Annette Søndergaard Bukh

State of the art molecular markers for fecal pollution source tracking in water

Most environmental waters are susceptible to fecal contamination from animal and/or human pollution sources. To attenuate or eliminate such contamination, it is often critical that the pollution sources are rapidly and correctly identified. Fecal pollution source tracking (FST) is a promising research area that aims to identify the origin(s) of fecal pollution in water. This mini-review focuses on the potentials and limitations of library independent molecular markers that are exclusively or strongly associated with fecal pollution from humans and different animals. Fecal-source-associated molecular markers include nucleic acid sequences from prokaryotes and viruses associated with specific biological hosts, but also sequences such as mitochondrial DNA retrieved directly from humans and animals. However, some fecal-source-associated markers may not be absolutely specific for a given source type, and apparent specificity and frequency established in early studies are sometimes compromised by new studies suggesting variation in specificity and abundance on a regional, global and/or temporal scale. It is therefore recommended that FST studies are based on carefully selected arrays of markers, and that identification of human and animal contributions are based on a multi-marker toolkit with several markers for each source category. Furthermore, future FST studies should benefit from increased knowledge regarding sampling strategies and temporal and spatial variability of marker ratios. It will also be important to obtain a better understanding of marker persistence and the quantitative relationship between marker abundance and the relative contribution from individual fecal pollution source types. A combination of enhanced pathogen screening methods, and validated quantitative source tracking techniques could then contribute significantly to future management of environmental water quality including improved microbial risk assessment.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark

OBJECTIVES The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). METHODS The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. RESULTS Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. CONCLUSIONS Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Application of mussels as biosamplers for characterization of faecal pollution in coastal recreational waters

Sources of faecal pollution in coastal recreational waters may be identified by analysing different host associated microorganisms or molecular markers. However, the microbial targets are often present at low numbers in moderately impacted waters, and often exhibit significant temporal and spatial variability in waters with fluctuating faecal loads. This patchy occurrence can limit successful detection of relevant targets in microbial source tracking studies. In this study, we explored the possibility for using the blue mussel (Mytilus edulis) as a biosampler for accumulation of faecal bacteria relevant for microbial source tracking. Non-contaminated blue mussels were transferred to three coastal recreational waters affected by faecal pollution of unknown origin. Molecular markers associated with animal and human waste were targeted by PCR and compared in seawater and mussel samples. The results demonstrated that transplanted mussels in simple enclosures accumulated and retained elevated levels of molecular markers associated with different types of faecal pollution. The targets included a novel putative human associated E. coli subgroup B2 VIII clone, and animal and human associated markers in enterococci (esp, M19, M66, M90, and M91). Human (sewage) associated markers including esp and M66 were sometimes not detectable in seawater samples despite known wastewater contamination, whereas the markers were detectable in mussels. We suggest that transplanted mussels should be considered as potential biosamplers in studies focusing on identifying source of faecal pollution in low or moderately impacted recreational waters. Bioaccumulation of molecular markers in mussels for several days may represent the water quality better than traditional grab samples from the water column.

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

Aims:  To evaluate the potential for using a novel chemiluminescence‐based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste.