Characterization of flagella produced by clinical strains of stenotrophomonas maltophilia

Emerging Infectious Diseases, Sept, 2002 by Doroti de Oliveira-Garcia, Monique Dall'Agnol, Monica Rosales, Ana C.G.S. Azzuz, Marina B. Martinez, Jorge A. Giron

Stenotrophornonas maltophilia is an emerging nosocomial pathogen associated with opportunistic infections in patients with cystic fibrosis, cancer, and HIV. Adherence of this organism to abiotic surfaces such as medical implants and catheters represents a major risk for hospitalized patients. The adhesive surface factors involved in adherence of these bacteria are largely unknown, and their flagella have not yet been characterized biochemically and antigenically. We purified and characterized the flagella produced by S. maltophilia clinical strains. The flagella filaments are composed of a 38-kDa subunit, S[M.sub.FliC], and analysis of its N-terminal amino acid sequence showed considerable sequence identity to the flagellins of Serratia marcescens (78.6%), Escherichia coli, Proteus mirabilis, Shigella sonnei (71.4%), and Pseudornonas aeruginosa (57.2%). Ultrastructural analysis by scanning electron microscopy of bacteria adhering to plastic showed flagellalike structures within the bacterial clusters, suggesting that flagella are produced as the bacteria spread on the abiotic surface.

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Stenotrophomonas (formerly Pseudomonas and Xanthomonas) maltophilia is a widespread environmental microorganism that has become an important opportunistic pathogen associated with nosocomial colonization and infection (1-7). These organisms have been recovered from water faucets, water traps, respirometers, sinks, suction catheters, and occasionally, from cultures of the hands of hospital personnel (5,8). Infection and colonization of implantable medical devices such as catheters and intravenous cannulae represent a major risk for hospitalized patients. S. maltophilia can cause septicemia, endocarditis, conjunctivitis, mastoiditis, meningitis, post-operative wounds, abscesses, urinary tract infections, and pneumonia (6,9-11). The isolation rates of S. maltophilia from the respiratory tracts of patients with cystic fibrosis and from cancer and HIV-infected patients with opportunistic infections is increasing (4,12,13). Adhesion of these bacteria to abiotic surfaces such as those of medical implants and catheters suggests the development of a biofilm that protects bacteria from natural immune defenses or from the action of antimicrobial compounds. Biofilms are made up of a community of bacteria immobilized and embedded in an organic polymer matrix composed of polysaccharides and proteins of bacterial origin (14-16). Management of infection and successful clinical outcome by means of antimicrobial therapy are complicated by the intrinsic resistance of the bacteria to multiple antimicrobial agents, including carbapenems, and to the natural protection that biofilms confer to the enclosed bacteria (8,14). Besides the ability to adhere to plastic, to survive and multiply within total parenteral nulrition and other types of intravenous infusions, and to produce extracellular enzymes (4,8), little information is available regarding virulence factors associated with the pathogenesis of these bacteria. Production of a protease and elastase appears to be important in the pathogenesis of S. maltophilia--associated infections (5,17).

While for some bacteria the expression of flagella does not clearly relate to pathogenesis, for a variety of bacterial pathogens, such as Proteus mirabilis, Salmonella enterica, and Yersinia enterocolitica, the participation of flagella in adherence and invasion has been documented (18-20). In addition, the role of flagella in the formation and development of biofilm has recently been investigated in Pseudomonas, Escherichia coli, and Vibrio cholerae (21-24). Jucker et al. reported that nonspecific adhesion and biofilm formation by S. maltophilia to glass and Teflon may be attributed to the net positive surface charge of the bacteria (23). As with a variety of microorganisms, other surface determinants may confer the adhesive attributes necessary for S. maltophilia-specific adhesion. Although biofilm formation by S. maltophilia has been documented, no surface molecule or structure such as flagella or fimbrial adhesins implicated in adherence to plastic or eukaryotic cells has yet been characterized in detail (4,23,25). To characterize the surface appendages produced by S. maltophilia, we purified flagella from a clinical isolate and used specific anti-flagella antibodies to test for the presence of these structures in a collection of clinical isolates. In addition, we studied the kinetics of adhesion and performed ultrastructural studies by scanning electron microscopy of bacteria adhering to plastic. These studies showed structures resembling flagella, suggesting that these structures may be important for the adherence phenomenon.

Materials and Methods

Bacterial Strains and Growth Conditions

We included in this study 46 clinical isolates of S. maltophilia obtained from patients admitted to four institutions in the City of Silo Paulo, Brazil: Instituto Dante Pazzanese de Cardiologia, Hospital das Clinicas, Laboratorio Fleury, and Hospital Universitario (Universidade de Sao Paulo). Most of these clinical strains were isolated from respiratory tract secretions 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% defibrinated sheep blood (Oxoid, Basingstoke, England) at 37[degrees]C for 48 h.