Automated flow cytometry as a flexible tool for comparing disinfection characteristics of indigenous bacterial communities and pure cultures.

Bacterial inactivation efficiency of chlorine varies with organisms and environmental conditions. The comparability of different samples/studies, especially comparing indigenous bacterial communities with pure cultures, is impeded by inconsistent experimental conditions and analytical methods used in various studies. We tested a novel 96-well plate FCM experimental and automated analytical approach, where bacterial communities and pure cultures were suspended in the same natural water matrix prior to chlorination directly in the plate. We demonstrated the ability to rapidly monitor the efficiency of 32 different combinations of chlorine concentration and time (i.e. chlorine exposure) on bacterial pure cultures and indigenous aquatic communities, which enabled correct comparison of the data from different samples under the exact same experimental conditions. In this study, the 96-well plate automated FCM approach enabled large sets (896) of independent chlorination experiments to be carried out in a short time period. To our knowledge, this is the largest dataset of chlorination experiments which consumed least time (within 18 h after sampling) until now. Staining with SYBR Green I (SG) and SG combined with propidium iodide (SGPI) was used to assess cellular damage during chlorination. The results showed that with the same chlorine exposure, a higher chlorine concentration with a shorter contact time is favorable for inactivation of bacteria. Our research provides a promising framework to compare disinfection characteristics of various microorganism and can be further developed to diagnose effect of antimicrobial products.

360-Degree Distribution of Biofilm Quantity and Community in an Operational Unchlorinated Drinking Water Distribution Pipe.

In the present study, triplicate rings of 360° pipe surfaces of an operational drinking water distribution pipe were swabbed. Each ring was equally divided into 16 parts for swabbing. The collected swabs were grouped into 3 sections and compared with the biofilm samples sampled by sonication of specimens from the same pipe. The results showed that the biofilm is unevenly distributed over the 16 parts and the 3 sections of the pipe surface. Both the active biomass and the number of observed OTUs increased as the measurements proceeded from the top to the bottom of the pipe. The bacterial community was dominated in all sections by Proteobacteria. At the genus level, Nitrospira spp., Terrimonas spp., and Hyphomicrobium spp. were dominant in all sections. Gaiella spp. and Vicinamibacter spp. dominated in S-I, Blastopirellula spp. and Pirellula spp. dominated in S-II, while Holophaga spp. and Phaeodactylibacter spp. dominated in S-III. When swabbing and pipe specimen sonication were compared, the results showed that the sampling strategy significantly influences the obtained biofilm bacterial community. A consistent multisectional swabbing strategy is proposed for future biofilm sampling; it involves collecting swabs from all sections and comparing the swabs from the same position/section across locations.

Inactivation of Antibiotic Resistant Bacteria and Resistance Genes by Ozone: From Laboratory Experiments to Full-Scale Wastewater Treatment.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

Kinetics and yields of pesticide biodegradation at low substrate concentrations and under conditions restricting assimilable organic carbon.

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter(-1). We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.

Continuous monitoring of enzymatic reactions on surfaces by real-time flow cytometry: sortase a catalyzed protein immobilization as a case study.

Only a few techniques, such as quartz crystal microbalance and surface plasmon resonance spectroscopy, enable the analysis of dynamic processes on solid supports. Here we have developed a straightforward assay based on flow cytometry to continuously follow enzymatic reactions directly on microparticle surfaces. We applied this real-time flow cytometry (RT-FCM) approach to study the covalent immobilization of green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the transpeptidase sortase A (SrtA) from Staphylococcus aureus. Though commonly treated as functionally identical catalysts, the SrtA variants SrtAΔ59 and SrtAΔ25, in which the N-terminal amino acid residues 1-59 and 1-25 of the native enzyme are truncated, were shown to perform very differently with regard to this particular immobilization reaction. While SrtAΔ59 efficiently catalyzed the covalent attachment of GFPuv to the surface (as indicated by a linear increase of microbead fluorescence), SrtAΔ25 was essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the hexahistidine tag at either the N- or C-terminus affected the efficiency of enzymatic protein immobilization. Apart from three enzyme variants containing the native core structure of SrtA, we also included three recently evolved mutants of SrtA in this comparative study. With these mutants we observed a rapid initial attachment of the GFPuv target protein to the microbeads. However, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevailed over the coupling reaction, consequently causing a decline of microbead fluorescence. In general, the RT-FCM approach used herein represents a powerful analytical tool for qualitative dynamic studies of many heterogeneous enzymatic reactions or other binding events that influence the fluorescence properties of microparticle surfaces.

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 x 10(3) to 7.74 x 10(5) for total cells and from 1.66 x 10(3) to 4.31 x 10(5) 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.

The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies.

Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.

Chemical extraction of microorganisms from water-saturated, packed sediment.

Microbial characterization of aquifers should combine collection of suspended and attached microorganisms (biofilms). This study investigated chemical extraction of microorganisms from water-saturated, packed sediment containing established biofilms. It compares the use of different detachment-promoting agent (DPA) solutions with tap water as eluent in column experiments. Extraction efficiency was determined from cell concentrations in the column effluent. Adenosine triphosphate concentrations were measured to confirm cell extraction and as an indicator of cell membrane integrity. Quality of extracted bacterial communities was assessed by comparing their terminal restriction fragment length polymorphism profiles with destructively sampled sediment-community profiles. Extraction efficiency increased more than 8-fold when deionized water, D-amino acids, or enzymes were used as a DPA. Community profiles recovered by individual DPA solutions showed more pronounced differences at the level of rare microbial groups, whereas abundant groups appeared ubiquitous across treatments. These results suggest that comparison of communities extracted by different DPAs can provide improved information on the occurrence of rare microbial groups in biofilms.

Legionella relative abundance in shower hose biofilms is associated with specific microbiome members.

Legionella are natural inhabitants of building plumbing biofilms, where interactions with other microorganisms influence their survival, proliferation, and death. Here, we investigated the associations of Legionella with bacterial and eukaryotic microbiomes in biofilm samples extracted from 85 shower hoses of a multiunit residential building. Legionella spp. relative abundance in the biofilms ranged between 0-7.8%, of which only 0-0.46% was L. pneumophila. Our data suggest that some microbiome members were associated with high (e.g. Chthonomonas, Vrihiamoeba) or low (e.g. Aquabacterium, Vannella) Legionella relative abundance. The correlations of the different Legionella variants (30 Zero-Radius OTUs detected) showed distinct patterns, suggesting separate ecological niches occupied by different Legionella species. This study provides insights into the ecology of Legionella with respect to: (i) the colonization of a high number of real shower hoses biofilm samples; (ii) the ecological meaning of associations between Legionella and co-occurring bacterial/eukaryotic organisms; (iii) critical points and future directions of microbial-interaction-based-ecological-investigations.

Development and laboratory-scale testing of a fully automated online flow cytometer for drinking water analysis.

Accurate and sensitive online detection tools would benefit both fundamental research and practical applications in aquatic microbiology. Here, we describe the development and testing of an online flow cytometer (FCM), with a specific use foreseen in the field of drinking water microbiology. The system incorporated fully automated sampling and fluorescent labeling of bacterial nucleic acids with analysis at 5-min intervals for periods in excess of 24 h. The laboratory scale testing showed sensitive detection (< 5% error) of bacteria over a broad concentration range (1 × 10(3) -1 × 10(6) cells mL(-1) ) and particularly the ability to track both gradual changes and dramatic events in water samples. The system was tested with bacterial pure cultures as well as indigenous microbial communities from natural water samples. Moreover, we demonstrated the possibility of using either a single fluorescent dye (e.g., SYBR Green I) or a combination of two dyes (SYBR Green I and Propidium Iodide), thus broadening the application possibilities of the system. The online FCM approach described herein has considerable potential for routine and continuous monitoring of drinking water, optimization of specific drinking water processes such as biofiltration or disinfection, as well as aquatic microbiology research in general.

Variable Legionella Response to Building Occupancy Patterns and Precautionary Flushing.

When stay-at-home orders were issued to slow the spread of COVID-19, building occupancy (and water demand) was drastically decreased in many buildings. There was concern that widespread low water demand may cause unprecedented Legionella occurrence and Legionnaires' disease incidence. In lieu of evidenced-based guidance, many people flushed their water systems as a preventative measure, using highly variable practices. Here, we present field-scale research from a building before, during, and after periods of low occupancy, and controlled stagnation experiments. We document no change, a > 4-log increase, and a > 1.5-log decrease of L. pneumophila during 3- to 7-week periods of low water demand. L. pneumophila increased by > 1-log after precautionary flushing prior to reoccupancy, which was repeated in controlled boiler flushing experiments. These results demonstrate that the impact of low water demand (colloquially called stagnation) is not as straight forward as is generally assumed, and that some flushing practices have potential unintended consequences. In particular, stagnation must be considered in context with other Legionella growth factors like temperature and flow profiles. Boiler flushing practices that dramatically increase the flow rate and rapidly deplete boiler temperature may mobilize Legionella present in biofilms and sediment.

Potential probiotic approaches to control Legionella in engineered aquatic ecosystems.

Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven "probiotic" control approach against Legionella. We focus on: (i) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (ii) exploitative and interference competitive interactions between Legionella and other microorganisms; and (iii) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach.

Bacterial colonization of pellet softening reactors used during drinking water treatment.

Pellet softening reactors are used in centralized and decentralized drinking water treatment plants for the removal of calcium (hardness) through chemically induced precipitation of calcite. This is accomplished in fluidized pellet reactors, where a strong base is added to the influent to increase the pH and facilitate the process of precipitation on an added seeding material. Here we describe for the first time the opportunistic bacterial colonization of the calcite pellets in a full-scale pellet softening reactor and the functional contribution of these colonizing bacteria to the overall drinking water treatment process. ATP analysis, advanced microscopy, and community fingerprinting with denaturing gradient gel electrophoretic (DGGE) analysis were used to characterize the biomass on the pellets, while assimilable organic carbon (AOC), dissolved organic carbon, and flow cytometric analysis were used to characterize the impact of the biological processes on drinking water quality. The data revealed pellet colonization at concentrations in excess of 500 ng of ATP/g of pellet and reactor biomass concentrations as high as 220 mg of ATP/m(3) of reactor, comprising a wide variety of different microorganisms. These organisms removed as much as 60% of AOC from the water during treatment, thus contributing toward the biological stabilization of the drinking water. Notably, only a small fraction (about 60,000 cells/ml) of the bacteria in the reactors was released into the effluent under normal conditions, while the majority of the bacteria colonizing the pellets were captured in the calcite structures of the pellets and were removed as a reusable product.

Stagnation leads to short-term fluctuations in the effluent water quality of biofilters: A problem for greywater reuse?

A key characteristic of decentralized greywater treatment and reuse is high variability in both nutrient concentrations and flow. This variability in flow leads to stagnant water in the system and causes short-term fluctuations in the effluent water quality. Automated monitoring tools provide data to understand the mechanisms underlying the dynamics and to adapt control strategies accordingly. We investigated the fluctuations in a building-scale greywater treatment system comprising a membrane bioreactor followed by a biological activated carbon filter. Short-term dynamics in the effluent of the biological activated carbon filter were monitored with automated flow cytometry and turbidity, and the impact of these fluctuations on various hygiene-relevant parameters in the reuse water was evaluated. Continuous biofilm detachment into the stagnant water in the biological activated carbon filter led to temporarily increased turbidity and cell concentrations in the effluent after periods of stagnation. The fluctuations in cell concentrations were consistent with a model assuming higher detachment rates during flow than during times with stagnant water. For this system, total cell concentration and turbidity were strongly correlated. We also showed that the observed increase in cell concentration was not related to either an increase of organic carbon concentration or the concentration of two opportunistic pathogens, P. aeruginosa and L. pneumophila. Our findings demonstrate that turbidity measurements are sensitive to changes in the effluent water quality and can be used to monitor the fluctuations caused by intermittent flow. Intermittent flow did not lead to an increase in opportunistic pathogens, and this study provides no indications that stagnant water in biological activated carbon filters need be prevented.

The value of human data annotation for machine learning based anomaly detection in environmental systems.

Anomaly detection is the process of identifying unexpected data samples in datasets. Automated anomaly detection is either performed using supervised machine learning models, which require a labelled dataset for their calibration, or unsupervised models, which do not require labels. While academic research has produced a vast array of tools and machine learning models for automated anomaly detection, the research community focused on environmental systems still lacks a comparative analysis that is simultaneously comprehensive, objective, and systematic. This knowledge gap is addressed for the first time in this study, where 15 different supervised and unsupervised anomaly detection models are evaluated on 5 different environmental datasets from engineered and natural aquatic systems. To this end, anomaly detection performance, labelling efforts, as well as the impact of model and algorithm tuning are taken into account. As a result, our analysis reveals the relative strengths and weaknesses of the different approaches in an objective manner without bias for any particular paradigm in machine learning. Most importantly, our results show that expert-based data annotation is extremely valuable for anomaly detection based on machine learning.

Evaluating the growth potential of pathogenic bacteria in water.

The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.

Critical evaluation of the volumetric "bottle effect" on microbial batch growth.

We have analyzed the impact of surface-to-volume ratio on final bacterial concentrations after batch growth. We examined six bottle sizes (20 to 1,000 ml) using three independent enumeration methods to quantify growth. We found no evidence of a so-called volumetric bottle effect, thus contradicting numerous previous reports.

Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications.

Rapid detection of microbial cells is a challenge in microbiology, particularly when complex indigenous communities or subpopulations varying in viability, activity and physiological state are investigated. Flow cytometry (FCM) has developed during the last 30 years into a multidisciplinary technique for analysing bacteria. When used correctly, FCM can provide a broad range of information at the single-cell level, including (but not limited to) total counts, size measurements, nucleic acid content, cell viability and activity, and detection of specific bacterial groups or species. The main advantage of FCM is that it is fast and easy to perform. It is a robust technique, which is adaptable to different types of samples and methods, and has much potential for automation. Hence, numerous FCM applications have emerged in industrial biotechnology, food and pharmaceutical quality control, routine monitoring of drinking water and wastewater systems, and microbial ecological research in soils and natural aquatic habitats. This review focuses on the information that can be gained from the analysis of bacteria in water, highlighting some of the main advantages, pitfalls and applications.

Substrate Pre-loading Influences Initial Colonization of GAC Biofilter Biofilms.

Microbial community composition and stability affect pollutant removal for biological/granular activated carbon (BAC/GAC) processes. Here, we pre-loaded the organic carbon substrates sucrose, lactose, and Lysogeny Broth (LB) medium onto new GAC prior to use and then tested whether this substrate pre-loading promoted development of biofilms with high coverage that remained stable for prolonged operational periods. Temporal dynamics of the biomass and microbial community on the GAC were monitored via flow cytometry (FCM) and sequencing, respectively, in both batch and continuous-flow experiments. In comparison with the non-loaded GAC (control), the initial biofilm biomass on substrate-loaded GAC was 3-114 times higher, but the initial richness was considerably lower (only accounting for 13-28% of the control). The initial community compositions were significantly different between batch and continuous-flow column experiments, even when loaded with the same substrates. In the continuous-flow column experiments, both biomass and microbial community composition became remarkably similar to the control filters after 64 days of operation. From these findings, we conclude that substrate-loaded GAC could enhance initial colonization, affecting both biomass and microbial community composition. However, the biomass and composition did not remain stable during long-term operation due to continuous dispersal and competition from influent bacteria.