Development of a Scorpion probe-based real-time PCR for the sensitive quantification of Bacteroides sp. ribosomal DNA from human and cattle origin and evaluation in spring water matrices.

Spring water from alpine catchments are important water resources but they can be vulnerable against faecal contamination. Potential faecal contamination sources are wildlife populations, pasturing activities, or alpine tourism. Unfortunately, no faecal source tracking method is available to date which is sensitive enough for appropriate spring water monitoring and source allocation. Our purpose was to develop a Duplex Scorpion real-time PCR approach for the specific and sensitive quantification of Bacteroides sp. 16S rDNA fragments from human and cattle origin. By the developed approach, detection of plasmids, carrying the respective biomarker sequence, was possible over a range of more than seven orders of magnitudes down to six copy numbers per PCR assay. Furthermore, the Duplex Scorpion real-time PCR allowed the specific quantification down to 50 targets in plasmid spiked spring water matrices. Results indicate that microbial source tracking appears feasible in spring water habitats by probe-based real-time PCR technologies. However, preliminary testing of the established approach on faecal samples collected from a representative alpine habitat did not allow unambiguous source allocation in all cases. In the future, the available sequence database has thus to be widened to allow reliable source tracking in alpine spring watersheds and even expand this approach to other potential faecal sources.

Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water.

AIMS: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water METHODS AND RESULTS: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338/EUB338-II/EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77-96%) bacteria and 11% (range 3-22%) identified as Archaea. CONCLUSIONS: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.