live / dead discrimination of
BIOFiLM bacteria from A drinking water pilot distribution system
Karlsruhe Institute of
Technology – Campus North, Institute of Functional Interfaces IFG, Microbiology
of Natural and Technical Interfaces Department,
jessica.villarreal@kit.edu
Formation
of biofilms in drinking water distribution networks,
including pipelines of households and food industries, are of great concern. Biofilms are potential habitats for all kinds of bacteria,
including pathogens, and may be responsible for contaminations of bulk water
systems.
Nowadays,
DNA-based methods are used for the detection and characterization of bacteria.
One of the major disadvantages of these techniques is that they can not
distinguish between DNA from live and dead cells. A battery of methods to face
this problematic is presented in this work.
Conditioned
surface water disinfected with ozone/ClO2 flowed through a pilot
scale built up with different pipe materials for biofilm
formation. Bacterial population analysis was done by PCR-DGGE, comparing direct
samples (total DNA) and samples pre-treated with Propidium
monoazide or DNase I (DNA
from live cells). Shifts in the DNA patterns observed after DGGE analysis,
demonstrated: (i) the applicability of PMA and DNase I treatment in natural biofilm
investigation; (ii) detection of DNA from dead bacteria and eDNA
was blocked by pretreatment with PMA or DNase I; and
(iii) DNase I treatment demonstrated a clearer effect
on live/dead differentiation. Traditional cultivation methods and qPCR completed the biofilm
analysis.
The
results of the bacterial population analysis and the results of the
quantification methods that provide an overview of the different physiological
states of bacteria: live cells, total amount of cells, and cultivable cells,
are presented here.