Downloaded from www.microbiologyresearch.org by IP: 54.144.197.59 On: Sat, 16 Apr 2016 14:36:50 J. Med. Microbiol. - Vol. 35 (1991), 330-337 0 1991 The Pathological Society of Great Britain and Ireland Contact- haemolysin production by entero-invasive Escherichia cok and shigellae KHALEDA HAIDER, M. J. ALBERT, A. HOSSAIN* and S. NAHAR Laboratory Sciences Division, International Centre for Diarrhoea1 Disease Research, GPO Box 128, Dhaka 1000, and Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Summary. Entero-invasive Escherichia coli (EIEC) and shigellae were tested for contact- haemolysin (CH) with red blood cells (RBCs) of guinea-pig, rabbit, rat, mouse, monkey, man, sheep and chicken; all bacteria showed the best lysis with guinea-pig RBCs. The best culture medium for CH activity of shigellae was tryptic soy broth, and for EIEC it was casamino acid-yeast extract broth with 1 mM CaC12.CH production by all species was best at the slightly alkaline pH which is optimal for growth; it was also dependent on the presence of a large (140-Mda) plasmid. Pre-treatment of bacteria with homologous antisera inhibited CH activity. Various treatments of bacterial cells and RBCs suggested that CH may be a protein molecule, and that a chitotriose-like moiety may serve as CH receptor. RBCs that were incubated with bacteria at 4"C, or with heat-killed bacteria at 37"C, were not lysed; also, isolated cell-surface components (lipopolysaccharideand outer-membrane protein) did not lyse RBCs. This suggests that metabolically active cells are required for CH activity. Production of CH by both EIEC and shigellae is consistent with a common mechanism for the virulence of these organisms. Introduction Disease caused by shigellae involves a complex process of invasion and multiplication in the mucosa of the human colon. The process of invasion requires entry of the bacterium into the enterocytes, and lysis of the membranous vacuole which surrounds it after invasion, to allow free multiplication of the pathogen within the cytoplasm. This leads to spread of the organisms to the lamina propria, triggering a severe inflammatory response in the gut mucosa with ab- scesses and ~lceration.~~ All these features are regulated by a large non-self-conjugative plasmid of 120-140 Mda called the virulence plasmid. Entero- invasive Escherichia coli (EIEC) produces dysentery like that caused by shigellae; it also possesses a 140- Mda plasmid which is associated with vir~lence.~. Sansonetti et a1.6 described a virulence-plasmid- mediated, thermoregulated contact-haemolysin (CH) in Shigella, which was active against sheep red blood cells (SRBC). Close contact was required between virulent bacteria and SRBC which was achieved by centrifugation. The production of CH correlated with its capacity to destroy the membranous vacuole surrounding the bacteria after invasion of HeLa cells, and with efficient intracellular multiplication. CH was also demonstrated in S. dysenteriae and S. sonnei strains in another s t ~ d y . ~ Received 19 Dec. 1990; revised version accepted 3 April 1991. The large virulence-plasmids of EIEC and all species of Shigella have extensive homologous sequences and are functionally exchangeable.' As the pathogenesis of EIEC and Shigella spp. is similar, this study was initiated to investigate whether CH was also produced by S. boydii and EIEC, other species of Shigella being included for comparison. Factors influencing the production and activity of CH were investigated by the use of virulent and avirulent pairs. Preliminary experiments were also performed to study the chemical nature of CH. Materials and methods Bacterial strains and culture conditions The bacterial strains are shown in table I. Dr D. N. Taylor, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, kindly provided 26 strains of EIEC from patients with invasive diarrhoea in Thailand. Five similar isolates were from central Australia (2), Bangladesh (2) and Hungary (l).9 The avirulent EIEC strain 2/33 is an isogenic mutant strain of virulent EIEC strain 2, isolated in Hungary ;9 these were kindly supplied by Dr P. Echeverria (AFRIMS, Bangkok). E. coli strain 36 000 was obtained from the Centers for Disease Control, Atlanta, GA, USA. All Shigella strains were isolated from patients at the Clinical Research Centre, International Centre for Diarrhoeal Disease Research, Bangladesh. The CH 330