Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Tipo de documento: | Tese |
Idioma: | eng |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da USP |
Texto Completo: | http://www.teses.usp.br/teses/disponiveis/60/60137/tde-19122019-083346/ |
Resumo: | Urinary tract infection (UTI) is the most common hospital acquired pathological process and indwelling urinary catheters increase the risk of bacteriuria, which can progress to a serious condition. This infection usually follows formation of biofilm on both the internal and external catheter surface. The uropathogenic bacteria Escherichia coli is the most common infecting microorganism on catheter and its biofilms have been studied as a platform to select strategies to control UTIs. The present study examined whether a nano delivery system containing chitosan affected the growth of uropathogenic biofilms of E. coli. This work was divided in two stages, the first involved comparing adhesion surfaces most used in in vitro biofilm models and the second evaluated the susceptibility of free cells and biofilm of E. coli to a nanostructured lipid carrier coated with chitosan (NLC-chitosan). Thus, E. coli biofilms were formed on catheter, glass slides or tissue culture plates for 5 days and the composition of biofilm was evaluated. In the second stage of the work, NLC-chitosan was prepared using the emulsion and sonication method, and further characterized with respect to particle size, polydispersity index, and zeta potential. After determining the minimum inhibitory (MIC) and bactericidal concentrations (MBC), E. coli biofilms were grown on catheter specimens. At the 48, 72, 96, and 120 hours of growth, biofilms were exposed to 0.9% NaCl solution (negative control), 0.12% chlorhexidine solution (positive control), or NLC-chitosan (final chitosan concentration of 0.28%). After 24 hours of treatment, the biofilms were collected to analyze their bacterial viability. Data were statistically analyzed by Tukey-Kramer or Tukey test with a level of significance of 5%. Bacterial colony viability was higher in catheter compared to glass slides or plates (p<0.05) and the lowest bacterial count was observed for glass slide (p<0.05). Although concentrations of carbohydrate were lower in biofilm formed on catheter (p<0.05), no differences were observed between catheter and plate (p>0.05) as well as glass slides and plate (p>0.05) for protein quantification. Regarding second work stage, NLC-chitosan preparation had bimodal particle size distribution with mean size of 292.9 ± 2.5 nm and polydispersity index of 0.24 ± 0.03, and positive zeta potential (+19.1 ± 0.2) indicating the nanoparticle coating by chitosan. Analysis of MIC and MBC values revealed that formulation inhibited bacterial growth and exerted bactericidal action at concentrations 100 times higher than those required for chlorhexidine digluconate (positive control). Compared with the control groups, NLC-chitosan affected bacterial colony viability of biofilms at all ages studied (p<0.05). The results suggest that catheter is a proper surface to study E. coli biofilm compared to either glass slides or polystyrene plates. In addition, both free cells and biofilms of E. coli were significantly affected by NLC-chitosan, which can be a feasible approach for studies using uropathogenic bacteria. In future, urinary catheter can be used as model to study simulated UTIs, using mixed populations of bacteria, and the effect of NLC-chitosan or its association with other antimicrobial agents evaluated. |
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Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coliEfeito de carreador lipídico nanoestruturado contendo quitosana em células livres e biofilme de Escherichia coliBiofilmBiofilmeCarreador lipídico nanoestruturadoCateterCatheterChitosanNanostructured lipid carrierQuitosanaUropathogenUropatógenoUrinary tract infection (UTI) is the most common hospital acquired pathological process and indwelling urinary catheters increase the risk of bacteriuria, which can progress to a serious condition. This infection usually follows formation of biofilm on both the internal and external catheter surface. The uropathogenic bacteria Escherichia coli is the most common infecting microorganism on catheter and its biofilms have been studied as a platform to select strategies to control UTIs. The present study examined whether a nano delivery system containing chitosan affected the growth of uropathogenic biofilms of E. coli. This work was divided in two stages, the first involved comparing adhesion surfaces most used in in vitro biofilm models and the second evaluated the susceptibility of free cells and biofilm of E. coli to a nanostructured lipid carrier coated with chitosan (NLC-chitosan). Thus, E. coli biofilms were formed on catheter, glass slides or tissue culture plates for 5 days and the composition of biofilm was evaluated. In the second stage of the work, NLC-chitosan was prepared using the emulsion and sonication method, and further characterized with respect to particle size, polydispersity index, and zeta potential. After determining the minimum inhibitory (MIC) and bactericidal concentrations (MBC), E. coli biofilms were grown on catheter specimens. At the 48, 72, 96, and 120 hours of growth, biofilms were exposed to 0.9% NaCl solution (negative control), 0.12% chlorhexidine solution (positive control), or NLC-chitosan (final chitosan concentration of 0.28%). After 24 hours of treatment, the biofilms were collected to analyze their bacterial viability. Data were statistically analyzed by Tukey-Kramer or Tukey test with a level of significance of 5%. Bacterial colony viability was higher in catheter compared to glass slides or plates (p<0.05) and the lowest bacterial count was observed for glass slide (p<0.05). Although concentrations of carbohydrate were lower in biofilm formed on catheter (p<0.05), no differences were observed between catheter and plate (p>0.05) as well as glass slides and plate (p>0.05) for protein quantification. Regarding second work stage, NLC-chitosan preparation had bimodal particle size distribution with mean size of 292.9 ± 2.5 nm and polydispersity index of 0.24 ± 0.03, and positive zeta potential (+19.1 ± 0.2) indicating the nanoparticle coating by chitosan. Analysis of MIC and MBC values revealed that formulation inhibited bacterial growth and exerted bactericidal action at concentrations 100 times higher than those required for chlorhexidine digluconate (positive control). Compared with the control groups, NLC-chitosan affected bacterial colony viability of biofilms at all ages studied (p<0.05). The results suggest that catheter is a proper surface to study E. coli biofilm compared to either glass slides or polystyrene plates. In addition, both free cells and biofilms of E. coli were significantly affected by NLC-chitosan, which can be a feasible approach for studies using uropathogenic bacteria. In future, urinary catheter can be used as model to study simulated UTIs, using mixed populations of bacteria, and the effect of NLC-chitosan or its association with other antimicrobial agents evaluated.A infecção do trato urinário (ITU) é a infecção mais comum em nível hospitalar, sendo os cateteres urinários responsáveis por desenvolver o risco de bacteriúria, o que pode agravá-la. Esta infecção geralmente está relacionada a formação de biofilme na superfície interna e externa do cateter. A bactéria uropatogênica Escherichia coli continua sendo o microrganismo mais isolado em cateteres e seus biofilmes são estudados de forma a desenvolver estratégias de controle das ITUs. O presente estudo examinou o efeito de um sistema de liberação nanoestruturado contendo quitosana no crescimento de biofilmes uropatogênicos de E. coli. Este trabalho foi dividido em duas etapas, sendo a primeira uma comparação de biofilmes crescidos nas superfícies mais utilizadas nos modelos de biofilme in vitro e a segunda a avaliação da suscetibilidade de células livres e biofilme de E. coli exposto a um carreador lipídico nanoestruturado contendo quitosana (CLN-quitosana). Assim, biofilmes de E. coli foram formados em cateter, lâminas de vidro ou placas de cultura de células por 5 dias, sendo a composição do biofilme avaliada. Na segunda etapa do trabalho, a CLN-quitosana foi preparada usando o método de emulsão e sonicação, sendo caracterizada em relação ao tamanho de partícula, índice de polidispersividade e potencial zeta. Após a determinação das concentrações inibitórias mínimas (CIM) e bactericidas (CBM), os biofilmes de E. coli foram crescidos em cateter. Após 48, 72, 96 e 120 horas de crescimento, os biofilmes foram expostos a solução de NaCl a 0,9% (controle negativo), solução de clorexidina a 0,12% (controle positivo) e CLN-quitosana (concentração final de quitosana de 0,28%). Após 24 horas de tratamento, os biofilmes foram coletados para análise de viabilidade bacteriana. Os dados foram analisados estatisticamente pelo teste de Tukey-Kramer ou Tukey, com nível de significância de 5%. A viabilidade bacteriana foi maior no cateter em comparação com lâminas de vidro ou placas de cultura (p <0,05) e a menor contagem bacteriana foi observada na lâmina de vidro (p <0,05). Embora as concentrações de carboidratos tenham sido menores no biofilme formado no cateter (p <0,05), não foram observadas diferenças estatisticamente significativas na quantificação de proteínas para os grupos cateter e placa de cultura (p>0,05) ou entre as lâminas de vidro e a placa (p> 0,05). Em relação à segunda etapa do trabalho, a preparação de CLN-quitosana apresentou distribuição bimodal de tamanho de partícula com tamanho médio de 292,9 ± 2,5 nm, índice de polidispersividade de 0,24 ± 0,03 e potencial zeta positivo (+19,1 ± 0,2), indicando o revestimento de nanopartículas pela quitosana. A análise dos valores de CIM e CBM revelou que a formulação inibiu o crescimento bacteriano e exerceu ação bactericida em concentrações 100 vezes maior do que a necessária para o digluconato de clorexidina (controle positivo). Comparado com os grupos controle, a CLN-quitosana afetou a viabilidade bacteriana dos biofilmes em todas as idades avaliadas (p<0,05). Sendo assim, os resultados sugerem que o cateter é a superfície adequada para estudar biofilmes de E. coli. Tanto as células livres quanto os biofilmes foram afetados pelo CLN-quitosana. No futuro, o cateter urinário pode ser utilizado como modelo para estudar ITUs com populações mistas de bactérias e o efeito de CLN-quitosana ou de sua associação com outros antimicrobianos poderá ser avaliado.Biblioteca Digitais de Teses e Dissertações da USPGarbellini, Carolina Patricia AiresOsungunna, Michael Oluwole2019-10-02info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttp://www.teses.usp.br/teses/disponiveis/60/60137/tde-19122019-083346/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2021-12-18T12:55:57Zoai:teses.usp.br:tde-19122019-083346Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212021-12-18T12:55:57Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false |
dc.title.none.fl_str_mv |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli Efeito de carreador lipídico nanoestruturado contendo quitosana em células livres e biofilme de Escherichia coli |
title |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
spellingShingle |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli Osungunna, Michael Oluwole Biofilm Biofilme Carreador lipídico nanoestruturado Cateter Catheter Chitosan Nanostructured lipid carrier Quitosana Uropathogen Uropatógeno |
title_short |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
title_full |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
title_fullStr |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
title_full_unstemmed |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
title_sort |
Effect of nanostructured lipid carrier containing chitosan on free cells and biofilm of Escherichia coli |
author |
Osungunna, Michael Oluwole |
author_facet |
Osungunna, Michael Oluwole |
author_role |
author |
dc.contributor.none.fl_str_mv |
Garbellini, Carolina Patricia Aires |
dc.contributor.author.fl_str_mv |
Osungunna, Michael Oluwole |
dc.subject.por.fl_str_mv |
Biofilm Biofilme Carreador lipídico nanoestruturado Cateter Catheter Chitosan Nanostructured lipid carrier Quitosana Uropathogen Uropatógeno |
topic |
Biofilm Biofilme Carreador lipídico nanoestruturado Cateter Catheter Chitosan Nanostructured lipid carrier Quitosana Uropathogen Uropatógeno |
description |
Urinary tract infection (UTI) is the most common hospital acquired pathological process and indwelling urinary catheters increase the risk of bacteriuria, which can progress to a serious condition. This infection usually follows formation of biofilm on both the internal and external catheter surface. The uropathogenic bacteria Escherichia coli is the most common infecting microorganism on catheter and its biofilms have been studied as a platform to select strategies to control UTIs. The present study examined whether a nano delivery system containing chitosan affected the growth of uropathogenic biofilms of E. coli. This work was divided in two stages, the first involved comparing adhesion surfaces most used in in vitro biofilm models and the second evaluated the susceptibility of free cells and biofilm of E. coli to a nanostructured lipid carrier coated with chitosan (NLC-chitosan). Thus, E. coli biofilms were formed on catheter, glass slides or tissue culture plates for 5 days and the composition of biofilm was evaluated. In the second stage of the work, NLC-chitosan was prepared using the emulsion and sonication method, and further characterized with respect to particle size, polydispersity index, and zeta potential. After determining the minimum inhibitory (MIC) and bactericidal concentrations (MBC), E. coli biofilms were grown on catheter specimens. At the 48, 72, 96, and 120 hours of growth, biofilms were exposed to 0.9% NaCl solution (negative control), 0.12% chlorhexidine solution (positive control), or NLC-chitosan (final chitosan concentration of 0.28%). After 24 hours of treatment, the biofilms were collected to analyze their bacterial viability. Data were statistically analyzed by Tukey-Kramer or Tukey test with a level of significance of 5%. Bacterial colony viability was higher in catheter compared to glass slides or plates (p<0.05) and the lowest bacterial count was observed for glass slide (p<0.05). Although concentrations of carbohydrate were lower in biofilm formed on catheter (p<0.05), no differences were observed between catheter and plate (p>0.05) as well as glass slides and plate (p>0.05) for protein quantification. Regarding second work stage, NLC-chitosan preparation had bimodal particle size distribution with mean size of 292.9 ± 2.5 nm and polydispersity index of 0.24 ± 0.03, and positive zeta potential (+19.1 ± 0.2) indicating the nanoparticle coating by chitosan. Analysis of MIC and MBC values revealed that formulation inhibited bacterial growth and exerted bactericidal action at concentrations 100 times higher than those required for chlorhexidine digluconate (positive control). Compared with the control groups, NLC-chitosan affected bacterial colony viability of biofilms at all ages studied (p<0.05). The results suggest that catheter is a proper surface to study E. coli biofilm compared to either glass slides or polystyrene plates. In addition, both free cells and biofilms of E. coli were significantly affected by NLC-chitosan, which can be a feasible approach for studies using uropathogenic bacteria. In future, urinary catheter can be used as model to study simulated UTIs, using mixed populations of bacteria, and the effect of NLC-chitosan or its association with other antimicrobial agents evaluated. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-10-02 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://www.teses.usp.br/teses/disponiveis/60/60137/tde-19122019-083346/ |
url |
http://www.teses.usp.br/teses/disponiveis/60/60137/tde-19122019-083346/ |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
|
dc.rights.driver.fl_str_mv |
Liberar o conteúdo para acesso público. info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Liberar o conteúdo para acesso público. |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
|
dc.publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da USP instname:Universidade de São Paulo (USP) instacron:USP |
instname_str |
Universidade de São Paulo (USP) |
instacron_str |
USP |
institution |
USP |
reponame_str |
Biblioteca Digital de Teses e Dissertações da USP |
collection |
Biblioteca Digital de Teses e Dissertações da USP |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP) |
repository.mail.fl_str_mv |
virginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.br |
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1809090877697556480 |