Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25°C after 3 days of incubation. Incubation at 37°C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25°C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.Salmonella enterica serovar Typhimurium is a causative agent of gastrointestinal salmonellosis in humans. Phage type 104 (definitive type 104 [DT104]) strain of S. enterica serovar Typhimurium has recently emerged with resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline in the United States (3), the United Kingdom (25), France, Denmark (2), and Japan (23). This multidrug resistance has posed a major problem in the treatment of complications resulting from these Salmonella infections. Moreover, new strains with resistance to additional antibiotics, such as trimethoprim and fluoroquinolones, are beginning to emerge (18,26). The Centers for Disease Control and Prevention has reported that DT104 was identified in 32% of the S. enterica serovar Typhimurium strains isolated from humans in 1996, an increase from 28% in 1995 and 7% in 1990. DT104 is now only the second most prevalent type of Salmonella after serovar Enteritidis phage type 4 in the United Kingdom (13).During our study on S. enterica serovar Typhimurium DT104, we have observed a rugose phenotype, which has also been described for Vibrio cholerae after serial passages in alkaline peptone media (14,19,28) or in response to starvation (17,27,29). This phenotype of V. cholerae was defined by corrugated colony morphology, which was associated with the formation of exopolysaccharide (EPS) and cell aggregation.Because there was a question of whether the rugose variant of V. cholerae was virulent, a study was performed and showed that the rugose c...

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37°C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acidcontaining CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37°C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25°C than at 37°C. Since the CP was shown to be produced at both 37°C and 25°C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.

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