A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data.

The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.

Fungal Diversity and Composition of the Continental Solar Saltern in Añana Salt Valley (Spain).

The Añana Salt Valley in Spain is an active continental solar saltern formed 220 million years ago. To date, no fungal genomic studies of continental salterns have been published, although DNA metabarcoding has recently expanded researchers' ability to study microbial community structures. Accordingly, the aim of this present study was to evaluate fungal diversity using the internal transcribed spacer (ITS) metabarcoding at different locations along the saltern (springs, ponds, and groundwater) to describe the fungal community of this saline environment. A total of 380 fungal genera were detected. The ubiquity of Saccharomyces was observed in the saltern, although other halotolerant and halophilic fungi like Wallemia, Cladosporium, and Trimmatostroma were also detected. Most of the fungi observed in the saltern were saprotrophs. The fungal distribution appeared to be influenced by surrounding conditions, such as the plant and soil contact, cereal fields, and vineyards of this agricultural region.

Genotyping Study of Salmonella 4,[5],12:i:- Monophasic Variant of Serovar Typhimurium and Characterization of the Second-Phase Flagellar Deletion by Whole Genome Sequencing.

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

Altererythrobacter muriae sp. nov., isolated from hypersaline Añana Salt Valley spring water, a continental thalassohaline-type solar saltern.

A novel salt-tolerant alpha-proteobacterium, designated SALINAS58T, was isolated from Santa Engracia hypersaline spring water in the Añana Salt Valley, Álava, Spain. The isolate was Gram-negative, aerobic, non-motile, catalase-positive, oxidase-negative, rod-shaped and formed orange colonies on marine agar. Optimal growth was observed at pH 6.0-6.5, at 30 °C and in the presence of 1% (w/v) NaCl. The main cellular fatty acids (>20%) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The major respiratory quinone was ubiquinone Q-10 and the major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidilglycerol, four unidentified glycolipids and one unidentified phospholipid. Strain SALINAS58T had the highest 16S rRNA gene sequence similarity to Altererythrobacter marensis MSW-14T (96.6%), Altererythrobacter aquaemixtae JSSK-8T (96.5%) and Pontixanthobacter luteolus SW-109T (96.5%) followed by Altererythrobacter atlanticus 26DY36T (96.4%). Results of the phylogenetic analysis, based on 16S rRNA gene sequences, and phylogenetic approaches based on whole genome nucleotide differences, showed that strain SALINAS58T could be distinguished from recognized species of the genus Altererythrobacter. The genomic DNA G+C content was 61.4 mol%. Digital DNA-DNA hybridization, average nucleotide identity and average aminoacid identity values between the genome of strain SALINAS58T and A. marensis MSW-14T were 18.4, 73.1 and 68.1%, respectively. Based on data from this polyphasic characterization, strain SALINAS58T (=CECT 30029T=LMG 31726T) is considered to be classified as representing a novel species in the genus Altererythrobacter, for which the name Altererythrobacter muriae sp. nov. is proposed.

Virulotyping of Salmonella enterica serovar Typhi isolates from Pakistan: Absence of complete SPI-10 in Vi negative isolates.

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates.

Antimicrobial susceptibility and MLVA analysis of S. Typhimurium strains isolated from human and poultry samples in Tunisia.

INTRODUCTION: Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. METHODOLOGY: This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). RESULTS: About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson's diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. CONCLUSIONS: Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Draft Genome Sequence of an Oceanobacillus sp. Strain Isolated from Soil in a Burial Crypt.

We present the draft genome of an Oceanobacillus sp. strain isolated from spores found in soil samples from a burial crypt of the Cathedral of Sant'Antonio Abate in Castelsardo, Italy. The data obtained indicated the closest relation of the strain with Oceanobacillus caeni.

Online exercise for the design and simulation of PCR and PCR-RFLP experiments.

BACKGROUND: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) are extensively used molecular biology techniques. An exercise for the design and simulation of PCR and PCR-RFLP experiments will be a useful educational tool. FINDINGS: An online PCR and PCR-RFLP exercise has been create that requires users to find the target genes, compare them, design primers, search for restriction endonucleases, and finally to simulate the experiment. Each user of the service is randomly assigned a gene from Escherichia coli; to complete the exercise, users must design an experiment capable of distinguishing among E. coli strains. By applying the experimental procedure to all completely sequenced E. coli, a basic understanding of strain comparison and clustering can also be acquired. Comparison of results obtained in different experiments is also very instructive. CONCLUSIONS: The exercise is freely available at http://insilico.ehu.es/edu.

Intra- and inter-laboratory evaluation of an improved multiplex-PCR method for detection and typing of Salmonella.

INTRODUCTION: We developed and evaluated a multiplex-PCR method for rapid detection of the most common Salmonella serovars in both developed and developing countries. Additionally, the stability of the premixed reagents at high room temperature was studied. METHODOLOGY: Fifty-two Salmonella strains belonging to the collections of the University of Sassari, Italy, and to the University of the Basque Country, Spain, and a collection of a hundred blinded strains, were used to evaluate the multiplex-PCR. Primers targeting genes STY1599 and fliC were selected, and the method was evaluated both intra and inter-laboratories. RESULTS: The inter-laboratory reproducibility was 95.92%, with a kappa index of 0.757 that indicates a substantial agreement and a high accuracy (80.81%). The sensitivity, specificity, accuracy and precision indexes for the Salmonella genus and S. Typhi targets were maximum, although the targets for Paratyphi A, Typhimurium and Enteritidis showed less accuracy. During a seven-week period, hot-start multiplex-PCR runs were performed with reagents mixed with wax to test their stability at 30ºC, and no significant variation in the patterns of amplification was observed. CONCLUSIONS: An improved multiplex-PCR for rapid detection of the most common serovars of Salmonella operable in both developed and developing countries has been designed and tested intra and inter-laboratories. Following a careful optimization protocol will not only allow the confirmation of any suspicious colony by the amplification of the Salmonella genus target, but also the preliminary adscription to the prevalent serovars. Premixed reagents with wax facilitate the throughput and stability of reagents at high room temperatures.

What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis

Dos secciones incluyen los genes y moléculas relacionadas con la absorción de nutrientes, la señalización y las regulaciones metabólicas implicadas en la virulencia, incluyendo enzimas, como las serin-proteasas (alp/asp f 13, alp2 y asp f 18), metaloproteasas (mep/asp f 5, mepB y mep20), aspártico-proteasas (pep/asp f 10, pep2 y ctsD), dipeptidilpeptidasas (dppIV y dppV) y fosfolipasas (plb1-3 y fosfolipasa C); sideróforos y la adquisición de hierro (sidA-G sreA, ftrA, fetC, mirB-C y amcA); adquisición de zinc (zrfA-H, zafA, y pacC); biosíntesis de aminoácidos, absorción de nitrógeno, y regulación por Cross-pathway Control (areA, rhbA, mcsA, lysF, cpcA/gcn4p y cpcC/gcn2p); vías de biosíntesis generales (pyrG, hcsA, y pabaA) y biosíntesis de trehalosa (tpsA y tpsB); otras vías de regulación, como MAP quinasas (sakA/hogA, mpkA-C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2 y sho1), proteínas G (gpaA, sfaD y cpgA), AMPc-PKA (acyA, gpaB, pkaC1 y pkaR), histidin-quinasas (fos1 y tcsB), señalización de Ca2+(calA/cnaA, crzA, gprC y gprD), familia Ras (rasA, rasB y rhbA), y otros (ace2, medA, y srbA). Por último, también se comentan los efectos de los alérgenos de A. fumigatus (Asp f 1 a Asp f 34) en la AI. Los datos obtenidos generan un complejo rompecabezas, cuyas piezas serían factores de virulencia o diferentes actividades del hongo, que se deben reunir para obtener una visión conjunta de la virulencia de A. fumigatus. Los estudios de expresión mediante microarrays de ADN podrían ser útiles para entender esta compleja virulencia, y para detectar dianas para desarrollar métodos rápidos de diagnóstico y nuevos agentes antifúngicos. Aspergillus fumigatus es un patógeno oportunista que causa el 90% de las aspergilosis invasoras (AI) con un 50–95% de mortalidad. Se ha postulado la existencia de factores de virulencia característicos, pero en A. fumigatus existe una gran variabilidad de factores de virulencia «no clásicos». Todos los estudios han demostrado que la virulencia de este hongo es multifactorial, asociada a su estructura, su capacidad de crecimiento y adaptación a condiciones de estrés, sus mecanismos de evasión del sistema inmune y su capacidad de causar daños en un huésped. En esta revisión se pretende dar una visión general de los genes y moléculas que intervienen en el desarrollo de la AI. La sección de termotolerancia incluye cinco genes relacionados con la capacidad de que el hongo crezca a más de 30°C (thtA, cgrA, afpmt1, kre2/afmnt1 y hsp1/asp f 12). En las siguientes secciones se discuten las moléculas y los genes relacionados con la interacción con el huésped y con la respuesta inmune. Estas secciones incluyen el β-glucano, el α-glucano, la quitina, el galactomanano, galactomanoproteinas (afmp1/asp f 17 y afmp2), hidrofobinas (rodA/hyp1 y rodB), la DHN-melanina, sus respectivas enzimas sintasas (fks1, rho1-4, ags1-3, chsA-G, och1-4, mnn9, van1, anp1, glfA, pksP/alb1, arp1, arp2, abr1, abr2 y ayg1) y enzimas modificantes (gel1-7, bgt1, eng1, ecm33, afpigA, afpmt1-2, afpmt4, kre2/afmnt1, afmnt2-3, afcwh41 y pmi), varias enzimas relacionadas con la protección del estrés oxidativo como catalasas (catA, cat1/catB, cat2/katG, catC y catE), superóxido dismutasas (sod1-2, sod3/asp f 6 y sod4), oxigenasas de ácidos grasos (ppoA-C), glutatión transferasas (gstA-E) y otros (afyap1, skn7 y pes1), y los transportadores de moléculas (mdr1-4, atrF, abcA-E y msfA-E)...(AU)

Two sections cover genes and molecules related with nutrient uptake, signaling and metabolic regulations involved in virulence, including enzymes, such as serine proteases (alp/asp f 13, alp2, and asp f 18), metalloproteases (mep/asp f 5, mepB, and mep20), aspartic proteases (pep/asp f 10, pep2, and ctsD), dipeptidylpeptidases (dppIV and dppV), and phospholipases (plb1–3 and phospholipase C); siderophores and iron acquisition (sidA–G, sreA, ftrA, fetC, mirB–C, and amcA); zinc acquisition (zrfA–H, zafA, and pacC); amino acid biosynthesis, nitrogen uptake, and cross-pathways control (areA, rhbA, mcsA, lysF, cpcA/gcn4p, and cpcC/gcn2p); general biosynthetic pathway (pyrG, hcsA, and pabaA), trehalose biosynthesis (tpsA and tpsB), and other regulation pathways such as those of the MAP kinases (sakA/hogA, mpkA–C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2, and sho1), G-proteins (gpaA, sfaD, and cpgA), cAMP-PKA signaling (acyA, gpaB, pkaC1, and pkaR), His kinases (fos1 and tcsB), Ca2+ signaling (calA/cnaA, crzA, gprC and gprD), and Ras family (rasA, rasB, and rhbA), and others (ace2, medA, and srbA). Finally, we also comment on the effect of A. fumigatus allergens (Asp f 1–Asp f 34) on IA. The data gathered generate a complex puzzle, the pieces representing virulence factors or the different activities of the fungus, and these need to be arranged to obtain a comprehensive vision of the virulence of A. fumigatus. The most recent gene expression studies using DNA-microarrays may be help us to understand this complex virulence, and to detect targets to develop rapid diagnostic methods and new antifungal agents. Aspergillus fumigatus is an opportunistic pathogen that causes 90% of invasive aspergillosis (IA) due to Aspergillus genus, with a 50–95% mortality rate. It has been postulated that certain virulence factors are characteristic of A. fumigatus, but the “non-classical” virulence factors seem to be highly variable. Overall, published studies have demonstrated that the virulence of this fungus is multifactorial, associated with its structure, its capacity for growth and adaptation to stress conditions, its mechanisms for evading the immune system and its ability to cause damage to the host. In this review we intend to give a general overview of the genes and molecules involved in the development of IA. The thermotolerance section focuses on five genes related with the capacity of the fungus to grow at temperatures above 30°C (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12)... (AU)

Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant.

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID]=0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID=0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.

What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis.

Aspergillus fumigatus is an opportunistic pathogen that causes 90% of invasive aspergillosis (IA) due to Aspergillus genus, with a 50-95% mortality rate. It has been postulated that certain virulence factors are characteristic of A. fumigatus, but the "non-classical" virulence factors seem to be highly variable. Overall, published studies have demonstrated that the virulence of this fungus is multifactorial, associated with its structure, its capacity for growth and adaptation to stress conditions, its mechanisms for evading the immune system and its ability to cause damage to the host. In this review we intend to give a general overview of the genes and molecules involved in the development of IA. The thermotolerance section focuses on five genes related with the capacity of the fungus to grow at temperatures above 30°C (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12). The following sections discuss molecules and genes related to interaction with the host and with the immune responses. These sections include ß-glucan, α-glucan, chitin, galactomannan, galactomannoproteins (afmp1/asp f 17 and afmp2), hydrophobins (rodA/hyp1 and rodB), DHN-melanin, their respective synthases (fks1, rho1-4, ags1-3, chsA-G, och1-4, mnn9, van1, anp1, glfA, pksP/alb1, arp1, arp2, abr1, abr2, and ayg1), and modifying enzymes (gel1-7, bgt1, eng1, ecm33, afpigA, afpmt1-2, afpmt4, kre2/afmnt1, afmnt2-3, afcwh41 and pmi); several enzymes related to oxidative stress protection such as catalases (catA, cat1/catB, cat2/katG, catC, and catE), superoxide dismutases (sod1, sod2, sod3/asp f 6, and sod4), fatty acid oxygenases (ppoA-C), glutathione tranferases (gstA-E), and others (afyap1, skn7, and pes1); and efflux transporters (mdr1-4, atrF, abcA-E, and msfA-E). In addition, this review considers toxins and related genes, such as a diffusible toxic substance from conidia, gliotoxin (gliP and gliZ), mitogillin (res/mitF/asp f 1), hemolysin (aspHS), festuclavine and fumigaclavine A-C, fumitremorgin A-C, verruculogen, fumagillin, helvolic acid, aflatoxin B1 and G1, and laeA. Two sections cover genes and molecules related with nutrient uptake, signaling and metabolic regulations involved in virulence, including enzymes, such as serine proteases (alp/asp f 13, alp2, and asp f 18), metalloproteases (mep/asp f 5, mepB, and mep20), aspartic proteases (pep/asp f 10, pep2, and ctsD), dipeptidylpeptidases (dppIV and dppV), and phospholipases (plb1-3 and phospholipase C); siderophores and iron acquisition (sidA-G, sreA, ftrA, fetC, mirB-C, and amcA); zinc acquisition (zrfA-H, zafA, and pacC); amino acid biosynthesis, nitrogen uptake, and cross-pathways control (areA, rhbA, mcsA, lysF, cpcA/gcn4p, and cpcC/gcn2p); general biosynthetic pathway (pyrG, hcsA, and pabaA), trehalose biosynthesis (tpsA and tpsB), and other regulation pathways such as those of the MAP kinases (sakA/hogA, mpkA-C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2, and sho1), G-proteins (gpaA, sfaD, and cpgA), cAMP-PKA signaling (acyA, gpaB, pkaC1, and pkaR), His kinases (fos1 and tcsB), Ca(2+) signaling (calA/cnaA, crzA, gprC and gprD), and Ras family (rasA, rasB, and rhbA), and others (ace2, medA, and srbA). Finally, we also comment on the effect of A. fumigatus allergens (Asp f 1-Asp f 34) on IA. The data gathered generate a complex puzzle, the pieces representing virulence factors or the different activities of the fungus, and these need to be arranged to obtain a comprehensive vision of the virulence of A. fumigatus. The most recent gene expression studies using DNA-microarrays may be help us to understand this complex virulence, and to detect targets to develop rapid diagnostic methods and new antifungal agents.

Validation of double digest selective label database for sequenced prokaryotic genomes.

SUMMARY: A database for simulation of double digest selective label (DDSL) typing technique has been created and validated against a sequenced strain (Salmonella enterica serovar Typhimurium strain LT2). In silico bands were in agreement with experimental, and the technique was able to discriminate among strains belonging to the same species. When compared with other strain discrimination techniques, DDSL showed a higher discriminatory power. The database contains precomputed data which may be searched to retrieve experimental conditions for typing all up-to-dated sequenced prokaryotic microorganisms. AVAILABILITY: This is a new resource for molecular biology freely available on the Internet at http://insilico.ehu.es/DDSL.

Use of DNA microarray technology and gene expression profiles to investigate the pathogenesis, cell biology, antifungal susceptibility and diagnosis of Candida albicans.

The use of DNA microarrays is becoming the method of choice for assaying gene expression, particularly as costs and complexity are being reduced as the technology becomes more widespread and better standardized. A DNA array is nothing but a collection of probes fixed on a solid support. The probes can be PCR products of ORFs or short intragenic oligonucleotides deposited or synthesized in situ by photolithographic methods. To date, sequencing projects for fungal genomes have yielded 10 complete genomes and 21 whole shotgun sequences, including Candida albicans strain SC5314. Sequencing of the C. albicans genome has led to the construction of whole-genome DNA microarrays for in vitro transcription profiling by several universities and companies. The use of microarray or DNA chip techniques for Candida research has started recently but the number of studies using this technology is increasing rapidly, in order to address important remaining questions about pathogenesis, cell biology, antifungal susceptibility, and diagnosis.

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA.

UNLABELLED: We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting. AVAILABILITY: This free online tool is available at http://www.in-silico.com/DDF/.

Genes and molecules involved in Aspergillus fumigatus virulence.

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as beta-(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu, ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 degrees C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infec tion. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.

In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction.

UNLABELLED: We have developed a website, www.in-silico.com, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP-PCR) and endonuclease restriction. For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed. Plasmids of sequenced species may be included in the analysis. Theoretical AFLP-PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome. Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome. If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated. Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences. AVAILABILITY: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com

Colony variation in Candida glabrata isolates from patients with vaginitis.

The ability of Candida glabrata to switch between different colony phenotypes on a Sabouraud's-chloramphenicol agar supplemented with phloxine B was assessed in 14 C. glabrata isolates. Three phenotypes (pale pink smooth, dark pink smooth and fuchsia petite) were observed in vitro and two of them (pale pink smooth and dark pink smooth) in fresh clinical isolates plated directly from vaginal specimens from patients with C. glabrata vaginitis and patients colonized with C. glabrata. The pale pink smooth phenotype was the predominant and the remaining phenotypes can be derived from it. No changes in susceptibility to different antifungals were observed in the pale pink smooth, dark pink smooth, fuchsia petite phenotypes but they showed differences in cell wall antigens.