Vibrio alginolyticus PEPCK Mediates Florfenicol Resistance through Lysine Succinylation Modification.

Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.

Genome-wide expanding of genetic evolution and potential pathogenicity in Vibrio alginolyticus.

Vibrio alginolyticus, an emergent species of Vibrio genus, exists in aquatic and marine environments. It has undergone genetic diversification, but its detailed genomic diversity is still unclear. Here, we performed a multi-dimensional comparative genomic analysis to explore the population phylogeny, virulence-related genes and potential drug resistance genes of 184 V. alginolyticus isolates. Although genetic diversity is complex, we analysed the population structure using three sub-datasets, including the subdivision for three lineages into sublineages and the distribution of strains in the marine ecological niche. Accessory genes, most of which reclassified V. alginolyticus genomes as different but with relatively close affinities, were nonuniformly distributed among these isolates. We demonstrated that the spread of some post-evolutionary isolates (mainly L3 strains isolated from Chinese territorial seas) was likely to be closely related to human activities, whereas other more ancestral strains (strains in the L1 and L2) tended to be locally endemic and formed clonal complex groups. In terms of pathogenicity, the potential virulence factors were mainly associated with toxin, adherence, motility, chemotaxis, and the type III secretion system (T3SS). We also found five types of antibacterial drug resistance genes. The prevalence of ß-lactam resistance genes was 100%, which indicated that there may be a potential risk of natural resistance to ß-lactam drugs. Our study reveals insights into genomic characteristics, evolution and potential virulence-associated gene profiles of V. alginolyticus.

Roles of virulence regulator ToxR in viable but non-culturable formation by controlling reactive oxygen species resistance in pathogen Vibrio alginolyticus.

Under adverse circumstances, bacteria enter the viable but non-culturable (VBNC) state, a dormancy-like state for survival. The altered gene regulation underlying the entry of the VBNC state has not yet been well elucidated. Here, we reported that a subpopulation of cells (23.8 %) in Vibrio alginolyticus cultures enters the VBNC state in response to nutrient limitation at alkaline pH. The proteolysis of pivotal virulence regulator ToxR at these conditions is associated with VBNC formation. Meantime, ToxR abrogation impaired the mobility and the expression of virulence-associated genes, resulting in attenuated virulence in V. alginolyticus. RNA-seq and ChIP-seq analyses of the cells grown in VBNC-inducing conditions revealed that ToxR directly controls the expression of ∼8 genes including ahpC and dps involved in reactive oxygen species (ROS) resistance. ToxR binds to the promoter regions of kdgR, ppiC, ahpC, and dps and further controls their respective expression under oxidative stress conditions. The cells with impaired ToxR accumulated detrimental intracellular ROS. Moreover, these genes contribute to bacterial culturability as their in-frame deletion strains exhibiting severely decreased plate counts and the complementary strain showed rescued viability. Collectively, this study revealed the role of ToxR in switching on the VBNC state by sensing unfavorable environmental signals such as endogenous ROS (hydrogen peroxide, H2O2) in V. alginolyticus and provided mechanistic insights into Vibrio lifestyle adaptation in the marine environment.

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 µM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

Vanillic acid combats Vibrio alginolyticus by cell membrane damage and biofilm reduction.

Antibiotics are the most powerful weapon against bacterial infectious diseases in aquaculture. However, the indiscriminate usage of antibiotics often culminates in the emerging development of antibiotic-resistant bacteria, making it imperative to search for novel types of antimicrobial agents. This study investigated the antibacterial and antivirulence effects of vanillic acid (VA) against the fish pathogen, Vibrio alginolyticus. We showed that VA had a good anti-Vibrio activity with minimal inhibitory concentration (MIC) of 1.0 mg/ml. In addition, VA wielded its antibacterial action in a dose-/time-dependent manner by causing cell membrane damage and increasing membrane permeability, which is evidenced by increasing the conductivity and malondialdehyde content in the treated cell cultures and the scanning electron microscopy images. Furthermore, VA significantly reduced the biofilm-forming capability, mobility and exotoxin production (protease and exopolysaccharide) and downregulation of the expression of biofilm- and virulence-associated genes (sypG, fliS, fliK, lafA, lafK, asp and luxR) was seen in the V. alginolyticus that exposed to VA at subinhibitory concentrations. Overall, our findings suggested that VA may be of interest for treating V. alginolyticus-associated infections in aquaculture.

Synergy of alanine and gentamicin to reduce nitric oxide for elevating killing efficacy to antibiotic-resistant Vibrio alginolyticus.

The present study explored the cooperative effect of both alanine (Ala) and gentamicin (Gent) on metabolic mechanisms by which exogenous Ala potentiates Gent to kill antibiotic-resistant Vibrio alginolyticus. To test this, GC-MS-based metabolomics was used to characterize Ala-, Gent- and both-induced metabolic profiles, identifying nitric oxide (NO) production pathway as the most key clue to understand metabolic mechanisms. Gent, Ala and both led to low, lower and lowest activity of total nitric oxide synthase (tNOS) and level of NO, respectively. NOS promoter L-arginine and inhibitor NG-Monomethyl-L-arginine inhibited and promoted the killing, respectively, with the elevation and decrease of NOS activity and NO level. The present study further showed that CysJ is the enzyme-producing NO in V. alginolyticus. These results indicate that the cooperative effect of Ala and Gent causes the lowest NO, which plays a key role in Ala-potentiated Gent-mediated killing.

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.

Pectin of cacao pod husk, an efficient immunostimulant for white shrimp, Litopenaeus vannamei.

The disposal of cacao pod husk, a byproduct of cacao bean processing, can cause serious adverse environmental impacts, motivating scientist to explore and develop potential beneficial applications of this resource. Dried cacao pod husk was extracted with ethanol to obtain a 10.6% pectin of cacao pod husks (pCPH), and its effects on the immunocompetence of Litopenaeus vannamei were estimated. Measured variables included total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, as well as phagocytic activity and clearance efficiency against Vibrio alginolyticus after receiving pCPH at 0, 1.5, 3, and 6 µg shrimp-1 for 0, 1, 3 and 7 days via injection, and their resistance to thermal stress and V. alginolyticus infection were further evaluated. No significant differences were observed in total haemocyte count, differential haemocyte count, and respiratory bursts in shrimp receiving pCPH at 1.5 µg shrimp-1 for 1 day; however, these variables were significantly elevated after 3 days of injection, compared to the control group. The significantly increased phenoloxidase activity was assessed in shrimp receiving pCPH at 1.5, 3 and 6 µg shrimp-1 within 3 days, and activity returned to the baseline after 7 days. Furthermore, the reduced phenoloxidase activity per granulocytes or respiratory bursts per haemocytes maintained homeostasis following the variation of haemogram. For gene expression assessments in haemocytes, the immune-related genes of the lipopolysaccharide and ß-1,3-glucan binding protein, prophenoloxidase II and anti-lipopolysaccharide factor as well as innate immune signaling pathway-related genes of toll-like receptors 1 and 3 significantly increased after shrimp received pCPH for 1 day. The increases in phagocytic activity and clearance efficiency were only detected in shrimp receiving pCPH at 6 µg shrimp-1 within 7 days, compared to the control. There was no significant difference in the mortality ratio of shrimp against hyperthermal stress when they received pCPH for 1 day, and the significant higher resistance to hypothermal stress and V. alginolyticus infection were found in shrimp received pCPH at 6 µg shrimp-1 for 1 days than those in the other treatments. It is therefore found that pCPH triggers immune responses serving as an immunostimulant capable of enhancing resistance against V. alginolyticus and hypothermal stress.

Na+-NQR Confers Aminoglycoside Resistance via the Regulation of l-Alanine Metabolism.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

Comprehensive depiction of novel heavy metal tolerant and EPS producing bioluminescent Vibrio alginolyticus PBR1 and V. rotiferianus PBL1 confined from marine organisms.

The current study depicts the isolation of luminescent bacteria from fish and squid samples that were collected from Veraval fish harbour. From Indian mackerel, total 14 and from squid, total 23 bioluminescent bacteria were isolated using luminescence agar medium. Two bioluminescent bacteria with highest relative luminescence intensity PBR1 and PBL1 were selected. These two isolates were subjected to detailed biochemical characterization and were tested positive for 5 out of 13 biochemical tests. Furthermore, both PBR1 and PBL1 were able to ferment cellobiose, dextrose, fructose, galactose, maltose, mannose, sucrose and trehalose with acid production. Based on 16S rRNA partial gene sequence analysis, PBR1 was identified as Vibrio alginolyticus and PBL1 as V. rotiferianus. Antibiotic susceptibility test using paper-disc method showed that PBR1 and PBL1 were sensitive to chloramphenicol, ciprofloxacin, co-trimoxazole, gatifloxacin, levofloxacin, linezolid ad roxithromycin out of 18 antibiotics tested. Moreover, both strains were evaluated for their exopolysachharide (EPS) producing ability where PBR1 and PBL1 were able to yield 1.34 g% (w/v) and 2.45 g% (w/v) EPS respectively from 5 g% (v/v) sucrose concentration. Heavy metal toxicity assessment was carried out using agar well diffusion method with eight heavy metals and both the strains were sensitive to As(III), Cd(II), Ce(II), Cr(III), Cu(II), Hg(II) and while they showed resistance to Pb(II) and Sr(II). Based on these results, a study was conducted to demonstrate bio-removal of Pb and Sr by EPS of PBR1 and PBL1. Fourier transform infrared (FTIR) spectra revealed the functional groups of EPS involved in interaction with the heavy metals. Owing to the sensitivity for the remaining heavy metals, these bioluminescent bacteria can be used further for the development of luminescence-based biosensor.

Reduced ROS-mediated antibiotic resistance and its reverting by glucose in Vibrio alginolyticus.

Antibiotic-resistant Vibrio alginolyticus poses a big challenge to human health and food safety. It is urgently needed to understand the mechanisms underlying antibiotic resistance to develop effective approaches for the control. Here we explored the metabolic difference between gentamicin-resistant V. alginolyticus (VA-RGEN ) and gentamicin-sensitive V. alginolyticus (VA-S), and found that the reactive oxygen species (ROS) generation was altered. Compared with VA-S, the ROS content in VA-RGEN was reduced due to the decreased generation and increased breakdown of ROS. The decreased production of ROS was attributed to the decreased central carbon metabolism, which is associated with the resistance to gentamicin. As such a mechanism, we exogenously administrated VA-RGEN with the glucose that activated the central carbon metabolism and promoted the generation of ROS, but decreased the breakdown of ROS in VA-RGEN . The gentamicin-mediated killing was increased with the elevation of the ROS level by a synergistic effect between gentamicin and exogenous glucose. The synergistic effect was inhibited by thiourea, a scavenger of ROS. These results reveal a reduced ROS-mediated antibiotic resistance mechanism and its reversal by exogenous glucose.

Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Metabolic modulation of redox state confounds fish survival against Vibrio alginolyticus infection.

Vibrio alginolyticus threatens both humans and marine animals, but hosts respond to V. alginolyticus infection is not fully understood. Here, functional metabolomics was adopted to investigate the metabolic differences between the dying and surviving zebrafish upon V. alginolyticus infection. Tryptophan was identified as the most crucial metabolite, whose abundance was decreased in the dying group but increased in the survival group as compared to control group without infection. Concurrently, the dying zebrafish displayed excessive immune response and produced higher level of reactive oxygen species (ROS). Interestingly, exogenous tryptophan reverted dying rate through metabolome re-programming, thereby enhancing the survival from V. alginolyticus infection. It is preceded by the following mechanism: tryptophan fluxed into the glycolysis and tricarboxylic acid cycle (TCA cycle), promoted adenosine triphosphate (ATP) production and further increased the generation of NADPH. Meanwhile, tryptophan decreased NADPH oxidation. These together ameliorate ROS, key molecules in excessive immune response. This is further supported by the event that the inhibition of pyruvate metabolism and TCA cycle by inhibitors decreased D. reiro survival. Thus, our data indicate that tryptophan is a key metabolite for the host to fight against V. alginolyticus infection, representing an alternative strategy to treat bacterial infection in an antibiotic-independent way.

Bio-inspired antibacterial cellulose paper-poly(amidoxime) composite hydrogel for highly efficient uranium(vi) capture from seawater.

A bio-inspired cellulose paper-poly(amidoxime) composite hydrogel is explored via UV-polymerization. This hydrogel has a highly efficient uranium capture capacity of up to 6.21 mg g-1 for WU/Wdry gel and 12.9 mg g-1 for WU/Wpoly(amidoxime) in seawater for 6 weeks, due to its enhanced hydrophilicity, good hydraulic/ionic conductivity and broad-spectrum antibacterial performance.

Marine bacteria associated with shallow hydrothermal systems in the Gulf of California with the capacity to produce biofilm inhibiting compounds.

Shallow hydrothermal systems are extreme environments. The sediments and fluids emitted from the vents present unusual physical and chemical conditions compared to other marine areas, which promotes unique biodiversity that has been of great interest for biotechnology for some years. In this work, a bioprospective study was carried out to evaluate the capacity of bacteria associated with shallow hydrothermal vents to produce biofilm-inhibiting compounds. Degradation assays of N-acyl homoserine lactone (AHL) autoinducers (C6HSL) involved in the quorum sensing process were carried out on 161 strains of bacteria isolated from three shallow hydrothermal systems located in Baja California Sur (BCS), Mexico. The biosensor Chromobacterium violaceum CV026 was used. Twenty-three strains showed activity, and organic extracts were obtained with ethyl acetate. The potential of the extracts to inhibit the formation of biofilms was tested against two human pathogenic strains (Pseudomonas aeruginosa PAO1 and Aeromonas caviae ScH3), a shrimp pathogen (Vibrio parahaemolyticus M8), and two marine strains identified as producing biofilms on submerged surfaces (Virgibacillus sp C29 and Vibrio alginolyticus C96). The results showed that Vibrio alginolyticus and Brevibacillus thermoruber, as well as some thermotolerant strains (mostly Bacillus), produce compounds that inhibit bacterial biofilms (B. licheniformis, B. paralicheniformis, B. firmus, B. oceanizedimenis, B. aerius and B. sonorensis).

Engineering siderophores.

Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale.

Highly oxygenated polyketides produced by Trichoderma koningiopsis QA-3, an endophytic fungus obtained from the fresh roots of the medicinal plant Artemisia argyi.

Eight new highly oxygenated fungal polyketides, namely, 15-hydroxy-1,4,5,6-tetra-epi-koninginin G (1), 14-hydroxykoninginin E (2), koninginin U (3), 4'-hydroxykoninginin U (4), koninginin V (5), 14-ketokoninginin B (6), 14-hydroxykoninginin B (7), and 7-O-methylkoninginin B (8), together with six known related analogues (9-14), were isolated from Trichoderma koningiopsis QA-3, a fungus obtained from the inner root tissue of the well known medicinal plant Artemisia argyi. All these compounds are bicyclic polyketides, with compound 1 contains unusual hemiketal moiety at C-5 and compounds 2-14 having ketone group at C-1 and double bond at C-5(6). The structures and absolute configurations of the new compounds were established by spectroscopic analysis, X-ray crystal diffraction, modified Mosher's method, and ECD calculation. The absolute configurations of the known compounds 9, 10, and 12 were determined by X-ray crystal diffractions for the first time. The antimicrobial activities against human pathogen, marine-derived aquatic bacteria, and plant-pathogenic fungi of compounds 1-14 were evaluated, and compound 1 showed remarkable activity against aquatic pathogen Vibrio alginolyticus with MIC value 1 µg/mL, which is as active as that of the positive control.

Molecular Identification of Vibrio alginolyticus Causing Vibriosis in Shrimp and Its Herbal Remedy.

Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.

Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus.

Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus (VA-RGEN ) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing, indicating the metabolic shift in VA-RGEN. Thus, metabolic profile was investigated by GC-MS based metabolomics. Glucose was identified as a crucial biomarker, whose abundance was decreased in VA-RGEN . Further analysis with iPath, and gene expression and enzyme activity of the pyruvate cycle (the P cycle) demonstrated a global depressed metabolic pathway network in VA-RGEN . Consistently, NADH, sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) system, membrane potential and intracellular gentamicin were decreased in VA-RGEN . These findings indicate that the reduced redox state contributes to antibiotic resistance. Interestingly, exogenous glucose potentiated gentamicin to efficiently kill VA-RGEN through the promotion of the P cycle, NADH, membrane potential and intracellular gentamicin. The potentiation was further confirmed in a zebrafish model. These results indicate that the gentamicin resistance reduces the P cycle and Na(+)-NQR system and thereby decreases redox state, membrane potential and gentamicin uptake, which can be reversed by exogenous glucose.

Bioactive Compounds of Pseudoalteromonas sp. IBRL PD4.8 Inhibit Growth of Fouling Bacteria and Attenuate Biofilms of Vibrio alginolyticus FB3.

Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.