Methodological approaches for studying the microbial ecology of drinking water distribution systems

The study of the microbial ecology of drinking water distribution systems (DWDS) has traditionally been based on culturing organisms from bulk water samples. The development and application of molecular methods has supplied new tools for examining the microbial diversity and activity of environmental samples, yielding new insights into the microbial community and its diversity within these engineered ecosystems. In this review, the currently available methods and emerging approaches for characterising microbial communities, including both planktonic and biofilm ways of life, are critically evaluated. The study of biofilms is considered particularly important as it plays a critical role in the processes and interactions occurring at the pipe wall and bulk water interface. The advantages, limitations and usefulness of methods that can be used to detect and assess microbial abundance, community composition and function are discussed in a DWDS context. This review will assist hydraulic engineers and microbial ecologists in choosing the most appropriate tools to assess drinking water microbiology and related aspects

High-performance liquid chromatography-ToxPrint: Chromatographic analysis with a novel (geno)toxicity detection

In order to aid the monitoring of the overall quality of (surface) waters a new analytical approach has been developed, combining on-line solid-phase extraction, HPLC separation and effect-related detection. Compounds present in surface water or wastewater samples are extracted on-line with Oasis [poly(divinylbenzene-co-N-vinylpyrrolidone)] material and directly fractionated by reversed-phase HPLC. The eluent of the total chromatogram is collected on a microtitre plate in fractions of 1 min each. After evaporation and re-dissolvation in a suitable solvent, the (geno)toxicity of the individual fractions before and after enzymatic activation with S9, is determined with the umu test. In this way, harmful compounds can be detected and localized in the HPLC-diode array detection trace even without their identity and exact concentration being known at that moment. The method was developed using two test compounds, 4-nitroquinoline-N-oxide and 2-aminoanthracene. Compounds with mutagenic properties comparable to those of the test compounds can be detected from 0.1 μg/l, which is a concentration relevant for surface waters. The new analytical approach was successfully applied to various types of model samples, as well as real wastewater.</p

Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas

Flow-through real time bacterial biosensor for toxic compounds in water

A flow-through fiber-optic-based bacterial monitoring system for online monitoring of toxic pollutants in water has been developed. Two bacterial strains containing fusions of recA (DNA damage) and grpE (heat-shock) promoters to the lux operon (CDABE) were immobilized on a fiber optic and tested for their ability to detect pollutants in flowing tap water and surface water. Conditions for running the system for 24 h were optimized and first experiments with the system show (1-h) response times and response heights similar to the previous static systems. Responses were related to the doses and the sensitivity is good (comparable to static systems), but needs to be increased to be able to monitor whether also the low guideline values are exceeded by pollutants. 24-h measurements in tap water demonstrate the ability of the device to run for such a time period, but in river water loss of functionality of the bacteria was observed. This flow-through fiber-optic-based monitoring system has proven to be a useful next step in the development of a simple on-line real time sensor for relevant human toxicants in flowing water.</p

Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas

Surveillance into zoonoses in veal calfs 2022

Dieren kunnen ziekteverwekkers bij zich dragen waarvan mensen ook ziek kunnen worden. De ziekten die hierdoor worden veroorzaakt, noemen we zoönosen. In 2022 is de mest en neus van vleeskalveren op 180 Nederlandse bedrijven onderzocht. Ook is bij 55 kalverhouders, gezinsleden en medewerkers onderzocht of ze de ziekteverwekkers bij zich dragen. Het RIVM, de NVWA(Nederlandse Voedsel- en Warenautoriteit) en WFSR(Wageningen Food Safety Research ) (Wageningen Food Safety Research) hebben dit onderzoek gedaan. Het onderzoek richt zich op verschillende ziekteverwekkende bacteriën. De belangrijkste zijn Campylobacter, STEC(Shigatoxineproducerende E. coli-stammen), Listeria en Salmonella. Daarnaast is gekeken naar ESBL(Extended spectrum beta-lactamases)-producerende bacteriën, colistineresistente bacteriën en MRSA(Methicilline-resistente Staphylococcus aureus ). Deze laatste zijn belangrijk omdat veel soorten antibiotica daar niet tegen werken. De meeste van deze ziekteverwekkers kunnen bij mensen diarree veroorzaken, maar bij mensen met een kwetsbare gezondheid kunnen infecties ernstiger verlopen. De ziekteverwekkers zitten meestal in de darmen van de dieren en dus ook in de mest. Het vlees kan tijdens de slacht besmet raken. Het is daarom belangrijk om kalfsvlees goed gaar te eten. Campylobacter kwam het meeste voor en is op 96 procent van de bedrijven gevonden. Bij de veehouders en gezinsleden is deze bacterie bij 5 personen gevonden. Het waren wel andere typen Campylobacterbacteriën dan die de dieren op de bijbehorende bedrijven bij zich droegen. Deze mensen kunnen op een andere manier met Campylobacter besmet zijn geraakt, bijvoorbeeld via voedsel of andere dieren. STEC-bacteriën, Listeria en Salmonella kwamen wat minder vaak voor, namelijk op 66 procent (STEC), 20 procent (Listeria) en 15 procent (Salmonella) van de bedrijven. Deze drie bacteriën zijn vaker gevonden bij bedrijven met zogenoemde rosé kalveren dan bij bedrijven met blanke kalveren. Twee personen droegen STEC bij zich en één deelnemer Listeria. Salmonella is niet bij de deelnemers gevonden. ESBL-producerende bacteriën zijn op 27 procent van de bedrijven gevonden en bij 3 deelnemers. Het percentage bij de deelnemers is ongeveer hetzelfde als bij de Nederlandse bevolking. Veegerelateerde MRSA is op 25 procent van de bedrijven gevonden, vaker bij bedrijven met blanke kalveren, en bij 13 procent van de deelnemers; dit is hoger dan MRSA bij de Nederlandse bevolking. Ten slotte zijn op 2 procent van de bedrijven colistine-resistente bacteriën gevonden, maar niet bij de deelnemers. Bij de bevolking is dat 0,8 procent.Animals can carry pathogens that can also cause disease in humans. Such diseases are known as zoonoses. In 2022 manure and nose swabs of veal calves was investigated at 180 Dutch farms. In addition, 55 livestock farmers, family members and employees were also tested for these pathogens. The study was carried out by RIVM, the Netherlands Food and Consumer Product Safety Authority (Nederlandse Voedsel- en Warenautoriteit, NVWA) and WFSR (Wageningen Food Safety Research). The study focuses on multiple pathogenic bacteria. The most important are Campylobacter, STEC, Listeria and Salmonella. In addition ESBL-producing bacteria, colistin-resistent bacteria and MRSA are investigated. These bacteria are important because they are resistant to multiple groups of antibiotics. Most of these pathogens usually cause diarrhea in humans, but the infections can sometimes be more severe in vulnerable populations. The pathogens are usually present in the animal’s intestines and therefore end up in the manure as well. Meat can become contaminated during slaughter. It is therefore important to only eat veal that has been thoroughly cooked. Of the investigated pathogens, Campylobacter was found most frequently, namely at 96% of the farms. Among livestock farmers and family members, Campylobacter was found in 5 persons. The types of Campylobacter carried by the humans were different from the types found in the calves on their farms. These people could be infected by Campylobacter by other routes, for example via food or other animals. Shigatoxigenic Escherichia coli (STEC) bacteria, Listeria and Salmonella were found less, on 66% (STEC), 20% (Listeria) and 15% (Salmonella) of the farms. These three bacteria were found more often on farms with so called rosé calves then on farms with white calves. Two persons carried STEC and one person Listeria. Salmonella was not found in human participants. ESBL-producing bacteria were found on 27% of the farms and in 3 human participants. The percentage in human participants is comparable to the percentage in the general Dutch population. MRSA was found on 25% of the farms, more often in farms with white calves and in 13% of the human participants. This is higher than the percentage of MRSA in the general Dutch population. Finally, in 2% of the farms colistin resistant bacteria were found, but not in human participants. In the general population this is 0,8%