A heat-stable hemolysin from clinical Enterobactercloacae Strain

SIMI, S.(1), CARBONELL, G.V.(1), DARINI, A.L.(3), GATTI, M.S.V.(1), FALCÓN, R.(1), TOYAMA, M.H.(2), MARANGONI, S.(2), YANO, T.(1)

Departamento de Microbiologia e Imunologia(1) e Bioquímica(2) do Instituto de Biologia, UNICAMP, SP, e Departamento de Análises Clínicas, Toxicológicas e Bromatológicas(3), Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, SP.

Introduction. Enterobacter spp. have been recognized as increasingly important pathogens in recent years. Toxins of E. cloacae has been described, however the relevance of these toxins remains to be established. In this study, we describe some properties of a hemolysin produced by clinical E. cloacae.

Methods. E. cloacae  was cultured inTSB with 0.5% glucose with stationary incubation. The culture supernatant was assayed for hemolytic activity with different species of erythrocytes. Crude hemolysin was partially purified by ultrafiltration, acetone precipitation, and reversed phase chromatography (mBondapak C₁₈), HPLC. Physicochemical properties of hemolysin such as heat and pH stability, effect of chemical reduction and proteolytic enzymes were determined. Biological assays including rat intestinal loop, suckling mouse test, skin permeability test in rabbits were done with hemolysin.

Results. E. cloacae hemolysin was active on horse, sheep, guinea pig and human erythrocytes. The hemolysin presented an estimated molecular mass smaller than 10 kDa, was resistant to proteolic enzymes and 2-mercaptoethanol, was heat stable, soluble in organic solvents and stable to acid pHs but not above pH 6. The hemolysin caused hemorrhage and fluid accumulation in the rat intestinal loop test, however gave a negative reaction in the suckling mouse assay and in the rabbit skin permeability test.

Conclusions. E. cloacae hemolysin showed some physicochemical properties similar to other heat-stable Enterobacteriaceae enterotoxins. This study, is the first description of a low molecular mass heat-stable hemolysin that causes hemorrhagic fluid secretion in ligated rat ileal loops, which may contribute to our understanding of the pathogenesis of this species.

CORRESPONDENCE TO:

Silvia Simi, Rua Francisco Glicério, No 1664, Apto. 1303, Campinas, SP, CEP: 13012-100, Brasil, Email: silviasimi@hotmail.com