Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines.     

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10−⁷ to 10² g⁻¹ (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.     

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic Escherichia coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.     

Identification of airborne bacterial and fungal community structures in an urban area by T-RFLP analysis and quantitative real-time PCR

This study explores the characteristics of bacterial and fungal communities of total suspended particles (TSP) in the atmosphere by using various molecular methods. TSP samples were collected on a glass fiber filter at an urban location in the middle of the Korean Peninsula (Seoul) between middle autumn and early winter in 2007. From the aerosol samples, DNA could be extracted and DNA sequences were determined for bacteria and fungi. Terminal restriction length polymorphism (T-RFLP) analysis was applied to analyze the community structure of them. To estimate the concentration of DNA originating from bacterial and fungal communities, we used the quantitative real-time polymerase chain reaction (Q-PCR). Sequence analyses were also used to determine the identity of biological organisms. The number of bacteria and fungi in the air were between 5.19 × 10¹ and 4.31 × 10³ cells m^− 3 and from 9.56 × 10¹ to 4.22 × 10⁴ cells m^− 3, respectively and bacterium/fungus ratios ranged from 0.09 to 0.76 across the seven sampling dates. Most of the bacterial sequences found in our TSP samples were from Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The fungal sequences were characteristic for Ascomycota, Basidiomycota, and Glomeromycota which are known to actively discharge spores into the atmosphere. The plant sequences could be also detected. We found that large shifts in the community structure of bacteria and fungi were present in our TSP samples collected on different dates. The results demonstrated that in our TSP samples collected at the urban site; (1) there were very diverse bacterial and fungal groups including potential pathogens and allergens and (2) there were temporal shifts in both bacterial and fungal communities in terms of both diversity and abundances across an inter-seasonal period.     

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.     

DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples

A small scale DNA microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by microarray hybridization without the need for PCR amplification. Specifically, the microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses--an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures.     

Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR

A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets. Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures. The m-PCR assay was specific and rapid, with a turnaround time of < 12 h. The detection limit of the assay for the bacterial targets was estimated at 10(0)-10(2) cfu. Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E. coli counts. Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality.     

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.     

Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.     

土石坝安全实时评价系统研究与开发

大坝安全管理的实时性与风险特点越来越明显,工程安全是水库防洪调度安全的基础,也是发挥大坝防洪减灾与洪水资源利用的根本保障。针对水库防洪调度实时性需求,对大坝的实时安全性进行了分析,研究提出了大坝工程安全实时评价的分析框架及系统设计要求,就工程结构的实时计算方法进行了实现,并成功应用于青山水库防洪安全调度中。     

Development of an electromagnetic hydrocyclone separator for purification of wastewater

A number of various organic and inorganic contaminants are commonly present in the technological fluids hindering production quality. Importantly, waste influents must be purified to meet the appropriate environmental standards for their disposal. This paper deals with the development of a treatment technique for technological and wastewater streams allowing the separation of microscopic droplets of water, sand, oil and other organic and inorganic contaminants. Some existing approaches and processes for treatment of contaminated fluids are briefly reviewed and the design of an electromagnetic hydrocyclone separator is presented in this paper. The effect of the main forces on the separation of small‐sized impurities (<10 μm) is analysed. The experimental tests demonstrated the efficiency of the developed apparatus in purifying the industrial wastewaters of the textile dyeing industry.     

A bacterial bioassay for assessment of wastewater toxicity

A bioassay using freeze-dried Nitrobacter as the test organism has been shown to successfully detect various toxicants in municipal and industrial wastewaters. The test is simple, sensitive, rapid and inexpensive; as a result, this test shows potential as a quantitative measurement technique for wastewater toxicity.The bioassay technique was applied in the analysis of a wastewater treatment system at a fiberboard plant near Portland, Oregon. The fiberboard is manufactured from a slurry of chipped wood, shavings and sawdust.Wastewater from the process is treated at an on-site facility consisting of settling ponds, activated sludge basin, clarifier and holding ponds. Treated water is recycled to the mill for reuse. Recently the removal of BOD by the activated sludge system dropped significantly and soluble and particulate organics began to accumulate. The influent and effluent flows for the treatment facility were tested with the Nitrobacter bioassay and both were determined to have significant toxicity. Further tests at points in the process showed that the toxicity was not associated with a single waste stream and was prevalent throughout the entire wastewater treatment system. In an effort to identify the toxicant, toxicity tests were conducted for known chemicals used in the process. The wastewater was treated with various physical and chemical unit processes to determine the most effective method for toxicity removal.Due to the complexity of the wastewater composition, no specific agents have been identified as solely responsible for the observed toxic response, however, several possible explanations for the apparent toxicity are discussed.     

Development and implementation of an asset management framework for wastewater collection networks

This paper presents a framework to develop, implement and communicate a multi-perspective asset management plan for wastewater collection networks. The framework takes into account four strategic perspectives – socio-political, financial, operational/technical, and regulatory – and devises four strategic themes for sustainable wastewater collection systems.The asset management strategic themes, perspectives, and strategic objectives were developed from the proceedings of collaborative working sessions held at the first Canadian National Asset Managers workshop in 2007 in Hamilton, Ontario. The themes and strategic objectives are illustrated in a strategy map and detailed in the modified balanced scorecard model. A case study based on real data presents the use of business intelligence tools to implement, monitor, and report various components of the proposed framework.The proposed framework provides a unified gateway for efficient and effective management of wastewater collection systems. It can be adapted to devise and implement strategic asset management plans in comparable organizations, and to comply with the new legislative requirements that demand increasing accountability to meet stakeholders’ expectations, protection of public health and environment, efficient allocation of funds, and greater disclosure.     

Behaviour of pathogenic and indicator bacteria during urban wastewater treatment and sludge composting, as revealed by quantitative PCR

Two enteric pathogens, Salmonella spp. and Campylobacter jejuni, and two bacteria commonly used as indicators, Escherichia coli and Clostridium perfringens, were monitored using quantitative real-time PCR during municipal wastewater treatment and sludge composting. The results were compared with those obtained using standard culture methods. A reduction of all bacteria was observed during wastewater treatment and during the thermophilic phase of composting. However, the bacterial groups studied behaved differently during the process, and the main differences were observed during biological treatment in activated sludge basins. In particular, Salmonella spp. and C. jejuni survived better during activated sludge treatment than E. coli. C. jejuni was the most resistant to wastewater treatment among the four bacterial groups. Overall, differences in survival were observed for all bacteria studied, when submitted to the same environmental pressure. This holds both for differences between indicators and pathogenic bacteria and between pathogenic bacteria. These results show the difficulty in defining reliable indicators.     

Characterization of fecal concentrations in human and other animal sources by physical,culture-based,and quantitative real-time PCR methods

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.     

Improved strategies and optimization of calibration models for real-time PCR absolute quantification

Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed ‘pooled’ and ‘mixed’ for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the ‘pooled’ model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the ‘mixed’ model achieved uncertainty estimates similar to the ‘single’ model while increasing the number of available reaction wells per instrument run.     

Development of an expanded-bed GAC reactor for anaerobic treatment of terephthalate-containing wastewater

An expanded-bed granular activated carbon (GAC) anaerobic reactor was developed to treat terephthalate-containing wastewater. Terephthalate inhibits biological anaerobic degradation of terephthalate and methane production when present at a concentration of more than 150 mg/L. In the GAC anaerobic reactor developed here, degradation of terephthalate and other organic compounds occurred smoothly and stably with removal and methane fermentation ratios of more than 90% under a chemical oxygen demand (COD) loading rate of 4 kg COD/(m³ d) and a terephthalate loading rate of 1 kg terephthalate/(m³ d).     

Flame and smoke detection method for early real-time detection of a tunnel fire

This paper presents an image-processing technique for automatic real-time flame and smoke detection in a tunnel environment. To avoid the large-scale damage caused by fire occurring in different environments, there are many studies about discovering and minimizing an incident as fast as possible. However, we need an original algorithm specialized for a tunnel fire, because this environment is quite different and it is difficult to apply existing fire detection algorithms to a tunnel environment. Therefore, this paper proposes an original algorithm that applies to a tunnel environment. Color and motion information are used to minimize false detections in tunnels, and this information enables us to detect the exact position of an event at an early stage, by detection, test, and verification procedures. The experimental results show the validity and efficiency of the proposed algorithms by a comparison of the characteristics of each algorithm.     

Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species

To study the ecology of ammonia-oxidising bacteria (AOB), quantitative techniques are essential. Real-time PCR assays based on the 16S rRNA or on the structural amoA gene are routinely used. The CTO primer set rooted on the 16S rRNA gene has a number of mismatches with some of the cultures of AOB. To examine if these mismatches have an effect on the outcome of real-time PCR assays, the assay was tested with DNA from a number of closely related isolates of AOB. Standard curves of known amounts of initial DNA were similar among most of the tested cultures of AOB, except for the standard curves of Nitrosomonas strain AL212 and Nitrosospira strain NpAV. Nitrosomonas strain AL212 had 3 mismatches with the CTO primer set. Adaptation of the CTO primer set in order to perfectly match the Nitrosomonas strain AL212 gave a standard curve similar to the majority of the AOB tested. As Nitrosospira strain NpAV has no mismatches with the original CTO primer set, there must be another reason for the less efficient amplification than the sequence itself. Application of an existing sigmoidal mathematical model gave no other results with respect to the standard curves of Nitrosomonas europaea and Nitrosomonas strain AL212, but also demonstrated that primer mismatches can seriously underestimate the initial target concentration. It was concluded that in general correct interpretation of real-time PCR results requires knowledge of the target community composition, in particular of the target sequences of the dominant community members.     

DNA signature-based approaches for bacterial detection and identification

During the late eighties, environmental microbiologists realized the potential of the polymerase chain reaction (PCR) for the design of innovative approaches to study microbial communities or to detect and identify microorganisms in diverse and complex environments. In contrast to long-established methods of cultivation-based microbial identification, PCR-based techniques allow for the identification of microorganisms regardless of their culturability. A large number of reports have been published that describe PCR-inspired methods, frequently complemented by sequencing or hybridization profiling, to infer taxonomic and clonal microbial diversity or to detect and identify microorganisms using taxa-specific genomic markers.Typing methods have been particularly useful for microbial ecology-driven studies; however, they are not suitable for diagnostic purposes, such as the detection of specific species, strains or clones. Recently, comprehensive reviews have been written describing the panoply of typing methods available and describing their advantages and limitations; however, molecular approaches for bacterial detection and identification were either not considered or only vaguely discussed. This review focuses on DNA-based methods for bacterial detection and identification, highlighting strategies for selecting taxa-specific loci and emphasizing the molecular techniques and emerging technological solutions for increasing the detection specificity and sensitivity. The massive and increasing number of available bacterial sequences in databases, together with already employed bioinformatics tools, hold promise of more reliable, fast and cost-effective methods for bacterial identification in a wide range of samples in coming years. This tendency will foster the validation and certification of these methods and their routine implementation by certified diagnostic laboratories.