Sub-minimal inhibitory concentrations of cefmetazole enhance serum bactericidal activity in vitro by amplifying poly-C9 deposition

Abstract

Serum-resistant organisms grown in sub-minimal inhibitory concentrations (subMICs) of antibiotics in vitro may be rendered sensitive to complement-mediated, serum bactericidal activity. We measured 125I-C3 and 125I-C9 deposition on genetically serum resistant Salmonella montevideo SH5770 (SH5770) that was rendered serum sensitive by growth in sub-MICs of cefmetazole (CMZ), a parenteral, second generation, cephamycin-group antibiotic. Three times as much C3 and over six times as much C9 bound to SH5770 grown in one-fourth the MIC of CMZ compared to broth-grown bacteria. SDS-PAGE analysis and autoradiography showed that neither the ratio of C3b:iC3b (approximately 1:2.5) nor the nature of the C3-bacterial bond was changed by growing the organisms in CMZ. Large amounts of complement membrane attack complexes containing poly-C9 were seen only on CMZ-grown SH5770 by SDS-PAGE and autoradiography. Poly-C9 was also detected only on CMZ-grown bacteria by indirect immunofluorescence and ELISA using a murine monoclonal antibody directed against a neoantigen of poly-C9. Bacterial hydrophobicity increased after growth in CMZ, and transmission electron micrographs of CMZ-grown SH5770 showed cell wall disruption and blebbing. These results indicate that growth in subMICs of CMZ increases bacterial hydrophobic domains available for interacting with the membrane attack complex, C5b-9, allowing formation and stable insertion of bactericidal complexes containing poly-C9.