El objetivo de esta investigación fue evaluar ”in vitro” la actividad antibacteriana de dos pastas: 3 Mix-MP y Calen PMCC® como medicación intraconducto en un biofilm formado por 3 cepas: Porphyromona Gingivalis, Enterococcus Faecalis y Peptostreptococcus Anaerobius, presentes en Periodontis apical crónica; se utilizaron 32 piezas dentarias (premolares) a las cuales se les aplicó el mismo protocolo: fueron instrumentadas con sistema Mtwo hasta la lima 40.04, luego 22 piezas fueron seccionadas mesiodistalmente y 10 no seccionadas, fueron esterilizadas y contaminadas manteniéndolas en caldo BHI vitaminado por un lapso de 7 días. El proceso se dividió en 2 fases: en la primera fase se usaron 12 piezas seccionadas; a las que se les hizo el raspado en toda la superficie sembrándose en Agar Shaedler por 7 días; luego se realizaron 4 pozos de 5mm de diámetro por cada placa donde se colocaron las pastas de 3Mix-MP, Calen PMCC®, Hidróxido de calcio con suero fisiológico (control positivo) y glicerina (control negativo) dichas placas se incubaron por 7 días en anaerobiosis y se procedió a la lectura de los halo de inhición bacteriana. En la segunda fase siguiendo el protocolo anterior se usaron las 20 piezas restantes (10 seccionadas y 10 no seccionadas a las que se les colocó la pasta 3Mix-MP, la pasta Calen PMCC®, hidróxido de calcio con suero fisiológico (control positivo), se realizó el raspado y sembrado en agar shaedler manteniéndolo en anaerobiosis por 7 días para finalmente realizar la lectura de las unidades formadoras de colonias (ufc) presentes. El resultado observado con respecto a los halos de inhibición mostró que fue mayor para la pasta 3Mix - MP (40mm.) en comparación con la pasta Calen PMCC® (7mm.), con respecto a la lectura no se pudo recuperar colonias en las muestras de 3Mix-MP a diferencia de Calen PMCC® que se obtuvo 04 ufc.; concluyéndose que la pasta 3Mix-MP tiene mayor efecto antibacteriano como medicación intraconducto frente a la pasta Calen PMCC®. / Tesis

Indiana University-Purdue University Indianapolis (IUPUI) / Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

Hintergrund und Studienziele: Endoskopisch eingebrachte Kunststoffstents, die zur Behandlung von Gallenwegsobstruktionen verwendet werden, okkludieren mit der Zeit infolge einer mikrobiellen Besiedlung und Bildung von Biofilmen. Die antimikrobielle Behandlung der Stent-assoziierten Cholangitis ist unter klinischen Gesichtspunkten oftmals unvollständig, da nicht hinreichend wirksame Substanzen eingesetzt werden oder antimikrobielle Resistenzen vorliegen. Um die aktuelle Ätiologie von Stent-assoziierten Biofilmen in Bezug auf die Stentverweildauer bei Patienten mit endoskopischer Gallenwegsdrainage am Universitätsklinikum Leipzig näher zu charakterisieren, wurde eine prospektive Kohortenstudie durchgeführt. Zusammenfassung: Die vorliegende Arbeit gibt Hinweise darauf, dass die Besiedlung mit Enterokokken und Candida spp. einen relevanten Kofaktor bei der Bildung von Biofilmen auf Gallestents und der nachfolgenden Stentokklusion darstellt. Daher sollte die empirische antibiotische Therapie von Patienten mit Stent-assoziierter Cholangitis Enterokokken, Enterobakterien, Streptokokken, Candida-Spezies und Anaerobier umfassen. Die Betrachtung der Stentverweildauer erbrachte in unserer Arbeit nur für den Nachweis von Staphylokokken und Enterobacterales einen signifikanten Unterschied, sodass dieses Kriterium bei der Auswahl einer empirischen antibiotischen Therapie nur eine untergeordnete Rolle spielt. Die Analyse der klinischen Parameter zeigt jedoch, dass vor Beginn einer empirischen antibiotischen Therapie auf lokale Unterschiede und patientenspezifische Gegebenheiten (vorheriger Einsatz antimikrobieller Substanzen, Immunsuppression, Nieren- und Leberfunktion sowie Arzneimittelallergien) geachtet werden muss, um eine optimale Therapie zu gewährleisten und die Ausbreitung multiresistenter Erreger zu begrenzen. Daher sollte bei der Behandlung ein multidisziplinärer Ansatz unter Einbeziehung von Gastroenterologen, Infektiologen, Mikrobiologen und klinischen Apothekern verfolgt werden. Für den Nachweis von Krankheitserregern ist eine genaue mikrobiologische Analyse der extrahierten Stents inkl. Sonifikation erforderlich.

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Horizontal gene transfer transforms commensal E. faecalis into multidrug resistance (MDR) opportunistic pathogens causing diseases such as infective endocarditis (IE), septicemia, and urinary tract infections (UTI) (4,1). E. faecalis are among the top three leading causes of hospital-acquired infections and pheromone responsive plasmids (pCF10) are the most extensively characterized conjugative plasmids in E. faecalis infection (2,4). E. faecalis is a potential future public health concern because of the co-occurrence factors of antibiotic resistance and virulence traits (6)Plasmid-free commensal E. faecalis form homogenous biofilms that have a uniform distribution of the bacterial cell and a fluid-like movement (22). The introduction of the pheromone responsive plasmid pCF10 leads to the formation of heterologous rigid structures within the biofilm (22). In the current work, the timeline of biofilm tower formation was characterized. Tower formation was not observed in the commensal strain. The pCF10-containing bacteria formed a rigid base layer on day 1 and small aggregates on day 1. pCF10-containing biofilm forms heterologous towers on days two and three. Interestingly, mixed biofilms with both plasmid-containing and plasmid-free bacteria developed tower-like structures as early as day 1 and had larger resulting structures by day three. In the mixed population, we hypothesize that the induction of aggregation substance and cell clumping during plasmid transfer may further contribute to structure formation (5,10). Plasmid-free mCherry-labeled bacteria could be observed in the viscous biofilms between heterologous rigid structures; however, the rigid structures were predominantly composed of plasmid-containing cells. Occasionally, mCherry cells were observed in the rigid structures, we hypothesize that these cells represent transconjugants, where pCF10 was transferred by conjugation to mCherry-plasmid-free OG1RF. The formation of rigid structures can protect bacteria from antibiotics by reducing the penetration of the antibiotic but binding and sequestration of the antibiotic in the outer layers. Antibiotic resistance increased in the pCF10-containing biofilms as rigid structures were formed. We hypothesize that underflow, like that found in the gastrointestinal tract, the heterologous rigid structures may form protected microenvironments for sensitive regions of the biofilms. In future studies, fluorescently labeled antibiotics will be used to access the formation of protected microenvironments in biofilms underflow. Previous studies in the laboratory demonstrated that the presence of pCF10 protects E. faecalis from hydrogen peroxide oxidative stress. E. faecalis produces hydrogen peroxide. Higher levels of hydrogen peroxide can be detected in rigid structures. The presence of pCF10 is known to increase the size of heart vegetations during endocarditis and hydrogen peroxide is a known activator of platelet activation (12,19). In these studies, the presence of pCF10 increased platelet activation in pCF10 containing biofilms. Software toolboxes are currently being developed to quantitate visual observations. The role of hydrogen peroxide is supported in our preliminary experiment revealing catalase treatment reduced platelet activation. Studies are ongoing to mutate the aroc and menB gene of E. faecalis, which contribute to hydrogen peroxide production (35). We will compare platelet activation in knockout (double or single) E. faecalis and the wild-type strain. For future studies, several of the preliminary data need to be repeated to further the study. We will repeat quantitative hydrogen peroxide production in the catalase experiments. We will also finish knocking out the aroc and men B gene of E. faecalis responsible for hydrogen peroxide and then compare platelet activation to the control strain. / Microbiology and Immunology

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This thesis evaluates the performance of a deammonification system designed as a two-step tech-nology consisting of an initial partial nitritation followed by an Anammox process. Operation of a technical-scale pilot plant at the Himmerfjärden Wastewater Treatment Plant (Grödinge, Swe-den) has been assessed. Oxygen Uptake Rate (OUR) to evaluate the respiration activity of nitrifi-ers in the system and batch tests to assess reaction rates have also been applied in the study. It was found that the total inorganic nitrogen elimination strongly depended on the nitrite-to-ammonium ratio in the influent to the Anammox reactor, which was correlated with the per-formance of the partial nitritation phase. Therefore, a control strategy for oxidation of ammo-nium to nitrite has been proposed. Controlled oxygen supply to the partial nitritation reactor is obligatory to obtain a proper pH drop indicating oxidation of ammonia to nitrite at the adequate ratio. A very high nitrogen removal efficiency (an average of 84%) and stable operation of the system have been reached. Conductivity measurements were also used to monitor the system influent nitrogen load and the nitrogen removal in the Anammox reactor. The data gathered from the operation of the pilot plant enabled the use of multivariate data analysis to model the process behaviour and the assessment of the covariances between the process parameters. The options for full-scale implementation of the Anammox systems have been proposed as a result of the study. / QC 20101115

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