Influence of Sub-inhibitory Concentrations of Cephalosporins on Surface Properties of Klebsiella Pneumoniae Important in Infection

Abstract

A method was devised to remove iron from complex culture media. The envelope properties and growth kinetics of bacteria in iron-depleted media were studied. The production of siderophores was reduced when several strains of Klebsiella pneumoniae and Escherichia coli were grown under iron-depleted conditions in the presence of ceftriaxone, cefuroxime, CGP 17520, cephalexin, or cefotaxime. Changes in surface properties were observed in encapsulated strains of K. pneumoniae when the bacteria were grown in the presence of all cephalosporins. The surface hydrophobicity of K. pneumoniae increased significantly when the bacteria were grown in either iron-sufficient or iron-depleted media in the presence of sub-MICs of all cephalosporins. Rapid agglutination was observed when cells grown in the presence of antibiotics were reacted with antisera raised against a non-capsulated strain of K. pneumoniae. No agglutination was observed when the cells were grown in the absence of antibiotics. The antigenicity of the OMPs of a clinical and laboratory strain of K. pneumoniae grown in the presence or absence of ceftriaxone or CGP 17520 was investigated using immunoblotting. The encapsulated clinical strain had three proteins (60K, 35.5K, and 32.5K) exposed on the surface. Mutants lacking capsule (K-0+) and O-antigen (K-0-) derived from an encapsulated strain (K+0+) had a number of protein antigens, including a 32.5K protein exposed on the surface. Growth of all strains of K. pneumoniae in the presence of either CGP 17520 or ceftriaxone resulted in the exposure of a greater number of protein antigens in the range of 26K to 83K. The effect of sub-inhibitory levels of cephaloridine and CGP 17520 on a strain of K. pneumoniae in experimental infections was investigated. Three high molecular weight proteins (69K, 70K, and 78K) were induced when bacterial cells were cultivated in the peritoneal cavity of rabbits, compared to six to seven observed in vitro. Addition of iron to the growth medium, either in vivo or in vitro, completely repressed the expression of these proteins. A difference in immunological recognition of the OM antigens of cells grown in vitro and in vivo was observed. The 26K, 28K, and 30K proteins present in vitro-grown cells were immunologically recognized by homologous antisera, but were not detected in in vivo-grown cells. Antisera raised against a non-capsulated K. pneumoniae caused rapid agglutination of cells grown in vivo in the presence of cephalosporins, but no agglutination was observed when the same strain was grown in the absence of cephalosporins. The reduction in siderophore and capsule production among the bacteria used in this study represents novel modes of action of cephalosporins and are likely significant factors in their clinical practice.