Effect of climate change on nature and human health with a special focus on infectious diseases in the Mediterranean region.

INTRODUCTION: In this short review, the effect of climate change on nature and human health with a special focus on infectious diseases in the Mediterranean region is discussed. This research is a part of the Mediterranean Convention of Human Rights project, which is an organizational work on human rights issues that was established in cooperation with civil society and the national authorities of the Mediterranean Region. METHODOLOGY: Previously published data were collected by retrieving published literature from PubMed, Google Scholar, and Web of Science using "climate change", "the Mediterranean region", "infections in Mediterranean Region", "infectious diseases", "biodiversity", and "the Mediterranean Sea" as keywords. The collected data were then evaluated and reviewed. The recommendations and guidelines were analysed by the preferred reporting items for systematic reviews and meta-analyses (PRISMA). CONCLUSIONS: The Mediterranean region presents a typical example witnessing a dramatic change in climate events and their adverse impact on biodiversity, ecosystems and public health are multiple. This negative impact is in part due to the geographical particularities, and sociocultural and geopolitical conflicts that are progressively worsening the burden of climate change. While most of these changes cannot be totally avoided, many of the health risks related to climate change could be monitored. This can be done by establishing health systems with policies to reduce and prevent the risks of infectious diseases and to recover and support the affected areas, which may identify priority and management of high-risk events.

Bacterial Genomics and Epidemiology.

Innovative technologies for Whole-Genome Sequencing (WGS) help to improve our understanding of the epidemiology and pathogenesis of bacterial infectious diseases and are becoming affordable for most microbiological laboratories [...].

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.

Molecular Epidemiology, Antimicrobial Surveillance, and PK/PD Analysis to Guide the Treatment of Neisseria gonorrhoeae Infections.

The aim of this study was to apply molecular epidemiology, antimicrobial surveillance, and PK/PD analysis to guide the antimicrobial treatment of gonococci infections in a region of the north of Spain. Antibiotic susceptibility testing was performed on all isolates (2017 to 2019, n = 202). A subset of 35 isolates intermediate or resistant to at least two antimicrobials were selected to search for resistance genes and genotyping through WGS. By Monte Carlo simulation, we estimated the probability of target attainment (PTA) and the cumulative fraction of response (CFR) of the antimicrobials used to treat gonorrhea, both indicative of the probability of treatment success. In total, 2.0%, 6.4%, 5.4%, and 48.2% of the isolates were resistant to ceftriaxone, cefixime, azithromycin, and ciprofloxacin, respectively. Twenty sequence types were identified. Detected mutations were related to antibiotic resistance. PK/PD analysis showed high probability of treatment success of the cephalosporins. In conclusion, multiple populations of N. gonorrhoeae were identified. We can confirm that ceftriaxone (even at the lowest dose: 250 mg) and oral cefixime are good candidates to treat gonorrhea. For patients allergic to cephalosporins, ciprofloxacin should be only used if the MIC is known and ≤0.125 mg/L; this antimicrobial is not recommended for empirical treatment.

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.

Aspergillus fumigatus transcriptome response to a higher temperature during the earliest steps of germination monitored using a new customized expression microarray.

Aspergillus fumigatus is considered to be the most prevalent airborne pathogenic fungus and can cause invasive diseases in immunocompromised patients. It is known that its virulence is multifactorial, although the mechanisms of pathogenicity remain unclear. With the aim of improving our understanding of these mechanisms, we designed a new expression microarray covering the entire genome of A. fumigatus. In this first study, we analysed the transcriptomes of this fungus at the first steps of germination after being grown at 24 and 37 °C. The microarray data revealed that 1249 genes were differentially expressed during growth at these two temperatures. According to our results, A. fumigatus modified significantly the expression of genes related to metabolism to adapt to new conditions. The high percentages of genes that encoded hypothetical or unclassified proteins differentially expressed implied that many as yet unknown genes were involved in the establishment of A. fumigatus infection. Furthermore, amongst the genes implicated in virulence upregulated at 37 °C on the microarray, we found those that encoded proteins mainly related to allergens (Asp F1, Asp F2 and MnSOD), gliotoxin biosynthesis (GliP and GliZ), nitrogen (NiiA and NiaD) or iron (HapX, SreA, SidD and SidC) metabolism. However, gene expression in iron and nitrogen metabolism might be influenced not only by heat shock, but also by the availability of nutrients in the medium, as shown by the addition of fresh medium.

Reprint of "Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)".

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray).

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

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.

The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR.

Invasive aspergillosis (IA) is a serious nosocomial infection caused by Aspergillus spp. which has a high mortality rate due to the fact, among other factors, that it is difficult to diagnose. Within the Aspergillus genus, A. fumigatus is the main species causing IA. We propose a virulence factor, the aspHS gene, as a novel target for the specific detection of A. fumigatus by quantitative real-time PCR (qPCR). This target gene encodes a haemolysin, which is overexpressed in vivo during infection. We have designed specific primers and hydrolysis (Taqman) probes for the detection of this target and a chimeric internal amplification control (IC), designed to detect false negative results due to PCR inhibition. This qPCR assay was tested with DNA extracted from a wide collection of microorganisms, tissues from infected mice, and human bronchoalveolar lavage (BAL) samples. Results showed that it, together with the DNA extraction method, could detect A. fumigatus with high specificity. Furthermore, it can distinguish between germinated (first step to the development of infection) and non-germinated conidia (not detected). Our data indicate that these techniques could be sufficiently sensitive and rapid to help clinicians establish an earlier diagnosis, but the presence of PCR inhibitors in clinical samples such as BAL fluids needs to be addressed.

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.