and the different whole-cell antisera varied according to the bacterial group analyzed. In the case of V. anguillarum , it was found that by using two sera against serotypes 1 and 2, it was possible to detect most of the strains causing vibriosis regardless of their origin. Cross-reactions were not detected between either both serotypes or with other pathogenic or environmental vibrios. In general, the whole-cell antisera from P. piscicida, A. salmonicida, Y. ruckeri and E. tarda detected all the strains within each species. However, a more heterogeneous pattern was exhibited by the motile Aeromonas species. The majority of A. hydrophila and A. sobria isolates did not agglutinate with the anti A. caviae serum, indicating that this antiserum is not adequate for identifying all motile Aeromonas strains. The antiserum against A. salmonicida subsp. masoucida displayed weak cross-reactions with some A. salmonicida subsp. salmonicida and A. hydrophila whole-cell antigens. Strong cross-agglutinations occurred also between types I and II of Y. ruckeri as well as between E. tarda and E. ictaluri . These cross-reactions were eliminated by using the respective thermostable somatic “O” antigens, which indicates that common thermolabile antigens are shared by these strains. The slide agglutination test, using anti whole-cell sera and two antigen preparations (whole-cells and “O” antigen) for each strain, is useful for a rapid detection of fish pathogens, with the additional advantage of its applicability for serotyping studies. Furthermore, some cross-reactions using the whole-cell antigens are reliable in identifying a wide range of strains causing similar diseases with a small number of anti whole-cell sera.

Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot

In this study the effectiveness of a toxoid-enriched whole-cell bacterin (ET-2) in cultured turbot (Scophthalmus maximus) against Enterococcus sp. was evaluated by bath immersion and intraperitoneal (i.p.) injection. In addition, the influences of fish size as well as enrichment of the standard semimoist diet with the commercial preparation Trouvitol plusR, which contains β-glucan from yeast, were also investigated. Regardless of fish size, type of diet, and bacterial dose employed in the experimental challenges, our Enterococcus bacterin ET-2 proved to be very effective when it was delivered by injection. The RPS (relative percent of survival) values achieved by this route ranged from 89 to 100 (for 45-g turbot) and from 67 to 86 (for 150-g turbot), depending on the bacterial levels and time of experimental challenge. Moreover, this strong degree of protection lasted for at least 1 year. No circulating antibodies were detected in the vaccinated turbot. However, the injected bacterin, irrespective of the diet utilized, elicited a significant (P < 0.01) increase of the phagocytic activity in the spleen. Although the glucan alone also induced an enhancement of the non-specific defence mechanisms, this stimulation was not correlated with the protection of turbot against Enterococcus infection.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Virulence factors of bacteria pathogenic for coldwater fish

Abstract The importance of cell surface associated properties and extracellular products (ECP) as virulence factors varies among the fish pathogens considered in this review. Among members of the genus Vibrio, V. salmonicida, and V. ordalii do not secrete proteases, hemolysins, or cytotoxins, while V. anguillarum, V. vulnificus, and V. damsela are strong exotoxin producers. In V. salmonicida, the presence of a hydrophobic surface antigen, VS-P1 (40 Kd), has been described as a possible virulence determinant protecting the bacterium against the action of host serum. Although in V. anguillarum surface properties related to adherence and invasiveness, as well as different exoenzymes (i.e. hemolysins, cytotoxins, and dermatotoxins) can contribute to the development of infections, metallo-proteases, and undetermined low MW substances are the main toxins responsible for the lethality of their ECP. Whereas V. vulnificus seems to have a different mechanism of virulence for eels and mice, with the presence of a capsule being associated only with its pathogenicity for mammals, all V. damsela strains possess similar virulence determinants for poikilotherm and homoiothermic hosts secreting a potent lethal phospholipase toxin with hemolytic and cytotoxic activities. Although cell surface characteristics linked to the A layer can play a role in disease produced by Aeromonas salmonicida, the pathogenesis of furuncle formation in vivo is due to a combined effect of two enzymes, a 70 Kd serine protease and a 25 Kd phospholipase. Pseudomonas anguilliseptica, Flavobacterium branchiophilum, and the gliding bacteria (Flexibacter-Cytophage spp) are producers of proteolytic enzymes, but the involvement of ECP in the first attack of external fish tissues has only been demonstrated in Flavobacterium. In P. anguilliseptica, however, a relationship between the presence of K antigens and virulence has been clearly confirmed. Yersinia ruckeri lacks most of the cell-surface virulence properties present in other pathogenic Yersinia spp., with proteases and/or hemolysins being the main toxins responsible of the lethal effects of the ECP. In contrast, ECP from Renibacterium salmoninarum are devoid of proteolytic, hemolytic, and cytotoxic activities, and are not lethal for fish. The virulence of R. salmoninarum is strongly correlated with the nature and properties of the cell envelope such as hydrophobicity, autoaggregation, hemagglutination, and the presence of a 57 Kd antigenic protein. Although in some of these bacteria, the iron-acquisition systems can play a role in their pathogenicity, only in V. anguillarum has it been conclusively demonstrated that a direct relationship exists between virulence for fish and the possesion of two siderophore-mediated iron transport mechanisms coded, respectively, by plasmidic and chromosomal genes.

Antigenic and Molecular Characterization of Yersinia ruckeri Proposal for a New Intraspecies Classification

Summary The serological and molecular properties of a group of 17 Yersinia ruckeri strains isolated from cultured rainbow trout in Spain were compared with a total of 36 collection strains isolated from fish in other geographic areas. The serological relationship among isolates, as well as the study of their antigenic determinants (LPS and membrane proteins) allow to propose a new serotyping scheme with four O-serotypes (from O1 to O4). Moreover, serotypes O1 and O2 were divided in two and three groups respectively based mainly on minor differences observed in LPS and membrane protein profiles. The patterns obtained in the analysis of the LPS and proteins present in the extracellular products also support the serological scheme proposed. The strains showed a great genetic homogeneity, displaying similar DNA fingerprint patterns regardless of their serotype. In contrast, only the serotype O1 isolates harbour a plasmid of about 50 MDa, while the strains of the other serotypes presented no extrachromosomical DNA or only small plasmids (less than 10 MDa).

Homology of Vibrio anguillarum strains causing epizootics in turbot, salmon and trout reared on the Atlantic coast of Spain

Abstract In this paper we report the physiological, biochemical, serological, and molecular properties of pathogenic Vibrio anguillarum strains isolated from turbot, salmon and trout reared at several seawater hatcheries on the Galician coast (N.W. Spain). These characteristics were compared with reference strains isolated from different fish or in other geographic areas. The role of extracellular virulence factors in the pathogenesis of vibriosis was also investigated. Agglutination reactions and LPS patterns revealed that most of our isolates belonged to serotype 1, and only the strain responsible for a turbot epizootic in 1985 belonged to serotype 2. The main differential phenotypic traits between these pathogenic vibrios were indol reaction, growth at 37°C and acid production from arabinose, galactose and sorbitol. All the isolates displayed strong protease, amylase, and phospholipase activities, and produced haemolysins against human, sheep and trout erythrocytes. In addition, these strains exhibited similar drug resistance patterns, being sensitive to nitrofuranes, flumequine, oxolinic acid, oxytetracycline and chloramphenicol, and highly resistant to ampicillin. Although the extracellular products from our Vibrio isolates displayed strong cytotoxic activity on the five fish cell lines tested, a non-pathogenic reference strain also showed a positive toxic effect, which indicates that a relationship between fish virulence and cytotoxicity cannot be established for all V. anguillarum strains. Whereas the Northwest Spain isolates belonging to serotype 1 shared one plasmid (47 Md) similar to the virulence plasmid pJM1, in the pathogenic vibrios assigned to serotype 2, no plasmids were detected and, hence, their virulence properties are chromosome-coded.