Biological characterization of endotoxins released from antibiotic-treated Pseudomonas aeruginosa and Escherichia coli

Abstract

The supernatants taken from Pseudomonas aeruginosa and Escherichia coli cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those taken from the same cultures in the presence of imipenem (IPM) yielded a very low level of endotoxin. The biological activities of endotoxin in the supernatants were compared with those of phenol water-extracted lipopolysaccharide (LPS). The endotoxin released from the organisms as a result of CAZ treatment (CAZ-released endotoxin) contained a large amount of protein. The protein, however, lacked endotoxic activity, since the endotoxin did not show any in vivo toxic effects in LPS-hyporesponsive C3H/HeJ mice sensitized with D-(+)-galactosamine (GalN) or any activation of C3H/HeJ mouse macrophages in vitro. The activities of CAZ- and IPM-released endotoxin (as assessed by a chromogenic Limulus test) were fundamentally the same as those of P. aeruginosa LPS, since their regression lines were parallel. The CAZ-released endotoxin was similar to purified LPS with respect to the following biological activities in LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice: lethal toxicity in GalN-sensitized mice, in vitro induction of tumor necrosis factor- and NO production by macrophages, and mitogen-activated protein kinase activation in macrophages. The macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on the presence of serum factor and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A).

FIG. 1

Kinetics of bacterial growth and antibiotic-induced release of endotoxin from P. aeruginosa and E. coli incubated in synthetic M9 medium. The organisms were cultured in M9 medium in the presence or absence of antibiotics; 0.5× the MIC of CAZ (▵) or IPM (▴), 2× the MIC of CAZ (□) or IPM (▪), 4× the MIC of CAZ (⊠), and no antibiotic (○).

FIG. 2

Bacterial growth and antibiotic-induced release of endotoxin from P. aeruginosa and E. coli incubated in M9 medium or PBS. The organisms were cultured in M9 medium (○ and □) or PBS (• and ▪) in the presence of 2× the MIC of CAZ (□ and ▪) or with no drug (○ and •).

FIG. 3

Regression lines of the ES test with antibiotic-induced endotoxins. P. aeruginosa (closed symbols) or E. coli (open symbols) was cultured in M9 medium in the presence of 2× the MIC of CAZ (• and ○) or IPM (▴ and ▵) or with no drug (▪ and □) for 8 h. As controls, the activities of standard solutions containing known amounts of E. coli O111:B4 LPS (⊙) and P. aeruginosa LPS (⊠) were assessed.

FIG. 4

Serum dependency of TNF, IL-6, and NO production by C3H/HeN macrophages of C3H/HeN mice stimulated with phenol-water-extracted LPS or antibiotic-released endotoxin from P. aeruginosa (P.a.). Thioglycolate-elicited macrophages were cultured in the presence (•) or absence (○) of 2% FBS with LPS or CAZ-released endotoxin that had been prepared from bacteria exposed to 2× the MIC of CAZ for 8 h. Culture supernatants were collected at 4 h for the TNF assay (A and B), 16 h for the IL-6 assay (C and D), and 48 h for the NO assay (E and F). Each point represents the mean ± standard error of the mean. The data represent the results of one of two or three experiments with similar results.

FIG. 5

TNF, IL-6, and NO production by LPS-responsive C3H/HeN mouse macrophages and LPS-hyporesponsive C3H/HeJ mouse macrophages stimulated with antibiotic-released endotoxin. Thioglycolate-elicited macrophages from C3H/HeN mice (•) or C3H/HeJ mice (○) were cultured with P. aeruginosa (P.a.) LPS (A, C, and E) or endotoxin from bacteria exposed to 2× the MIC of CAZ for 8 h (B, D, and F) in the presence of 2% FBS. The culture supernatants were collected at 4 h for the TNF assay (A and B), 16 h for the IL-6 assay (C and D), and 48 h for the NO assay (E and F), and the activities were assessed. Each point represents the mean ± standard error of the mean. The data represent the results of one of two experiments with similar results.

FIG. 6

Induction of protein tyrosine phosphorylation and tyrosine phosphorylation of MAP kinases in LPS-responsive C3H/HeN mouse macrophages and LPS-hyporesponsive C3H/HeJ mouse macrophages stimulated with CAZ-released endotoxin from an 8-h culture. Macrophages from C3H/HeN mice (lanes 1 to 7) or C3H/HeJ mice (lanes 8 to 14) were stimulated for 15 min with the following stimulants: RPMI 1640 (lanes 1 and 8), M9 medium (lanes 2 and 9), endotoxin from P. aeruginosa exposed to 2× the MIC of CAZ for 8 h (290 ng/ml; lanes 3 and 10), endotoxin from E. coli exposed to 2× the MIC of CAZ for 8 h (390 ng/ml; lanes 4 and 11), P. aeruginosa LPS (270 ng/ml; lanes 5 and 12), glutaraldehyde-killed P. aeruginosa (5 × 10⁵/ml; lanes 6 and 13), and phenol-chloroform-petroleum ether-extracted Ra-LPS (1 μg/ml) plus mouse recombinant gamma interferon (20 U; Shionogi Pharmaceutical Co., Osaka, Japan) (lanes 7 and 14). Macrophage expression of protein tyrosine phosphorylation and tyrosine phosphorylation of MAP kinases 1 and 2 was detected by Western blot analysis with MAb PY-20 (A) and MAb Z033 (B), respectively. p-MAP1 and pMAP2, tyrosine-phosphorylated MAP kinases 1 and 2, respectively.

FIG. 7

TNF production by J774.1 cells and J7.DEF3 cells stimulated with LPS or endotoxin. J774.1 cells (•) and J7.DEF3. cells (○) were incubated with P. aeruginosa (P.a.) LPS or endotoxin (endotoxin from bacteria exposed to 2× the MIC of CAZ for 8 h) in the presence (A and B) or absence (C and D) of 2% FBS for 4 h. The data represent the results of one of two experiments with similar results.