Dead/alive bacteria detection using an all-fibre optical ...

Dead/alive bacteria detection using an all-fibre optical system. E. Bogomolnya, S. Swiftb, M. Cheng,b S. van Binsbergenc, F. Vanholsbeecka* a

Department of Physics, The University of Auckland, New Zealand. School of Medical Sciences, The University of Auckland, New Zealand c Optical Sciences Group, Department of Science and Technology, Twente University, The Netherlands *corresponding author: [email protected]. b

ABSTRACT Accurate monitoring of microbial viability plays an essential role in pharmacodynamic studies such as in estimating the efficiency of antimicrobial agents. Traditionally, bacterial viability is determined by their ability to form colonies on solid growth medium or to proliferate in liquid nutrient broths but, with these culture-based methods, the live bacterial population can only be estimated retrospectively. To address this challenge, we have employed differential fluorescence staining and an all-fiber optical system developed by our group. The detection is based on the collection of the fluorescence from commercial dyes that produce a substantially increased signal upon binding with bacterial nucleic acids. The dyes allow discrimination between alive and dead cells through differential membrane permeability and fluorescence wavelength. The respective fluorescence signal is correlated to the number of bacterial cells present in the sample. Our setup uses DPSS lasers and a sensitive CCD-based spectrometer over the 400-800 nm wavelength range. A laser shutter allows the sample exposure time and acquisition time to be synchronized to minimize the effect of photobleaching. As a model, bacteria (Escherichia coli or Staphylococcus aureus) killed with isopropyl alcohol were mixed with live cells at different ratios. The population ratios of alive and dead cells were accurately quantified by our optical setup providing a rapid method for the estimation of bactericidal treatments. In summary, our optical system may offer a robust, accurate and fast alternative for detection of dead/alive bacteria in turbid solution opening the new avenues for pharmacodynamic studies. Keywords: dead/alive bacterial detection, optical system, fluorescence, fibres, nucleic acid dyes.

1. INTRODUCTION Over the last few years, much concern has been raised regarding the optimization of antibiotic use, owing to the worrying increase of bacterial resistance and to the scarcity of new antibiotic classes under development [1]. In this context, progress in the field of anti-infective pharmacology has led to the emergence of a new need to understand the relationships between the efficiency of bactericidal factors and drug concentrations and/or the time of drug exposure. The death of a bacterial cell has long been defined as the inability of a cell to grow to a visible colony on bacteriological media. Using culturing methods to assess cell death has many limitations, the major one being that one can observe bacterial death only retrospectively after at least 24 hours of culturing [2]. The LIVE/DEAD BacLight kit (Invitrogen) was released about 10 years ago [3] to address the increasing demand among researchers in various fields for assessing bacterial viability without the need for culturing methods. The kit consists of two stains, propidium iodide (PI) and SYTO 9, which both stain nucleic acids. Green fluorescing SYTO 9 is able to enter all cells and is used for assessing total cell counts, whereas red fluorescing PI enters only cells with damaged cytoplasmic membranes, and is assumed to stain dead cells [4]. The emission properties of the stain mixture bound to DNA change due to the displacement of one stain by the other and quenching by fluorescence resonance energy transfer [5]. LIVE/DEAD staining is effective not only for bacteria but also with archaea or eukaryotic cells, making this a universal stain [6,7].

Frontiers in Biological Detection: From Nanosensors to Systems VI, edited by Benjamin L. Miller, Philippe M. Fauchet, Brian T. Cunningham, Proc. of SPIE Vol. 8933, 89330F · © 2014 SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2039680 Proc. of SPIE Vol. 8933 89330F-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 12/07/2014 Terms of Use: http://spiedl.org/terms

This LIVE/DEAD kit can be used with a few fluorescence technologies like a flow cytometry, epifluorescence microscopy and fluorospectrometry that have shown promising results to answer industrial and/or research needs [8-10]. Difficulties exist, however, in part from requirements for sensitivity, specificity and accuracy. Here, we suggest using an all fibre spectroscopic device (Optrode) in combination with the LIVE/DEAD kit to asses in situ and in real time cell viability. The major advantages offered by our optical system are [11-14]: a) Sensitivity. This is due to the efficient delivery of 10mW of light for in situ fluorescence excitation and detection as well as the use of a sensitive CCD spectrometer, stable excitation laser sources, and an appropriate collection volume of the fibre probe. The system can detect as low as 1 CFU/ml. b) Accuracy. By monitoring the laser power, we take into account the laser power fluctuations while measuring the fluorescence signal. Also a synchronized laser shutter allows us to acquire a signal with a high signal to noise ratio (SNR) with minimal integration time, thereby reducing the photobleaching effect. Computerized data processing and multivariate analysis further decrease the measurement variance. c) Portability. The current model has a footprint of 483mm x 335mm with a height of 128 mm and a weight of 12 kg. d) Near real time data acquisition. e) Ability to detect a wide range of bacteria concentrations without using dilution or filtration. The dynamic range examined in this study is 1-108 CFU/ml.

2. METHODOLOGY 2.1 Optical layout. A schematic diagram of our fibre optic fluorescence system is shown in Figure 1 and described elsewhere [1114]. Light from the laser is delivered into a 2x2 multi-mode 50/50 fibre coupler by a coupling stage. A laser shutter, controlled by a data acquisition card (DAQ), is placed in front of the fibre coupling stage to synchronize the exposure time of the sample to the acquisition time of the spectrometer so that the effect of photobleaching is minimized. Half the excitation light is guided to the sample via one of the output arms of the coupler while the balance is directed through the other output arm to a photodiode for monitoring the laser noise. A fraction of the fluorescence from the illuminated sample is collected at the tip of the excitation fibre and guided to a compact fibre-coupled spectrometer via the 2x2 multi-mode fibre coupler. A laser line emission filter blocks the remaining of the excitation light from reaching the spectrometer. The spectrometer is connected via a USB connection to the computer that is used for data acquisition and spectral analysis. Diode pumped solid state lasers (DPSS, CNI Tech) at wavelengths of 473 nm and 532 nm were used as excitation sources. They have an output power of 100 mW with power fluctuations lower than 5% and a noise level lower than 3%. The delivery of excitation light and the collection of the fluorescent emission is achieved using high OH–, multimode, optical fibre (CF04406-03; it has excellent characteristics in efficient power transmission from the UV to the visible light range). The fibre core, cladding and coating diameters are 200, 220 and 250 μm respectively with a numerical aperture (NA) of 0.22. Attenuation across the visible range is less than 0.014dB/m and the total fibre length was less than 5 m. After end-fire fibre coupling, the fibre coupler, and power adjustment using the collimating lens, 10mW of excitation light is delivered to the sample. To control the excitation light, we use a custom made shutter (OZ Optics) to only let the laser light through when acquiring data. This helps to minimize the sample photobleaching, and improves the laser stability. A scientific-grade 16-bit spectrometer (QE65000, Oceanoptics) with thermal electric cooling (–15 °C) was used for this low-light level application. The detection unit is a 2-dimensional charge-coupled device (CCD) array with a sensitivity of 0.065 counts/e-. The quantum efficiency is greater than 80% in the range of the interest (505-550 nm). The dark noise of the spectrometer is 3 root mean square (RMS) counts. A 16 bit DAQ card (USB1608FS, Measurement Computing) was used to synchronize the shutter with the spectrometer’s acquisition time.

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DPSS 100mW

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USB cable Optical fibre Electric cable

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setup controlled by Labview code

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Figure 1. (A) Schematic diagram of the all-fibre spectroscopic system. (B) Picture of the optical setup.


2.2 Data Acquisition, signal processing and fluorescence measurements A graphical interface code was written in Labview (National Instruments Co). It allows users to monitor the laser power, to adjust acquisition parameters and to perform initial signal processing (background subtraction, averaging, and normalization according to illumination power). The interface is coded to allow a simple control and synchronization of the DAQ card trigger and the spectrometer. The code saves the raw data and scans for the desirable SNR with an appropriate integration time. These data can be displayed in real time for acquisition rates up to 250 Hz. 2.3 Samples Two types of laboratory grown bacteria were utilized in our study: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538). The bacteria were grown to late log phase at 37 oC in tryptic soy broth. Micro-organisms were harvested by centrifugation at 10,000 × g for 10 minutes, supernatant removed, and the pelleted cells resuspended in saline (0.85% w/v) and divided into two tubes. Isopropyl alcohol was added to one tube, to a final concentration of 70% by volume, to provide killed bacteria. Both tubes were incubated at room temperature for 1 hour, with mixing every 15 minutes. The killed bacteria in the tube with isopropyl alcohol were pelleted by centrifugation as before, washed twice with saline and then resuspended in saline. Using these tubes, the live and dead-cell suspensions were mixed to achieve various live/dead bacteria ratios within 1 ml samples containing approximately 108 bacterial cells. 2.4 Staining protocol and dyes The two dye components provided with the LIVE/DEAD BacLight Bacterial Viability Kits have been added to the samples. 1 µl of dyes (PI and SYTO 9) in DMSO solution were added to a microfuge tube that contained the 1 ml of the sample. The dye was mixed for 3 minutes by vortexing and kept in the dark at room temperature for 15 minutes to allow binding. Micro-organisms were pelletted in a microcentrifuge at 10,000 rpm for 5 minutes. The supernatant containing unbound dye particles was removed and the bacterial pellets were resuspended in 1 ml of sterile water by 3 min of vortexing before being taken to the optical setup.

3. RESULTS First, it was essential to examine optical acquisition parameters for double stained bacteria to acquire high quality spectra (a high SNR) and minimize the photobleaching effect. The photostability of the fluorescent dyes is important to ensure statistical accuracy of the quantitative fluorescence measurements.


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time [ms] Figure 2. Photobleaching of the doubled stained bacteria under excitation by 473 nm (blue) or 532 nm laser light and with the maximal fluorescence peaks at 574 nm and 637 nm respectively.

Figure 2 demonstrates fluorescence intensity versus exposure time of the lasers. The fluorescence intensity decreases exponentially and exhibits photobleaching half-life times of 1.53 and 1.7 s at the maxima intensity of 573 nm and 637 nm for the 473 nm and 532 nm lasers respectively (see figure 2). Thereby, by using 8ms as an integration time for our measurements, the photobleaching would not decrease the fluorescence intensity by more than 1% while keeping the SNR above 50.


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Wavelength[nm] Figure 3. The normalized fluorescence spectra of double stained microorganisms. (A) E..coli (B) S. aureus.

The typical fluorescence spectra are shown in figure 3. Similar fluorescence patterns are observed for both bacterial species, with two distinct fluorescence peaks at 574 nm and 637 nm. The fluorescence signals were normalized to the maxima of these peaks for non-mixed dead/alive samples at 108 CFU/ml concentrations corresponding to 532 nm/473 nm excitation wavelength respectively. This normalization allows us to reduce the variance due to variations in bacteria number measured by single fibre probe and corrects for different binding efficiency of the nucleic acids dyes.


Normalized fluorescence ratio Fex 532 nm/Fex 473 nm

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Staphylococcus a. E.coli R2=0.975 R2=0.944

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Dead/alive bacteria mixture ratio [%] Figure 4. The quantified assessment of dead/alive population double stained with PI/SYTO 9

In the next step we assessed the bacterial viability by producing a supervised mixture of dead/ alive volumes (at least 3 measurements for each sample). Figure 4 shows that there is clear linear regression fit between the proportional volumes of the dead/ alive bacteria and their corresponding normalized fluorescence spectra. Thereby the population ratios of alive and dead cells were accurately quantified by our optical setup providing a rapid method for the estimation of bactericidal treatments.

4. CONCLUSIONS In summary, our all-fibre optical system offers a robust method for assessment of bacterial viability and presents a real alternative from the conventional methods. The major advantages of the all-fibre optical system are portability, sensitivity, near real time measurements and the ability to detect a high dynamic range of bacteria concentrations between 1 and 108 CFU/ml without any need for dilution.

REFERENCES [1] Bosso J.A., “The antimicrobial armamentarium: Evaluating current and future treatment options,” Pharmacotherapy 25 (10P2), 55S-62S (2005). [2] Postgate, J. R., “A microbial way of death,” New Sci. 122, 43–47 (1989). [3] Boulos, L., Prevost, M., Barbeau, B., Coallier, J. & Desjardins, R.,” LIVE/DEAD (R) BacLight (TM): application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water,” J Microbiol Meth 37, 77-86 (1999). [4] Berney, M., Weilenmann, H. U. & Egli, T. “Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS),” Microbiology 152, 1719-1729 (2006). doi:DOI 10.1099/mic.0.28617-0 [5] Stocks, S. M., “Mechanism and use of the commercially available viability stain, BacLight,” Cytom Part A 61A, 189-195 (2004). [6] Zhang, T., and H. H. Fang. , “Quantification of Saccharomyces cerevisiae viability using BacLight,” Biotechnol. Lett. 26, 989–992 (2004).


[7] Leuko, S., A. Legat, S., Fendrihan, H., Stan-Lotter. “Evaluation of the LIVE/DEAD BacLight kit for detection of extremophilic archaea and visualization of microorganisms in environmental hypersaline samples,” Appl. Environ. Microbiol. 70, 6884-6886 (2004). [8] Berney, M., Hammes, F., Bosshard, F., Weilenmann, H.U., and Egli, T., “Assessment and interpretation of Bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry,” Applied and Environ. Microbiology, 73(10), 3283-3290 (2007). [9] Zetsche, E. M. & Meysman, F. J. R., “Dead or alive? Viability assessment of micro- and mesoplankton,” J Plankton Res. 34, 493-509 (2012). [10] Corich, V., Soldati, E. & Giacomini, “A. Optimization of fluorescence microscopy techniques for the detection of total and viable lactic acid bacteria in whey starter cultures,” Ann. Microbiol. 54, 335-342 (2004). [11] Bogomolny, E., Swift, S. & Vanholsbeeck, F., “Total viable bacterial count using a real time all-fibre spectroscopic system,” The Analyst 138, 4112-4119 (2013). [12] Patel A., Bogomolny E., Cheng M., and Vanholsbeeck F., “Sensitivity improvement of an all-fibre computerized optical fluorescence setup using dual fibre probes,” European Conferences on Biomedical Optics, 87980F-87980F-5 (2013). [13] Bogomolny E., Swift S., Patel A., Cheng M., and Vanholsbeeck F., “Near real time accurate bacterial enumeration in aquatic environment using an all-fibre optical system,” European Conferences on Biomedical Optics, 87980N-87980N-4 (2013). [14] Hewitt B.M., Singhal N., Elliot R.G., Chen A.Y., Kuo J.Y., Vanholsbeeck F., and Swift S., “Novel fiber optic detection method for in situ analysis of fluorescently labeled biosensor organisms,” Environ Sci Technol, 46(10), 5414-5421 (2012).
