Serological and structural features of Hafnia alvei lipopolysaccharides containing D-3-hydroxybutyric acid.

The serological heterogeneity of Hafnia alvei lipopolysaccharides from strains ATCC 13337, 1187, 1221, 114/60, 1211 and 1216, that contain D-3-hydroxybutyric acid, was analyzed by rocket immunoelectrophoresis, immunoblotting and passive hemagglutination. The significance of D-3-hydroxybutyric acid component for their cross-reactivity has been discussed. The results obtained allowed us to place four H. alvei strains (ATCC 13337, 1187, 1221 and 114/60) in one serotype (A) and to consider two other strains (1211 and 1216) as separate serotypes (B and C, respectively).

Strains of Hafnia alvei have been isolated from faeces of men and animals, water, soil and dairy products; however many cases of nosocomial infections with Hafnia have also been reported [1].
In our previous paper [2] the preliminary chemical characterization of H. alvei lipopolysac-charides (LPS) isolated from 33 strains was described. Among these strains a group of six lipopolysaccharides containing D-3-hydroxybutyric acid was identified. Recently the structures of O-antigens from strains ATCC 13337, 1187 and 1211 have been established [3,4]. The O-specific polysaccharides (PS) of 114/60 and ATCC 13337 lipopolysaccharides proved to be identical [5]; on the other hand the O-specific polysaccharide of strain 1221 has the same structure as the de-Oacetylated form of ATCC 13337 polysaccharide (paper in preparation).
The aim of the present work was the immuno-

Materials and Methods
H. alvei standard strain ATCC 13337 and four strains 1187, 1221, 1211 and 1216 derived from the collection of the Pasteur Institute (Paris) and strain 114/60 received from the National Institute of Hygiene (Warsaw) were used in the studies. The origin of the remaining H. alvei strains was described in paper [2]. The growth of bacteria in liquid medium, isolation and purification of the lipopolysaccharides and preparation of the O-specific polysaccharides were carried out as described elsewhere [6].
The antisera were prepared by immunization of rabbits with bacteria suspended in phosphatebuffered saline (PBS). The animals were injected first subcutaneously with a dose of 100 /zg dry bacteria ml 1 PBS and then intravenously twice a week with increasing amounts of the bacteria (100-6400/xg ml-1 PBS). One week after the last injection the rabbits were bled and the sera collected.
Rocket immunoelectrophoresis was carried out by the method of Weeke [7] with a 1% agarose gel in 0.02 M barbital buffer, pH 8.6, containing 2% polyethylene glycol 6000. The antibody gel contained 5% of the appropriate antiserum. After electrophoresis, gel was washed to rid excess reagent, dried and photographed directly or after staining with 0.5% amido black 10B.
For immunoblotting the separated lipopolysaccharides were transblotted from the gel into nitrocellulose (Schleicher-Schuell pore size 0.45 /xm) [8]. Electrophoretic transfer was carried out in 10 mM Tris-150 mM glycine buffer containing 20% methanol, pH 8.3, at 100 mA for 1 h. After transfer, the nitrocellulose was blocked with 3% gelatin in 20 mM Tris, 50 mM NaC1, pH 7   Hafnia alvei [2]. Its content in the LPS preparations and the respective O-specific polysaccharides is given in Table 1.
The serological relationships between the lipopolysaccharides were studied by several methods, like rocket immunoelectrophoresis, immunoblotting and passive hemagglutination.
Rocket immunoelectrophoresis of H. alvei lipopolysaccharides isolated from 32 strains with anti-ATCC 13337 and anti-1187 sera is shown in Fig. 1A Immunoblotting experiments confirmed the results obtained in the immunoelectrophoresis. A ladder-like pattern of transblotted lipopolysaccharides of ATCC 13337, 1187, 1221 and 114/60 strains after their reaction with anti-ATCC 13337, anti-l187 and anti-114/60 sera ( Fig. 2A,B,C) show evidence that they have a common epitope which is located in their O-specific polysaccharide chain.
Lipopolysaccharide of 1211 strain reacted with the homological serum only (Fig. 2D).
For quantitative evaluation of the cross-reactivity of H. alvei lipopolysaccharides a passive hemagglutination test was employed. It is clearly visible from the results presented in Table 2 that the lipopolysaccharides of four strains (ATCC 13337, 1187, 1221 and 114/60) cross-reacted distinctly, whereas no reactivity was shown with lipopolysaccharides of strains 1211 and 1216.
As the serological results showed, the antisera used are directed mainly to the O-specific region of the lipopolysaccharides. In some of the sera a minute portion of anti-core antibodies is also present which could be observed in immunoblotting (Fig. 2B,C,D), but not in rocket immunoelectrophoresis (Fig. 1).
For a better understanding of the serological relations within the group of LPS preparations containing i>3-hydroxybutyric acid, the structures of their repeating units are shown in Fig. 3. As can be seen, the base chains of the O-specific polysaccharides of strains ATCC 13337, 1187, 1221 and 114/60 are identical. Additional a-glucosyl and O-acetyl side chains occur in ATCC 13337 and 114/60 strains but only a-glucosyl side chains in the 1221 strain. This is in good agreement with the serological evidence on a common Table 2 Passive hemagglutination of the lipopolysaccharides isolated from H. ah,ei strains with the homological and heterological antisera From the results presented above it may be suggested that H. alvei strains containing D-3-hydroxybutyric acid in their O-antigens can be divided into 3 serotypes. Four strains (ATCC 13337, 1187, 114/60 and 1221) belong to one serotype (A) and their O-antigens have the common basic structure containing D-3-hydroxybutyric acid. Their antigenic structures are not uniform, however. The differences in the length and density of O-specific chains in the LPS molecules correspond to the serological microheterogeneity within this serotype.
11. alvei strains 1211 and 1216 differ serologically and represent two separate serotypes (B and C, respectively). The structures of H. alvei 1211 and 1216 O-specific polysaccharides are unique and very much different from those of the four polysaccharides (serotype A) mentioned above as well as amongst themselves.