Adhesive properties and slime formation of vibrios and their relationship to haemagglutination.

RELATION BETWEEN ADHESIVE PROPERTIES OF VIBRIOS AND HAEMAGGLUTINATION 329 Adhesive Properties and Slime Formation of Vibrios and Their Relationship to Haemagglutination by K. N. NEOGY, Associate Professor of Bacteriology, School of Tropical Medicine, Calcutta, India, and S. N. SANYAL, Lecturer in Bacteriology, School of Tropical Medicine, Calcutta, India The formation of slime by strains of Vibrio cholerae has been observed and reported earlier by Neogy & Sanyal.a This communication reports the results of tests carried out on comparatively large numbers of different vibrio species to investigate the production of slime and their adhesive properties in culture plates. Direct bacterial haemagglutination was also performed according to the standard method. A total of 67 strains of different vibrio species was included in this study: this total included 26 strains of El Tor vibrio (18 from cases, 8 from water), 20 non-agglutinable (NAG) vibrios (10 from cases, 10 from water) and 11 strains of classical V. cholerae (from cases) and 10 strains of rough, classical V. cholerae (laboratory-induced). All the strains were tested for direct bacterial haemagglutination on glass slides by the method of Barua & Mukherjee.b Slime formation and the adhesive properties of the strains were noted in special media-phosphate peptone water (PPW) and phosphate bile-salt agar (PBA). For the preparation of PPW, the ingredients of alkaline peptone water and sodium taurocholate (final concentration 0.5 %) were added to phosphate buffer solution at pH 8.0. PBA was prepared by adding the ingredients of bile-salt agar to the same buffer solution. Both the media were sterilized in a steamer for 1 hour after their pHs had been adjusted to pH 9.2 and 8.4 respectively. Two loopfuls of a fresh agar culture were inoculated into 10 ml of PPW and were incubated at 370C for 48 hours. Later the PBA plates were flooded with the PPW cultures, the excess being removed with a pipette. The plates were incubated at 37°C for 48-72 hours to allow the formation of slime. The adhesive properties of the vibrios were noticed while trying to pick up portions of the growth from culture plates with a stiff Nichrome loop. Slime formation was noted in the same plate, about 15 min a Neogy, K. N. & Sanyal, S.N. (1967) Bull. Calcutta Sch. trop. Med.. 15, 106. b Barua, D. & Mukherjee, A. C. (1963) Bull. Calcutta Sch. trop. Med., 11, 85. after the addition of about 5 ml of normal saline, by gently rubbing the moistened portion with a platinum loop. Results All the 11 strains of classical V. cholerae, and 6 of the 10 strains ofNAG vibrios from cases, produced abundant slime in PBA. All the slime-producing vibrios (except one classical V. cholerae strain) produced a thick growth which could not be scraped off with the Nichrome loop and were thus shown to have adhesive properties. The growths of slime producers were smooth and adhered strongly to the medium. It was only after the addition of normal saline that the organisms could be picked up as a mucilaginous mass. None of the El Tor vibrios from cases or from water showed such properties of adhesion or slime formation. Slime formation was noticed in some NAG vibrios from cases and from water and in some of the rough variants of classical V. cholerae strains. Haemagglutination tests on El Tor vibrios were uniformly positive whereas in the case of the other vibrios the tests gave variable results. The results of the tests for haemaggluti- nation, slime formation and adhesive properties of the different strains are shown in the accompanying table. Discussion For the primary attachment of the pathogenic organism at the site of the infection, tissue virulence of the organism is one of the important factors. By tissue virulence is meant the anchoring or adhesive properties of the organism. This mechanism of adhesion is particularly important for enteropatho- genic organisms like V. cholerae because the normal peristaltic processes will always try to expel the organism before it is able to anchor and multiply. A clue to a possible mechanism of vibrio attachment was gained from several reports that El Tor vibrios are capable of direct agglutination of erythrocytes of many animal species. Classical V. cholerae strains can acquire such haemagglutinating activity only 2302F 330 NOTES RELATION BETWEEN HAEMAGGLUTINATION REACTION AND SLIME PRODUCTION IN THE VIBRIO STRAINS TESTED Strai ns Haemagglutination HaemagglutinationStrains ~~~~~positive negative Slime Slime Slime SlimeType No. positive negative positive negative Classical V. cholerae 11 - - 11 (100 %) El Tor (from cases) 18 - 18 (100 %) - NAG (from cases) 10 - 6 (60 %) 4 (40 %) - NAG (from water) 10 - 5 2 3 El Tor (from water) 8 - 8 (100 %) - - Classical V. cholerae (rough) 10 4 2 2 2 after serial subcultures in nutrient broth.C It was necessary, therefore, to devise some method to demonstrate the anchoring powers of classical V. cholerae under artificial conditions of growth. In our experiments the smooth strains of classical V. cholerae, which had been isolated from cases, all produced the characteristic slime and all but one became anchored to the special culture medium. It is also interesting to note that 60% of theNAG vibrios which had been isolated from cases caused haemagglutination whereas the remaining 40% were c Neogy, K. N. & Sanyal, S. N. (1967) Lancet, 2, 307. slime producers and had adhesive properties. It is not known whether the slime produced by the V. cholerae is directly responsible for its patho- genicity but the production of slime may have some bearing on its tissue virulence. In our opinion, Lankford & Legsomburana's postulation '1 that slime production and adhesive properties may be important factors in the pathogenicity of V. cholerae strains seems to be correct. d Lankford, C. E. & Legsomburana, U. (1965) In: Pro- ceedings of the Cholera Research Symposium, Honolulu, Washington, D.C., US Government Printing Office, p. 109.