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.

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-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.

Indole analogues decreasing the virulence of Vibrio campbellii towards brine shrimp larvae.

Indole signalling has been proposed as a potential target for the development of novel virulence inhibitors to control bacterial infections. However, the major structural features of indole analogues that govern antivirulence activity remain unexplored. Therefore, we investigated the impact of 26 indole analogues on indole-regulated virulence phenotypes in Vibrio campbellii and on the virulence of the bacterium in a gnotobiotic brine shrimp model. The results demonstrated that 10 indole analogues significantly increased the fluorescence of indole reporter strain Vibrio cholerae S9149, 21 of them decreased the swimming motility of V. campbellii, and 13 of them significantly decreased the biofilm formation of V. campbellii. Further, we found that 1-methylindole, indene, 2,3-benzofuran, thianaphthene, indole-3-acetonitrile, methyl indole-3-carboxylate, 3-methylindole, and indole-2-carboxaldehyde exhibited a significant protective effect on brine shrimp larvae against V. campbellii infection, resulting in survival rates of challenged brine shrimp above 80%. The highest survival of shrimp larvae (98%) was obtained with indole-3-acetonitrile, even at a relatively low concentration of 20 µM. Importantly, the indole analogues did not affect bacterial growth, both in vitro and in vivo. These results indicate the potential of indole analogues in applications aiming at the protection of shrimp from vibriosis.

Halogenated Indoles Decrease the Virulence of Vibrio campbellii in a Gnotobiotic Brine Shrimp Model.

Indole signaling is viewed as a potential target for antivirulence therapy against antibiotic-resistant pathogens because of its link with the production of virulence factors. This study examined the antimicrobial and antivirulence properties of 44 indoles toward Vibrio campbellii. Based on the results, 17 halogenated indole analogues were selected, as they significantly improved the survival of brine shrimp larvae challenged with V. campbellii. Specifically, 6-bromoindole, 7-bromoindole, 4-fluoroindole, 5-iodoindole, and 7-iodoindole showed a high protective effect, improving the survival of brine shrimp to over 80% even at a low concentration of 10 µM. To explore the impact of selected indole analogues on bacterial virulence phenotypes, swimming motility, biofilm formation, protease activity, and hemolytic activity of V. campbellii were determined. The results showed that all of the 17 selected indole analogues decreased swimming motility at both 10 µM and 100 µM. Most of the indole analogues decreased biofilm formation at a concentration of 100 µM. In contrast, only a slightly decreased protease activity and no effect on hemolytic activity were observed at both concentrations. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system, and the results demonstrate the potential of these compounds in applications aiming at the protection of shrimp from vibriosis, a major disease in aquaculture. IMPORTANCE Bacterial diseases are a major problem in the aquaculture industry. In order to counter this problem, farmers have been using antibiotics, and this has led to the evolution and spread of antibiotic resistance. In order for the aquaculture industry to further grow in a sustainable way, novel and sustainable methods to control diseases are needed. We previously reported that indole signaling is a valid target for the development of novel therapies to control disease caused by Vibrio campbellii and related bacteria, which are among the major bacterial pathogens in aquaculture. In the present study, we identified indole analogues that are more potent in protecting brine shrimp (a model organism for shrimp) from V. campbellii. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system.

Deciphering the virulent Vibrio harveyi causing spoilage in muscle of aquatic crustacean Litopenaeus vannamei.

The muscle of aquatic crustaceans is perishable and susceptible to environmental contamination. Vibrio harveyi is a widely occurring pathogen in aquatic animals. Here, bath treatment with a virulent V. harveyi strain (which was added directly in the rearing water to imitate environmental contamination) isolated from the muscle of the whiteleg shrimp, Litopenaeus vannamei, caused the muscle of Li. vannamei to display a whitish-opaque appearance due to microscopic changes including muscle lysis, muscle fiber damage and microbial colonization. When administered orally by incorporating this isolate in feed (which is an imitation of infection via natural route), rather than direct invasion followed by colonization in the muscle, this isolate indirectly stimulated severe muscle necrosis in Li. vannamei via steering the enrichment of two important (human) pathogens, V. cholerae and V. vulnificus, and one environmental bacterium Pseudomonas oleovorans, based on the meta-taxonomic analyses. In addition to the scientifically proven viral diseases, our research proved that bacterial agents are also capable of causing muscle spoilage in crustaceans via changing the microbial composition, and that the crustaceans might be exploited as the wide-spectrum sensitive bio-detector to indicate the extent of microbial contamination.

Indole decreases the virulence of the bivalve model pathogens Vibrio tasmaniensis LGP32 and Vibrio crassostreae J2-9.

Indole signaling plays an important role in bacterial pathogenesis. In this study, the impact of indole on biofilm formation, swimming and swarming motility were explored in Vibrio tasmaniensis LGP32 and Vibrio crassostreae J2-9, two model pathogens of bivalves. The results showed that indole decreased swimming and swarming motility in both strains, and decreased biofilm formation in V. crassostreae J2-9. Furthermore, indole affected a large number of genes at RNA level, including genes related to metabolism, ABC transporters, flagellar assembly, chemotaxis, and response regulators. Finally, the bacterial virulence towards mussel larvae was decreased by pretreatment with indole in both V. tasmaniensis LGP32 and V. crassostreae J2-9. After 5 days, the survival rate of mussel larvae increased 2.4-fold and 2.8-fold in mussel larvae challenged with V. tasmaniensis LGP32 pretreated with 200 µM and 500 µM indole, respectively. The survival rate of mussel larvae increased 1.5-fold and 1.9-fold in mussel larvae challenged with V. crassostreae J2-9 pretreated with 200 µM and 500 µM indole, respectively. These data indicate that indole has a significant impact on the virulence of V. tasmaniensis LGP32 and V. crassostreae J2-9, and indole signaling could be a promising target for antivirulence therapy.

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.

One health pathogen surveillance demonstrated the dissemination of gut pathogens within the two coastal regions associated with intensive farming.

BACKGROUND: Intensive aquaculture farming has caused significant degradation of coastal wetlands and has been proposed as a reservoir for pathogenic Vibrio spp. RESULTS: Gut pathogens including Vibrio spp., Salmonella spp., and Klebsiella spp. were isolated from bird feces, shrimp and wetland water in two typical coastal regions of China in 2015 and 2017 and were subsequently subjected to whole-genome sequencing. Meanwhile, local patient isolates were also selected to confirm the epidemiological links. Bacterial community composition analyses of the sediments that were sampled in 2015 and 2017 were conducted by the hypervariable region 4 of the 16S rRNA gene. Together with the local clinical isolates, we observed highly related Vibrio isolates from waterbirds, wetlands and shrimp. Phylogenetic genome comparisons also demonstrated that sequence types ST3 and ST2414 Vibrio parahaemolyticus isolates obtained from aquatic animals were clonally related to patient isolates. Likewise, three Salmonella typhimurium isolates were also genomically related to one clinical strain. The results showed that farming activities significantly altered the community composition and resulted in the emergence of several pathogens, including Acinetobacter, Mycobacterium and Legionella. CONCLUSIONS: In conclusion, our results demonstrated that intensive shrimp farming in wetlands has two devastating impacts: pathogen dissemination from aquatic animals into migratory birds and transmission of foodborne pathogens into local communities.

Probiotics: their action against pathogens can be turned around.

Probiotics when applied in complex evolving (micro-)ecosystems, might be selectively beneficial or detrimental to pathogens when their prophylactic efficacies are prone to ambient interactions. Here, we document a counter-intuitive phenomenon that probiotic-treated zebrafish (Danio rerio) were respectively healthy at higher but succumbed at lower level of challenge with a pathogenic Vibrio isolate. This was confirmed by prominent dissimilarities in fish survival and histology. Based upon the profiling of the zebrafish microbiome, and the probiotic and the pathogen shared gene orthogroups (genetic niche overlaps in genomes), this consequently might have modified the probiotic metabolome as well as the virulence of the pathogen. Although it did not reshuffle the architecture of the commensal microbiome of the vertebrate host, it might have altered the probiotic-pathogen inter-genus and intra-species communications. Such in-depth analyses are needed to avoid counteractive phenomena of probiotics and to optimise their efficacies to magnify human and animal well-being. Moreover, such studies will be valuable to improve the relevant guidelines published by organisations such as FAO, OIE and WHO.

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.

The blue mussel inside: 3D visualization and description of the vascular-related anatomy of Mytilus edulis to unravel hemolymph extraction.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Media Optimization, Strain Compatibility, and Low-Shear Modeled Microgravity Exposure of Synthetic Microbial Communities for Urine Nitrification in Regenerative Life-Support Systems.

Urine is a major waste product of human metabolism and contains essential macro- and micronutrients to produce edible microorganisms and crops. Its biological conversion into a stable form can be obtained through urea hydrolysis, subsequent nitrification, and organics removal, to recover a nitrate-enriched stream, free of oxygen demand. In this study, the utilization of a microbial community for urine nitrification was optimized with the focus for space application. To assess the role of selected parameters that can impact ureolysis in urine, the activity of six ureolytic heterotrophs (Acidovorax delafieldii, Comamonas testosteroni, Cupriavidus necator, Delftia acidovorans, Pseudomonas fluorescens, and Vibrio campbellii) was tested at different salinities, urea, and amino acid concentrations. The interaction of the ureolytic heterotrophs with a nitrifying consortium (Nitrosomonas europaea ATCC 19718 and Nitrobacter winogradskyi ATCC 25931) was also tested. Lastly, microgravity was simulated in a clinostat utilizing hardware for in-flight experiments with active microbial cultures. The results indicate salt inhibition of the ureolysis at 30 mS cm-1, while amino acid nitrogen inhibits ureolysis in a strain-dependent manner. The combination of the nitrifiers with C. necator and V. campbellii resulted in a complete halt of the urea hydrolysis process, while in the case of A. delafieldii incomplete nitrification was observed, and nitrite was not oxidized further to nitrate. Nitrate production was confirmed in all the other communities; however, the other heterotrophic strains most likely induced oxygen competition in the test setup, and nitrite accumulation was observed. Samples exposed to low-shear modeled microgravity through clinorotation behaved similarly to the static controls. Overall, nitrate production from urea was successfully demonstrated with synthetic microbial communities under terrestrial and simulated space gravity conditions, corroborating the application of this process in space.

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.

Virulence-inhibitory activity of the degradation product 3-hydroxybutyrate explains the protective effect of poly-ß-hydroxybutyrate against the major aquaculture pathogen Vibrio campbellii.

The bacterial storage compound poly-ß-hydroxybutyrate, a polymer of the short-chain fatty acid 3-hydroxybutyrate, has been reported to protect various aquatic animals from bacterial disease. In order to obtain a better mechanistic insight, we aimed to (1) investigate whether 3-hydroxybutyrate is released from poly-ß-hydroxybutyrate within sterile brine shrimp larvae, (2) determine the impact of 3-hydroxybutyrate on the virulence of Vibrio campbellii to brine shrimp larvae and on its cell density in the shrimp, and (3) determine the impact of this compound on virulence factor production in the pathogen. We detected 3-hydroxybutyrate in poly-ß-hydroxybutyrate-fed brine shrimp, resulting in 24 mM 3-hydroxybutyrate in the intestinal tract of shrimp reared in the presence of 1000 mg l-1 poly-ß-hydroxybutyrate. We further demonstrate that this concentration of 3-hydroxybutyrate does not affect the growth of V. campbellii, whereas it decreases the production of different virulence factors, including hemolysin, phospholipase and protease activities, and swimming motility. We hypothesize that by affecting all these virulence factors at once, 3-hydroxybutyrate (and thus also poly-ß-hydroxybutyrate) can exert a significant impact on the virulence of V. campbellii. This hypothesis was confirmed in a challenge test showing that 3-hydroxybutyrate protected gnotobiotic brine shrimp from pathogenic V. campbellii, without affecting the number of host-associated vibrios.

Quorum-Sensing Systems as Targets for Antivirulence Therapy.

The development of novel therapies to control diseases caused by antibiotic-resistant pathogens is one of the major challenges we are currently facing. Many important plant, animal, and human pathogens regulate virulence by quorum sensing, bacterial cell-to-cell communication with small signal molecules. Consequently, a significant research effort is being undertaken to identify and use quorum-sensing-interfering agents in order to control diseases caused by these pathogens. In this review, an overview of our current knowledge of quorum-sensing systems of Gram-negative model pathogens is presented as well as the link with virulence of these pathogens, and recent advances and challenges in the development of quorum-sensing-interfering therapies are discussed.