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Vibrio cholerae is the causative agent of cholera, which in its most severe form is characterized by profuse diarrhea, vomiting, and muscle cramps. V. cholerae strains have been divided into two groups, O1 and non-O1, based on their ability to cause cholera epidemics. To date, there have been seven recorded pandemics of this severe dehydrating diarrheal disease caused by V. cholerae strains of serotype O1, and it was therefore assumed that only this serotype has epidemic potential. The new serogroup, designated O139 synonym Bengal, is the first recorded serogroup other than O1 to cause epidemic cholera. V. cholerae O139 closely resembles V. cholerae O1 biotype El Tor strains of the seventh pandemic (5, 12, 21, 40). The major differences between V. cholerae O139 and O1 are the composition and lengths of the O side chains of the cell wall lipopolysaccharide (LPS) and the presence of a capsular polysaccharide (CPS) in O139 strains that is not found in V. cholerae O1 strains (7, 15, 20). Serological and genetic studies suggested that CPS of O139 V. cholerae has the same repeating unit as the O antigen (8, 39).

V. cholerae strains are natural inhabitants of brackish water and estuarine systems (6). As a response to nutrient depletion, copiotrophic (32) heterotrophic bacteria may undergo considerable morphological, physiological, and chemical changes (11, 22, 23, 26, 27, 29). In fact, to survive energy- and nutrient-deprived conditions, non-spore-forming, heterotrophic bacteria are known to undergo an active adaptation program (29). Wai et al. (38) reported that V. cholerae O1 TSI-4 can shift to a rugose colony morphology associated with the expression of an amorphous exopolysaccharide (EPS) that promotes biofilm formation, and they also indicated that rugose strains displayed resistance to osmotic and oxidative stress.

Many microorganisms produce EPSs which are located outside the cell wall, either attached to it in the form of capsules or secreted into the extracellular environment in the form of slime. Extracellular polysaccharide excretion (slime or capsule) is a common phenomenon for many bacteria following the exhaustion of the nitrogen supply under otherwise nutrient-sufficient conditions (28). Bacterial cells initiate the process of irreversible adhesion by binding to the surface by using EPSs, glycocalyx polymers, and the development of biofilms. A biofilm is a functional consortium of microorganisms organized within an extensive exopolymer matrix comprised mainly of hydrated polysaccharides (43). Biofilms are produced by a wide variety of environmentally and medically important microorganisms, including Staphylococcus, Pseudomonas, Desulfovibrio, Thermococcus, and Methanosarcina (4, 9, 10, 18, 35, 36). The production of biofilm may enhance the survival of cells in dynamic environments by allowing the formation of colonies containing thousands of cells.

View larger version (96K): [in this window] [in a new window]   FIG. 1.   Photomicrograph of V. cholerae O139 MO10/SPR (arrow) and MO10/NSPS (arrow head) colonies. Bacteria were incubated on an L agar plate at 37°C for 18 h.

This study demonstrates that V. cholerae O139 MO10 is able to shift to a phenotype having a rugose colony morphology associated with the excretion of slime in response to starvation. This form promotes biofilm formation. Interestingly, the antiserum against V. cholerae O1 TSI-4 EPS (38) is reactive with the slime produced by V. cholerae O139 rugose strains. It may support the hypothesis that V. cholerae O139 arose from an O1 El Tor strain.

Isolation of the rugose strain of V. cholerae O139 MO10. V. cholerae O139 opaque encapsulated MO10 (40) was used in this study. The original isolate of strain MO10 had a smooth colony morphology. Cells of MO10 were routinely grown at 37°C with shaking or in a static condition in Luria (L) broth (25). MO10 cells were incubated to mid-log phase, which corresponded to an A₆₀₀ of 0.4. The cells were then harvested by centrifugation (13,000 × g for 10 min), washed three times with cold M9 salts (37), resuspended in starvation medium (M9 salts) to give a final concentration of approximately 5 × 10⁷ cells per ml, and incubated at 4°C without shaking. Strain MO10 exhibits a shift of colony morphology to the rugose form under starvation conditions at 2 weeks after inoculation. To establish the criteria for slime production and rugose colony morphology, V. cholerae O139 MO10 strains have been classified as either slime-producing rugose type (MO10/SPR) or non-slime-producing smooth type (MO10/NSPS). MO10/SPR grown overnight with shaking in L broth produced MO10/NSPS colonies at a frequency of 1.5 × 10⁵. Colony counts for rugose strains represent the number of particles not the number of cells (34). This makes the determination of a frequency of phase variation difficult. Two distinct colony morphologies are shown in Fig. 1. The larger size of the smooth colonies is due to the difference of the growth rates of smooth and rugose colonies. Both colony types were tested for agglutination with anti-O139 Bengal sera (Denka, Seiken, Co. Ltd., Tokyo, Japan) and showed positive reactions. The antiserum against V. cholerae O1 TSI-4 EPS (38) agglutinated MO10/SPR, whereas it did not agglutinate MO10/NSPS.

Thin-section electron microscopy. To determine the nature of the colony morphology differences, bacterial pellets were stained with polycationic ferritin, and thin sections were observed by electron microscopy as described previously (38). Both strains were surrounded by relatively thin electron-dense capsule (Fig. 2). Slime materials released by MO10/SPR were recognized as a heavy, electron-dense ferritin-stained layer surrounding the cell in addition to a thin electron-dense layer of capsule (Fig. 2A and B), but MO10/NSPS did not appear to have this slime layer surrounding its cells (Fig. 2C).

View larger version (98K): [in this window] [in a new window]   FIG. 2.   Thin sections of V. cholerae O139 stained with polycationic ferritin showing a thick, electron-dense slime layer in addition to a thin electron-dense layer of capsule surrounding MO10/SPR cells (A and B) and no slime layer surrounding MO10/NSPS (C). Bars, 0.5 µm.

Immunoelectron microscopy. Immunoelectron microscopy was performed with anti-EPS serum of the rugose form of V. cholerae O1 TSI-4 as described previously (38). The antiserum against V. cholerae O1 TSI-4 EPS (38) was reactive only with V. cholerae O139 MO10/SPR and not with MO10/NSPS (Fig. 3A and B). The gold particles were specifically bound to the slime layer surrounding MO10/SPR cells and at the intercellular spaces (Fig. 3A).

View larger version (120K): [in this window] [in a new window]   FIG. 3.   Immunoelectron micrographs of the surface labeling of V. cholerae O139 MO10/SPR (A) and MO10/NSPS (B) with antiserum against EPS of rugose V. cholerae O1 TSI-4. Bars, 0.5 µm.

Outer membrane and LPS profiles. The outer membrane was prepared from a broth culture of V. cholerae O139 MO10/NSPS or MO10/SPR according to the method of Filip et al. (13). LPS was prepared from 1 ml of an overnight culture (11). LPS and outer membrane samples were electrophoresed and detected by silver staining as previously described (16). No outer membrane protein or LPS differences between cell types were detected (data not shown).

Biofilm growth of V. cholerae O139 MO10/SPR and scanning electron microscopy. V. cholerae O139 MO10/SPR was cultured overnight in L broth at 37°C without shaking. The biofilms growing on the upper surface of the L broth and on the wall of a culture tube were sampled and prepared for scanning electron microscopy as described previously (38). The specimens were examined with a scanning image-observing device (ASID) equipped with a JEOL JEM 2000EX electron microscope. Figure 4 shows a biofilm examined by scanning electron microscopy; the surface of the film was completely covered with a layer of rod cells, rounded cells, and filamentous cells embedded within a polymeric matrix. Throughout the biofilm, cells were interconnected by a finger-like glycocalyx matrix that extended from the substratum to the outer boundaries of the biofilm. Interestingly, some of the surface of the biofilm was covered by a twisting long filamentous growth of bacteria.

View larger version (146K): [in this window] [in a new window]   FIG. 4.   Scanning electron micrographs of biofilm formation by V. cholerae O139 MO10/SPR. (A) Most of the surface has been colonized by rod cells, rounded cells, and twisting filamentous cells, and finger-like projections of extracellular polymeric material are present. Bar, 1 µm. (B) High magnification shows extracellular polymeric materials on the surface of bacterial cells and long twisting filamentous cells. Bar, 1 µm.