Materials and Methods Bacterial Strains and Growth Conditions We included in this study 46 clinical isolates of S. malto- philia obtained from patients admitted to four institutions in the City of São Paulo, Brazil: Instituto Dante Pazzanese de Cardiologia, Hospital das Clínicas, Laboratório Fleury, and Hospital Universitário (Universidade de São Paulo). Most of these clinical strains were isolated from respiratory tract secre- tions obtained from intubated patients with pneumonia; in most cases, S. maltophilia was the only infectious agent found (25). S. maltophilia ATCC 13637 is a reference strain also used in our studies. For expression of flagella, bacteria were grown on trypticase soy agar supplemented with 5% defibri- nated sheep blood (Oxoid, Basingstoke, England) at 37°C for 48 h. Transmission and Scanning Electron Microscopy We analyzed the presence of flagella by negative staining and transmission electron microscopy. Bacteria were nega- tively stained for 2 min with 1% phosphotungstic acid (pH 7.4) on carbon-Formvar (Electron Microscopy Sciences, Fort Washington, PA) copper grids as previously described (26,27). For ultrastructural analysis, bacterial specimens were fixed in 2% formalin and processed for scanning electron microscopy. Briefly, glass or plastic coverslips containing the adherent bac- teria were postfixed with 1% osmium tetraoxide, dehydrated by sequential ethanol concentrations, dried to critical point, and coated with a mixture of gold and paladium (27). The specimens were examined in a high-resolution Hitachi (Tokyo, Japan) scanning electron microscope. Isolation of Flagella For purification of flagella, clinical isolate S. maltophilia SMDP92 was grown on 100 blood agar plates and harvested in 100 mL of 10 mM phosphate-buffered saline (PBS), pH 7.4. The flagella were detached from the bacterial cells by vigorous shaking, and the supernatant containing the sheared flagella was separated by centrifugation at 8,000 x g for 30 min (26). The flagella were separated from outer membrane proteins and other contaminants by precipitation with 60% saturation of ammonium sulfate for 18 h at 4°C. After centrifugation at 12,000 x g for 30 min, the flagella were resuspended in PBS, and insoluble contaminants were removed by a similar centrif- ugation step. The supernatant was subjected to a second cycle of 20% ammonium sulfate precipitation (26). After dialysis with distilled water to remove excess salts, the purity of the preparations was monitored by sodium dodecyl-sulfate poly- acrylamide gel electrophoresis (SDS-PAGE) (28) and electron microscopy (26). Western Blotting and N-Terminal Amino Acid Sequence Analysis For SDS-PAGE and Western blot, whole bacterial cell extracts or flagellar extracts were denatured and separated in RESEARCH 14% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA) (27). The blot was reacted with anti-flagella antibodies and secondary anti-rabbit immunoglobulin (Ig) G conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO). The reaction was developed with a mixture of diamino benzidine and 30% hydrogen peroxide (Sigma). A 38-kDa protein band of interest was excised from the PVDF mem- brane and subjected to N-terminal amino acid sequence analy- sis at the Instituto de Química, Universidade de São Paulo. Sequence analysis and homology studies with published flagellin sequences were performed by using the EMBL/Gen- Bank (BLAST of National Center for Biotechnology Informa- tion, Bethesda, MD) software. Anti-Flagella Antibodies Antibodies against S. maltophilia flagella were raised by immunization of New Zealand rabbits with the flagellin pro- tein (38-kDa band) excised from Coomassie-blue stained gels. The bands were dried and homogenized in complete Freund’s adjuvant for the first dose and in incomplete adjuvant for the subsequent three weekly doses. Blood was collected at each immunization, and the presence of antibodies was monitored by Western blot. Antibodies against flagella obtained from E. coli E2348/69 (O127:H6), Shigella flexneri, and S. sonnei were available from previous studies (26,27,29). Adhesion to Inert Surfaces Adhesion to abiotic surfaces was studied at different times by using polystyrene 24-well plates (Nunc, Naperville, IL) with or without glass coverslips (21). Twenty microliters of an overnight culture of bacteria grown in Luria-Bertani broth was added to the wells containing 1 mL of Dulbecco’s minimal essential medium (D-MEM), supplemented with high glucose, and incubated for 15 and 30 min and 1, 2, 3, 4, 5, 6, 18, 48, and 72 h. After three washes with PBS, the bound bacteria were fixed with methanol, stained with crystal violet, and visualized under a light microscope. Replica samples were fixed with 2% formalin for scanning electron microscopy as described. All experiments were conducted in triplicate and were repeated at least three times. To quantitate bacterial adherence over time (from 30 min to 72 h), we performed an adherence assay in 96-well plates as described and measured the uptake of crystal violet staining by reading optical density at 620 nm (22). Results Characteristics of S. maltophilia Flagella Growth of the bacteria in blood agar plates at 37°C resulted in a condition favorable for flagella expression. Anal- ysis by electron microscopy demonstrated that while some organisms had only one polar flagellum, others had several flagellar structures (Figure 1). The flagella filaments, ~45 nm in width and >15 µm long, are indistinguishable from other Emerging Infectious Diseases • Vol. 8, No. 9, September 2002 919