Molecular diagnostics of the bacterial response to antibiotic therapy
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Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Brennecke, Johannes
Metadata
Abstract
Bacterial bloodstream infections (BSIs) are a major healthcare problem causing
high mortality and economic cost. BSIs require an immediate initiation of
antibiotic therapy as any delay is associated with a mortality increase. With the
emergence of antimicrobial resistance, the choice of the appropriate antibiotic
becomes increasingly difficult, thus creating an urgent need for new diagnostics,
ideally to be done at the point of care. The current gold standard is blood culture
with subsequent susceptibility testing although several molecular methods have
recently entered the market. However, in many instances there is a discrepancy
between the in-vitro data provided by the test and the outcome of antimicrobial
therapy in-vivo because current diagnostics fail to take into account the impact of
the environment in the patient such as the immune system, pharmacokinetics
and pharmacodynamics or bacterial fitness. In this thesis, it was hypothesised
that the measurement of the bacterial gene expression after the beginning of
antibiotic therapy might be a more accurate indicator of the therapy outcome
because it reflects the bacterial response under in-vivo conditions.
In the first part of the thesis the expression of a set of pre-defined mRNA markers
was investigated under various conditions. Experiments conducted with clinical
E. coli isolates incubated in human whole blood revealed an excellent correlation
between the gene expression, the treatment outcome, the antibiotic susceptibility
and the genetic background for three different classes of antimicrobial drugs.
The second part of the thesis describes the extraction of bacterial RNA from
human whole blood specimen. The effect of different agents for the lysis of human
blood cells and the impact of co-purified human RNA were analysed and a method
for high yield extraction of undegraded bacterial RNA was established.
The third part of the thesis investigates two methods for the sensitive
measurement of the bacterial gene expression. This is relevant because the
bacterial loads in BSI patients are extremely low. For genes with high gene
expression levels both methods yielded reliable results but were unable to
quantify the expression of the previously investigated mRNA markers due to their
low copy numbers. Other approaches, especially those based on single cell
measurements, might be able to overcome the problem in the future and should
be explored in greater detail.
Overall, the foundations for a future diagnostic test based on the measurement of
the bacterial gene expression have been laid in this work. Future work should
address the mRNA quantification and further evaluate the connection between
gene expression and therapy outcome, e.g. in animal models. A future diagnostic
test should also fulfil point-of-care requirements. This will include integrated
sample preparation and quantification as well as a time-to-result in the range of
a few minutes.