Browsing by Author "Nocker, Andreas"
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Item Open Access The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure(Springer, 2018-03-15) Ameh, Ekwu M.; Tyrrel, Sean; Harris, Jim A.; Ignatiou, Athanasios; Orlova, Elena; Nocker, AndreasDisinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication.Item Open Access Characterisation and control of the biosolids storage environment: Implications for E. coli dynamics(Elsevier, 2020-08-15) Fane, Sarah Elizabeth; Nocker, Andreas; Vale, Peter C. J.; Rivas Casado, Monica; Cartmell, Elise; Harris, Jim A.; Bajón Fernández, Yadira; Tyrrel, Sean F.E. coli survival in biosolids storage may present a risk of non-compliance with guidelines designed to ensure a quality product safe for agricultural use. The storage environment may affect E. coli survival but presently, storage characteristics are not well profiled. Typically biosolids storage environments are not actively controlled or monitored to support increased product quality or improved microbial compliance. This two-phased study aimed to identify the environmental factors that control bacterial concentrations through a long term, controlled monitoring study (phase 1) and a field-scale demonstration trial modifying precursors to bacterial growth (phase 2). Digested and dewatered biosolids were stored in operational-scale stockpiles to elucidate factors controlling E. coli dynamics. E. coli concentrations, stockpile dry solids, temperature, redox and ambient weather data were monitored. Results from ANCOVA analysis showed statistically significant (p < 0.05) E. coli reductions across storage periods with greater die-off in summer months. Stockpile temperature had a statistically significant effect on E. coli survival. A 4.5 Log reduction was measured in summer (maximum temperature 31 °C). In the phase 2 modification trials, covered stockpiles were able to maintain a temperature >25 °C for a 28 day period and achieved a 3.7 Log E. coli reduction. In winter months E. coli suppression was limited with concentrations >6 Log10 CFU g−1 DS maintained. The ANCOVA analysis has identified the significant role that physical environmental factors, such as stockpile temperature, has on E. coli dynamics and the opportunities for controlItem Open Access Chlorine disinfection of drinking water assessed by flow cytometry: new insights(Elsevier, 2020-07-10) Cheswick, Ryan; Moore, Graeme; Nocker, Andreas; Hassard, Francis; Jefferson, Bruce; Jarvis, PeterThe efficacy of chlorine disinfection was assessed for the first time over a range of disinfection conditions using flow cytometry (FCM) to provide new insights into disinfection processes. Inactivation was assessed for pure culture bacteria (Escherichia coli) and micro-organisms in real treated water from operational water treatment works (WTWs). A dose dependent increase in inactivation rate (k) was observed for both test matrices, with values of 0.03 to 0.26 and 0.32 to 3.14 L/mg min for the WTW bacteria and E. coli, respectively. After 2 min, E. coli was reduced by 2 log for all chlorine doses (0.12 to 1.00 mg/L). In the case of the WTW filtrate bacteria, after 2 min log reductions were between 0.54 and 1.14 with increasing chlorine concentration, reaching between 1.32 and 2.33 after 30 min. A decrease in disinfection efficacy was observed as temperature decreased from 19 to 5 °C for both microbial populations. With respect to chlorination at different pH (pH 6, 7, 8), membrane damage was more pronounced at higher pH. This was not consistent with the higher disinfection efficacy seen at lower pH. when culture based methods are used to assess bacterial reductions. This provides evidence that more understanding into the fundamental mechanisms of chlorine disinfection are required and that methodological alterations may be required (e.g. pH standardisation) to fully utilise FCM over the entire range of chlorination conditions observed in operational environmentsItem Open Access Comparing flow cytometry with culture-based methods for microbial monitoring and as a diagnostic tool for assessing drinking water treatment processes(Elsevier, 2019-06-18) Cheswick, Ryan Ashley; Cartmell, Elise; Lee, Susan; Upton, Andrew; Weir, Paul; Moore, Graeme; Nocker, Andreas; Jefferson, Bruce; Jarvis, PeterFlow cytometry (FCM) and the ability to measure both total and intact cell populations through DNA staining methodologies has rapidly gained attention and consideration across the water sector in the past decade. In this study, water quality monitoring was undertaken over three years across 213 drinking water treatment works (WTW) in the Scottish Water region (Total n = 39,340). Samples subject to routine regulatory microbial analysis using culture-based methods were also analysed using FCM. In addition to final treated water, the bacterial content in raw water was measured over a one-year period. Three WTW were studied in further detail using on-site inter-stage sampling and analysis with FCM. It was demonstrated that there was no clear link between FCM data and the coliform samples taken for regulatory monitoring. The disinfectant Ct value (Ct = mg·min/L) was the driving factor in determining final water cell viability and the proportion of intact cells (intact/total cells) and the frequency of coliform detections in the water leaving the WTW. However, the free chlorine residual, without consideration of treatment time, was shown to have little impact on coliform detections or cell counts. Amongst the three treatment trains monitored in detail, the membrane filtration WTW showed the greatest log removal and robustness in terms of final water intact cell counts. Flow cytometry was shown to provide insights into the bacteriological quality of water that adds significant value over and above that provided by traditional bacterial monitoring.Item Open Access Disruption of cells in biosolids affects E. coli dynamics in storage(IWA Publishing, 2019-05-22) Fane, Sarah Elizabeth; Madureira, D.; Nocker, Andreas; Vale, Peter C. J.; Rivas Casado, Monica; Wilson, A.; Bajón Fernández, Yadira; Harris, Jim A.; Cartmell, Elise; Tyrrel, SeanAchieving microbial compliance during biosolids storage can be complicated by the unpredictable increase of Escherichia coli. Thermal treatment during anaerobic digestion (AD) and the effects of dewatering may be a significant factor contributing to indicator survival. Shear forces present during dewatering may promote cell damage, releasing nutrient for E. coli growth. The effect of cell damage on E. coli survival was assessed in laboratory-scale thermal and physical disruption experiments. E. coli growth curves for disrupted treatments were compared with control conditions and quantified using flow cytometry and membrane filtration techniques. A significant difference (p < 0.05) in the level of damaged cells between control and disrupted conditions was observed. For thermal and physical disruption treatments, the peak of E. coli concentration increased significantly by 1.8 Log and 2.4 Log (CFU (colony forming units) g−1 DS), respectively, compared with control treatments. Research findings contribute to the understanding of bacterial growth and death dynamics in biosolidsItem Open Access Effect of temperature on bacteriophage-mediated lysis efficiency with a special emphasis on bacterial temperature history(University of Mohammed Premier, Oujda, Morocco, 2022-09-30) Ameh, E. M.; Nocker, Andreas; Tyrrell, Sean; Harris, Jim A.; Orlova, E. V.; Ignatiou, A.Despite the great potential of phages as biocontrol agents, there is much uncertainty about the environmental factors influencing lysis efficiency. In this study we investigated the effect of temperature using three distinct lytic E. coli phages that were isolated from a single environmental water sample. All three were identified as dsDNA phages belonging to the Myoviridae family. Whereas the optimal growth temperature of E. coli is well known to be 37 ˚C and exposure of phages (prior to mixing with bacteria) to temperatures between 4 and 37˚C did not affect their infectivity, plaque sizes and numbers greatly decreased with increasing incubation temperature (20˚C, 30˚C, 37˚C) of the phage-host mix. At 37˚C, no visible plaques were observed. Results suggest that temperature sensitivity of the phage-host interaction is distinct from the temperature susceptibility of the two players and corroborate previous reports that highest lysis rates are obtained at temperatures approximate with ambient conditions of the phage environment. Infectivity was however found not only to depend on the incubation temperature of the phage-host mix, but also on the bacterial temperature history. Moreover, exposure of bacteria to heat stress prior to phage challenge resulted in a phage-resistant phenotype raising the question whether bacterial pathogens shed from warm-blooded hosts might be less susceptible to phages adapted to environmental temperature conditions.Item Open Access Effect of turbidity on water disinfection by chlorination with the emphasis on humic acids and chalk(Taylor and Francis, 2019-03-21) Léziar, Tangui; Dutheil de la Rochere, Pierre-Marie; Cheswick, Ryan; Jarvis, Peter; Nocker, AndreasChlorine is globally the most widely used chemical for water disinfection. Whereas disinfection efficiency is well known to depend on water pH and temperature, the effect of turbidity is less well studied. Although turbidity is measured online in most drinking water works and most countries where regulations exist have set limits of <1 NTU for water leaving the works, the composition of turbidity is typically unknown. Given the heterogeneous nature of substances contributing to turbidity, the aim of this work was to study the effect of selected compounds on chlorination efficacy. The effect of humic acids and chalk on the inactivation of the indicator bacteria Escherichia coli and Enterococcus faecalis was assessed at neutral pH at different turbidity levels using both plate counting and flow cytometry in combination with membrane integrity staining. For humic acids, a turbidity of 1 NTU (corresponding to 2 mg L−1) was identified as a critical threshold, which when exceeded was found to have a negative impact on chlorine disinfection. Chalk, on the other hand, had no measurable impact up to 5 NTU. The observation applied to both bacterial species with identical conclusions from the two diagnostic methods. Results corroborate that different turbidity causing substances affect chlorination efficiency to very different extents with chlorine demand by organic material probably being the most important determinant. In the case of turbidities >1 NTU, turbidity measurement benefits from the consideration of the organic content as mere NTU values do not allow predicting an impact on chlorination efficiency.Item Open Access Evaluation of engineered nanoparticle toxic effect on wastewater microorganisms: current status and challenges(Elsevier, 2013-06-14) Eduok, Samuel; Martin, R.; Villa, Raffaella; Nocker, Andreas; Jefferson, Bruce; Coulon, FredericThe use of engineered nanoparticles (ENPs) in a wide range of products is associated with an increased concern for environmental safety due to their potential toxicological and adverse effects. ENPs exert antimicrobial properties through different mechanisms such as the formation of reactive oxygen species, disruption of physiological and metabolic processes. Although there are little empirical evidences on environmental fate and transport of ENPs, biosolids in wastewater most likely would be a sink for ENPs. However, there are still many uncertainties in relation to ENPs impact on the biological processes during wastewater treatment. This review provides an overview of the available data on the plausible effects of ENPs on AS and AD processes, two key biologically relevant environments for understanding ENPs–microbial interactions. It indicates that the impact of ENPs is not fully understood and few evidences suggest that ENPs could augment microbial-mediated processes such as AS and AD. Further to this, wastewater components can enhance or attenuate ENPs effects. Meanwhile it is still difficult to determine effective doses and establish toxicological guidelines, which is in part due to variable wastewater composition and inadequacy of current analytical procedures. Challenges associated with toxicity evaluation and data interpretation highlight areas in need for further research studies.Item Open Access Exploring the use of flow cytometry for understanding the efficacy of disinfection in chlorine contact tanks(Elsevier, 2022-04-06) Cheswick, Ryan; Nocker, Andreas; Moore, Graeme; Jefferson, Bruce; Jarvis, PeterA pilot scale chlorine contact tank (CCT) with flexible baffling was installed at an operational water treatment plant (WTP), taking a direct feed from the outlet of the rapid gravity filters (RGF). For the first time, disinfection efficacy was established by direct microbial monitoring in a continuous reactor using flow cytometry (FCM). Disinfection variables of dose, time, and hydraulic efficiency (short circuiting and dispersion) were explored following characterisation of the reactor's residence time distributions (RTD) by tracer testing. FCM enabled distinction to be made between changes in disinfection reactor design where standard culture-based methods could not. The product of chlorine concentration (C) and residence time (t) correlated well with inactivation of microbes, organisms, with the highest cell reductions (N/N0) reaching <0.025 at Ctx of 20 mg.min/L and above. The influence of reactor geometry on disinfection was best shown from the Ct10. This identified that the initial level of microbial inactivation was higher in unbaffled reactors for low Ct10 values, although the highest levels of inactivation of 0.015 could only be achieved in the baffled reactors, because these conditions enabled the highest Ct10 values to be achieved. Increased levels of disinfection were closely associated with increased formation of the trihalomethane disinfection by-products. The results highlight the importance of well-designed and operated CCT. The improved resolution afforded by FCM provides a tool that can dynamically quantify disinfection processes, enabling options for much better process control.Item Open Access Flow cytometry for rapid analysis of microbiological water quality(Cranfield University, 2014-03) Lipphaus, Patrick; Nocker, AndreasItem Open Access Flow cytometry-based evaluation of the bacterial removal efficiency of a blackwater reuse treatment plant and the microbiological changes in the associated non-potable distribution network(Elsevier, 2018-07-26) Whitton, Rachel; Fane, Sarah Elizabeth; Jarvis, Peter; Tupper, Martyn; Raffin, Marie; Coulon, Frederic; Nocker, AndreasThe study evaluated the changes in bacterial numbers across a full-scale membrane bioreactor (MBR) blackwater reuse system. Flow cytometry was used to quantify total and intact bacterial concentrations across the treatment train and during distribution of the recycled water. Membrane passage reduced bacterial numbers by up to 5-log units resulting in coliform-free permeate. A 2-log increase in bacterial cell concentration was subsequently observed after the granular activated carbon unit followed by a reduction in intact cells after chlorination, which corresponds to an overall intact bacteria removal of 3.4-log units. In the distribution network, the proportion of intact cells greatly depended on the free chlorine residual, with decreasing residual enabling regrowth. An initial target of 0.5 mg L−1 free chlorine ensured sufficient suppression of intact cells for up to 14 days (setting the time intervals for system flushes at times of low water usage). Bacterial regrowth was only observed when the free chlorine concentration was below 0.34 mg L−1. Such loss of residual chlorine mainly applied to distant points in the distribution network from the blackwater reuse treatment plant (BRTP). Flushing these network points for 5 min did not substantially reduce cell numbers. At points closer to the BRTP, on the other hand, flushing reduced cell numbers by up to 1.5-log units concomitant with a decreasing proportion of intact cells. Intact cell concentrations did not correlate with DOC, total nitrogen, or soluble reactive phosphate, but it was shown that dead biomass could be efficiently converted into new biomass within seven days.Item Open Access Impact of treated sewage effluent on the microbiology of a small brook using flow cytometry as a diagnostic tool(Springer Verlag, 2016-01-22) Harry, Ibiye S. K.; Ameh, Ekwu; Coulon, Frederic; Nocker, AndreasFlow cytometry was applied to assess the microbiological impact of treated sewage effluent discharge into a small brook carrying surface runoff water. Increases in dissolved organic carbon and soluble reactive phosphorous were accompanied by increases in counts of intact bacteria by up to eightfold. Effluent ingress furthermore resulted in a pronounced shift of bacterial clusters. Whereas brook water upstream of the discharge point was characterised by a bacterial cluster with low nucleic acid (LNA) content, downstream water showed a shift to bacteria with high nucleic acid (HNA) content. Changes in the LNA/HNA ratio were largely maintained along the course of the brook. Results suggest that the LNA/HNA ratio can under certain conditions serve as an indicator of anthropogenic nutrient impact. Measuring impact on this low trophic level might be more sensitive and straightforward than measuring macroindicators. More evidence will however be required to assess the usefulness of LNA/HNA measurements to assess the ecological nutrient status of natural waters and the impact of nutrient pollution.Item Open Access Lysis performance of bacteriophages with different plaque sizes and comparison of lysis kinetics after simultaneous and sequential phage addition(Mary Ann Liebert, 2020-09-16) Ameh, Ekwu M.; Tyrrel, Sean; Harris, Jim A.; Pawlett, Mark; Orlova, Elena V.; Ignatiou, Athanasios; Nocker, AndreasBackground: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.Item Open Access Pollution prevention in wastewater networks: development of a biological early warning device(Cranfield University, 2016-06) Black, Gary; Dotro, Gabriela; Nocker, Andreas; Coulon, FredericA biological early warning system (EWS) was developed to screen wastewater containing nitrification inhibitors and identify nitrifying bacteria activity reduction without relying on absolute values of sensor signals. To do so, numerous sensors were evaluated using a tiered approach to aid the analysis and made it easier to convey the current state of the technology. The research then produced a framework for the development of an EWS and the applicability of sensors to the wastewater matrix. The research identified a need for the development of a strategy and guidance that can help in the prevention and detection of nitrification inhibitors. Initial tests focussed on sewer biofilm N2O emissions, however, despite average nitrification rates of 19.5 g-NH4 + - N.m- 2 .d- 1 the response was unreliable due to inadequate control. To address this, a circulating floating bed biofilm reactor (CFBBR) was designed as a sidestream. The CFBBR biofilm’s toxicity response was compared to the sewer biofilm, a 2850 mg.L- 1 MLSS culture and a 10.5 mg.L- 1 MLSS culture (with equivalent biomass concentration to the CFBBR biofilm). The cultures responded differently with an inhibitory effect scale of Cu2+ > ATU > Ni2+ > Cr6+ for CFBBR biofilm, ATU > Cu2+ > Ni2+ > Cr6+ for 2850 mg L- 1 MLSS, ATU > Ni2+ > Cr6+ > Cu2+ for 10.5 mg.L- 1 MLSS and ATU > Cu2+ > Cr6+ > Ni2+ for sewer biofilm. This was firstly attributed to suspended growth nitrification stimulation by Cu2+ doses up to ~45 mg.L- 1 resulting in a lower inhibitory effect. Secondly, very high Cr6+ and Ni2+ doses were required for biofilm nitrification inhibition, due to diffusion limitations and slow transport through cell membranes. The CFBBR biofilm response to heavy metals was characterised through N2O and CO2 spikes and a post shock emissions recovery period was observed with the trend Ni2+ > Cr6+ > Cu2+ . A 10 minute hydraulic retention time allowed quick detection and steady state nitrification rates of 0.4 g-NH4 + -N.m- 2 .d- 1 despite high organic loading rates. Additionally, a suspended growth based monitor (Nitritox) was assessed as an inlet works toxicity detector. Incorporation of a Nitritox with a CFBBR based sewer monitor offered increased robustness over a CFBBR only system and was shown to be viable system in catchments >200,000 population equivalent. This information is useful to water utilities so that they can plan for and experiment with upset early warning protocols. It is also useful to manufacturers as they can determine product performance needs.Item Open Access Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR.(Elsevier Science B.V., Amsterdam., 2011-05-31T00:00:00Z) Yáñez, M. Adela; Nocker, Andreas; Soria-Soria, Elena; Múrtula, Raquel; Martínez, Lorena; Catalán, VicenteOne of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cellsItem Open Access Turbidity composition and the relationship with microbial attachment and UV inactivation efficacy(Elsevier, 2017-12-27) Farrell, Charlotte; Hassard, Francis; Jefferson, Bruce; Leziart, Tangui; Nocker, Andreas; Javis, PeterTurbidity in water can be caused by a range of different turbidity causing materials (TCM). Here the characteristics and attachment of bacteria to TCMs was assessed and the resultant impact on UV disinfection determined. TCMs represent potential vehicles for bacterial penetration of water treatment barriers, contamination of potable supplies and impact on subsequent human health. The TCMs under investigation were representative of those that may be present in surface and ground waters, both from the source and formed in the treatment process. The TCMs were chalk, Fe (III) hydroxide precipitate, kaolin clay, manganese dioxide and humic acids, at different turbidity levels representative of source waters (0, 0.1, 0.2, 0.4, 1, 2, and 5 NTU). Escherichia coli and Enterococcus faecalis attachment followed the order of Fe(III) > chalk, with little to no attachment seen for MnO2, humic acids and clay. The attachment was postulated to be due to chalk and Fe(III) particles having a more neutral surface charge resulting in elevated aggregation with bacteria compared to other TCMs. The humic acids and Fe(III) were the TCMs which influenced inactivation of E. coli and E. faecalis due to decreasing UV transmittance (UVT) with increasing TCM concentration. The presence of the Fe(III) TCM at 0.2 NTU resulted in the poorest E. coli inactivation, with 2.5 log10 reduction at UV dose of 10 mJ cm− 2 (kd of − 0.23 cm2 mJ− 1) compared to a 3.9 log10 reduction in the absence of TCMs. E. faecalis had a greater resistance to UV irradiation than E. coli for all TCMs. Effective disinfection of drinking water is a priority for ensuring high public health standards. Uniform regulations for turbidity levels for waters pre-disinfection by UV light set by regulators may not always be appropriate and efficacy is dependent on the type, as well as the amount, of turbidity present in the water.Item Open Access The use of bacteriophages as natural biocontrol agents against bacterial pathogens(Cranfield University, 2016-03) Ameh, Ekwu Mark; Nocker, Andreas; Tyrrel, SeanBacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.Item Open Access When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining(Taylor & Francis: STM, Behavioural Science and Public Health Titles, 2016-12-05) Nocker, Andreas; Cheswick, R.; Dutheil de la Rochere, P. M.; Denis, M.; Léziart, T.; Jarvis, PeterFlow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered ‘dead’. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant.