title: Monolayer culture systems with respiratory epithelial cells for evaluation of bacterial invasiveness.
abstract: Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm(2) filter membranes with 3.0 microm pores, and were incubated at 37 degrees C under 5% CO(2) for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 +/- 16.8 to 628.8 +/- 66.3 Omega cm(2)) and Calu-3 cells (TER values: 490.5 +/- 25.2 to 547.8 +/- 21.6 Omega cm(2)) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.
authors.affiliation: Department of Infection Control and Laboratory Diagnostics, Tohoku University Graduate School of Medicine, Sendai, Japan. hiraichi@mail.tains.tohoku.ac.jp
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Hirakata
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Yano
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Arai
authors.forename: Shiro
authors.initials: S
authors.lastname: Endo
authors.forename: Hajime
authors.initials: H
authors.lastname: Kanamori
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Aoyagi
authors.forename: Ayako
authors.initials: A
authors.lastname: Hirotani
authors.forename: Miho
authors.initials: M
authors.lastname: Kitagawa
authors.forename: Masumitsu
authors.initials: M
authors.lastname: Hatta
authors.forename: Natsuo
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kunishima
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Kawakami
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kaku
mesh_terms: D000818:Animals; D001419:Bacteria / Q000472:pathogenicity*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002469:Cell Separation; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology* / Q000382:microbiology*; D006801:Humans; D012137:Respiratory System / Q000166:cytology* / Q000382:microbiology*