Resistance to the antibiotic bacitracin in Enterococcus faecalis strain AR01/DGVS is conferred by the genes bcrABD that are under the control of the regulatory protein BcrR. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins and topological modelling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR is a novel transmembrane transcriptional regulator which acts to both sense bacitracin and initiate transcription of bcrABD. The transcription of bcrABD in the presence of bacitracin results in the expression of a putative ABC transporter, BcrAB, that may function by the efflux of bacitracin from the cell membrane. The aim of this study was to further characterise the mechanism of bacitracin resistance in E. faecalis and to conduct structural and functional studies with BcrR. A series of bcrA-lacZ transcriptional fusions were created to investigate the regulation of bcrABD transcription by BcrR and map the bcrABD promoter. We determined that BcrR activates bcrA-lacZ expression in response to bacitracin directly and not indirectly via the build-up of cell wall stress or cell wall precursors. A 69-bp region of the bcrABD promoter was required for activation of bcrA-lacZ expression. The introduction of mutations into this sequence demonstrated that two inverted repeat regions (viz. 5�-CTGACA(N)₆GTGTC-3�) were required for bcrA-lacZ expression. Furthermore, the creation of a bcrR::kan insertion mutant and a bcrR-lacZ transcriptional fusion revealed that bcrR is required for high-level bacitracin resistance in AR01/DGVS and that BcrR does not act to auto-regulate bcrR-lacZ expression. To study BcrR function, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni�⁺-NTA affinity and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes and BcrR binding to bcrABD promoter DNA was analysed using gel-shift assays. We demonstrated that BcrR binds the bcrABD promoter in both the absence and presence of Zn�⁺-bacitracin and that the presence of the inverted repeat regions is necessary for binding. Purification of BcrR has also enabled us to commence structural studies toward obtaining the three dimensional structure of BcrR. We obtained crystals of BcrR that diffracted poorly to a resolution of 19 Å. To characterise the function of the putative ABC type transporter (exporter) of bacitracin, BcrAB, we conducted transport assays with purified bacitracin A, fluorescently labelled FITC-bacitracin A, and radioactive ⁶�Ni�⁺-bacitracin. Transport assays conducted with radioactive ⁶�Ni�⁺-bacitracin and inverted membrane vesicles of E. coli, in which BcrABD had been recombinantly overproduced, displayed uptake of ⁶�Ni�⁺-bacitracin, suggesting that BcrAB functions as a transporter of bacitracin. We conclude that BcrR is a transmembrane DNA-binding protein that functions as both a bacitracin sensor and regulator of bcrABD expression. We propose that BcrR binds to inverted repeat regions in bcrABD promoter to regulate bcrABD expression. Expression of bcrABD results in the production of BcrAB, which appears to be capable of bacitracin efflux (i.e. uptake in inverted membrane vesicles), conferring high-level bacitracin resistance to E. faecalis.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 119-139).

Resistance to the antibiotic bacitracin in Enterococcus faecalis strain AR01/DGVS is conferred by the genes bcrABD that are under the control of the regulatory protein BcrR. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins and topological modelling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR is a novel transmembrane transcriptional regulator which acts to both sense bacitracin and initiate transcription of bcrABD. The transcription of bcrABD in the presence of bacitracin results in the expression of a putative ABC transporter, BcrAB, that may function by the efflux of bacitracin from the cell membrane. The aim of this study was to further characterise the mechanism of bacitracin resistance in E. faecalis and to conduct structural and functional studies with BcrR. A series of bcrA-lacZ transcriptional fusions were created to investigate the regulation of bcrABD transcription by BcrR and map the bcrABD promoter. We determined that BcrR activates bcrA-lacZ expression in response to bacitracin directly and not indirectly via the build-up of cell wall stress or cell wall precursors. A 69-bp region of the bcrABD promoter was required for activation of bcrA-lacZ expression. The introduction of mutations into this sequence demonstrated that two inverted repeat regions (viz. 5�-CTGACA(N)₆GTGTC-3�) were required for bcrA-lacZ expression. Furthermore, the creation of a bcrR::kan insertion mutant and a bcrR-lacZ transcriptional fusion revealed that bcrR is required for high-level bacitracin resistance in AR01/DGVS and that BcrR does not act to auto-regulate bcrR-lacZ expression. To study BcrR function, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni�⁺-NTA affinity and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes and BcrR binding to bcrABD promoter DNA was analysed using gel-shift assays. We demonstrated that BcrR binds the bcrABD promoter in both the absence and presence of Zn�⁺-bacitracin and that the presence of the inverted repeat regions is necessary for binding. Purification of BcrR has also enabled us to commence structural studies toward obtaining the three dimensional structure of BcrR. We obtained crystals of BcrR that diffracted poorly to a resolution of 19 Å. To characterise the function of the putative ABC type transporter (exporter) of bacitracin, BcrAB, we conducted transport assays with purified bacitracin A, fluorescently labelled FITC-bacitracin A, and radioactive ⁶�Ni�⁺-bacitracin. Transport assays conducted with radioactive ⁶�Ni�⁺-bacitracin and inverted membrane vesicles of E. coli, in which BcrABD had been recombinantly overproduced, displayed uptake of ⁶�Ni�⁺-bacitracin, suggesting that BcrAB functions as a transporter of bacitracin. We conclude that BcrR is a transmembrane DNA-binding protein that functions as both a bacitracin sensor and regulator of bcrABD expression. We propose that BcrR binds to inverted repeat regions in bcrABD promoter to regulate bcrABD expression. Expression of bcrABD results in the production of BcrAB, which appears to be capable of bacitracin efflux (i.e. uptake in inverted membrane vesicles), conferring high-level bacitracin resistance to E. faecalis.

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 31-37).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 31-37). Also available in print.

Univ., Diss., 2009--Kassel.

Thesis (M.Sc.)--University of Alberta, 2010. / Title from PDF file main screen (viewed on Apr. 30, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Technology, Department of Agricultural, Food and Nutritional Science, University of Alberta. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 170-182).

Thesis (M.S.)--Ohio State University. / Bibliography: leaves 57-63. Available online via OhioLINK's ETD Center.

SANTIAGO, Adriana Kelly de Sousa. Avaliação in vitro da efetividade de diferentes pas antibióticas utilizadas para curativos endodônticos sobre o E. faecalis. 2013. 48 f. Dissertação (Mestrado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2013-10-21T11:31:49Z No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-10-21T13:51:55Z (GMT) No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) / Made available in DSpace on 2013-10-21T13:51:55Z (GMT). No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) Previous issue date: 2013 / There is an agreement that in dental treatments with incomplete rizogenese it is necessary an intracanal medication with high antibacterial effect because the use of files and standard protocol based on instrumentation must be avoided to protect the fragile dental element from extra hazard. Several endodontic dressings have been used with this purpose, alone or associated to a copious irrigation procedure. However, it is not yet standardized in the literature a unique medication with protracted and excellent antimicrobial effect, associated to a feasible handling and insertion into the duct. The purpose of this study was to evaluate the in vitro effectiveness of different antimicrobial pastes currently used as intracanal medication against the pathogen Enterococcus faecalis by means of agar disk diffusion method. Accordingly, it was created a polypropylene device to simulate the release characteristics of the main root canal. The efficacy tests were performed over a period of 30 days. The formulations studied were distributed in triplicate just as follows: Triantibiotic Paste (metronidazole, ciprofloxacin and minocycline); Biantibiotic Paste (metronidazole and ciprofloxacin); Ciprofloxacin Paste; Amoxilin Paste; Calcium hydroxide paste; 0.9% saline (control group). All pastes were still allotted into 2 categories: open and closed apex. The devices remained in the medium at 37ºC over 30 days (static). Samples used for the measurement of biologic response were collected in pre-fixed times: H1(1st hour), H6(6th hour), H24(24th hour), D3(3rd day), D7(7th day), D14(14th day) and D30(30th day) in the course of thirty days. At every settled time, 20μL of solution was removed from each device and embedded on sterile paper discs with 6mm of diameter, being the equivalent volume replaced with fresh release medium. After, the discs were transferred with sterile tweezersg to the surface of agar-BHI medium previously inoculated with bacteria over petri plaques. From the devices with closed apex, it was removed 20μL of solution from above the paste surface, avoiding any directly touch on it. This collect aimed to evaluate the antimicrobial effect of different preparations inside the root canal. From the devices with open apex it was removed 20μL of the solution in which the plastic device was submerged, in order to measure the action of the diffused antibiotic to the periapical region through the apex. From the inhibition halos obtained in each case it was possible to rank the formulations in order of effectiveness. The Amoxicillin paste presented an initial antimicrobial effect more robust, keeping it throughout the experiment. However from the second week there was no more significant difference between this and the other antibiotic pastes (triantibiotic and biantibiotic). These pastes had an initial effect less representative than amoxicillin, however the effect became similar from the second week. Nevertheless, calcium hydroxide paste had a discreet effect initially and it was completely ceased already after the 3rd day. Even if Amoxicillin paste presented a superior result among the studied pastes, it was noticed color changes associated to degradation signs after two weeks. The findings show that these antibiotics pastes posed an excellent pharmacological effect, apart from calcium hydroxide. It can be concluded that all the antibiotics pastes, among them the pastes of amoxicillin, triantibiotic and biantibiotic were effective against E. faecalis. As a result its use as dressing for teeth with incomplete risogenesis in may be a good alternative. Among the advantages it is the microbial effectiveness, as well as the feasible manipulation during the clinical procedure. When comparing biantibiotic and triantibiotic pastes, the first showed to be almost equally effective, being a great choice for the clinical treatments of anterior teeth, where the pigmentation caused by minocycline antibiotic could present a drawback in the final result of the treatment. / Existe um consenso de que no tratamento de dentes com rizogênese incompleta é necessária uma medicação intracanal com máximo efeito antibacteriano, pois o uso de limas e o protocolo convencional de instrumentação devem ser evitados para que não torne o elemento dental ainda mais frágil. Diversos curativos endodônticos têm sido utilizados com este objetivo, bem como o uso de uma irrigação mais copiosa e com irrigante mais efetivo. No entanto, ainda não está padronizada na literatura uma medicação com efeito antibiótico prolongado e efetivo, além de fácil manipulação e inserção no conduto. O objetivo deste trabalho foi avaliar in vitro a eficácia de diferentes pastas antimicrobianas utilizadas como medicação intracanal sobre o patógeno Enterococcus faecalis utilizando o método de disco - difusão em Ágar. Para isso, foi confeccionado um dispositivo de polipropileno que possui características semelhantes ao canal radicular principal. Os testes de eficácia foram realizados por um período de 30 dias. Foram analisadas as seguintes formulações: Pasta triantibiótica (metronidazol, ciprofloxacino e minociclina); Pasta biantibiótica (metronidazol e ciprofloxacino); Pasta de Ciprofloxacino; Pasta de Amoxicilina; Pasta de hidróxido de cálcio; Solução fisiológica 0,9% (grupo controle). Todas as pastas foram ainda alocadas em duas categorias: ápice aberto e ápice fechado e ensaiadas em triplicata. Os dispositivos permaneceram em estufa a 37ºC durante 30 dias (sem agitação), sendo as coletas para aferição da resposta biológica realizadas em períodos preestabelecidos: H1(1ª hora), H6(6ª hora), H24(24ª hora), D3(3º dia), D7(7º dia), D14(14º dia) e D30(30º dia) no decorrer de 30 dias. A cada coleta foram retirados de cada dispositivo 20µL de solução e depositados sobre discos de papel de filtro estéreis com 6 mm de diâmetro. Em seguida, os discos de papel embebidos foram transferidos com pinça estéril para a superfície das placas de petri previamente semeadas com a bactéria. Dos dispositivos com ápice fechado foram removidos 20µL da solução que estava sobre a superfície da pasta, tendo o cuidado para não tocá-la. Este procedimento teve o intuito de medir a ação antimicrobiana direta dos diferentes preparados no interior do conduto radicular. A partir dos dispositivos com ápice aberto foram retirados 20µL da solução na qual o dispositivo plástico encontrava-se imerso, com o intuito de medir a ação do preparado difundido à região periapical. A partir dos halos de inibição obtidos, foi possível observar quais preparados foram mais eficazes com respeito ao seu efeito antibacteriano. A pasta de amoxicilina apresentou o maior efeito antimicrobiano inicial, mantendo-o durante todo o experimento, no entanto, a partir da segunda semana não houve mais diferença estatística entre as pastas antibióticas e o efeito tornou-se semelhante. As pastas triantibiótica e biantibiótica tiveram efeito inicial menor do que o da Amoxicilina, porém o efeito foi igualado a partir da segunda semana. Por outro lado, a pasta de hidróxido de cálcio teve um efeito discreto inicialmente, o qual foi totalmente cessado já após o 3° dia. Ainda que a pasta de Amoxicilina tenha apresentado os melhores resultados dentre as pastas estudadas, foi evidenciado o aparecimento de cor escurecida associada a sinais de degradação após duas semanas. Este estudo mostrou que as pastas antibióticas tiveram um excelente efeito farmacológico, ao contrário da pasta de hidróxido de cálcio. Pode-se concluir que as pastas antibióticas, dentre elas as pastas de Amoxicilina, triantibiótica e biantibiótica apresentaram um excelente efeito sobre o micro-organismo E. faecalis. Portanto, seu uso como medicação intracanal nos casos de dentes com rizogênese incompleta pode ser uma ótima alternativa, estando ainda a facilidade de manipulação durante o procedimento clínico atrelada à eficácia microbicida. Quando comparadas, a pasta biantibiótica mostrou-se quase que igualmente efetiva a triantibiótica, podendo ser uma ótima escolha em casos clínicos de tratamento de dentes anteriores, sempre e quando a pigmentação causada pelo fármaco Minociclina possa comprometer o resultado final do tratamento. Pôde-se concluir que as pastas antibióticas, dentre elas as pastas de amoxicilina, triantibiótica e biantibiótica apresentaram excelente efeito inibitório sobre o micro-organismo E. faecalis. Portanto, o seu uso como medicação intracanal nos casos de dentes com rizogênese incompleta pode ser uma excelente alternativa.