Drinking water utilities currently rely on a range of microbiological detection techniques to evaluate the quality of their drinking water (DW). However, microbiota profiling using culture-free 16S rRNA gene next-generation sequencing (NGS) provides an opportunity for improved monitoring of the microbial ecology and quality of DW. Here, we evaluated the utility of a previously validated microbiota profiling platform (MYcrobiota) to investigate the microbial dynamics of a full-scale, non-chlorinated DW distribution system (DWDS). In contrast to conventional methods, we observed spatial and temporal bacterial genus changes (expressed as operational taxonomic units - OTUs) within the DWDS. Further, a small subset of bacterial OTUs dominated with abundances that shifted across the length of the DWDS, and were particularly affected by a post-disinfection step. We also found seasonal variation in OTUs within the DWDS and that many OTUs could not be identified, even though MYcrobiota is specifically designed to reduce potential PCR sequencing artefacts. This suggests that our current knowledge about the microbial ecology of DW communities is limited. Our findings demonstrate that the user-friendly MYcrobiota platform facilitates culture-free, standardized microbial dynamics monitoring and has the capacity to facilitate the introduction of microbiota profiling into the management of drinking water quality.

The emergence of clinical enterococcal isolates that are resistant to both ampicillin and vancomycin is a cause of great concern, as therapeutic alternatives for the treatment of infections caused by such organisms are becoming limited. Aquatic environments could play a role in the dissemination of antibiotic resistant enterococci. This study investigated the presence of ampicillin and vancomycin resistant enterococci in the treated effluent of six wastewater treatment plants (WWTPs) and in surface water used as a source for drinking water production in the Netherlands. Membrane filtration in combination with selective media with ampicillin or vancomycin was applied to determine the presence of ampicillin resistant Enterococcus (ARE) and vancomycin resistant Enterococcus (VRE) species. Ampicillin resistant E. faecium (minimal inhibitory concentration (MIC) >16 µg/ml; n=1033) was observed in all studied WWTP effluents. In surface water used for drinking water production (intake locations), no ARE or VRE were observed. At both types of location, intrinsic vancomycin resistant Pediococcus spp., Leuconostoc spp. and Lactobacillus spp. were isolated with the vancomycin medium. The ampicillin resistant E. faecium (AREfm) isolates (n=113) did not contain the vanA or vanB gene, but MIC testing for vancomycin showed intermediate vancomycin resistance (2 to 8 µg ml -1 ) to occur in these AREfm strains. This study documents the discharge of ampicillin resistant E. faecium strains with intermediate vancomycin resistance by the WWTPs into the surface water, but no presence of these strains downstream at intake locations for drinking water production.

Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) hasincreasingly been used for rapid and reliable identification of clinically relevant micro-organisms. To establish the applicability of this technique in (drinking) water quality analysis, the MALDI-TOF MS identification results for Enterococcus spp. isolated from various water environments were compared with those obtained using the commercial Rapid 32 ID Strep system. One hundred and one bacterial isolates were isolated from various types of water and determined as enterococci on the basis of their growth on Slanetz-Bartley agar in typical colonies. The isolates were identified by MALDI-TOF MS and the commercial biochemical test Rapid 32 ID Strep. Isolates yielding in discrepant identifications were genotyped using 16S rRNA gene sequence analysis. For 86 isolates (85%), the results of Rapid ID 32 Strep were identical to those obtained with MALDI-TOF MS. Six isolates were impossible to be classified by means of the Rapid 32 ID Strep test. And for eight out of a total of nine discrepant results (89%), the 16S analyses confirmed the MALDI-TOF MS identification. MALDI-TOF MS produced highly reproducible results. These results indicated that the use of two different culture media had no effect on the identification. In addition, no significant differences (p ¼ 0.32; n ¼ 20)were evident between the scores obtained from a 20-fold measurement of the same isolate. The results of this study showed that MALDI-TOF MS identification (Bruker) is a reliable method for identifying E. faecium, E. faecalis, E. durans, E. hirae and E. casseliflavus isolated from water samples. E. mundtii and E. moraviensis were not included in the Rapid 32 ID Strep database and could therefore not be identified using that test set. However, MALDI-TOF MS and 16S identified all six isolates as members of these species.

Soil passage through sand dunes has previously been shown to remove enteric micro-organisms very effectively, and hence is used for the production of drinking water. However, enterococci have occasionally been isolated from abstracted water (after dune passage) in one of the dune infiltration areas in the Netherlands. E. moraviensis was the most frequently isolated species. Until now, no faecal sources of this species have been reported and the potential for growth under certain environmental conditions was reported for other Enterococcus species. The aim of this study was to determine the ability of E. moraviensis to grow in habitats present in the dune passage process (dune vegetation, sediment from abstraction wells, biofilm developed using abstracted water and soil). Different concentrations of boiled and filtered (0.45 µm) plant extracts obtained from dune vegetation supported growth (up to 6 log), with maximum concentrations after four to six days at 15 ˚C. Although E. moraviensis was shown to be able to attach to the biofilm, no growth was observed in biofilm or in sediment and soil. These observations confound the use of E. moraviensis as a faecal indicator.