Analyse antigénique de la membrane externe de Legionella pneumophila serogroupes 1 à 8

Les protéines de la membrane externe de Leqionella pneumophila sérogroupes 1 à 8 ont été préparées, à partir d'un lysat bactérien, par solubilisation sélective au lauryl sarcosinate de sodium. Les protéines ainsi obtenues ont été séparées par électrophorèse sur gel de polyacrylamide en présence de dodécyl sulfate de sodium (SDS-PAGE) et transférées sur des feuilles de nitrocellulose. Des antisérums de lapin dirigés contre chacun des 8 sérogroupes de L. pneumophila ont été obtenus en immunisant chaque animal avec des bactéries vivantes. Les protéines tranfé- rées ont été révélées avec ces antisérums et des immunoglobulines de porc anti-immunoglobulines de lapins marquées à la peroxidase. Des déterminants antigéniques communs aux 8 sérogroupes ont été trouvés dans au moins 2 antigènes de la membrane externe (29 Kd et 45 Kd). Cependant, des expériences d'absorptions croisées ont révélé que ces antigènes auraient des déterminants antigéniques différents selon le sérogroupe. Ces résultats ne sont pas incompatibles avec ce que l'on connaît des protéines de la membrane externe des autres bactéries. Toutefois, certains indices nous laissent croire que notre préparation de protéines a pu être contaminée avec du lipopolysaccharide. Ceci expliquerait pourquoi les relations antigéniques observées avec les bandes de 29 et 45 Kd correspondent bien avec celles observées par immunofluorescence.Montréal Trigonix inc. 201

Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

BACKGROUND: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. RESULTS: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. CONCLUSION: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes

Gut metagenome profile of the Nunavik Inuit youth is distinct from industrial and non-industrial counterparts

Comparative metagenomics studies have highlighted differences in microbiome community structure among human populations over diverse lifestyles and environments. With their unique environmental and historical backgrounds, Nunavik Inuit have a distinctive gut microbiome with undocumented health-related implications. Using shotgun metagenomics, we explored the taxonomic and functional structure of the gut microbiome from 275 Nunavik Inuit ranging from 16 to 30-year-old. Whole-metagenome analyses revealed that Nunavik Inuit youths have a more diverse microbiome than their non-industrialized and industrialized counterparts. A comparison of k-mer content illustrated the uniqueness of the Nunavik gut microbiome. Short-chain fatty acids producing species, and carbohydrates degradation pathways dominated Inuit metagenomes. We identified a taxonomic and functional signature unique to the Nunavik gut microbiome contrasting with other populations using a random forest classifier. Here, we show that the Nunavik Inuit gut microbiome exhibits high diversity and a distinct community structure

Génomique et bioterrorisme

Les attaques terroristes récentes utilisant des spores de Bacillus anthracis ont révélé les lacunes des organismes de santé publique. Pourtant, l’utilisation des bactéries comme arme n’est pas nouvelle et il faudra développer notre capacité à y faire face. Il est important de mettre au point de nouvelles méthodes de diagnostic moléculaire afin d’identifier les bactéries en moins d’une heure. Notre laboratoire utilise la génomique pour créer des outils qui promettent de révolutionner la médecine.The use of biological weapons has been recorded throughout history. However, the anthrax-tainted letters of the fall of 2001 caused shock and panic in several countries. Knowledge of the principal bacteriological weapons allows design of novel rapid DNA-based diagnostic tests that should help defuse the impact of future bioterrorist attacks. Less than one-hour real-time PCR identification of bacteria and their associated antibiotic resistance genes will revolutionize the practice of medicine

Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences

Background: Streptococci are divided into six phylogenetic groups, i.e, anginosus, bovis, mitis, mutans, pyogenic, and salivarius, with the salivarius group consisting of only three distinct species. Two of these species, Streptococcus salivarius and Streptococcus vestibularis, are members of the normal human oral microflora whereas the third, Streptococcus thermophilus, is found in bovine milk. Given that S. salivarius and S. vestibularis share several physiological characteristics, in addition to inhabiting the same ecosystem, one would assume that they would be more closely related to each other than to S. thermophilus. However, the few phylogenetic trees published so far suggest that S. vestibularis is more closely related to S. thermophilus. To determine whether this phylogenetic relationship is genuine, we performed phylogenetic inferences derived from secA and secY, the general secretion housekeeping genes, recA, a gene from a separate genetic locus that encodes a major component of the homologous recombinational apparatus, and 16S rRNA-encoding gene sequences using other streptococcal species as outgroups. Results: The maximum likelihood (ML) and maximum parsimony (MP) phylogenetic inferences derived from the secA and recA gene sequences provided strong support for the S. vestibularis/S. thermophilus sister-relationship, whereas 16S rRNA-encoding and secY-based analyses could not discriminate between alternate topologies. Phylogenetic analyses derived from the concatenation of these sequences unambiguously supported the close affiliation of S. vestibularis and S. thermophilus. Conclusion: Our results corroborated the sister-relationship between S. vestibularis and S. thermophilus and the concomitant early divergence of S. salivarius at the base of the salivarius lineage.Botany, Department ofScience, Faculty ofNon UBCReviewedFacult

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention

Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs

Rapid Detection of Shiga Toxin-Producing Bacteria in Feces by Multiplex PCR with Molecular Beacons on the Smart Cycler

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes

Rapid Detection of Clostridium difficile in Feces by Real-Time PCR

Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile (n = 14), including Clostridium sordellii for which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdA and tcdB were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficile directly from feces samples

Modular ultrasonic Lysis system for rapid nucleic acid extraction and sample transfer of Bacillus spores

This paper describes the design, functioning and use of an ultrasonic modular system intended for rapid extraction and fragmentation of DNA from microbial organisms following sample collection in the field. PCR assessment of the DNA extracts revealed that the system can disrupt Bacillus atrophaeus spores, a simulant for Bacillus anthracis, in less than 1 min, providing a DNA yield equivalent to that of a commercial nucleic acid extraction method. Simulation of the transfer from a contaminated to a secure area confirmed that the sample remained confined within the module while the exterior surface can be decontaminated through immersion in a disinfectant solution.Peer reviewed: YesNRC publication: Ye