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

Purification of Saccharomyces cerevisiae cystathionine gamma-lyase (gamma-CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that they are coded for by CYS3 (CYI1) and MET17 (MET25), respectively, leading to the conclusion that CYS3 is the structural gene for gamma-CTLase and that the contaminant is O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified gamma-CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3-disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of gamma-CTLase from this strain. Purified gamma-CTLase had cystathionine gamma-synthase (gamma-CTSase) activity if O-succinylhomoserine, but not O-acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae gamma-CTLase and Escherichia coli gamma-CTSase.

Cystathionine γ‐lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ‐synthase activity

Vibrio vulnificus biotype 2, a primary eel pathogen, is also an opportunistic pathogen for humans. The strains in this biotype secrete a cytolysin into the culture medium. The cytolysin from the strain CDC B3547 (ATCC 33817), which was originally isolated from a human leg wound, can disrupt various kinds of eukaryotic cells including erythrocytes and mast cells, and artificial vesicles, liposomes. The cytolysin is a 50 kDa single‐chain protein and is categorized into the pore‐forming toxins. After binding tightly to the cell‐membrane cholesterol in a temperature‐independent manner, the toxin molecules assemble each other in a temperature‐dependent manner, forming a small transmembrane pore. When incubated with a metalloprotease from the same species, the cytolysin is converted to the nicked toxin composed of some peptide chains, joined with disulfide bond(s). This nicked toxin is more hydrophilic while maintaining comparable cytolytic activity.

An Exocellular Cytolysin Produced by Vibrio vulnificus CDC B3547, a Clinical Isolate in Biotype 2 (Serovar E)

This study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens. Active surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes. Of the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5–10 years age group (33%). Shigellosis was more in children from 2–5 years of age (19%), whereas children <2 years appeared to be susceptible for infection caused by EAEC (16%). When tested for the pathogenicity, the EAEC strains colonized well and caused inflammatory infection in the gut mucosa of BALB/C mice. This hospital-based surveillance revealed prevalence of large number of inflammatory diarrhea. EAEC was the suspected pathogen and <2 years children appeared to be the most susceptible age group. BALB/C mice may be a suitable animal model to study the EAEC-mediated pathogenesis.

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Inflammatory diarrhea due to enteroaggregative Escherichia coli : evidence from clinical and mice model studies

Cholera is an epidemic diarrhoeal disease caused by the Gram-negative bacterium Vibrio cholerae (Faruque et al., 1998, J Microbiol Mol Biol Rev 62: 1301±1314). Until recently, only the O1 serotype of V. cholerae has been believed to cause epidemic cholera. However, one of the non-O1 serotypes, O139, is now shown to cause epidemic cholera, and it is suggested that this serotype originates from the O1 serotype. We investigated whether Vibrio cholerae contains a retroelement, called retron, which is responsible for production of a unique RNA±DNA complex, multicopy singlestranded DNA (msDNA) (Inouye and Inouye, 1993, Curr Opin Genet Dev 3: 713±718). The retron is a genetic element consisting of ret, the gene for bacterial reverse transcriptase (RT), msr, a coding region for the RNA part of msDNA, and msd, a coding region for the DNA part of msDNA. Although the function of RT and msDNA remains elusive, the overproduction of msDNA has been shown to impair the DNA mismatch repair system by sequestration of MutS function resulting in a high frequency of mutations in a genome (Maas et al., 1996, Mol Microbiol 19: 505± 509; Mao et al., 1996, FEMS Microbiol Lett 144: 109± 115). We found that some Vibrio spp., such as V. cholerae O139, V. parahaemolyticus and V. mimicus contain msDNA (unpublished data). The msDNA from V. cholerae O139 was isolated and its DNA sequence, determined using the method of Maxam and Gilbert, indicates that it has a typical msDNA structure consisting of a stable stem-loop structure (not shown). Subsequently, we examined a partial genomic sequence of V. cholerae O1 (biotype El Tor, strain N16961) carried out by the Institute of Genomic Research and found that the bacterial genome contains a sequence completely matching the msDNA sequence from V. cholerae O139. Furthermore, the sequence for msDNA is followed by three open reading frames (ORFs) (Fig. 1A). The ®rst ORF is homologous to all bacterial RTs found to date (Fig. 1B). Inverted repeat sequences (a1-a2 sequences), which are essential for msDNA synthesis are also identi®ed. The length of the DNA part of msDNA is predicted to be 95 bases (Fig. 1C). Thus the retron, the msDNA and RT from V. cholerae O1 are termed as retron-Vc95, msDNA-Vc95 and RT-Vc95 respectively. The msDNA-Vc95 has sequence similarity to msDNA-Ec78 from enteropathogenic Escherichia coli (Lima and Lim, 1997, Plasmid 38: 25±33), and has one mismatched basepair in its secondary structure. RT-Vc95 has the highest similarity with RT-Ec83 (47% identity) (Lim, 1992, Mol Microbiol 6: 3531±3542) and RT-Ec78 (47% identity) (Lima and Lim, 1997, Plasmid 38: 25±33), both from pathogenic E. coli. The second ORF is similar to the second ORF of retron-Ec83, which is a presumed ATP-binding protein. The function of the third ORF is unknown. The present ®ndings suggest a genetic relationship between V. cholerae and pathogenic E. coli. LeClerc et al. reported that, in pathogenic E. coli and Salmonella, there are a large number of populations of hypermutable strains due to a defective mismatch repair system (LeClerc et al., 1996, Science 273: 1208±1211). It would be interesting to examine whether this phenotype is correlated to the ability of msDNA production in these strains. Although the toxin genes ctxABr of V. cholerae are parts of the lysogenized genome of CTXF ®lamentous bacteriophage, and V. cholerae produces the infectious CTXF phage particles (Waldor and Mekalaros, 1996, Science 272: 1910±1914), retron-Vc95 does not seem to be linked to the CTXF genome, as judged from the sequence data.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.1999.01531.x

A retroelement in Vibrio cholerae.

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.

Sumio Shinoda

Papers

Cystathionine γ‐lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ‐synthase activity

An Exocellular Cytolysin Produced by Vibrio vulnificus CDC B3547, a Clinical Isolate in Biotype 2 (Serovar E)

Inflammatory diarrhea due to enteroaggregative Escherichia coli : evidence from clinical and mice model studies

A retroelement in Vibrio cholerae.

Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus