Rôle des facteurs de virulence des Escherichia coli pathogènes aviaires dans la colibacillose

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Identification et caractérisation de gènes impliqués dans la virulence de Salmonella typhi suite à une analyse globale par biopuces de l'infection de macrophages humains en culture

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence.

The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30-60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.

Molecular Typing, Virulence Traits and Antimicrobial Resistance of Diabetic Foot Staphylococci

Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes.

Genomics and virulence factors of Fusobacterium necrophorum

Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.

Caractérisation fonctionnelle de facteurs de virulence chez "Streptococcus suis"

Les progrès technologiques dans l’industrie de la viande ont des répercussions considérables sur les agents pathogènes de ces environnements. Parmi ceux-ci, Streptococcus suis occupe une place prédominante dans l’industrie porcine. En effet, S. suis, colonisateur naturel des voies respiratoires et digestives du porc, peut infecter son hôte en provoquant des méningites, septicémies, endocardites, arthrites ou pneumonies. De surcroît, S. suis peut également infecter l’humain en provoquant majoritairement des méningites et septicémies, et a notamment été la cause de deux épidémies en Chine en 1998 et 2005. La pathogenèse des infections à S. suis demeure partiellement connue à l’heure actuelle, rendant difficile le contrôle des infections. Il est par conséquent essentiel de caractériser les facteurs de virulence chez S. suis puisqu’ils pourraient représenter des cibles d’intérêt pour des applications préventives ou thérapeutiques. Ce projet de doctorat consiste donc en la caractérisation fonctionnelle de facteurs de virulence chez S. suis. Dans un premier temps, la capacité de S. suis à moduler son potentiel pro-inflammatoire en présence de concentrations sous-inhibitrices d’amoxicilline a été mise en évidence. Dans un second temps, la caractérisation plus avancée de la hyaluronate lyase de S. suis a permis de démontrer que son activité ne contribue pas à la virulence de la bactérie étant donné son absence au sein de souches les plus virulentes, mais que les interactions avec l’acide hyaluronique pourraient moduler la virulence de S. suis. Par la suite, l’étude fonctionnelle d’une DNase de S. suis a permis de démontrer son implication comme facteur de virulence et suggère son intérêt dans le développement de vaccins. Finalement, le dernier objectif du projet a permis la mise en évidence de la production de microvésicules fortement immunogéniques par S. suis. La présence de facteurs de virulence dans leur contenu protéique représente un élément encourageant dans le développement de vaccins contre l’agent pathogène. Ce projet a donc permis d’élargir les connaissances sur le potentiel néfaste de l’utilisation des antibiotiques à faible concentration dans l’industrie porcine, sur le rôle des activités hyaluronate lyase et DNase dans la virulence de S. suis, et de découvrir un nouveau mécanisme impliqué dans la virulence de la bactérie par le biais des microvésicules.

Rice-Infecting Pseudomonas Genomes Are Highly Accessorized and Harbor Multiple Putative Virulence Mechanisms to Cause Sheath Brown Rot

Sheath rot complex and seed discoloration in rice involve a number of pathogenic bacteria that cannot be associated with distinctive symptoms. These pathogens can easily travel on asymptomatic seeds and therefore represent a threat to rice cropping systems. Among the rice-infecting Pseudomonas, P. fuscovaginae has been associated with sheath brown rot disease in several rice growing areas around the world. The appearance of a similar Pseudomonas population, which here we named P. fuscovaginae-like, represents a perfect opportunity to understand common genomic features that can explain the infection mechanism in rice. We showed that the novel population is indeed closely related to P. fuscovaginae. A comparative genomics approach on eight rice-infecting Pseudomonas revealed heterogeneous genomes and a high number of strain-specific genes. The genomes of P. fuscovaginae-like harbor four secretion systems (Type I, II, III, and VI) and other important pathogenicity machinery that could probably facilitate rice colonization. We identified 123 core secreted proteins, most of which have strong signatures of positive selection suggesting functional adaptation. Transcript accumulation of putative pathogenicity-related genes during rice colonization revealed a concerted virulence mechanism. The study suggests that rice-infecting Pseudomonas causing sheath brown rot are intrinsically diverse and maintain a variable set of metabolic capabilities as a potential strategy to occupy a range of environments.

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E. coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E. coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Study of Lomentospora prolificans pathogenicity: Host immune response and fungal virulence factors.

188 p.

Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence

Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilmgrowing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

2011

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.

Assessment of virulence factors characteristic of human Escherichia coli pathotypes and antimicrobial resistance in O157:H7 and non-O157:H7 isolates from livestock in Spain

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.