Delayed antibiotic-induced lysis of Escherichia coli in vitro is correlated with enhancement of LPS release.

Abstract

A kinetic turbidimetric Limulus amebocyte lysate (LAL) assay was used to study the effects of gentamicin, amoxycillin and ciprofloxacin (16 x MIC) upon release of lipopolysaccharide at different stages of a growing Escherichia coli 055:B5:H culture in vitro. In this model a linear correlation was present between the logarithms of colony counts and free LAL activities. Untreated E. coli grew from log values of 4.9 +/- 0.15 (low inoculum) and 6.8 +/- 0.08 cfu/ml (high inoculum) at t = 0 to 8.9 +/- 0.05 and 9.1 +/- 0.13 cfu/ml at t = 6 h, respectively. The log values of basal free LAL activities at low and high inoculum sizes were 1.9 +/- 0.07 and 3.3 +/- 0.14 endotoxin units/ml, increasing 2100- and 69-fold, respectively during a 6-h growth. Amoxycillin-induced lysis was not significantly associated with an increase in free LAL activity. Efficacy of bacterial killing by gentamicin was high, but free LAL activity increased only 3.2- and 7.7-fold at the low and high inoculum experiments, respectively. Ciprofloxacin induced cell filamentation during the experiments. At low and high inoculum conditions exposure to ciprofloxacin induced a 43- and 68-fold increase in free LAL activities, respectively. Our data indicate that (a) LPS is released as long as E. coli remain structurally intact; (b) LPS release is enhanced when bacterial biomass increases; and (c) are taken as evidence against the concept of lysis-correlated LPS release.