Delayed antibiotic-induced lysis of Escherichia coli in vitro is correlated with enhancement of LPS release.
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.
Van Den Berg
C
C
Laboratory for Medicines and Medical Devices, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
de Neeling
A J
AJ
Schot
C S
CS
Hustinx
W N
WN
Wemer
J
J
de Wildt
D J
DJ
D000658:Amoxicillin / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology*; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000378:metabolism*
D005839:Gentamicins; D008070:Lipopolysaccharides; D002939:Ciprofloxacin; D000658:Amoxicillin