Aitor Rementeria

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

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.

Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of Salmonella in Human Clinical Samples

ABSTRACT We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:−. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohens kappa index.

Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities.

Kefir is a fermented milk beverage. The milk fermentation is achieved by the of kefir grains, a cluster of microorganisms held together by a polysaccharide matrix named kefiran. Kefir grains are an example of symbiosis between yeast and bacteria. They have been used over years to produce kefir, a fermented beverage that is consumed all over the world, although its origin is Caucasian. A vast variety of different species of organisms forming the kefir grains, comprising yeast and bacteria, have been isolated and identified. Kefir is a probiotic food. Probiotics have shown to be beneficial to health, being presently of great interest to the food industry. Kefir has been accredited with antibacterial, antifungal and antitumoural activities among other beneficial attributes. This review includes a critical revision of the microbiological composition of kefir along with its beneficial properties to human health.

DNA Microarray-Based Typing of an Atypical Monophasic Salmonella enterica Serovar

ABSTRACT A multidrug-resistant fljB-lacking Salmonella enterica serovar [4,5,12:i:−] emerged in Spain in 1997. We analyzed the genome from four strains of this serovar using a microarray containing almost all the predicted protein coding regions of serovar Typhimurium strain LT2, including the pSLT plasmid. Only a few differences from serovar Typhimurium LT2 were observed, suggesting the serovar to be Typhimurium as well. Six regions of interest were identified from the microarray data. Cluster I was a deletion of 13 genes, corresponding to part of the regulon responsible for the anaerobic assimilation of allantoin. Clusters II and IV were associated with the absence of the Fels-1 and Fels-2 prophage. Cluster III was a small group of Gifsy-1 prophage-related genes that appeared to be deleted or replaced. Cluster V was a deletion of 16 genes, including iroB and the operon fljAB, which is reflected in the serovar designation. Region VI was the gene STM2240, which appears to have an additional homologue in these strains. The regions spanning the deletions involving the allantoin operon and the fljAB operon were PCR amplified and sequenced. PCR across these regions may be an effective marker for this particular emergent serovar. While the microarray data for all isolates of the new serovar were essentially identical for all LT2 chromosomal genes, the isolates differed in their similarity to pSLT, consistent with the heterogeneity in plasmid content among isolates of the new serovar. Recent isolates have acquired a more-complete subset of homologues to this virulence plasmid. In general, microarrays can provide useful complementary data to other typing methods.

Genotypic characterisation by PFGE of Salmonella enterica serotype Enteritidis phage types 1, 4, 6, and 8 isolated from animal and human sources in three European countries

A total of 101 strains of Salmonella Enteritidis phage types (PT) 1, 4, 6, and 8 from Denmark, England and Spain were studied by PFGE to elucidate genetic relationships among strains isolated from animal, human and environmental sources between 1983 and 1997. Analysis with Xba I, Bln I and Spe I enzymes showed that the power of discrimination of this method was increased by the combination of the three enzymes (D=0.802), subdividing the strains into 28 genomic groups or genotypes. Many of the PT1, PT4, and PT6 strains from the three countries shared the same PFGE combination profile A1-A1-A1, confirming the close relationship among these phage types and the protracted spread of a single clone over a large geographical area. In general, strains from Denmark showed more variation in their PFGE profiles than those from England and Spain. PT4 strains exhibited genetic homogeneity in the three countries independently of their sources and period of isolation. Spe I gave the highest index of discrimination among PT6 strains as evidenced by a variety of PFGE profiles. The data clearly confirmed that PT8 strains isolated in the three countries were of a unique clonal origin, and the PFGE combination profile A10-A10-A1 was predominant and specific for this phage type. It is concluded that PFGE, in combination with phage typing, represents a suitable tool for the epidemiological typing of Salmonella Enteritidis strains which could be used for investigations or surveillance of the international spread of these clones.

Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains

Phage typing (PT) combined with pulsed-field gel electrophoresis (PFGE) and a random amplified polymorphic DNA (RAPD) fingerprinting method was used to characterize Salmonella enteritidis strains. Twenty-four epidemiologically unrelated isolates, sampled from diverse ecological niches and fifteen isolates from four well-defined outbreaks of foodborne gastroenteritis, were studied. Seven phage types, with a predominance of PT 4 (63% of isolates), were observed when analysing the epidemiologically unrelated group. PT 4 was detected in all of the ecological niches studied, including food and fecally polluted river and beach water. The discriminatory power for phage typing, the average probability that the typing system will assign a different type to two unrelated strains randomly sampled in the microbial population, was 0.62. Ten PFGE pattern types were obtained with Xba I restriction endonuclease enzyme among the unrelated isolates; thirteen isolates belonged to PFGE pattern type 1 and the rest of the PFGE types were assigned to one or two isolates. The Dice coefficient clustered the similarities of the PFGE patterns between 80-100%. PFGE showed a discriminatory power of 0.72. Five clearly distinct RAPD patterns were observed with the OPS-19 oligonucleotide, but the discrimination obtained was low (0.46). The combination of the three typing methods increased the number of types to seventeen, giving high discrimination (0.92). Seven of the isolates recovered from various ecological niches belonged to the combination PT 4/PFGE 1/RAPD A and other combinations were unique or included only two strains. The four epidemiologically well-defined foodborne outbreaks were associated with the PT 4 phage type. In two of the outbreaks, other phage types (PT 7a and RDNC) were also observed in two isolates. Most of the isolates belonging to the foodborne outbreaks had an identical PFGE pattern (PFGE pattern type 1), but a difference in a restriction band was observed in an isolate belonging to an outbreak. Two RAPD patterns were observed in the outbreaks; RAPD pattern type A was detected in three of the four outbreaks. When the combined typing method was applied to the study, high concordance was observed and most of the outbreak strains belonged to the combination PT 4/PFGE 1/RAPD A. It is concluded that the combination of phage type with PFGE and RAPD provides a powerful discriminatory tool for the epidemiological analysis of unrelated and related strains of S. enteritidis.

Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis

ABSTRACT Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D= 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI,BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance ofSalmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

A Fungicidal Monoclonal Antibody Protects against Murine Invasive Candidiasis

ABSTRACT Mice infected by Candida albicans and treated with monoclonal antibody C7 survived longer than saline-treated animals. A prozone-like effect was observed. The in vitro candidacidal activity of macrophages was strongly enhanced when C. albicans was opsonized by C7 and complete murine serum was present.

Detection of a Salmonella enterica Serovar California Strain Spreading in Spanish Feed Mills and Genetic Characterization with DNA Microarrays

ABSTRACT We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.