Identification of hemolysin BL from Bacillus cereus of patients with endophthalmitis and its biological activity detection in vitro

Abstract

Objective To identify the hemolysin BL(HBL) gene from Bacillus cereus of patients with endophthalmitis comfirmed by API bacterial identification test strip,and detect its biological activity in vitro.Methods Three pairs of specific primers were designed according to the gene sequence of HBL(B,L1 and L2 components),then the PCR assay were established through condition optimization,and to further detect five Bacillus cereus strains isolated from clinical patients with endophthalmitis.HBL with biological activity was extracted by salt fractionation from a randomly selected strain,and a series of different concentrations of HBL were prepared and acted on sheep red blood cells(SRBC),Vero cells and Hela cells; virulence of HBL was also assessed through observating lethal effect of which on mice with intraperitoneal injection.Results Three genes of HBL were detected in all B.cereus strains from clinical patients; Strong hemolytic activity of HBL showed obvious time-and dose-dependent.In the study,we found the morphological changes of Vero and Hela cells caused by HBL were different,but cell death were the same result with contents released; Within 48 h after infection,lethality of HBL for mice showed 100％ with the concentration of more than 2.0 HU/ml,and was also in a time-and dose-dependent manner.Conclusion HBL isolated from B.Cereus had high hemolysis activity and low concentration.The expression of all BL genes provided a strong basis for the clinical feature of B.Cereus infection,which was developed rapidly and with a poor prognosis.It also provided a new method for rapid diagnosis and molecular epidemiology investigation in clinical. Key words: Endophthalmitis; Bacillus cereus; Hemolysin