Klebsiella spp. are coliform bacteria that cause mastitis in dairy cattle and account for high mortality rates in infected cows leading to a significant financial loss. Recent outbreaks indicate that within farms a single strain can be responsible for clinical signs in multiple animals. Identification of the virulence of factors enabling Klebsiella spp. survival in the mammary glands of multiple animals may provide insight into host adaptation. In this study, Klebsiella spp. strains were evaluated for their ability to evade neutrophil killing, the primary immune defense in the bovine mammary gland. Our research focused on capsule and biofilm production by Klebsiella spp. when strains were grown in Luria Broth or skim milk to examine the effects on evasion of neutrophil killing. Biofilm production was not significantly related to the ability to resist neutrophil killing nor was capsule (P = 0.29). Farm (P < 0.001), media type (P < 0.005), and strain type by cow (P < 0.001) were found to play significant roles in neutrophil evasion. This suggests farm of origin, media type used, and cow all may play a role in evasion of neutrophils by Klebsiella spp. Further evaluation of virulence factor expression in different media types and the role of individual cow immune responses may provide insight into ability of Klebsiella spp. to cause outbreaks of mastitis in multiple animals. / Master of Science

<p>Poznavanje i razumevanje adhezivne sposobnosti i formiranja biofilma patogenih bakterija, kao i njihovog odnosa prema faktorima koji mogu stimulisati ili inhibirati razvoj biofilma, je od fundamentalnog značaja za iznalaženje mera za njihovu efikasnu prevenciju i eliminaciju.<br />Imajući u vidu navedenu činjenicu kao i da je Salmonella enterica serotip Enteritidis epidemiolo&scaron;ki najfrekventniji serotip cilj ovog istraživanja je bio da se ispita: sposobnost različitih sojeva Salmonella Enteritidis izolovanih iz kliničkog materijala, hrane za životinje i odabranog referentnog soja da formiraju biofilm, adherentnost na povr&scaron;ine od stakla i nerđajućeg čelika, sposobnost preživljavanja odabranih biofilm produkujućih sojeva kao i mogućnost primene konfokalne laserske i skening elektronske mikroskopije u vizuelizaciji trodimenzionalne strukture biofilma.<br />Određivanjem morfotipa kolonija na Kongo red agaru na temperaturi inkubiranja od 25&deg;C među testiranim izolatima detektovana su tri morfotipa RDAR (red, dry and rough), BDAR (brown dry and rough) i SAW (smooth and white). Polovina testiranih izolata je eksprimirala RDAR morfotip. Izolati koji su eksprimirali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.<br />Uporednom analizom rezultata primenjenih skrining testova za utvrđivanje sposobnosti formiranja biofilma pri temperaturi inkubiranja od 25&deg;C ustanovljena je korelacija između pojave određenog morfotipa na Kongo crvenom agaru i sposobnosti formiranja biofilma u kristal violet i pelikula testu. Međutim, sa povećanjem temperature inkubiranja na 37oC, ova korelacija nije ustanovljena, sa izuzetkom izolata SE8.<br />Svi testirani izolati su pokazali sposobnost adherencije na povr&scaron;inu stakla i nerđajućeg čelika, ali u različitoj meri. Na sposobnost adherencije povoljniji uticaj je imala temperatura inkubiranja od 25&deg;C (p&lt;0,05), sa izuzetkom izolata SE3 (p&gt;0,05). Između stepena adherencije izolata na povr&scaron;ine stakla i nerđajućeg čelika nisu ustanovljene statistički značajne razlike (p&gt;0,05).<br />Praćenjem stope preživljavanja tokom 28 dana u uslovima isu&scaron;ivanja evidentirana je znatno veća stopa preživljavanja ćelija izolata RDAR morfotipa u odnosu na stopu preživljavanja ćelija BDAR morfotipa (p&lt;0,05). Praćenjem stope preživljavanja tokom 90 dana u uslovima povremene dostupnosti hranljivih materija, zabeležena je veća stopa preživljavanja u odnosu na stopu preživljavanja u uslovima isu&scaron;ivanja. U uslovima povremene dostupnosti hranljivih materija nakon devedeset dana ispitivanja kod obe grupe izolata procenat vijabilnih ćelija je iznosio vi&scaron;e od 50%.<br />Primenjenim mikroskopskim tehnikama (CLSM i SEM) omogućena je detaljna vizualizacija formiranih biofilmova. Na modelu izolata SERDAR morfotipa, ustanovljeno je da se formiranje biofilma pod primenjenim eksperimentalnim uslovima, odvija u tri faze: 1) inicijalna adhezija za povr&scaron;inu i formiranje manjih ćelijskih agregata (24h); 2) formiranje većih ćelijskih agregata uz produkciju EPS (48h); 3) sazrevanje biofilma uz značajnu produkciju EPS &scaron;to omogućava formiranje stabilne trodimenzionalne strukture biofilma (96h).<br />Nasuprot karakteristikama koje bakterije pokazuju tokom rasta u medijumima koji obiluju hranljivim materijama, bakterije u biofilmovima pokazuju drugačije osobine u pogledu ekspresije gena i karakteristika rasta. Zahvaljujući ovim razlikama, bakterije u biofilmovima pokazuju povećanu rezistenciju na antibiotike i dezinficijense, zbog čega se konstantno razvijaju nove kontrolne strategije u cilju iznalaženja potencijalnih biolo&scaron;kih re&scaron;enja koja pored različitih enzima, faga, antimikrobnih jedinjenja proizvedenih od strane mikroorganizama uključuju i antimkrobna jedinjenja biljnog porekla kao &scaron;to su biljni ekstrakti, etarska ulja i različiti začini. Stoga, je u okviru drugog segmenta ovog istraživanja ispitivan hemijski sastav etarskih ulja, antimikrobni efekat etarskih ulja (O. heracleoticum , O. vulgare , Th. vulgaris i Th. serpyllum) i pojedinačnih komponenti etarskog ulja (karvakrola i timola) na bujonske kulture testiranih sojeva Salmonella Enteritidis kao i uticaj odabranih koncentracija etarskih ulja na inicijalnu adheziju i već formirani biofilm odabranih sojeva Salmonella Enteritidis.<br />Etarska ulja je karakterisao visok zbirni udeo glavnih fenolnih komponenti karvakrola i timola: O. heracleoticum (71,6%), O. vulgare (63,6%), Th. vulgaris (59,77%), Th. serpyllum (40,04%). Etarska ulja su ispoljila antimikrobni efekat sledećim redosledom: O. heracleoticum &gt;O. vugare =Th. vulgaris &gt;Th. serpyllum. Antimikrobni efekat etarskih ulja je bio direktno srazmeran zbiru fenolnih komponenti (karvakrola i timola) u etarskom ulju. U odgovoru na tretman etarskim uljima između izolata S. Enteritidis nisu ustanovljene razlike.<br />Etarska ulja, karvakrol i timol su pokazali inhibitorni efekat na inicijalnu adheziju i posledično na formiranje biofilma testiranih izolata S. Enteritidis na dozno zavisan način. Upoređivanjem uticaja etarskih ulja na inhibiciju inicijalne adhezije i metabolitičke aktivnosti ćelija između izolata RDAR i BDAR morfotipa ustanovljene razlike nisu bile statistički značajne (p&gt;0,05).<br />Ispitivanjem uticaja etarskih ulja, karvakrola i timola na ukupnu biomasu biofilma i metabolitičku aktivnost ćelija dokazano je da etarska ulja u primenjenim koncentracijama ispoljavaju uticaj na redukciju ukupne biomase formiranog biofilma i metaboličke aktivnosti bakterijskih ćelija na dozno zavisan način u funkciji vremena. Znatno veća efikasnost primenjenih tretmana je pokazana u slučaju njihove primene na biofilmove formirane od strane izolata BDAR morfotipa (p&lt;0,05).</p> / <p><span style="font-size:10px;">Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination.<br />Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure.<br />Determination of colony morphotype on Congo red agar at incubation temperature of 25&deg;C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface.<br />Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25&ordm;C. In the case of assays conducted at 37&deg;C, this correlation was not established, except for the isolate SE8.<br />All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25&ordm;C had more favorable effect on the adherence, with the exception of isolate SE3 (p&gt;0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p&gt;0.05).<br />Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was</span> <span style="font-size:10px;">obtained for RDAR than BDAR morphotype isolates (p&lt;0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates.<br />Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h).<br />Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices.<br />Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates.<br />Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum &gt; O. vugare = Th. vulgaris &gt; Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference.<br />Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p&gt;0.05).<br />Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity</span> <span style="font-size:10px;">of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p&lt;0.05).</span></p>

Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is of major clinical importance due to its ability to significantly affect pregnancy outcome and enhance the transmission and acquisition of HIV. BV is characterized by a dramatic shift in the vaginal microflora; in most BV cases, the predominant bacterial species is Gardnerella vaginalis. It has been demonstrated that G. vaginalis forms an adherent biofilm on the vaginal epithelium of women with BV. Furthemore, evidence suggests that the high rate of recurrence associated with BV is related to incomplete eradication of the biofilm. The overall goal of this study was to characterize G. vaginalis virulence properties, including biofilm formation, in order to better understand the pathogenesis of BV and to improve available treatment methods. In an effort to tease apart the uncertain etiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. Only G. vaginalis demonstrated all three virulence properties, including robust biofilm formation. It has been shown that the biofilm phenotype allows its constituent bacteria to be resistant to many negative environmental stimuli. Therefore, we studied the susceptibilities of biofilm vs. planktonic cultures to H2O2 and lactic acid. Biofilms tolerated higher concentrations of both chemicals; however, when the biofilm was proteolytically disrupted, sensitivity to the chemicals returned to planktonic levels. Since our data suggested a critical role for a protein in biofilm formation, a partial genome sequence of G. vaginalis was searched for sequence homology to known biofilm adhesins using the tBLASTn program. This revealed an open-reading frame encoding a hypothetical protein with significant homology to the staphylococcal Bap protein. Antibody towards a portion of the identified gene product was produced in rabbits by inoculation of a recombinant peptide to an antigenic region of the protein. Antibody inhibition assays against biofilm formation, adherence, initial adherence and aggregation were conducted. Relative expression levels of the biofilm-associated protein were analyzed under different conditions by western blot analysis. Finally, the protein was expressed in heterologous hosts and analyzed for an increase in biofilm formation.

La bactérie aquatique Shewanella oneidensis est capable, en condition statique et en présence d'oxygène, de former un biofilm à l'interface air-liquide, appelé pellicule. Mon travail a porté sur la biogenèse de la pellicule.Il a été montré dans le groupe que le régulateur de réponse du système chimiotactique, la protéine CheY3, était impliqué dans la biogenèse de la pellicule. Cette protéine est essentielle dans les étapes précoces et tardives de sa formation alors que son partenaire habituel, CheA3, semble ne jouer un rôle que dans les étapes tardives. Mon travail s'est focalisé sur la recherche de partenaires de CheY3.J'ai introduit une banque d'ADN génomique de S. oneidensis dans la souche ΔcheY3 et j'ai cherché des gènes dont la surexpression permettait de restaurer la formation de la pellicule. Cette approche a révélé deux gènes pdgA et pdgB. J'ai montré que les protéines PdgA et PdgB étaient capables de synthétiser du di-GMPc, suggérant que ce messager secondaire est impliqué dans la biogenèse de la pellicule. L'hydrolyse du di-GMPc par des enzymes dédiées empêche en effet sa formation.J'ai montré que l'opéron mxd, contrôlant la synthèse d'exopolysaccharides dans les biofilms de surface, était impliqué dans la formation de la pellicule. La première protéine codée par cet opéron, MxdA, est capable de lier le di-GMPc. Des expériences de pontage chimique et de double hybride ont révélé que MxdA, CheY3, PdgA et PdgB, formaient un réseau de régulation gouvernant la biogenèse de la pellicule.J'ai montré que les systèmes à deux composants BarA/UvrY et ArcS/ArcA contrôlant la transcription de l'opéron mxd sont aussi impliqués dans la formation du biofilm flottant. / The aquatic bacterium Shewanella oneidensis is able to form, under static conditions and in the presence of oxygen, a biofilm at the air-liquid interface, called pellicle. My work was focused on the biogenesis of this pellicle.It was previously shown in the team that, surprisingly, the CheY3 protein, the response regulator of the chemotactic regulatory system, is involved in the biogenesis of the pellicle. This protein was shown to be essential both in early and late steps of pellicle formation whereas its usual partner, the kinase CheA3, seems to play a role in the late steps only. I was therefore looked for the partners of the CheY3 protein for pellicle formation.For this purpose, I have introduced a multi-copy genomic library in the ΔcheY3 strain and searched for genes whose overexpression allowed pellicle restoration. Strikingly, this approach revealed two genes pdgA and pdgB. Interestingly, we showed that PdgA and PdgB proteins are able to synthesize c-di-GMP, suggesting a role for this second messenger in pellicle biogenesis. Indeed, c-di-GMP hydrolysis by dedicated enzymes blocks pellicle formation.We also showed that the mxd operon, controlling the exopolysaccharides synthesis in biofilm associated with a solid surface, is also involved in pellicle formation. Moreover, the first protein encoded by this operon, MxdA, is able to bind c-di-GMP. Cross-linking and bacterial two-hybrid experiments revealed that MxdA, CheY3, PdgA and PdgB, form a complex regulatory pathway governing the biogenesis of the pellicle.Finally, we have shown that the two-component systems BarA/UvrY and ArcS/ArcA, controlling the mxd transcription, are also involved in pellicle formation.

Die Mechanismen von Immobilisierungsprozessen radioaktiver Schwermetall-Ionen innerhalb von Biofilmen sind noch weitgehend unerforscht. Das liegt an der Komplexität der Biofilme, welche häufig diskrete geochemische Mikromilieus bilden, die sich vom umgebenden Milieu („Bulk Solution“) in Bezug auf dessen Biozönose (der mikrobiellen Diversität), den darin herrschenden geochemischen Bedingung (z.B. Red/Ox-Potential u./o. gelöster Sauerstoffmenge), aber auch in der Konzentration möglicher Komplexbildner (z.B. Metaboliten u./o. EPS-Komponenten) deutlich unterscheiden. Alle diese Faktoren können die Speziation der Radionuklide verändern und damit auch deren Transportverhalten. Für ein besseres Prozessverständnis zu den Wechselwirkungen von Radionukliden mit natürlichen, in Uran-kontaminierten Milieus lebende Mikroorganismen und den damit verbunden Stoffen wurde die Biozönose in Biofilmen und im Grubenwasser des ehem. WISMUT-Uranbergwerkes Königstein nach klassischen mikrobiologischen- und molekularbiologischen Methoden bestimmt. Aus einem Vergleich der Chemie im Biofilm mit der Chemie der umgebenden Lösung wird der Einfluss der Biofilme auf das Migrationsverhalten von Radionukliden in der Natur beurteilt. Die Identifizierung und Quantifizierung von Prokaryoten erfolgte u.a. mit der CARD FISH Methode. Die selektive Visualisierung der EPS-Komponenten in der Matrix der Biofilme wurde mit Hilfe der Konfokalen Laser Scanning Mikroskopie (CLSM) bewerkstelligt. Zur Untersuchung der Speziation von fluoreszierenden Schwermetall-Ionen wie U(VI) kam die zeitaufgelöste, laser-induzierte Fluoreszenzspektroskopie (TRLFS) zum Einsatz. Um diese Methode auch im mikroskopischen Bereich anwenden zu können, wurde sie weiter zum CLSM hin entwickelt: Da ein 80-MHz-MaiTai-Laser zur Verfügung stand, wurde durch im kHz-Bereich alternierendes Beugen des Anregungslaserstrahls von der Probe weg (und wieder zu ihr hin) mittels akusto-optischem Modulator (AOM) eine quasi-gepulste Laseranregung im kHz-Bereich erreicht. Durch Einbindung von Frequenzvervielfachern („Harmonixx“ von APE Berlin und „Inspire“ von Spectra-Physics) konnte so eine gepulste Anregung innerhalb eines breiten Wellenlängenbereiches (ca. 230-1090 nm) ermöglicht werden. Für die Auswertung des als äußerst schwach zu erwartenden Fluoreszenzsignales (entsprechend des mikroskopisch kleinen Anregungsraumes) wurde die Time-Correlated Single-Photon Counting Methode (TCSPC) – auch „zeitbezügliche Einzelphotonenzählungs-Methode“ – an das Laser-Anregungssystem angepasst. Die Fluoreszenzlebenszeitkurve des Fluoreszenzsignals von U(VI) Species, die sich an der Oberfläche von den Protozoen Euglena Mutabilis befanden, konnte z.B. auf diese Art mit Hilfe der TCSPC ermittelt werden.

Biofilms are formed in aquatic environments by the attachment of bacteria to submerged surfaces, to the air/liquid interface, and to each other. Although biofilms are associated with disease and biofouling, the robust nature of biofilms; for example, their ability to tolerate chemical and physical stresses, makes them attractive for beneficial biotechnology applications such as bioremediation and biofuels. Based on an understanding of diverse signals and regulatory networks during biofilm development, biofilms can be engineered for these applications by manipulating extracellular/intercellular signals and regulators. Here, we rewired the global regulator H-NS of Escherichia coli to control biofilm formation using random protein engineering. H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared with wild-type H-NS (wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it) via its interaction with the nucleoid-associated proteins Cnu and StpA. H-NS K57N leads to enhanced excision of the defective prophage Rac and results in cell lysis through the activation of a host killing toxin HokD. We also engineered another global regulator, Hha, which interacts with H-NS, to disperse biofilms. Hha variant Hha13D6 was obtained that causes nearly complete biofilm dispersal by increasing cell death by the activation of proteases. Bacterial quorum sensing (QS) systems are important components of a wide variety of engineered biological devices, since autoinducers are useful as input signals because they are small, diffuse freely in aqueous media, and are easily taken up by cells. To demonstrate that biofilms may be controlled for biotechnological applications such as biorefineries, we constructed a synthetic biofilm engineering circuit to manipulate biofilm formation. By using a population-driven QS switch based on the LasI/LasR system and biofilm dispersal proteins Hha13D6 and BdcAE50Q (disperses biofilms by titrating cyclic diguanylate), we displaced an existing biofilm and then removed the second biofilm. Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Here, we engineered toxin MqsR with greater toxicity and demonstrated that the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.

L’origine de ce travail est principalement les effets biologiques potentiels des ondes électromagnétiques de la gamme non ionisante rencontrés dans la littérature et quelques faits observés par des clients de la technologie commercialisée par la société Planet Horizon SA. Les essais de couplage du traitement aux ondes électromagnétiques et les procédés de traitements des eaux usées par boues actives sont réalisés à niveau industriel au sein de la STEP de Penthaz (Suisse) compose de 2 lignes parallèles et sur des pilotes de laboratoire en mode SBR. Le traitement électromagnétique de très basse fréquence (Antennes émettant deux fréquences harmoniques F1 et F2 < 10 kHz) est appliqué via 5 antennes dans le bassin d’aération ou une antenne sans le SBR directement. Dans une autre expérience , des tubes, émettant les mêmes fréquences, sont utilisés pour le recyclage de la liqueur mixte avec toujours une ligne de référence. Au niveau de la STEP, dans le bassin d’aération le traitement électromagnétique a permis une réduction de la quantité de biomasse produite au niveau de l’essai de l’ordre de 42,5% par rapport à ligne témoin. De plus, ce traitement électromagnétique n’affecte pas la qualité de l’eau épurée puisque la quantité de DCO résiduelle est la même à la sortie des deux ligne (REF et EM) et le rendement d’abattement de la matière organique est de l’ordre de 94% pour les deux lignes. Par contre à l’échelle du laboratoire, les résultats obtenus après le couplage du traitement électromagnétique n’ont aucun effet sur la quantité de biomasse produite dans les différents essais avec antenne ou bobine comme étant des émetteurs d’ondes EM. De même, on ne remarque aucun effet significatif sur les rendements de dépollution. La deuxième partie concerne l’application de ces mêmes ondes sur la formation de biofilm microbien. Les champs électromagnétiques visés sont des faibles champs de quelques dizaines de milli-teslas au niveau du générateur (tension de l’ordre de la dizaine de volts et intensité de l’ordre d’un ampère) avec des fréquences comprises entre 0 et 10 kHz; deux ondes de fréquences harmoniques sont imposées simultanément. Le biofilm est quantifié au cours du temps grâce à la mesure de différents paramètres (densité optique, DCO, protéines, ATP, exopolysaccharides). Les résultats expérimentaux démontrent que l’onde électromagnétique limite la formation du biofilm si la continuité hydraulique est assurée entre la bobine électromagnétique et les supports de biofilm ; la quantité de biofilm mesurée est alors deux fois plus faible environ. Cependant, l’effet semble bactériostatique et non bactéricide : l’application de l’onde ne détruit pas le biofilm mais réduit sa formation car dès l’arrêt de l’onde, la vitesse augmente. Des signaux électriques de quelques millivolts et dans le domaine des très basses fréquences ont été détectés seulement dans la cuve en continuité hydraulique avec le générateur d’ondes et située à une distance de 1m environ. Des mesures de champs électromagnétiques dans l’air donnent des intensités de champs très faibles ne pouvant expliquer un effet thermique ou ionisant sur le biofilm. / The first part of this work concerns the coupling of VLF electromagnetic waves treatment and the processes of wastewater treatment by activated sludge in order to reduce excess sludge. Experiments are made at real -scale ( Penthaz , Switzerland),containing two parallel lines and at lab-scale with SBR pilots. The electromagnetic treatment of very low frequency ( antennaemitting harmonic frequencies F1 and F2 <10 kHz) is applied via 5 (2,3x1m) antennas in the aeration basin of the real plant. Other type of antenna (30x 5 cm) was configurated for one of the labscale assays. In the other lab-scale experiment, tubes emitting the same frequency are used for recycling the mixed liquor. With the WWTP, the amount of excess sludge produced in the treated line is reduced by about 42.5%, compared to the control line. In addition, the electromagnetic treatment does not affect the quality of treated water. In this case, outlet COD concentration was similar for the the two lines (REF and EM) and the reduction of or ganic matter yield was about 94% for the two lines. Electromagnetic treatment had no effect on the amount of excess biomass produced in all tests conducted at lab-scale. No significant effect has been noticed regarding depollution yields. The second part aims to better understand the interaction between an electromagnetic wave of very low frequency and biological material and, in particular, the effects on the development of microbial biofilms. Electromagnetic fields are covered weak fields a few milli-Tesla (voltage of the order of tens of volts and intensity of the order of one ampere) with frequencies between 0 and 10 kHz, two waves of harmonic frequencies are transmitted simultaneously. The biofilm was quantified over time by measuring different parameters (optical density, COD, protein, ATP, exopolysaccharides). The experimental results how that the electromagnetic wave limit biofilm formation ( close to 50% of untreated biofilm) if the hydraulic continuity is ensured between the electromagnetic coil. However, the effect appears bacteriostatic and no antibacterial : the application of the wave does not destroy the biofilm but reduced its establishment and upon discontinuation of the wave, the growth increases. Measurements and modeling of electromagnetic fields in the air give intensities of very weak fields that can explain a thermal or ionizing effect on the biofilm.

Dans la nature, les micro-organismes sont organisés en communautés agrégées dénommées biofilms, particulièrement adaptées à la survie en milieu hostile. Les difficultés pour prévenir la formation ou éliminer des biofilms matures par des stratégies conventionnelles ont encouragé le développement de nouvelles approches inspirées des mécanismes de compétition entre différents micro-organismes au sein de biofilms naturels. Au cours de ce travail, nous nous sommes intéressés à l'effet anti-biofilm de bactéries bénéfiques appartenant aux genres Lactobacillus et Bifidobacterium. Dans un premier temps, nous avons testé l'effet anti-biofilm de surnageants neutralisés vis-à-vis de deux pathogènes Klebsiella pneumoniae et Staphylococcus epidermidis dans un modèle expérimental statique. Si les extraits des quelques souches de Bifidobacterium testées stimulaient la formation de biofilm par K. pneumoniae sur surface abiotique, la majorité de ceux des 140 souches de Lactobacillus exerçait un effet inhibiteur et nous avons retenu une des souches dont le surnageant de culture entraînait une inhibition majeure (70%), Lactobacillus plantarum CIRM653. Cet extrait s'est également avéré capable de disperser des biofilms préformés à K. pneumoniae sur surface abiotique mais aussi d’inhiber la formation de biofilms sur surface biotique, et ce indépendamment d’un effet bactéricide. La formation de biofilms mixtes formés par L. plantarum et K. pneumoniae dans des modèles expérimentaux cinétiques a permis, comparativement à l'observation de biofilms mono-espèce à K. pneumoniae, de mettre en évidence des défauts de structuration du biofilm associés à une diminution de la biomasse de K. pneumoniae et une augmentation de celle de L. plantarum. Grâce à une approche transcriptionnelle ciblée, nous avons montré que L. plantarum induisait, par le biais de son surnageant, des modifications de l’expression de gènes impliqués dans la formation de biofilm chez K. pneumoniae. Quatre gènes impliqués dans le quorum-sensing (opérons lsr) étaient sous-exprimés et trois gènes de structure du pilus de type 3 étaient sur-exprimés. L'augmentation de la production de pili de type 3 fonctionnels a été validée par Western-blot et des tests d’hémagglutination. Cette surexpression est probablement responsable du niveau élevé des capacités d’adhésion sur surface abiotique d'agrégats de K. pneumoniae issus de la dispersion induite par L. plantarum.Le comportement des deux souches a également été testé in vivo, dans un modèle murin de colonisation intestinale par K. pneumoniae avec administration orale quotidienne de L. plantarum. Le dénombrement du pathogène dans les selles des animaux a montré qu'en présence de L. plantarum, K. pneumoniae maintient des niveaux de colonisation élevés, contrairement au contrôle (sans Lactobacillus) où une diminution graduelle est observée.Enfin, nous avons initié le développement d'un modèle expérimental tripartite permettant d'associer les deux partenaires bactériens avec des cellules épithéliales dans un système en flux continu. La réponse spécifique des cellules eucaryotes a également été abordée : nous avons pu mettre en évidence que L. plantarum exerçait un effet inhibiteur vis-à-vis de la réponse inflammatoire épithéliale pulmonaire induite par K. pneumoniae. En conclusion, la description d'une activité anti-biofilm in vitro ne serait pas synonyme d'une réduction in vivo de la colonisation de surfaces biotiques, mais à une plus grande capacité de dissémination. Ces observations démontrent l’importance d’une expertise précise de l’action des bactéries bénéfiques et de la maitrise du ratio bénéfice-risque pour leur utilisation. / In the natural environment microorganisms are organized in aggregated communities called biofilms, which are particularly adapted to the survival in harsh conditions. The difficulties to prevent the formation or elimination of mature biofilms by conventional strategies have encouraged the development of new approaches inspired by competition mechanisms occurring between microorganisms within natural biofilms.In this work, we looked for anti-biofilm effects of beneficial bacteria belonging to Lactobacillus and Bifidobacterium genus. We first tested the anti-biofilm effect of neutralized supernatants against both pathogens Klebsiella pneumoniae and Staphylococcus epidermidis in a static experimental model. The few Bifidobacterium extracts tested led to an increase in biofilm formation by K. pneumoniae on abiotic surface, whereas the majority of the 140 strains of Lactobacillus exerted an inhibitory effect. Lactobacillus plantarum CIRM653 was selected for further experiments because its culture supernatant displayed major inhibition (70%). This extract was also capable of dispersing preformed biofilms of K. pneumoniae on abiotic surface, but also able to inhibit biofilm formation on biotic surface, independently of a bactericidal effect. The formation of mixed biofilm containing L. plantarum and K. pneumoniae in kinetic experimental models highlighted the biofilm structure defects associated with a decrease of K. pneumoniae biomass and an increase of that of L. plantarum, compared to a monospecies K. pneumoniae biofilm. Targeted transcriptional approach was used to assess changes in the expression of genes involved in biofilm formation by K. pneumoniae after contact with L. plantarum supernatant. Four genes involved in quorum-sensing (operons lsr) were under-expressed and three type 3 pili structural genes were over-expressed. The increase of functional surface located type 3 pili was validated by Western blotting and hemagglutination tests. This overexpression was probably responsible for the observed high level of adhesion capacity to abiotic surfaces of K. pneumoniae aggregates recovered after dispersion induced by L. plantarum.The behavior of the two strains was also tested in vivo in a K. pneumoniae murine intestinal colonization model with daily oral administration of L. plantarum. Viable cells counting of the pathogen in the animals’ feces showed that K. pneumoniae maintained high levels of colonization in the presence of L. plantarum, unlike the control (without Lactobacillus) where a gradual decrease was observed.Finally, we initiated the development of a tripartite experimental model allowing the combination of the two bacterial partners with epithelial cells in a continuous flow system. In parallel, the specific response of eukaryotic cells to these bacteria was addressed: L. plantarum exerted an inhibitory effect on the pulmonary epithelial inflammatory response induced by K. pneumoniae.In conclusion, these results highlight the discrepancy between in vitro anti-biofilm activity of L. plantarum and its in vivo behavior leading to increased dissemination of the pathogen. Substantial expertise of beneficial bacteria is therefore necessary to fully assess their benefit-risk ratio.

Le Quorum sensing (QS) est un système de communication bactérienne capable de réguler divers processus cellulaires qui dépendent de la densité de la population microbienne. Chez les bactéries à Gram négatif, cela se produit par production de molécules de signalisation auto-inductrices (AI), les acyl homosérine lactones (AHL). La libération d´AHL à l'extérieur de la cellule est détectée par la population bactérienne provoquant en réponse la régulation de l'expression de certains gènes (régulon QS).Notre laboratoire a étudié et identifié un système QS fonctionnel dans la souche Acidithiobacillus ferrooxidans ATCC 23270T. En outre, nous avons montré que des analogues synthétiques d´AHL modulent l´adhésion d´At. ferrooxidansT sur un substrat minéral, tels des coupons de soufre.Dans ce projet de recherche, nous nous proposons d'identifier les gènes qui sont régulés par le QS chez At. ferrooxidansT, en particulier ceux impliqués dans la biogénèse du biofilm. Notre hypothèse est que des analogues synthétiques d’AHL induisent le système QS. Ainsi, nous nous proposons de moduler l´adhésion sur substrat minéral grâce à l'utilisation de ces molécules. L'utilisation de ces AHL permettra de caractériser le régulon QS dans cette souche bactérienne.L'identification d'analogues synthétiques d´AHL qui favorisent l'adhésion à des coupons de soufre nous a permis d'étudier le transcriptome d´At. ferrooxidansT dans des conditions où le régulon QS est stimulé. Puces à ADN d´At. ferrooxidansT avec/sans ces analogues synthétique d´AHLs nous a permis de caractériser le régulon QS et les gènes impliqués dans la biogénèse du biofilm dans les conditions utilisées. / Quorum sensing (QS) is a bacterial communication system capable of controlling several cellular processes dependent on the density of the microbial population. In Gram-negative bacteria, it occurs mainly through the production by bacteria of small diffusible signaling molecules, termed autoinducers (AI), of the acyl homoserine lactones type (AHLs). The release of AHLs outside the cell is detected by the bacterial population generating the regulation of the expression of several genes (regulon QS).Our laboratory has studied and identified a functional QS system in the Acidithiobacillus ferrooxidans ATCC 23270T type strain. Besides, by using synthetic analogs of AHLs, we have shown that AHL-type QS molecule analogs modulate adhesion of At. ferrooxidansT to minerals, such as sulfur coupons. In this research, we propose to identify the genes that are regulated by QS in At. ferrooxidansT, particularly those that are associated with biofilm formation. For this, we propose to modulate the adhesion of At. ferrooxidansT to mineral substrate through the use of a synthetic AHL analog. Our working hypothesis postulates that AHLs molecules induce the QS system, and that their use will allow the characterization of the QS regulon of this bacterial strain by transcriptomic analysis.The identification of synthetic AHLs improving adherence of At. ferrooxidansT on sulfur coupons allowed us to study the transcriptome of this organism in conditions in which QS regulon is stimulated. DNA microarrays of At. ferrooxidansT with/without one of these AHLs synthetic analogues allowed us to identify the QS regulon and to determine genes involved in biofilm formation.

Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui peut développer deux modes d’infection. Les infections aigües sont associées à la production et à la sécrétion de toxines qui peuvent avoir un effet cytotoxique, alors que dans le contexte d’une infection chronique la bactérie a tendance à s’établir sous la forme d’un biofilm. Un biofilm est une population de microorganismes organisée en une communauté et attachée sur une surface. Cette surface peut être biotique ou abiotique. Les biofilms bactériens ont des caractéristiques intrinsèques qui les rendent plus résistants à des conditions environnementales difficiles (pH, oxygène, UV, flux, etc…). Dans le cadre d’une infection l’établissement du biofilm résulte aussi en une population bactérienne difficile à éliminer par le système immunitaire mais également résistante aux antibiotiques. Des déterminants moléculaires majeurs qui interviennent dans la formation du biofilm sont le flagelle, les pili de type IV et les fimbriae Cup pour l’attachement, ou bien encore les exopolysaccharides qui sont avec l’ADN des composants essentiels de la matrice extracellulaire du biofilm qui englobe la population bactérienne. Chez P. aeuginosa, si l’alginate est le polysaccharide majeur, il a été montré que des souches non-mucoides forment également des biofilms et que dans ce contexte la synthèse et la sécrétion du polysaccharide majeur de la matrice sont dépendantes des gènes pel. Mon travail a consisté dans l’étude de l’organisation structurale du système Pel et le contrôle de son activité. / Pseudomonas aeruginosa is a human opportunistic pathogen, and in the course of an infection can develop two lifestyles. One of them is involved in secretion of toxins and results in acute infection, and the other one causes chronic infections and is characterized by formation of a biofilm. A biofilm is a highly organized bacterial population, organized as a complex community that is attached to a surface. Bacterial biofilm shows higher resistance to different enviromental factors including physical factors, antimicrobials or host immune response. Major components taking part in biofilm formation are flagella, type IV pili, cup fimbriae as well as exopolysaccharides. The latter provides a protective matrix for the biofilm, together with proteins and DNA. In non-mucoid strains the major exopolysaccharide of biofilm matrix is synthetised and secreted by pel gene cluster. My work concentrates on the structural organisation of Pel polysaccharide secretion machinery, regulation of its activity as well as detailed characterisation of the Pel polysaccharide itself.