Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

<Emphasis Type="Italic">Coxiella burnetii</Emphasis> Nine Mile II proteins modulate gene expression of monocytic host cells during infection

Background  

Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

A candidate gene association study on muscat flavor in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>L.)

Background  

The sweet, floral flavor typical of Muscat varieties (Muscats), due to high levels of monoterpenoids (geraniol, linalool and nerol), is highly distinct and has been greatly appreciated both in table grapes and in wine since ancient times. Muscat flavor determination in grape (Vitis vinifera L.) has up to now been studied by evaluating monoterpenoid levels through QTL analysis. These studies have revealed co-localization of 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) with the major QTL positioned on chromosome 5.     

Exome sequencing identifies a novel missense variant in <Emphasis Type="Italic">RRM2B</Emphasis> associated with autosomal recessive progressive external ophthalmoplegia

Background  

Whole-exome sequencing using next-generation technologies has been previously demonstrated to be able to detect rare disease-causing variants. Progressive external ophthalmoplegia (PEO) is an inherited mitochondrial disease that follows either autosomal dominant or recessive forms of inheritance (adPEO or arPEO). AdPEO is a genetically heterogeneous disease and several genes, including POLG1 and C10orf2/Twinkle, have been identified as responsible genes. On the other hand, POLG1 was the only established gene causing arPEO with mitochondrial DNA deletions. We previously reported a case of PEO with unidentified genetic etiology. The patient was born of a first-cousin marriage. Therefore, the recessive form of inheritance was suspected.     

Genetic diversity of <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>strains isolated in northeastern Brazil as revealed by DNA sequencing,PCR-based analyses and molecular karyotyping

Background  

Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.     

Analysis of a <Emphasis Type="Italic">c</Emphasis><Subscript>0</Subscript><Emphasis Type="Italic">t-1</Emphasis> library enables the targeted identification of minisatellite and satellite families in <Emphasis Type="Italic">Beta vulgaris</Emphasis>

Background  

Repetitive DNA is a major fraction of eukaryotic genomes and occurs particularly often in plants. Currently, the sequencing of the sugar beet (Beta vulgaris) genome is under way and knowledge of repetitive DNA sequences is critical for the genome annotation. We generated a c ₀ t-1 library, representing highly to moderately repetitive sequences, for the characterization of the major B. vulgaris repeat families. While highly abundant satellites are well-described, minisatellites are only poorly investigated in plants. Therefore, we focused on the identification and characterization of these tandemly repeated sequences.     

An effective approach for identification of <Emphasis Type="Italic">in vivo</Emphasis> protein-DNA binding sites from paired-end ChIP-Seq data

Background  

ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites.     

Identification of a novel <Emphasis Type="Italic">Plasmopara halstedii</Emphasis> elicitor protein combining <Emphasis Type="Italic">de novo</Emphasis> peptide sequencing algorithms and RACE-PCR

Background  

Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).     

Evaluation of the <Emphasis Type="Italic">OPTC</Emphasis> gene in primary open angle glaucoma: functional significance of a silent change

Background  

We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients.     

Transcriptome analysis of the mobile genome ICE<Emphasis Type="Italic">clc</Emphasis> in <Emphasis Type="Italic">Pseudomonas knackmussii</Emphasis> B13

Background  

Integrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICEclc element of Pseudomonas knackmussii strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol.     

Ori-Finder: A web-based system for finding <Emphasis Type="Italic">oriC</Emphasis> s in unannotated bacterial genomes

Background  

Chromosomal replication is the central event in the bacterial cell cycle. Identification of replication origins (oriCs) is necessary for almost all newly sequenced bacterial genomes. Given the increasing pace of genome sequencing, the current available software for predicting oriCs, however, still leaves much to be desired. Therefore, the increasing availability of genome sequences calls for improved software to identify oriCs in newly sequenced and unannotated bacterial genomes.     

Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

Background  

Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing.     

Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> populations based on <Emphasis Type="Italic">spa</Emphasis> polymorphisms

Background  

For typing of Staphylococcus aureus, DNA sequencing of the repeat region of the protein A (spa) gene is a well established discriminatory method for outbreak investigations. Recently, it was hypothesized that this region also reflects long-term epidemiology. However, no automated and objective algorithm existed to cluster different repeat regions. In this study, the Based Upon Repeat Pattern (BURP) implementation that is a heuristic variant of the newly described EDSI algorithm was investigated to infer the clonal relatedness of different spa types.     

<Emphasis Type="Italic">Gen</Emphasis><Superscript><Emphasis Type="Italic">Htr</Emphasis></Superscript>: a tool for comparative assessment of genetic heterogeneity in microbial genomes generated by massive short-read sequencing

Background  

Microevolution is the study of short-term changes of alleles within a population and their effects on the phenotype of organisms. The result of the below-species-level evolution is heterogeneity, where populations consist of subpopulations with a large number of structural variations. Heterogeneity analysis is thus essential to our understanding of how selective and neutral forces shape bacterial populations over a short period of time. The Solexa Genome Analyzer, a next-generation sequencing platform, allows millions of short sequencing reads to be obtained with great accuracy, allowing for the ability to study the dynamics of the bacterial population at the whole genome level. The tool referred to as Gen ^Htr was developed for genome-wide heterogeneity analysis.     

Potential for <Emphasis Type="Italic">luxS</Emphasis> related signalling in marine bacteria and production of autoinducer-2 in the genus <Emphasis Type="Italic">Shewanella</Emphasis>

Background  

The autoinducer-2 (AI-2) group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat.     

<Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish sheep: confirmation by DNA sequencing

Background  

The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing.     

Comparison of homoeolocus organisation in paired BAC clones from white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and microcolinearity with model legume species

Background  

White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.