Detection of Bacterial Cells without Cultivation on Membrane Filter by Novel Enzymatic Cycling Method Using Pyruvate Ort

1. Detection of Bacterial Cells without Cultivation on Membrane Filter by Novel Enzymatic Cycling Method Using Pyruvate Orthophosphate Dikinase and Firefly Luciferase Thank you very much, for invitation and for kind introduction. It is a great honor to have a chance for presentation in this session of American Society for Photobiology. Today, I would like to make a presentation concerning detection of bacterial cells without cultivation on membrane filter by novel enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase. Thank you very much, for invitation and for kind introduction. It is a great honor to have a chance for presentation in this session of American Society for Photobiology. Today, I would like to make a presentation concerning detection of bacterial cells without cultivation on membrane filter by novel enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase.

2. Today’s presentation is consisting of three sections. Today’s presentation is consisting of three sections.

3. Amplification of Firefly Bioluminescence by Coupling Pyruvate Orthophosphate Dikinase (PPDK) First of all, I will introduce Amplification of firefly Biluminescence by coupling pyruvate orthophosphate dikinase. PPDK is an abbreviation for Pyruvate Orthophosphate Dikinase. The aim of the of this section is to obtain highly amplified luminescence with low background luminescence.First of all, I will introduce Amplification of firefly Biluminescence by coupling pyruvate orthophosphate dikinase. PPDK is an abbreviation for Pyruvate Orthophosphate Dikinase. The aim of the of this section is to obtain highly amplified luminescence with low background luminescence.

4. This is Enzymatic Properties of pyruvate orthophosphate dikinase. Regarding enzyme reaction, AMP, Phosphoenolpyruvate and pyrophosphate are converted to ATP and pyruvate and phosphate in the presence of Mg ion. The origin of PPDK is Microbispora subsp. Aerata. (Actinomyces) The properties of PPDK is as shown. It is specific for AMP and the Michaelis constants are as shown. This is Enzymatic Properties of pyruvate orthophosphate dikinase. Regarding enzyme reaction, AMP, Phosphoenolpyruvate and pyrophosphate are converted to ATP and pyruvate and phosphate in the presence of Mg ion. The origin of PPDK is Microbispora subsp. Aerata. (Actinomyces) The properties of PPDK is as shown. It is specific for AMP and the Michaelis constants are as shown.

5. Firefly Luciferase Bioluminescent Reaction This figure shows Bioluminescence reaction of firefly luciferase. In this system, ATP is degraded to AMP and pyrophosphate in the presence of Mg ion. Luciferin and oxygen are converted to oxyluciferin and carbon dioxide with the concomitant generation of light. ATP is measured with high sensitivity by measuring the intensity of the generated light. Since ATP is an essential metabolite in all living organisms, the luciferase reaction has been used to measure the number of living bacteria by determining the intracellular ATP. However, the amount of light is limited depending on the ATP amount, because the light decays with the consumption of ATP. This figure shows Bioluminescence reaction of firefly luciferase. In this system, ATP is degraded to AMP and pyrophosphate in the presence of Mg ion. Luciferin and oxygen are converted to oxyluciferin and carbon dioxide with the concomitant generation of light. ATP is measured with high sensitivity by measuring the intensity of the generated light. Since ATP is an essential metabolite in all living organisms, the luciferase reaction has been used to measure the number of living bacteria by determining the intracellular ATP. However, the amount of light is limited depending on the ATP amount, because the light decays with the consumption of ATP.

6. Principle of Bioluminescent Cycling Reaction Using PPDK and Luciferase This figure shows principle of bioluminescence cycling reaction using PPDK and luciferese. To obtain highly amplified light, PPDK was combined with firefly luciferase system. PPDK is an enzyme which catalyzes the reaction of conversion of AMP and Pyrophosphate to ATP in the presence of Mg ion and phosphoenol pyruvate. In this system, AMP and pyrophosphate produced by luciferase reaction were directly converted back into ATP by PPDK reaction and recycled. In this way, highly amplified light was obtained. This figure shows principle of bioluminescence cycling reaction using PPDK and luciferese. To obtain highly amplified light, PPDK was combined with firefly luciferase system. PPDK is an enzyme which catalyzes the reaction of conversion of AMP and Pyrophosphate to ATP in the presence of Mg ion and phosphoenol pyruvate. In this system, AMP and pyrophosphate produced by luciferase reaction were directly converted back into ATP by PPDK reaction and recycled. In this way, highly amplified light was obtained.

7. Novel Background Reduction System with Adenosine phosphate deaminase (APD) This slide shows schematic drawing for reduction of background luminescence with adenosine phosphate deaminase which is abbreviated to APD. PPDK BL reagent had high background luminescence derived from intrinsic ATP and AMP in the reagent. Adenosine phosphate deaminase is an enzyme which catalyzes deamination of ATP, ADP, AMP and adenosine derivatives. By adding adenosine phosphate deaminase into the reagent, intrinsic ATP and AMP were converted into Inosine Tri Phosphate and Inosine Mono Phosphate, respectively which are not active to luciferase, and the background luminescence was reduced effectively. APD inhibitor coformycin was added to the PPDK BL reagent prior to measurement to prevent luminescence decrease.This slide shows schematic drawing for reduction of background luminescence with adenosine phosphate deaminase which is abbreviated to APD. PPDK BL reagent had high background luminescence derived from intrinsic ATP and AMP in the reagent. Adenosine phosphate deaminase is an enzyme which catalyzes deamination of ATP, ADP, AMP and adenosine derivatives. By adding adenosine phosphate deaminase into the reagent, intrinsic ATP and AMP were converted into Inosine Tri Phosphate and Inosine Mono Phosphate, respectively which are not active to luciferase, and the background luminescence was reduced effectively. APD inhibitor coformycin was added to the PPDK BL reagent prior to measurement to prevent luminescence decrease.

8. This figure shows reduction of background luminescence of PPDK bioluiminescence reagent with adenosine phosphate deaminase. The PPDK bioluminescence reagent had 28,000 RLU of background luminescence before adding adenosine phosphate deaminase. The background luminescence was dramatically reduced to 90 RLU by adding adenosine phosphate deaminase. Background luminescence was reduced to about 1/310 of initial background luminescence. This figure shows reduction of background luminescence of PPDK bioluiminescence reagent with adenosine phosphate deaminase. The PPDK bioluminescence reagent had 28,000 RLU of background luminescence before adding adenosine phosphate deaminase. The background luminescence was dramatically reduced to 90 RLU by adding adenosine phosphate deaminase. Background luminescence was reduced to about 1/310 of initial background luminescence.

9. This figure shows the luminescence pattern of PPDK bioluminescence reagent containing various concentration of PPDK. Luciferase concentration in each reagent were constant and the concentration of PPDK only were varied. The figure on the left is for ATP and the figure on the right is for AMP. In the case of reagent without PPDK added with ATP, the luminescence pattern decayed very rapidly. In the case of reagent without PPDK added with AMP, no luminescence was observed. As the concentration of the PPDK in the reagent was increased, the luminescece intensity of stable state was increased, and the lag time to the stable state was decreased. This figure shows the luminescence pattern of PPDK bioluminescence reagent containing various concentration of PPDK. Luciferase concentration in each reagent were constant and the concentration of PPDK only were varied. The figure on the left is for ATP and the figure on the right is for AMP. In the case of reagent without PPDK added with ATP, the luminescence pattern decayed very rapidly. In the case of reagent without PPDK added with AMP, no luminescence was observed. As the concentration of the PPDK in the reagent was increased, the luminescece intensity of stable state was increased, and the lag time to the stable state was decreased.

10. This table shows Amplification of bioluminescence by proposed Cycling method. Amplified luminescence was calculated by dividing the integrated luminescence amount for 5 min in the presence of PPDK by that in the absence of PPDK. Amplified luminescence increased as the concentration of the PPDK was increased and reached to 13.2 folds when the PPDK concentration of 80 µg/ml was employed. This table shows Amplification of bioluminescence by proposed Cycling method. Amplified luminescence was calculated by dividing the integrated luminescence amount for 5 min in the presence of PPDK by that in the absence of PPDK. Amplified luminescence increased as the concentration of the PPDK was increased and reached to 13.2 folds when the PPDK concentration of 80 µg/ml was employed.

11. This figure shows typical standard curves for ATP and AMP using the PPDK BL reagent. The linearity and reproducibility were good in the both cases of ATP and AMP. This figure shows typical standard curves for ATP and AMP using the PPDK BL reagent. The linearity and reproducibility were good in the both cases of ATP and AMP.

12. Cultivation-Free Bacteria Enumeration by PPDK BL Reagent Next, I will talk about Cultivation-Free Bactria Enumeration by PPDK BL reagent. Next, I will talk about Cultivation-Free Bactria Enumeration by PPDK BL reagent.

13. This figure illustrates MicroStar Rapid Microbiology System developed by Millipore. MicroStar instrument is composed of computer, image processor, image controller, and detector. Right figure shows precise constitution of detector. The grid PVDF filter used for MicroStar has 650 of special hydrophobic grids which prevent running of the bioluminescence spots. Bioluminescence derived from bacterial intracellular ATP on the grid PVDF filter is lead through a tapered fiber and amplified in a image intensifier. Finally, the image is captured by a CCD camera and displayed on a monitor. This figure illustrates MicroStar Rapid Microbiology System developed by Millipore. MicroStar instrument is composed of computer, image processor, image controller, and detector. Right figure shows precise constitution of detector. The grid PVDF filter used for MicroStar has 650 of special hydrophobic grids which prevent running of the bioluminescence spots. Bioluminescence derived from bacterial intracellular ATP on the grid PVDF filter is lead through a tapered fiber and amplified in a image intensifier. Finally, the image is captured by a CCD camera and displayed on a monitor.

14. This figure shows comparison of luminescence pattern. Red line shows the luminescence pattern of PPDK BL reagent for PPDK-Rapid Microbe Detection System (RMDS) and the blue line is for traditional BL reagent for current MicroStar. PPDK BL reagent for PPDK-RMDS was improved to obtain maximum Integrated luminescence by increasing luciferase concentration. adenosine phosphate deaminase inhibitor Coformycin was not used because it caused gradual increase of background luminescence. Luminescence pattern decayed slightly because of active adenosine phosphate deaminase added to the reagent to reduce background luminescence. The rate of luminescence decrease was about 11% per 1 min. Integrated luminescence was amplified 42.9 times in 5 min compared to that of the traditional BL reagent without PPDK. This figure shows comparison of luminescence pattern. Red line shows the luminescence pattern of PPDK BL reagent for PPDK-Rapid Microbe Detection System (RMDS) and the blue line is for traditional BL reagent for current MicroStar. PPDK BL reagent for PPDK-RMDS was improved to obtain maximum Integrated luminescence by increasing luciferase concentration. adenosine phosphate deaminase inhibitor Coformycin was not used because it caused gradual increase of background luminescence. Luminescence pattern decayed slightly because of active adenosine phosphate deaminase added to the reagent to reduce background luminescence. The rate of luminescence decrease was about 11% per 1 min. Integrated luminescence was amplified 42.9 times in 5 min compared to that of the traditional BL reagent without PPDK.

15. This is the image of ATP spots on membrane filter corresponding to single bacterial cell obtained with PPDK BL reagent. 0.31 to 5.0 amol/spot (amol corresponds to 10-18 mol) were detected with 42.9 times luminescence amplification in 5 min. This is the image of ATP spots on membrane filter corresponding to single bacterial cell obtained with PPDK BL reagent. 0.31 to 5.0 amol/spot (amol corresponds to 10-18 mol) were detected with 42.9 times luminescence amplification in 5 min.

16. This flow chart shows procedure for PPDK- Rapid Microbe Detection System which is abbreviated to RMDS in the presence of sugars as carbon source for keeping intracellular ATP. Bacterial cell was washed and suspended in bacteria suspension buffer containing sugars and used as sample. After sample filtration, the filter was washed with washing buffer containing sugars. The filter was dried and sprayed with extraction reagent and then sprayed with PPDK BL reagent. The image was detected by MicroStar instrument for 5 min. This flow chart shows procedure for PPDK- Rapid Microbe Detection System which is abbreviated to RMDS in the presence of sugars as carbon source for keeping intracellular ATP. Bacterial cell was washed and suspended in bacteria suspension buffer containing sugars and used as sample. After sample filtration, the filter was washed with washing buffer containing sugars. The filter was dried and sprayed with extraction reagent and then sprayed with PPDK BL reagent. The image was detected by MicroStar instrument for 5 min.

17. These are images of various kinds of bacteria. This is the image of Escherichia coli. Staphylococcus aureus. Pseudomonas aeruginosa. And Lactobacillus brevis. All kinds of bacteria were detected without cultivation by using PPDK BL reagent and RMDS instrument. These are images of various kinds of bacteria. This is the image of Escherichia coli. Staphylococcus aureus. Pseudomonas aeruginosa. And Lactobacillus brevis. All kinds of bacteria were detected without cultivation by using PPDK BL reagent and RMDS instrument.

18. This figure shows correlation curve between the number of luminescence spots obtained by PPDK-RMDS and traditional colony forming units (CFU) upon cultivation in the case of E. coli. Significant correlation was observed and R2 was 0.972. R stands for coefficient of correlation. This figure shows correlation curve between the number of luminescence spots obtained by PPDK-RMDS and traditional colony forming units (CFU) upon cultivation in the case of E. coli. Significant correlation was observed and R2 was 0.972. R stands for coefficient of correlation.

19. This table shows comparison of time to result. Traditional colony counting method using agar plate takes 1 day to 1 week to form visible colony depending on the speed of colony formation. MicroStar using traditional BL reagent takes about 1/3 to ¼ time to result compared to traditional colony counting method. PPDK-RMDS took only 30 min because it did not depend on cultivation. This table shows comparison of time to result. Traditional colony counting method using agar plate takes 1 day to 1 week to form visible colony depending on the speed of colony formation. MicroStar using traditional BL reagent takes about 1/3 to ¼ time to result compared to traditional colony counting method. PPDK-RMDS took only 30 min because it did not depend on cultivation.

20. Practical sample tends to contain no nutrients and bacteria in low energy state. So, behavior of intracellular adenine nucleotide levels was measured to investigate the relationship between the strength of luminescence spots, intracellular adenine nucleotide content, and the energy state of the cell in the presence or absence of sugars. Practical sample tends to contain no nutrients and bacteria in low energy state. So, behavior of intracellular adenine nucleotide levels was measured to investigate the relationship between the strength of luminescence spots, intracellular adenine nucleotide content, and the energy state of the cell in the presence or absence of sugars.

21. This flow chart shows procedure for PPDK-RMDS in the absence of sugars. Bacteria suspension buffer and washing buffer lacking sugars were used to get rid of the effect of sugars as carbon source for keeping intracellular ATP.This flow chart shows procedure for PPDK-RMDS in the absence of sugars. Bacteria suspension buffer and washing buffer lacking sugars were used to get rid of the effect of sugars as carbon source for keeping intracellular ATP.

22. This figure shows comparison of images of E. coli in the presence of sugars and in the absence of sugars. Strength of the luminescence spots were almost the same in both cases. The reason why will be discussed after now by measuring adenine nucleotide content in the presence of sugars and in the absence of sugars.This figure shows comparison of images of E. coli in the presence of sugars and in the absence of sugars. Strength of the luminescence spots were almost the same in both cases. The reason why will be discussed after now by measuring adenine nucleotide content in the presence of sugars and in the absence of sugars.

23. Principle of BL Reagent for Measuring ATP+ADP+AMP This figure shows priciple of BL reagent for measuring ATP+ADP+AMP. Pyruvate kinase is an enzyme which catalyzes the synthesis of ATP from ADP and phosphoenol pyruvate in the presence of Mg ion. By combining pyruvate kinase with PPDK BL reagent, ADP was converted into ATP and measured. In this way, ATP, ADP, and AMP were measured simultaneously with this BL reagent. This figure shows priciple of BL reagent for measuring ATP+ADP+AMP. Pyruvate kinase is an enzyme which catalyzes the synthesis of ATP from ADP and phosphoenol pyruvate in the presence of Mg ion. By combining pyruvate kinase with PPDK BL reagent, ADP was converted into ATP and measured. In this way, ATP, ADP, and AMP were measured simultaneously with this BL reagent.

24. These are the formulas for obtaining ADP and AMP amount. ADP amount was obtained by subtracting ATP+AMP from ATP+ADP+AMP. AMP amount was obtained by subtracting ATP from ATP+AMP. These are the formulas for obtaining ADP and AMP amount. ADP amount was obtained by subtracting ATP+AMP from ATP+ADP+AMP. AMP amount was obtained by subtracting ATP from ATP+AMP.

25. These tables show composition of each BL reagent for measuring adenine nucleotides. BL reagent for ATP+ADP+AMP contained luciferase, PPDK, and pyruvate kinase. BL reagent for ATP+AMP contained luciferase and PPDK. BL reagent for ATP contained luciferase. Components other than colored enzymes are almost the same among these three types of BL reagent.These tables show composition of each BL reagent for measuring adenine nucleotides. BL reagent for ATP+ADP+AMP contained luciferase, PPDK, and pyruvate kinase. BL reagent for ATP+AMP contained luciferase and PPDK. BL reagent for ATP contained luciferase. Components other than colored enzymes are almost the same among these three types of BL reagent.

26. This figure shows luminescence pattern of each BL reagent The sample was 20 f mol (each) mixture of ATP, ADP, and AMP. All three types of BL reagent showed stable luminescence pattern. Furthermore, only the concentration of the intended adenine nucleotides were detected with each type of BL reagent. BL reagent for ATP+ADP+AMP detected all three kinds of adenine nucleotide in the mixture. BL reagent for ATP+AMP detected ATP and AMP in the mixture, while did not detect ADP in the mixture. BL reagent for ATP detected only ATP in the mixture. This figure shows luminescence pattern of each BL reagent The sample was 20 f mol (each) mixture of ATP, ADP, and AMP. All three types of BL reagent showed stable luminescence pattern. Furthermore, only the concentration of the intended adenine nucleotides were detected with each type of BL reagent. BL reagent for ATP+ADP+AMP detected all three kinds of adenine nucleotide in the mixture. BL reagent for ATP+AMP detected ATP and AMP in the mixture, while did not detect ADP in the mixture. BL reagent for ATP detected only ATP in the mixture.

27. This figure shows calibration curve of each BL reagent with same amount of mixture of ATP, ADP, and AMP. Lumitester K-100 was used to record BL intensity. Good linearity and reproducibility were obtained. CV values were between 1.7% and 5.3%. Calibration curves of three kind of BL reagent were parallel. This figure shows calibration curve of each BL reagent with same amount of mixture of ATP, ADP, and AMP. Lumitester K-100 was used to record BL intensity. Good linearity and reproducibility were obtained. CV values were between 1.7% and 5.3%. Calibration curves of three kind of BL reagent were parallel.

28. This flow chart shows procedure for measuring intracellular adenine nucleotide content. Bacteria was suspended in the bacteria suspension buffer containing sugars or lacking sugars. This sample was mixed with 10% of trichloroacetic acid containing 4 mM EDTA to extract intracellular ATP, ADP, and AMP. After 2 min later, mixed with 4.9 ml of 25 mM Tricine (pH7.8) for neutralization and diluted 10 fold. And then, measured with each BL reagent. This flow chart shows procedure for measuring intracellular adenine nucleotide content. Bacteria was suspended in the bacteria suspension buffer containing sugars or lacking sugars. This sample was mixed with 10% of trichloroacetic acid containing 4 mM EDTA to extract intracellular ATP, ADP, and AMP. After 2 min later, mixed with 4.9 ml of 25 mM Tricine (pH7.8) for neutralization and diluted 10 fold. And then, measured with each BL reagent.

29. This figure shows comparison of intracellular adenine nucleotide content of various kinds of bacteria in the presence or absence of sugars. In the cases of E. coli and L. brevis in the absence of sugars, ATP content dramatically decreased, while AMP content dramatically increased compared to that in the presence of sugars. ADP content was not so different in both cases. On the other hand, S. aureus and P aeruginosa showed almost the same tendencies of adenine nucleotide content irrespective of in the presence or absence of sugars. ATP content was higher than ADP and AMP in both cases, irrespective of in the presence or absence of sugars. This figure shows comparison of intracellular adenine nucleotide content of various kinds of bacteria in the presence or absence of sugars. In the cases of E. coli and L. brevis in the absence of sugars, ATP content dramatically decreased, while AMP content dramatically increased compared to that in the presence of sugars. ADP content was not so different in both cases. On the other hand, S. aureus and P aeruginosa showed almost the same tendencies of adenine nucleotide content irrespective of in the presence or absence of sugars. ATP content was higher than ADP and AMP in both cases, irrespective of in the presence or absence of sugars.

30. Comparison of Intracellular Adenine Nucleotide Content This figure illustrates comparison of intracellular adenine nucleotides content. E. Coli and L. brevis showed starvation state in the absence of sugars, while showed active state in the presence of sugars. However, ATP+AMP content were almost the same irrespective of presence or absence of sugars. S aureus and P aeruginosa showed similar active energy state and ATP+AMP content irrespective of presence or absence of sugars. PPDK-RMDS could detect bacteria irrespective of energy state and nutrient condition since it is based on ATP+AMP content, which is stable compared to ATP content.This figure illustrates comparison of intracellular adenine nucleotides content. E. Coli and L. brevis showed starvation state in the absence of sugars, while showed active state in the presence of sugars. However, ATP+AMP content were almost the same irrespective of presence or absence of sugars. S aureus and P aeruginosa showed similar active energy state and ATP+AMP content irrespective of presence or absence of sugars. PPDK-RMDS could detect bacteria irrespective of energy state and nutrient condition since it is based on ATP+AMP content, which is stable compared to ATP content.

31. Finally, this is the Summary. 1.Novel bioluminescent enzymatic cycling reagent using PPDK and firefly luciferase was developed. It had highly amplified luminescence with low background luminescence. The luminescence was amplified 42.9 times in 5 min. (PPDK BL reagent) 2.Various kinds of bacteria were detected without cultivation using PPDK BL reagent and MicroStarTM instrument in 5 min. (PPDK-RMDS) 3.PPDK-RMDS could detect bacteria irrespective of energy state and nutrient condition since it is based on ATP+AMP content, which is stable compared to ATP content. That’s all for my presentation. Thank you very much for your attention.Finally, this is the Summary. 1.Novel bioluminescent enzymatic cycling reagent using PPDK and firefly luciferase was developed. It had highly amplified luminescence with low background luminescence. The luminescence was amplified 42.9 times in 5 min. (PPDK BL reagent) 2.Various kinds of bacteria were detected without cultivation using PPDK BL reagent and MicroStarTM instrument in 5 min. (PPDK-RMDS) 3.PPDK-RMDS could detect bacteria irrespective of energy state and nutrient condition since it is based on ATP+AMP content, which is stable compared to ATP content. That’s all for my presentation. Thank you very much for your attention.