title: Evaluation of Pseudomonas aeruginosa (PAO1) adhesion to human alveolar epithelial cells A549 using SYTO 9 dye.
abstract: 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. aeruginosa to bronchial epithelial cells. To meet our goal we optimized a staining protocol using the vital dye SYTO 9 and P. 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. 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. 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.
authors.affiliation: Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain. marlarrosa@cebas.csic.es
authors.forename: Mar
authors.initials: M
authors.lastname: Larrosa
authors.forename: Pilar
authors.initials: P
authors.lastname: Truchado
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Espín
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Tomás-Barberán
authors.forename: Ana
authors.initials: A
authors.lastname: Allende
authors.forename: María Teresa
authors.initials: MT
authors.lastname: García-Conesa
mesh_terms: D001422:Bacterial Adhesion; D002460:Cell Line; D004396:Coloring Agents; D004847:Epithelial Cells / Q000502:physiology*; D006801:Humans; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011650:Pulmonary Alveoli / Q000166:cytology
chemical_list: D004396:Coloring Agents; D009930:Organic Chemicals; C103389:SYTO 9