The release of cells from a biofilm to the surrounding environment is poorly understood and the importance of this stage of biofilm development has only recently been realized. A key part of this process is the generation of phenotypic variants in the biofilm dispersal population. This thesis reports on the characterization of biofilm development of Serratia marcescens MG1, the analysis of the biofilm dispersal population, and the identification of the conditions that trigger phenotypic diversification. Furthermore, it provides an insight into the molecular understanding of how phenotypic variation is being generated, and demonstrates the clinical and environmental implications of phenotypic diversification during bacterial pathogenesis and bacterial persistence. Characterization of the microcolony biofilm development of S. marcescens revealed that the S. marcescens biofilm develops through a process involving microcolony formation, hollowing of mature microcolonies, and a sudden biofilm expansion within a very short period (&lt 24h) resulting in an increase in biofilm biomass with a radiation of biofilm structures at days 3 to 4. The biofilm expansion phase consistently correlated to an increase in the number of dispersal variant morphotypes. Studies of variant induction in planktonic cultures and biofilm flow cells demonstrated that phenotypic diversification in S. marcescens is not only a biofilm-specific phenomenon, but also involves biofilm-specific morphotypes. These morphological variants can only be isolated from the microcolony biofilm morphotype and not from the filamentous biofilms, leading to the hypothesis that there is a strong diversifying selection that is specific to the microcolony biofilms. To further explore how these variants were generated, molecular analyses revealed that exopolysaccharides and lipopolysaccharides are important moieties that are involved in phenotypic variation in S. marcescens biofilms. The etk gene, encoding a tyrosine protein kinase within the exopolysaccharide biosynthesis operon, was found to contain single nucleotide polymorphisms (SNPs) that were present in the 'sticky' variants but not in the 'non-sticky' wild-type or the 'non sticky' small colony variants. Furthermore, infrequent-restriction-site PCR (IRS-PCR), BIOLOG metabolic profiling, and gene sequence analyses, suggest that phenotypic diversification in S. marcescens is likely to involve mutational hotspots in specific genes. The biofilm-derived morphotypic variants differed extensively in cell ultrastructure properties, and exhibited specialized colonization and virulence traits, such as attachment, biofilm formation, swimming and swarming motilities, protease production, and hemolysin production. It was also demonstrated that phenotypic diversification contributed to a varying degree of resistance to protozoan predation, and bacterial pathogenecity in Caenorhabditis elegans, highlighting the complexity of the dispersal populations from S. marcescens biofilms. Furthermore, mixed-culture experiments involving multiple variant isolates (with or without the parental wild-type) showed that the persistence and virulence potential of S. marcescens can be synergistically enhanced in the Acanthamoeba castellanii grazing model and in the C. elegans infection model, respectively. This indicates that the different bacterial morphotypes work in concert to provide S. marcescens with enhanced protection against environmental perturbations and a competitive edge during the infection process. It was proposed that phenotypic diversification is not only an integral part of S. marcescens biofilm life-cycle, but also represents an important strategy for bacteria to greatly enhance its survival and persistence in different environments, ranging from aquatic and soil ecosystems, to those of the infected hosts.

Serratia entomophila, the causative agent of amber disease, is an endemic bacterium used for the biocontrol of New Zealand grass grub larvae. Although the available biopesticide is effective, its use is limited to areas where sub-surface application is feasible, and is also impacted by soil conditions such as moisture levels and osmolarity. The aim of this study was to elucidate the responses of S. entomophila to osmotic and desiccation stresses in relation to challenges encountered during production, storage and soil application, with the goal of developing a more robust and versatile biocontrol agent. RpoS is a key factor in the stress response of many enteric bacteria. In order to dissociate the effect of RpoS from subsequent cellular stress studies, an rpoS mutant was constructed by site-directed mutagenesis. Assessment of the rpoS mutant showed that RpoS was not implicated in NaC1 or desiccation tolerance of S. entomophila. The rpoS mutant was instead found to have enhanced salt tolerance and could be distinguished from the wild-type by the ability to ferment arabinose, a phenotype that was confirmed through complementation. Complete abolition of the amber disease process was observed using an rpoS strain also missing the Sep virulence genes, suggesting that RpoS is a regulator of the S. entomophila anti-feeding prophage (Afp). These findings indicate a subtle interplay between NaC1 tolerance, virulence and RpoS-mediated regulation of amber disease in S. entomophila. A transposon mutagenesis screen was carried out to identify genes associated with NaC1 tolerance in S. entomophila. Fourteen mutants displaying NaC1 sensitivity were identified, two of which had mutations in genes with potential implications for the formulation of the bacterium as a biocontrol agent. The gene leuO that encodes a LysR-family transcriptional regulator was found to be essential for S. entomophila NaC1 tolerance. The toxicity of increased cellular LeuO from an over-expression vector led to the investigation of the effects of leuO mutation on the proteome. Multiple protein changes observed by two-dimensional gel analysis suggested that LeuO may be a global regulator in S. entomophila, as has been hypothesised for Salmonella species. A second NaC1-sensitive mutant contained an insertion in afp15, the product of which is thought to be involved in assembly of the Afp. As well as being sensitive to NaC1, the afp15 mutant was unable to induce the anti-feeding component of amber disease, again highlighting the link between stress tolerance and virulence in S. entomophila. This study also determined that pre-exposure to NaC1 in conjunction with the provision of exogenous glycine betaine significantly enhanced the survival of S. entomophila either in a desiccated state or after application to soil, regardless of the soil moisture content. The implication of this finding on the future formulation of S. entomophila led to investigation of the underlying genetic mechanisms involved in glycine betaine synthesis and NaC1 tolerance. The genes involved in glycine betaine biosynthesis from choline were identified through genomic comparison, degenerate PCR and primer walking. A 6.5 kb region was sequenced and found to contain four genes with homology and similar chromosomal arrangement to the E. coli bet genes (betTIBA). The S. entomophila betIBA genes comprised an operon, flanked by the divergently-transcribed betT gene whose product is responsible for choline transport. To ascertain the relative transcription levels of components of the bet operon, quantitative RT-PCR was performed. Results of qRT-PCR showed that choline in conjunction with NaC1 induced the greatest levels of bet gene transcription, and that levels of the betA transcript were significantly lower than those of the other bet genes. Examination of the betA 5� non-coding region identified a previously undetected hairpin region, possibly accounting for the observed decrease in betA transcript levels. The findings of this study have significantly advanced our understanding of how S. entomophiia responds to stress, and will contribute to the development of formulation strategies for the production of a robust product capable of application to pasture by a range of teclmiques. In addition, there is significant potential to utilise these findings in the development of other bacterial inocula for a range of biotechnological applications.

The release of cells from a biofilm to the surrounding environment is poorly understood and the importance of this stage of biofilm development has only recently been realized. A key part of this process is the generation of phenotypic variants in the biofilm dispersal population. This thesis reports on the characterization of biofilm development of Serratia marcescens MG1, the analysis of the biofilm dispersal population, and the identification of the conditions that trigger phenotypic diversification. Furthermore, it provides an insight into the molecular understanding of how phenotypic variation is being generated, and demonstrates the clinical and environmental implications of phenotypic diversification during bacterial pathogenesis and bacterial persistence. Characterization of the microcolony biofilm development of S. marcescens revealed that the S. marcescens biofilm develops through a process involving microcolony formation, hollowing of mature microcolonies, and a sudden biofilm expansion within a very short period (&lt 24h) resulting in an increase in biofilm biomass with a radiation of biofilm structures at days 3 to 4. The biofilm expansion phase consistently correlated to an increase in the number of dispersal variant morphotypes. Studies of variant induction in planktonic cultures and biofilm flow cells demonstrated that phenotypic diversification in S. marcescens is not only a biofilm-specific phenomenon, but also involves biofilm-specific morphotypes. These morphological variants can only be isolated from the microcolony biofilm morphotype and not from the filamentous biofilms, leading to the hypothesis that there is a strong diversifying selection that is specific to the microcolony biofilms. To further explore how these variants were generated, molecular analyses revealed that exopolysaccharides and lipopolysaccharides are important moieties that are involved in phenotypic variation in S. marcescens biofilms. The etk gene, encoding a tyrosine protein kinase within the exopolysaccharide biosynthesis operon, was found to contain single nucleotide polymorphisms (SNPs) that were present in the 'sticky' variants but not in the 'non-sticky' wild-type or the 'non sticky' small colony variants. Furthermore, infrequent-restriction-site PCR (IRS-PCR), BIOLOG metabolic profiling, and gene sequence analyses, suggest that phenotypic diversification in S. marcescens is likely to involve mutational hotspots in specific genes. The biofilm-derived morphotypic variants differed extensively in cell ultrastructure properties, and exhibited specialized colonization and virulence traits, such as attachment, biofilm formation, swimming and swarming motilities, protease production, and hemolysin production. It was also demonstrated that phenotypic diversification contributed to a varying degree of resistance to protozoan predation, and bacterial pathogenecity in Caenorhabditis elegans, highlighting the complexity of the dispersal populations from S. marcescens biofilms. Furthermore, mixed-culture experiments involving multiple variant isolates (with or without the parental wild-type) showed that the persistence and virulence potential of S. marcescens can be synergistically enhanced in the Acanthamoeba castellanii grazing model and in the C. elegans infection model, respectively. This indicates that the different bacterial morphotypes work in concert to provide S. marcescens with enhanced protection against environmental perturbations and a competitive edge during the infection process. It was proposed that phenotypic diversification is not only an integral part of S. marcescens biofilm life-cycle, but also represents an important strategy for bacteria to greatly enhance its survival and persistence in different environments, ranging from aquatic and soil ecosystems, to those of the infected hosts.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.

PARENTE, T.M.L Perfil de resistência a antibióticos e a terapia fotodinâmica antimicrobiana exibida por isolados ambientais, orais e extra-orais de Serratia marcescens. 2010. 90 f. Dissertação (MESTRADO EM BIOTECNOLOGIA) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2010. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2017-09-04T10:52:07Z No. of bitstreams: 1 2010_dis_tmlparente.pdf: 2557651 bytes, checksum: 75c39a01d93208de5800a785cfa1c2ee (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2017-10-03T14:53:42Z (GMT) No. of bitstreams: 1 2010_dis_tmlparente.pdf: 2557651 bytes, checksum: 75c39a01d93208de5800a785cfa1c2ee (MD5) / Made available in DSpace on 2017-10-03T14:53:42Z (GMT). No. of bitstreams: 1 2010_dis_tmlparente.pdf: 2557651 bytes, checksum: 75c39a01d93208de5800a785cfa1c2ee (MD5) Previous issue date: 2010 / Serratia marcescens is widely distributed in nature, but has emerged in the last years as important nosocomial pathogen with resistance of many antimicrobial drugs. This study aimed to verify the susceptibility of Serratia marcescens isolates from environment, from oral infections and from extra-oral infections to different antibiotics and evaluate the antimicrobial effect of photodynamic antimicrobial therapy as biotechnology tools reducing bacterial growth in planktonic cells and biofilm. E-test® were performed for fifty-five strains and the PACT for the thirty strains more resistant to antimicrobials tested. The antimicrobial effect of toluidine blue O, associated with 4,72 J cm-2 of a light-emitting diode , was evaluated. Before and after the treatments, bacterial inocula were analysed with regard to the number of colony- forming units. For antimicrobials, we observed that the 55 strains analyzed, 13 (23.63%) were resistant to doxycycline, but only one (1.81%) isolate showed resistance to ciprofloxacin, another to tobramycin and another to cefotaxime, 24 ( 43.63%) strains had intermediate sensitivity to doxycycline, all were sensitive to imipenem and most were sensitive to ciprofloxacin, tobramycin and cefotaxime Statistical analysis showed no significant differences in resistance of samples of different origins for drugs DX, CT, and IP. Considering the resistance to CI, the environmental samples were significantly more resistant than samples oral and extra-oral. For the drug TM, the oral samples were significantly more sensitive than the other samples. The irradiation of planktonic and biofilm cultures in the absence of TBO (L+S-), incubation with TBO alone (L-S+) and untreated control group (L-S-) had no significant effect on the viability of strains of S. marcescens studied (p <0.05). Significant decreases in bacterial viability was observed only when planktonic and biofilm culture of environmental strains, oral and extra-oral S. marcescens were exposed to toluidine blue O and LED light at the same time (L+S+). Significant reductions in bacterial counts were observed by antimicrobial photodynamic therapy ranging from 10-11 to 10-7.The association of TBO and light, with energy density 4,72 J cm-2, was effective in reducing the viability of bacterial strains in environmental, oral and extra-oral S. marcescens and can be a useful biotechnological tool in the control of bacterial resistance. / Serratia marcescens se encontra largamente distribuída na natureza, mas tem emergido nos últimos anos como um importante patógeno nosocomial resistente a diversos antimicrobianos. Este estudo teve como objetivo verificar a susceptibilidade de isolados ambientais, orais e extra-orais de Serratia marcescens a diferentes antibióticos e avaliar a terapia fotodinâmica antimicrobiana na redução do crescimento bacteriano em culturas de células planctônicas e biofilme. O teste de susceptibilidade antimicrobiano E-test® foi realizado para as 55 cepas e o TFA para as 30 cepas mais resistentes aos antimicrobianos testados. O efeito antimicrobiano do azul de o-toluidina associado com 4,72 J cm-2 de luz emitida por um diodo (LED) foi avaliado. Antes e após os tratamentos, os inóculos bacterianos foram analisados com consideração do número de unidades formadoras de colônias. Considerando o perfil antimicrobiano observamos que das 55 cepas analisadas, 13 (23,63%) apresentaram resistência à doxiciclina, mas apenas um (1,81%) isolado apresentou resistência ao ciprofloxacino, outro à tobramicina e outro à cefotaxima; 24 (43,63%) cepas apresentaram sensibilidade intermediária à doxiciclina, todas foram sensíveis ao imipenem e a maioria foi sensível ao ciprofloxacino, à tobramicina e à cefotaxima. A análise estatística demonstrou não haver diferenças significativas no perfil de resistência das amostras de diferentes origens em relação as drogas DX, CT e IP. Considerando a resistência a CI, as amostras ambientais foram significativamente mais resistentes do que as amostras orais e extra-orais. Para a droga TM, as amostras orais foram significantemente mais sensíveis do que as demais amostras. A irradiação das culturas planctônicas e biofilmes na ausência de TBO (L+C-), a incubação com TBO sozinho (L-C+) e o grupo controle não tratado (L-C-) não apresentou efeitos significativos na viabilidade das cepas de S. marcescens estudadas (p < 0,05). Decréscimos significativos na viabilidade bacteriana foram observados somente quando cultura planctônica e biofilme de cepas ambientais, orais e extra-orais de S. marcescens foram expostas ao azul de orto toluidina e luz LED ao mesmo tempo (L+C+). Reduções significativas nas contagens bacterianas foram observadas pela Terapia Fotodinâmica Antimicrobiana com variação de 10-11 a 10-7. A associação de TBO e LED, com densidade de energia de 4,72 J cm-2 , foi efetivo na redução da viabilidade bacteriana em cepas ambientais, orais e extra-orais de S. marcescens podendo ser uma ferramenta biotecnológica útil no controle da resistência bacteriana.

Made available in DSpace on 2014-06-12T15:52:02Z (GMT). No. of bitstreams: 2 arquivo2808_1.pdf: 3862930 bytes, checksum: e749256848081d690dbce7c04ef3958f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Prodigiosinas é uma família de pigmentos naturais, de cor vermelha caracterizado por um esqueleto comum pirrolilpirrometano, produzido por várias bactérias, porém primeiro produzido por Serratia marcescens. Este pigmento é uma droga promissora, devido às suas características de atividade antifúngica, imunossupressores e antiproliferativa. As condições ótimas para o aumento do crescimento em S. marcescens está relacionada ao aumento da produção do pigmento, sob o ponto de vista industrial. Neste trabalho, foram utilizados os meios convencionais Peptona glicerol e Manitol, bem como os meios alternativos, Caldo de arroz, de gergelim e de amendoim, visando à produção de prodigiosina pela bactéria isolada do solo semi-árido, Serratia marcescens UCP 1549, utilizando fermentação em estado sólido, a 280 C, durante 48 horas de cultivo. A produção da prodigiosina foi observada nos meios convencionais, principalmente meio Manitol, sendo obtidos 1,2g/g de biomassa, porém não foi detectada nos meios alternativos. O pigmento foi purificado por cromatografia de exclusão, empregando-se Sephadex LH-20, obtendo-se 96 frações que foram reunidas, sendo caracterizada por espectrofotometria e espectrometria de massa (GC-MS), sendo sugerido ser Undecilprodigiosina. Estudos foram realizados com a atividade citotóxica para Artemia salina demonstrando uma CL50 de 78,33&#956;g/mL. A fitoxidade para sementes de alface (Lactuca sativa) e pimentão (Capsicum annuum) com inibição da germinação das sementes a partir de concentrações superiores a 40&#956;g/mL, representando mais de 50% de inibição. Os resultados obtidos sugerem alto potencial biotecnológico na produção de Undecilprodigiosina pela nova linhagem de S. marcescens UCP 1549, como também indica como promissores os resultados com o meio Manitol em estado sólido, os processos de extração e purificação do pigmento

Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-01T16:30:03Z (GMT). No. of bitstreams: 1 Silva_CristinaFerraz_M.pdf: 18209191 bytes, checksum: f414e61f8115923d3061ee387b9f0283 (MD5) Previous issue date: 2002 / Resumo: Biosurfactantes são compostos produzidos por microrganismos os quais possuem em sua molécula uma porção hidrofilica (solúvel em água) e uma porção hidrofóbica (insolúvel em água). Essas moléculas são capazes de reduzir a tensão superficial e interfacial em ambas soluções aquosas e misturas de hidrocarbonetos, as quais fazem desses compostos potenciais candidatos para aumentar a recuperação de óleo e processos de deemulsificação. No presente trabalho foi estudada a produção de biosurfactante por uma linhagem de bactéria. O microrganismo considerado foi pré-selecionado como produtor de biosurfactante em trabalhos anteriores e identificado nesta dissertação como Serratía marcescens. As melhores condições para produção do biosurfactante em agitador rotativo foram determinadas através do processo de otimização utilizando Planejamento Experimental. Além disso, foram estudadas algumas propriedades do biosurfactante, como por exemplo, capacidade emulsificante e estabilidade em diferentes pHs e temperaturas. Finalmente, testou-se a aplicação do biosurfactante produzido avaliando-se o efeito da sua adição na atividade de lipase de Rhízopus sp. quando comparado ao efeito produzido por surfactantes químicos. / Abstract: Biosurfactants are compounds produced by microrganisms those molecules include a hydrophilic portion (water soluble) and a hydrophobic portion (water insoluble). These molecules are capable of reducing surface and interfacial tensions in both aqueous solutions and hydrocarbon mixtures, which makes them potential candidates for enhancing oil recovery and deemulsification processes. Biosurfactant production by a bacterial strain was studied in this work. The microrganism considered was isolated in previous studies and identified in this work as Serratia marcescens. The best conditions for biosurfactant production in shake flasks were determined through an optimization process using an Experimental Design. Móreover, some properties of the biosurfactant were studied, for example, its emulsifying capacity and stability at different pH values and temperatures. Finally, the application of the biosurfactant produced was tested evaluating the effect of its addition on the activity of Rhizopus sp. Lipase, as compared to the effect produced by chemical surfactants. / Mestrado / Mestre em Ciência de Alimentos

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Serratia marcescens se encontra largamente distribuÃda na natureza, mas tem emergido nos Ãltimos anos como um importante patÃgeno nosocomial resistente a diversos antimicrobianos. Este estudo teve como objetivo verificar a susceptibilidade de isolados ambientais, orais e extra-orais de Serratia marcescens a diferentes antibiÃticos e avaliar a terapia fotodinÃmica antimicrobiana na reduÃÃo do crescimento bacteriano em culturas de cÃlulas planctÃnicas e biofilme. O teste de susceptibilidade antimicrobiano E-test foi realizado para as 55 cepas e o TFA para as 30 cepas mais resistentes aos antimicrobianos testados. O efeito antimicrobiano do azul de o-toluidina associado com 4,72 J cm-2 de luz emitida por um diodo (LED) foi avaliado. Antes e apÃs os tratamentos, os inÃculos bacterianos foram analisados com consideraÃÃo do nÃmero de unidades formadoras de colÃnias. Considerando o perfil antimicrobiano observamos que das 55 cepas analisadas, 13 (23,63%) apresentaram resistÃncia à doxiciclina, mas apenas um (1,81%) isolado apresentou resistÃncia ao ciprofloxacino, outro à tobramicina e outro à cefotaxima; 24 (43,63%) cepas apresentaram sensibilidade intermediÃria à doxiciclina, todas foram sensÃveis ao imipenem e a maioria foi sensÃvel ao ciprofloxacino, à tobramicina e à cefotaxima. A anÃlise estatÃstica demonstrou nÃo haver diferenÃas significativas no perfil de resistÃncia das amostras de diferentes origens em relaÃÃo as drogas DX, CT e IP. Considerando a resistÃncia a CI, as amostras ambientais foram significativamente mais resistentes do que as amostras orais e extra-orais. Para a droga TM, as amostras orais foram significantemente mais sensÃveis do que as demais amostras. A irradiaÃÃo das culturas planctÃnicas e biofilmes na ausÃncia de TBO (L+C-), a incubaÃÃo com TBO sozinho (L-C+) e o grupo controle nÃo tratado (L-C-) nÃo apresentou efeitos significativos na viabilidade das cepas de S. marcescens estudadas (p < 0,05). DecrÃscimos significativos na viabilidade bacteriana foram observados somente quando cultura planctÃnica e biofilme de cepas ambientais, orais e extra-orais de S. marcescens foram expostas ao azul de orto toluidina e luz LED ao mesmo tempo (L+C+). ReduÃÃes significativas nas contagens bacterianas foram observadas pela Terapia FotodinÃmica Antimicrobiana com variaÃÃo de 10-11 a 10-7. A associaÃÃo de TBO e LED, com densidade de energia de 4,72 J cm-2 , foi efetivo na reduÃÃo da viabilidade bacteriana em cepas ambientais, orais e extra-orais de S. marcescens podendo ser uma ferramenta biotecnolÃgica Ãtil no controle da resistÃncia bacteriana. / Serratia marcescens is widely distributed in nature, but has emerged in the last years as important nosocomial pathogen with resistance of many antimicrobial drugs. This study aimed to verify the susceptibility of Serratia marcescens isolates from environment, from oral infections and from extra-oral infections to different antibiotics and evaluate the antimicrobial effect of photodynamic antimicrobial therapy as biotechnology tools reducing bacterial growth in planktonic cells and biofilm. E-test were performed for fifty-five strains and the PACT for the thirty strains more resistant to antimicrobials tested. The antimicrobial effect of toluidine blue O, associated with 4,72 J cm-2 of a light-emitting diode , was evaluated. Before and after the treatments, bacterial inocula were analysed with regard to the number of colony- forming units. For antimicrobials, we observed that the 55 strains analyzed, 13 (23.63%) were resistant to doxycycline, but only one (1.81%) isolate showed resistance to ciprofloxacin, another to tobramycin and another to cefotaxime, 24 ( 43.63%) strains had intermediate sensitivity to doxycycline, all were sensitive to imipenem and most were sensitive to ciprofloxacin, tobramycin and cefotaxime Statistical analysis showed no significant differences in resistance of samples of different origins for drugs DX, CT, and IP. Considering the resistance to CI, the environmental samples were significantly more resistant than samples oral and extra-oral. For the drug TM, the oral samples were significantly more sensitive than the other samples. The irradiation of planktonic and biofilm cultures in the absence of TBO (L+S-), incubation with TBO alone (L-S+) and untreated control group (L-S-) had no significant effect on the viability of strains of S. marcescens studied (p <0.05). Significant decreases in bacterial viability was observed only when planktonic and biofilm culture of environmental strains, oral and extra-oral S. marcescens were exposed to toluidine blue O and LED light at the same time (L+S+). Significant reductions in bacterial counts were observed by antimicrobial photodynamic therapy ranging from 10-11 to 10-7.The association of TBO and light, with energy density 4,72 J cm-2, was effective in reducing the viability of bacterial strains in environmental, oral and extra-oral S. marcescens and can be a useful biotechnological tool in the control of bacterial resistance.

Orientador: Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T13:27:08Z (GMT). No. of bitstreams: 1 Orsi_DanielaCastilho_D.pdf: 2624783 bytes, checksum: add51ff0035724efe97b8947a93c9d67 (MD5) Previous issue date: 2008 / Resumo: A sacarose é o principal açúcar utilizado no processamento de alimentos, contudo, o consumo excessivo e não balanceado de alimentos com alto teor de sacarose contribui para a prevalência de doenças como obesidade e cáries dentárias. Nas últimas décadas, tem ocorrido um aumento do interesse pela produção de novos açúcares como alternativa para substituir a sacarose. A isomaltulose (O-a-D-glicopiranosil-1,6-frutofuranosídeo) é um açúcar pouco cariogênico e isômero estrutural da sacarose, encontrada naturalmente no mel em pequenas quantidades. A bactéria Serratia plymuthica ATCC 15928 produz a enzima glicosiltransferase e catalisa a conversão da sacarose em isomaltulose. Neste trabalho, utilizou-se a metodologia de superfície de resposta para estudar o efeito dos componentes do meio de cultivo na produção de glicosiltransferase pela bactéria Serratia plymuthica em frascos sob agitação a 200 rpm e 30ºC. Foi obtida alta produção de glicosiltransferase (14,26 UA/mL, média dos pontos centrais) utilizando-se o meio de cultivo 1, composto de 40 g/L de melaço de cana de açúcar, 15 g/L de peptona bacteriológica da BiobrásÒ e 20 g/L de extrato de levedura Prodex Lac SDÒ. O meio de cultivo 2 (40 g/L de melaço de cana de açúcar e 20 g/L de extrato de levedura Prodex Lac SDÒ), além de render ótima produção de glicosiltransferase (13,54 UA/mL, média dos pontos centrais), teve seu custo reduzido por ser formulado sem a adição do componente peptona bacteriológica da BiobrásÒ. Foi estudado o efeito da temperatura (26ºC, 28ºC e 30ºC) na fermentação da bactéria Serratia plymuthica para produção de massa celular e de glicosiltransferase em fermentador de 6,6 L. A maior produção de glicosiltransferase ocorreu após 6 horas de fermentação na temperatura de 26ºC, sendo obtida atividade enzimática de 25,97 UA/mL. As células livres da bactéria Serratia plymuthica foram utilizadas para a conversão de sacarose em isomaltulose. Utilizando-se concentração de massa celular úmida de 20% (p/v) e concentração de solução de sacarose de 25% (p/v) obteve-se alta porcentagem de isomaltulose (84,33%, valor médio dos meios de cultivo 1 e 2) após 2 horas de reação a 27ºC, em frascos sob agitação a 180 rpm. As células livres cultivadas em meio de cultivo 2 (sem adição de peptona bacteriológica da BiobrásÒ) foram reutilizadas por nove bateladas sucessivas e obteve-se eficiente conversão de sacarose em isomaltulose (75,20%, média das bateladas). Foi estudada a produção de isomaltulose a partir de sacarose por células da bactéria Serratia plymuthica imobilizadas em alginato de cálcio. A conversão de sacarose em isomaltulose pelas células imobilizadas foi feita em bioreatores de leito empacotado mantidos a temperatura de 25°C. O tratamento das células imobilizadas em alginatoSynthÒ 2% com glutaraldeído aumentou a atividade enzimática, sendo obtida conversão de sacarose em isomaltulose acima de 64% por 15 dias. A goma gelana KELCOGELÒ F foi utilizada como suporte para imobilização das células de Serratia plymuthica. As células imobilizadas em goma gelana tratadas com glutaraldeído foram secas por 36 horas, sob refrigeração a 10°C. As células imobilizadas foram transferidas para bioreatores mantidos a 25ºC e usadas na conversão contínua de sacarose em isomaltulose. Quando as células imobilizadas secas foram utilizadas no processo contínuo, a conversão de sacarose em isomaltulose manteve-se acima de 69% por 15 dias. Esse estudo demonstrou a possibilidade do uso da goma gelana KELCOGELÒ F como suporte para imobilização das células de Serratia plymuthica. O suporte utilizado combina a simplicidade na técnica de imobilização celular, boa estabilidade operacional e altas taxas de bioconversão / Abstract: Sucrose is the main sweetener used in food processing, but, the excessive and imbalanced consumption of high-sucrose foods is a contributory factor in obesity and dental caries. In the last few decades, the production of new sweeteners as alternatives to sucrose has aroused great interest. Isomaltulose (O-a-D-glucopyranosyl-1,6- fructofuranose) is a low cariogenic sweetener and a structural isomer of sucrose, naturally present in honey in small quantities. The bacteria Serratia plymuthica ATCC 15928 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose into isomaltulose. In this work, response surface methodology was applied to study the effect of culture medium components in the production of glucosyltransferase by Serratia plymuthica in shaken flasks at 200 rpm and 30ºC. Higher glucosyltransferase production (14.26 UA/mL, average of the central points) was obtained in culture medium 1, composed of 40 g/L of sugar cane molasses, 15 g/L of BiobrásÒ bacteriological peptone and 20 g/L of Prodex Lac SDÒ yeast extract. Culture medium 2 (40 g/L of sugar cane molasses and 20 g/L of Prodex Lac SDÒ yeast extract, formulated without the component BiobrásÒ bacteriological peptone, resulted in a low cost medium and optimized glucosyltransferase production (13.54 UA/mL, average of the central points). The influence of temperature (26ºC, 28ºC and 30ºC) on the growth of the bacterium Serratia plymuthica for cell mass and glucosyltransferase production in a 6.6 L bioreactor, was also studied. The highest production of glucosyltransferase (25.97 UA/mL) was obtained in culture medium 1 after 6 hours at 26ºC. Free Serratia plymuthica cells were used for the conversion of sucrose into isomaltulose. A higher isomaltulose production (84.33%, mean value for culture media 1 and 2) was obtained at a temperature of 27ºC, 20% (w/v) wet cell mass and 25% (w/v) sucrose solution after 2 hours of reaction in shaken flasks at 180 rpm. The free cells cultivated in the culture medium 2 (without BiobrásÒ bacteriological peptone) were reused for nine successive batches, with efficient conversion of sucrose into isomaltulose (75.20%, average of the batches). Furthermore, the conversion of sucrose into isomaltulose using Serratia plymuthica cells immobilized in calcium alginate was also studied. The continuous production of isomaltulose by immobilized cells was accomplished in packed bed bioreactors maintained at 25°C. The treatment of cells immobilized in 2% SynthÒ alginate with glutaraldeyde increased enzyme activity obtaining an isomaltulose production of over 64% for 15 days. The gellan gum KELCOGELÒ F was also used as a support in the immobilization of Serratia plymuthica cells. The cells immobilized in gellan gum and treated with glutaraldeyde, were dried for approximately 36 hours at 10°C and used for the continuous production of isomaltulose. The immobilized cells were packed into bioreactors maintained at 25°C. When dry immobilized cells were used in the continuous process, the conversion of sucrose into isomaltulose was over 69% for about 15 days. This study demonstrated the feasibility of using KELCOGELÒ F gellan gum as a support in the immobilization of Serratia plymuthica cells. This support used combines the simplicity of the immobilization technique with good operational stability and high levels of bioconversion / Doutorado / Doutor em Ciência de Alimentos

With the increase of antibiotic resistant bacteria strains, the need to determine the mechanisms of antimicrobial resistance is similarly rising. <i>Serratia marcescens</i>, a ubiquitous, Gram-negative opportunistic pathogen is known to have strong, natural resistance to diverse antimicrobial agents including antibiotics and antimicrobial peptides. Recently, we identified <i>S. marcescens</i> as one of the few bacteria resistant to antimicrobial compounds produced by <i>Stemphylium vesicarium</i>, an isolated fungal spinach endophyte. To identify the mechanism of antimicrobial resistance to the unknown <i>Stemphylium</i> antimicrobial compounds, we designed a transposon mutant screen identifying mutants sensitive to antimicrobial inhibition of bacterial growth. A transposon mutant library was constructed using the Tn5 EZ-Transposome (Epicentre) system and contains 1,824 individual mutants with 127 being identified as having a decreased resistance to the <i>Stemphylium</i> antimicrobial compounds. The transposon growth inhibition screen initially evaluates the mutants for reduced growth in the presence of 25% fungal metabolite over 24 hours. The growth phenotype is then confirmed in triplicate in a 12-hour time course growth experiment. Identification of the genomic insertion site of the Tn5 transposon utilized a multi-step modified nested-PCR protocol, termed TAIL-PCR. Following PCR purification, nanodrop spectroscopy and gel electrophoresis were performed to ensure the amplification purity of the extracted DNA and was subsequently sequenced via WideSeq analysis. BLAST identified insertions in genes necessary for membrane biogenesis, drug transport, pili formation, and iron metabolism. Future work is aimed at confirming these results and understanding the role of iron sequestration. Not only will this research contribute to our understanding of <i>S. marcescens</i> antimicrobial resistance mechanisms, but it aids in our understanding of the mechanisms of antimicrobial resistance development in other human pathogens.