Production of common enterobacterial antigen by pigment-producing, gram-negative bacilli.

A previously overlooked antigen shared by numerous enterobacteriaceae was described in 1962 by Kunin et al. (2). This common antigen, referred to as CA for the sake of brevity, was identified by means of hemagglutination of antigenically modified erythrocytes and Escherichia coli 014 antiserum. CA present in cultures of E. coli 014 is highly immunogenic in rabbits, whereas CA of other enterobacteriaceae is not, although the antigenic determinant is identical. Only one strain of Serratia was tested by Kunin regarding the presence ofCA (personal communication), and the results were negative. This study was undertaken to determine the production of CA by pigment-producing, gram-negative bacilli. Fifty strains, isolated in our laboratories from 46 patients hospitalized between 1965 and 1968, were used; 31 strains originated from the respiratory tract, nine from various exudates, five from the urinary tract, and five from miscellaneous sources. Strains of Serratia, Enterobacter cloacae, and Pectobacterium were identified according to the criteria used by the National Communicable Disease Center (1, 4). Flavobacterium sp. was identified on the basis of the following characteristics: gram-negative rods, the formation of bright-yellow colonies on nutrient-agar, and failure to produce acid from carbohydrates. Production of CA was determined according to procedures previously described in detail (7). Briefly, the microorganisms were grown on veal brain-agar in Kolle flasks for 18 hr at 37 C. The organisms were suspended in phosphate buffer (pH 7.3; Difco); the suspension was heated at 100 C for 1 hr and then centrifuged at 23,500 X g. The clear supernatant fluid was used for modification of sheep erythrocytes. E. coli 014 antiserum containing CA antibodies in high titer, in serial twofold dilutions, was mixed with the antigenically modified erythrocytes. The mixtures were incubated at 37 C for 30 min, and the resulting hemagglutination was read after centrifugation at 1,300 x g for 2 min. To confirm the specificity, hemagglutination-inhibition tests were carried out by the use of CA from an unrelated microorganism, Salmonella typhimurium. The results obtained with 50 strains of chromogenic, gramnegative bacilli are summarized in Table 1. It is evident that all strains of Serratia, Enterobacter cloacae, and Pectobacterium produced CA. In contrast, strains of Flavobacterium, belonging to the family Achromobacteriaceae, did not. All 12 strains of nonchromogenic Serratia, obtained from the National Communicable Disease Center, Atlanta, Ga., or isolated in our laboratories from clinical material, also produced CA. In order to determine whether CA produced by pigment-producing, gram-negative bacilli is immunogenic, four groups of three rabbits each were immunized with supernatant fluids of cultures; two strains of Serratia and two strains of Pectobacterium were used. Three intravenous injections (1 ml) of supernatant fluid in dilutions of 1:20, 1:10, and 1:10 were given 3 to 4 days apart. The mean antibody titers against CA remained below 1 :10 in animals injected with Serratia and increased from <1:10 to 1:14 only in those immunized with Pectobacterium. Thus, these strains were, at best, poorly immunogenic. It was shown previously (5, 8) that supernatant fractions of cultures of enterobacteriaceae other than E. coli 014, while not engendering circulating CA antibodies in substantial titers, nonetheless stimulate (prime) rabbits to antibody production after a booster injection of isolated CA. Therefore, the rabbits were given an intravenous injection of a subeffective dose (1 ml) of ethyl alcohol-soluble CA of S. typhimurium (6). Four days later, the mean CA antibody titers rose to 1 :110 in rabbits primed with supernatant fluids of cultures of Serratia and to 1:396 in those immunized with supernatant fluids of Pectobacterium. The same dose of isolated CA given to nonprimed animals resulted in a mean antibody titer of 1:20 only. Therefore, CA produced by Serratia and Pectobacterium primes rabbits to CA antibody production. From these observations, it is concluded that pigment-producing Serratia, Enterobacter cloacae, and Pectobacterium all produce CA, whereas