Cytotoxic activity induced by culture filtrates of Stenotrophomonas maltophiliaclinical isolates

Figueirêdo*, P.; Furumura, M. and Yano, T.

Depto. Microbiologia e Imunologia, IB, Universidade Estadual de Campinas, SP, Brazil

Stenotrophomonasmaltophilia previously known as Pseudomonas maltophilia or Xanthomonasmaltophilia, has gained to prominence in recent years as an important nosocomial pathogen associated with significant case/fatality ratios in debilitated or immunosuppressed patients. This species has also been implicated in an increasing numbers of infections, such as bacteremia, endocarditis, ophthalmological syndromes, skin lesions, and urinary, respiratory tract and gastrointestinal infections. Despite this clinical importance, little is known about putative virulence factors involved in the pathogenicity of Stenotrophomonasmaltophilia and in this study the cytotoxic effects induced by culture filtrates of Stenotrophomonas in Vero and Hep-2 cells were investigated. Five strain of S. maltophilia were cultivated in tryptic soy broth (TSB), were inoculated and incubated in shaker  at 37°C. After 20h incubation, cultures were centrifuged and the supernatants filtered through 0.22mm membranes. Culture filtrates were assayed on Vero and Hep-2 cell monolayers that were grown in Eagle minimal essential medium (EMEM) with 10% fetal bovine serum. Monolayers were established in plastic plates containing 96 wells, using 0.1 mL of cell culture (2 X 10⁵ cells mL^-1).To each well with cells, 0,1 mL of culture filtrate was added. Cultures were incubated at 37°C in 5% CO2 atmosphere and the morphological changes in cells were observed after 24 and 48h of inoculation. The viability of cultured mamamlian cells was determined after 24h of treatment with bacterial supernatants, the medium was removed and 0.2mL of MEM containing 50mg of neutral red ml^–1 was added to each  well followed by incubation for 3h at 37°C. After washing with formol-calcium, 0.2 mL of na acetic acid-ethanol mixture was added to each well. The plates were kept at room temperature for 15 min and then read in a spectrophotometer at 540 nm. Filtrates from the culture supernatants of five strains of S. maltophilia caused rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells after 24 h. In Vero cells, the cytotoxic effects were characterized by initial rounding, vigorous endocytosis and cell agregation within 30 min. of incubation, with complete destruction and cell death after 72 h. These cellular alterations were similar to those found in viral and intracellular membrane fusion proteins that contain a minimal set of domains that must be deployed at the appropriate time during the fusion process. Membrane fusion is a crucial event mediated by a fusigenic protein that binds the target membrane receptors, thereby causing conformational changes in the lipid bilayer. The cytotoxins present in culture supernatants of S. maltophilia have yet to be characterized. S. maltophilia produces many extracellular enzymes mainly proteases and lipases, that may play a role in the pathogenesis of S. maltophilia because its presents ability to use animal proteins as a source of carbon and nitrogen.

FINANCIAL SUPPORT: CNPq

Correspondence to: patty_marry@yahoo.com