A correlation between the virulence and the adhesion of Listeria monocytogenes to silicon nitride: An atomic force microscopy study

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium that is widely distributed in the environment. Despite being pathogenic at the species level, L. monocytogenes in fact comprises a diversity of strains from pathogenic ones that can result in disease and/or mortality to others that are relatively avirulent. The main goal of the current study was to answer the question on whether enhanced binding or attachment of L. monocytogenes to inert surfaces bears any relationship to pathogenicity in food-borne isolates. To answer this question, the nanoscale adhesion forces of eight L. monocytogenes strains that vary in their pathogenicity levels to a model surface of silicon nitride were quantified using atomic force microscopy. The strains used were the highly pathogenic (EGDe, 874, 1002, ATCC 19115), the intermediate pathogenic (ATCC 19112, ATCC 19118), and the non pathogenic (ATCC 15313 and HCC25). Our results indicate that the average nanoscale adhesion (in nN) and the 50% lethal dose (LD50) of strain virulence quantified in mice are logarithmically correlated according to: (nN) = −0.032 ln (LD50) + 1.040, r² = 0.96. Such correlation indicates that nanoscale adhesion could potentially be used as a design criterion to distinguish between virulent and avirulent L. monocytogenes strains. Finally, stronger adhesion of virulent strains to inert surfaces modeled by silicon nitride might be a way for pathogenic strains to survive better in the environment and thus increase their likelihood of infecting animals or humans.     

A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL⁻¹ and 13 CFU mL⁻¹ respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL⁻¹ and 25 CFU mL⁻¹, respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices.     

Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: Determination of Listeria monocytogenes in milk samples

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME GC–MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1–1.5 × 10² CFU ml⁻¹ of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.     

A xanthone and a polyketide derivative from the leaves of Cassia obtusifolia (Leguminosae)

A new xanthone, 1,8-dihydroxy-3-methoxy-6-methylxantone and a new polyketide derivative, (4R^∗,5S^∗,6E,8Z)-ethyl-4-((E)-but-1-enyl)-5-hydroxypentdeca-6,8-dienoate, together with 20 known secondary metabolites, including 2 steroids, 4 xanthones, 10 anthraquinones, 2 triterpenoids, 1 fatty ester, and (E)-eicos-14-enoic acid, were isolated from the leaves of Cassia obtusifolia. To the best of our knowledge, the last compound was isolated from a natural source for the first time. The structures of all the compounds were elucidated on the basis of 1D and 2D NMR experiments. Some of the compounds were tested against Salmonella typhi, Staphylococcus aureus, Candida albicans ATCC 9002, and Candida tropicalis, they did not show any activity.     

Euryjanicin A: a new cycloheptapeptide from the Caribbean marine sponge Prosuberites laughlini

A new proline-containing cycloheptapeptide, euryjanicin A (1), has been isolated from the marine sponge Prosuberites laughlini indigenous to Puerto Rico, and its structure established by an X-ray crystal structure determination. The absolute configuration of each amino acid residue was determined by Marfey’s method.     

Covalent selection of the thiol proteome on activated thiol sepharose: A robust tool for redox proteomics

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M_r thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.     

Capisterones A and B from the tropical green alga Penicillus capitatus: unexpected anti-fungal defenses targeting the marine pathogen Lindra thallasiae

The mechanism by which marine algae show resistance to pathogenic microorganisms remains poorly understood. To examine the possible role that algal secondary metabolites play in the prevention of infection, we examined the abundant green alga Penicillus capitatus, one of the major shallow water algae found in the Tropical Atlantic Ocean. Both aqueous and EtOAc extracts of this alga were found to be potent inhibitors of the well-known marine algal pathogen Lindra thallasiae. Using L. thallasiae in bioassay-guided fractionation, we isolated two new triterpene sulfate esters, capisterones A (1) and B (2). The capisterones are potent inhibitors of L. thallasiae at natural and below natural concentrations. The structures of the capisterones, with relative stereochemistry only, were assigned by comprehensive spectral analyses that relied heavily on 2D NMR methods.     

Sinutriangulin A, a novel diterpenoid from the soft coral Sinularia triangula

A novel compound, named sinutriangulin A (1), was isolated from the soft coral Sinularia triangula. The compound possesses a new carbon skeleton that is derived from the cubitane skeleton. The structure was determined by extensive spectroscopic analyses. In addition, compound 1 exhibited weak cytotoxicity toward human tumor cell lines CCRF-CEM and DLD-1.     

Rumphellaone A, a novel caryophyllane-related derivative from the gorgonian coral Rumphella antipathies

A novel 4,5-seco-caryophyllane sesquiterpenoid derivative, rumphellaone A (1), which was found to possess an unprecedented γ-lactone moiety, was isolated from the gorgonian coral Rumphella antipathies. The structure of 1 was elucidated by spectroscopic method. A plausible biosynthetic pathway of compound 1 was proposed.     

A crotofolane-type diterpenoid and a rearranged nor-crotofolane-type diterpenoid with a new skeleton from the stems of Croton cascarilloides

From the stems of Croton cascarilloides collected in the Okinawa Islands, a structurally rare crotofolane-type diterpenoid (1) and a rearranged nor-crotofalane, a new skeletal diterpenoid (2) were isolated. The structures were determined by X-ray crystallographic analyses, establishing their absolute stereostructures for the first time. Compound 2 was probably biosynthesized from 1 through several steps, such as decarboxylation, oxidation, C-C bond migration.     

The alkaloids of the mersinine group: a new subclass of the monoterpenoid indole alkaloids from Kopsia

A total of 16 alkaloids, exemplified by mersinine A and its congeners, and constituting a new subclass of the monoterpenoid indoles, were isolated exclusively and for the first time from Kopsia singapurensis. The structures of these alkaloids were established by spectroscopic methods and in some instances confirmed by X-ray diffraction analysis. A possible biogenetic route from an aspidofractinine precursor is proposed. Compounds 1, 2, 3, 9, and 10 were found to reverse multidrug-resistance in drug-resistant KB cells.     

A methodology for the determination of reductive sulphur in optical and technical glass

This paper describes an analytical method for the determination of reductive sulphur (S(IV), S(-II)) in glass. The glass sample is dissolved in hydrofluoric/hydrochloric acid mixture and the sulphur is separated via distillation in an apparatus made of polyfluoralkoxyethylene (PFA). The distilled hydrogen sulphide is trapped in buffered boric acid-zinc acetate solution and subsequently determined after conversion to an ethylene blue dye. The range of the method lies within a range of 2-1200 μg g⁻¹ reductive sulphur. The quantification limit for reductive sulphur is 2 μg g⁻¹.Different analysed glass types show either no detectable reductive sulphur or up to 30% of the total sulphur content reductive sulphur. The inter-laboratory standard deviation shown by a round robin test performed is excellent (±4 μg g⁻¹; average 59 μg g⁻¹). Sources of error of the methodology are discussed.     

Gloriosaols A and B, two novel phenolics from Yucca gloriosa: structural characterization and configurational assignment by a combined NMR-quantum mechanical strategy

Gloriosaols A (1) and B (2), two novel phenolic derivatives characterized by unusual spirostructures made up of two C₁₅ units linked via a γ-lactone to a central stilbenic portion were isolated from the roots of Yucca gloriosa. On the basis of an extensive NMR analysis, the same basic structure was established for the two compounds but no further information about their structural difference could be deduced. Thus two hypotheses were formulated: (1) gloriosaols A and B could be atropisomers caused by a restriction of the free rotation around the double bond due to a steric congestion of the bulky phenolic portions; (2) gloriosaols A and B could be two configurational isomers, indicating, in this case, a nonstereoselective biogenetic formation of the stereogenic center C-2. Semi-empirical calculations of the potential energy surfaces on gloriosaols A and B, together with the ¹H NMR spectra recorded at various temperatures, allowed us to unambiguously exclude the hypothesis of two restricted rotational conformers of a single configurational isomer. Finally, quantum mechanical calculations of the geometries and of the ¹H chemical shifts on the gloriasols A and B in combination with the analysis of the ROE data allowed us to deduce a diastereomeric relation between the two compounds and to assess the relative configuration of the two diastereomers.     

Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4

We describe the characterization of polyhydroxyalkanoate (PHA)-producing bacteria isolated from an ammunition-polluted soil in Kitakyushu City, Japan. Over 270 strains were evaluated for PHA accumulation based on a colony staining method using Nile red. Of these, nine strains were selected based on the intensity of Nile red fluorescence and the cells were quantitatively analyzed for PHA by gas chromatography. PHA accumulation was observed in five strains, all of which are inferred to be close to the Bacillus cereus group according to 16S rDNA sequence analysis. Interestingly, these strains produced a PHA copolymer, poly(3-hydroxybutyrae-co-3-hydroxyvalerate) [P(3HB-co-3HV)], with a 3HV fraction up to 2 mol% with glucose as a carbon source. Further characterization was performed on one isolate, B. cereus YB-4. Gel permeation chromatography analysis revealed that the number of average molecular weights of PHA accumulated in B. cereus YB-4 drastically changed from 722,000 to 85,000 over a 72-h cultivation period. Furthermore, the PHA synthase genes were cloned and the deduced amino acid sequences were determined. This study provides new insights into PHA biosynthesis by members of the B. cereus group.     

Rumphellclovane A, a novel clovane-related sesquiterpenoid from the gorgonian coral Rumphella antipathies

A novel clovane-related derivative, rumphellclovane A (1), which was found to possess a new carbon skeleton, and a new natural clovane, 2β-hydroxyclovan-9-one (2), were isolated from the gorgonian coral Rumphella antipathies. The structures of metabolites 1 and 2 were elucidated by spectroscopic analysis and by comparison of the spectral data with those of related clovane analogues. A plausible biosynthetic pathway of 1 was proposed.     

Natural product inhibitors of fatty acid biosynthesis: synthesis of the marine microbial metabolites pseudopyronines A and B and evaluation of their anti-infective activities

Total syntheses of the title natural products, pseudopyronines A (1) and B (2), have been achieved using methyl β-oxo carboxylic ester starting materials. The natural products and a small set of structurally related compounds were evaluated for growth inhibitory activity against a range of pathogenic microorganisms and were found to exhibit good potency (IC₅₀≥0.46 μg/mL) and selectivity towards Leishmania donovani. Several of the compounds inhibited recombinant fatty acid biosynthesis enzymes from both Plasmodium falciparum and Mycobacterium tuberculosis, validating these targets in the search for new anti-infective agents.     

Banyasin A and banyasides A and B, three novel modified peptides from a water bloom of the cyanobacterium Nostoc sp.

Three new modified peptides banyasin A, banyaside A and banyaside B were isolated from the hydrophilic extract of a natural bloom of the cyanobacterium Nostoc sp. The planar structure of the new compounds was determined by homonuclear and inverse-heteronuclear 2D NMR techniques as well as high-resolution mass spectrometry. Banyasides A and B, are structurally closely related to the cyanobacteria metabolite, suomilide and to the sponge derived, dysinosins. The absolute configuration of the asymmetric centers was studied using Marfey's method for HPLC. Banyaside A and B were found to be trypsin and thrombin inhibitors.     

Holophyllin A,a rearranged abietane-type diterpenoid from the trunk of Abies holophylla

Holophyllin A (1), a novel rearranged abietane-type diterpenoid was isolated, together with a new diterpene glycoside, holophyllin B (2), from the trunk of Abies holophylla. The structures of 1 and 2 were established by extensive spectroscopic analyses and their absolute configurations were determined by ECD calculation. All the isolates were tested for their inhibitory effects on NO production in LPS-activated murine microglial cells.     

Asymmetric synthesis of a diastereomer of the structure proposed for amphidinolide A and the determination of its absolute configuration

An asymmetric synthesis of a diastereomer (2) of the structure (1) proposed for amphidinolide A, a cytotoxic macrolide from the cultured dinoflagellate Amphidinium sp., has been accomplished. The absolute configuration of amphidinolide A was established as 3 from comparison of NMR data, HPLC analysis, and [α]_D values of amphidinolide A, and comparison with the synthetic diastereomers 2 and 3, the latter of which was synthesized previously by Trost's group.     

Saikachinoside A, a novel 3-prenylated isoguanine glucoside from seeds of Gleditsia japonica

A new isoguanine glucoside designated saikachinoside A has been isolated from the seeds of Gleditsia japonica. The structure was determined as 7-β-d-glucopyranosyl-3-[(Z)-4-hydroxy-3-methyl-2-butenyl]isoguanine by interpretation of the spectroscopic data and was confirmed by X-ray crystallographic analysis. Saikachinoside A accelerated acetylcholinesterase activity.