RESEARCH Figure 1. Electron micrographs showing expression of flagella by SMDP92. Stenotrophomonas maltophilia strains can have one (A) to several flagella (B,C). The flagella on these bacteria show a polar dis- position. Bars, 0.5 µm Figure 2. Analysis of flagella purified from SMDP92. (A) sodium dode- cyl-sulfate polyacrylamide gel electrophoresis of purified flagella, show- ing the 38-kDa flagellin subunit. Lane 1, molecular weight standards; lane 2, purified SMFliC. (B) Immunoblotting and reactivity of purified fla- gella with anti-SMFliC antibodies. The 38-kDa flagellin is indicated by an arrow. (C) Electron microscopy of purified flagella visualized by nega- tive staining. Bar, 0.37 µ m. unsheathed flagella such as those produced by E. coli or Sal- monella (30,31). After purification of flagella by repeated ammonium sulfate precipitations, a peptide band that migrated with an apparent mass of 38 kDa was visualized in SDS-PAGE Coomasie blue-stained gels (Figure 2A). Antibodies obtained against the excised 38-kDa putative flagellin reacted with this polypeptide in immunoblots (Figure 2B). The flagella prepara- tion was rich in flagellar filaments as determined by negative staining and electron microscopy (Figure 2C). Sequence and Antigenic Relatedness of S. maltophilia Flagellin to Other Flagellins These results suggested that the 38 kDa is the major struc- tural component (FliC) of the flagella filament. Thus, this polypeptide was subjected to N-terminal amino acid sequence analysis, which showed that the 38-kDa protein is in fact the flagellin structural protein, which is highly homologous to other bacterial flagellins. The S. maltophilia FliC protein, SMFliC, showed considerable identity in its first 14 amino acid residues to the flagellins of E. coli, P. mirabilis, and Shigella sonnei (71.4%), and 78.6% identity to the flagellin of Serratia marcescens. The flagella produced by P. aeruginosa showed the lowest level of identity (57.2%) with SMFliC (Figure 3). Because of these sequence similarities, we were then inter- ested in determining if SMFliC shared any common epitopes with the other flagellins. This antigenic cross-reactivity was investigated by using several antisera against flagellins of E. coli, Shigella, P. aeruginosa, and P. mirabilis. Among these, only antibodies against flagella of P. mirabilis and anti-FlaA and anti-FlaB of P. aeruginosa reacted in immunoblots with the s. maltophilia flagellins, although to differing levels of reaction (Figure 4). Anti-SMFliC antibodies reacted with the SMFliC proteins produced by both S. maltophilia strains tested (Figure 4). Expression of Flagella by Clinical S. maltophilia Isolates We investigated SMFliC in fresh isolates of S. maltophilia. Forty-six S. maltophilia clinical isolates and S. maltophilia ATCC 13637 were studied by immunoblot, with antibodies against SMFliC of SMDP92. A preparation of purified flagella was used in all reactions as a positive control. All the isolates tested produced the ~38-kDa flagellin that reacted with anti- bodies against SMFliC (Figure 5). However, the molecular mass of the flagellin produced by some of the isolates differed slightly, and doublet bands were seen in some cases. We also performed negative staining and transmission electron micros- copy in these isolates to confirm expression of flagella (Figure 1). These results show that the production of the 38-kDa flagellin and flagella is a common feature of reference and fresh clinical isolates of S. maltophilia. Kinetics of Adherence to Plastic As early as 30 min, individual bacteria were seen attaching to the glass surface and forming small clumps (Figure 6A). As the incubation time extended to 1, 2, and 4 h, the number of attached bacteria increased throughout the abiotic surface (Figure 6B–D). At 6 h, the adhering bacterial monolayer pro- gressed into three-dimensional bacterial clumps (Figure 6E). After 18 h, extended areas of the glass surface were covered with large bacterial clumps (Figure 6F). No obvious Figure 3. N-terminal amino acid sequence analysis. The first 14 resi- dues of SMFliC showed considerable identity to other flagellins. The highest degree of identity was found with the Serratia marcescens flagellin (78.6%). Stenotrophomonas maltophilia flagellin also showed identity to flagellins of Shigella sonnei, Escherichia coli, and Proteus mirabilis (71.4%), and the lowest identity was found with Pseudomonas aeruginosa flagellin (57.2%). The numbers on top indicate the amino acid position. Figure 4. Reactivity of Stenotrophomonas maltophilia flagellin with dif- ferent antibodies. Lane 1, SMDP92 strain; lane 2, ATCC 13637. Blots containing whole cells extracts of SMDP92 and ATCC 13637 were reacted with antibodies against SMFliC, flagella of Proteus mirabilis, and anti-FlaA and anti-FlaB of Pseudomonas aeruginosa. 920 Emerging Infectious Diseases • Vol. 8, No. 9, September 2002