In the UK, there are up to 3,000 cancer patients who have undergone total laryngectomy and use voice prostheses (VPs), for speech rehabilitation. VPs are inserted between the eosphagus and trachea to provide a 'voice' but, as with other semi-in-dwelling devices, such as nasal and gastric tubes, they invariably fail due to occlusion by microbial biofilms. The requirement for frequent replacement is financially costly to the NHS and impacts patient well-being. Replacement frequency varies by patient, from 7 weeks to 6 months but the reasons for this variation are not clearly understood. By designing and implementing a study of microbial colonisation of VPs and oral rinse samples submitted by 14 Speech and Voice Clinic patients over 13 months, this study explored whether specific microbes or patient factors, including the use of antacids, antibiotics and nystatin, along with denture-use, were potential predictors of device longevity. Focussing on the role of the commonly isolated fungi, we sought to understand the role of diet or the presence of the bacterium, S. aureus, in biomass accumulation. We also asked whether biofilm regulation pathways are shared across the fungi and could constitute a potential target for therapeutics. Microbial isolation from 66 VPs showed the predominant species as described in previous studies, but each participant had a unique profile which persisted over time, with half of the microbes originating from the oral flora. Clinic-based participants experienced fewer problems, primarily due to the device type used, and carried fewer species of Gram negative bacteria than the long term users. Statistical analysis showed that patient medication influences biofilm composition and dietary sugars differentially affect biomass formation. In vitro experiments showed that the ability to treat Candida biofilms with nystatin was improved in the presence of S. aureus. Expression analysis showed that regulation of biofilms in C. parapsilosis was the closest to that of C. albicans but that the extent of protein homology to C. albicans regulators was not a predictor of expression levels. It is therefore unlikely that a single therapeutic could be developed to target biofilm gene regulation. This work provides new insights into the complexity of biofilm formation in voice prosthesis users but reveals associations between microbes, diet, prosthesis type and medications that might be used to advise patients and help to reduce the stress and cost of frequent device failure and replacement.

Orientador: Ana Claúdia Pavarina / Banca: Eunice Teresinha Giampaolo / Banca: Ana Paula Dias Ribeiro / Resumo: A necessidade de superar o desafio criado pelos biofilmes resistentes aos tratamentos antimicrobianos convencionais tem levado à busca por tratamentos alternativos, como terapia fotodinâmica antimicrobiana (aPDT). Este estudo avaliou in vitro a eficácia da aPDT na inativação de biofilmes de Staphylococcus aureus suscetíveis e resistentes à meticilina (MRSA e MSSA), mediado pelos fotossensibilizadores (PSs) Curcumina (Cur) e Photodithazine® (PDZ). Biofilmes foram formados e tratados com diferentes concentrações de Cur (0, 20, 40 e 80 μM) e PDZ (0, 50 e 75 mg/L), e iluminados ou não por fonte de luz LED (Cur 455 ± 3 nm/ 5,28 J/cm2; PDZ 660 ± 3 nm/ 5,28 J/cm2 ou 50 J/cm²). Os grupos Controle Positivo (CP) não receberam nenhum PS e também não foram iluminados. A viabilidade dos micro-organismos após a aPDT foi avaliado pelo número de colônias viáveis, pelo ensaio de XTT e pela utilização do kit LIVE/DEAD® na Microscopia Confocal de Varredura à Laser (MCVL). Os resultados foram avaliados por análises de variância de dois fatores de efeitos fixos (ANOVA) e complementados por comparações múltiplas de médias pelo teste de Tukey. Para ambas as cepas, todas as concentrações de Cur e PDZ testadas reduziram significativamente a atividade metabólica e o UFC/mL para ambos micro-organismos quando comparado com os grupos CN (p0,05). Os resultados foram otimizados para a Cur quando utilizou-se a maior concentração (80 μM), para a PDZ, a maior redução nos micro-organismos foi observada quando associou-se a maior concentração de PDZ (75 mg/L) com a maior dose de luz (50 J/cm²). Os biofilmes submetidos a aPDT demostraram pela MCVL um maior número de células coradas em vermelho, indicando que a aPDT foi eficaz para promover danos ou morte às células bacterianas. Assim, a aPDT pode ser considerada promissora para atuar de forma sinérgica no tratamento de infecções bacterianas / Abstract: The need to overcome the challenge created by biofilms regarding conventional antimicrobial approaches has lead to search of alternative treatments such as Antimicrobial Photodynamic Therapy (aPDT). This in vitro study evaluated the efficacy of aPDT using the photosensitizer (PS) Curcumin (Cur) and Photodithazine® (PDZ) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA). Biofilms were treated with different Cur (0, 20, 40 or 80 μM of Cur) and PDZ concentrations (0, 50 or 75 mg/L) and illuminated or not with LED source (Cur 455 ± 3 nm/ 5.28 J/cm2; PDZ 660 ± 3 nm/ 5.28 J/cm2 or 50 J/cm²). Positive control samples were not exposed to PS or light. The microorganisms viability after aPDT were evaluated by counting the number of colonies, the XTT assay and LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). The results were evaluated by analysis of variance, two-factor fixed effects (ANOVA) and complemented by multiple comparisons by Tukey test. For both strains, all the tested Cur and PDZ concentrations reduced significantly both biofilm metabolic activity and CFU/mL compared to the negative control (p0.05). Moreover, the results were optimized for Cur when the higher concentration was used (80 μM); For PDZ, the best results were obtained when it was associated a higher concentration of PDZ (75 mg/L) with the higher dose of light (50 J/cm²). Biofilms submitted to aPDT showed a large number of red-stained colonies, indicating that this therapy was efficient in disrupting the bacterial membrane. It can be concluded that PS was efficient in reducing viable colonies of both S. aureus strains by damaging cell membrane and causing cell death. Thus, the aPDT is can be considered promising to act synergistically in the treatment of bacterial infections / Mestre

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Diane S. Herson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M. Sc.)(Microbiology)--University of Pretoria, 2005. / Includes summary. Includes bibliographical references (leaves 87-108). Available on the Internet via the World Wide Web.

Thesis (M.S. in chemical engineering)--Washington State University, December 2009. / Title from PDF title page (viewed on Jan. 21, 2010). "Department of Chemical Engineering." Includes bibliographical references (p. 28-31).

To effectively minimize biofilms, which are prevalent in chlorinated drinking water distribution systems, the effect of biofilm age on antimicrobial tolerance and resistance must be investigated. It was our hypothesis that antimicrobial tolerance emerges quickly during biofilm development and that both antimicrobial tolerance and resistance increase as the biofilm ages. We further hypothesized that antimicrobial tolerance and resistance vary among the individual community members. In this study, young and mature biofilms of Pseudomonas aeruginosa, a model biofilm organism, were grown and exposed to antimicrobial agents in several different treatments. Results showed that the increased antimicrobial tolerance of intact biofilms compared to planktonic cells arises early (i.e., within hours) in biofilm development. The short-term tolerance of resuspended mature biofilm cells to antimicrobial agents peaked at a biofilm age of 14 days and subsequently declined; the peak and decline in antimicrobial tolerance may be related to periodic detachment events in the biofilm. The antimicrobial resistance of resuspended mature biofilm cells continuously exposed to antimicrobial agents increased with biofilm age. Furthermore, individual members in mature biofilm communities exhibited variation in antimicrobial tolerance, thereby highlighting divergence of the biofilm community from the original parent strain.

published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

No description available.

Nine distinctive bacteria were isolated from a diclofop-methyl-degrading-consortium biofilm and their genomic DNA extracted for genetic characterization. With one exception, all the isolates contained plasmid(s). The consortium degrades diclofopmethyl producing a number of metabolites that are intermediates or substrates for bacteria that degrade chlorinated aromatic compounds. Accordingly, specific catabolic gene PCR primers for chlorinated degradation pathways were designed and tested to determine if these genes are involved in diclofop-methyl degradation. DNA homology analysis between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates following the 16S rDNA sequencing and the identification of each of the isolates. These probes were successfully used in whole cell hybridization and will be used in subsequent studies of the microbial ecology of the consortium.

The aim of this project was to determine the possible role of the extracellular polymeric substances (EPS) of <I>Staphylococcus epidermidis</I> in biomaterial-centered infections. The first part of the project involved the production, isolation, and characterisation of <I>S.epidermidis</I> EPS. A novel method for the isolation of EPS from complex medium was developed which was able to eliminate the problems associated with contamination of bacterial EPS by medium components. Using this method, samples of EPS were obtained from <I>S.epidermidis</I> ATCC 35984 and clinical isolates. In the second part of the project, studies were carried out in order to determine the effect of EPS-eradicating treatments on (a) the susceptibility of stationary phase planktonic cultures to antibiotics, and (b) the integrity of intact biofilms. Eradication of EPS was achieved by exposure of planktonic stationary phase cultures of a clarithromycin-resistant strain of <I>S.epidermidis</I> (Clar^r No.6) to clarithromycin^*. This antibiotic had no effect on the growth or viability of this strain, however, exposure to clarithromycin resulted in a dose-dependent reduction in the dry weight of EPS obtained (5 μg ml^-1 = 50% reduction, 10 μg ml^-1 = 65% reduction). This result was also achieved with stationary phase cultures of a clarithromycin-sensitive strain of <I>S.epidermidis</I> (16595A). The effect of clarithromycin exposure (EPS eradication) on the subsequent efficacies of the antibiotics teicoplanin, cefuroxime and ciprofloxacin, towards stationary phase planktonic cultures of <I>S.epidermidis</I> Clar^r No.6 was determined by total and viable cell counts. No effect on cell viability was observed for combinations of clarithromycin and teicoplanin or ciprofloxacin. However, the 2 to 3 log reduction in viability that was observed for combinations of clarithromycin and cefuroxime suggested that these two antibiotics may have been working in synergy. ^* Clarithromycin is a macrolide antibiotic which is thought to act on the 50 S ribosomal subunit of bacteria which will then interfere with protein synthesis and in turn lead to inhibition of bacterial growth.