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Doctoral Thesis
DOI
https://doi.org/10.11606/T.82.2017.tde-03102017-154010
Document
Author
Full name
Natália Helena Mendes
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2015
Supervisor
Committee
Pizzolitto, Elisabeth Loshchagin (President)
Storti, Anísio
Caminaga, Raquel Mantuaneli Scarel
Capote, Ticiana Sidorenko de Oliveira
Rollo, João Manuel Domingos de Almeida
Title in Portuguese
Desenvolvimento do biofilme bacteriano em superfícies de metais puros
Keywords in Portuguese
Biofilme
Citotoxicidade
Metais puros
Microscopia de força atômica
Microscopia eletrônica de varredura
Abstract in Portuguese
Biofilmes são aglomerados complexos de células microbianas que crescem na superfície de um material sólido, como um metal. As superfícies metálicas são amplamente utilizadas em dispositivos biomédicos cirúrgicos e em superfícies de mobiliário intra-hospitalares as quais podem ser infectadas por bactérias epidemiologicamente importantes e permitir o desenvolvimento de um biofilme. O objetivo desse trabalho foi avaliar a superfície topográfica dos metais puros, incluindo: chumbo, cromo, estanho, ferro e níquel, avaliar a aderência bacteriana nestas superfícies, com a consequente formação de biofilme e a potencial citotoxicidade dos metais por meio de microscopia de força atômica (MFA), microscopia óptica e microscopia eletrônica de varredura (MEV). A metodologia constou de observações microscópicas e procedimentos bacteriológicos. A aderência bacteriana foi verificada por meio de MEV e a viabilidade celular bacteriana por contagem de Unidades Formadoras de Colônia (UFC). A citotoxicidade dos metais foi avaliada frente a células CHO-K1 por ensaio XTT. As bactérias selecionadas foram: Escherichia coli, Pseudomonas aeruginosa (gram-negativos); Staphylococcus epidermidis e Staphylococcus aureus (gram-positivos) Para realizar o estudo, foram preparados corpos de prova dos metais puros e colocados em contato com cada uma das bactérias (da ordem 108 UFC/mL). Os resultados mostraram a formação de biofilme em cada um dos corpos de prova. A contagem das células viáveis demonstrou a recuperação de 105 UFC/mL para Escherichia coli, Pseudomonas aeruginosa e Staphylococcus aureus após contato com os metais chumbo, cromo, estanho, ferro e níquel, e para Staphylococcus epidermidis após contato com chumbo, níquel e cromo houve uma redução bacteriana de 103 UFC/mL. Para Staphylococcus epidermidis após contato com estanho foram recuperadas 1,14 x 102 UFC/mL e para o ferro, houve recuperação de 1,3 x 103 UFC/mL. A MEV demonstrou nestas superfícies metálicas, os bacilos e cocos aderidos e agrupados em uma massa amorfa formando biofilme. Os resultados da rugosidade (Ra) de cada uma destas superfícies obtidos por MFA em varredura em 2 microns da superfície, mostram que o Ra do chumbo foi de 38.258 μm; estanho 13.481 μm; níquel 3.929 μm; ferro 3.689 μm e cromo 2.097 μm. Os resultados do teste XTT, após contato com as células CHO-K1, mostram que o ferro foi citotóxico para estas células (p<0,05) diferença estatisticamente significante em relação ao controle negativo. Os metais puros avaliados nas condições experimentais do estudo mostram que as superfícies dos metais puros não impedem a aderência bacteriana e formação de biofilme das bactérias selecionadas.
Title in English
Bacterial biofilm development onto pure metals surface
Keywords in English
Atomic force microscopy
Biofilm
Cytotoxicity
Pure metals
Scanning electron microscopy
Abstract in English
Biofilms are complex microbial cell clusters that are growing on the solid material surface, as a metal. The metalic surfaces are widely used in surgical biomedical devices and hospital furniture surfaces which can be infected by important bacteria and to allow biofilm development. The aim of this work was to evaluate the topographic surface of the lead, chromium, tin, iron and nickel pure metals, evaluate bacterial adhesion in these surfaces with subsequent biofilm formation and the potential cytotoxicity of metals through force microscopy atomic (AFM), optical microscopy and scanning electron microscopy (SEM). The methodology consisted of microscopic observations and bacteriological procedures. Bacterial adherence was observed by SEM and the bacterial cell viability by colony forming units colony counts (CFU). The cytotoxicity of metals was evaluated using CHO-K1 cells by XTT assay.The isolated bacteria were: Escherichia coli, Pseudomonas aeruginosa (gram-negative), and Staphylococcus epidermidis and Staphylococcus aureus (gram positive). In order to realize this study, samples of pure metals were prepared and put in touch with each bacteria (in 108 CFU/mL). The results show biofilm formation in each of the specimens. The counting of viable cells demonstrated a recovery 105 CFU/mL for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus after contact with lead, chromium, tin, iron and nickel metals and Staphylococcus epidermidis after contact with lead, nickel and chromium there was a bacterial reduction of 103 CFU/mL. For Staphylococcus epidermidis after contact with tin were recovered 1.14 x 102 CFU / mL and the iron, there was a recovery of 1.3 x103 CFU/mL. The data show that there was a bacterial reduction of 103 CFU/mL viable cells. Staphylococcus epidermidis on contact with tin were recovered 1,14 x 102 and the iron recovery was 1,3 x 103 UFC/mL. SEM showed the metal surfaces, bacilli and coccus adhered and grouped in an amorphous mass forming biofilm. The results of the roughness (Ra) of each of the surfaces obtained by AFM scan of 2 microns from the surface, show that the RA lead was 38,258 uM; Tin 13,481 uM; Nickel 3,929 uM; iron 3,689 μm and chrome 2,097 μm. The results of XTT, after contact with the CHO-K1 cells, show that iron was cytotoxic to these cells (p <0.05) statistically significant difference compared to the negative control. Pure metals evaluated in the experimental conditions of the study show that the surfaces of pure metals do not prevent bacterial adherence and biofilm formation of the bacteria selected.
 
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Publishing Date
2017-10-03
 
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