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

In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal α-specific suppressors of it. By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-BglII restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the α-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form. From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed.

Relationship between chromosomal α-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-α,β,γ,δ-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.

The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source.

The properties and roles of adenosine triphosphate (ATP) -hydrolyzing enzymes in the membrane of Vibrio parahaemolyticus were investigated. At least two (perhaps three) types of ATP-hydrolyzing enzyme was present in everted membrane vesicles. The adenosine triphosphatase (ATPase) activity of one of the enzymes was sensitive to dicyclohexylcarbodiimide and the activity was lost when the membranes were washed with buffer containing ethylenediaminetetraacetic acid. This enzyme seems to be an H +-translocating ATPase. The other ATP-hydrolyzing activity was due to a tightly bound enzyme that could not be removed from the membranes by the washing. This enzyme required higher concentrations of Mg2+ (10 to 20 mM) and Cl - (100 mM) for maximal activity. Zn2+ strongly inhibited the activity. The substrate specificity of this enzyme activity showed that it was a 5 '-nucleotidase.

Properties of adenosine triphosphate-hydrolyzing enzymes in membrane vesicles of Vibrio parahaemolyticus.

A flow-injection system was described for continuous monitoring of total nitrogen in water samples. The present method exploited a series of chemical reactions to transform all nitrogen compounds into nitrite that is then coupled with N-(1-naphthyl) ethylenediammonium dichloride to form a highly colored diazodye and selectively monitored at 540 nm. When 200 μl of standard solution (potassium nitrate) was injected at a sampling frequency of 20/h, the determination range was 0.02-10 mg/l nitrogen. The detection limit and relative standard deviation of the method were 0.02 mg/l nitrogen and 1.2% (n=12), respectively. Utilization of a high-purity distilled water attained a detection limit of 0.002 mg/l and determination range of 0.002-10 mg/l nitrogen. Since the flow-injection system was operated by a high-precision pump in a reproducible manner and any vaporization, loss and matrix effect of samples can be perfectly prevented in the system, the reliability of monitoring results appeared to be far better than that obtained by the standard manual method. The flow-injection system, which is coupled with a sampling system, fulfills the essential functions of continuous monitoring in near-real time. The monitoring system has been designed with emphasis on high sampling rate, long-term stability, low reagent consumption and minimum maintenance, and is therefore suitable for use in water quality management to control eutrophication in an aquatic environment.

Sumio Shinoda

Papers

Relationship between chromosomal α-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment

The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source.

Properties of adenosine triphosphate-hydrolyzing enzymes in membrane vesicles of Vibrio parahaemolyticus.

A Flow-injection System for Monitoring of Total Nitrogen and Control of Eutrophication in an Aquatic Environment

Immobilization of Nitrifying Bacteria and Its Application for Wastewater Treatment