Figure 5. Identification of the 38-kDa flagellin protein SMFliC in clinical isolates of Stenotrophomonas maltophilia. Lane 1, SMDP14; lane 2, SMDP275; lane 3, SMHC176; lane 4, SMHC181; lane 5, SMDP315; lane 6, SMDP314; and lane 7, SMHC179. Lane 8, the purified SMFliC, was used as positive control. The immunoblot shows the presence of the 38-kDa flagellin protein in all the isolates. Doublet bands were seen in some of the isolates. Molecular weight standards and the 38-kDa flagellin protein are indicated by arrows. Figure 6. Kinetics of Stenotrophomonas maltophilia adherence to plas- tic. (A) As early as 30 min, individual bacteria have attached to the plas- tic surface and formed small clumps (arrows). (B–D) As incubation time proceeds for 1 (B), 2 (C), and 4 (D) h, the number of attached bacteria increases throughout the abiotic surface. (E) At 6 h, the bacterial mono- layers progress into three-dimensional microcolonies (arrows). (F) After 18 h, the microcolonies have formed true bacterial communities. No obvious differences were noted beyond this incubation period. Magnifi- cation 400x. differences were observed at incubation periods >18 h of infection (data not shown). The kinetics of adhesion were also monitored by quantification of crystal violet–stained-bacteria adhering to the 96-well plates. In correlation with the light microscopy micrographs shown above (Figure 6), a time- dependent adherence profile was obtained that reached a max- imum level at 18 h (Figure 7), with no substantial increase in adherence beyond this period (data not shown). Furthermore, analysis by scanning electron microscopy of SMDP92 adhering to the plastic showed structures resembling flagella on the adhering bacteria (Figure 8). These filaments were seen protruding from the bacteria, apparently forming physical bridges between them. Thus, these filaments may play some yet-undefined role in adherence to plastic. High-power magnification of adhering bacteria showed flagella-like fila- ments (40–50 µm in width) and thin fibrillar structures (5–7 µm in width) resembling pili interconnecting bacteria and mediat- ing adhesion of the bacteria to the abiotic surface (Figure 8). Discussion Although adherence to abiotic surfaces is a property of both environmental and clinical S. maltophilia isolates, little information has been available to elucidate the nature of the surface factors involved in this phenomenon. Flagella have RESEARCH been associated with biofilm formation in other bacteria (18,20–22), where they can perform three basic roles: a) act as an adhesin promoting intimate attachment to the surface; b) generate force to subjugate the repulsive forces between bacte- ria and surface; and c) promote spread of the bacteria through- out the surface (20). In 1983, Montie and Stover purified flagella from several pseudomonads, including P. maltophilia strain B69 (now referred to as Stenotrophomonas maltophilia) (32), and found that B69 produced a flagellin and had a molec- ular mass of 33 kDa. They found that antisera against flagella of P. aeruginosa and P. cepacia did not agglutinate P. malto- philia bacteria, suggesting absence of antigenic cross-reactiv- ity between these flagella. No further biochemical characterization of S. maltophilia flagella has been done. In this paper, we describe the purification and characterization of S. maltophilia flagella; we raised specific antibodies to study the production of flagella in a collection of clinical isolates. The flagella produced by S. maltophilia strains are composed of a 38-kDa flagellin subunit, SMFliC. The identity of this polypeptide was demonstrated by N-terminal amino acid sequencing analysis and by immunodetection assays using antibodies raised against the purified flagellin. The discrep- ancy between the molecular mass of SMFliC and the flagellin (33 kDa) found previously in B69 could be attributed to differ- ences in the electrophoresis conditions and molecular mass standards used, as well as to differences in the strains per se. Nevertheless, we did find molecular mass differences among flagellins produced by clinical isolates. The comparison between the N-terminal amino acid sequence obtained from this 38-kDa polypeptide (14 residues) showed that SMFliC shares important identity with several known flagellins: 71.4% identity to FliC of E. coli, P. mirabi- lis, and S. sonnei, and 78.6% identity to FliC of Serratia marcescens. Stenotrophomonas was previously considered a pseudomonad (2,3); however, the identity between the FliC of Figure 7. Graph showing kinetics of adherence by SMPD92 and ATCC 13637. Bacteria were allowed to bind to the plastic for 72 h and then were stained with crystal violet. Bacterial uptake of the dye was mea- sured at 620 nm. Closed and open circles represent SMDP92 and ATCC13637, respectively. Emerging Infectious Diseases • Vol. 8, No. 9, September 2002 921