A library of 18 coliform bacteria strains was obtained from different sampling points in the drinking water distribution system in Lexington, KY, over a three month period in 2006. To investigate the cause of the coliform occurrence we conducted a microbial source tracking study using phenotypic (API 20E, Biolog, and Vitek) and genotypic (pulsed field gel electrophoresis (PFGE) and ribotyping) analyses to determine the degree of genetic variation among isolates. Characterization of isolates by PFGE and ribotyping showed that coliform events in the distribution system were related and a regrowth problem may exist due to biofilm formation. The ability of a persistent Enterobacter cloacae strain to adhere and form biofilm was found to depend on environmental conditions such as temperature, pipe material, soiled surface, chlorine and nutrient levels with higher temperatures and nutrient levels promoting adherence. Considerable variation in adherence and biofilm formation was observed among representative Enterobacter isolates.

Lactobacillus- Spezies, die zur Gattung der Milchsäurebakterien gehören, haben bereits hemmende Eigenschaften gegen Candida albicans gezeigt. Dieser dimorphe Hefepilz ist einer der bedeutendsten Erreger von Pilzinfektionen beim Menschen und einer der häufigsten Verursacher Katheter- assoziierter Infektionen. Eine bedeutende Rolle bei der Pathogenität von C. albicans spielt die Biofilmbildung, die sowohl die körpereigene Abwehr als auch die antimykotische Therapie einer invasiven Infektion erheblich erschwert. Zu den Virulenzfaktoren zählt eine Vielzahl von Genen, darunter auch die sekretorischen Aspartylproteasen (SAPs), die zur Infektion sowohl in vitro als auch in vivo beitragen. In der vorliegenden Arbeit wurde der Einfluss verschiedener Lactobacillus- Stämme auf die Biofilmbildung des invasiv pathogenen C. albicans SC 5314 und des in der Pathogenität abgeschwächten Stammes ATCC 10231 untersucht, sowohl phänotypisch als auch hinsichtlich der metabolischen Aktivität durch den semi- quantitativen XTT- Reduktions- Assay. Zudem erfolgten Expressionsanalysen ausgewählter Gene von C. albicans, deren Zusammenhang mit der Biofilmbildung und Pathogenität bekannt ist. Dabei konnte gezeigt werden, dass L. johnsonii DSM 10533 die metabolische Aktivität beider C. albicans- Stämme erheblich verringern kann (um bis zu 80%) und auch einen phänotypisch drastisch reduzierten Biofilm verursacht. In Anwesenheit dieses Stammes kam es zu stark verringerter Aktivität der beobachteten SAP- Gene vor allem des invasiven Stammes C. albicans SC 5314. Andere Pathogenitäts- assoziierte Gene wie Als 3 und Hwp 1 wurden dagegen eher hochreguliert. L. rhamnosus DSM 20021 und ein klinisches Isolat verursachten ebenfalls eine Verringerung der metabolischen Aktivität, sorgten phänotypisch aber eher für vermehrte Hyphenbildung des Pilzes. Ersterer verursachte eine deutlich reduzierte Aktivität von Hwp 1 und Ume 6 bei C. albicans ATCC 10231. L. reuteri DSM 20016 zeigte keinen signifikanten Einfluss auf Biofilmbildung, Aktivität und Genexpression der beobachteten C. albicans- Stämme. Diese Ergebnisse zeigen deutlich, dass unterschiedliche Lactobacillus- Stämme sich in ihrem Einfluss auf C. albicans erheblich unterscheiden. Auch die Reaktion verschiedener C. albicans- Stämme auf Lactobacillus- Spezies ist sehr verschieden. In dieser Arbeit zeigte L. johnsonii DSM 10533 ein deutliches Potential, C. albicans in der Biofilmbildung und Expression von Pathogenitätsfaktoren zu hemmen. Dieser Stamm erscheint damit für weiterführende Untersuchungen hinsichtlich probiotischen Potentials geeignet. Die Ergebnisse einer Lactobacillus Spezies können nicht generell auf andere Lactobacillus Spezies übertragen werden. Ob sich innerhalb einer Spezies alle Stämme gleichermaßen verhalten, bedarf ebenfalls der Überprüfung. Die Ergebnisse dieser Arbeit werfen auch die Frage auf, ob Lactobacillus Spezies sogar die Pathogenität von C. albicans erhöhen können.

Salmonella enterica is an important cause of food poisoning and is responsible for approximately a billion human infections each year. Disease manifestation in humans varies from severe systemic enteric (typhoid) fever to self-limiting gastroenteritis depending upon the infecting S. enterica serovar. S. Typhimurium is responsible for acute gastroenteritis in humans but causes a typhoid-like disease in mice and thus serves as an important model for studying the pathogenesis of systemic salmonellosis. Following ingestion, S. Typhimurium employs a variety of virulence mechanisms to survive within its host and establishes infection in the intestinal tract by invading the epithelial cells. Recent studies have revealed the importance of sulfur compounds in the intestine, such as tetrathionate and thiosulfate for the disease progression. S. Typhimurium is capable of utilising these sulfur compounds as terminal electron acceptors for its anaerobic respiration and thus gains a growth advantage over host microbiota during infection. However, the regulation of sulfur availability within S. Typhimurium and the mechanisms involved in mitigating cellular sulfide toxicity are not well-defined. During this study, we have identified the sstRA operon in S. Typhimurium encoding a deduced SmtB/ArsR family of transcriptional regulatory protein (SstR) and a deduced rhodanese-family sulfurtransferase (SstA) and demonstrated a role in mitigating the effects of cellular sulfide toxicity. SstR has been confirmed to act as a transcriptional repressor from the sstRA operator-promoter and the SstR-dependent repression is alleviated by low pH and sulfide stress (sodium thiosulfate), consistent with a role for SstR in sensing sulfide stress to trigger gene expression. Electrophoretic mobility shift assays confirm binding of purified SstR to the sstRA operator-promoter region. Furthermore, a conserved pair of cysteine residues within SstR was identified to be crucial for alleviating SstR-mediated repression, with the substitution of either cysteine causing constitutive repression. This is consistent with SstR inducer-responsiveness involving a thiol-based redox switch. Importantly, S. Typhimurium mutants lacking the sstRA operon have reduced tolerance to sulfide stress, consistent with the sstRA operon having a role in cellular sulfide detoxification. Work is continuing to further characterise the roles of sstR and sstA in S. Typhimurium on their contributions to infections.

Proteins can often adsorb irreversibly at fluid/fluid interfaces; the understanding of the adsorption mechanism has relevance across a variety of industrial (e.g. the creation of stable emulsions) and biological (e.g. biofilm formation) processes. I performed molecular dynamics simulations of two surfactant proteins as they interact with air/water and oil/water interfaces, describing the origin of the surface activity, the adsorption dynamics and the conformational changes that these proteins undergo at the interface. BslA is an amphiphilic protein that forms a highly hydrophobic coat around B. subtilis biofilms, shielding the bacterial community from an external aqueous solution. By investigating the behaviour of BslA variants at oil/water interfaces via coarse-grained molecular dynamics, I show that BslA represents a biological example of an ellipsoidal Janus nanoparticle, whose surface interactions are controlled by a local conformational change. All-atom molecular dynamics simulations then reveal the details of the conformational change of the protein upon adsorption, and the self-assembly into a two-dimensional interfacial crystal. Ranaspumin-2 is one of the main components of the tungara frog foam nest. Contrary to most surfactant proteins, its structure lacks any sign of amphiphilicity. All-atom simulations show that the adsorption proceeds via a two-step mechanism where firstly the protein binds to the interface through its flexible N-terminal tail and then it undergoes a large conformational change in which the hydrophobic core becomes exposed to the oil phase. I then developed a simple structure-based coarse-grained model that highlights the same adsorption mechanism observed in all-atom simulations, and I used it to compare the dynamics of adsorption and the underlying free energy landscape of several mutants. These results agree with and are used to rationalise the observations from Langmuir trough and pendant drop experiments. Colloids can often be considered simpler versions of proteins that lack conformational changes. I performed coarse-grained simulations of the compression of interfacial monolayers formed by rod-like particles. These simulations show a rich behaviour characterised by the flipping of adsorbed rods, nematic ordering and bilayer formation. I report the series of transitions that take place as the rod aspect ratio is increased from 3 to 15.

Resumo: Os cateteres venosos centrais são muito usados nas Unidades de Terapia Intensiva (UTIs). O seu uso está freqüentemente associado a complicações incluindo infecções fatais. Durante o período de janeiro de 2004 a janeiro de 2005, foram analisadas 118 pontas de cateteres e 42 amostras de sangue provenientes de 100 pacientes hospitalizados em Unidade de Terapia Intensiva de um hospital da região Noroeste do estado de São Paulo. O objetivo deste estudo foi analisar, por meio de cultura e microscopia eletrônica de varredura, a colonização do cateter intravenoso central de vialon Intracath de lúmem único. Para detectar a produção de slime dos microrganismos isolados foi usado o método de Christensen et al., (1985) e o perfil de sensibilidade aos antimicrobianos de acordo com o (NCCLS-M2-A7-2000). Das 118 pontas de cateteres estudadas pelo método semi-quantitativo, 34 (28,8%) estavam colonizadas (d15 UFC/placa) em que foram isolados 55 microrganismos. Desses, 32 (58,2%) foram classificados como Gram-positivos com freqüência maior para 15 (27,3%) Staphylococcus aureus, seis (10,9%) Staphylococcus epidermidis; 19 (34,5%) classificados como Gram-negativos com freqüência maior para seis (10,9%) Acinetobacter baumannii, três (5,4%) Pseudomonas aeruginosa, Enterobacter aerogenes respectivamente e ainda quatro (7,3%) classificadas como leveduras sendo duas (3,6%) Candida albicans e duas (3,6%) Candida parapsilosis...(Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The aim of the present study was to evaluate the hepatotoxicity, pharmacokinetic parameters and biotransformation of isoniazid when rats were treated with isoniazid (INH); rifampicin (RMP); and INH + RMP. Daily doses of the tuberculostatic drugs were administrated intragastrically to the animals (Wistar rats) for one period of 21 days as follow: sterile water (group I, control); INH (100mg/Kg) (group II), RMP (100mg/Kg) (group III); INH (100mg/Kg) + RMP (100mg/Kg) (group IV). The serum levels of the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined before the administration of the drugs (basal) and after the 21 days treatments. On day 21, blood samples were obtained before and 15þ; 30þ; 45þ; 60þ; 1,5; 3h; 6h; 12h and 24 hours after the dose. (five animals for each point). The blood samples were deproteinized with 10% trichloroacetic acid, derivatized by 1% cinnamaldehyde and analyzed by liquid chromatograph. For the determination of the acetylated metabolites acetylisoniazid (AcINH) and acetylhydrazine (AcHz) a previous hydrolysis with 6 M hydrochloride acid was performed. The results are presented as mean and SEM. ...(Complete abstract, click electronic address below). / Orientador: Elisabeth Loshchagin Pizzolitto / Coorientador: Antonio Carlos Pizzolitto / Banca: Taís Maria Bauab / Banca: Sérgio Aparecido Torres / Banca: Sergio Eduardo Longo Fracalanza / Banca: Beatriz Ernestina Cabilio Guth / Doutor

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Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-08-22T17:02:49Z No. of bitstreams: 2 Dissertação - Suellen Rocha Araújo Castilho - 2016.pdf: 2584243 bytes, checksum: b73318ec0bcbfffa7eb7b840a656cda0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2016-08-22T17:05:00Z (GMT) No. of bitstreams: 2 Dissertação - Suellen Rocha Araújo Castilho - 2016.pdf: 2584243 bytes, checksum: b73318ec0bcbfffa7eb7b840a656cda0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-22T17:05:00Z (GMT). No. of bitstreams: 2 Dissertação - Suellen Rocha Araújo Castilho - 2016.pdf: 2584243 bytes, checksum: b73318ec0bcbfffa7eb7b840a656cda0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The species Acinetobacter baumannii is the most important representative of the genus, being globally considered the pathogen of larger relevance, within the complex A. baumannii – A. calcoaceticus, to healthcare institutions worldwide. Its clinical significance has increased in recent years partly due to its notable capacity of adherence to surfaces due to presence of structures such as pili, production of EPS (exopolysaccharides) and presence of genes that favor biofilm formation, providing larger adhesion between the cells and consequent resistance to the antimicrobials. Another important characteristic is its capacity to regulate or to acquire antibiotic resistance, specifically through oxacilinases, encoded by the gene OXA, that presents potent hydrolytic activity against penicilinase-resistant penicillins. Until the moment, 250 types of OXA were already described. The expression of OXA genes can be increased in the presence of the insertion element ISAba1, which when located upstream of the gene, confer a selective advantage for the isolate when this depends on the efficient expression of a variety of resistance genes. In its absence, the resistance is usually expressed in the presence of two or more OXA genes. The capacity of biofilm formation and the presence of the genes blaPER-1, OXA-23, OXA-40, OXA- 51, OXA-58 and ISAba1 were investigated among 84 isolates from 64 patients of the Intensive Care Units of Goiânia. It was observed that 78.5% of the isolates formed biofilm, of which 23% presented the gene blaPER-1. The presence of OXA-51 gene was detected in all isolates, whereas 64.3% of the isolates had the OXA-23 gene and 24.0% of the isolates had the OXA-58 gene. The ISAba1 gene was detected upstream of the OXA-51, OXA-23 and OXA-58 genes in 33.3% (28/84), 92.6% (50/54) and 10.0% (2/20) of the isolates, respectively. None of the isolates studied presented the gene OXA-40. Of the 62 isolates resistant to imipenem and meropenem, 76.0% possessed the OXA-23 gene while 24.2% of the isolates possessed the OXA-58 gene. Of these isolates, 79.0% (49/62) presented the gene ISAba1. In the present study, the blaPER-1 gene presence was not associated with the biofilm formation, while the presence of OXA genes was related to resistance in Acinetobacter baumannii. / A espécie Acinetobacter baumannii é a representante mais importante do gênero, sendo considerado o patógeno de maior relevância, dentro do complexo A. baumannii – A. calcoaceticus, para as instituições de saúde mundialmente. Sua importância clínica tem aumentado nos últimos anos em parte devido a sua notável capacidade de aderência a superfícies devido a presença de estruturas como pili, produção de EPS (exopolissacarídeo) e presença de genes que favorecem a formação de biofilme, proporcionando maior adesão entre as células e consequente resistência aos antimicrobianos. Outro destaque é sua capacidade de regular ou adquirir resistência, em particular por meio das oxacilinases, codificadas pelo gene OXA, que apresentam atividade hidrolítica potente contra penicilinas resistentes às penicilinases. Até o momento, 250 tipos de OXA já foram descritas. A expressão de genes OXA pode ser aumentada na presença do elemento de inserção ISAba1, localizado à montante do gene, conferindo uma vantagem seletiva para o isolado quando este depende da expressão eficiente de uma variedade de genes de resistência a antibióticos. Na sua ausência, a resistência geralmente é expressa na presença de dois ou mais genes OXA. A capacidade de formar biofilme e a presença dos genes blaPER-1, OXA-23, OXA-40, OXA-51, OXA-58 e ISAba1 foram investigados em 84 isolados provenientes de 64 pacientes de UTIs de Goiânia. Observou-se que 78,5% dos isolados formaram biofilme, dos quais 23% apresentaram o gene blaPER-1. A presença do gene OXA-51 foi detectada em todos os isolados, enquanto que 64,3% dos isolados apresentavam o gene OXA-23, e em 24% dos isolados foi encontrado o gene OXA-58. O gene ISAba1 foi detectado à montante dos genes OXA-51, OXA-23 e OXA-58, em 33,3% (28/84), 92,6% (50/54) e 10% (2/20) dos isolados, respectivamente. Nenhum isolado estudado apresentou o gene OXA-40. Dos 62 isolados resistentes ao imipenem e meropenem, 76% dos isolados possuíam o gene OXA-23 enquanto que 24,2% possuíam o gene OXA-58. Dentre estes isolados, 79% (49/62) apresentaram a sequência de inserção ISAba1. No presente estudo, a presença do gene blaPER-1 não foi associada com a formação de biofilmes, enquanto que a presença de genes OXA estava relacionada à resistência em Acinetobacter baumannii.

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<p dir="ltr">Mycobacterium abscessus (Mab) is a type of mycobacterium that is known for its remarkable resistance to a variety of antibiotics. This pathogen poses a significant risk for individuals with weakened immune systems as it can cause skin and soft tissue infections, pulmonary disease and disseminated infections. Mab's ability to expel antibiotics through efflux pumps and form strong biofilms makes it even more challenging to treat infections. Lipids form a major part of the extracellular matrix of Mab biofilms. Efflux pumps have been shown to export lipids to the cell surface. Despite ongoing research into Mab's antibiotic tolerance, there is still much to learn about the impact of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis during biofilm development in Mab. In this study, we investigated the impact of the EPIs; CCCP (carbonyl cyanide m-chlorophenyl hydrazone), piperine (PIP), reserpine (RES), berberine (BER), and verapamil (VER) on efflux activity, biofilm formation, antibiotic resistance, and lipid biosynthesis in Mab during planktonic and biofilm growth conditions. We found that Mab cells had a higher tolerance to EPIs in biofilm-stimulating medium and that the presence of EPIs led to a decrease in minimum inhibitory concentrations of frontline antibiotics, reduced efflux activity within Mab cells, and significantly inhibited biofilm formation. We examined the effects of EPIs that inhibited biofilm formation on lipid metabolism in Mab using radiolabeling with 14C?palmitic acid and 14C-acetic acid which are precursors of lipid biosynthesis. We observed that the EPI berberine inhibited the incorporation of 14C-palmitic acid into glycopeptidolipids in the surface lipids of planktonic cells and increased cellular glycopeptidolipid (GPL) in biofilm cells. Verapamil-treated cells showed a 55 % increase in cellular trehalose monomycolate (TMM) compared to controls. Piperine-treated cells exhibited a 50 % increase in cardiolipin. The incorporation of 14C-acetate into biofilm cells showed that piperine-treated biofilm cells showed a 146 % increase in surface glycopeptidolipids. Overall, our study enhances our understanding of lipid biosynthesis in Mab, the effects of EPIs on Mab biofilms, efflux mechanisms, and antibiotic resistance and offers insights for combating Mab-related infections.</p>