There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

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Probiotic Bacteria as Biological Control Agents in Aquaculture

Polyphasic taxonomy, a consensus approach to bacterial systematics.

Summary: Over the past decade, the genus Aeromonas has undergone a number of significant changes of practical importance to clinical microbiologists and scientists alike. In parallel with the molecular revolution in microbiology, several new species have been identified on a phylogenetic basis, and the genome of the type species, A. hydrophila ATCC 7966, has been sequenced. In addition to established disease associations, Aeromonas has been shown to be a significant cause of infections associated with natural disasters (hurricanes, tsunamis, and earthquakes) and has been linked to emerging or new illnesses, including near-drowning events, prostatitis, and hemolytic-uremic syndrome. Despite these achievements, issues still remain regarding the role that Aeromonas plays in bacterial gastroenteritis, the extent to which species identification should be attempted in the clinical laboratory, and laboratory reporting of test results from contaminated body sites containing aeromonads. This article provides an extensive review of these topics, in addition to others, such as taxonomic issues, microbial pathogenicity, and antimicrobial resistance markers.

The Genus Aeromonas: Taxonomy, Pathogenicity, and Infection

Summary: High-affinity iron acquisition is mediated by siderophore-dependent pathways in the majority of pathogenic and nonpathogenic bacteria and fungi. Considerable progress has been made in characterizing and understanding mechanisms of siderophore synthesis, secretion, iron scavenging, and siderophore-delivered iron uptake and its release. The regulation of siderophore pathways reveals multilayer networks at the transcriptional and posttranscriptional levels. Due to the key role of many siderophores during virulence, coevolution led to sophisticated strategies of siderophore neutralization by mammals and (re)utilization by bacterial pathogens. Surprisingly, hosts also developed essential siderophore-based iron delivery and cell conversion pathways, which are of interest for diagnostic and therapeutic studies. In the last decades, natural and synthetic compounds have gained attention as potential therapeutics for iron-dependent treatment of infections and further diseases. Promising results for pathogen inhibition were obtained with various siderophore-antibiotic conjugates acting as “Trojan horse” toxins and siderophore pathway inhibitors. In this article, general aspects of siderophore-mediated iron acquisition, recent findings regarding iron-related pathogen-host interactions, and current strategies for iron-dependent pathogen control will be reviewed. Further concepts including the inhibition of novel siderophore pathway targets are discussed.

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Siderophore-Based Iron Acquisition and Pathogen Control

Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity using Human Keratinocyte Cells

N1-Acetylnorspermidine [CH3CONH(CH2)3NH(CH2)3NH3] was identified in Vibrio parahaemolyticus, which contains norspermidine as a major polyamine. This is the first example for the natural occurence of monoacetylated unusual polyamine. The N1-acetylnorspermidine content was the highest 4 h after inoculation. Incubation of norspermidine and acetyl CoA with a cell extract from V. parahaemolyticus produced N1-acetylnorspermidine. A remarkable increase in specific activity of the acetyltransferase was observed at the exponential phase of growth. Spermidine also served as a substrate for the enzyme, with the formation of two isomers of the acetylspermidines (N1-acetylspermidine was predominant), but the reaction rate was less than 50% of that with norspermidine. These results suggest that norspermidine in V. parahaemolyticus may be associated with the cell growth and its role may be controlled through acetylation, as reported for spermidine in Escherichia coli.

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation.

L-2, 4-Diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1, 3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13048 was cloned and characterized. Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis. Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteriaceae. Such a relatively wide occurrence of the ddc homologs implies biological singificance of DABA DC and its product DAP.

Cloning and Characterization of the ddc Homolog Encoding L-2, 4-Diaminobutyrate Decarboxylase in Enterbacter aerogenes

Specific antiserum against Vibrio vulnificus hemolysin was prepared by immunizing rabbits with a liposome suspension bearing the hemolysin. A cholesterol-phosphatidylcholine-liposome suspension was incubated with crude hemolysin preparation, washed well and then injected intravenously (i.v.) into rabbits. Of the various proteins in the crude preparation, the hemolysin was the only protein which was able to bind to the liposomes, because V. vulnificus hemolysin possesses specific cholesterol-binding activity. Therefore, the antiserum obtained could react only with the hemolysin. Specific antiserum was thus prepared by a simple method, with no need for tedious treatment to purify the immunizing antigen.

Preparation of specific antiserum against Vibrio vulnificus hemolysin by immunization with hemolysin-bound liposomes

The effects of various synthetic triamines having a general structure, H2N(CH2)xNH(CH2)yNH2, where x=2-5 and y=2-8 (abbreviated, x-y; with 3-4 being spermidine itself), on poly(U)-directed polypeptide synthesis of Escherichia coli and on growth of its polyamine-requiring mutants were examined in comparison with those of spermidine. Except for 2-2 and 2-3, all of the triamines stimulated more or less polypeptide synthesis at suboptimal Mg2+ concentrations, but the Mg2+ concentration required for the maximal stimulatory effect was different for each triamine. The degree of maximal stimulation caused by 3-3 (norspermidine), 4-4 (homospermidine), or 4-5 was nearly comparable with that by spermidine. The acetylspermidines were inactive, however, they inhibited the spermidine-stimulated polyphenylalanine synthesis. Many of the triamines examined reduced the ratio of leucine to phenylalanine incorporation into polypeptides during poly(U)-directed translation, and the degree of this effect did not necessarily correspond with that of the stimulatory effect. Moreover, 2-4, 2-5, 3-3 and 4-4 could stimulate the growth of a polyamine auxotroph of E. coli, MA 261, as effectively as did spermidine. However, 3-3 was the only triamine which could fully replaced spermidine in promoting growth of a mutant strain, KK 101, which is more dependent on polyamines than MA 261. Thus, these results demonstrated that some synthetic triamines were as active as spermidine in eliciting these effects, and also that there were some differences among these effects in the structural requirement for triamine.

Sumio Shinoda

Papers

Identification of N1-acetylnorspermidine in Vibrio parahaemolyticus and an enzyme activity responsible for its formation.

Cloning and Characterization of the ddc Homolog Encoding L-2, 4-Diaminobutyrate Decarboxylase in Enterbacter aerogenes

Preparation of specific antiserum against Vibrio vulnificus hemolysin by immunization with hemolysin-bound liposomes

Effects of various triamines on cell-free polypeptide synthesis of Escherichia coli and on growth of its polyamine auxotrophs.

Immunological identification of Vibrioparahaemolyticus: Staphylococcal coagglutination test and enzyme-linked immunosorbent assay