Antimicrobial Susceptibility Assays Based on the Quantification
of Bacterial Lipopolysaccharides
via a Label Free Lectin Biosensor
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Abstract
A label free lectin biosensor developed
in our laboratory that
can quantitatively measure the binding between the lectin immobilized
at the carbohydrate sensor surface and the lipopolysaccharide (LPS)
on Gram-negative bacteria was demonstrated for an antibiotic susceptibility
assay. The biosensor utilizes a polythiophene interface containing
fused quinone moieties glycosylated to form a carbohydrate platform
for the immobilization of Concanavalin A (Con A) and is capable of
LPS binding measurements via orthogonal quartz crystal microbalance
and electrochemical readouts (EQCM). Such orthogonal transduction
provides cross-validation, better sensor sensitivity, and a large
dynamic range of the measurements. We have applied this label free
lectin biosensor for a new antibiotic susceptibility assay by characterizing
the antimicrobial activities of various antibiotics (i.e., ciprofloxacin,
ceftriaxone, and tetracycline) against Escherichia
coli W1485 as a model system. The label free biosensor
allows both end point and real time measurements of antibiotic effects
on the bacterial cell surface LPS, which is shown to correlate to
their antibiotic effects. At the end point, after 18 h incubation
of bacterial cells with these three antibiotics respectively, the
bacterial LPS binding signal was reduced to 23%, 27%, and 38%, respectively,
for the three antibiotics, indicating that ciprofloxacin is the most
effective against this E. coli strain.
Real time measurements at the 1 h time point showed a similar trend
with a reduction of binding to 91%, 93%, and 95%, respectively. From
the binding kinetics of these measurements, the relaxation time (τ)
was obtained, where higher τ value means slow binding interactions
between the lectin and the bacterial LPS. The obtained order of τ,
(i.e., τ<sub>ciprofloxacin</sub> > τ<sub>ceftriaxone</sub> > τ<sub>tetracycline</sub>) again indicated that ciprofloxacin
has more bactericidal activity than the other two antibiotics with
the same concentrations. Thus, we are able to establish that the reduction
in the binding of LPS with the lectin Con A sensor upon exposure to
various antibiotics has a direct relation with the antibiotic dosages
making this label free biosensor assay promising for therapeutic management
of these drugs as well as for applications in antibiotic research
and development
