Stefan Kötzsch

Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method.

Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15 min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing.

Microbiological tap water profile of a medium-sized building and effect of water stagnation

Whereas microbiological quality of drinking water in water distribution systems is routinely monitored for reasons of legal compliance, microbial numbers in tap water are grossly understudied. Motivated by gross differences in water from private households, we applied in this study flow cytometry as a rapid analytical method to quantify microbial concentrations in water sampled at diverse taps in a medium size research building receiving chlorinated water. Taps differed considerably in frequency of usage and were located in laboratories, bathrooms, and a coffee kitchen. Substantial differences were observed between taps with concentrations (per mL) in the range from 6.29×103 to 7.74×105 for total cells and from 1.66×103 to 4.31×105 for intact cells. The percentage of intact cells varied between 7% and 96%. Water from taps with very infrequent use showed the highest bacterial numbers and the highest proportions of intact cells. Stagnation tended to increase microbial numbers in water from those taps which were otherwise frequently used. Microbial numbers in other taps that were rarely opened were not affected by stagnation as their water is probably mostly stagnant. For cold water taps, microbial numbers and the percentage of intact cells tended to decline with flushing with the greatest decline for taps used least frequently whereas microbial concentrations in water from hot water taps tended to be somewhat more stable. We conclude that microbiological water quality is mainly determined by building-specific parameters. Tap water profiling can provide valuable insight into plumbing system hygiene and maintenance.

Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry

Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation‐independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 μm), (2) then cells are resuspended with sterile filtered buffer and double‐stained with FITC‐ and Alexa‐conjugated Legionella‐specific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell‐counting correlated well.

A new method to assess the influence of migration from polymeric materials on the biostability of drinking water

After having produced drinking water of high quality it is of vital interest to distribute the water without compromising its quality neither by recontamination nor by microbial regrowth. To minimize regrowth, the strategy of distributing biostable water is followed in several European countries. This implies on one hand the production of water that has a low level of growth-supporting nutrients, in particular organic carbon compounds, and, on the other hand, using materials for storage/distribution that have a low biofilm formation potential and from which only low amounts of total organic carbon (TOC) leach into the water phase. Currently, the approval of materials in contact with drinking water relies on two tests, a migration test and a biofilm formation test. Here we describe an extended migration testing procedure that allows to obtain information not only on the amount of chemical compounds but also on the amount of growth-supporting compounds leaching into the water. In short, the test developed combines several migration cycles and subsequent measurement of the TOC with a novel, fast and reliable test method for determining the assimilable organic carbon (AOC) in the migration waters. AOC gives an indication on the growth-supporting properties of the material. Thus, an initial characterisation of a material with respect to its suitability for usage in contact with drinking water can be performed in a single assay. Results obtained with the new assay for a number of materials typically used in drinking water and sanitary installations are reported.

Biofilms in shower hoses – choice of pipe material influences bacterial growth and communities

Flexible polymeric pipe materials are commonly used as shower hoses or connections to faucets in the last meters of building plumbing, but these tend to leach high concentrations of carbon that encourage bacterial growth. Here we compared the microbiological impact of six such materials, with both a short-term material comparison test and a daily shower simulator operating for eight months. The materials ranked differently in the migration potential and biomass formation potential assays of the comparison test, but overall these results correlated (R2 > 0.77) with long-term biofilm development in the shower simulator. The biofilm concentration after eight months ranged from 2 × 106 cells per cm2 on the control material (PE-Xc) to 2 × 108 cells per cm2 on a typical shower hose (PVC-P). However, the differences in four of the six materials were much less pronounced after eight months than in the early months. The communities were characterized with 16S rRNA amplicon sequencing clustered with both material and time (R2 = 0.31; R2 = 0.25), and correlated strongly with the biofilm concentration (R2 > 0.74). A universal core consisting of 7 genera accounted for 44% of all sequences, and accounted for more of young and high-biomass biofilms. Genera containing opportunistic pathogens were more common in low-biomass pipes. We conclude that choice of materials is not only critical for determining biofilm concentration, but also community composition. Our results show that a seemingly small choice in plumbing material in the final meters of distribution can make a considerable difference in the building plumbing ‘exposome’.

BioMig—A Method to Evaluate the Potential Release of Compounds from and the Formation of Biofilms on Polymeric Materials in Contact with Drinking Water

In contact with water, polymeric materials (plastics) release compounds that can support suspended microbial growth and/or biofilm formation. The different methods presently used in the European Union to test plastics take 7-16 weeks to obtain a result. In industry, this delays material and product development as well as quality testing. Therefore, we developed a method package (BioMig) that allows testing of plastic materials with high reproducibility in 2 weeks for their potential biofilm (or biomass) formation and release of carbonaceous migration products when in contact with water. BioMig consists of (i) an extended migration potential test (seven times for 24 h at 60 °C), based on the European norm EN 12873-1 and the German UBA (Umweltbundesamt) guideline, and (ii) a biomass formation potential (BFP) test (14 days at 30 °C), which is a modified version of the Dutch biofilm production potential test. In the migration potential test, the amount of carbon released into water by the specimen is quantified by monitoring total and assimilable organic carbon over time; furthermore, the modular design of the test also allows one to assess additional parameters such as pathogen growth potential on the migration water or toxic effects on microbial growth. Flow cytometry (FCM)-based total cell counting (TCC) is used to quantify microbial growth in suspension and on surfaces after removal with mild sonication without affecting cell integrity. The BFP test allows one to determine both the planktonic (pBFP) and the sessile (sBFP) cell fractions. The sBFP consists of surface-attached cells after removal (>90% efficiency). Results for four standard test materials (PE-Xa, PE-Xc, EPDM 2%, and EPDM 20%), plus positive (PVC-P) and negative (glass) controls are presented. FCM-based TCC demonstrates that the release of growth-supporting carbon and proliferation of surface-attached cells stops increasing and stabilizes after 14 days of incubation; this allows for faster assessment of growth-supporting properties of plastics with BioMig compared to established tests.

Cryptosporidium spp. in drinking water. Samples from rural sites in Switzerland.

In most rural areas and small communities in Switzerland the drinking water is supplied to the consumers after a minimum or even no treatment at all. However, it is just in these areas where drinking water from sources of agricultural activities can be contaminated by liquid manure and faeces of pasturing animals. The Swiss drinking water regulations are limited to the monitoring of E. coli, Enterococcus spp. and total plate counts only. Hence, resistant pathogens, as for example Cryptosporidium spp., remain unnoticed. During a drinking water survey, which lasted from June 2003 to December 2004, water samples were collected from 3 selected rural sites in Switzerland. The drinking water was investigated for Cryptosporidium spp., E. coli, Enterococcus spp., Clostridium perfringens and other parameters. In all samples oocysts of Cryptosporidium spp. were detected at elevated concentrations of up to 0.18 oocysts/l. Between 28% and 75% of the oocysts were found to be vital by the excystation method. Sampled oocysts collected from the three sites were subjected to genotyping and in one case the isolate was found to belong to the genotype of C. parvum. No evidence for increased incidents of diarrhoea in the past years was noted by local authorities.