L’hidrosadénite suppurée (HS) ou maladie de Verneuil, est une maladie cutanée orpheline fréquente qui touche 1 % de la population générale. Elle se manifeste par des lésions inflammatoires récidivantes ou chroniques des plis axillaires, inguinaux et périnéaux. La sévérité clinique de la maladie varie selon les patients. Les lésions les moins sévères (lésions de stade 1 dans la classification de sévérité clinique de Hurley) sont des nodules inflammatoire centimétriques pouvant évoluer vers l’abcédation. Les lésions les plus sévères (lésions de stade 2 et 3 de Hurley) sont des lésions suppurées étendues et chroniques. Sur le plan histologique, la lésion primitive de l’HS semble être une hyperplasie de l’épithélium du follicule pileux. La physiopathologie de la maladie est mal connue et probablement multifactorielle, incluant des facteurs génétiques, hormonaux, infectieux et dys-immunitaires. Il a été montré récemment qu’une antibiothérapie à large spectre pouvait permettre d’obtenir une rémission clinique prolongée des lésions inflammatoires de l’HS. Objectif du travail : L’objectif principal de ce travail était d’identifier par des techniques de culture classique et de métagénomique bactérienne les espèces ou les flores bactériennes spécifiquement associées aux lésions d’HS des trois stades de sévérité clinique. Résultats : Nous avons identifié par culture bactérienne deux profils bactériens lésionnels. Le premier était représenté par Staphylococcus lugdunensis, et plus rarement par d’autres espèces bactériennes de la flore cutanée commensale (Propionibacterium acnes, staphylocoques à coagulase négative et Staphylococcus aureus). Le second correspondait à une flore anaérobie composée de bactéries anaérobies stricts, d’Actinomycetes et de streptocoques du groupe milleri. L’approche métagénomique a permis d’identifier les germes anaérobies stricts associés aux lésions : des cocci à Gram positif de la flore cutanée (principalement Anaerococcus spp., Peptoniphilus spp., Finegoldia spp.) des bacilles à Gram négatif anaérobies n’appartenant pas à la flore cutanée (Prevotella spp., Porphyromonas spp., Fusobacterium spp), Veillonellaceae et Corynebacteriaceae. Ce profil était caractéristique des lésions suppurées chroniques de stade 2 et 3 et était également associé à certaines lésions de stade 1. Les lésions des stades 2 et 3 présentaient une diversité bactérienne supérieure à celle des lésions de stade 1, avec un nombre plus élevé de taxons très minoritaires dans la flore cutanée (Fusobacteria, Bacteroidetes, Peptococcaceae, Erysipelotrichales). Conclusion : Cette étude démontre que certaines espèces bactériennes sont spécifiquement associées aux lésions d’HS. Ces espèces sont impliquées dans des infections cutanées, mais aussi dans des infections sévères, ce qui témoigne de leur pathogénicité. L’efficacité des antibiotiques chez les patients et les résultats de cette étude suggèrent qu’un processus infectieux participe à la présentation clinique de l’HS. Notre hypothèse est que ces infections surviennent en raison d’une anomalie primitive de la barrière cutanée folliculaire. / Hidradenitis suppuratiav (HS) is an orphan skin inflammatory disease disease characterized by chronic or recurrent inflammatory lesions localized in the armpits, the inguinal and perineal folds. With a 1% prevalence of a general population, HS is an public health issue. The clinical severity of the disease is heterogeneous among patients. Most patients present the mild form of the disease with inflammatory nodules and abscesses (Hurley stage 1 lesions). More severe patients show extended chronically suppurating lesions (Hurley stage 2 and Hurley stage 3 lesions). The primary histological lesion of HS is characterized by epidermal follicular hyperplasia and perifollicular inflammation. The physiopathology of HS remains unclear. HS is probably a multifactorial disease, involving genetical, immunological and infectious factors. Indeed, wide-spectrum antibiotic treatments can significantly improve or induce prolonged clinical remissions of HS inflammatory lesions. Objective: The main objective of this work was to identify the bacterial species or flora specifically associated with Hurley stage 1, 2 and 3, using prolonged aerobic and anaerobic culture and bacterial metagenomics (454 sequencing of 16Sr DNA libraries). Results. Using bacterial culture, we identified two bacterial profiles associated with HS lesions . The first one was represented by Staphylococcus lugdunensis and rarely by other skin commensals (Propionibacterium acnes, coagulase negative staphylococci and Staphylococcus aureus). Results. The second one corresponded to a mixted anaerobic flora including strict anaerobes, Actinomycetes and milleri group streptococci. The metagenomic approach allowed to identify the anaerobic flora associated with lesions : Gram positive cocci from the cutaneous flora (mainly Anaerococcus spp., Peptoniphilus spp., Finegoldia spp.) and Gram negative rods which do not belong to the cutaneous microbiota (Prevotella spp., Porphyromonas spp., Fusobacterium spp), Veillonellaceae and Corynebacteriaceae. This profile was typically associated with Hurley stage 2 and 3 lesions but was also observed in Hurley stage 1 lesions. Hurley stage 2 and 3 lesions presented an increased bacterial diversity as compared to Hurley stage 1 lesions, with a higher number of taxa taxa rarely associated with normal skin microbiota (Fusobacteria, Bacteroidetes, Peptococcaceae, Erysipelotrichales). Conclusion. This study demonstrate that particular bacterial species are specifically associated with HS lesions. These species are cause soft tissue and skin infections, but also in severe infections arguing for their pathogenicity. These data provide a rationale for antibiotic use in HS, and suggests that the disease may be due to a primitive immune defect of the follicular skin barrier.

The Streptococcus Anginosus/Milleri Group (SMG) is made up of three closely related but distinct bacterial species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. The SMG are recovered from about one-third of healthy, asymptomatic individuals. Despite this, the SMG cause more incidences of invasive streptococcal disease than Group A and Group B Streptococcus combined. Members of this group are somehow able to live a dual lifestyle. Little work has been conducted on the molecular pathogenicity of the SMG and host factors that contribute to host susceptibility to this group have been under-investigated. My research works towards discovering how the host recognizes the SMG as well as what enables the SMG to evade clearance by the immune system. I hypothesize that: 1) recognition of the SMG by toll-like receptor 2 (TLR2) plays a key role in triggering a cytokine response by the innate immune branch (which coordinates the immune response to the SMG), 2) the expression of cytolysins and extracellular polysaccharides by members of the SMG enables evasion of innate immune recognition and cytokine responses. hTLR2 reporter and monocyte-like cell lines as well as human blood samples from healthy donors were used to investigate the host factors that contribute to SMG infection. Five clinical reference SMG strains and a transposon mutant library were used to probe the contributing bacterial factors. It was found that TLR2 activation plays an important role in the cytokine response to the SMG, but there is heterogeneity between strains in their ability to activate TLR2. It was also found that intermedilysin expression by S. intermedius strains enables evasion of recognition; however, different hosts display varying susceptibility to this cytolysin. This study reveals that investigation of both host and microbial factors is essential to build an understanding of the mechanisms of SMG transition from commensalism to pathogenicity. / Thesis / Master of Science (MSc) / The Streptococcus Miller/Anginosus Group (SMG) is a group of bacteria comprised of three species. Members of this group are recovered from roughly one-third of healthy individuals. However, the SMG are also found in samples collected from patients with invasive disease. It is not well understood why some human-SMG relationships are pathogenic and others are not. However, it is likely that the combination of both human and SMG factors determine the nature of the relationship formed between the two. In this study, the human and SMG factors that contribute to infection were investigated. The ways by which human cells recognize members of the SMG and defend themselves from damage was explored. Additionally, SMG factors that potentially contribute to infection were probed to discover their effect on human cells. By investigating both the bacterial and host factors that lead to infection, disease treatments and preventative strategies can be tailored to individual cases.

<p>The Streptococcus Milleri/Anginosus group (SMG) includes Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius. The SMG is found in healthy individuals but these bacteria are most known clinically for being associated with invasive disease and more recently, airway infections including cystic fibrosis (CF). The SMG like many other streptococci are naturally competent, being able to actively bind, uptake and integrate extracellular DNA. Competence regulation involves a competence-stimulating peptide (CSP) derived from the ComC precursor and a two- component signaling system (a histidine kinase ComD and its response regulator ComE). In this study, I examined the distribution of CSP/ComD sequences and competence in 170 SMG clinical isolates from CF airways and invasive disease. Five predicted CSP sequences were observed; one represented a newly predicted CSP and two arose from frameshift mutations in comC and appeared to be non-functional. The three CSPs fall into two functional groups that do not cross-activate due to receptor specificity. In addition, I observed that the Streptococcus constellatus subspecies pharyngis strains could not be transformed. However, I demonstrated that the pharyngis strains possess a functional ComCDE pathway, suggesting that the CSP regulates genes other than those involved in natural transformation. For many strains, I observed high endogenous competence levels that were only marginally induced by added peptide. These strains appear to be constitutively competent during exponential growth. The high basal level of expression and the heterogeneity in the SMG competence systems could impact how the SMG evolve during colonization and infections and specifically acquire antibiotic resistance and virulence genes.</p> / Master of Science (MSc)

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The Rodent Hill Locality is a fossil-bearing site that is part of the Cypress Hills Formation, and is located roughly 15 km northwest of the town of Eastend, Saskatchewan. A number of fossil mammal and other vertebrate taxa are present at Rodent Hill; the primary objective of this project was to identify the fossil rodents of the families Sciuridae, Aplodontidae, Castoridae, Heliscomyidae, Heteromyidae, Florentiamyidae and Zapodidae. These taxa were correlated with rodents from other North American faunas to establish the age of the Rodent Hill Locality. <p>The species Haplomys cf. H. liolophus, Dakotallomys cf. D. pelycomyoides, Kirkomys milleri, Proheteromys nebraskensis, Agnotocastor cf. A. praetereadens, and possibly Cedromus cf. C. wilsoni support the Whitneyan age designation of the Rodent Hill Locality. Taxa that are described from Rodent Hill that are better known from earlier-age sites include Heliscomys vetus and H. hatcheri, Ecclesimus sp. and Oligotheriomys sp. Taxa that are younger than Whitneyan but have been recovered at Rodent Hill include Parallomys sp., Plesiosminthus sp., Protospermophilus sp., and Nototamias sp. Two new species in the genus Sciurion, and one new species in the genus Pseudallomys are described, and a new species of Heliscomys is identified but not formally named. <p>The rodents from the Rodent Hill Locality support the Whitneyan age assignment of the site. This is based on the presence of Whitneyan taxa, and the in situ co-occurrence of older and younger taxa within the site.

The Rodent Hill Locality is a fossil-bearing site that is part of the Cypress Hills Formation, and is located roughly 15 km northwest of the town of Eastend, Saskatchewan. A number of fossil mammal and other vertebrate taxa are present at Rodent Hill; the primary objective of this project was to identify the fossil rodents of the families Sciuridae, Aplodontidae, Castoridae, Heliscomyidae, Heteromyidae, Florentiamyidae and Zapodidae. These taxa were correlated with rodents from other North American faunas to establish the age of the Rodent Hill Locality. <p>The species Haplomys cf. H. liolophus, Dakotallomys cf. D. pelycomyoides, Kirkomys milleri, Proheteromys nebraskensis, Agnotocastor cf. A. praetereadens, and possibly Cedromus cf. C. wilsoni support the Whitneyan age designation of the Rodent Hill Locality. Taxa that are described from Rodent Hill that are better known from earlier-age sites include Heliscomys vetus and H. hatcheri, Ecclesimus sp. and Oligotheriomys sp. Taxa that are younger than Whitneyan but have been recovered at Rodent Hill include Parallomys sp., Plesiosminthus sp., Protospermophilus sp., and Nototamias sp. Two new species in the genus Sciurion, and one new species in the genus Pseudallomys are described, and a new species of Heliscomys is identified but not formally named. <p>The rodents from the Rodent Hill Locality support the Whitneyan age assignment of the site. This is based on the presence of Whitneyan taxa, and the in situ co-occurrence of older and younger taxa within the site.