Hemadsorption and enzyme-linked immunosorbent assay nitrocellulose replica methods for identification of colonization factor antigen (CFA)-positive Escherichia coli colonies and for isolation of CFA-negative mutants.

Methods were developed that allow demonstration of individual colonies carrying colonization factor antigen (CFA) I or CFA/II or E8775-type antigen in mixed bacterial cultures on solid media. These methods are based on mannose-resistant hemadsorption or CFA enzyme-linked immunosorbent assay (ELISA) on nitrocellulose replicas of the cultures allowing simultaneous analysis of up to 200 colonies per plate. The sensitivity and specificity of the CFA ELISA nitrocellulose replica method were 97 and 99%, respectively, for CFA/I-carrying colonies and 99 and 100% for CFA/II-positive colonies; corresponding figures for the quicker and simpler hemadsorption modification were somewhat lower. Both methods seem to be useful for studying excretion of CFA-carrying bacteria in feces, as indicated by studies in rabbits infected with enterotoxin-producing Escherichia coli in a nonligated-intestine model. By initially absorbing CFA-carrying bacteria on erythrocytes and then performing nitrocellulose replicas of agar colonies of the nonabsorbed bacteria, CFA-deficient mutants could be identified by the hemadsorption method, as well as by the CFA ELISA. Treatment of CFA-carrying bacteria with antiserum against CFA and complement also resulted in enrichment of spontaneous CFA-deficient mutants that could be identified by the replica methods. Several stable CFA-deficient mutants from enterotoxin-producing E. coli carrying CFA/I, CFA/II, or E8775 were isolated by these approaches.