Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1988 Jul;26(7):1343–1348. doi: 10.1128/jcm.26.7.1343-1348.1988

Congo red binding and salt aggregation as indicators of virulence in Shigella species.

F Qadri 1, S A Hossain 1, I Ciznár 1, K Haider 1, A Ljungh 1, T Wadstrom 1, D A Sack 1
PMCID: PMC266606  PMID: 3045151

Abstract

Smooth strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii, and Shigella sonnei which form pigmented colonies (Pcr+) on Congo red agar were virulent in the Sereny test. Smooth variants unable to bind Congo red (Pcr-) were avirulent. Measurements of dye uptake from solution showed that S. dysenteriae type 1 bound the most dye, followed in order of uptake by S. flexneri, S. boydii, and S. sonnei. Using the salt aggregation test (SAT) to determine cell surface hydrophobicity, we found the same order of species. The SAT could not, however, detect differences in surface properties between Pcr+ and Pcr- pairs of isogenic smooth strains. Enteroinvasive Escherichia coli strains used in the study showed SAT and Congo red-binding properties which were similar to those of the S. flexneri strains. A direct correlation was found between pigment-binding ability and the presence of the large 140-megadalton plasmid in S. flexneri, enteroinvasive E. coli, and S. boydii but not in S. dysenteriae type 1 or S. sonnei strains. Congo red interacted with outer membranes and outer membrane proteins of S. dysenteriae type 1 but not with lipopolysaccharides. However, rough mutants of Shigella species deficient in lipopolysaccharides bound Congo red and formed pigmented colonies, showing that dye binding as a virulence assay may be misinterpreted in such cases. There was complete correlation of the Pcr+ phenotype with virulence in the smooth strains in this study, suggesting that Congo red binding can be utilized as a quick and reliable alternative to the Sereny test.

Full text

PDF
1343

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamus G., Mulczyk M., Witkowska D., Romanowska E. Protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of Shigella spp. Infect Immun. 1980 Nov;30(2):321–324. doi: 10.1128/iai.30.2.321-324.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BURROWS T. W., JACKSON S. The pigmentation of Pasteurella pestis on a defined medium containing haemin. Br J Exp Pathol. 1956 Dec;37(6):570–576. [PMC free article] [PubMed] [Google Scholar]
  3. Bhaduri S., Conway L. K., Lachica R. V. Assay of crystal violet binding for rapid identification of virulent plasmid-bearing clones of Yersinia enterocolitica. J Clin Microbiol. 1987 Jun;25(6):1039–1042. doi: 10.1128/jcm.25.6.1039-1042.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Binns M. M. Molecular genetics of virulence in Shigella. Microbiol Sci. 1985 Sep;2(9):275–278. [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Daskaleros P. A., Payne S. M. Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype. Infect Immun. 1986 Nov;54(2):435–443. doi: 10.1128/iai.54.2.435-443.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daskaleros P. A., Payne S. M. Cloning the gene for Congo red binding in Shigella flexneri. Infect Immun. 1985 Apr;48(1):165–168. doi: 10.1128/iai.48.1.165-168.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Formal S. B., Gemski P., Baron L. S., Labrec E. H. A Chromosomal Locus Which Controls the Ability of Shigella flexneri to Evoke Keratoconjunctivitis. Infect Immun. 1971 Jan;3(1):73–79. doi: 10.1128/iai.3.1.73-79.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gemski P., Jr, Sheahan D. G., Washington O., Formal S. B. Virulence of Shigella flexneri hybrids expressing Escherichia coli somatic antigens. Infect Immun. 1972 Aug;6(2):104–111. doi: 10.1128/iai.6.2.104-111.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glazer A. N. On the prevalence of "nonspecific" binding at the specific binding sites of globular proteins. Proc Natl Acad Sci U S A. 1970 Apr;65(4):1057–1063. doi: 10.1073/pnas.65.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hale T. L., Guerry P., Seid R. C., Jr, Kapfer C., Wingfield M. E., Reaves C. B., Baron L. S., Formal S. B. Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1. Infect Immun. 1984 Nov;46(2):470–475. doi: 10.1128/iai.46.2.470-475.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hale T. L., Oaks E. V., Formal S. B. Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli. Infect Immun. 1985 Dec;50(3):620–629. doi: 10.1128/iai.50.3.620-629.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ishiguro E. E., Ainsworth T., Trust T. J., Kay W. W. Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface protein array involved in virulence. J Bacteriol. 1985 Dec;164(3):1233–1237. doi: 10.1128/jb.164.3.1233-1237.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnston K. H., Gotschlich E. C. Isolation and characterization of the outer membrane of Neisseria gonorrhoeae. J Bacteriol. 1974 Jul;119(1):250–257. doi: 10.1128/jb.119.1.250-257.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kabir S., Ali S. Characterization of surface properties of Vibrio cholerae. Infect Immun. 1983 Mar;39(3):1048–1058. doi: 10.1128/iai.39.3.1048-1058.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kopecko D. J., Washington O., Formal S. B. Genetic and physical evidence for plasmid control of Shigella sonnei form I cell surface antigen. Infect Immun. 1980 Jul;29(1):207–214. doi: 10.1128/iai.29.1.207-214.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Labrec E. H., Schneider H., Magnani T. J., Formal S. B. EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP IN THE PATHOGENESIS OF BACILLARY DYSENTERY. J Bacteriol. 1964 Nov;88(5):1503–1518. doi: 10.1128/jb.88.5.1503-1518.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leive L., Shovlin V. K., Mergenhagen S. E. Physical, chemical, and immunological properties of lipopolysaccharide released from Escherichia coli by ethylenediaminetetraacetate. J Biol Chem. 1968 Dec 25;243(24):6384–6391. [PubMed] [Google Scholar]
  20. Ljungh A., Wadström T. Salt aggregation test for measuring cell surface hydrophobicity of urinary Escherichia coli. Eur J Clin Microbiol. 1982 Dec;1(6):388–393. doi: 10.1007/BF02019940. [DOI] [PubMed] [Google Scholar]
  21. Magnusson K. E., Davies J., Grundström T., Kihlström E., Normark S. Surface charge and hydrophobicity of Salmonella, E. coli, Gonococci in relation to their tendency to associate with animal cells. Scand J Infect Dis Suppl. 1980;Suppl 24:135–140. [PubMed] [Google Scholar]
  22. Maurelli A. T., Baudry B., d'Hauteville H., Hale T. L., Sansonetti P. J. Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect Immun. 1985 Jul;49(1):164–171. doi: 10.1128/iai.49.1.164-171.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maurelli A. T., Blackmon B., Curtiss R., 3rd Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun. 1984 Jan;43(1):397–401. doi: 10.1128/iai.43.1.397-401.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meyers J. A., Sanchez D., Elwell L. P., Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol. 1976 Sep;127(3):1529–1537. doi: 10.1128/jb.127.3.1529-1537.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mulczyk M. Hemagglutinating properties of Shigella bacilli. Arch Immunol Ther Exp (Warsz) 1967;15(4):636–639. [PubMed] [Google Scholar]
  26. OSBORN M. J. STUDIES ON THE GRAM-NEGATIVE CELL WALL. I. EVIDENCE FOR THE ROLE OF 2-KETO- 3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM. Proc Natl Acad Sci U S A. 1963 Sep;50:499–506. doi: 10.1073/pnas.50.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oaks E. V., Hale T. L., Formal S. B. Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp. Infect Immun. 1986 Jul;53(1):57–63. doi: 10.1128/iai.53.1.57-63.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Okamura N., Nagai T., Nakaya R., Kondo S., Murakami M., Hisatsune K. HeLa cell invasiveness and O antigen of Shigella flexneri as separate and prerequisite attributes of virulence to evoke keratoconjunctivitis in guinea pigs. Infect Immun. 1983 Feb;39(2):505–513. doi: 10.1128/iai.39.2.505-513.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Payne S. M., Finkelstein R. A. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect Immun. 1977 Oct;18(1):94–98. doi: 10.1128/iai.18.1.94-98.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reed W. P., Williams R. C., Jr Bacterial adherence: first step in pathogenesis of certain infections. J Chronic Dis. 1978 Feb;31(2):67–72. doi: 10.1016/0021-9681(78)90091-7. [DOI] [PubMed] [Google Scholar]
  31. Rozgonyi F., Szitha K. R., Hjertén S., Wadström T. Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species. J Appl Bacteriol. 1985 Nov;59(5):451–457. doi: 10.1111/j.1365-2672.1985.tb03345.x. [DOI] [PubMed] [Google Scholar]
  32. SERENY B. Experimental shigella keratoconjunctivitis; a preliminary report. Acta Microbiol Acad Sci Hung. 1955;2(3):293–296. [PubMed] [Google Scholar]
  33. Sansonetti P. J., Hale T. L., Dammin G. J., Kapfer C., Collins H. H., Jr, Formal S. B. Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri. Infect Immun. 1983 Mar;39(3):1392–1402. doi: 10.1128/iai.39.3.1392-1402.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sansonetti P. J., Kopecko D. J., Formal S. B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun. 1982 Mar;35(3):852–860. doi: 10.1128/iai.35.3.852-860.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seltmann G., Pál T., Tschäpe H. Surface hydrophobicity of plasmid-carrying virulent Shigella flexneri and their avirulent variants. J Basic Microbiol. 1986;26(5):283–287. doi: 10.1002/jobm.3620260508. [DOI] [PubMed] [Google Scholar]
  36. Surgalla M. J., Beesley E. D. Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl Microbiol. 1969 Nov;18(5):834–837. doi: 10.1128/am.18.5.834-837.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wadström T., Adegbola R. A., Baloda S. B., Ljungh A., Sethi S. K., Yuk Y. R. Non-haemagglutinating fimbriae of enteropathogenic Escherichia coli (EPEC). Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Jul;261(4):417–424. doi: 10.1016/s0176-6724(86)80073-6. [DOI] [PubMed] [Google Scholar]
  38. Watanabe H., Timmis K. N. A small plasmid in Shigella dysenteriae 1 specifies one or more functions essential for O antigen production and bacterial virulence. Infect Immun. 1984 Jan;43(1):391–396. doi: 10.1128/iai.43.1.391-396.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES