Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (φSPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (φSPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T01:56:06Z (GMT). No. of bitstreams: 1 Mantovani_RebecaPassarelli_M.pdf: 3691454 bytes, checksum: 62da767a8ae513cf2860f407c915aae6 (MD5) Previous issue date: 2018-08-18T22:55:56Z / Resumo: Fibrose cística (FC) é uma doença genética, autossômica recessiva, caracterizada por anormalidade no transporte eletrolítico. Achromobacter xylosoxidans é um bacilo Gram-negativo, não-fermentador e nos últimos anos tem sido encontrado em pacientes com FC, levando à doença pulmonar crônica. Neste estudo foram analisadas 17 amostras de A.xylosoxidans, isoladas de pacientes do Ambulatório de Fibrose Cística do Hospital das Clínicas (HC) da UNICAMP-SP. Os isolados foram analisados quanto as suas propriedades de virulência: atividade hemolítica, presença de hemaglutinação, atividade enzimática (caseinase, esterase, lecitina e hidrolise de elastina), adesão em célula, formação de biofilme e produção de atividade citotóxica. As amostras produziram hemolisinas, hemaglutininas e atividades enzimáticas. Catorze amostras de A.xylosoxidans (78%) formaram biofilme em microplacas, o que sugere o seu potencial em formar biofilme em cateteres e sondas. Entretanto essas bactérias não aderiram em células de pulmão NCI-H292 nas condições utilizadas. Os sobrenadantes de cultura de A.xylosoxindas apresentaram atividade citotóxica termoestável sobre célula NCI-H292, induzindo a formação de vacúolos, perda de contato celular, condensação da cromatina, núcleos picnoticos e morte celular. Foi verificado que a atividade citotóxica foi mantida mesmo após o tratamento dos sobrenadantes com polimixina B, sugerindo não ser uma endotoxina Ensaios de ELISA demonstraram significativa produção de IL-6 e IL-8 pelas células NCI-H292 após 24 horas de incubação com a fração >50kDa do sobrenadante de cultura da bacteria. Os resultados sugerem que o fator citotóxico produzido por A. xylosoxidans apresenta um importante papel na virulência, o qual provavelmente está associado à inflamação pulmonar crônica em pacientes com FC, levando a danos teciduais e declínio da função pulmonar / Abstract: Cystic fibrosis is a genetic disease, autosomal recessive, characterized by abnormal electrolyte transport. Achromobacter xylosoxidans is a Gram-negative non-fermenter and in recent years has been found in CF patients, leading to chronic lung disease. These studies were analyzed 17 samples of A.xylosoxidans isolated from patients in the Cystic Fibrosis Clinic, HC UNICAMP-SP. The isolates were analyzed for their virulence properties, such as haemolytic activity, the presence of hemagglutination, enzyme activity (caseinase, esterase, lecithin and hydrolysis of elastin), cell adhesion, biofilm formation and production of cytotoxic activity. The presence of hemolysins, hemagglutinins and enzymatic activities were not detected. Fourteen samples (78%) of A.xylosoxidans formed biofilms in microplates, suggesting the ability of bacteria to form biofilms on catheters and probes, but there was no adhesion in NCI-H292 cell lung. The culture supernatants of A.xylosoxindas isolated produced a heatstable cytotoxic factor in NCI-H292 cells, inducing the formation of vacuoles, loss of cell contact, chromatin condensation, pyknotic nuclei and cell death. ELISA assays to verify the release of IL-6 and IL-8, showed great production of these proinflammatory cytokines by NCI-H292 cells after 24 hours of incubation with the fraction> 50 kDa of culture supernatant of bacteria. It was also verified that the cytotoxic activity was maintained even after treatment with polymyxin B, suggesting may be not an endotoxin. Therefore, this cytotoxic factor produced by A. xylosoxidans may represent an important virulence factor, which is probably associated with chronic pulmonary inflammation in CF patients, leading to tissue damage and decline in lung function, decreasing the survival of these patients / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-13T18:09:59Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Silva, Keila de Cássia Ferreira de Almeida [Dissertação, 2016].pdf: 1387702 bytes, checksum: 5161fb19d344833ea4e10e115c322cf4 (MD5) / Made available in DSpace on 2017-03-13T18:09:59Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Silva, Keila de Cássia Ferreira de Almeida [Dissertação, 2016].pdf: 1387702 bytes, checksum: 5161fb19d344833ea4e10e115c322cf4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pseudomonas aeruginosa é um dos principais patógenos causadores de infecções graves em pacientes queimados, estando associado a elevadas taxas de mortalidade e morbidade. A alta plasticidade de seu genoma confere a este microrganismo a capacidade de tornar-se multirresistente a antimicrobianos, dificultando o tratamento devido à redução de opções terapêuticas. Este estudo teve como objetivo analisar 35 cepas de P. aeruginosa isoladas de pacientes queimados e da mesa onde era realizada a balneoterapia em um centro de tratamento de queimados (CTQ), determinando o perfil de resistência aos antimicrobianos, os fatores genéticos relacionados à virulência e resistência antimicrobiana, a capacidade de produzir biofilme e ação de biocidas sobre o mesmo, além de realizar tipificação molecular das cepas envolvidas. A suscetibilidade antimicrobiana foi verificada utilizando o método de disco-difusão, segundo os critérios do Clinical and Laboratory Standards Institute (CLSI). A pesquisa de genes de resistência que codificassem β-lactamases (blaPER-1, blaCTX-M, blaOXA-10, blaGES-1, blaVIM, blaIMP, blaSPM-1, blaKPC, blaNDM e blaSIM) e dos genes de virulência exoS e exoU, foi realizada através da técnica de Polymerase Chain Reaction (PCR). O teste fenotípico Carba NP, foi utilizado com a finalidade de detectar atividade hidrolítica enzimática à carbapenemas. A capacidade de formação de biofilme foi avaliada em placa para microtitulação e em cupom de aço inoxidável, sendo este último utilizado para verificar a eficácia de três agentes biocidas (hipoclorito de sódio 1%, peróxido de hidrogênio 5% e clorexidina 4%) na remoção dos biofilmes formados. As cepas foram tipificadas através da técnica de Multilocus Sequence Typing (MLST). De acordo com o teste de suscetibilidade antimicrobiana, foi observado um elevado padrão de resistência, com 71,5% (25/35) das cepas sendo resistentes a multidrogas (MDR). O maior percentual de resistência foi para o ciprofloxacino (94,3%; 33/35), seguido da gentamicina (88,6%; 31/35). Também foi observada resistência aos β-lactâmicos, inclusive à carbapenemas (22,9%; 8/35). Com relação aos genes de resistência pesquisados foi encontrado em 34,3% (12/35) das cepas o blaGES-1 e em uma única cepa o blaCTX-M. Nenhum gene codificador de carbapenemases pesquisado foi encontrado. Da mesma forma, nenhuma atividade hidrolítica enzimática ao imipenem foi detectada através do teste Carba NP, sugerindo que outros mecanismos de resistência possam estar envolvidos, como superexpressão de bomba e perda de porina de membrana externa (OprD). O gene de virulência exoS foi o mais prevalente estando presente em 71,4% (25/35) das cepas, enquanto o gene exoU foi detectado em 14,3% (5/35), porém todas as cepas que abrigavam este gene eram carbapenemas resistentes. Onze (31,4%) das 35 cepas, foram formadoras de biofilme utilizando a placa de microtitulação, sendo estas em sua maioria (81,8%) pertencentes ao clone A (obtido através de tipificação por PFGE em estudo prévio), o qual era o mais prevalente infectando/colonizando pacientes e contaminando a mesa de balneoterapia. A remoção do biofilme formado em superfície de cupom de aço inoxidável foi eficaz para os três biocidas testados (hipoclorito de sódio 1%, peróxido de hidrogênio 5% e clorexidina 4%), não havendo contagem de células viáveis das cepas analisadas após contato com os mesmos. A tipificação por MLST originou dois tipos de sequenciamentos novos, ST2236 e ST2237. Conclui-se com este estudo, que a redução de opções terapêuticas associada à presença de genes de virulência, pode agravar a situação destes pacientes. Assim como a presença de genes como blaGES-1 e blaCTX-M é preocupante, pois podem ser difundidas entre as demais cepas por transmissão horizontal, se não houver um controle adequado. Entretanto, o teste de remoção dos biofilmes mostrou que se a desinfecção for realizada de maneira correta, a contaminação cruzada entre pacientes pode ser evitada. / Pseudomonas aeruginosa is major causative pathogens of serious infections in burn patients and is associated with high mortality and morbidity rates. The high plasticity of its genome confers to this organism the ability to become multiresistant to antibiotics, difficulting the threathment due to reduced therapeutic options. This study aimed to analyze 35 strains of P. aeruginosa isolated from burn patients and the tank where balneotherapy was held in a burn treatment center (BTC), determining the resistance profile to antimicrobial agents, genetic factors related to virulence and antimicrobial resistance, the ability to produce biofilms and biocide action thereon and perform molecular typing of the strains involved. Antimicrobial susceptibility was assessed using the disk diffusion method, according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). The research for resistance genes encoding β-lactamases (blaPER-1, blaCTX-M, blaOXA-10, blaGES-1, blaVIM, blaIMP, blaSPM-1, blaKPC, blaNDM and blaSIM) and the virulence genes exoS and exoU was performed using the Polymerase Chain Reaction (PCR). The phenotypic test Carba NP, was used in order to detect enzymatic hydrolytic activity to carbapenems. Biofilm formation ability was evaluated through plate for microtitration and stainless steel coupon, the latter being used to verify the efficacy of three biocides (sodium hypochlorite 1%, hydrogen peroxide 5% and chlorhexidine 4%) to remove formed biofilms. The strains were typed by Multilocus Sequence Typing technique (MLST). According to the antimicrobial susceptibility test, a high resistance pattern was observed in which 71.5% (25/35) of strains were multidrug resistant (MDR). The highest percentage of resistance was to ciprofloxacin (94.3%; 33/35), followed by gentamicin (88.6%; 31/35). It was also observed resistance to β-lactams antibiotics, including the carbapenems (22.9%; 8/35). Regarding the resistance genes investigated were found the blaGES-1 in 34,3% (12/35) of the strains and the blaCTX-M in a single strain. None of the searched genes encoding carbapenemases was found. Similarly, no enzymatic hydrolytic activity to imipenem was detected by Carba NP test, suggesting that other resistance mechanisms may be involved, such as pump over expression and loss of outer membrane porin (OprD). The exoS virulence gen was the most prevalent being present in 71.4% (25/35) of the strains, while exoU was detected in 14.3% (5/35), however all strains that harbored this gene were carbapenems resistant. Eleven (31.4%) of 35 isolates were biofilm formers using the plate for microtitration, which mostly (81.8%) belonging to clone A (obtained in a previous study by PFGE) that was the most prevalent infecting/ colonizing patients and contaminating balneotherapy tank. Removal of biofilm formed in stainless steel coupon surface was effective for all three biocides tested (sodium hypochlorite 1%, hydrogen peroxide 5% and chlorhexidine 4%), with no viable cell count after contact with biocides solutions. The MLST originated two new sequencing types, ST2236 and ST2237. It is concluded from this study that the reduction of therapeutic options associated with the presence of virulence genes, can aggravate the situation of these patients. As well as the presence of genes as blaGES-1 and blaCTX-M is worrying as they may be spread among the other strains by horizontal transmission, if there is no adequate control. However, the removal test of biofilms showed that, if the disinfection is carried out in a correct way, cross-contamination between patients can be avoided.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-07T17:48:18Z No. of bitstreams: 1 José, Kelly Fernanda Campos [Dissertação, 2014].pdf: 955086 bytes, checksum: 84564fe3de6240b8248fb1dbad2cf5ec (MD5) / Made available in DSpace on 2017-04-07T17:48:18Z (GMT). No. of bitstreams: 1 José, Kelly Fernanda Campos [Dissertação, 2014].pdf: 955086 bytes, checksum: 84564fe3de6240b8248fb1dbad2cf5ec (MD5) / O pescado é um alimento de excelente valor nutritivo devido às suas vitaminas, proteínas e ácidos graxos insaturados, no entanto, pode veicular uma variedade de microrganismos patogênicos ao homem, aumentando a preocupação com a qualidade sanitária. Nesse contexto, a formação de biofilme na indústria de alimentos representa uma preocupação por sua potencialidade em resistir a tratamentos sanitizantes, além da possibilidade de perda da qualidade por deterioração e/ou veiculação de patógenos, justificando assim, a importância de uma correta higienização na indústria. Neste estudo objetivou-se identificar microrganismos presentes nas superfícies de processamento de pescado, assim como avaliar a capacidade de formação de biofilmes bacterianos em equipamentos e sinalizar maneiras de evitar sua formação. Para tal, foi realizada a avaliação de superfícies através do método “swab”, com coleta de amostras das superfícies do tanque de lavagem, esteira de evisceração e descabeçamento e tanque de armazenamento de molho de cobertura, em uma indústria de pescado do Estado do Rio de Janeiro, através de três visitas aleatórias realizadas em diferentes meses do ano. As referidas amostras foram encaminhadas sob refrigeração para o Laboratório de Higiene e Microbiologia de Alimentos da Faculdade de Farmácia – UFF, onde foram processadas conforme métodos e recomendações oficiais. Em relação aos resultados foram constatadas não conformidades na lista de verificação, com situação mais crítica observada no item manipuladores. Na análise bacteriológica evidenciou-se elevadas contagens de bactérias heterotróficas aeróbias mesófilas, presença de Salmonela spp., Listeria spp., Bacillus spp., coliformes totais e Escherichia coli. Demonstrou-se capacidade de formação de biofilme por cepas de E. coli isoladas nas três visitas realizadas. Na avaliação da eficácia de sanitizantes de uso industrial, foram observados melhores resultados na exposição à biguanida polimérica e taxas elevadas de resistência ao quaternário de amônio. Diante dos resultados, constatou-se ausência de condições higiênico-sanitárias adequadas e circunstâncias propícias para formação de biofilmes bacterianos / Fish is a food of great nutricional value due to their vitamins, proteins and unsaturated fatty acids, however, can convey a variety of pathogenic microorganism to man increasing the concern about ensuring health standards. In this context, the formation of biofilms in the food industry represent a preoccupation because of its potencial to resist sanitizing treatments, besides the possibility of causing quality loss due to deterioration and/or transmission of pathogens, justifying the importance of proper hygiene in industry. This study aimed to identify microorganisms present on the surfaces of fish processing and assess the ability of formation of microbial biofilms on equipament and to signal ways to avoid their formation. In this sense, the evaluation of surfaces contact was performed from samples collected on the surfaces of the washing tank, gutting and beheading mat and of storing tank coverage sauce, at seafood industry in a state of Rio de Janeiro, through three randomized visits in different months of the year. These samples were sent under refrigeration to the Laboratory of Food Microbiology and Hygiene, Faculty of Pharmacy – UFF, which were processed by methods and official recommendations. Regarding the results, non-conformities were found by checklist, with more critical situation in the item handlers. Bacteriological analysis revealed a high count of aerobic mesophilic, presence of Salmonella spp., Listeria spp., Bacillus spp., total coliforms and Escherichia coli. It was demonstrated ability of biofilm formation by strains of E.coli isolated in three visits. In evaluating the effectiveness of sanitizers industrial use was observed better results in exposure to polymeric biguanide and high rates resistance to quaternary ammonium. Considering the results, it has been found a lack of adequate sanitary conditions and circumstances conducive to the formation of bacterial biofilms

Two batch mesocosms were created on site in Da Nang, Vietnam to reduce nutrients in wastewater from fish processing factories. The mesocosms contained either activated carbon or coconut fibre which in earlier studies has shown promising results in wastewater treatment. Three aspects of the materials were compared; Chemical content, which measured levels of COD, total-nitrogen and total-phosphorus. Rate of biofilm formation, where biofilm were measured visually and through weight. The last aspect was microbiological presence where fours species of microorganisms were cultivated. The experiment showed no obvious difference between the materials but concludes that this is an experiment that could and should be developed further.

Une approche in vitro a consisté à étudier la formation de biofilm de souches d’origine du genre Lactobacillus d’intérêt probiotique. Nous nous sommes également attachés à évaluer l’impact de conditions de stress mimant l’environnement intestinal sur la formation du biofilm pour l’ensemble de ces souches. Les effets antagonistes des surnageants de cultures en biofilm ou en planctonique contre des agents pathogènes alimentaires ont été appréhendés. Non seulement toutes les souches testées forment des biofilms mais ce mode de croissance génère un effet antagoniste accentué pour certaines d’entre elles. Parmi les critères de sélection des bactéries à intérêt probiotique, les effets immunomodulateurs des probiotiques sont souvent recherchés. L. casei ATCC334 connue pour ses effets anti-inflammatoires a été retenue pour notre étude. A l’aide du modèle de lignée cellulaire THP-1 et en présence de LPS, le surnageant de culture de L. casei ATCC334 cultivée en biofilm s’est avéré présenter un effet anti-inflammatoire bien supérieur à celui des cultures planctoniques. Une approche utilisant des techniques biochimique et immunologique a permis d’identifier un des principes actifs responsable de l’effet anti-inflammatoire de cette souche. L’utilisation du modèle poisson zèbre a permis de montrer la colonisation de l’intestin des larves et de confirmer le rôle anti-inflammatoire de L. casei ATCC334 avec une diminution de la production des interleukines pro-inflammatoires et une augmentation de la production d'IL-10. Le recrutement des macrophages fluorescents mesuré en cytométrie de flux est également atténué chez la larve nourrie auparavant par le probotique en présence d’un agent inflammatoire. Le résultat majeur de cette étude est l’identification de la protéine GroEL qui contribue de façon significative à l’effet anti-inflammatoire de la souche L. casei ATCC334 lorsque qu’elle est cultivée en biofilm. / An in vitro approach was used to study biofilm formation by bacterial strains with probiotic properties and belonging to the Lactobacillus genus. We also evaluated the impact of stress conditions mimicking the intestinal environment on biofilm formation for all of these strains. The antagonistic effects of supernatants from cultures in biofilm or planktonic conditions against food-borne pathogens were apprehended. This growth mode generates an antagonistic effect accentuated for some of them. Among the selection criteria of interest probiotic bacteria, the immunomodulatory effects of probiotics are often sought. L. casei ATCC334 known for its anti-inflammatory effects was selected for our study. Using the model cell line THP-1 and in the presence of LPS, the culture supernatant of L. casei ATCC334 grown in biofilm was found to have an anti-inflammatory effect much greater than planktonic cultures. An approach using immunological and biochemical techniques has allowed the identification of the active substances responsible for the anti-inflammatory effect of this strain. Using the zebrafish model, we showed the colonization of the gut of the larvae and confirmed the anti-inflammatory role of L. casei ATCC334 with a decreased production of pro-inflammatory interleukins, and increased IL-10 production. Recruitment of fluorescent macrophages measured by flow cytometry was also mitigated in larvae fed previously by probotic in the presence of an inflammatory agent. The major result of this study is the identification of the GroEL protein that contributes significantly to anti-inflammatory effect of the strain L. casei ATCC334 when it is grown in biofilm.

Urinary catheters are indispensable in healthcare and, with an ageing population, their use will continue to increase. However, they are commonly associated with colonisation and urinary tract infections (UTIs) caused by the attachment of bacteria to the catheter surface. Application of a novel cationic compound as a catheter coating may have a significant impact on the costs associated with treatment of UTIs and reduce the need for catheter replacement, as well as decreasing the number of UTI associated morbidity and mortality. Cationic compounds in particular are known to interact with the negatively charged outer membrane of bacteria, therefore have a broad spectrum of activity. The purpose of this study was to source and evaluate a novel cationic antimicrobial for use as a potential coating to impede biofilm formation on urinary catheters, and to investigate the cellular response to the selected lead compound. This research has demonstrated that the antimicrobial activity of commercially available Byotrol™ was superior to that of polyamines and quaternary ammonium compounds that were screened. Using high-throughput antimicrobial assays, such as the minimum inhibitory concentration and microtitre plate biofilm forming assays, the inhibitory concentrations of Byotrol™ were found to range from 3 µg/mL to 15 µg/mL for planktonic cultures, and 3 µg/mL to 20 µg/mL for the biofilm growth of uropathogenic bacteria. Furthermore, the minimum biofilm eradication concentration assay demonstrated that 200-1000 µg/mL Byotrol™ was able to eradicate an established biofilm. Byotrol™ may also have significant potential as a device coating, as pre-coating data on glass slides and microtitre plates with the compound inhibited bacterial growth on the surface at concentrations of 400 µg/mL for E. coli, and 1000 µg/mL K. pneumoniae. Atomic force microscopy validated the expectation that higher concentrations of Byotrol™ coated a surface more evenly than lower concentrations. Using two-dimensional gel electrophoresis, the metabolic protein tryptophanase was seen to be significantly over-expressed when E. coli K12 was treated with sub-inhibitory concentrations of Byotrol™. A transcriptomic approach using RNA-Seq demonstrated that a majority of the differentially expressed genes were identified in cells that were challenged with 4 times the minimum inhibitory concentration of Byotrol™. Genes associated with protein synthesis and stress response were significantly up-regulated. Interestingly, the global gene regulators AI-2 and indole were significantly up-regulated, which may have an influence on the expression of genes related to motility, biofilm formation and acid-resistance. Genes associated with chemotaxis and motility, acid-resistance and iron transport were significantly down-regulated, particularly in cells challenged with Byotrol™.Byotrol™ displayed antimicrobial activity both in suspension and as a coating. Identification of differentially expressed genes and proteins, when the bacteria were treated and challenged with Byotrol™, has, for the first time, revealed the bacterial cell’s response to this biocide. The findings may enable the development of strategies to prevent or better manage catheter associated urinary tract infection (CAUTI).

Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Coral diseases were unknown in the scientific community fifty years ago. Since the discovery of a coral disease in 1965, there has been an exponential increase in the number of known coral diseases, as the abundance, prevalence, distribution, and number of host species affected has also significantly increased. Coral diseases are recognized as contributing significantly to the dramatic losses of coral cover on a global basis, particularly in the Caribbean. The apparent sudden emergence of coral diseases suggests that they may be a symptom of an overall trend associated with changing environmental conditions. However, not much evidence has been gathered to address this question. The following studies were designed to build a comprehensive argument to support this hypothesis for one important coral disease – black band disease (BBD). A meta-analysis of clone libraries identifying the microbial communities associated with BBD reveal important information including that a single cyanobacterial operational taxonomic unit (OTU) was by far the most prevalent OTU in diseased samples, and that the alphaproteobacteria, which include some of the most common bacteria in marine waters, were the most diversely represented. The analysis also showed that samples exhibited regional similarities. An fine and ultrastructural characterization of the disease revealed that the cyanobacteria are prolific borers through the coral skeleton, and that the cyanobacteria penetrate coral tissue, leading to their presence ahead of the main migrating disease band. It was further found that apparently healthy corals exposed to toxins found in BBD, exhibited similar tissue degradation to those infected with BBD. Comparing the disease progression to biofilm formation, it was determined that scouting cyanobacteria may contribute to the migration of the disease through progressive biofilm development over intact coral tissue. Together, these studies provide significant evidence for the hypothesis that BBD is an opportunistic disease, caused by common environmental bacteria, facilitated by the changing environmental conditions associated with climate change.

New federal regulations with regards to ammonia in wastewater effluent discharge will require over 1000 existing wastewater treatment facilities to be upgraded. Although biological treatment is the most common and economical means of wastewater ammonia removal, nitrification rates can be completely impeded at cold temperatures. Moving bed biofilm reactors (MBBR) have shown promise as an upgrade nitrifying unit at pilot-scale and full-scale applications with respect to low temperature nitrification. MBBR technologies offfer the advantages of less space requirement, utilizing the whole tank volume, no sludge recycling, and no backwashing, over other attached growth systems. Two laboratory MBBRs were used in this study to investigate MBBR nitrification rates at 20deg.C, after long-term exposure to 1deg.C, and at the kinetic threshold temperature of 5deg.C. Furthermore, the biologically produced solids from the MBBR system 20deg.C and after long-term exposure to 1deg.C, and the Arrhenius temperature correction models used to predict nitrification rates after long-term exposure to 1deg.C. The nitrification rates at 1deg.C over a four month exposure period as compared to the rate at 20deg.C were 18.7 + 5.5% and 15.7 + 4.7% for the two reactors. The nitrification rate at 5deg.C was 66.2 + 3.9% and 64.4 + 3.7% compared to the rate measured at 20deg.C for reactors 1 and 2, respectively, and as such was identified as the kinetic temperature threshold. The quantity of solids detached from the nitrifying MBBR biocarriers was low and did not vary significantly at 20deg.C and after long-term exposure to 1deg.C. Lastly, a temperature correction model based on exposure time to cold temperatures, developed by Delatolla et al. (2009) showed a strong correlation to the calculated ammonia removal rates relative to 20deg.C following a gradual acclimatization period to cold temperatures. Biofilm morphology along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope imaging (VPSEM) and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant changes. Hence, this study observed higher cell activities at warm temperatures and a slightly greater quantity of biomass with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, 'Nitrosomonas' and 'Nitrosospira' (ammonia oxidizers)as well as 'Ntrospira' (nitrite oxidizer) were identified in which no population shift was observed during 20deg.C and after long-term exposure to 1deg.C. Furthermore, a number of non-nitrifiers were identified int he biofilm during warm and cold temperatures presenting the possibility that their presence may have provided some form of protection to the nitrifiers during long-term temperature exposure.