Dr. Michael Lebuhn
Bavarian State Research Center for Agriculture (LfL)
Abteilung Qualitätssicherung und Untersuchungswesen Mikro- und Molekularbiologie

Prof. Dr. Stefan Wuertz
University of California, Davis
Department of Civil & Environ. Engineering (DCEE)

The concept of microbial fecal indicators is not applicable to many environmental settings when it comes to protecting public health. Indicator organisms but no pathogens can present in samples and vice versa. The cooperation will involve joint development of molecular and ultrafiltration technologies to quantify pathogens (e.g. human adenovirus 40/41, enteroviruses, pathogenic protozoa) in aqueous environmental samples. TUM-WGA (Technical University of Munich, Institute of Water Quality Control and Waste Management, Dr. M. Lebuhn) will develop high-efficiency extraction systems for nucleic acids and specific low-level assessment of pathogens by (Reverse-Transcription) Real-Time Quantitative PCR in the presence of PCR-inhibitors. Conventional indicators will be assessed in parallel. UCD-DCEE (University of California, Davis, Department of Civil & Environmental Engineering, Prof. S. Wuertz) will develop adequate concentration and ultrafiltration steps of high-volume aqueous samples. The development of suitable Hi-Tech approaches and identification of suitable indicators and concentration levels may help to improve bathing water directives.

Final report:

In a transcontinental microbial source tracking (MST) approach, the efficiency of commercial DNA extraction kits (FastDNA® spin kit for soil (1), QIAamp stool mini kit (2), modified QIAamp maxi spin kit (3)) was compared using four Bacteroidales assays based on Quantitative Real-Time PCR (qPCR). Universal, human-, cow, and dog-specific Bacteroidales qPCR assays were tested to determine their specificity, their applicability on different continents. The four assays were tested with 3 cow, 3 dog, and 3 cat feces samples, primary influent of a wastewater treatment plant (WWTP) as human fecal samples, a WWTP sample after the UV treatment unit, and with Isar river samples from up- and downstream of the WWTP inlet area to assess impacts of the wastewater treatment and to identify the major source of fecal contamination. Concentration of bacteria by a novel hollow fiber ultrafiltration system was successfully applied for river water samples.

With 93 % DNA recovery rate, as compared to 35 % and 17 % for method (2) and (3), respectively, method (1) was most effective to quantify Bacteroidales spp., and resulted in superior downstream performance in qPCR. For pure water samples, omission of the extraction step was similarly successful. Eventual PCR inhibition could be revealed by template dilution qPCR. The assessment of the amount and source of fecal pollution in environmental samples by molecular MST can thus be affected by the choice of the DNA extraction method. The limit of quantification was 20 - 30 gene copies per qPCR reaction. As expected, the least human Bacteroidales were found upstream the WWTP inlet and the highest in the WWTP influent. Human Bacteroidales DNA was still detectable downstream of the UV treatment unit.

10⁷ – 10¹¹ gene copies / g dry matter were found for the samples with the corresponding host-specific assays, but non-specific amplification occurred for the human assay with 2 dog and 1 cat feces samples, and for the dog assay with 1 cow and 1 cat feces samples. Due to genetic variation of target DNA, the specificity of the genotype assays must therefore be improved for transcontinental application.