live / dead discrimination of
bacteria via DNase/Proteinase treatment
Karlsruhe
Institute of Technology – Campus North, Institute of Functional Interfaces IFG,
Microbiology of Natural and Technical Interfaces Department,
jessica.villarreal@kit.edu
DNA-based
molecular biology techniques are very sensitive, but have some limitations to
discriminate DNA coming from live, injured and dead cells as well as extracellular
DNA (eDNA) in natural and technical systems. Regarding
this dilemma, DNase I combined with proteinase K
treatment was used for a better discrimination between live and dead bacteria. DNase I is an endonuclease that non-specifically cleaves
single and double stranded DNA. This enzyme was tested in order to analyze its
capacity of digesting eDNA and DNA coming from dead/injured
cells with damaged cell membranes, leaving DNA from living and VBNC cells unaffected
and available for DNA-based detection methods. For this, an optimized DNase I/Proteinase K (DNase/PK)
protocol was established using mixtures of living cells, dead cells and free
genomic DNA. The efficiency of the DNase/PK method
was compared with the already established propidium monoazide (PMA) technique. In both approaches RealTime PCR was used to assess the efficiencies of the DNase/PK and PMA application.
Additionally,
the DNase/PK method was applied to natural drinking
water biofilm samples from a German waterworks where ozone/ClO2 was used for
disinfection. Shifts in the DNA patterns observed after PCR-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 treatment with PMA
or DNase I; and (iii) DNase/PK
treatment demonstrated a discrimination of live and dead bacteria. Conventional
cultivation methods, staining techniques and qPCR
completed the biofilm analysis.