Evaluation of Pseudomonas aeruginosa (PAO1) adhesion to human alveolar epithelial cells A549 using SYTO 9 dye

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• 作者：Mar Larrosa ; ¹ ; ^{marlarrosa@cebas.csic.es} ; Pilar Truchado¹ ; ^{ptruchado@cebas.csic.es} ; Juan Carlos Espí ; n ^{jcespin@cebas.csic.es} ; Francisco A. Tomá ; s-Barberá ; n ^{fatomas@cebas.csic.es} ; Ana Allende ^{aallende@cebas.csic.es} ; Marí ; a Teresa Garcí ; a-Conesa ^{mtconesa@cebas.csic.es}
• 关键词：Bacterial adhesion ; Vital staining ; Fluorescence microplate reader ; Epifluorescence microscopy ; Genistein
• 刊名：Molecular and Cellular Probes
• 出版年：2012
• 期刊代码：104_08908508
• 类别：imm
• 出版时间：June, 2012
• 卷：26
• 期：3
• 页码：121-126
• 文件大小：601 K

摘要

Pseudomonas aeruginosa is an aerobic Gram-negative bacterium characterized by a natural resistance to several antibiotics. It is a major cause of nosocomial infections in patients with compromised host defence mechanisms mainly related to the respiratory tract. P. aeruginosa infection first step is the adhesion of the bacteria to the host cells and thus, the development of techniques that can easily assess adhesion of bacteria strains and of bacteria isolated from biological samples is fundamental. The aim of our work was to develop a fast and effective method to evaluate the adhesion of P.聽aeruginosa to bronchial epithelial cells. To meet our goal we optimized a staining protocol using the vital dye SYTO 9 and P.聽aeruginosa PAO1. We established the appropriate dying conditions as well as the stability of the stained bacteria. Adhesion was first measured using the traditional plate counting method and then, adhesion values were compared to those obtained using a fluorescence microplate reader and epifluorescence microscopy. Our results show that the use of SYTO 9 does not interfere with the bacteria viability, bacteria cell growth, and adhesion of P.聽aeruginosa to A549 epithelial cells. Both the fluorescence microplate reader and the epifluorescence microscopy gave similar results to those attained with the plate counting method, however, the epifluorescence microscopy also allowed for simultaneous discrimination of damaging effects on the human cells. Overall, our data indicate that the use of SYTO 9 combined with a fluorescence microplate reader or an epifluorescence microscope provides a rapid method to evaluate the adhesion of P.聽aeruginosa to human epithelial cells. However, to show unequivocally that a specific drug or compound has a truly inhibitory effect on the bacterial adhesion without affecting the number of human cells, the epifluorescence microscopy is recommended.