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

Specific quorum sensing-disrupting activity (A(QSI)) of thiophenones and their therapeutic potential

Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (A(QSI)) to describe specific quorum sensing activity. A(QSI) is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of A(QSI) allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 mu M, with A(QSI) higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10

Norepinephrine and dopamine increase motility, biofilm formation and virulence of Vibrio harveyi

Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V harveyi, norepinephrine (NE) and dopamine (Dopa) increased growth in serum-supplemented medium, siderophore production, swimming motility, and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V harveyi. Further, NE-induced effects could be neutralized by alpha-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by beta-adrenergic or dopaminergic antagonists. Dopa-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesize that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host

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

Quroum sensing disruption and the use of short-chain fatty acids and polyhydroxyalkanoates to control luminescent vibriosis

Luminescent vibrios are amongst the most important pathogens in aquaculture. As a result of the massive (mis)use of antibiotics in order to treat infections, these pathogens have acquired resistance to most of the antibiotics that are used. Consequently, antibiotics are not effective anymore to cure luminescent vibriosis disease and in addition, the transfer of resistance determinants constitutes a threat to public health. Therefore, alternative methods to control the disease are urgently needed. In the first part of this work, the disruption of quorum sensing, bacterial cell-to-cell communication, was studied as a possible new strategy to control luminescent vibriosis. First of all, it was shown that quorum sensing regulates the virulence of Vibrio harveyi towards gnotobiotic brine shrimp (Artemia franciscana) nauplii. Subsequently, halogenated furanones were shown to disrupt quorum sensing in these bacteria by decreasing the DNA-binding activity of the quorum sensing master regulator protein LuxR. Interestingly, the addition of 20 mg l-1 of the furanone to the culture water of gnotobiotic Artemia nauplii resulted in a significantly increased survival in challenge tests with different pathogenic luminescent vibrio isolates. Unfortunately, the furanone also appeared to be toxic to the brine shrimp, as high mortality was observed after the addition of 50 mg l-1 to the culture water. The second part of this work describes the application of short-chain fatty acids and polyhydroxyalkanoates. First, it was shown that formic, acetatic, propionic, butyric and valeric acid inhibit growth of luminescent vibrios in vitro in a pH-dependent way. Furthermore, the addition of 20 mM of the short-chain fatty acids to the culture water of gnotobiotic Artemia nauplii resulted in a significantly increased survival in challenge tests with a pathogenic luminescent Vibrio campbellii strain. Subsequently, the application of the homopolymer of the short-chain fatty acid β-hydroxybutyrate, the well-known bacterial storage compound poly-β-hydroxybutyrate, was shown to offer a more efficient protection. Finally, it was shown that poly-β-hydroxybutyrate containing bacteria can be used to protect brine shrimp from luminescent vibriosis. The addition of poly-β-hydroxybutyrate containing Brachymonas bacteria (yielding a poly-β-hydroxybutyrate concentration of 10 mg l-1) resulted in a complete protection from the pathogenic Vibrio campbellii strain

Early mortality syndrome outbreaks : a microbial management issue in shrimp farming?

A recent disease of farmed Penaeid shrimp, commonly referred to as ‘‘early mortality syndrome’ ’ (EMS) or more technically known as ‘‘acute hepatopan-creatic necrosis disease’ ’ (AHPND), was first reported in southern China in 2010 and subsequently in Vietnam, Thailand, and Malaysia [1]. The EMS/AHPND disease typically affects shrimp postlarvae within 20–30 days after stocking and frequently causes up to 100 % mortality. The Global Aquaculture Alliance [2] has estimated that losses to the Asian shrimp culture sector amount to USD 1 billion. The causative agent of EMS/AHPND ha

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

The bacterial storage compound poly-beta-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-beta-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-beta-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-beta-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-beta-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