Characterization and Potentiating Effects of the Ethanolic Extracts of the Red Seaweed Gracillaria sp. on the Activity of Carbenicillin against Vibrios.

ß-lactam-resistant Vibrio strains are a significant clinical problem, and ß-lactamase inhibitors are generally coadministered with ß-lactam drugs to control drug-resistant bacteria. Seaweed is a rich source of natural bioactive compounds; however, their potential as ß-lactamase inhibitors against bacterial pathogens remains unknown. Herein, we evaluated the potential ß-lactamase inhibitory effect of the ethanolic extracts of the red seaweed Gracilaria sp. (GE) against four Vibrio strains. The minimum inhibitory concentration, half-maximal inhibitory concentration, checkerboard assay results, and time-kill study results indicate that GE has limited antibacterial activity but can potentiate the activity of the ß-lactam antibiotic carbenicillin against Vibrio parahemolyticus and V. cholerae. We overexpressed and purified recombinant metallo-ß-lactamase, VarG, from V. cholerae for in vitro studies and observed that adding GE reduced the carbenicillin and nitrocefin degradation by VarG by 20% and 60%, respectively. Angiotensin I-converting enzyme inhibition studies demonstrated that GE did not inhibit VarG via metal chelation. Toxicity assays indicated that GE exhibited mild toxicity against human cells. Through gas chromatography and mass spectrometry, we showed that GE comprises alkaloids, phenolic compounds, terpenoids, terpenes, and halogenated aromatic compounds. This study revealed that extracts of the red seaweed Gracillaria sp. can potentially inhibit ß-lactamase activity.

Phenolic Compound Ethyl 3,4-Dihydroxybenzoate Retards Drug Efflux and Potentiates Antibiotic Activity.

The World Health Organization indicated that antibiotic resistance is one of the greatest threats to health, food security, and development in the world. Drug resistance efflux pumps are essential for antibiotic resistance in bacteria. Here, we evaluated the plant phenolic compound ethyl 3,4-dihydroxybenzoate (EDHB) for its efflux pump inhibitory (EPI) activity against drug-resistant Escherichia coli. The half-maximal inhibitory concentration, modulation assays, and time-kill studies indicated that EDHB has limited antibacterial activity but can potentiate the activity of antibiotics for drug-resistant E. coli. Dye accumulation/efflux and MALDI-TOF studies showed that EDHB not only significantly increases dye accumulation and reduces dye efflux but also increases the extracellular amount of antibiotics in the drug-resistant E. coli, indicating its interference with substrate translocation via a bacterial efflux pump. Molecular docking analysis using AutoDock Vina indicated that EDHB putatively posed within the distal binding pocket of AcrB and in close interaction with the residues by H-bonds and hydrophobic contacts. Additionally, EDHB showed an elevated postantibiotic effect on drug-resistant E. coli. Our toxicity assays showed that EDHB did not change the bacterial membrane permeability and exhibited mild human cell toxicity. In summary, these findings indicate that EDHB could serve as a potential EPI for drug-resistant E. coli.

Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli.

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.

A Novel Cooperative Metallo-ß-Lactamase Fold Metallohydrolase from Pathogen Vibrio vulnificus Exhibits ß-Lactam Antibiotic-Degrading Activities.

Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with ß-lactamase activity, it is important to identify and characterize ß-lactamases with clinical significance. In this study, the protein sequence of the metallo-ß-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, ß-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against ß-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with kcat/Km values ranging from 6.23 × 102 to 1.02 × 104 M-1 s-1. The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.

Determination of Drug Efflux Pump Efficiency in Drug-Resistant Bacteria Using MALDI-TOF MS.

Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB's efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB's substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.

Secretory Production of Functional Grouper Type I Interferon from Epinephelus septemfasciatus in Escherichia coli and Bacillus subtilis.

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.

Brown and Red Seaweeds Serve as Potential Efflux Pump Inhibitors for Drug-Resistant Escherichia coli.

Multidrug-resistant pathogens are a significant clinical problem. Efflux pump inhibitors (EPIs) can restore the activities of existing antibiotics by interfering with drug efflux pumps located in bacterial cell membranes. Seaweeds are important sources of biologically active metabolites of natural origin; however, their potential as EPIs remains uninvestigated. Here, functional extracts from the brown seaweeds Laminaria japonica and Sargassum horneri and the red seaweeds Gracilaria sp. and Porphyra dentata were evaluated as potential EPIs against drug-resistant Escherichia coli. All these extracts were found to potentiate the activities of drugs in modulation tests, although not to the same extent. Synergistic effects of the extracts and the drug clarithromycin were observed from the onset of Time-kill assays, with no evidence of bacterial regrowth. Ethidium bromide accumulation studies revealed that the efflux decreased in the presence of each extract, as indicated by the presence of EPIs. Most identified EPIs that have been discovered to date have aromatic structures, and the seaweed extracts were found to contain various terpenes, terpenoids, phenolic compounds, indoles, pyrrole derivatives, alkaloids, and halogenated aromatic compounds. Our study highlights the potential of these compounds of the seaweeds as drug EPIs.

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

The Vibrio cholerae var regulon encodes a metallo-ß-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor.

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-ß-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have ß-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of ß-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by ß-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer ß-lactams that would prove more beneficial to the bacterium in light of current selective pressures.

Effects of Low-Molecular-Weight Fucoidan and High Stability Fucoxanthin on Glucose Homeostasis, Lipid Metabolism, and Liver Function in a Mouse Model of Type II Diabetes.

The combined effects of low-molecular-weight fucoidan (LMF) and fucoxanthin (Fx) in terms of antihyperglycemic, antihyperlipidemic, and hepatoprotective activities were investigated in a mouse model of type II diabetes. The intake of LMF, Fx, and LMF + Fx lowered the blood sugar and fasting blood sugar levels, and increased serum adiponectin levels. The significant decrease in urinary sugar was only observed in LMF + Fx supplementation. LMF and Fx had ameliorating effects on the hepatic tissue of db/db mice by increasing hepatic glycogen and antioxidative enzymes, and LMF was more effective than Fx at improving hepatic glucose metabolism. As for glucose and lipid metabolism in the adipose tissue, the expression of insulin receptor substrate (IRS)-1, glucose transporter (GLUT), peroxisome proliferator-activated receptor gamma (PPARγ), and uncoupling protein (UCP)-1 mRNAs in the adipose tissue of diabetic mice was significantly upregulated by Fx and LMF + Fx, and levels of inflammatory adipocytokines, such as adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were significantly modulated only by LMF + Fx supplementation. The efficacy of LMF + Fx supplementation on the decrease in urinary sugar and on glucose and lipid metabolism in the white adipose tissue of db/db mice was better than that of Fx or LMF alone, indicating the occurrence of a synergistic effect of LMF and Fx.

Low-molecular-weight fucoidan and high-stability fucoxanthin from brown seaweed exert prebiotics and anti-inflammatory activities in Caco-2 cells.

BACKGROUND: The aim of this study is to investigate the anti-inflammatory effects of low-molecular-weight fucoidan (LMF) and high-stability fucoxanthin (HS-Fucox) in a lipopolysaccharide-induced inflammatory Caco-2 cell line co-culture with B. lactis. METHODS: We used various methods such as transepithelial resistance (TER) assay, cytokine secretion assay, and tight junction protein mRNA expression assay to examine LMF and HS-Fucox anti-inflammatory properties. RESULTS: LMF and HS-Fucox activated probiotic growth and reduced the inflammation of the intestinal epithelial cells. Moreover, the combination of LMFHS-Fucox dramatically enhanced the intestinal epithelial barrier and immune function against the lipopolysaccharide effect by inhibiting IL-1ß and TNF-α and promoting IL-10 and IFN-γ. CONCLUSION: These findings suggested that LMF and HS-Fucox, alone or in combination, could be the potential natural compounds to enhance the immune system and have an anti-inflammatory effect on the intestinal cells.

Extracellular production of a novel endo-ß-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.

The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type ß-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.