Personal exposure to traffic-related air pollutants and relationships with respiratory symptoms and oxidative stress: A pilot cross-sectional study among urban green space workers.

Exposure to ambient air pollution has been associated with various adverse health effects including respiratory, cardiovascular and neurological diseases. Exposure data for some specific pollutants and settings are however still insufficient and mechanisms underlying negative health outcomes are not fully elucidated. This pilot study aimed to assess individual exposure to three traffic-related air pollutants, black carbon (BC), polycyclic aromatic hydrocarbons (PAHs) and benzene, and the relationship with respiratory and oxidative stress outcomes in a cross-sectional sample of 48 green space workers in Brussels, Belgium. Participants were followed during four consecutive working days in 2016-2017 during which their individual exposure to BC, PAHs, benzene and more generally air pollution was measured using aethalometers, urinary biomarkers (1-hydroxypyrene, 1-naphthol, 2-naphthol, S-phenylmercapturic acid) and questionnaires. Data on respiratory health and oxidative stress were collected using questionnaires and respiratory/urinary biomarkers (exhaled nitric oxide [NO], 8-hydroxydeoxyguanosine [8-OHdG]). Associations between exposure and health outcomes were investigated using comparison tests and linear regression models, after stratification by present-day smoking status. Spatial variation in BC exposure was high, with concentrations varying between 0.26 and 5.69 µg/m3. The highest levels were recorded during transport and, to a lesser extent, in green spaces located in the vicinity of roads with high traffic intensity. Concentrations of PAHs and benzene biomarkers did not systematically exceed the limits of detection. Among smokers, respiratory inflammation increased linearly with exposure to BC measured over the four days of follow-up (ß = 8.73, 95% CI: 4.04, 13.41). Among non-smokers, oxidative stress increased linearly with BC measured on the fourth day (ß = 2.88, 95% CI: 1.52, 4.24). Despite some limitations, this work supports the hypothesis that BC induces respiratory inflammation and oxidative stress. It also highlights the value of this compound as well as exhaled NO and urinary 8-OHdG biomarkers to detect early/mild effects of air pollution.

Respiratory medication sales and urban air pollution in Brussels (2005 to 2011).

BACKGROUND: We investigated the associations between daily sales of respiratory medication and air pollutants in the Brussels-Capital Region between 2005 and 2011. METHODS: We used over-dispersed Poisson Generalized Linear Models to regress daily individual reimbursement data of prescribed asthma and COPD medication from the social security database against each subject's residential exposure to outdoor particulate matter (PM10) or NO2 estimated, by interpolation from monitoring stations. We calculated cumulative risk ratios (RR) and their 95% confidence intervals (CI) for interquartile ranges (IQR) of exposure for different windows of past exposure for the entire population and for seven age groups. RESULTS: Median daily concentrations of PM10 and NO2 were 25µg/m(3) (IQR=17.1) and 38µg/m(3) (IQR=20.5), respectively. PM10 was associated with daily medication sales among individuals aged 13 to 64y. For NO2, significant associations were observed among all age groups except >84y. The highest RR were observed for NO2, among adolescents, including three weeks lags (RR=1.187 95%CI: 1.097-1.285). CONCLUSION: The associations found between temporal changes in exposure to air pollutants and daily sales of respiratory medication in Brussels indicate that urban air pollution contributes to asthma and COPD morbidity in the general population.

Biofouling ecology as a means to better understand membrane biofouling.

Despite more than a decade of worldwide research on membrane fouling in membrane bioreactors, many questions remain to be answered. Biofouling, which is referred to as the unwanted deposition and growth of biofilms, remains the main problem. Due to its complexity, most of the existing anti-biofouling strategies are not completely successful. To unravel this complexity and finally to developed well-adapted control strategies, a microbial-based description of the biofouling development is needed. Therefore, in this review, the biofouling formation will be described as a typical biofilm formation in five steps including the formation of a conditioning film, the bacterial attachment, the production of extracellular polymeric substances, the biofilm maturation, and the bacterial detachment. Moreover, important processes such as hydrodynamics and bacterial communication or quorum sensing will be taken into account. It is finally discussed whether biofouling formation is an active or inactive biofilm process together with suggestion for further research.

Spherical nucleic acid enhanced FO-SPR DNA melting for detection of mutations in Legionella pneumophila.

A home-built fiber optic surface plasmon resonance platform (FO-SPR) was applied to directly screen PCR amplified DNA for mutations. The FO-SPR sensor was used for real-time monitoring of DNA duplex melting during high resolution temperature cycling. The signal of the DNA melting was enhanced by means of gold nanoparticle labels. This FO-SPR genetic assay allowed for detection of single-point mutations (SNP) in less than 20 min. The concept was demonstrated for the analysis of 9 different serogroups of the bacterium Legionella pneumophila, a common human pathogen responsible for atypical pneumonia. FO-SPR allowed us to detect genetic mutations inhibiting PCR, which could lead to amplification bias when molecular diagnostics are applied for L. pneumophila detection. All serogroups were found to display unique melting temperatures, indicating that mutations have accumulated in the target sequence. In a next step, clinical samples of L. pneumophila were analyzed using the FO-SPR sensor. This technology was proven to be reliable for the detection of mutations for those samples that previously displayed ambiguous qPCR quantification results. When these results were benchmarked, FO-SPR results were found to be consistent with Sanger sequencing but not with fluorescence based DNA melting. The presented results convincingly advocate the advantages of FO-SPR as a high resolution and fast genetic screening tool that can compete with the current standard techniques for SNP detection.

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration.

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.

Novel magnetically induced membrane vibration (MMV) for fouling control in membrane bioreactors.

Conventional submerged membrane bioreactors (MBRs) rely on the coarse bubbles aeration to generate shear at the liquid-membrane interface to limit membrane fouling. Unfortunately, it is a very energy consuming method, still often resulting in a rapid decrease of membrane permeability and consequently in higher expenses. In this paper, the feasibility of a novel magnetically induced membrane vibration (MMV) system was studied in a lab-scale MBR treating synthetic wastewater. The effects on membrane fouling of applied electrical power of different operation strategies, of membrane flux and of the presence of multiple membranes on one vibrating engine on membrane fouling were investigated. The filtration performance was evaluated by determining the filtration resistance profiles and critical flux. The results showed clear advantages of the vibrating system over conventional MBR processes by ensuring higher fluxes at lower fouling rates. Intermittent vibration was found a promising strategy for both efficient fouling control and significant energy saving. The optimised MMV system is presumed to lead to significant energy and cost reduction in up-scaled MBR operations.

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods.

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene. Microbial community fingerprints were assessed by automated ribosomal intergenic spacer analysis. The resulting community profiles were analyzed with canonical correspondence analysis. Our results clearly demonstrate that direct DNA extraction methods can significantly influence the DNA quantity, purity, and observed community patterns of microbiota in activated sludge. Fast and Mobio generated high amounts of good quality DNA compared to Bead and Qiagen. Mobio also resulted in the detection of the highest number of species while Fast scored the best in discriminating between the community patterns of different activated sludge types. With respect to the characterization of community profiles, our analyses demonstrated a strong sludge type dependent variability among methods. Taking into account our results, we recommend Fast as the most suitable DNA extraction method for activated sludge samples used for bacterial community studies.

Evaluation of process parameters of ultrasonic treatment of bacterial suspensions in a pilot scale water disinfection system.

In this study, several process parameters that may contribute to the efficiency of ultrasound disinfection are examined on a pilot scale water disinfection system that mimics realistic circumstances as encountered in an industrial environment. The main parameters of sonication are: (i) power; (ii) duration of treatment; (iii) volume of the treated sample. The specific energy (E(s)) is an indicator of the intensity of the ultrasound treatment because it incorporates the transferred power, the duration of sonication and the treated volume. In this study, the importance of this parameter for the disinfection efficiency was assessed through changes in volume of treated water, water flow rate and electrical power of the ultrasonic reactor. In addition, the influences of the initial bacterial concentration on the disinfection efficiency were examined. The disinfection efficiency of the ultrasonic technique was scored on a homogenous and on a mixed bacterial culture suspended in water with two different types of ultrasonic reactors (Telsonic and Bandelin). This study demonstrates that specific energy, treatment time of water with ultrasound and number of passages through the ultrasonic reactor are crucial influential parameters of ultrasonic disinfection of contaminated water in a pilot scale water disinfection system. The promising results obtained in this study on a pilot scale water disinfection system indicate the possible application of ultrasound technology to reduce bacterial contamination in recirculating process water to an acceptable low level. However, the energy demand of the ultrasound equipment is rather high and therefore it may be advantageous to apply ultrasound in combination with another treatment.

Evaluation of power ultrasound for disinfection of both Legionella pneumophila and its environmental host Acanthamoeba castellanii.

The objectives of this study were to (1) examine the effect of power ultrasound on the viability of both Legionella pneumophila and Acanthamoeba castellanii trophozoites and cysts, (2) investigate if intracellular Legionella replication in trophozoites positively affects bacterial resistance to ultrasound and (3) study if Legionella renders viable but non-culturable (VBNC) due to ultrasound treatments. Using laboratory scale experiments, microorganisms were exposed for various time periods to power ultrasound at a frequency of 36 kHz and an ultrasound power setting of 50 and 100%. Due to a fast destruction, trophozoite hosts were not able to protect intracellular Legionella from eradication by ultrasound, in contrast to cysts. No significant effects of ultrasound on cyst viability could be detected and power settings of 100% for 30 min only made intracellular Legionella concentrations decrease with 1.3 log units. Due to intracellular replication of Legionella in trophozoites, ultrasound no longer affected bacterial viability. Concerning the VBNC state, ultrasound treatments using a power setting of 50% partly induced Legionella (+/-7%) to transform into VBNC bacteria, in contrast to power settings of 100%. Promising results obtained in this study indicate the possible application of power ultrasound in the control of both Legionella and Acanthamoeba concentrations in anthropogenic water systems.

Biofilms: the environmental playground of Legionella pneumophila.

Legionella pneumophila, the aetiological agent of 90% of legionellosis cases, is a common inhabitant of natural and anthropogenic freshwater environments, where it resides in biofilms. Biofilms are defined as complex, natural assemblages of microorganisms that involve a multitude of trophic interactions. A thorough knowledge and understanding of Legionella ecology in relation to biofilm communities is of primary importance in the search for innovative and effective control strategies to prevent the occurrence of disease cases. This review provides a critical update on the state-of-the-art progress in understanding the mechanisms and factors affecting the biofilm life cycle of L. pneumophila. Particular emphasis is given to discussing the different strategies this human pathogen uses to grow and retain itself in biofilm communities. Biofilms develop not only at solid-water interfaces (substrate-associated biofilms), but also at the water-air interface (floating biofilms). Disturbance of the water surface can lead to liberation of aerosols derived from the floating biofilm into the atmosphere that allow transmission of biofilm-associated pathogens over considerable distances. Recent data concerning the occurrence and replication of L. pneumophila in floating biofilms are also elaborated and discussed.

Replication of Legionella pneumophila in biofilms of water distribution pipes.

Biofilms similar to those present in water distribution pipes of anthropogenic aquatic systems were simulated in a rotating annular reactor using a non-Legionella community consisting of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve and Pseudomonas aeruginosa. The impact of this community and Acanthamoeba castellanii on the replication of Legionella pneumophila was investigated. Despite the presence of 10(7) non-Legionella bacteria, culture and real-time polymerase chain reaction (PCR) results clearly showed that biofilm-associated Legionella bacteria only increased after intracellular replication in A. castellanii. Fluorescent in situ hybridization (FISH) staining of biofilm samples revealed that 48 h after addition of amoebae to the reactor, the amoeba population was lysing and replicated Legionella bacteria were released into the bulk water. This study demonstrated that amoebae like A. castellanii can play a crucial role in the increase and spread of L. pneumophila in anthropogenic aquatic systems and thus in the occurrence of Legionnaires' disease.

The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water.

In order to reduce the risks of Legionnaires' disease, caused by the bacterium Legionella pneumophila, disinfection of tap water systems contaminated with this bacterium is a necessity. This study investigates if electrochemical disinfection is able to eliminate such contamination. Hereto, water spiked with bacteria (10(4)CFU Escherichia coli or L. pneumophila/ml) was passed through an electrolysis cell (direct effect) or bacteria were added to tap water after passage through such disinfection unit (residual effect). The spiked tap water was completely disinfected, during passage through the electrolysis cell, even when only a residual free oxidant concentration of 0.07 mg/l is left (L. pneumophila). The residual effect leads to a complete eradication of cultivable E. coli, if after reaction time at least a free oxidant concentration of 0.08 mg/l is still present. Similar conditions reduce substantially L. pneumophila, but a complete killing is not realised.

Replication of Legionella pneumophila in floating biofilms.

Biofilms are a major source of human pathogenic Legionella pneumophila in aquatic systems. In this study, we investigated the capacity of L. pneumophila to colonize floating biofilms and the impact of Acanthamoeba castellanii on the replication of biofilm-associated Legionella. Biofilms were grown in Petri dishes and consisted of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve, and Pseudomonas aeruginosa. Six hours following inoculation, Legionella were detected in floating biofilms in mean concentrations of 1.4 x 10(4) cells/cm(2 )(real-time polymerase chain reaction) and 8.3 x 10(2) CFU/cm(2 )(culture). Two-way analysis of variance tests and fluorescent in situ hybridization clearly proved that increased biofilm-associated L. pneumophila concentrations were the result of intracellular replication in A. castellanii. Forty-eight hours after the introduction of A. castellanii in the Petri dishes, 90 +/- 0.8% of the amoebae (infection rate) were completely filled with highly metabolic active L. pneumophila (mean infection intensity).

Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments.

Floating biofilms develop at the water-air interface and harbor numerous microorganisms, some of which are human pathogens like Legionella pneumophila. The presence of Legionella spp. and especially L. pneumophila in such biofilms was investigated. In parallel, the occurrence of Naegleria spp., Acanthamoeba spp., Willaertia spp., Vahlkampfia spp. and Hartmanella spp. was determined and it was examined whether Acanthamoeba spp. isolates were naturally infected with L. pneumophila bacteria. Eight anthropogenic and 37 natural aquatic environments were sampled between June and August 2005. Both Legionella spp. and L. pneumophila were present in 100% of the floating biofilms of the anthropogenic aquatic systems. Eighty-one percent of all natural floating biofilm samples were positive for Legionella spp. and 70% of these samples were positive for L. pneumophila. Legionella concentrations were in the range of 10(1)-10(2)cells/cm(2). Naegleria spp. and Acanthamoeba spp., two well-known L. pneumophila amoeba hosts, were present in 50-92% and 67-72% of floating biofilm samples, respectively. Acanthamoeba spp. isolates appeared to be naturally infected with L. pneumophila bacteria as proved by fluorescent in situ hybridization.

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples.

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples.

A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (<200 N. fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

Survey for the presence of specific free-living amoebae in cooling waters from Belgian power plants.

Free-living amoebae (FLA) are distributed ubiquitously in aquatic environments with increasing importance in hygienic, medical and ecological relationships to man. In this study, water samples from Belgian industrial cooling circuits were quantitatively surveyed for the presence of FLA. Isolated, thermotolerant amoebae were identified morphologically as well as using the following molecular methods: enzyme-linked immunosorbent assay and isoenzyme electrophoresis and PCR. Thermophilic amoebae were present at nearly all collection sites, and the different detection methods gave similar results. Naegleria fowleri was the most frequently encountered thermotolerant species, and concentrations of thermotolerant FLA were correlated with higher temperatures.

Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

Detection of legionella in various sample types using whole-cell fluorescent in situ hybridization.

The human pathogenic Legionella bacteria are found ubiquitously in natural and human-made aquatic environments as residents in biofilms, where close interactions with other microorganisms like protozoa are possible. Nosocomial legionellosis already has been linked frequently to Legionella-contaminated artificial water supplies. For this reason, a rapid and accurate detection and quantification of these bacteria in environmental and clinical samples, combined with more information about their behavior in complex microbial communities and diverse ecosystems, is of importance. More insight into the ecology of the Legionella bacteria can lead to new methods to suppress their high numbers in human-made aquatic systems. Fluorescent in situ hybridization (FISH), based on ribosomal ribonucleic acid-targeted oligonucleotide probes, combines the precision and specificity of a molecular technique with the power to visualize individual cells without prior cultivation. In this chapter, the use of FISH for the detection and quantification of Legionella in water samples and in the visualization of these bacteria inside protozoa and biofilms is described in detail.

Detection and quantification of Legionella pneumophila in water samples using competitive PCR.

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.