Isolation and Molecular Identification of Fungi with Magnesite Enrichment Potential from KÜMAŞ Quarries in Turkey

Abstract

Magnesite is an important raw material used in various industrial applications, especially the production of high-temperature resistant materials. Due to its high reactant nature, magnesite ore is not found in pure form and it contains a great variety of pollutants such as calcium compounds, which restrict its use when exceeding 1% of the ore. Thus, the development of efficient strategies for the removal of pollutants remains a crucial step for magnesite utilization. In this regard, our present work was conducted to isolate and identify active fungal strains that remove calcium pollutants without changing the main magnesium content of the ore. For this aim, magnesite ore samples were collected from two quarries (Turanoca?? and Ortaocak) of KÜMA? Magnesite Inc. and fungal isolation studies were done by using the ore’s flora. Active isolates were chosen according to their CaCO₃ and MgCO₃ dissolving capabilities and identified by using conventional light microscopy and molecular characterization techniques. 71 fungal isolates were obtained from the isolation step and 14 of them were chosen as active isolates that solve calcium compounds while not affecting the magnesium component. The data of the microscopic examination and 18S rDNA gene sequence analysis showed that 14 active strains with magnesite enrichment potential grouped in Aspergillus alliaceus (3), Aspergillus flavus (2), Aspergillus leporis (1), Aspergillus nomius (1), Fusarium tricinctum (2), Penicillium chrysogenum (1) and Penicillium sp. (4).     

Isolation and Identification of l-Menthyl-β-d-glucoside from Shubi

Quinoxalines derived from d-galactose with o-phenylenediamine (OPD) in acidic media under reflux were studied by using GLC and NMR measurements. Four quinoxaline derivatives were obtained from the reaction mixture, and were identical with those derived from d-glucose. The yields of 2-(D-lyxo-tetrahydroxybutyl)quinoxaline (GA-III), and the stereoisomeric derivative of GA-III, i.e., 2-(D-arabino-tetrahydroxybutyl)quinoxaline (ATBQ), were 13.2 and 5.3–, respectively. The ratio of GA-III to ATBQ derived from d-galactose was reciprocally coincident with that from d-glucose. Some proposals are made on the relationship between the isomerization of these sugars and the formation of quinoxaline derivatives.     

Isolation and Identification of α-Methyloctanylcarnitines from Human Urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron inpact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of δ⁶-octenylcarnitine, 2-methyloct-anylcarnitine and 2-methyl-δ⁶-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of α-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Isolation and Identification of Polycyclic Aromatic Hydrocarbon–Degrading Bacteria from Crude Oil Exploration Bore Well Sludge

Two bacterial strains capable of degrading polycyclic aromatic hydrocarbons were isolated from the crude oil exploration bore well sludge and identified by 16s rRNA gene sequencing as Pseudomonas stutzeri and Bacillus subtilis. The bacterial strains Pseudomonas stutzeri and Bacillus subtilis were able to degrade 95.1% and 99.4% of naphthalene (100 mg L^?1) and 99.5% and 94.6% of anthracene (50 mg L^?1), respectively, as a sole carbon and energy source in the liquid phase within a period of 6 days. The specific growth rate was determined for both the species and found to be 0.169 and 0.124 day^?1.     

Isolation and Identification of γ-l-Glutamyl-l-glutamic Acid from Tobacco Cells in Suspension Culture

The Maillard Reaction (MR) rate below the glass transition temperature (T_g) for various model glassy food systems was studied at temperatures between 40 °C and 70 °C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T_g was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD₂₈₀), and the MR rate (k₂₈₀) was determined as a pseudo zero order reaction rate from the time course of OD₂₈₀. Although k₂₈₀ was described by the Arrhenius plot, the temperature dependence of k₂₈₀ was almost the same and the intercept was different among the matrices. From the comparison of k₂₈₀, it was suggested that the MR rate in glassy matrix was affected not only by the T_g, but also by the hydrogen bonding between MR reactants and glassy matrix.     

Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K _m?=?0.355 mM [pNPG]; K _i?=?15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.     

Isolation and Identification of α-Ketoaldehydes in Calf and Rabbit Livers

Carbonyl compounds in calf liver stored at 4°C for 5~6 hr after slaughter were investigated, with emphasis on α-ketoaldehydes. After conversion of carbonyls into their 2,4-dinitrophenylhydrazones (2,4-DNPs), they were fractionated by preparative thin-layer chromatography (TLC); at least, twenty-three fractions were separated, among which eighteen fractions contained 2,4-DNPs of α-dicarbonyls. From some of the fractions, crystalline 2,4-DNPs were isolated, and identified by TLC and infrared spectra: pyruvaldehyde, 3-deoxypentosulose, xylosulose, 3-deoxyglucosulose, 2,3-diketogulonic acid or/and dehydroascorbic acid, and formaldehyde were identified, and glucosulose was tentatively identified.

The carbonyl pattern, obtained from rabbit liver frozen in liquid nitrogen immediately after slaughter, was also similar as that from calf liver, and further, the amount of each α-ketoaldehydes remained at the almost same level during storage at 4°C for 24 hr. These results indicate that the identified α-ketoaldehydes are constituents or metabolic intermediates in calf and rabbit livers.

The formation mechanisms of the carbonyls in tissues are discussed.     

Isolation and Identification of 5′-Methylthioadenosine Sulfoxide from Human Urine

Abstract

An adenine nucleoside isolated from human urine has been identified by mass spectra and other techniques as 5′-deoxy-5′-methyl-thioadenosine sulfoxide. Elevated levels (3–5 nmols/μmol creatinine) were noted in two children with severe combined immunodeficiency.     

Isolation of Phosphate Solubilizing Bacteria from Maize Rhizosphere and Their Potential for Rock Phosphate Solubilization–Mineralization and Plant Growth Promotion

Phosphate solubilizing bacteria (PSB) play a significant role in plant P nutrition by their effect on soil P dynamics and their subsequent ability to make P available to plants via solubilization and mineralization processes. This study aimed to evaluate the effect of separate and combined use of indigenous PSB, poultry manure (PM) and compost on solubilization and mineralization of rock phosphate (RP) and their subsequent effect on growth and P accumulation of maize (Zea mays L.). A group of fifty seven bacteria were isolated from the rhizosphere/rhizoplane of maize that had been grown in soils collected from varying altitudes (655–2,576 m) of the mountain region of Rawalakot, Azad Jammu and Kashmir, Pakistan. After screening, the capacity of eleven isolates to solubilize mineral phosphate was quantitatively evaluated using insoluble Ca₃(PO₄)₂ in culture medium as a time course study through spectrometer. The growth hormone producing (IAA) capacity of the isolates was also determined. Furthermore, five potential isolates were tested for their ability to increase P release capacity (mineralization) of insoluble RP in an incubation study. The effect of PSB inoculation on maize was determined in a completely randomized greenhouse experiment where root and shoot biomass and P accumulation in plants were assessed. The P solubilization index of selected isolates varied from 1.94 to 3.69, while the P solubilization efficiency ranged between 94.1% and 269.0%. The isolates MRS18 and MRS27 displayed the highest values. The P solubilization in the liquid medium was maximum at 6 and 9 days of incubation ranging between 9.91 and 44.04 µgmL^?1 and the isolates MRS27 and MRS34 exhibited the highest solubilization. Six isolates showed additional capability of producing IAA ranging between 2.66 and 28.41 µgmL^?1. Results of the incubation study indicated that P release capacity (P mineralization) of RP-amended soil varied between 6.0 and 11.8 µgPg^?1 that had been significantly increased to 30.6–36.3 µgPg^?1 (maximum value) when PSB were combined with RP. The combined application of PSB and organic amendments (PM, compost) with RP further increased P mineralization by releasing a maximum of 37.7 µgPg^?1 compared with separate application of RP (11.8 µgPg^?1) and organic amendments (21.5 and 16.5 µgPg^?1). The overall effect of PSB (as a group) with RP over RP alone on maize growth showing a relative increase in shoot length 21%, shoot fresh weight 42%, shoot dry weight 24%, root length 11%, root fresh weight 59%, root dry weight 35% and chlorophyll content 32%. This study clearly indicates that use of PSB, and organic amendments with insoluble RP could be a promising management strategy to enhance P availability in soil pool and improve plant growth in intensive cropping systems.     

Isolation and Identification of Tubaic Acid and β-Tubaic Acid from Derris Roots

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10^-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco plants.     

Isolation and Identification of Two β-Hydroxy-pyridine Derivatives from the Nondialyzable Melanoidin-hydrolyzate

The hydrolyzate of the melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was fractionated into the two fractions of non-adsorbate and adsorbate on Amberlite IR-120 (H⁺-form). In the present paper, the adsorbed fraction (Fraction B) was examined.

Paper chromatographic examination of the Fraction B indicated the presence of at least eight compounds positive to diazotized sulphanilic acid reagent. The two compounds of them (indicated orange and orange-yellow color) were isolated and identified as 2-methyl-5-hydroxy-pyridine and 2-hydroxymethyl-5-hydroxy-pyridine, respectively.

It is probable that these compounds would loosely be bound as a small moiety in the melanoidin molecule.     

Isolation and Identification of α-Spinasterol and α-Spinasteryl D-Glucoside from Sugar Beet Pulp

A sterol and a steryl glucoside were isolated from dried beet pulp. The sterol was identified with α-spinasterol, the glucoside possessed chemical and physical properties such as follows: The molecular formula C₃₅H₅₈O₆, m.p. 292°, [ α]¹⁹_D-34.1°, acetate; m.p. 168°, benzoate; m.p. 175-177°, and positive for Molish and Lieber-mann-Burchard reactions. When it was hydrolyzed with 1% sulfuric acid, the crystal of α-spinasterol and D-glucose detectable by paper chromatography were obtained. These results gave evidence that the glucoside was in question to be α-spinasteryl D-glucoside.     

Molecular Cloning and Expression of an α-Amylase Inhibitor from Rye with Potential for Controlling Insect Pests

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new -amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of -amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic -amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.     

Identification and Molecular Characterization of a Novel Type of α-galactosidase from Pyrococcus furiosus

An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.     

Isolation and Identification of (+)-β-Methyl Adipic Acid from “Shubi”

The response surface methodology (RSM) was used to optimize the extraction conditions for the acetylcholinesterase (AchE) inhibitory activity and extraction yield from Camellia japonica seed cake. Predicted values for AchE inhibition and extraction yield were 19.41 and 13.35%, respectively, which are in good agreement with the experimental values from validation, suggesting that RSM may provide a useful tool to optimization processes.     

Schistosomajaponicum： Isolation and Identification of Peptides Mimicking Ferritin Epitopes from Phage Display Library

Although schistosomicidal drug and other control measures (including hygiene and snail control) are availableschistosomiasis continues to afflict an estimated 200 million and kill 20 thousand people every year, new approachefor controlling this disease are urgently needed. Cocktail vaccine, such as multiple antigen peptidvaccine, emerged as the most potentially powerful meansFor multiple antigen peptide vaccine that bases on domainor subdomains of antigens, the key is to obtain antigeniepito…     

Isolation and Identification of (−)- Methylcitric Acid and 2-Methyl-cis-aconitic Acid from the Culture of Candida lipolytica

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc₂ (3.4%), Man₂FucGlcNAc₂ (2.1%), Man₃FucGlcNAc₂ (2.5%), Man₃FucXylGlcNAc₂ (42.5%), two isomers of Man₂FucXylGlcNAc₂ (39.1%), Man₃XylGlcNAc₂ (6.5%), and Man₂XylGlcNAc₂ (3.9%).     

Isolation of VanA-Type Vancomycin-Resistant Enterococcus Strains from Domestic Poultry Products with Enrichment by Incubation in Buffered Peptone Water at 42°C

Eight VanA-type enterococcal strains were isolated from 8 of 171 domestic poultry products by using enrichment by incubation in buffered peptone water at 35°C and 42°C. The pulsed-field gel electrophoresis patterns of all six VanA-type Enterococcus faecalis isolates were nearly indistinguishable, indicating the presence of a specific clone in Japan.The use of avoparcin as a growth promoter and prophylactic agent in feed has been associated with the occurrence of vancomycin-resistant enterococci (VRE) in farm animals in the European Union (EU) (2, 3, 4, 11). In Japan, avoparcin was also used as a food additive on animal farms for approximately 7 years until it was banned in March 1997 (10, 22). However, there has been no documentation in the literature on the isolation of VanA-type VRE from domestic poultry products in Japan (8, 10). Several traditional methods for detecting enterococci, including VRE, in poultry products include an enrichment step using buffered peptone water (BPW) (7, 12, 14, 21). Although BPW is typically incubated at 35 to 37°C, based on the reported optimal growth temperature for enterococci of 35°C (19, 20), the growth of VRE at this temperature can be inhibited by antagonistic activities of background microflora in samples contaminated with low levels of VRE, as 35 to 37°C is also the optimal growth temperature for most mesophilic microorganisms. Several investigators have demonstrated that incubation at an elevated temperature using selective enrichment medium was useful in the isolation of enterococcal strains from retail meat (9, 17, 18). To our knowledge, however, no study has evaluated cultivation temperature during the enrichment process in the isolation of VanA-type VRE from poultry products to examine the influence of background microorganisms on VanA-type VRE growth in BPW. In this study, we carried out isolation of VanA-type VRE from retail Japanese domestic chicken products by using two different enrichment temperatures (35°C and 42°C) and characterized VanA-type VRE isolates by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). We also assessed the effects of incubation temperature on the growth of VanA-type VRE under pure culture conditions, on the rates of recovery of inoculated VanA-type VRE strains from artificially contaminated poultry samples, and on the inhibition of suspected enterococcal strains by background microorganisms in poultry samples.A total of 171 retail domestic poultry products obtained in Osaka, Japan, between September 2008 and April 2009 were examined for the presence of VanA-type VRE. Twenty-five grams of each sample was removed aseptically and placed in a sterile plastic bag (30 by 19 cm), to which 225 ml of BPW (Eiken Chemical Co., Ltd., Tokyo, Japan) was added. The sample was then incubated for 24 h at either 35°C or 42°C. Ten microliters of the culture supernatants was spread plated on Enterococcosel agar (Becton, Dickinson and Company, Sparks, MD) plates containing 4 μg/ml vancomycin (Wako Pure Chemical Industries, Ltd., Hyogo, Japan) and incubated at 36°C for 48 h. Screening for the vanA gene was done using the colony-sweep PCR method with phosphate-buffered saline (PBS) washed-cell suspension, Z Taq polymerase (Takara Biochemicals, Shiga, Japan), and previously described primers (1, 16). PCR-positive sweeps were streaked for isolated VanA-type VRE colonies on an Enterococcosel agar plate containing 32 μg/ml vancomycin. The plates were incubated at 36°C for 48 h, and suspected VRE colonies were confirmed as enterococci by conventional biochemical methods (8). In addition, identification of Enterococcus faecalis and Enterococcus faecium harboring the vanA gene was performed by a multiple PCR assay previously described by Depardieu et al. (6). For the five VanA-positive poultry product samples obtained in October 2008 and April 2009, the number of VanA-type VRE was determined by plating 1-ml volumes of samples diluted 10-fold in sterile peptone saline onto Enterococcosel agar plates containing 32 μg/ml vancomycin. Colonies were counted after incubation at 36°C for 48 h. MICs of vanA-genotype VRE isolates to vancomycin and teicoplanin were determined using the Etest method (AB Biodisk, Solna, Sweden) in accordance with the criteria of the Clinical and Laboratory Standards Institute (5). Subtyping of VanA-type VRE isolates was performed by PFGE with SmaI-digested chromosomal DNA (8, 15). PFGE was performed on a 1.0% SeaKem Gold agarose gel (Cambrex Bio Science, Rockland, ME) and a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, CA). The PFGE standard strain Salmonella enterica serovar Braenderup H9812 was obtained from the PulseNet program, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, through the National Institute of Infectious Diseases (Japan).The two representative VanA-type VRE strains were precultured overnight in Trypticase soy broth (Becton, Dickinson and Company) at 36°C. Each culture medium was diluted in BPW to obtain 2 log CFU/ml, and 100 μl of the culture dilution was inoculated into 40 ml of brain heart infusion (BHI) broth (Becton, Dickinson and Company) in a 200-ml Erlenmeyer flask. The flask was incubated at 35°C or 42°C with constant shaking (160 rpm) for 24 h, and the optical density at 600 nm (OD₆₀₀) of the cultures was monitored every 30 min using an OD-Monitor A & S (Taitec Co., Ltd., Saitama, Japan). Each strain was examined in triplicate. All VanA-type isolates in this study were inoculated onto the poultry samples to compare the effect of BPW incubation at 42°C on the isolation of VanA-type VRE with the effect of incubation at 35°C. The strains were precultured at 36°C for 24 h under static conditions and then serially diluted in BPW to obtain a cell concentration of approximately 3 log CFU/ml. One hundred microliters of each bacterial cell suspension was individually inoculated onto two 25-g portions of each poultry sample in sterile plastic bags. Each inoculated poultry sample was added to 225 ml of BPW and homogenized for 1 min. One sample was incubated at 35°C for 24 h, and the second was incubated at 42°C for 24 h. Colony-sweep PCR analyses with Enterococcosel agar containing 4 μg/ml vancomycin for the presence of the vanA gene were then performed as described above. The artificially contaminated poultry sample experiment was performed in triplicate. To evaluate the success of cultivation using the different enrichment temperatures, data regarding the identification of the vanA gene from the colony-sweep PCR were analyzed using Fisher''s exact test. To examine the effect of enrichment temperature on inhibition of suspected enterococcal strains by background microflora, growth kinetics were compared among these microorganisms in three different poultry samples. For each sample, two 25-g portions were placed into sterile plastic bags to which 225 ml of BPW was added. After homogenization, the samples were incubated at 35°C and 42°C. Each culture was sampled every 3 h for the initial 15 h and after incubation for 24 h. The number of background microorganisms was determined by plating 1-ml volumes of samples serially diluted in PBS onto standard agar (Eiken) plates. Colonies were counted after incubation at 36°C for 24 h. The growth of enterococcal strains was monitored by plating 100-μl volumes of appropriate dilutions onto EF agar plates (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and characteristic enterococcal colonies were counted after incubation at 36°C for 48 h.Colony-sweep PCR analyses revealed that 8 of 171 domestic poultry samples were positive for the vanA gene. Isolation of VRE strains from the PCR-positive sweeps resulted in the identification of six vanA-genotype E. faecalis strains and two vanA-genotype E. faecium strains. Of the eight vanA-genotype VRE-positive samples, five were identified from only the 42°C BPW enrichment culture, while only a single isolate was uniquely identified from incubation at 35°C (Table ​(Table1).1). The number of VanA-type VRE was beneath the detection limit (i.e., less than 10 CFU/g) in each of the five VRE-positive poultry samples obtained in October 2008 and April 2009, suggesting that VanA-type VRE levels might be markedly low in domestic chickens in Japan. All vanA-genotype E. faecalis and E. faecium strains had vancomycin and teicoplanin MICs of >256 μg/ml and ≥32 μg/ml, respectively, confirming that these strains corresponded to the VanA phenotype (Table ​(Table1).1). Although the PFGE patterns of two VanA-type E. faecium strains were distinct (Fig. ​(Fig.1,1, lanes 7 and 8), the patterns of all E. faecalis isolates were almost indistinguishable (lanes 1 to 6). Given that the six VanA-type E. faecalis strains were isolated from distinct poultry samples that were obtained from 5 different retail shops, these results indicated that a specific E. faecalis clone harboring the vanA gene may be widely disseminated in Japan. The dominant species of VanA-type VRE isolated from poultry sources has been shown to differ depending on region. In New Zealand, greater than 80% of VRE isolates from broiler fecal samples were identified as vanA-genotype E. faecalis strains with very similar PFGE patterns, indicating that once established, a single, dominant clonal strain is capable of widespread contamination, and supporting the idea of clonal VRE dissemination rather than horizontal transfer of vancomycin resistance elements (13). Our findings also suggest that the epidemiological features of VanA-type VRE strains associated with poultry production environments in Japan may be similar to those observed in New Zealand. To our knowledge, this is the first report of the isolation of VanA-type VRE from retail domestic poultry products in Japan.Open in a separate windowFIG. 1.DNA fingerprinting of VanA-type VRE (E. faecalis and E. faecium strains) isolated from retail domestic poultry samples. Isolated genomic DNA of each isolate was digested with SmaI and subjected to PFGE. Lane 1, E. faecalis Ha8; lane 2, E. faecalis Ha16; lane 3, E. faecalis Ha36; lane 4, E. faecalis Ha55; lane 5, E. faecalis Ha58; lane 6, E. faecalis Ha61; lane 7, E. faecium Ha20; lane 8, E. faecium Ha26; lanes M, XbaI-digested PFGE patterns of DNA size standard Salmonella enterica serovar Braenderup H9812. Numbers to the left of the gel image indicate the molecular size in kilobase pairs.

TABLE 1.

Characteristics of vancomycin-resistant enterococcal strains isolated from domestic poultry samples in this study

Identification of Highly Potent α-Glucosidase Inhibitors from Artocarpus integer and Molecular Docking Studies

A new natural Diels-Alder adduct ( 3 ) was isolated from the leaves and stem bark of Artocarpus integer, along with seventeen known compounds ( 1 , 2 , and 4 – 18 ). Structural elucidation was conducted using NMR and HR-ESI-MS data, and comparisons were made with previous studies. Deoxyartonin I ( 3 ) exhibited the most potent α-glucosidase inhibition (IC₅₀ 7.80±0.1 μM), outperforming the acarbose positive control. This was mixed-mode inhibition, as indicated by the intersect in the second quadrant of each respective plot. An in silico molecular docking model and the pharmacokinetic features of 3 suggest that it is a potential inhibitor of enzyme α-glucosidase, and is therefore a lead candidate as a drug against diabetes mellitus.