The O polysaccharide lipopolysaccharide (LPS) layer of gram-negative bacteria contributes to the virulence of pathogens by providing resistance to host defenses such as complement-mediated lysis and phagocytosis. An intact O polysaccharide region of LPS gives colonies of gram-negative bacteria the property of smoothness. Loss or removal of the O polysaccharide results not only in a decrease in virulence of these bacteria but also a rough colony appearance (2).
The ability to bind the dye Congo red is also recognized as a phenotype that differentiates between virulent and avirulent colony types of many bacteria, including Yersinia pestis, Aeromonas salmoncida, Neisseria meningitidis, and Vibrio cholerae (1–3). For Shigella species, smooth colonies that bind Congo red are virulent, while smooth colonies that do not bind the dye are avirulent (2, 3). Although rough colony types of Shigella are avirulent, they are able to bind Congo red because loss of the hydrophilic O polysaccharide from the LPS during the smooth to rough transition makes the cell surface more hydrophobic and increases binding of the dye molecules to the lipid A core of the LPS (3).
For the images above, Shigella sonnei ATCC strain 29930, an invasive pathogen which causes bacillary dysentery in humans, was rehydrated in trypticase soy broth and streaked for isolation onto trypticase soy agar containing 0.003% Congo red. Plates were incubated at 37°C for 24 hours. This initial culture was designated as the first passage. For subsequent passages, well-isolated smooth colonies from the previous passage were subcultured onto Congo red agar plates at 24-hour intervals and incubated as above. Passages were numbered consecutively.
FIG. 1. First passage of S. sonnei on Congo red agar. Smooth colonies are round, slightly raised, and have deep red centers. All of the colonies depict the virulent phenotypes of smoothness and Congo red binding.
FIG. 2. First passage of S. sonnei on Congo red agar. Smooth colonies are round, slightly raised, and have deep red centers. All of the colonies depict the virulent phenotypes of smoothness and Congo red binding. This is a close-up view of Fig. 1. In the labeled view, arrows indicate smooth colony types.
FIG. 3. Second passage of S. sonnei on Congo red agar. A few colonies exhibit the rough colony phenotype of flattened dull colonies with irregular margins.
FIG. 4. Second passage of S. sonnei on Congo red agar. This is a close-up view of Fig. 3. In the labeled view, arrow 1 indicates the smooth colony phenotype, and arrow 2 indicates the rough colony phenotype.
FIG. 5. Third passage of S. sonnei on Congo red agar. Although a smooth colony was chosen from a second passage plate, the image shows the growth of an abundance of flattened, rough colonies with irregular margins. Although avirulent, the rough S. sonnei colonies still bind Congo red because loss of the hydrophilic O polysaccharide allows binding of the hydrophobic dye to the lipid A core of the LPS (3).
FIG. 6. Third passage of S. sonnei on Congo red agar. This is a close-up view of Fig. 5. In the labeled view, arrow 1 indicates the smooth colony phenotype, and arrow 2 indicates the rough colony phenotype.
References.
1. Daskaleros, P. A., and S. M. Payne. 1985. Cloning the gene for Congo red binding in Shigella flexneri. Infect. Immun. 48:165–168.
2. Maurelli, A. T., and P. J. Sansonetti. 1988. Genetic determinants of Shigella pathogenicity. Annu. Rev. Microbiol. 42:127–150.
3. Qadri, F., S. A. Hossain, I. Ciznar, K. Haider, A. Ljungh, T. Wadstrom, and D. A. Sack. 1988. Congo red binding and salt aggregation as indicators of virulence in Shigella species. J. Clin. Microbiol. 26:1343–1348.
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