Escherichia coli and enterococci are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources.

AIMS: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. METHODS AND RESULTS: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7-7·0CFU g(-1) ) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0-8·4CFU g(-1) ). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3-7·7 CFUg(-1) . To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi-parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment (Reischer et al. 2008; Environ Microbiol 10:2598-2608). CONCLUSION: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.

Development of a novel amplified fragment length polymorphism (AFLP) typing method for enterococci isolates from cattle faeces and evaluation of the single versus pooled faecal sampling approach.

A novel and highly reproducible amplified fragment length polymorphism (AFLP) typing approach was developed for typing of Enterococcus strains from the environment. Pooled and corresponding single faecal sample isolates were analysed to test the efficiency and coverage of dominant isolates for future sampling procedures. AFLP development was based on the selection of appropriate restriction enzymes and the design of adaptors and primers which was supported by in silico optimisation of selective bases using Enterococcus spp. genome data. Three optimal combinations of selective bases at the 3' end of the designed primers (i.e., CC, GG, CG) could be determined. AFLP fragment analysis using a capillary sequencer and intralane standardisation resulted in excellent methodical stability (> or =98% similarity for GG and > or =94% similarity for CC). Furthermore, the developed typing method was evaluated on 16 type trains of the genera Enterococcus and Streptococcus and 398 faecal isolates of cow pats from five alpine pastures in a karstic catchment area. Statistical analysis revealed a discrimination capacity of DI > or =0.95 (Simpson Diversity Index) and a reproducibility level of > or =94% similarity indicating the methods high typing capacity and robustness. Results of the comparative analysis of single and pooled faecal samples indicate that for a "strain to strain" based faecal source tracking, pooled faecal samples rather than single faecal samples are likely to be the most efficient sampling strategy for collecting the abundant corresponding strains.