Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.     

Mixotrophic production of phycoerythrin and exopolysaccharide by the microalga Porphyridium cruentum

The ability of the unicellular rhodophyte Porphyridium cruentum to grow mixotrophically on the soluble fraction of Solarium tuberosum meal was tested. At the beginning of stationary phase Porphyridium cruentum produced 7 μg ml⁻¹ of phycoerythrin and 129 μg ml⁻¹ of total soluble exopolysaccharide when cultured autotrophically. When cultured mixotrophically with the soluble fraction of Solanum tuberosum meal, the productivity increased to 10 μg ml⁻¹ of phycoerythrin and 330 μg ml⁻¹ of total soluble exopolysaccharide. When the soluble fraction of S. tuberosum meal was supplied together with nitrate and phosphate, the productivity of phycoerythrin increased to 21 μg ml⁻¹ while the production of total soluble exopolysaccharide decreased to 195 μg ml⁻¹. Results demonstrate that the soluble fraction of S. tuberosum meal can be used as substrate for the production of phycoerythrin and exopolysaccharide by P. cruentum improving the results obtained with the autotrophic culture medium.     

GABA/BABA priming: a means for enhancing abiotic stress tolerance potential of plants with less energy investments on defence cache

Abiotic stress is one of the major factors limiting plant growth and yield globally. Though substantial progress has been made in breeding and genetic manipulation of plants to enhance abiotic stress tolerance, the task remains as a challenge even today. Investigations on the priming activity of various chemicals in plants for enhancing abiotic stress tolerance have been undertaken over the past few years. Priming with γ-amino butyric acid (GABA) and β-amino butyric acid (BABA) gains greater attention, because priming with these non-protein amino acids equips the plants to resist abiotic stresses effectively without suffering costly energy investments in operating defence mechanisms. It is well documented that the protective effect of non-protein amino acids like BABA and GABA on plants is due to a potentiation of natural defence mechanisms against abiotic stresses but at the same time not activating the complete defence arsenal before the stress exposure. The exact mode of action of priming with GABA/BABA in plants is still a puzzle, though their importance as signaling molecules during stress is undoubtful. The better understanding of molecular, physiological, and ecological aspects of GABA/BABA priming might lead to the emergence of this technique as a successful strategy for enhancing the abiotic stress(es) tolerance potential of plants in the field, without compromising much on productivity.     

Herbicidal activity of volatile oils from Eucalyptus citriodora against Parthenium hysterophorus

The herbicidal effect of volatile oils from leaves of Eucalyptus citriodora against the noxious weed Parthenium hysterophorus was tested. In a laboratory bioassay, seed germination and seedling length, chlorophyll content and respiratory activity of Parthenium decreased with increased concentration of eucalypt oils from 0.2 to 5.0 nL mL^‐1. Germination was completely inhibited at 5.0 nL mL^‐1 eucalyptus oils. Further, for 4‐week‐old plants of Parthenium sprayed with different concentrations of volatile oils, visible damage increased and chlorophyll content and respiratory activity decreased with increased concentration from 0 to 100 μL mL^‐1, the week after spraying. At concentrations up to 50 μL mL^‐1, plants showed some recovery over time but plants sprayed with 75 and 100 μL mL^‐1 died 2 weeks after treatment. Plants sprayed with 50 μL mL^‐1 and higher concentrations of eucalypt oils were desiccated and wilted in appearance. At concentrations of 5–75 μL mL^‐1, eucalypt oils caused a rapid electrolyte leakage from the Parthenium plants thereby indicating an effect on membrane integrity. It is concluded that volatile oils from E. citriodora possess weed‐suppressing ability and could be used as a potential bioherbicide for future weed management programmes.     

Priming of the Arabidopsis pattern‐triggered immunity response upon infection by necrotrophic Pectobacterium carotovorum bacteria

Boosted responsiveness of plant cells to stress at the onset of pathogen‐ or chemically induced resistance is called priming. The chemical β‐aminobutyric acid (BABA) enhances Arabidopsis thaliana resistance to hemibiotrophic bacteria through the priming of the salicylic acid (SA) defence response. Whether BABA increases Arabidopsis resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) is not clear. In this work, we show that treatment with BABA protects Arabidopsis against the soft‐rot pathogen Pcc. BABA did not prime the expression of the jasmonate/ethylene‐responsive gene PLANT DEFENSIN 1.2 (PDF1.2), the up‐regulation of which is usually associated with resistance to necrotrophic pathogens. Expression of the SA marker gene PATHOGENESIS RELATED 1 (PR1) on Pcc infection was primed by BABA treatment, but SA‐defective mutants demonstrated a wild‐type level of BABA‐induced resistance against Pcc. BABA primed the expression of the pattern‐triggered immunity (PTI)‐responsive genes FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1 (FRK1), ARABIDOPSIS NON‐RACE SPECIFIC DISEASE RESISTANCE GENE (NDR1)/HAIRPIN‐INDUCED GENE (HIN1)‐LIKE 10 (NHL10) and CYTOCHROME P450, FAMILY 81 (CYP81F2) after inoculation with Pcc or after treatment with purified bacterial microbe‐associated molecular patterns, such as flg22 or elf26. PTI‐mediated callose deposition was also potentiated in BABA‐treated Arabidopsis, and BABA boosted Arabidopsis stomatal immunity to Pcc. BABA treatment primed the PTI response in the SA‐defective mutants SA induction deficient 2‐1 (sid2‐1) and phytoalexin deficient 4‐1 (pad4‐1). In addition, BABA priming was associated with open chromatin configurations in the promoter region of PTI marker genes. Our data indicate that BABA primes the PTI response upon necrotrophic bacterial infection and suggest a role for the PTI response in BABA‐induced resistance.     

Investigation of the antibacterial activity of 3-O-octanoyl-(-)-epicatechin

Aims: To measure antibacterial activity of the semi-synthetic flavonoid 3-O-octanoyl-(–)-epicatechin and investigate the mechanism of action. Methods and Results: MICs determined by the broth microdilution method were 50 μg ml⁻¹ for β-lactam sensitive and resistant Staphylococcus aureus, and 100 μg ml⁻¹ for vancomycin sensitive and resistant enterococci. In time-kill studies, 100 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin reduced colony forming unit numbers of antibiotic sensitive and methicillin-resistant Staph. aureus below detectable levels within 120 min. Bacterial aggregation was not observed when cells exposed to 3-O-octanoyl-(–)-epicatechin were examined by light microscopy. It was also shown that 50 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin is capable of reducing colony forming unit numbers of high cell density Staph. aureus populations by 80-fold within 60 min incubation, and inducing leakage of 50% of their internal potassium within just 10 min. Conclusions: 3-O-Octanoyl-(–)-epicatechin is active against Gram-positive bacteria, has bactericidal activity against both antibiotic sensitive and resistant strains, and is likely to exert its primary antibacterial effect by damaging the cytoplasmic membrane. Significance and Impact of the Study: 3-O-Octanoyl-(–)-epicatechin has significant antibacterial activity and additional structural modification and/or formulation studies may allow this to be potentiated.     

Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico

Aims: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Methods and Results: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed‐field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 μg ml^?1) and trimethoprim (MIC of >32 μg ml^?1); few were resistant to clindamycin (MIC₉₀ 4 μg ml^?1), erythromycin (MIC₉₀ 8 μg ml^?1) and tetracycline (MIC₉₀ 8 μg ml^?1); but none was resistant to azithromycin (MIC₉₀ 4 μg ml^?1), ciprofloxacin (MIC₉₀ 1 μg ml^?1) or gentamycin (MIC₉₀ 4 μg ml^?1). Most strains restricted with SmaI, but a combination of SmaI–KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST‐2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco‐2 cells, medium cytotoxicity with INT‐407 and Hep‐2 cells and high cytotoxicity with CHO cells. Conclusion: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. Significance and Impact of the Study: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.     

Elimination of apple stem grooving virus by chemotherapy and development of an immunocapture RT-PCR for rapid sensitive screening

Ombuin (7,4′-dimethyl quercetin) (10 μg ml^-1, for 12 wk), glycyrrhizin/quercetin (80 μg ml^-1and 10 μg ml^-1respectively, for 18 wk), ribavirin (10 μg ml^-1, for 12 wk) and quercetin/ribavirin (10 μg ml^-1each, for 9–12 wk) reduced the titre of apple stem grooving virus (ASGV) when applied in vitro to infected tissue cultures of Nicotiana occidentalis obliqua Wheeler, and/or Malus domestica. ASGV was not detectable in both plant species after the quercitin/ribavirin treatment when tested by ISEM, herbaceous host indexing, RT-PCR, and immunocapture RT-PCR. A sensitive immunocapture RT-PCR procedure for the detection of ASGV was developed for the screening of treated samples to assess antiviral activity.     

Bio-analytically fluorimetric method for estimation of ertapenem in real human plasma and commercial samples; application to pharmacokinetics study

Ertapenem (EPM) has been recently approved by the United States Food and Drug Administration (US-FDA) as an antimicrobial drug. EPM has a broad spectrum of action against different bacterial strains and is most commonly prescribed in Egypt for the treatment of Klebsiella pneumonia. In this study, EPM was estimated using a sensitive and selective spectrofluorimetric method for human plasma and pharmaceutical vials. The measured fluorescence (at 540 nm) was obtained from reaction of EPM with 0.05% w/v benzofurazan (NBD-Cl) using 0.1 M borate buffer pH 8.8 after excitation at 460 nm. The fluorometric linear range was stable from 10 to 350 ng ml⁻¹. The lower limit of detection and the lower limit of quantitation were found to be 2.13 and 6.47 ng ml⁻¹ respectively. Many factors such as pH, temperature, heating time, and NBD-Cl concentration were optimized. The presented work was validated according to International Council for Harmonisation guidelines and bio-analytically validated using FDA recommendations. The significant finding of this study, sensitivity, was successfully applied in Egypt for a pharmacokinetic application and commercial vials. Pharmacokinetic parameters were studied and the result, recorded as C_max of EPM, was found to be 83.60 μg ml⁻¹ after infusion of 0.5 g of Invanz® for 30 min. AUC_0-∞ was found to be 320 ± 30.2 μ.h ml⁻¹.     

Fluorescence detection of carbofuran in aqueous extracts based on dual-emission SiO2@Y2O3:(Eu3+,Tb3+)@MIP core-shell structural nanoparticles

A novel double-windows fluorescence sensor for carbofuran (CF) detection was successfully developed based on rare-earth Eu,Tb-doped Y₂O₃@SiO₂-based molecularly imprinted nanoparticles (MINs) with a multilayer core-shell structure. The recognition process of the MINs for CF was fairly fast and needed only ~8 min to reach a dynamic equilibrium. Interestingly, one fluorescence attenuation window was found with an increase in CF concentration (Q) from 0.1 to 10 μg ml⁻¹ and with a limit of detection (LOD) of 0.04 μg ml⁻¹ at 544 nm belonging to the Tb³⁺ emission, as well as another fluorescence enhanced window within the CF concentration range 10–100 μg ml⁻¹ (LOD = 4 μg ml⁻¹) at 617 nm of Eu³⁺ emission in the dispersed rare-earth-doped MIN colloidal aqueous solution. Luminescence resonance energy transfer from CF to Eu³⁺ and an inner filter effect of CF towards Tb³⁺, as well from the two independent detection windows were clearly observed simultaneously. The competition experiment displayed hardly any marked interference during detection of CF following addition of its analogues (carbaryl, isoprocarb, aldicarb, methomyl, and etofenprox). Moreover, the MINs could also be applied to accurately detect CF in rhubarb and wolfberry samples with recoveries of 85.7–92.2%. This sensing system has high specific recognition and a wide detection range for CF and provides new opportunities for pesticide detection.     

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences

Aims: The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). Methods and Results: The bactericidal effect of PHMG on E. coli was investigated based on β‐galactosidase activity assay, fluorescein‐5‐isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 μg ml^?1) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 μg ml^?1) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Conclusions: Dose‐dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 μg ml^?1) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. Significance and Impact of the Study: The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.     

Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high‐performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study

A direct fluorometric high‐performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100 μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and 4‐tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ‐H chiral column based on cellulose tris(4‐methylbenzoate) coated on 5 μm silica‐gel, 250 x 4.6 mm at 22 °C with a mobile phase composed of n‐hexane, 2‐propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1 ml min⁻¹. A fluorometric detector was set at: λ_excit. = 220 nm and λ_emis. = 290 nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within‐run and between‐run precision and accuracy. The LLOQ for the two enantiomers was 0.125 μg ml⁻¹ in plasma, 0.09 μg g⁻¹ in brain, and 0.25 μg g⁻¹ in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35 μg ml⁻¹ for plasma, 0.09–1.44 μg g⁻¹ for brain, and 0.25–20 μg g⁻¹ for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10 mg kg⁻¹ of racemate.     

Picolinic acid spray stimulates the antioxidative metabolism and minimizes impairments on photosynthesis on wheat leaves infected by Pyricularia oryzae

Fungal pathogens produce toxins that are important for their pathogenesis and/or aggressiveness towards their hosts. Picolinic acid (PA), a non‐host selective toxin, causes lesions on rice leaves resembling those originated from Pyricularia oryzae infection. Considering that non‐host selective toxins can be useful for plant diseases control, this study investigated whether the foliar spray with PA on wheat (Triticum aestivum L.) plants, in a non‐phytotoxic concentration, could increase their resistance to blast, stimulate the anti‐oxidative metabolism, and minimize alterations in photosynthesis. The PA spray at concentrations greater than 0.1 mg ml^?1 caused foliar lesions, compromised the photosynthesis and was linked with greater accumulation of hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂^??). Fungal mycelial growth, conidia production and germination decreased by PA at 0.3 mg ml^?1. Blast severity was significantly reduced by 59 and 23%, respectively, at 72 and 96 h after inoculation for plants sprayed with PA (0.1 mg ml^?1) at 24 h before fungal inoculation compared to non‐sprayed plants. Reduction on blast symptoms was linked with increases on ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.8.1.7), glutathione‐S‐transferase (EC 2.5.1.18), peroxidase (EC 1.11.1.7), and superoxide dismutase (EC 1.15.1.1) activities, lower H₂O₂ and O₂^?? accumulation, reduced malondialdehyde production as well as less impairments to the photosynthetic apparatus. A more efficient antioxidative metabolism that rapidly scavenges the reactive oxygen species generated during P. oryzae infection, without dramatically decreasing the photosynthetic performance, was a remarkable effect obtained with PA spray.