BactiQuant-Water Technical Paper

1 Mycometer, Inc, 3218 Wallcraft Avenue Tampa, Florida 33611, USA Whitepaper: BactiQuant® Attain Microbiological Quality and Biological Stability in Water: Detect, Assess and Respond. Copyright © July 2008, Mycometer A/S 2 Content Introduction………………………………………………………… Page 3 Current technology and the consequences ……………………… Page 3 Mycometers technology and the advantages……………………. Page 3 Application of the BactiQuant® method to water treatment operations Page 6 Laboratory validation studies with Mycometers BactiQuant®… Page 6 Field validation studies with Mycometers BactiQuant®……….. Page 8 Sensitivity and specificity of the method………………………… Page 9 Summary…………………………………………………………… Page 10 Comparison of current method vs. Mycometer BactiQuant®….. Page 10 References…………………………………………………………. Page 11 3Introduction Mycometer is a world leader in developing rapid and robust microbiological methods, to detect and quantify microbiological contamination, based on a well proven highly sensitive fluorescence technology. No other technology allows quantification of bacteria and fungi in air, surface, bulk and liquid samples. This paper briefly discusses Mycometer’s new method BactiQuant®, how it works, how it is applied, and how it solves problems in Water Works. Current Technology and the Consequences Currently, Heterotrophic Plate Counts (HPC) is used for monitoring overall water quality. In both disinfected and non-disinfected systems, sudden increases in HPC above normal baseline levels can indicate a change in raw water quality or a problem such as bacterial re-growth in the distribution system. Measuring HPC levels in water upon leaving the treatment plant or during treatment is used by plant operators to monitor plant operation. High HPC measurements indicate low biological stability of the water. Bacterial re-growth can promote corrosion, foul-tasting or discoloured water and harbour secondary respiratory pathogens as well as increase the demand for disinfectant (Health Canada, 2006). The existing conventional testing methods have several drawbacks: ? The process is labour intensive. ? The test methods are sensitive to errors during performance of the tests ? The results are generally not available for two to four days. Among these undesirable attributes, the consequences of time delayed feedback of results are generally the most significant and include processes (sampling and analysis) that are to episodic and slow for rapid detection and action. Mycometer’s Technology and the Advantages Mycometer’s technology has been developed to mitigate these consequences for the professional consultants and the industry. Specifically, the method facilitates detection and quantification of microbial contaminants in near real time. The methods are based on measuring microbial enzyme activities by use of a highly sensitive fluorescence technique. The use of this technology for environmental sampling and analysis is well documented in the scientific literature (Miller et al., 1998; Krause and Hammad 2002; Reeslev et al, 2003; Krause, 2003; Corfitzen et al., 2006). There are several advantages of targeting microbial enzyme activities: ? Method is culture-independent ? High versatility – many enzyme targets ? Easy adjustment of sensitivity. 4The advantage of using fluorescent enzyme substrates: ? No alteration of bacterial cell necessary ? Simple chemistry ? Low detection limits Mycometer’s BactiQuant® method detects and quantifies enzyme activity in bacteria. The fluorescence signal produced is directly proportional to the amount of bacteria present in a given water sample. With the BactiQuant® (BQ) system, bacterial contaminants can be concentrated from large volumes of water allowing low analysis time (10 – 30 minutes) even at relatively low bacterial concentrations. As an additional benefit, the method can also be used to detect bacteria on surfaces and in air samples. Mycometer Technology Mycometer methods are based on a fluorescence technique that makes use of methylumbelliferyl (4-MU) labelled enzyme substrates. These enzyme substrates consist of a derivative of an enzyme specific moiety and a fluorophore in this case 4-MU. When the enzyme specific moiety is recognized by the enzyme, the chemical bond between the enzyme specific moiety and 4-MU is cleaved, thereby releasing free 4-MU (Figure 1). When a fluorophore is excited by monochromatic light it emits energy in the form of photons. The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitivity of fluorescence detection techniques. Figure 1. Schematic representation of chemical reaction In practice, a sample containing bacteria is saturated with enzyme substrate. The bacterial enzyme reacts with the enzyme substrates and 4-MU is released. The 4-MU is excited by photons of wavelength 365 nm. The 4-MU absorbs the photons and enters an excited state S1’(higher level of energy). This excited state is unstable and only exists for a very short interval (nanoseconds). When the excited state returns to the more stable ground state (S0’), a photon is emitted. Due to energy dissipation during the brief excited-state lifetime, the energy of the emission photon is lower and therefore, of longer wavelength than the excitation photon (Richard P. Haugland, 2002). The photons (fluorescence) of 4-MU are measured at 445 nm. Bacteria Enzyme 5The absorption and fluorescence spectra of the unhydrolysed enzyme substrate and the 4-MU are shown below in Figures 2 and 3 (adapted from Wilson and Goulding, 1986). The figures indicate that if the assay system is irradiated with 350 to 400 nm light virtually all of the fluorescence measured around 450-500 nm is due to the 4-MU product of the enzyme reaction. Figure 2. Absorption spectra of enzyme substrate and 4-MU. Figure 3. Fluorescence spectra of enzyme substrate and 4-MU For over ten years, Mycometer has been collaborating with the US manufacturer of fluorescence instruments, Turner Biosystems (TBS, Sunnyvale, California). The high performance instrumentation technology TBS provides for the fluorescence detection in the Mycometer methods consists of: 1) Excitation source which emit light at a wavelength that excites the 4-MU (wavelength 365 nm) 2) Wavelength filters to isolate emission photons (445 nm) from excitation photons and 3) Detector that registers emission photons and produces a recordable output. 0102030405060708090100280 340 400Wavelength (nm)Relative extinction4-MU 0102030405060708090100300 400 500Wavelength (nm)Relative fluorescence Enzyme substrate (substrate + 4-MU moiety) 4-MU Enzyme substrate (substrate + 4-MU moiety) 6The handheld instrumentation is light weight and battery operated for use on location. It has a menu driven keypad for easy use and LCD display. TBS instruments and Mycometer technology have met the demanding specifications of the US military for mold detection in buildings. Other Mycometer products include microbial contamination monitoring by air sampling, using generic air sampling equipment and the new air sample protocol for fungi and bacteria. Mycometer technology can also be used for surface tests for bacteria and for fungi as well as in bulk material samples. Application of the BactiQuant® Method to Water Treatment Operations The BQ method is a flexible tool that enhances the ability in the Water Works to detect, assess and respond to changes in the biological stability of water. The BactiQuant® delivers a robust system to monitor source water quality at intake, treatment efficiency, distribution system integrity and as a quality assurance following renovation of water systems. The BactiQuant® system offers significant advantages to the waterworks: ? Low supply and human resource demand ? Applicable in chlorinated water ? Parameter is directly linked to biological stability of water High frequency monitoring with the BactiQuant® allows for timely trending to spot problems early and lowers the chances of contamination. As opposed to on-line monitoring systems that are installed at fixed control points, BactiQuant® can be used in emergencies by rapid response teams for flexible and rapid surveys of the whole water system. The multiple sampling modes allow sampling in areas with difficult access. The flexibility in sampling facilitates a rapid sampling strategy in case of contamination and the ability to rapidly determine where the leading edge of the contamination is located as well as delineate the extent of contamination. Practical uses of the BactiQuant® in US water works. 1) Monitoring the efficiency of the water treatment process, including disinfection 2) Assessing changes in finished water quality during distribution and storage 3) Assessing microbial growth on materials used in the construction of potable water treatment and distributions systems 4) Measuring bacterial regrowth or aftergrowth potential in treated drinking-water 5) Monitoring bacterial growth changes following treatment modifications, such as a change in the type of disinfectant used. Laboratory Validation Studies with Mycometer’s BactiQuant® The BactiQuant® system has been evaluated in laboratory experiments. Figure 4 shows a scatter plot of HPC counts and results of the BactiQuant® analysis from an experiment simulating organic pollution in drinking water. The drinking water was diluted 101,102,103,104 and 5x105. The scatter 7plot shows a very strong correlation between HPC counts and the results of the BactiQuant® analysis. Figure 4. Comparison of BactiQuant® to HPC results BactiQuant® results will differ with growth conditions and bacterial species. Therefore these results should be interpreted with caution when applied to more complex environmental samples. Figure 5 shows data from a similar experiment conducted in collaboration with the Technical University of Denmark. The figure shows the dynamic response pattern of HPC counts, DAPI microscopic counts and the results of BactiQuant® analysis, in response to a simulated organic pollution of drinking water. Figure 5. Results of HPC, DAPI, BactiQuant® in complex environmental samples The results are consistent with a strong correlation between bacterial presence and analysis results obtained with the BactiQuant® method. 1101001000100001000001000000100000001 10 100 1000 10000 100000 1000000BactiQuant analysis result (fu/ml)HPC - DS6222 (cfu/ml)02000004000006000008000000 50 100 150 200 250 300Incubation (hours)HPC / Microscopical counts (kim/ml)0102030405060BactiQuant analysis result (fu/ml)HPCDAPIBQ 8Field Validation Studies with Mycometer’s BactiQuant® The BactiQuant® system was evaluated at the Copenhagen Water Works (KE – Water) in 2005 and 2006. Water samples (n = 974) were collected from more than 200 sampling sites. Parallel analysis was conducted on all water samples. Parallel analysis included the traditional standard method (DS/EN ISO 6222) for enumeration of culturable micro-organisms at 22OC and 36OC on yeast extract agar and the BactiQuant® analysis conducted according to the standard protocol for drinking water analysis described in the BactiQuant® user manual. The water samples were collected from the source (ground) water, treatment plant (WTP) outlet (after aeration, precipitation and sand filtration), before and after water pipe renovations and from water taps in private and public buildings. Results of parallel analysis of HPC and BactiQuant® showed a good correlation (r = 0,75, n =975). Excerpts of the baseline data are shown in Figure 6. The figure shows an increase in enzyme activity from source ground water (with expected low bacterial numbers) to the outlet at WTP and further downstream in the water distribution system where increasing HPC counts were expected. Figure 6. Excerpts of baseline data 01002003004005001 20 39 58 77 96 115 134 153 172 191 210Sample numberBactiQuant (FE/250ml/30min)Ground w aterWTP ouletDistribution system The extensive data set allowed operative threshold values to be established based on the empirical distribution of analysis data. The threshold values were organized with a level indicating a normal and acceptable condition, an intermediary level between normal and critical and finally an unacceptable level of bacteria in the water samples. An example of how categories were defined at the Copenhagen Waterworks (KE-Water) is shown below: Best quality – no activation of control measures. BQ values = 40 Between best quality and unacceptable quality – control measures may be activated. 40 < BQ values = 200 Unacceptable quality – high risk of exceeding tolerance limits for total bacteria in water – control measures activated. BQ values > 200 9Sensitivity and Specificity of the Method The sensitivity of the method can be adjusted to fit the needs of the user. Protocols are available for drinking water and ultra pure water. The target enzyme activity in the assay is a hydrolytic enzyme belonging to the enzyme group of hydrolases. The enzyme activity that is measured with the method is not unique to bacteria, however has been shown to be preferential to bacteria. The target enzyme activity, used in the BactiQuant® method, has been shown to have utility as an indicator of microbial population density and biomass in freshwater sediments (Saylor et al., 1979); Utility water systems are dominated by bacteria hence in utility water systems the activity is of a predominantly bacterial origin. The good correlation with HPC in water samples shows that the target enzyme activity is a good indicator of bacterial presence and activity in water utility systems. The enzyme activity is widely distributed in bacteria. The table below shows examples of bacteria that posses the target enzyme. The bacteria in the table represent a wide range of bacterial phylogenetic groups including both gram negative and gram positive. Organism Gram stain Phyllum/ class Athrobacter sp. Negative Actinobacteria / grp. 20 Bacillus cereus Positive Firmicutes / grp. 18 Bacillus subtilis Positive Firmicutes / grp. 18 Bacillus thuringiensis Positive Firmicutes / grp. 18 Clostridium tetani Positive Firmicutes / clostridia Desulfovibrio desulfouricans Positive Proteobacteria / d Enterobacter sp. Negative Proteobacteria / ? Enterococcous faecalis Positive Firmicutes / bacilli Eschericia coli Negative Proteobacteria / ? Escherichia fergusonii Negative Proteobacteria / ? Flavobacteria bacterium Negative Bacteroidetes / flavobacteria Flavobacterium johnsoniae Negative Bacteroidetes / flavobacteria Klebsiella pneumonia Negative Proteobacteria / ? Lactobacillus acidophilus Positive Firmicutes / bacilli Lactobacillus reuteri Positive Firmicutes / bacilli Methanococcus aeolicus Negative Euryarchaeota / methanococci Micrococcous sodonensis Positive Actinobacteria / actinobacteria Pseudomonas aeroginosa Negative Proteobacteria / ? Rhodobacter sphaeroides Negative Proteobacteria / a Serratia marcescens Positive Proteobacteria / ? Staphylococcous aureus Positive Firmicutes / bacilli Sphingomonas Wittichii Negative Proteobacteria / a Vibrio sp Negative Proteobacteria / ? Xanthomonas campestris Positive Proteobacteria / ? Yersinia pestis Positive Proteobacteria / ? Source: Enzyme database Brenda and ExPASy (Expert Protein Analysis System) Proteomic Server. 10 Summary BactiQuant® provides the professional a rapid, robust method to mitigate the consequences of current technology. With rapid detection onsite in less than 30 minutes, data collected is near real-time for fast response to biological instability in the system. The contamination can be isolated and delineated quickly to focus response resources effectively. The robustness of the method, based on scientific research and documentation, provides assurance that the data is repeatable and reliable. With BactiQuant®, water treatment operations can improve their performance, minimize contamination related maintenance, and exceed the expectations of the end user. Comparison of Current Method vs. Mycometer BactiQuant® The table below compares BactiQuant® against the current HPC method. Feature BactiQuant® HPC Consequence Time from measurement to results. 10 – 30 minutes Typically from two to four days Plate count processes are slow, not compatible with rapid reaction. Gives a historic recording of water quality. Rapid response teams cannot use plate counts for rapid surveys and delineation of contamination. Mode of detection Culture-independent, fluorescence Culture-dependent – visual The conventional method only detects culturable bacteria. BactiQuant detects bacteria in UV and MCA treated water. This provides the end users with a more comprehensive evaluation of the biological stability of drinking water. Time to set up sample 5 minutes Can be significant The conventional method requires high labour costs and advanced lab facilities. Human intervention Minimal High Human intervention creates more possibilities for inaccuracies and contamination of sample 11References Guidelines for Canadian Drinking Water Quality: Guideline Technical Document, Health Canada, Ottawa, Ontario, 2006 Miller et al. (1998) Applied and Environmental Microbiology 64, p. 613-617 Krause and Hammad. (2002) Proceedings Indoor Air 2002. p. 360-365. Reeslev et al. (2003) Applied and Environmental Microbiology 69(7), p. 3996-3998 Krause et al. (2003) Applied Occupational and Environmental Hygiene, 18 (7) p. 499-503 Corfitzen et al. (2006) Nordic Drinking Water Conference, Reykjavik, Iceland. Richard P. Haugland (2002) Handbook of Fluorescent Probes and Research Products, Ninth Edition, Molecular Probes Inc. Wilson and Goulding. (1986) Principles and Techniques of Practical Biochemistry Saylor et al. (1979) Applied and Environmental Microbiology. Nov, p. 922 – 927.