The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

Indole-3-acetic acid increases the survival of brine shrimp challenged with vibrios belonging to the Harveyi clade.

Vibrios belonging to the Harveyi clade (including closely related species such as Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus) are important pathogens of aquatic organisms. In this study, we investigated the use of indole-3-acetic acid to control disease caused by Harveyi clade vibrios. Indole-3-acetic acid, which can be produced by various seaweeds and microalgae, was added to the rearing water of brine shrimp larvae challenged with 12 different Harveyi clade Vibrio strains. Indole-3-acetic acid significantly decreased the virulence of 10 of the strains without any effect on their growth. The latter is important as it will minimize the selective pressure for resistance development. The survival rate of brine shrimp larvae increased from 1.2-fold to 4.8-fold upon treatment with 400 µM indole-3-acetic acid. Additionally, indole-3-acetic acid significantly decreased the swimming motility in 10 of the strains and biofilm formation in eight of the strains. The mRNA levels of the pirA and pirB toxin genes were decreased to 46% and 42% by indole-3-acetic acid in the AHPND-causing strain V. parahaemolyticus M0904. Hence, our data demonstrate that indole-3-acetic acid has the potential to be an effective virulence inhibitor to control infections in aquaculture.

Utilisation of probiotics for disease management in giant freshwater prawn (Macrobrachium rosenbergii): Administration methods, antagonistic effects and immune response.

The giant freshwater prawn (Macrobrachium rosenbergii) is a high-yielding prawn variety well-received worldwide due to its ability to adapt to freshwater culture systems. Macrobrachium rosenbergii is an alternative to shrimp typically obtained from marine and brackish aquaculture systems. However, the use of intensive culture systems can lead to disease outbreaks, particularly in larval and post-larval stages, caused by pathogenic agents such as viruses, bacteria, fungi, yeasts and protozoans. White tail disease (viral), white spot syndrome (viral) and bacterial necrosis are examples of economically significant diseases. Given the increasing antibiotic resistance of disease-causing microorganisms, probiotics have emerged as promising alternatives for disease control. Probiotics are live active microbes that are introduced into a target host in an adequate number or dose to promote its health. In the present paper, we first discuss the diseases that occur in M. rosenbergii production, followed by an in-depth discussion on probiotics. We elaborate on the common methods of probiotics administration and explain the beneficial health effects of probiotics as immunity enhancers. Moreover, we discuss the antagonistic effects of probiotics on pathogenic microorganisms. Altogether, this paper provides a comprehensive overview of disease control in M. rosenbergii aquaculture through the use of probiotics, which could enhance the sustainability of prawn culture.

Indole decreases the virulence of pathogenic vibrios belonging to the Harveyi clade.

AIM: Indole is a signaling molecule secreted by over 85 species of bacteria, including several Vibrio species, and it has been reported to affect different bacterial phenotypes such as biofilm formation, motility, and virulence. In this study, we aimed at investigating the inter-strain variability of the effect of indole in 12 different strains belonging to the Harveyi clade of vibrios. METHODS AND RESULTS: Indole reduced the virulence of all strains towards gnotobiotic brine shrimp larvae. The survival rate of brine shrimp larvae challenged with vibrios pretreated with indole was increased by 1.3-fold to 1.8-fold. Additionally, indole significantly decreased the biofilm formation in all of the strains, decreased the swimming motility in eight of the strains, and decreased swarming motility in five of the strains. When cultured in the presence of exogenous indole, the mRNA level of the pirA and pirB toxin genes were down-regulated to 65% and 46%, and to 62% and 55% in the AHPND-causing strains Vibrio parahaemolyticus M0904 and Vibrio campbellii S01, respectively. CONCLUSIONS: These data indicate that indole has a significant impact on the virulence of different strains belonging to the Harveyi clade of vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that indole signaling is a valid target for the development of novel therapeutics in order to control infections caused by Harveyi clade vibrios in aquaculture.

Insights into a Pyruvate Sensing and Uptake System in Vibrio campbellii and Its Importance for Virulence.

Pyruvate is a key metabolite in living cells and has been shown to play a crucial role in the virulence of several bacterial pathogens. The bioluminescent Vibrio campbellii, a severe infectious burden for marine aquaculture, excretes extraordinarily large amounts of pyruvate during growth and rapidly retrieves it by an as-yet-unknown mechanism. We have now identified the responsible pyruvate transporter, here named BtsU, and our results show that it is the only pyruvate transporter in V. campbellii. Expression of btsU is tightly regulated by the membrane-integrated LytS-type histidine kinase BtsS, a sensor for extracellular pyruvate, and the LytTR-type response regulator BtsR. Cells lacking either the pyruvate transporter or sensing system show no chemotactic response toward pyruvate, indicating that intracellular pyruvate is required to activate the chemotaxis system. Moreover, pyruvate sensing and uptake were found to be important for the resuscitation of V. campbellii from the viable but nonculturable state and the bacterium's virulence against brine shrimp larvae. IMPORTANCE Bacterial infections are a serious threat to marine aquaculture, one of the fastest growing food sectors on earth. Therefore, it is extremely important to learn more about the pathogens responsible, one of which is Vibrio campbellii. This study sheds light on the importance of pyruvate sensing and uptake for V. campbellii, and reveals that the bacterium possesses only one pyruvate transporter, which is activated by a pyruvate-responsive histidine kinase/response regulator system. Without the ability to sense or take up pyruvate, the virulence of V. campbellii toward gnotobiotic brine shrimp larvae is strongly reduced.

Rearing water microbiomes in white leg shrimp (Litopenaeus vannamei) larviculture assemble stochastically and are influenced by the microbiomes of live feed products.

The development of effective management strategies to reduce the occurrence of diseases in aquaculture is hampered by the limited knowledge on the microbial ecology of these systems. In this study, the dynamics and dominant community assembly processes in the rearing water of Litopenaeus vannamei larviculture tanks were determined. Additionally, the contribution of peripheral microbiomes, such as those of live and dry feeds, to the rearing water microbiome were quantified. The community assembly in the hatchery rearing water over time was dominated by stochasticity, which explains the observed heterogeneity between replicate cultivations. The community undergoes two shifts that match with the dynamics of the algal abundances in the rearing water. Source tracking analysis revealed that 37% of all bacteria in the hatchery rearing water were introduced either by the live or dry feeds, or during water exchanges. The contribution of the microbiome from the algae was the largest, followed by that of the Artemia, the exchange water and the dry feeds. Our findings provide fundamental knowledge on the assembly processes and dynamics of rearing water microbiomes and illustrate the crucial role of these peripheral microbiomes in maintaining health-promoting rearing water microbiomes.

Quorum sensing is required for full virulence of Vibrio campbellii towards tiger grouper (Epinephelus fuscoguttatus) larvae.

The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4 days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V. campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.

Does quorum sensing interference affect the fitness of bacterial pathogens in the real world?

Many bacterial pathogens rely on quorum sensing to control virulence gene expression. Based on numerous experiments conducted under well-defined conditions, quorum sensing interference is considered as a promising strategy to tackle infections and thus might have the potential to (partially) replace antibiotics. Despite the promising results in well-defined (artificial) laboratory experiments, there still is a lack of knowledge with respect to the impact of quorum sensing interference on the fitness of pathogens in more realistic scenarios, including interactions with a host, the external environment and complex microbial communities. In this article, we critically evaluate the current knowledge with respect to these three facets of the real world that can affect the fitness of quorum sensing bacterial pathogens. We argue that further research is needed in order to determine how these factors interplay with quorum sensing and to what extent they can influence the selective pressure that might be exerted by quorum sensing interference (and thus determine the risk of resistance development against quorum sensing interference). This kind of information is indispensable in order to optimize quorum sensing interference-based therapeutic strategies.

Indole signalling and (micro)algal auxins decrease the virulence of Vibrio campbellii, a major pathogen of aquatic organisms.

Vibrios belonging to the Harveyi clade are major pathogens of marine vertebrates and invertebrates, causing major losses in wild and cultured organisms. Despite their significant impact, the pathogenicity mechanisms of these bacteria are not yet completely understood. In this study, the impact of indole signalling on the virulence of Vibrio campbellii was investigated. Elevated indole levels significantly decreased motility, biofilm formation, exopolysaccharide production and virulence to crustacean hosts. Indole furthermore inhibited the three-channel quorum sensing system of V. campbellii, a regulatory mechanism that is required for full virulence of the pathogen. Further, indole signalling was found to interact with the stress sigma factor RpoS. Together with the observations that energy-consuming processes (motility and bioluminescence) are downregulated, and microarray-based transcriptomics demonstrating that indole decreases the expression of genes involved in energy and amino acid metabolism, the data suggest that indole is a starvation signal in V. campbellii. Finally, it was found that the auxins indole-3-acetic acid and indole-3-acetamide, which were produced by various (micro)algae sharing the aquatic environment with V. campbellii, have a similar effect as observed for indole. Auxins might, therefore, have a significant impact on the interactions between vibrios, (micro)algae and higher organisms, with major ecological and practical implications.

Bactericidal, quorum quenching and anti-biofilm nanofactories: a new niche for nanotechnologists.

Despite several conventional potent antibacterial therapies, bacterial infections pose a significant threat to human health because they are emerging as the leading cause of death worldwide. Due to the development of antibiotic resistance in bacteria, there is a pressing demand to discover novel approaches for developing more effective therapies to treat multidrug-resistant bacterial strains and biofilm-associated infections. Therefore, attention has been especially devoted to a new and emerging branch of science "nanotechnology" to design non-conventional antimicrobial chemotherapies. A range of nanomaterials and nano-sized carriers for conventional antimicrobial agents have fully justified their potential to combat bacterial diseases by reducing cell viability, by attenuating quorum sensing, and by inhibiting/or eradicating biofilms. This communication summarizes emerging nano-antimicrobial therapies in treating bacterial infections, particularly using antibacterial, quorum quenching, and anti-biofilm nanomaterials as new approaches to tackle the current challenges in combating infectious diseases.

Ureolytic Activity and Its Regulation in Vibrio campbellii and Vibrio harveyi in Relation to Nitrogen Recovery from Human Urine.

Human urine contains a high concentration of nitrogen and is therefore an interesting source for nutrient recovery. Ureolysis is a key requirement in many processes aiming at nitrogen recovery from urine. Although ureolytic activity is widespread in terrestrial and aquatic environments, very little is known about the urease activity and regulation in specific bacteria other than human pathogens. Given the relatively high salt concentration of urine, marine bacteria would be particularly well suited for biotechnological applications involving nitrogen recovery from urine, and therefore, in this study, we investigated ureolytic activity and its regulation in marine vibrios. Thirteen out of 14 strains showed ureolytic activity. The urease activity was induced by urea, since complete and very rapid hydrolysis, up to 4 g L-1 h-1 of urea, was observed in synthetic human urine when the bacteria were pretreated with 10 g L-1 urea, whereas slow hydrolysis occurred when they were pretreated with 1 g L-1 urea (14-35% hydrolysis after 2 days). There was no correlation between biofilm formation and motility on one hand, and ureolysis on the other hand, and biofilm and motility inhibitors did not affect ureolysis. Together, our data demonstrate for the first time the potential of marine vibrios as fast urea hydrolyzers for biotechnological applications aiming at nutrient recovery from human urine.

Photobacterium sanguinicancri sp. nov. isolated from marine animals.

Six strains were isolated from the hemolymph of the spider crab Maja brachydactyla, captured in Spain, and one from a diseased blue mussel, Mytilus edulis. The 16S rRNA gene sequences showed close similarity to the recently described Photobacterium swingsii (98.1 %) and to a lesser degree to Photobacterium aquimaris (97.8 %). MLSA analyses showed a monophyletic group including P. swingsii that form a new subclade. All genomic analyses (Average Nucleotide Identity, Average Amino Acid Identity, and in silico DNA-DNA) clearly separate the strains analysed from P. swingsii with values below the thresholds to delimit a new species. The phenotypic, genotypic and genomic data presented here clearly place these strains as a coherent group within the genus Photobacterium, for which we propose the name Photobacterium sanguinicancri sp. nov. Strain CAIM 1827(T) (=CECT 7579(T), =DSM 24670(T)) is proposed as the type strain of the species.

Quorum sensing positively regulates flagellar motility in pathogenic Vibrio harveyi.

Vibrios belonging to the Harveyi clade are among the major pathogens of aquatic organisms. Quorum sensing (QS) is essential for virulence of V. harveyi towards different hosts. However, most virulence factors reported to be controlled by QS to date are negatively regulated by QS, therefore suggesting that their impact on virulence is limited. In this study, we report that QS positively regulates flagellar motility. We found that autoinducer synthase mutants showed significantly lower swimming motility than the wild type, and the swimming motility could be restored by adding synthetic signal molecules. Further, motility of a luxO mutant with inactive QS (LuxO D47E) was significantly lower than that of the wild type and of a luxO mutant with constitutively maximal QS activity (LuxO D47A). Furthermore, we found that the expression of flagellar genes (both early, middle and late genes) was significantly lower in the luxO mutant with inactive QS when compared with wild type and the luxO mutant with maximal QS activity. Motility assays and gene expression also revealed the involvement of the quorum-sensing master regulator LuxR in the QS regulation of motility. Finally, the motility inhibitor phenamil significantly decreased the virulence of V. harveyi towards gnotobiotic brine shrimp larvae.

Isolation of AHL-degrading bacteria from micro-algal cultures and their impact on algal growth and on virulence of Vibrio campbellii to prawn larvae.

Inactivation of quorum sensing (QS) signal molecules, such as acylhomoserine lactones (AHLs) of pathogenic bacteria, has been proposed as a novel method to combat bacterial diseases in aquaculture. Despite the importance of micro-algae for aquaculture, AHL degradation by bacteria associated with micro-algal cultures has thus far not been investigated. In this study, we isolated Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from open cultures of the micro-algae Tetraselmis suecica and Chaetoceros muelleri, respectively. An AHL degradation assay showed that either monocultures or co-cultures of the isolates were able to degrade the AHL N-hexanoyl-L-homoserine lactone. In contrast, only Bacillus sp. NFMI-C was able to inactivate N-hydroxybutanoyl-L-homoserine lactone, the AHL produced by Vibrio campbellii. The isolated bacteria were able to persist for up to 3 weeks in conventionalized micro-algal cultures, indicating that they were able to establish and maintain themselves within open algal cultures. Using gnotobiotic algal cultures, we found that the isolates did not affect growth of the micro-algae from which they were isolated, whereas a mixture of both isolates increased the growth of Tetraselmis and decreased the growth of Chaetoceros. Finally, addition of Bacillus sp. NFMI-C to the rearing water of giant river prawn (Macrobrachium rosenbergii) larvae significantly improved survival of the larvae when challenged with pathogenic V. campbellii, whereas it had no effect on larval growth.

Host-induced increase in larval sea bass mortality in a gnotobiotic challenge test with Vibrio anguillarum.

Vibrio anguillarum is the major cause of haemorrhagic septicaemia, vibriosis, which is a severe disease affecting marine fish. In this work, it was found that the mortality of gnotobiotic sea bass larvae challenged with V. anguillarum was dependent on the number of dead fish in the vials at the moment of challenge. Based on this finding, the effect of dead hosts (homogenised sea bass larvae or brine shrimp) on the virulence of V. anguillarum towards sea bass larvae was further investigated. Addition of homogenised hosts led to significantly increased larval mortality of challenged larvae, and this was observed for 3 different V. anguillarum strains, i.e. 43, NB 10 and HI 610. In contrast, the addition of similar levels of tryptone had no effect on mortality. In line with this, the motility of all 3 V. anguillarum strains was significantly increased by the addition of homogenised hosts but not by tryptone. These results suggest that dead hosts increase infectivity of V. anguillarum, not merely by offering nutrients to the bacteria, but also by increasing virulence-associated activities such as motility.

The Effect of Caco-2 Cells on Sporulation and Enterotoxin Expression by Foodborne Clostridium perfringens.

Clostridium perfringens enterotoxin (Cpe)-producing strains cause gastrointestinal infections in humans and account for the second-largest number of all foodborne outbreaks caused by bacterial toxins. The Cpe toxin is only produced during sporulation; this process might be affected when C. perfringens comes into contact with host cells. The current study determined how the cpe expression levels and spore formation changed over time during co-culture with Caco-2 cells (as a model of intestinal epithelial cells). In co-culture with Caco-2 cells, total C. perfringens cell counts first decreased and then remained more or less stable, whereas spore counts were stable over the whole incubation period. The cpe mRNA level in the co-culture with Caco-2 cells increased more rapidly than in the absence of Caco-2 cells (3.9-fold higher levels in coculture than in the absence of Caco-2 cells after 8 h of incubation). Finally, we found that cpe expression is inhibited by a cue released by Caco-2 cells (8.3-fold lower levels in the presence of supernatants of Caco-2 cells than in the absence of the supernatants after 10 h of incubation); as a consequence, the increased expression in co-culture with Caco-2 cells must be caused by a factor associated with the Caco-2 cells.

The apparent quorum-sensing inhibitory activity of pyrogallol is a side effect of peroxide production.

There currently is more and more interest in the use of natural products, such as tea polyphenols, as therapeutic agents. The polyphenol compound pyrogallol has been reported before to inhibit quorum-sensing-regulated bioluminescence in Vibrio harveyi. Here, we report that the addition of 10 mg · liter(-1) pyrogallol protects both brine shrimp (Artemia franciscana) and giant river prawn (Macrobrachium rosenbergii) larvae from pathogenic Vibrio harveyi, whereas the compound showed relatively low toxicity (therapeutic index of 10). We further demonstrate that the apparent quorum-sensing-disrupting activity is a side effect of the peroxide-producing activity of this compound rather than true quorum-sensing inhibition. Our results emphasize that verification of minor toxic effects by using sensitive methods and the use of appropriate controls are essential when characterizing compounds as being able to disrupt quorum sensing.