Specificity of rabbit antisera against lipopolysaccharide of Acinetobacter

Abstract

Acinetobacter has been reported to be involved in hospital-acquired infections with increasing frequency. However, clinical laboratories still lack simple methods that allow the accurate identification of Acinetobacter strains at the species level. For this study, proteinase K-digested whole-cell lysates from 44 clinical and environmental isolates were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with hyperimmune rabbit sera to examine the possibility of developing a serotyping scheme based on the O antigen of Acinetobacter lipopolysaccharide (LPS). The antisera, obtained by immunization of rabbits with 13 of the heat-killed isolates investigated, were characterized by Western blotting and enzyme immunoassay by using proteinase K-digested whole-cell lysates and phenol-water-extracted LPS as antigens. In both assays, the antisera were shown to be highly specific for the homologous antigen. In addition, assignment of Acinetobacter LPS to the smooth or the rough phenotype was shown not to be reliable when it was based only on the results obtained with silver-stained gels. O-antigen reactivity, determined by Western blot analysis, was observed with 11 of the 31 isolates, most of which belonged to the species Acinetobacter baumannii (DNA group 2) and the unnamed DNA group 3. Interestingly, some O antigens were found in a DNA group different from that of the strain used for immunization. The results indicate that O serotyping of Acinetobacter strains is feasible and thus may provide a simple method for the routine identification of these opportunistic pathogens.

FIG. 1

Reactivities of proteinase K-digested whole-cell lysates of Acinetobacter strains used for immunization after separation by SDS-PAGE on a 10% separating gel and staining with alkaline silver nitrate (A) or with homologous rabbit antisera in Western blots (B). Lanes 1, strain 34; lanes 2, strain 57; lanes 3, strain 44; lanes 4, strain 24; lanes 5, strain 7; lanes 6, strain 108; lanes 7, strain 61; lanes 8, strain 65; lanes 9, strain 9; lanes 10, strain ATCC 17906; lanes 11, strain 90; lanes 12, strain ATCC 11171; lanes 13, strain 96.

FIG. 2

Reactivities of proteinase K-digested whole-cell lysates of Acinetobacter strains used for immunization after separation by SDS-PAGE on a 15% separating gel and staining with alkaline silver nitrate (A) or with homologous rabbit antisera in Western blots (B). Lane numbering is as described in the legend to Fig. 1.

FIG. 3

Reactivity of rabbit antiserum K320 in Western blots after separation of proteinase K-digested whole-cell lysates of Acinetobacter sp. strains 34 (homologous strain; lane 1), 36 (lane 2), 37 (lane 3), and 64 (lane 4) by SDS-PAGE on a 10% separating gel.