AUTOFLUORESCENCE BASED BACTERIA DETECTION USING AN OPTICAL FIBER Indu Saxena, Darin Files, Srivatsa Rao, Intelligent Optical Systems, Inc. 2520 W. 237th St. Torrance, CA 90505
[email protected] and W. J. Costerton, Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717 Abstract We report here on bacterial biofilm detection with an optical fiber probe and a compact detection system. This probe was tested on cells of the Pseudomonas aeruginosa and other species of bacteria in planktonic and sessile forms, and optical signal changes corresponding to the number density of the bacterial cells were measured.
1.0 INTRODUCTION
Bacteria are one of nature’s most abundant and viable life forms.[1,2] Sessile forms of these bacteria form biofilms on surfaces and can nurture additional pathogens. These biofilms often cause major health problems when infesting man-made medical delivery systems that should, ideally, be sterile. It is well established that biofilms are the main cause of contamination of water delivered by dental equipment.[3-9] The high surface-to-volume ratio in the tubing, and the fluid dynamics in narrow smooth-walled water lines, provide a unique environment for biofilm development in dental tubing. Williams et al. investigated the nature of biofilm bacterial species colonizing dental units[9] and found several, including Pseudomonas, Pasteurella escherichia, Legionella, and Flavobacterium species. These bacteria can cause a variety of infections and illnesses.[6, 10-13] The goal of this ongoing work is to develop a reliable on-line biofilm detection instrument to determine biofilm presence in a timely manner to enable countering the disease-causing effects of infesting bacteria. Reliable biofilm detection can be accomplished by detecting biomarker chemical species unique to biofilms,. Some biofilm markers are tryptophan and exopolysacharides (EPS), which indicate the presence of living bacteria. IOS has demonstrated an instrument, referred to as the Bioprobe, which is based on fluorescence-based detection using optical fibers for instant biofilm detection. We measured the auto fluorescence of bacteria with an optical fiber; interaction with the biofilm's inherent constituents without addition or modification of the bacteria caused changes in the optical signal. The sub-millimeter size of optical fibers allows them to be easily inserted into narrow dental unit water lines (DUWLs) for bacteria monitoring.
2.0 EXPERIMENTS The experiments used to establish the bacteria detection method proceeded in three stages, i) culturing the bacteria for testing; ii) Instrument design and assembly; iii) Measurement of bacterial biomarkers; iv) cross-validation and correlation of bacteria measurement with the optical fiber probe and with traditional bacteria counting methods. 2.1
Bacterial Culturing
2.1.1
Preparation of Cultures
Biofilms were cultured from Pseudomonas aeruginosa ERC-1 (ATCC Number: 700829) bacteria samples obtained from the Biofilms Systems Training Laboratory (BSTL) at the Center for Biofilm Engineering (CBE). This single strain of bacteria was used in all the bacterial studies reported here. 2.1.1.1
Preparation of Inoculum
A plate culture of P. aeruginosa ERC-1 in R2A agar was used for these studies. A small amount of bacterial mass was removed using a sterile inoculating loop. This bacterial inoculum was suspended in a sterile Erlenmeyer flask containing 300 mg/L tryptic soy broth (TSB). The flask was incubated at room temperature on a shaker overnight. In 24 hours, the bacteria multiplied in TSB and the solution became cloudy. This solution was used to make optical probe measurements (shown in figure 6) with the suspended (planktonic form) bacteria and also to inoculate the biofilm rotating disk reactor (RDR) containing polycarbonate coupons.
2.1.1.2 Rotating Disk Reactor (RDR) for Biofilm Cultures The experimental setup used to conduct these experiments is shown in Figure1. The rotating disk reactor (RDR) is a standard tool for studying the time dependent growth of biofilms under shear stress. The RDR is used because biofilms tend to fall off during recovery and processing in systems with no shear, and because biofilms made in zero shear lack tensile strength. RDR-grown films also more closely resemble biofilms that are encountered in practical applications, since most water/fluid delivery systems, including DUWLs, have some flow. The flow cell is equipped with recessed coupon slots machined into the flow surface (see Figure 2). Biofilm was grown on coupons in media recirculating via a peristaltic pump through the RDR. Fluorescence changes occurring at the surface of biofilm (on sample coupons) were measured regularly. The response of the device to different biofilm thicknesses was calibrated by measuring biofilms on each coupon at different elapsed times. The cross calibration of biofilm was done by direct measurement of cell count using standard "plating" techniques by members of the Montana State University Center for Biofilm Engineering team. Figure 1 shows the RDR system for growing biofilms. The RDR vessel is essentially a 1-liter glass beaker fitted with a drain spout. The bottom of the beaker contains a magnetically driven rotor with six 1.27 cm (0.5") diameter biofilm test-surface coupons. Coupons of any material can be used in these studies. For our studies, we used polycarbonate coupons. The rotor was constructed from a bottom star-head magnetic stir bar. A Teflon and silicone rubber disk is attached to the stir bar to hold the coupons. The coupons rotate continuously to provide fluid shear. Biofilm growth nutrients are pumped continuously into the vessel.
Figure 1 Rotating disk reactor showing test coupon holder with [15] growth coupons inserted.
Figure 2 Test coupons: disk on left is polycarbonate; disk on right is stainless steel.
Sample preparation The coupons were harvested by carefully removing the rotor from the reactor using sterile dental tools. The bacterial growth mass was scraped from the coupon and the coupon was rinsed with 1 ml of fresh dilution water to bring the final volume to 10 ml in the dilution tube. This dilution must be "disrupted" to form a single cell suspension. We used 15 seconds in a sonicating bath and then proceeded with the serial dilutions for the measurements of figure 6. The coupons were removed two at a time to obtain a calibrated data point for each reading. One of the two identical coupons was used to obtain the bacterial cell count (in cfu/ml) by using the plating method, while the other was probed to obtain an optical signal using a fiber probe. The serial dilutions were plated according to the drop plate method.[15] 2.2 Bioprobe Instrument Figure 3 shows IOS’s Bioprobe detection system for the optical fiber based on-line biofilm sensor. Excitation light is sent through a fiber coupler and transmits down the fiber to the Bioprobe to excite biofilm markers present in the bacteria. The Bioprobe collects the emitted autofluorescence from the bacteria and transmits the signal back along the fiber through the coupler and to the detection system.
Figure 3: Prototype Bioprobe instrument.
The instrument measures 6 × 6 × 10, and includes an additional reference input port and detector. The fiber-end that detects the autofluorescence signal is mounted in a laboratory fixture for ease of positioning.
2.3
Bioprobe Measurements with Biofilm Markers
The autofluorescence of biofilm biomarkers 1 and 2, which were measured in real time, are shown in Figures 4 and 5, respectively. Direct signals in real time are output, that have been calibrated to bacterial count by the drop plate count. In comparison to our measurements with bacteria, the lowest signal level (i.e., generated by 0.122 mM of marker 1) detected corresponds to that generated by approximately 1,000 cfu of the tested bacterial species.
Portable Bioprobe Signal (A.U.)
0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0
0.2
0.4 0.6 Marker 1 Concentration Factor
0.8
Figure 4 Marker 1 detection with Bioprobe (unit concentration = 2.82 mM).
1
Portable Bioprobe Signal (A.U.
1.6 1.4 1.2 1 0.8 0.6 0.4 0
0.2
0.4
0.6
0.8
1
Marker 2 Concentration Factor
Figure 5: Marker 2 detection with Bioprobe (unit concentration = 4.85 mM).
2.4 Cross Validation Bacterial Cell Counts Correlated with Fiber Probe Measurement
Each of the planktonic dilutions of the cultured bacteria that were measured with the fiber probe were plated for counting by the standard bacteria counting technique. The results of the plating count were then plotted as a function of the optical signal intensity shown in figure 6 below.
Figure 6 Planktonic bacterial biofilms were detected using a fluorescence-based optical bioprobe. Fluorescence intensity was measured by IOS’s Bioprobe, and was calibrated against the planktonic bacterial count determined independently by the plating method.
In Figure 6, the lowest count detectable with a 200 µm core fiber probe is 1 × 105 cfu/ml. The spot size of the excitation light at the end of the cuvette containing the planktonic bacteria was 3 mm, giving a sample volume of 0.07 cm3 as the fiber has a numerical aperture of 0.22. Hence, the number of planktonic bacteria interrogated were about 2500 cfu.
3.0
Summary
The basis of the technology presented is that bacteria can be detected by inducing autofluorescence of biofilm markers and has been shown by detecting strong characteristic fluorescence signals, in a bacterial cell concentrationdependent manner. Specifically, we reported the following: 1. Cultured the Pseudomonas aeruginosa species of bacteria, in planktonic and sessile forms, in rotating disk reactors (RDRs), to create shear (flow) conditions typical of dental unit water line environments in which biofilms have been observed to grow; 2.
Demonstrated the distinct on-line detection of P. aeruginosa bacteria film on polycarbonate tubes (coupons). IOS used a compact and flexible optical fiber to deliver excitation light and collect the autofluorescence of bacterial markers in planktonic and sessile bacteria with the same sub mm single fiber probe;
3.
Quantified bacterial detection by using existing plating methods for measuring bacterial numbers; and
4.
Designed and tested a cost effective portable prototype detection instrument to demonstrate the viability of this sensing and detection technology.
The prototype probe, which consists of an optical fiber to deliver the excitation light and a coupled fiber to collect the induced fluorescent light, was immersed in suspensions of planktonic (floating) cells of Pseudomonas aeruginosa. This prototype probe was used to induce and collect fluorescent light in suspensions of bacterial cells whose cell concentrations had been adjusted by dilution with fresh medium, so that they contained cell concentrations ranging from 105 to 108 cells/ml (see Figure 6). The fluorescent signals obtained from these suspensions of planktonic cells were found to be proportional to the number of cells present, with an excellent correlation to the data. These data show a clear relationship between numbers of cells and signal strength. We clearly demonstrated the ability of the prototype Bioprobe to quantitatively detect biofilms formed by cells of P. aeruginosa, showing that the Bioprobe system can easily detect colonies of bacteria in biofilms formed in conditions similar to those found in dental water lines.
ACKNOWLEDGEMENTS: THIS REPORT SUMMARIZES THE WORK PERFORMED BY INTELLIGENT OPTICAL SYSTEMS, INC. (IOS) FROM JULY 15, 2000 TO JANUARY 15, 2001 UNDER THE NIH SBIR PHASE I CONTRACT ENTITLED "IN SITU DETECTION OF DENTAL UNIT BIOFILMS", CONTRACT NO. 1R43DE1298901A1.
5.0
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