Species distribution of staphylococci from small wild mammals

A total of 197 isolates of Staphylococcus from small wild animals (insectivores and rodents) were identified by partial sequencing of the rpoB and dnaJ genes. Among the identified isolates the predominant species was S. succinus (28%), followed by S. xylosus (20.8%) and S. stepanovicii (18.3%). The other 14 Staphylococcus species were occasionally isolated. PCR-RFLP of the rpoB gene digested by Hpy8I was a fast and simple method to distinguish the two subspecies of S. succinus. More than 90% of the 55 S. succinus strains isolated belonged to S. succinus subsp. casei and only 9% to S. succinus subsp. succinus. Moreover, the present study describes the first ever isolation of S. fleurettii from healthy animals.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.     

Genetic characterization of Zostera asiatica on the Pacific Coast of North America

We gathered sequence information from the nuclear 5.8S rDNA gene and associated internal transcribed spacers, ITS-1 and ITS-2 (5.8S rDNA/ITS), and the chloroplast maturase K (matK) gene, from Zostera samples collected from subtidal habitats in Monterey and Santa Barbara (Isla Vista) bays, California, to test the hypothesis that these plants are conspecific with Z. asiatica Miki of Asia. Sequences from approximately 520 base pairs of the nuclear 5.8S rDNA/ITS obtained from the subtidal Monterey and Isla Vista Zostera samples were identical to homologous sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California. Similarly, sequences from the matK gene from the subtidal Zostera samples were identical to matK sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California, but differed from Z. asiatica sequences accessioned into GenBank. This suggests the subtidal plants are conspecific with Z. marina, not Z. asiatica. However, we found that herbarium samples accessioned into the Kyoto University Herbarium, determined to be Z. asiatica, yielded 5.8S rDNA/ITS sequences consistent with either Z. japonica, in two cases, or Z. marina, in one case. Similar results were observed for the chloroplast matK gene; we found haplotypes that were inconsistent with published matK sequences from Z. asiatica collected from Japan. These results underscore the need for closer examination of the relationship between Z. marina along the Pacific Coast of North America, and Z. asiatica of Asia, for the retention and verification of specimens examined in scientific studies, and for assessment of the usefulness of morphological characters in the determination of taxonomic relationships within Zosteraceae.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition

The concentration of phenolic compound was measured in the seagrass Posidonia oceanica when interacting with two Bryopsidophyceae, Caulerpa taxifolia and Caulerpa racemosa, between May 1999 and May 2000. These measurements were performed on adult and intermediate leaves and in sheaths of the seagrass. Sampling was carried out at three stations subject to increasing levels of interaction with Caulerpa. The number of tannin cells was also analysed. Five phenolic compounds were identified in P. oceanica, with a predominance of caffeic acid in the adult and intermediate leaves. For a given level of interaction (and for both caulerpa sp.), a significant seasonal variation in phenolic compounds was shown in the adult leaves (higher in November and lower in September and March for example for the interaction with C. taxifolia). Only for two compounds (corresponding to a mixture containing ferulic acid and the ester methyl 12-acetoxyricinoleate) were significant differences observed as a function of the level of interaction with C. taxifblia, and only in the adult leaves: higher concentrations of phenols were observed with increasing level of interaction. Thus,adult leaves gave values of 55.5 +/- 14.1 microg g(-1) dm without interaction (OCt) and 94.9 +/- 23.4 microg g(-l) dm with high interaction (2Ct),corresponding to an increase of 70%. No significant difference was observed with intermediate leaves and sheaths, or for interaction with C. racemosa. The number of tannin cells (supposed to produce the phenolic compounds) largely increased in the adult and intermediate leaves when the degree of interaction with C. taxifolia increased: 90 mm above the base of the sheath (in adult leaves), 16.7 +/- 10.6 tannin cells cm(-2) were found without interaction (OCt), and 57.8 + 21.2 tannin cells cm(-2) with high interaction (2Ct). No significant difference was found for C. racemosa interaction. It thus appears that when the seagrass P. oceanica is in interaction with C. taxifolia, it accelerates its production of secondary metabolites so as to limit invasion of the beds.     

Biology of Cryptosporidium from marsupial hosts

The majority of biological data on Cryptosporidium has been collected from humans and domestic animal hosts which creates a bias in knowledge on the biodiversity and evolution of this parasite genus. Further to understanding Cryptosporidium biology are studies encompassing broad hosts that represent diverse taxa sampled across wide geographic ranges. Marsupials represent a group of wildlife hosts from which limited information on Cryptosporidium is available. As marsupial hosts are an ancient mammalian lineage they represent an important group for studying parasite evolution. This review summarises information of the biology, epidemiology and evolution of Cryptosporidium in marsupial hosts, and discusses the importance of further understanding interactions in this parasite-host system.     

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.     

Dissection of lin-11 enhancer regions in Caenorhabditis elegans and other nematodes

The Caenorhabditis elegans LIM homeobox gene lin-11 plays crucial roles in the morphogenesis of the reproductive system and differentiation of several neurons. The expression of lin-11 in different tissues is regulated by enhancer regions located upstream as well as within lin-11 introns. These regions are functionally separable suggesting that multiple regulatory inputs operate to control the spatiotemporal pattern of lin-11 expression. To further dissect apart the nature of lin-11 regulation we focused on three Caenorhabditis species C. briggsae, C. remanei, and C. brenneri that are substantially diverged from C. elegans but share almost identical vulval morphology. We show that, in these species, the 5′ region of lin-11 possesses conserved sequences to activate lin-11 expression in the reproductive system. Analysis of the in vivo role of these sequences in C. elegans has led to the identification of three functionally distinct enhancers for the vulva, VC neurons, and uterine π lineage cells. We found that the π enhancer is regulated by FOS homolog FOS-1 and LIN-12/Notch pathway effectors, LAG-1 (Su(H)/CBF1 family) and EGL-43 (EVI1 family). These results indicate that multiple factors cooperate to regulate π-specific expression of lin-11 and together with other findings suggest that the mechanism of lin-11 regulation by LIN-12/Notch signaling is evolutionarily conserved in Caenorhabditis species. Our work demonstrates that 4-way comparison is a powerful tool to study conserved mechanisms of gene regulation in C. elegans and other nematodes.     

Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay

The ITS1 sequences for C. suis, C. belli, C. rivolta, C. felis, and C. ohioensis-like oocysts were determined and a diagnostic PCR-RFLP assay specific for Cystosisopora species was developed. Phylogenetic analysis of ITS1 sequences of Cystosisopora species along with ITS1 sequences for Toxoplasma, Neospora, Sarcocystis and Eimeria spp. using distance, minimum evolution and parsimony-based methods confirmed previous studies, which suggested that the genus Cystoisospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae.     

Biochemical activities of Iranian Mentha piperita L. and Myrtus communis L. essential oils

GC-MS analysis of essential oils of Iranian Mentha piperita and Myrtus communis extracted by hydrodistillation lead to identification of 26 and 32 compounds, respectively. The oils had good to excellent antimicrobial activities against Escherichia coli, Staphylococcus aureus and Candida albicans with the oil of M. piperita being more active. The findings suggest feasibility of application of M. piperita oil in treatment of the infections caused by C. albicans and E. coli. D-values on exposure to M. piperita and Myrtus communis oils were (2.14 and 2.8min), (1.4 and 12.8min) and (4.3 and 8.6min) for E. coli, S. aureus and C. albicans , respectively. The oils were screened for their possible antioxidant activities by two complementary test systems, namely DPPH free radical scavenging and beta-carotene/linoleic acid systems. M. piperirta oil exerted greater antioxidant activity than that of M. communis. Phytochemical and phytobiological characteristics of these oils may lead to extraction and production of active compounds in single or combined forms with useful applications.     

Identification of Vibrio spp. in vibriosis Penaeus vannamei using developed monoclonal antibodies

Immunohistochemical study using monoclonal antibodies specific to various shrimp viruses and Vibrio spp. was performed in shrimp samples died from unknown cause with symptoms of black stripes on lateral sides of cephalothorax or smoky body coloration. The positive results in muscular tissue were obtained with MAb VAL57 (specific to Vibrio spp.) and in hepatopancreas tissues with MAbs VVB158 (specific to V. vulnificus) and VPC701 (specific to V. parahaemolyticus). Twelve isolates of Vibrio spp. isolated from shrimp tissues were identified with various MAbs by dot blotting, biochemical tests and 16S rRNA gene. The results revealed three groups of V. vulnificus and one group of V. shilonii. All four groups of isolated Vibrio spp. were immunologically and biochemically different. None of the V. parahaemolyticus-like bacterium was isolated. The results demonstrated that the mortality in shrimp is accompanied by the presence of Vibrio spp.     

Comparative phytochemical analysis of four Mexican Nymphaea species

Four Mexican Nymphaea species, N. ampla, N. pulchella, N. gracilis and N. elegans belonging to subgenera Brachyceras were analyzed. In this work two 5-glycosyl isoflavones, 7,3',4'-trihydroxy-5-O-beta-D-(2'-acetyl)-xylopyranosylisoflavone (1) and 7,3',4'-trihydroxy-5-O-alpha-L-rhamnopyranosylisoflavone (2), were isolated from N. ampla and N. pulchella, respectively, together with other known 3-glycosyl flavones and triterpene saponins from the same four species. The structures were elucidated by 1D and 2D NMR, FABMS, and other spectroscopic analyses. These results confirmed that the four species were different from each other and established that N. pulchella represents a different taxa than N. ampla. In addition, the 5-glycosyl isoflavones could be considered as a taxonomic character of this group of plants.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Identification key for European strand-forming house-rot fungi

An identification key for 20 common strand-forming indoor wood decay fungi is given. The key is based on observations of material from affected buildings and on wood samples that have been incubated in the laboratory. The key is with macro- and microscopic photographs.     

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.