Engineered Water Systems Research Articles

Differential Bacterial Predation by Free-Living Amoebae May Result in Blooms of Legionella in Drinking Water Systems.

Intracellular growth of pathogenic Legionella in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how Legionella spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species, Willaertia magna, Acanthamoeba polyphaga, and Vermamoeba vermiformis regarding Legionella pneumophila and two Escherichia coli strains. Although all the studied FLA species supported intracellular growth of L. pneumophila, they avoided this bacterium to a certain degree in the presence of E. coli and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once L. pneumophila were intracellular, it inhibited digestion of co-occurring E. coli within the same trophozoites. Altogether, based on FLA–bacteria interactions and the shifts in microbial population dynamics, we propose that FLA’s feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of Legionella and creates a “forced-feeding” condition facilitating the internalization of Legionella into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of Legionella.

Sialic Acids: An Important Family of Carbohydrates Overlooked in Environmental Biofilms

Sialic acids in the structural matrix of biofilms developing in engineered water systems constitute a potential target in the battle against biofouling. This report focuses specifically on the presence of sialic acids as part of the extracellular polymeric substances (EPS) of biofilms forming in cooling towers and the potential effect of nutrient starvation on sialic acid presence and abundance. Two cooling water compositions were compared in parallel pilot-scale cooling towers, one poor in nutrients and one enriched in nutrients. Fresh deposits from the two cooling towers were collected after a five-week operation period. EPS extractions and analyses by Fourier transform infrared spectroscopy (FTIR) and high-resolution mass spectrometry (MS), along with 16S rRNA gene amplicon sequencing were performed. The results of MS analyses showed the presence of pseudaminic/legionaminic acids (Pse/Leg) and 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) in both biofilm EPS samples. FTIR measurements showed the characteristic vibration of sialic acid-like compounds ν(C=O)OH in the nutrient poor sample exclusively. Our findings, combined with other recent studies, suggest that bacterial sialic acids are common compounds in environmental biofilms. Additionally, the conservation of sialic acid production pathways under nutrient starvation highlights their importance as constituents of the EPS. Further in-depth studies are necessary to understand the role of sialic acids in the structural cohesion and protection of environmental biofilm layer.

Role of amoebae for survival and recovery of ‘non-culturable’ Helicobacter pylori cells in aquatic environments

ABSTRACT Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

Interfacial and Activation Energies of Environmentally Abundant Heterogeneously Nucleated Iron(III) (Hydr)oxide on Quartz.

Poorly crystalline iron(III) (hydr)oxide nanoparticles are ubiquitous in environmental systems and play a crucial role in controlling the fate and transport of contaminants. Yet, the thermodynamic and kinetic parameters, e.g., the effective interfacial (α') and apparent activation (Ea) energies, of iron(III) (hydr)oxide nucleation on earth-abundant mineral surfaces have not been determined, which hinders an accurate prediction of iron(III) (hydr)oxide formation and its interactions with other toxic or reactive ions. Here, for the first time, we report experimentally obtained α' and Ea for iron(III) (hydr)oxide nucleation on quartz mineral surfaces by employing a flow-through, time-resolved grazing incidence small-angle X-ray scattering (GISAXS). GISAXS enabled the in situ detection of iron(III) (hydr)oxide nucleation rates under different supersaturations (σ, achieved by varying pH 3.3-3.6) and temperatures (12-35 °C). By quantitative analyses based on classical nucleation theory, α' was obtained to be 34.6 mJ/m2 and Ea was quantified as 32.8 kJ/mol. The fundamental thermodynamic and kinetic parameters obtained here will advance our fundamental understanding of the surface chemistry and nucleation behavior of iron(III) (hydr)oxides in subsurface and water treatment systems as well as their effects on the fate and transport of pollutants in natural and engineered water systems. The in situ flow-through GISAXS method can also be adapted to quantify thermodynamic and kinetic parameters at interfaces for many important solid-liquid environmental systems.

Mesoporous activated carbon shows superior adsorption affinity for 11-nor-9-carboxy-\u03949-tetrahydrocannabinol in water

Increasing cannabinoid use with the incipient favorable public discourse raises concerns about their environmental release and potential impacts. Concentration of common cannabinoids and their metabolites (e.g., THC and THC-COOH) are already detected in source waters and engineered water systems across the globe with a rising trajectory. This study examines the adsorption of THC and THC-COOH onto activated carbons in surface water-relevant concentrations and investigates the effect of carbon’s physicochemical properties. At higher equilibrium concentration (i.e., Ce > 300 µg/L), adsorption of cannabinoids correlates with the sorbent-specific surface area. On the contrary, at lower concentrations (i.e., Ce = 0.1–100 µg/L), cannabinoid adsorption was predominantly controlled by pore size of the carbon. More specifically, when the surface area was not limited (i.e., dilution conditions), cannabinoid molecules appeared to have preferably attached within the mesopores. The adsorption mechanism deciphered in the study will facilitate in selection of commercial activated carbon to remove cannabinoids in drinking water treatment systems. This work will also provide a baseline for further research on removal of other cannabinoids (e.g., CBD), other structurally relevant drugs, their metabolites, and reaction byproduct from engineered treatment systems.

Assessing the chemical compositions and disinfection byproduct formation of biofilms: Application of fluorescence excitation-emission spectroscopy coupled with parallel factor analysis.

There are increased concerns over the contributions of biofilms to disinfection byproduct (DBP) formation in engineered water systems (EWS). However, monitoring the biomolecular characteristics of biofilms to understand their impacts on DBP formation has been a great challenge as it requires complex analytical techniques. This study aimed to examine the applicability of fluorescence excitation-emission matrices (EEMs) coupled with parallel factor analysis (PARAFAC) to assess the chemical compositions and DBP formation of biofilms. Biofilms were collected from reactors grown on R2A media, as well as two drinking water-related organic substrates such as humic substances and algal organic matter. The chemical composition and formation of carbonaceous and nitrogenous DBPs of biofilms were continuously monitored every 21 days for 168 days and correlated with the derived EEM-PARAFAC components. Results indicated that all biofilm samples comprised mostly of protein-like components (∼90%), and to a lesser extent, humic-like components (∼10%). Strong correlations were generally found between tryptophan-like substances and the studied DBP formation (R2min ≥ 0.76, P<0.05), indicating that they play a major role in producing biofilm-derived DBPs upon chlorination. Moreover, significant discrepancies between the chemical compositions and DBP formation of biofilms and their corresponding feed solutions were observed, likely due to biotransformation and biosorption processes. Overall, this work highlights that EEM-PARAFAC analysis is a promising tool to monitor the biomolecular characteristics of biofilm components and to predict the subsequent DBP formation in optimizing disinfection protocols for EWS.

Nontuberculous Mycobacteria Infection: Source and Treatment

This review offers current understanding of the source of nontuberculous mycobacteria (NTM) infections especially in relation to engineered water systems. It provides a summary of current treatment methods and highlights novel treatment options being developed. Nontuberculous mycobacteria (NTM) infections are thought to originate from inhalation or aspiration of NTM from contaminated drinking water. While significant NTM metagenomics analysis and culturing of environmental samples have been performed, the bias of culture independent and dependent techniques along with the ubiquity of NTM organisms in the environment has made connecting the pathogen reservoir and route of transmission difficult. Recent advances in NTM DNA extraction protocols and NTM high-throughput sequencing methods suggest inhalation and aspiration of drinking water remain a critical route of transmission. The development of pulmonary diseases associated with NTM infections is complex. Patients with CF and non-CF bronchiectasis are susceptible to NTM infections, whereas the development of bronchiectasis may be due to long-term exposure to NTM. Source attribution of NTM infections is critical, especially if the source includes engineered water systems such as drinking water distribution networks and clinical and non-clinical premise plumbing. However, source identification requires more work. Current NTM treatment therapies were developed for other indications. New and combined therapies are being developed, but are insufficient to address growing antibiotic resistance and diversity of NTM infections.

Long-term persistence of infectious Legionella with free-living amoebae in drinking water biofilms.

Prolific growth of pathogenic Legionella pneumophila within engineered water systems and premise plumbing, and human exposure to aerosols containing this bacterium results in the leading health burden of any water-related pathogen in developed regions. Ecologically, free-living amoebae (FLA) are an important group of the microbial community that influence biofilm bacterial diversity in the piped-water environment. Using fluorescent microscopy, we studied in-situ the colonization of L. pneumophila in the presence of two water-related FLA species, Willaertia magna and Acanthamoeba polyphaga in drinking water biofilms. During water flow as well as after periods of long-stagnation, the attachment and colonization of L. pneumophila to predeveloped water-biofilm was limited. Furthermore, W. magna and A. polyphaga showed no immediate interactions with L. pneumophila when introduced to the same natural biofilm environment. A. polyphaga encysted within 5-7 d after introduction to the tap-water biofilms and mostly persisted in cysts till the end of the study period (850 d). W. magna trophozoites, however, exhibited a time delay in feeding on Legionella and were observed with internalized L. pneumophila cells after 3 weeks from their introduction to the end of the study period and supported putative (yet limited) intracellular growth. The culturable L.pneumophila in the bulk water was reduced by 2-log over 2 years at room temperature but increased (without a change in mip gene copies by qPCR) when the temperature was elevated to 40 °C within the same closed-loop tap-water system without the addition of nutrients or fresh water. The overall results suggest that L. pneumophila maintains an ecological balance with FLA within the biofilm environment, and higher temperature improve the viability of L. pneumophila cells, and intracellular growth of Legionella is possibly cell-concentration dependent. Observing the preferential feeding behavior, we hypothesize that an initial increase of FLA numbers through feeding on a range of other available bacteria could lead to an enrichment of L. pneumophila, and later force predation of Legionella by the amoeba trophozoites results in rapid intracellular replication, leading to problematic concentration of L. pneumophila in water. In order to find sustainable control options for legionellae and various other saprozoic, amoeba-resisting bacterial pathogens, this work emphasizes the need for better understanding of the FLA feeding behavior and the range of ecological interactions impacting microbial population dynamics within engineered water systems.

Viability and infectivity of viable but nonculturable Legionella pneumophila strains induced at high temperatures

Thermal disinfection is commonly used to prevent the proliferation of culturable Legionella in engineered water systems (EWS). In response to such stress, culturable Legionella populations can switch into a viable but nonculturable (VBNC) state. The importance of such VBNC Legionella cells is currently hotly debated. Here, we investigated the stress response patterns and transitions of the bacteria to the VBNC state at 55 °C, 60 °C and 70 °C on two L. pneumophila strains for >80 days using a combination of cell-based viability indicators. Complete loss of culturability at 55 °C, 60 °C and 70 °C occurred after 3–8 h, 60 min and <2 min, respectively. In contrast, L. pneumophila strains required 9 days at 55 °C, 8 h at 60 °C and 20 min at 70 °C to achieve a 2 log reduction in cells with intact membranes and high esterase activity; a 4 log reduction was achieved only after 150, 8–15 and 1–4 days, respectively. In parallel, the presence of diagnostic outer-membrane epitopes (OMEs) and changes in the infectivity patterns of the two strains towards amoebae and THP-1 cells were assessed. OMEs were more persistent than viability indicators, showing their potential as targets for VBNC Legionella detection. L. pneumophila strains infected amoebae and THP-1 cells for at least 85 days at 55 °C and 60 °C and for up to 8 days at 70 °C. However, they did so with reduced efficiency, requiring prolonged co-incubation times with the hosts and higher Legionella cell numbers in comparison to culturable cells. Consequently, infection of amoebae by thermally induced VBNC L. pneumophila with lowered virulence can be expected in EWS. Although the gold standard method cannot detect VBNC Legionella, it provides important information about the most virulent bacterial subpopulations. Our results indicate that a prolonged thermal regime ≥60 °C at the central parts of warm water systems is not only effective against culturable L. pneumophila but in the long run even against VBNC cells.

Validation and in-field testing of a new on-site qPCR system for quantification of Legionella pneumophila according to ISO/TS 12869:2012 in HVAC cooling towers.

Legionella pneumophila, found in engineered water systems such as HVAC cooling towers, poses a significant public health risk. Culture, though routinely used to quantify L. pneumophila, has several disadvantages including long turnaround time, low sensitivity, and inter-laboratory variability. In this study, we validated the performance of an on-site quantitative polymerase chain reaction (qPCR) detection system for L. pneumophila in accordance with International Standards Organization Technical Specification 12869:2012. We evaluated specificity, limit of detection and quantification, and calibration curve linearity. Additionally, we evaluated whole system recovery and robustness using samples taken from taps and evaporative cooling towers. We then compared the system's performance against laboratory culture and laboratory qPCR across 53 cooling towers in a 12-week in-field study. We found that concordance between on-site qPCR and culture was both laboratory- and site/sample-dependent. Comparison of laboratory qPCR with on-site qPCR revealed that laboratory results were highly variable and showed little concordance. Some discordance may be explained by time delay between sample collection and testing ('shipping effect') which may lead to inaccurate reporting. Overall, our study highlights the value of on-site qPCR detection of L. pneumophila, demonstrates that laboratories are prone to misreporting results due to shipping effects, and reveals significant discordance between laboratory qPCR and culture.

Exploiting the Richness of Environmental Waterborne Bacterial Species to Find Natural Legionella pneumophila Competitors.

Legionella pneumophila is one of the most tracked waterborne pathogens and remains an important threat to human health. Despite the use of biocides, L. pneumophila is able to persist in engineered water systems with the help of multispecies biofilms and phagocytic protists. For few years now, high-throughput sequencing methods have enabled a better understanding of microbial communities in freshwater environments. Those unexplored and complex communities compete for nutrients using antagonistic molecules as war weapons. Up to now, few of these molecules were characterized in regards of L. pneumophila sensitivity. In this context, we established, from five freshwater environments, a vast collection of culturable bacteria and investigated their ability to inhibit the growth of L. pneumophila. All bacterial isolates were classified within 4 phyla, namely Proteobacteria (179/273), Bacteroidetes (48/273), Firmicutes (43/273), and Actinobacteria (3/273) according to 16S rRNA coding sequences. Aeromonas, Bacillus, Flavobacterium, and Pseudomonas were the most abundant genera (154/273). Among the 273 isolates, 178 (65.2%) were shown to be active against L. pneumophila including 137 isolates of the four previously cited main genera. Additionally, other less represented genera depicted anti-Legionella activity such as Acinetobacter, Kluyvera, Rahnella, or Sphingobacterium. Furthermore, various inhibition diameters were observed among active isolates, ranging from 0.4 to 9 cm. Such variability suggests the presence of numerous and diverse natural compounds in the microenvironment of L. pneumophila. These molecules include both diffusible secreted compounds and volatile organic compounds, the latter being mainly produced by Pseudomonas strains. Altogether, this work sheds light on unexplored freshwater bacterial communities that could be relevant for the biological control of L. pneumophila in manmade water systems.

Water Distribution System Failure Risks with Increasing Temperatures.

In the coming decades, ambient temperature increase from climate change threatens to reduce not only the availability of water but also the operational reliability of engineered water systems. Relatively little is known about how temperature stress can increasingly cause hardware components to fail, quality to be affected, and service outages to occur. Changes to the estimated-time-to-failure of major water system hardware and the probability of quality noncompliance were estimated for a modern potable water system that experiences hot summer temperatures, similar to Phoenix, AZ, and Las Vegas, NV. A fault tree model was developed to estimate the probability that consequential service outages in quantity and quality will occur. Component failures are projected to have a percent increase of 10-101% in scenarios where peak summer temperature has increased from 36 to 44 °C, which create the conditions for service outages to have a percent increase of 7-91%. Increased service outages due to multiple pumping unit failures and water quality noncompliances are the most notable concerns for water utilities. The most effective strategies to prevent temperature-related failures should focus on monitoring and correcting chlorine residual and disinfection byproduct concentration, and on cooling pumping unit motors and electronics.

Persistence of infectious Enterovirus within free-living amoebae – A novel waterborne risk pathway?

Free-living amoebae (FLA) are phagocytic protozoa found in natural and engineered water systems. They can form disinfectant-resistant cysts, which can harbor various human pathogenic bacteria, therefore providing them with a means of environmental persistence and dispersion through water distribution and other engineered water systems. The association of FLA with human viruses has been raised, but the limited data on the persistence of infectious virions within amoebae leaves this aspect unresolved. Enteroviruses can cause a wide range of illness and replicate in human respiratory and gastrointestinal tracts, both of which could be exposed through contact with contaminated waters if virus detection and removal are compromised by virion internalization in free-living protozoa. This is especially problematic for high-risk contaminants, such as coxsackieviruses, representative members of the Enterovirus genus that are likely infectious at low doses and cause a variety of symptoms to a vulnerable portion of the population (particularly infants). To investigate Enterovirus persistence within free-living amoebae we co-cultured an infectious clinical coxsackievirus B5 (CVB5) isolate, with the commonly reported tap water amoeba Vermamoeba vermiformis, after which we tracked virus localization and persistence in co-culture over time through a combination of advanced imaging, molecular and cell culture assays. Our results clearly demonstrate that infectious CVB5 can persist in all life stages of the amoebae without causing any visible injury to them. We also demonstrated that the amoeba generated vesicles containing virions that were expelled into the bulk liquid surroundings, a finding previously described for FLA-bacteria interactions, but not for FLA and human pathogenic viruses. Therefore, our findings suggest that the ability of CVB5 to persist in V. vermiformis could be a novel waterborne risk pathway for the persistence and dispersion of infectious human enteric viruses through water systems.

Differential development of Legionella sub-populations during short- and long-term starvation

Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5–69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03–55%, and the concentration of the highly esterase-active sub-population dropped to 0.01–1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.

Quantification of viable and non-viable Legionella spp. by heterogeneous asymmetric recombinase polymerase amplification (haRPA) on a flow-based chemiluminescence microarray

Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp. as well as Legionella pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase polymerase amplification (haRPA) was carried out on flow-based CL DNA microarrays. Detection limits of 87 genomic units (GU) µL−1 and 26GUµL−1 for Legionella spp. and Legionella pneumophila, respectively, were achieved. In this work, it was shown for the first time that the combination of a propidium monoazide (PMA) treatment with haRPA, the so-called viability haRPA, is able to identify viable Legionella on DNA microarrays. Different proportions of viable and non-viable Legionella, shown with the example of L. pneumophila, ranging in a total concentration between 101 to 105GUµL−1 were analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella spp. were found between 81% and 133%. With the combination of these two methods, there is a chance to replace culture-based methods in the future for the monitoring of engineered water systems like condensation recooling plants.

High Prevalence and Genetic Polymorphisms of Legionella in Natural and Man-Made Aquatic Environments in Wenzhou, China.

Natural and engineered water systems are the main sources of Legionnaires’ disease. It is essential from a public health perspective to survey water environments for the existence of Legionella. To analyze the main serogroups, genotypes and pathogenicity of the pathogen, a stratified sampling method was adopted to collect water samples randomly from shower water, cooling tower water, and local public hot springs in Wenzhou, China. Suspected strains were isolated from concentrated water samples. Serum agglutination assay and real-time PCR (Polymerase chain reaction) were used to identify L. pneumophila. Sequence-based typing (SBT) and pulsed-field gel electrophoresis (PFGE) were used to elucidate the genetic polymorphisms in the collected isolates. The intracellular growth ability of the isolates was determined through their interaction with J774 cells and plating them onto BCYE (Buffered Charcoal Yeast Extract) agar plates. Overall, 25.56% (46/180) of water samples were Legionella-positive; fifty-two strains were isolated and two kinds of serogroups were co-detected from six water samples from 2015 to 2016. Bacterial concentrations ranged from 20 CFU/100 mL to 10,720 CFU/100 mL. In detail, the Legionella-positive rates of shower water, cooling tower water and hot springs water were 15.45%, 13.33%, and 62.5%, respectively. The main serogroups were LP1 (30.69%) and LP3 (28.85%) and all strains carried the dot gene. Among them, 52 isolates and another 10 former isolates were analyzed by PFGE. Nineteen distinct patterns were observed in 52 strains isolated from 2015 to 2016 with three patterns being observed in 10 strains isolated from 2009 to 2014. Seventy-three strains containing 52 from this study and 21 former isolates were selected for SBT analysis and divided into 25 different sequence types in 4 main clonal groups belonging to 4 homomorphic types. Ten strains were chosen to show their abilities to grow and multiply in J744 cells. Taken together, our results demonstrate a high prevalence and genetic polymorphism of Legionella in Wenzhou’s environmental water system. The investigated environmental water sources pose a potential threat to the public where intervention could help to prevent the occurrence of Legionnaires’ disease.

Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems.

NO dismutation into N2 and O2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium "Ca Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.

Evaluation of timing of re-appearance of VBNC Legionella for risk assessment in hospital water distribution systems.

In this study we estimated the presence of Legionella species, viable but non-culturable (VBNC), in hospital water networks. We also evaluated the time and load of Legionella appearance in samples found negative using the standard culture method. A total of 42 samples was obtained from the tap water of five hospital buildings. The samples were tested for Legionella by the standard culture method and were monitored for up to 12 months for the appearance of VBNC Legionella. All the 42 samples were negative at the time of collection. Seven of the 42 samples (17.0%) became positive for Legionella at different times of monitoring. The time to the appearance of VBNC Legionella was extremely variable, from 15 days to 9 months from sampling. The most frequent Legionella species observed were Legionella spp and L. anisa and only in one sample L. pneumophila srg.1. Our study confirms the presence of VBNC Legionella in samples resulting negative using the standard culture method and highlights the different time to its appearance that can occur several months after sampling. The results are important for risk assessment and risk management of engineered water systems.

English

Legionnaires disease can be acquired through exposure to Legionella pneumophila, a gram-negative bacteria ubiquitous in both natural and engineered water systems. Over the years, a number of disinfection techniques notably, chlorination, ozonation, thermal, UV and copper-silver ionization have been employed across different kinds of engineered water systems with diverse measures of success. Available evidence portends, most of the aforementioned techniques often have to be combined to achieve long-term efficacy. Remarkably, albeit the extensive research and reportage on Legionnaires outbreak in the developed world, very few studies have been carried out with regards to Africa. We reviewed existing literature on the application of the aforementioned techniques in buildings. Our study concurs with earlier studies; most of the disinfection techniques will have to be combined to achieve the desired efficacy. We found very scanty studies on Legionella or reportage of its outbreak within Africa. Our study also found very little in terms of any of the techniques been applied with the specific aim of reducing Legionella proliferation in engineered water systems within Africa. This is alarming, especially, on a continent where several communities have little or no access to quality water and healthcare. In light of the above, stronger measures such as sensitization, properly managed water distribution systems, as well as policies aimed at enforcing national and international guidelines on Legionella control is recommended. Key words: Legionnaires disease, water disinfection, engineered water system, Africa.

The response of aggregated Pseudomonas putida CP1 cells to UV-C and UV-A/B disinfection.

UV radiation is a spread method used worldwide for the disinfection of water. However, much of the research on the disinfection of bacterial cells by UV has focused on planktonic cells. Many bacterial cells in nature are present in clumps or aggregates, and these aggregates, which are more resistant to disinfection than their planktonic counterparts, can be problematic in engineered water systems. The current research used Pseudomonas putida (P. putida) CP1, an environmental and non-pathogenic microorganism which autoaggregates when grown under certain conditions, as a model organism to simulate aggregated cells. The study investigated the response of both the planktonic and the aggregated forms of the bacterium to UV-C (λ=253.7nm) and UV-A/B (λ>300nm) disinfection at laboratory scale in a minimal medium. The planktonic cells of P. putida CP1 were inactivated within 60s by UV-C and in 60min by UV-A/B; however, the aggregated cells required 120min of UV-C treatment and 240min of UV-A/B radiation to become inactive. The size of the aggregate was reduced following UV treatment. Although all the cells had lost culturability, viability as measured by the LIVE/DEAD® stain and epifluorescence microscopy was not completely lost and the cells all demonstrated regrowth after overnight incubation in the dark.