The bacterial storage compound PHB protects <i>Artemia franciscana</i> from pathogenic <i>Vibrio campbellii</i>

Infections caused by luminescent vibrios can cause dramatic losses in aquaculture. These infections are often hard to treat with antibiotics because of the spread of resistant strains and therefore, alternative control strategies are urgently needed. We previously found that the short-chain fatty acid 3-hydroxybutyrate protects Artemia from pathogenic Vibrio campbellii. In this study, we investigated whether the homopolymer of the fatty acid, the well-known bacterial storage compound poly-3-hydroxybutyrate (PHB), could be used to protect the nauplii from the pathogen. A starvation experiment learned us that the addition of 1000mg.l-1 PHB particles (average diameter 30μm) to the culture water of starved Artemia nauplii significantly enhanced their survival. This indicated that the nauplii could obtain energy from the PHB. In order to provide the nauplii from energy, the PHB must have been at least partially degraded into water-soluble products (i.e. 3-hydroxybutyrate monomers and oligomers). Subsequently, an in vivo challenge test was performed with the PHB particles and Artemia nauplii challenged to Vibrio campbellii LMG21363. The addition of the PHB particles (at 100mg.l-1 and 1000mg l-1) to the Artemia culture water together with the pathogen significantly enhanced the survival of the infected nauplii. A complete protection (no significant difference in survival with uninfected nauplii) was observed at the highest concentration. If the PHB particles were added 1 day after the addition of the pathogen, a similar but less pronounced effect was noticed. In a second in vivo challenge test, we investigated the effect of the addition of a PHBaccumulating Brachymonas denitrificans strain on the survival of infected nauplii. The strain, added either untreated or pasteurised at 107CFU ml-1, completely protected the nauplii from the pathogen if it had a high PHB content (32% of the VSS). No protection was observed if the strain had a low PHB content

Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example

The massive (mis)use of antibiotics to control infections in aquaculture has resulted in the development of resistant strains, which have rendered antibiotic treatments ineffective. Moreover, the horizontal transfer of resistance determinants to human pathogens and the presence of antibiotic residues in aquaculture products for human consumption constitute important threats to public health. Therefore, to make the aquaculture industry more sustainable, new strategies to control infections are urgently needed.

The natural furanone (5<i>Z</i>)-4-bromo-5-(bromomethylene)-3-butyl-2(5<i>H</i>)-furanone disrupts quorum sensing-regulated gene expression in <i>Vibrio harveyi</i> by decreasing the DNA-binding activity of the transcriptional regulator protein luxR

This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)- furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxRVh mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxRVh response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxRVh protein also showed less shifts with furanone-treated LuxRVh, whereas the LuxRVh concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxRVh unable to bind to the promoter sequences of quorum sensing-regulated genes

Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder.

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated

Comamonas testosteroni colony phenotype influences exopolysaccharide production and coaggregation with yeast cells.

A Comamonas testosteroni strain was isolated from activated sludge on the basis of its ability to coaggregate with yeast cells. On agar plates the following two types of colonies were formed: colonies with a mucoid appearance and colonies with a nonmucoid appearance. On plates this strain alternated between the two forms, making sectored colonies. In liquid medium with constant agitation no such change was observed. In the absence of agitation and in contact with a glass surface a culture with predominantly nonmucoid-colony-forming cells very rapidly shifted to a culture dominated by mucoid-colony-forming cells. In liquid medium the reverse was observed under stress conditions imposed by hydrogen peroxide, sodium dodecyl sulfate, or starvation. Nonmucoid cells formed very rapidly settling flocs with yeast cells, while coaggregation of mucoid cells with yeast cells did not occur. These findings may be relevant to the behavior of activated sludge microbial communities

The impact of mutations in the quorum sensing systems of <i>Aeromonas hydrophila, Vibrio anguillarum</i> and <i>Vibrio harveyi</i> on their virulence towards gnotobiotically cultured <i>Artemia franciscana</i>

Disruption of quorum sensing, bacterial cell-to-cell communication by means of small signal molecules, has been suggested as a new anti-infective strategy for aquaculture. However, data about the impact of quorum sensing on the virulence of aquatic pathogens are scarce. In this study, a model system using gnotobiotically cultured Artemia franciscana was developed in order to determine the impact of mutations in the quorum sensing systems ofAeromonas hydrophila, Vibrio anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AV. harvey abolished virulence of the strain towards Artemia. Moreover, the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrV. harveyi system or the quorum sensing systems of Ae. Hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. Our results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harvey

Short-chain fatty acids protect gnotobiotic <i>Artemia franciscana</i> from pathogenic <i>Vibrio campbellii</i>

Infections caused by antibiotic resistant luminescent vibrios can cause considerable losses in aquaculture. In this study, different short-chain fatty acids were investigated as possible alternative biocontrol agents. The addition of 100 mM formic, acetic, propionic, butyric or valeric acid to the growth medium of a pathogenic Vibrio campbellii strain completely inhibited its growth at pH 6. At 10 mM, the growth of the pathogen was delayed, whereas at 1 mM, no effect could be observed. The growth-inhibitory effect was clearly pH-dependent and decreased with increasing pH. An in vivo challenge test with gnotobiotic Artemia franciscana nauplii revealed that all five short-chain fatty acids protected the shrimp from the pathogenic V. campbellii strain. The addition of 20 mM of the short-chain fatty acids to the culture water resulted in a significantly increased survival of infected nauplii, with no difference between the different fatty acids. In conclusion, our data indicate that short-chain fatty acids might be useful as alternative biocontrol agents to treat luminescent vibriosis

Use of selected bacteria and yeast to protect gnotobiotic <i>Artemia</i> against different pathogens

To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound ß-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios