African Journal of Microbiology Research Vol. 3(9) pp. 575-580, September, 2009 Available online http://www.academicjournals.

org/ajmr ISSN 1996-0808 2009 Academic Journals

Full Length Research Paper

Rapid detection of bacteria without cultivation with a portable bioluminescence sensor system
Baoshan He1,2, Xiaohong Liu2, Weiwei Yue2, Aiyu Zhou2, Jinping Luo2 and Xinxia Cai2*
1

School of Food Science and Technology, Henan University of Technology, Songshan Road 140#, Zhengzhou Henan 450052, PR China. 2 State Key Laboratory of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, P.O. Box 2652, Beijing 100080, PR China.
Accepted 13 August, 2009

Based on the principle of ATP bioluminescence reaction, we designed a portable bioluminescence sensor system to detect bacteria in samples rapidly, which included an optical sensing cell, a flow injection unit and a center processing unit. A fast ATP extraction technique was developed by using trichloroacetic acid (TCA) as extraction reagent. Experimental results showed that the intracellular ATP could be released from bacterial cells sufficiently within 5 min by TCA. In the presence of luciferase, the reaction between ATP and luciferin would generate light and the luminescence intensity (RLU) was proportional to the amount of bacteria exsited in the sample. The dynamic range of the sensor system 7 ranged from 10 - 10 CFU/ml. The linear regression coefficient was 0.976, and the detection limit was 10 CFU/ml. The whole testing time needed was about 10 min. Compared with the plate count method, bacteria concentration in samples could be detected rapidly by using this portable sensor system without cultivation. Key words: ATP, portable, bioluminescence sensor system, trichloroacetic acid, luminescence intensity. INTRODUCTION In pharmaceutical, brewing, and beverage companies, the plate count technique is a classical method for evaluating microbial quality, which has been used for more than a century. However, this technique requires incubation periods of 24 - 48 h and the detecting procedures are laborious and need skilled persons. Therefore, it is in urgent need to develop a rapid method that can detect bacterial count in food. Currently, the bioluminescence assay using the firefly ATP-dependent luciferin-luciferase system become a popular method for ATP determination due to its high sensitivity and specificity (Tu et al., 2000; Ishida et al., 2002; Velten et al., 2007). When luciferin-luciferase system is used to measure intracellular ATP, one of the most important steps is to extract ATP from intact cells. Most commonly used methods for ATP extraction include ethanol and butanol (St. John, 1970; Janaszek et al., 1987), boiling in buffer solutions (Loike et al., 1979; Kane et al., 1985), continuous DC voltage (Ramadan et al., 2006; Wang et al., 2006; Lee and Cho, 2007) and ultrasound (Law et al., 2003; Padilla et al., 2003; Zhang et al., 2007). Among the ATP extraction methods mentioned, the addition of ethanol and butanol would inhibit the activity of subsequent luciferase. Yang et al found that boiling water could accomplish two missions in one step for extracting intracellular ATP and inhibiting ATP-hydrolyzing enzymes simultaneously, which may deplete ATP content in the cell extract, resulting in low ATP bioluminescence, finally, the 4 yield of ATP was low and high content of cells about 10 5 10 cells/ml was required for ATP determination (Yang et al., 2002). Although, a survey of the literature showed that the extraction efficiency of DC voltage and ultrasound methods was high, these physical extraction methods demanded special equipments and restricted their applications on-site. To improve the extraction method, we found that cellular ATP could be extracted from intact cells conveniently and efficiently by using trichloroacetic acid (TCA). Because of its strong acid nature, TCA can cause increment of the cell wall pore size

*Corresponding author. E-mail: xxcai@mail.ie.ac.cn. Tel/Fax: +86 10 62563409.

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pore size and degradation of the bacterial membrane, cellular ATP could pass through cell wall easily and ATPhydrolyzing enzymes in bacterial cells could be inactivated simultaneously. After isometric neutralization, the solution could be neutralized easily without using a pH meter and TCA interfering with the luciferin-luciferase system would be eliminated entirely. Consequently, the TCA method was simple and convenient, and suited to be used in fast evaluation of microbial quality. Basing on the bioluminescence mechanism of ATP and the TCA extraction principles, a portable bioluminescence sensor system which included an optical sensing cell, a flow injection unit and a center processing unit as well as functions of data acquisition, conversion and dynamically displaying is proposed and established in the presented work. With this portable sensor system, total bacterial count (TBC) in samples could be detected rapidly without cultivation. The dynamic range, detection limit, and sensitivity of the detecting method were also evaluated.
MATERIALS AND METHODS Materials Luciferin and luciferase (EC 1.13.12.7, 14.9 mg/ml) were purchased from Promega (USA). Magnesium acetate tetrahydrate was obtained from Sigma (USA). ATP was purchased from Shanghai Bio. Life Science and Technology Co., Ltd. (China). Dithiothreitol (DTT), albumin bovine serum (BSA) and TCA were from Beijing Xin Hui Ze AO Science Technology Co., Ltd. (China). All other chemicals were of analytical grades obtained from commercial sources. A Tris (hydroxymethyl) aminomethane (Tris-acetate) buffer solution was prepared in our laboratory by dissolving 0.607 g hydroxymethyl aminomethane in 200 ml of deionized water (DW) and adjusting pH to 7.8 with acetic acid at 25 C. Construction of sensor system Figure 1 shows the block diagram of the sensor system: a portable sensor system TX2007-1, which was developed to measure the light emitted by the bioluminescent reaction in our study. A center processing unit (CPU) with digital/analog (D/A), analog/digital (A/D) converter interface was taken as main center of the system. The control signal was sent to a flow injection unit via the D/A interface, so that the luciferin-luciferase reagent was injected into an optical sensing cell automatically. With a weak light detector (photomultiplier, PMT), the light emitted by the bioluminescent reaction was acquired and converted to electric signals. The results were dynamically displayed via a display module. In order to reducing the influence of the nature light outside, the PMT and the optical sensing cell were packaged in a dark chamber. Bacterial culture and colony counting Escherichia coli (ATCC 25922, gram-negative bacteria) and Staphylococcus aureus (ATCC 26001, gram-positive bacteria) were selected to be used as target detected, which were provided from the Microbial Laboratory of National Center for Clinical Laboratory (Beijing, China). An aliquot of 50 l bacterial sample was suspended into 0.5 ml of sterilized Tris-acetate buffer solution containing 17 mmol/l NaCl. The culture was serially diluted by decade and an aliquot of 20 l culture which was 102 and 103 diluted was spread

on the selective plates (Tianjin Jinzhang Science Technology Co., Ltd., China). Each sample was enumerated in triplicate. The plates were incubated in an Incucell instrument (MMM, Germany) at 35 C for 24 h. Colonies on the plate were counted by a colony counter (Synbiosis, USA) automatically to obtain the number of CFU of bacteria per ml (CFU/ml) in the sample. ATP measurement procedure Series of standard ATP solution were prepared with sterilized Trisacetate buffer solution. In this protocol, aliquot of 30 l of the ATP solution was added to an optical sensing cell and aliquot of 270 l of luciferin-luciferase reagent (39 g/ml luciferase, 78 g/ml luciferin, 1.1 mmol/l EDTA 2Na, 11 mmol/l magnesium acetate tetrahydrate, 1.1 mg/ml BSA, 0.66 mmol/l DTT, 25 mmol/l Tris-acetate, pH 7.8) was added subsequently. The luminescence patterns and luminescence intensity were recorded by using the portable sensor system TX2007-1 simultaneously. Luminescence intensity was recorded as relative light unit (RLU). Total bacterial count measurement procedure In this protocol, aliquot of 30 l of the bacterial samples was added to the optical sensing cell and first treated with 30 l of cell extracting reagent containing 0.06 mol/l TCA. After an incubation time of 5 min at 25 , the extract was neutralized by addition of 30 l of the neutralization reagent containing 0.06 mol/l NaOH. Aliquot of 210 l of luciferin-luciferase reagent (58 g/ml luciferase, 116 g/ml luciferin, 1.7 mmol/l EDTA 2Na, 17 mmol/l magnesium acetate tetrahydrate, 1.7 mg/ml BSA, 1 mmol/l DTT, 25 mmol/l Trisacetate, pH 7.8) was added and the bioluminescence was immediately measured with the portable sensor system TX2007-1.

RESULTS AND DISCUSSION Selection of emission wavelength Since the wavelength range emitted by the bioluminescence reaction was different with various luciferinluciferase system, first of all, we obtained the wavelength scan of the bioluminescence sensor by using a fluorescence spectrophotometer (F-4500, Hitachi, Japan) at room temperature. In the wavelength range from 200 nm to 900 nm, a strong peak of emission spectra appeared at 550 nm, indicating that the light output of the bioluminescence sensor was entirely centered at 550 nm. Therefore, the emission wavelength of the bioluminescence reaction was selected at 550 nm. Effects of pH on bioluminescence reaction pH studies were performed by using the above standard ATP bioluminescence procedure adopting a standard ATP -8 solution with a concentration of 10 mol/l, and varying the pH of the Tris-acetate buffer through addition of concentrated TCA or NaOH solution. Measurements of pH were performed with a microprocessor pH meter (HANNA, Italy). The effects of pH in range of 4 to 10 in the ATP bioluminescence reaction were examined. Results revealed that pH was a significant factor in luciferinluciferase reactions and when pH was between 7 and 8

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Figure 1. Block diagram of the sensor system.

Figure 2. Effects of pH on the bioluminescence reaction. Each point represents the mean with a standard deviation of triplicate assays.

higher luminescence intensity was produced (Figure 2).

Strong acid or strong base would cause inactivation of

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Figure 3. Effects of TCA concentrations on the intracellular ATP bioluminescence response. Each point represents the mean with a standard deviation of triplicate assays.

luciferase. Therefore, Tris-acetate buffer of pH 7.8 was chosen for the following experiments. Effects of TCA concentrations A successful ATP extraction agent would be that which maximizes the extraction efficiency through minimizing the agent volume ratio thus, maximizing its yield. Through studying the extraction efficiency of saponin, Triton X-100 and TCA on bacterial cells, we found that both E. Coli and S. aureus could be extracted by TCA solution immediately, and the amount of ATP releasing from intact cells was the highest. Therefore, TCA was selected as the extraction agent, and the effect of TCA concentration and extraction time on the performance of the extraction system was studied. To determine an optimal concentration of TCA for the intracellular ATP extraction, a bacterial sample (E. coli, ATCC 25922) with a concentration of 7 1.59 10 CFU/ml was adopted, and various concentrations of TCA (0, 0.01, 0.03, 0.05, 0.07, 0.09, 0.20, 0.40, and 0.80 mol/l of TCA) were used in this experiment. The extraction time was 10 minutes. The neutralization reagents contained 0, 0.01, 0.03, 0.05, 0.07, 0.09, 0.20, 0.40, and 0.80 mol/l of NaOH to stop the extraction procedure. Experimental results showed that luminescence intensity increased in a TCA concentrationdependent manner (Figure 3). We could also obviously find that the luminescence intensity was near zero with 0 mol/l of TCA solution and increased remarkably if the extraction reagent TCA was introduced to the bacterial sample, and a peak was obtained at 0.03 mol/l of TCA. It

was definitely demonstrated that using TCA as extraction reagent could increase the cell wall pore size and degrade the bacterial membrane. Large amount of intracellular ATP was released meanwhile ATP-hydrolyzing enzymes of cells were denatured by TCA simultaneously. This result showed that intracellular ATP could be extracted effectively by TCA. A possible explanation for the decrease of luminescence intensity at higher TCA concentration would be the inhibition of the luciferinluciferase reaction by excess TCA. This result suggested that 0.03 mol/l of TCA was the best extraction agent with the highest extraction efficiency and was used for further study. Effects of extraction times We performed assays in the range of 0 min to 30 min with 0.03 mol/l of TCA (Figure 4). A bacterial sample (E. coli, 7 ATCC 25922) with a concentration of 1.30 x 10 CFU/ml was adopted in this experiment. From Figure 4 we could see that the luminescence intensity was almost zero in the initial extraction stage i.e. the bacterial cells were intact at this point. As time extension, the cell wall was damaged quickly by TCA, large number of ATP released from bacterial cells. In the presence of luciferase reagent, the rapid reaction between ATP and luciferin took place and the luminescence intensity increased quickly. At the time of 5 min the luminescence intensity reached the highest value and decreased subsequently. This result showed that most of target ATP from bacterial cells could be obtained adequately for at least 5 min. The reason for

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Figure 4. Effects of extraction times on the intracellular ATP bioluminescence reaction. Each point represents the mean with a standard deviation of triplicate assays.

the luminescence intensity decreased with longer extraction time was probably the intracellular ATP was hydrolyzed by some ATP-hydrolyzing enzymes of cells. Therefore, the extraction procedure was carried out for 5 min in the following experiments. Measurement of TBC Under the optimal experimental conditions, the determination of TBC in bacterial samples (E. coli, ATCC 25922) was accomplished according to the method described above on the portable bioluminescence sensor system TX2007-1 (Figure 1). Bacterial cells were extracted with 0.03 mol/l TCA. The extraction procedure was completed in 5 minutes. Using the luciferin-luciferase system, the light production was proportional to the amount of bacteria in the samples. By means of linear regression, a good linear relationship was observed between the luminescence intensity and the colony count with the plate count method (Figure 5). The equation derived to describe the lg-lg relationship was: LgRLU = 0.315lg [TBC] + 3.373 (1)

broadened significantly by using the TCA method with the bioluminescence sensor system. The whole testing time including the extraction procedure could be completed within 10 min. The results indicated that under the applied experimental conditions, this TBC detecting method could be used for fast evaluating the colony count of specimen without cultivation. In practice, the data measured in preliminary calibration were inputted into the computer to simulate the corresponding empirical formulas and to store it into the sensor system TX2007-1. Then, the response signals acquired in situ were used, and the concentrations of TBC in the specimen could be calculated from their empirical formulas. Conclusions In summary, our present results show that the bioluminescence sensor system discussed in this study may be employed as a practical method to detect bacterial count in samples without cultivation. Sufficient ATP could be obtained in 5 minutes using TCA as extraction reagent. Based on the ATP bioluminescence reaction, the luminescence intensity is proportional to the TBC of samples and the linearity is good over a wide range of bacterial 7 numbers between 10 and 10 CFU/ml. The detection limit is 10 CFU/ml and one test takes 10 min. Compared with the plate count method, the detection time is greatly shortened and the sensitivity is higher with this portable sensor system. We propose that the portable device discussed in this report is rapid and sensitive enough to used

The correlation coefficient (r) was 0.976. From Figure 5, we can see that the luminescence intensity is directly proportional to TBC in power 0.315. And the dynamic range 7 of the bioluminescence sensor ranges from 10 - 10 CFU/ml. As low as bacterial concentration of 10 CFU/ml could be detected. Compared with the boiling water method (Yang et al., 2002), the detection range was

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Figure 5. Calibration curve for determination of bacterial count in the analysis samples. Each point represents the mean with a standard deviation of triplicate assays.

in microbial quality control. ACKNOWLEDGMENTS This work was supported by Beijing Municipal Science, Technology Commission (Z0006302040191), the NSFC (No. 60576049), and the Hi-Tech R. and D. Program of China (2007AA042105). Grateful thanks were expressed to the microbial laboratory of National Center for Clinical Laboratory for the supply of the reference culture used in this study.
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