Quorum Sensing-Disrupting Brominated Furanones Protect the Gnotobiotic Brine Shrimp Artemia franciscana from Pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus Isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Quorum sensing and silencing in Vibrio parahaemolyticus

The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and virulence factors--the hallmarks of the organism--are repressed by this scheme of gene control, and quorum sensing seems to be silenced in many isolates. In these studies, we examine a swarming-proficient, virulent strain and identify an altered-function allele of the quorum regulator luxO that is demonstrated to produce a constitutively active mimic of LuxO～P. We find that LuxO* affects the expression of three small regulatory RNAs (Qrrs) and the activity of a translational fusion in opaR, the output regulator. Tests for epistasis showed that luxO* is dominant over luxO and that opaR is dominant over luxO. Thus, information flow through the central elements of the V. parahaemolyticus quorum pathway is proven for the first time. Quorum-sensing output was explored using microarray profiling: the OpaR regulon encompasses ～5.2% of the genome. OpaR represses the surface-sensing and type III secretion system 1 (T3SS1) regulons. One novel discovery is that OpaR strongly and oppositely regulates two type VI secretion systems (T6SS). New functional consequences of OpaR control were demonstrated: OpaR increases the cellular cyclic di-GMP (c-di-GMP) level, positively controls chitin-induced DNA competency, and profoundly blocks cytotoxicity toward host cells. In expanding the previously known quorum effects beyond the induction of the capsule and the repression of swarming to elucidate the global scope of genes in the OpaR regulon, this study yields many clues to distinguishing traits of this Vibrio species; it underscores the profoundly divergent survival strategies of the quorum On/Off phase variants.     

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh ⁺, ATCC33846-tdh ⁺, ATCC33847-tdh ⁺, ATCC17802-trh ⁺, and F13-environmental strain-tdh ⁺) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.     

Variability in biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and the distribution of the genes involved in biofilm formation

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.     

Temporal and Spatial Variation in the Abundance of Total and Pathogenic Vibrio parahaemolyticus in Shellfish in China

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g^-1 and 10,000 MPN g^-1, respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g^-1. The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.     

Biochemical characteristics and genetic diversity of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> strains isolated from the Lac of Bizerte (Tunisia)

This study characterized the phenotypic and genetic properties of Vibrio spp. and Aeromonas hydrophila strains isolated from seawater and mussels (Mytilus edulis and Crassostrea gigas) cultured in mollusc farm localized in the lac of Bizerte. The 37 strains (31 strains of V. alginolyticus, one strain of V. fluvialis, one strain of V. parahaemolyticus and four strains of A. hydrophila) typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) showed a high polymorphism. Most of the isolates were resistant to at least two antimicrobial agents. All the tested strains were resistant to ampicillin. PCR was used to detect the presence of eight Vibrio cholerae virulence genes in the genome of the Vibrio spp. isolates. The results showed a wide dissemination of these genes in the genome of all Vibrio spp. isolates tested. Differentiation of these strains with the ERIC 2-PCR technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern.     

Prevention of quorum-sensing-mediated biofilm development and virulence factors production in Vibrio spp. by curcumin

The increasing occurrence of disease outbreaks caused by Vibrio spp. and the emergence of antibiotic resistance has led to a growing interest in finding alternative strategies to prevent vibriosis. Since the pathogenicity of vibrios is controlled in part by quorum-sensing (QS) system, interfering with this mechanism would prevent the pathogenicity of vibrios without developing resistance. Hence, a non-toxic phytochemical curcumin from Curcuma longa was assessed for its potential in reducing the production of QS-dependent virulence factors in Vibrio spp. The obtained results evidenced 88 % reduction in bioluminescence of Vibrio harveyi by curcumin. Further, curcumin exhibited a significant inhibition in alginate, exopolysaccharides, motility, biofilm development and other virulence factors production in Vibrio parahaemolyticus, Vibrio vulnificus and V. harveyi. In in vivo analysis, curcumin enhanced the survival rate of Artemia nauplii up to 67 % against V. harveyi infection by attenuating its QS-mediated virulence.     

Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae

Type VI secretion is critical for Vibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V. cholerae type VI secretion system genes are encoded in one large and two small clusters. In V. cholerae, type VI secretion is controlled by quorum sensing, the cell–cell communication process that enables bacteria to orchestrate group behaviours. The quorum‐sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum‐sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs (Qrr sRNAs) that function between LuxO and HapR in the quorum‐sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA‐dependent regulation of the type VI secretion system is conserved in pandemic and non‐pandemic V. cholerae strains.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Caenorhabditis elegans Senses Bacterial Autoinducers

Pseudomonas aeruginosa uses virulence factors controlled by quorum sensing (QS) to kill Caenorhabditis elegans. Here we show that C. elegans is attracted to the acylated homoserine lactones (AHSLs) that mediate QS in P. aeruginosa. Our data also indicate that C. elegans can distinguish AHSLs and may use them to mediate aversive or attractive learning.     

The Vibrio parahaemolyticus effector VopC mediates Cdc42‐dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection

Vibrio parahaemolyticus is a Gram‐negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non‐phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC‐mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.