Transcriptomic analysis of Vibrio alginolyticus challenged by Rhizoma coptidis reveals mechanisms of virulence genes.

Rhizoma coptidis, a Chinese herbal medicine widely used to treat various bacterial infections, has the potential to develop antibiotic substitutes to overcome the drug resistance of Vibrio alginolyticus. To study the inhibitory effect of R. coptidis on V. alginolyticus, we sequenced the transcriptomes of three groups of samples of wild-type V. alginolyticus (CK) and V. alginolyticus, which were stressed by 5 mg/mL R. coptidis for 2 h (RC_2 h) and 4 h (RC_4 h). CK was compared with RC_2 h and RC_4 h, respectively, and a total of 1565 differentially expressed genes (DEGs) (988 up-regulated and 577 down-regulated) and 1737 DEGs (1152 up-regulated and 585 down-regulated) were identified. Comparing RC_2 h with RC_4 h, 156 DEGs (114 up-regulated and 42 down-regulated) were identified. The ability of biofilm formation and motility of V. alginolyticus altered upon with different concentrations of R. coptidis. Interestingly, relative expression patterns of virulence genes appeared statistically significantly varied, upon different concentrations of R. coptidis extract. DEGs were annotated to the Gene Ontology (GO) database for function enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the results showed that the main enriched pathways, was those related to the virulence of V. alginolyticus. This study provides a new perspective for understanding the complex pathogenic mechanism of V. alginolyticus. R. coptidis could potnetially be used as alternative or complimnetary to antibiotics to treat infections after further research.

Management and Mitigation of Vibriosis in Aquaculture: Nanoparticles as Promising Alternatives.

Vibriosis is one of the most common diseases in marine aquaculture, caused by bacteria belonging to the genus Vibrio, that has been affecting many species of economically significant aquatic organisms around the world. The prevention of vibriosis in aquaculture is difficult, and the various treatments for vibriosis have their limitations. Therefore, there is an imperative need to find new alternatives. This review is based on the studies on vibriosis, specifically on the various treatments and their limitations, as well as the application of nanoparticles in aquaculture. One of the promising nanoparticles is graphene oxide (GO), which has been used in various applications, particularly in biological applications such as biosensors, drug delivery, and potential treatment for infectious diseases. GO has been shown to have anti-bacterial properties against both Gram-positive and Gram-negative bacteria, but no research has been published that emphasizes its impact on Vibrio spp. The review aims to explore the potential use of GO for treatment against vibriosis.

Fish Oil Ameliorates Vibrio parahaemolyticus Infection in Mice by Restoring Colonic Microbiota, Metabolic Profiles, and Immune Homeostasis.

The effect of fish oil (FO) on colonic function, immunity, and microbiota was investigated in Vibrio parahaemolyticus (Vp)-infected C57BL/6J mice. Mice intragastrically presupplemented with FO (4.0 mg) significantly reduced Vp infection as evidenced by stabilizing body weight and reducing disease activity index score and immune organ ratios. FO minimized colonic pathological damage, strengthened the mucosal barrier, and sustained epithelial permeability by increasing epithelial crypt depth, goblet cell numbers, and tight junctions and inhibiting colonic collagen accumulation and fibrosis protein expression. Mechanistically, FO enhanced immunity by decreasing colonic CD3+ T cells, increasing CD4+ T cells, downregulating the TLR4 pathway, reducing interleukin-17 (IL-17) and tumor necrosis factor-α, and increasing immune cytokine IL-4 and interferon-γ levels. Additionally, FO maintained colonic microbiota eubiosis by improving microbial diversity and boosting Clostridium, Akkermansia, and Roseburia growth and their derived propionic acid and butyric acid levels. Collectively, FO alleviated Vp infection by enriching beneficial colonic microbiota and metabolites and restoring immune homeostasis.

Vibrio vulnificus pneumonia with multiorgan failure: a case report and review of the literature.

BACKGROUND: Vibrio vulnificus is a gram-negative bacterium causing three clinical syndromes namely, gastrointestinal symptoms, skin sepsis and primary sepsis. Primary sepsis exhibits mortality rates exceeding 50%, particularly in the immunocompromised. Vibrio vulnificus is transmitted via consumption of contaminated seafood and contaminated seawater skin exposure. We describe a rare case of an immunocompetent male presenting with an atypical Vibrio vulnificus infection, culminating in severe pneumonia requiring intensive care. CASE PRESENTATION: A 46 year old Indian male dockyard worker, a non-smoker and teetotaler, of Indian origin presented to the emergency treatment unit of a tertiary care hospital in Sri Lanka, with fever, productive cough with yellow sputum, pleuritic chest pain and tachypnea for five days. He had no gastrointestinal or skin manifestations. His respiratory rate was 38 breaths/min, pulse rate was 120 bpm, blood pressure was 107/75 mmHg and pulse oximetry was 85% on air. Chest X-ray revealed consolidation of the left lung. Empiric intravenous Piperacillin-tazobactam and Clarithromycin were commenced after obtaining blood and sputum cultures. Over the next 24 h, his oxygen requirement rose and as he required vasopressor support, he was admitted to the intensive care unit. He was intubated and bronchoscopy was performed on day two, which demonstrated thick secretions from left upper bronchial segments. His antibiotics were changed to intravenous ceftriaxone and doxycycline following a positive blood culture report of Vibrio vulnificus. He was ventilated for ten days and his intensive care stay was complicated with a non-oliguric acute kidney injury, with serum creatinine rising up to 8.67 mg/dL (0.81-0.44 mg/dL). He developed mild thrombocytopenia with platelets dropping to 115 × 103 /uL (150-450 × 103/uL) which resolved spontaneously. Vasopressors were weaned off by day eight and the patient was extubated on day ten. He was discharged from intensive care on day twelve and made a full recovery. CONCLUSIONS: Pneumonia itself is an atypical manifestation of Vibrio vulnificus and furthermore, this patient was immunocompetent and did not exhibit the classical gastro-intestinal and skin manifestations. This case highlights the occurrence of atypical Vibrio sp. infections in patients with high exposure risks and the need for early supportive and appropriate antibiotic therapies.

On-site Gram staining that increases a post-test probability of an ominous infection: a case of necrotizing fasciitis caused by Vibrio vulnificus: a case report.

BACKGROUND: Gram staining is a classic but standard and essential procedure for the prompt selection of appropriate antibiotics in an emergency setting. Even in the era of sophisticated medicine with technically developed machinery, it is not uncommon that a classic procedure such as Gram staining is the most efficient for assisting physicians in making therapeutic decisions in a timely fashion. CASE PRESENTATION: A 65-year-old Asian man with alcoholic cirrhosis complicated by esophageal varices was brought to the emergency division of Saga Medical School Hospital in early August, complaining of severe pain, redness, swelling, and purpura of the lower extremities. On physical examination he appeared in a critically ill condition suggestive of deep-seated soft tissue infection, raising a pre-test probability of streptococci, staphylococci, Vibrio sp., or Aeromonas sp. as a causative pathogen. A characteristic of his residency in an estuarine area is that raw seafood ingestion, as documented in this patient prior to the current admission, predisposes those who have a chronic liver disease to a life-threatening Vibrio vulnificus infection. Given the pathognomonic clinical features suggestive of necrotizing fasciitis, our immediate attempt was to narrow down the differential list of candidate pathogens by obtaining clinical specimens for microbiological investigation, thus inquiring about the post-test probability of the causative pathogen. The Gram stain of the small amount of discharge from the test incision of the affected lesion detected Gram-negative rods morphologically compatible with V. vulnificus. After two sets of blood culture, intravenous meropenem and minocycline were immediately administered before the patient underwent emergency surgical debridement. The next day, both blood culture and wound culture retrieved Gram-negative rods, which were subsequently identified as V. vulnificus by mass spectrometry, matrix-assisted laser desorption/ionization. The antibiotics were switched to intravenous ceftriaxone and minocycline. CONCLUSION: The pre-test probability of V. vulnificus infection was further validated by on-site Gram staining in the emergency division. This case report highlights the significance of a classic procedure.

Vibrio vulnificus sepsis after shrimp shelling in a patient with preexisting primary biliary cholangitis: a case report.

BACKGROUND: Vibrio vulnificus is typically present in seawater, fish, and shellfish, and is known to cause severe sepsis, particularly in patients with liver diseases such as cirrhosis. V. vulnificus is one of the most dangerous waterborne pathogens, and infection mainly occurs in western Japan during the summer, with an increased fatality rate. Herein, we report the case of a patient with primary biliary cholangitis and sepsis caused by V. vulnificus infection sustained through shrimp shelling. CASE PRESENTATION: An 82-year-old Japanese Asian woman with no medical history or underlying disease developed redness, swelling, and pain, which extended from the right fingers to the upper arm. A diagnosis of sepsis due to cellulitis was made. Blood culture detected V. vulnificus; thus, minocycline was administered in addition to meropenem. The disease course was uneventful, and the patient was discharged on day 28 of hospitalization. Symptoms in the right upper arm developed 1 day after the patient shelled a large number of shrimp; therefore, the infection route was assumed to be through wounds sustained during shrimp shelling. We suspected liver disease and measured serum anti-mitochondrial M2 antibody levels, leading to the diagnosis of primary biliary cholangitis. CONCLUSIONS: As in this case, small wounds caused by handling fish and shrimp are a potential source of infection. Patients with severe V. vulnificus infection should be thoroughly assessed for the presence of liver diseases such as primary biliary cholangitis.

Clinical and Epidemiologic Characteristics and Therapeutic Management of Patients with Vibrio Infections, Bay of Biscay, France, 2001-2019.

Noncholera vibriosis is a rare, opportunistic bacterial infection caused by Vibrio spp. other than V. cholerae O1/O139 and diagnosed mainly during the hot summer months in patients after seaside activities. Detailed knowledge of circulating pathogenic strains and heterogeneities in infection outcomes and disease dynamics may help in patient management. We conducted a multicenter case-series study documenting Vibrio infections in 67 patients from 8 hospitals in the Bay of Biscay, France, over a 19-year period. Infections were mainly caused by V. alginolyticus (34%), V. parahaemolyticus (30%), non-O1/O139 V. cholerae (15%), and V. vulnificus (10%). Drug-susceptibility testing revealed intermediate and resistant strains to penicillins and first-generation cephalosporins. The acute infections (e.g., those involving digestive disorder, cellulitis, osteitis, pneumonia, and endocarditis) led to a life-threatening event (septic shock), amputation, or death in 36% of patients. Physicians may need to add vibriosis to their list of infections to assess in patients with associated risk factors.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

New Atypical Manifestations and Prognostic Factors of Vibrio vulnificus Infection: a 10-Year Retrospective Study.

Vibrio vulnificus (V. vulnificus) infection is rare but potentially fatal. This study explored new atypical manifestations and prognostic factors of V. vulnificus-infected patients during hospitalization. We retrospectively reviewed the medical records of 33 patients diagnosed with V. vulnificus infection in Guangdong Province, China between 2010 and 2020. Multiple logistic regression and receiver operating characteristic (ROC) curve analyses were performed. The new atypical manifestations included cholangitis, urinary tract infection, and suppurative otitis media. Eleven of the 33 (33.3%) V. vulnificus-infected patients eventually died. Univariate analysis showed that patients with cardio-cerebrovascular diseases, lower platelet counts, and higher levels of C-reactive protein and procalcitonin (PCT) had statistically higher mortality. However, multivariate analysis showed that only the PCT level (P = 0.036) was statistically significant. In addition, the area under the ROC value estimate for PCT was 0.8816 (95% confidence interval (CI), 0.759-1.000; P = 0.0009). More than half of the patients with V. vulnificus infection died when PCT was > 20 ng/mL, while no patient died when PCT was ≤ 20 ng/mL. This study found new atypical manifestations of V. vulnificus infection. In addition, PCT was an effective and independent predictor of mortality in patients with V. vulnificus infection, allowing clinicians to conduct early risk stratification and determine the best therapeutic strategies.

The inhibitory effect of Ulva fasciata on culturability, motility, and biofilm formation of Vibrio parahaemolyticus ATCC17802.

The outbreak of vibriosis from Vibrio parahaemolyticus (V. parahaemolyticus) is one of common pathogenic diseases found in the mariculture environment. In this study, the inhibitory effect of Ulva fasciata (U. fasciata) on the culturability, motility, and biofilm formation of V. parahaemolyticus ATCC17802 was examined by co-culturing system. Results showed that both of secretion and live tissue of U. fasciata could convert culturable V. parahaemolyticus ATCC17802 to non-culturable, both reaching more than 99% of inhibition rate after 3-day co-culture, and higher density (12 g L-1) of U. fasciata exhibited stronger inhibition. The twitching behavior of V. parahaemolyticus ATCC17802 was more easily affected by U. fasciata than the swimming behavior after 3-day co-culture, with the inhibitory rates varying at the ranges of 1.70-30.29% (twitching behavior) and 10.06-44.86% (swimming behavior) under the different environmental factors (salinity, NO3--N and PO43--P concentrations), but no significant correlation was found. The greatest inhibition effect on V. parahaemolyticus ATCC17802 biofilm formation occurred at 12 h, with inhibition rates at the range of 11.03-67.10 %, while there was still no significant correlation between inhibition rate and the three environmental factors. The different environmental factors might induce U. fasciata to excrete different levels of secondary metabolites, which caused the various inhibitory effect on the cultivability, motility, and biofilm formation of V. parahaemolyticus ATCC17802.

Medicinal applications and molecular targets of dequalinium chloride.

For more than 60 years dequalinium chloride (DQ) has been used as anti-infective drug, mainly to treat local infections. It is a standard drug to treat bacterial vaginosis and an active ingredient of sore-throat lozenges. As a lipophilic bis-quaternary ammonium molecule, the drug displays membrane effects and selectively targets mitochondria to deplete DNA and to block energy production in cells. But beyond its mitochondriotropic property, DQ can interfere with the correct functioning of diverse proteins. A dozen of DQ protein targets have been identified and their implication in the antibacterial, antiviral, antifungal, antiparasitic and anticancer properties of the drug is discussed here. The anticancer effects of DQ combine a mitochondrial action, a selective inhibition of kinases (PKC-α/ß, Cdc7/Dbf4), and a modulation of Ca2+-activated K+ channels. At the bacterial level, DQ interacts with different multidrug transporters (QacR, AcrB, EmrE) and with the transcriptional regulator RamR. Other proteins implicated in the antiviral (MPER domain of gp41 HIV-1) and antiparasitic (chitinase A from Vibrio harveyi) activities have been identified. DQ also targets α -synuclein oligomers to restrict protofibrils formation implicated in some neurodegenerative disorders. In addition, DQ is a typical bolaamphiphile molecule, well suited to form liposomes and nanoparticules useful for drug entrapment and delivery (DQAsomes and others). Altogether, the review highlights the many pharmacological properties and therapeutic benefits of this old 'multi-talented' drug, which may be exploited further. Its multiple sites of actions in cells should be kept in mind when using DQ in experimental research.

Vibrio parahaemolyticus: Basic Techniques for Growth, Genetic Manipulation, and Analysis of Virulence Factors.

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for bacterial culture, genetic manipulation, and analysis of virulence phenotypes. Detailed protocols for growth assessment, generation of mutants, and phenotype assessment are included in this article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assessment of growth of V. parahaemolyticus Alternate Protocol 1: Assessment of growth of V. parahaemolyticus using a plate reader Basic Protocol 2: Swimming/swarming motility assay Basic Protocol 3: Genetic manipulation Alternate Protocol 2: Natural transformation Basic Protocol 4: Secretion assay and sample preparation for mass spectrometry analysis Basic Protocol 5: Invasion assay (gentamicin protection assay) Basic Protocol 6: Immunofluorescence detection of intracellular V. parahaemolyticus Basic Protocol 7: Cytotoxicity assay for T3SS2.

Treatment Trial of Nile Tilapia (Oreochromis niloticus) Experimentally Infected with Vibrio alginolyticus Isolated from Sea bass (Dicentrarchus labrax).

BACKGROUND AND OBJECTIVE: In Egypt, Nile tilapia represents the main cultured type due to its economical price, palatability and easy culturing. This study was aimed to elucidate the pathogenicity of V. alginolyticus isolated from diseased sea bass and experimentally infected healthy Nile tilapia fish. MATERIALS AND METHODS: Healthy Nile tilapia fish were injected I/P with V. alginolyticus isolated from diseased sea bass. Symptoms and mortality rates of infected Nile tilapia fish were recorded during the experimental period. Re-isolation of V. alginolyticus was done from infected tilapia fish by bacteriological methods. For confirmation the pathogenicity of Vibrio isolated either from marine fish or tilapia fish, PCR test was done using tdh and bla gens. Liver and kidney function tests with histopathological examinations of some organs were performed. Treatment trial was done according to the antibiotic sensitivity test. RESULTS: The isolated Vibrio is highly pathogenic to Nile tilapia fish causing deterioration in all parameters which finished by severe mortalities. Treatment with florfenicol, enrofloxacin, or oxytetracycline reduced the mortality rate and improved liver and kidney function parameters of infected Nile tilapia fish. CONCLUSION: V. alginolyticus can infect both marine and fresh water fish inducing a high mortality rate. Treatment of infected fish with florfenicol, enrofloxacin, or oxytetracycline reduces the mortality rate.

Comprehensive genome based analysis of Vibrio parahaemolyticus for identifying novel drug and vaccine molecules: Subtractive proteomics and vaccinomics approach.

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.

Vibrio campbellii chitoporin: Thermostability study and implications for the development of therapeutic agents against Vibrio infections.

Vibrio campbellii (formerly Vibrio harveyi) is a bacterial pathogen that causes vibriosis, which devastates fisheries and aquaculture worldwide. V. campbellii expresses chitinolytic enzymes and chitin binding/transport proteins, which serve as excellent targets for antimicrobial agent development. We previously characterized VhChiP, a chitooligosaccharide-specific porin from the outer membrane of V. campbellii BAA-1116. This study employed far-UV circular dichroism and tryptophan fluorescence spectroscopy, together with single channel electrophysiology to demonstrate that the strong binding of chitoligosaccharides enhanced thermal stability of VhChiP. The alanine substitution of Trp136 at the center of the affinity sites caused a marked decrease in the binding affinity and decreased the thermal stability of VhChiP. Tryptophan fluorescence titrations over a range of temperatures showed greater free-energy changes on ligand binding (ΔG°binding) with increasing chain length of the chitooligosaccharides. Our findings suggest the possibility of designing stable channel-blockers, using sugar-based analogs that serve as antimicrobial agents, active against Vibrio infection.

Optimising efficacy of antibiotics against systemic infection by varying dosage quantities and times.

Mass production and use of antibiotics has led to the rise of resistant bacteria, a problem possibly exacerbated by inappropriate and non-optimal application. Antibiotic treatment often follows fixed-dose regimens, with a standard dose of antibiotic administered equally spaced in time. But are such fixed-dose regimens optimal or can alternative regimens be designed to increase efficacy? Yet, few mathematical models have aimed to identify optimal treatments based on biological data of infections inside a living host. In addition, assumptions to make the mathematical models analytically tractable limit the search space of possible treatment regimens (e.g. to fixed-dose treatments). Here, we aimed to address these limitations by using experiments in a Galleria mellonella (insect) model of bacterial infection to create a fully parametrised mathematical model of a systemic Vibrio infection. We successfully validated this model with biological experiments, including treatments unseen by the mathematical model. Then, by applying artificial intelligence, this model was used to determine optimal antibiotic dosage regimens to treat the host to maximise survival while minimising total antibiotic used. As expected, host survival increased as total quantity of antibiotic applied during the course of treatment increased. However, many of the optimal regimens tended to follow a large initial 'loading' dose followed by doses of incremental reductions in antibiotic quantity (dose 'tapering'). Moreover, application of the entire antibiotic in a single dose at the start of treatment was never optimal, except when the total quantity of antibiotic was very low. Importantly, the range of optimal regimens identified was broad enough to allow the antibiotic prescriber to choose a regimen based on additional criteria or preferences. Our findings demonstrate the utility of an insect host to model antibiotic therapies in vivo and the approach lays a foundation for future regimen optimisation for patient and societal benefits.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Bactericidal effect on skin mucosa of dietary guava (Psidium guajava L.) leaves in hybrid tilapia (Oreochromis niloticus × O. mossambicus).

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the intensification practices in global aquaculture, fish are often confined in small volumes, which can results in outbreak diseases. In this context, the use of antibiotics is very usual. Thus, looking for natural substance able to reduce the use of the antibiotics is imperative. Among them, there is a great interest at present in the study of medicinal plants such as guava (Psidium guajava L.). These plants could help to develop a more sustainable aquaculture all over the world. The application of guava in traditional medicine dates for centuries and it is widely used in tropical countries for the treatment of diseases in human and animals. AIM OF THE STUDY: The purpose of this work was to study the effects of the dietary administration of dried leaves of Psidium guajava on the skin mucosal immunity of hybrid tilapia (Oreochromis niloticus × O. mossambicus). Furthermore, the ability of this plant to inhibit the bacterial load in different tissues after an experimental infection with Vibrio harveyi was studied. MATERIALS AND METHODS: P. guajava leaves collection and the experimentation was carried out in Dominican Republic. Fish were fed with a commercial diet supplemented with guava leaf at different concentrations (0%, 1.5% and 3%) for 21 days before being intraperitoneally injected with V. harveyi (1 × 104 cells mL-1). Thereafter, several immune activities were measured in fish skin mucus and after 48 h of injection, the skin, spleen and liver were collected to analyse the bactericidal activity of guava leaf and the gene expression of some immune related genes. RESULTS: The administration of P. guajava leaves significantly modulated some immune-related enzymes (protease, antiprotease and peroxidase) in the skin mucus of hybrid tilapia. In addition, the bacterial load after V. harveyi infection in skin, spleen and liver significantly reduced in fish supplemented with guava leaves. Finally, the expression profile of hepcidin gene in skin and liver was modulated in fish feed with control diet after V. harveyi infection. CONCLUSION: These results demonstrate that the dietary intake of guava leaves increases the skin mucosal barrier defences of hybrid tilapia and confers protection against V. harveyi colonization.