Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.

Résumé confidentiel / Résumé confidentiel

Uma infecção pulpar pode resultar na colonização microbiana de todo sistema de canais radiculares incluindo os túbulos dentinários. Estes microorganismos e seus produtos tóxicos são responsáveis pelo desenvolvimento e persistência da periodontite apical de origem endodôntica. O presente estudo objetivou avaliar a viabilidade de E. faecalis e C. albicans em túbulos dentinários após a aplicação de hidróxido de cálcio, clorexidina gel 2%, hidróxido de cálcio associado à clorexidina gel 2% e soro fisiológico, através da análise por cultura microbiológica e microscopia de fluorescência. Para tanto 120 raízes de dentes humanos foram padronizadas e autoclavadas, sendo posteriormente divididas em 2 grupos (n= 60) para contaminação com E. faecalis e C. albicans por 21 dias. Em seguida, foram divididas em 8 grupos (n= 15) para aplicação das substâncias antimicrobianas nos canais radiculares e posterior incubação em estufa por 14 dias. Amostras da dentina radicular na extensão de 0 - 100 µm e de 100 - 200 µm foram coletadas e submetidas à cultura microbiológica através do plaqueamento em meios de cultura. Após 48 horas de incubação promoveu-se a avaliação das UFC. Paralelamente, as amostras foram processadas para análise em microscopia de fluorescência com auxílio de marcadores fluorescentes específicos, a fim de se determinar a proporção de microorganismos viáveis e não viáveis. Outros 6 espécimes foram preparados para análise em MEV. Os resultados mostraram uma maior capacidade de penetração intratubular para E. faecalis quando comparado a C. albicans. A aplicação de medicação intracanal resultou em significativa redução da viabilidade dos microorganismos quando comparado ao grupo controle independente da medicação aplicada e em ambas as porções da dentina radicular avaliadas. Entretanto, ao compararmos individualmente as medicações, observamos o melhor desempenho da clorexidina gel 2 % e da associação de hidróxido de cálcio e clorexidina gel 2% sem diferença significante entre elas. O hidróxido de cálcio apresentou os piores resultados para desinfecção dos canais radiculares contaminados com E. faecalis e C. albicans. Estes achados foram confirmados tanto pela cultura microbiológica quanto pela microscopia de fluorescência. A cultura microbiológica e a microscopia de fluorescência são métodos adequados e complementares para avaliação da viabilidade de E. faecalis e C. albicans e a eficácia da clorexidina gel 2% e da associação hidróxido de cálcio e clorexidina gel 2% justificam seu uso em endodontia como medicação intracanal. / A pulp infection can result in a microbial colonization of the entire root canals system, including dentinal tubules. These microorganisms and their toxic product are responsible for the development and persistence of apical periondontitis from endodontic source. The present study aimed to evaluate E. faecalis and C. albicans viability in dentinal tubules after the application of calcium hydroxide, chlorhexidine gel 2%, calcium hydroxide associated to chlorhexidine gel 2% and physiological solution, through the analysis by microbiological culture and fluorescence microscopy. For that, 120 human teeth root were standardized and submitted to autoclave, and after ere divided into 2 groups (n=60) for E. faecalis and C. albicans contamination for 21 days. Following this, they were divided into 8 groups (n=15) for application of antimicrobial substances in the root canals and subsequent incubation for 14 days. Samples from root dentin with 0 - 100 µm and 100 - 200 µm of extension were collected and submitted to microbiological culture.After 48 hours of incubation, it was performed the evaluation of colony-forming units (CFU). At the same time, the samples were processed for fluorescence microscopic analysis with the assistance of specific fluorescent markers, in order to determine the proportion of viable and non-viable microorganisms. Other 6 specimens were prepared for the analysis of scanning electron microscopy. Results demonstrated a higher capacity of penetration in the tubules for E. faecalis than for C. albicans. The application of intracanal medication resulted in significant reduction of microorganisms viability when compared to the control groups independently of the medication used and in both evaluated portions of root dentin. However, when one compares individually the medications, it was observed a better performance of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% without a significant difference between them. Calcium hydroxide had the worst results for disinfection of root canal contaminated with E. faecalis and C. albicans. These findings were confirmed for the microbiological culture as well as for the fluorescence microscopy. The microbiological culture and the fluorescence microscopy are adequate methods and complementary for the evaluation of E. faecalis and C. albicans viability and the efficacy of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% warrant their use in Endodontics as an intracanal medication.

Die im Biogasprozess anfallenden Gärreste werden oftmals als Wirtschaftsdünger verwendet. Krankheitserreger, die sich in den Gärresten befinden können über die Düngung in die Lebensmittelkette gelangen. Die Möglichkeit einer Vermehrung von Bakterien in den Biogasanlagen sowie deren Ausbreitung schürt die Bedenken der Öffentlichkeit. Das Ziel dieser Arbeit war es, Nachweismethoden für die Untersuchung von Proben aus Biogasanlagen zu etablieren, die Praxistauglichkeit dieser anhand von Proben aus Biogasanlagen zu überprüfen und die mikrobielle Belastung dieser Proben hinsichtlich ausgewählter Indikatorkeime zu erfassen. Bei den Indikatorkeimen handelte es sich um Clostridium perfringens, Clostridium botulinum, Enterokokken, Escherichia coli, ESBL-bildende Enterobaceriaceae und Salmonellen. Für die Etablierung der bakteriologischen Nachweismethoden wurde autoklavierter Gärrest mit einer definierten Keimmenge beimpft und auf verschiedene Nährmedien aufgebracht. Diese wurden bebrütet, ausgezählt und die KbE/ml berechnet. Mittels Probitanalyse wurde für jedes Medium die untere Grenze für den Nachweis aus beimpftem Gärrest bestimmt. Bei den Nährmedien handelte es sich um Brilliance™ Salmonella Agar, XLT4 Agar und XLD Agar für den Nachweis von Salmonella spp. Für E. coli wurden Tergitol 7 Lactose TCC Agar und Brilliance™ E. coli/Coliform Selektiv Agar verwendet. Der Nachweis von Enterokokken erfolgte mittels Slanetz Bartley Agar und Enterococcus Selektivagar. Für die ESBL-bildenden Enterobacteriaceae wurde der Brilliance™ ESBL Agar eingesetzt. Die getesteten Nährmedien zum Nachweis von C. perfringens waren Membran Clostridium Perfringens (mCP) Selektivnährboden sowie Tryptose Sulphite Cycloserine (TSC) Agar überschichtet mit TSC Agarbasis. Für C. botulinum erfolgte der Nachweis auf Eigelb Laktose Agar. Darüber hinaus wurde eine PCR zur C. perfringens Toxintyp-Bestimmung nach dem Protokoll von VAN ASTEN et al. (2009) etabliert. Zum Nachweis von C. botulinum wurde die PCR nach dem Protokoll von HILL et al. (2010) eingesetzt. Bei der Untersuchung der Praxistauglichkeit wurden Proben aus zehn Biogasanlagen des Freistaates Sachsen entnommen und untersucht. Hierbei handelte es sich um Proben aus Abschnitten vor, während und nach der Fermentation. Anhand der ermittelten Nachweisgrenze sowie der Handhabung wurden die folgenden Nährmedien für die Untersuchung der Biogasanlagen-Proben ausgewählt: Brilliance™ Salmonella Agar, XLT4 Agar, Brilliance™ E. coli/Coliform Selektiv Agar, Slanetz Bartley Agar, Brilliance™ ESBL Agar, TSC Agar überschichtet mit TSC Agarbasis und Eigelb Laktose Agar. Für die Anzucht anaerober Bakterien wurden die Proben vor der Beimpfung der Agarplatten erhitzt. Zudem erfolgte eine Anreicherung des zuvor erhitzten Probenmaterials in TPYG Bouillon. Diese wurde genutzt, um daraus aufgereinigte DNA mittels PCR auf C. botulinum und C. perfringens zu untersuchen. Die verwendeten Nährmedien wurden im Praxistest positiv evaluiert. Die Ergebnisse für die Proben aus den Biogasanlagen zeigten, dass, mit Ausnahme von C. perfringens, alle Indikatororganismen während des Biogasprozesses einer Reduktion unterlagen. Die durchschnittliche anaerobe Lebendkeimzahl belief sich auf 107 bis 108 KbE/g Probe. E. coli erfuhr eine Reduktion um bis zu vier Zehnerpotenzen. Enterokokken wurden um 1 bis 2 log10 Stufen reduziert. ESBL-bildende Enterobacteriaceae konnten in sechs der zehn Biogasanlagen nachgewiesen werden. Hierbei handelte es sich überwiegend um E. coli und Klebsiella spp. In keiner der Proben konnten Salmonellen oder C. botulinum nachgewiesen werden. Typ A war der am häufigsten nachgewiesene C. perfringens-Toxintyp. Das β2-Toxin-Gen wurde in 20 Fällen nachgewiesen. Einmal konnte C. perfringens Typ C, β2-Toxin-Gen-positiv detektiert werden. Der hygienische Status der Gärreste entsprach in etwa dem hygienischen Status von Gülle. In Abhängigkeit vom Indikatorkeim war eine Verbesserung des Status durch eine Reduktion der Keimzahl festzustellen.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this study was to investigate the antibacterial efficacy of 0.12-percent and 2.0-percent chlorhexidine gluconate (CHX) on eliminating Enterococcus faecalis from dentinal tubules, and whether this antibacterial effect was enhanced by heat. To date there have been no published articles that describe the heating of 2.0-percent CHX and its antimicrobial efficacy and clinical relevance towards E. faecalis within dentinal tubules in root canal systems. Ninety-five human extracted, single rooted, maxillary, anterior teeth were used to prepare dentin disk specimens. After proper sterilization, a 2.5-mm ISO-sized diameter lumen was prepared, and then the canals were filled with brain-heart infusion (BHI) broth infected with E. faecalis. The BHI was removed and the specimens in equally divided groups were rinsed with sterile saline and filled with saline, or 0.12 percent CHX or 2.0 percent CHX at ambient temperature (24°C) or experimental temperature (46°C) and incubated at oral temperature (37°C) or the experimental temperature (46°C), respectively. The specimens were frozen to -70˚C and pulverized in liquid nitrogen. Serial dilutions were prepared of 1:100 and 1:1000 and spiral plated on BHI agar plates in duplicate. They were incubated, and the number of bacterial colonies was recorded 24 hours later for data analysis. A two-way analysis of variance (ANOVA), with factors for solution, solution temperature, and the solution-by-temperature interaction was used to determine antibacterial efficacy. Pair-wise comparisons between groups were examined for significance using the Fisher’s Protected Least Significant Differences Method. The E. faecalis CFU were log-transformed to satisfy the assumptions required for the ANOVA. The results of this investigation demonstrated no statistically significant difference with the addition of heat to either test irrigation solution regarding the elimination of E. faecalis from dentinal tubules within the root canal system. There was a statistically significant difference in the antibacterial efficacy of CHX against E. faecalis in comparison with the concentration tested. A higher concentration of 2.0-percent CHX demonstrated a significantly higher antibacterial efficacy against E. faecalis compared with 0.12-percent CHX, and likewise with the saline control. It can be concluded that the use of a higher concentration of 2.0-percent CHX is advantageous as a final irrigation solution after copious amounts of NaOCl and EDTA have been utilized for effective antimicrobial efficacy and substantivity.

No description available.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-31Bitstream added on 2014-06-13T20:01:21Z : No. of bitstreams: 1 cardoso_mg_dr_sjc.pdf: 573962 bytes, checksum: 3dafa4a7dc0b8d00f8736dd5f731d5cd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Primeiramente foi avaliada no estudo a ação antimicrobiana da água ozonizada frente suspensão composta por Candida albicans e Enterococcus faecalis, verificando o tempo necessário de ozonização da água para eliminação destes microrganismos. Nas próximas etapas, foi avaliada a eficiência da água ozonizada como agente irrigante, durante o preparo biomecânico, tanto na eliminação de Candida albicans e Enterococcus faecalis, assim como na neutralização de LPS de Escherichia coli inoculados no interior de canais radiculares. Foram utilizados 48 dentes humanos unirradiculados, sendo que nos canais radiculares de 24 espécimes, foram inoculados 20 mL de uma suspensão contendo C. albicans e E. faecalis, e nos outros 24 espécimes, foram inoculados 10 mL LPS de E. coli, verificando-se a ação da água ozonizada como agente irrigante durante o preparo biomecânico. Foram realizadas coletas das amostras (imediata e após sete dias da instrumentação), e os dados foram submetidos à análise estatística. Como resultados, pode-se observar que a ação antimicrobiana da água ozonizada por dez minutos foi efetiva frente à suspensão microbiana. Como agente irrigante durante o preparo biomecânico, a água ozonizada apresentou efetividade frente à suspensão de C. albicans e E. faecalis inoculada no interior dos canais radiculares na coleta imediata, porém, na segunda coleta apresentou pouco efeito residual. Na verificação da neutralização de endotoxina, a ação da água ozonizada como agente irrigante não apresentou efetividade na neutralização de LPS de E. coli inoculada nos canais radiculares, sendo que quantidades restantes de LPS apresentaram efeitos biológicos. Como conclusão, a água ozonizada foi eficiente na redução de C. albicans e E. faecalis inoculada no interior dos canais radiculares, mas não apresentou efeito sobre LPS de E. coli. / At first, the study was to evaluated the antimicrobial action of the ozonized water before the suspension composed of Candida albicans and Enterococcus faecalis, checking the necessary time of ozonization of the water in order to eliminate these microorganisms. In the next stages, the efficiency of ozonized water as an irrigant agent was evaluated, during the biomechanical preparation, as in the elimination of C. albicans and E. faecalis, as well as in the neutralization of Escherichia coli LPS inoculated into the root canals. Were used 48 single-rooted human teeth, seeing that in the root canals of 24 specimens, it were inoculated 20 mL of a suspension containing C. albicans and E. faecalis, and on the other 24 specimens, it were inoculated 10 mL of Escherichia coli LPS, having been checked the action of the ozonized water as an irrigant agent during the biomechanical preparation. Collections of samples (immediately and after seven days of the instrumentation) were carried out, and the data was submitted to statistical analysis. As a result, one could observe that the antimicrobial action of the ozonized water for ten minutes was effective before the microbial solution. As an irrigant agent during the biomechanical preparation, the ozonized water presented effectiveness before the suspension of C. albicans and E. faecalis inoculated into the root canals in the immediate collection, however, in the second collection it presented little residual effect. When verifying the endotoxin neutralization, the action of the ozonized water as an irrigant agent did not present effectiveness in the neutralization of E. coli LPS inoculated in the root canals, seeing that the remaining quantities of LPS presented biological effects. It was concluded that, the ozonized water was efficient in the reduction of C. albicans and E. faecalis inoculated inside the root canals, but it did not present any effect over E. coli LPS.

DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p> Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p> Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p> Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p> This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p> Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p> Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p> Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p> This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

Metabolic were used to evaluate Enterococcus as an indicator of faecal pollution. Enterococci were isolated using m-Enterococcus agar and speciated using conventional biochemical tests. Forty percent of the isolates were identified and metabolically characterized by the automated Biolog system. The biochemical test scheme recognized 16 enterococcal species, while Biolog recognized nine. Both methods identified E. faecalis at the greatest frequency. Overall species frequencies varied between the two methods. Biolog was unable to identify 31% of the isolates; 7% of the isolates were unidentified by the biochemical test scheme. Of the identified isolates, metabolic profiling with Biolog achieved speciation with 60 substrates. Unique profiles were obtained for 89% of the isolates. Isolates also demonstrated inter-trial differntial metabolism of substrates. This and the large number of unidentified isolates suggest great diversity among enterococci. Diversity and inter-trial metabolic inconsistencies will complicate use of enterococcal metabolic profiles as a source-tracking tool. / xxiii, 264 leaves ; 29 cm.