Infectious diseases are a leading cause of morbidity and mortality in hospitalized patients. This fact has placed a tremendous burden on the clinical microbiology laboratory to rapidly diagnose the agent responsible for patient’s infection and to effectively provide therapeutic guidance for eradication of the microorganisms. Laboratories are expected to perform these tasks in a cost-effective and efficient manner. Two common methodologies for antimicrobial susceptibility testing in a clinical laboratory are Kirby-Bauer disk diffusion and variations of broth microdilution. The principle is based on the detection of bacterium reproduction ability under the influence of antibiotics. Therefore the testing time is determined by the doubling time of tested bacteria. These methods then usually take from one day to weeks to complete the examination. The long incubation period is inevitable for these conventional methods. Such a waiting period is not short for clinical doctors who urgently need the information to adjust the therapeutic strategy. Therefore it is important to explore new template and technology to perform an antimicrobial susceptibility test.
Surface plasmon resonance biosensing technique is well known for its characteristics of label-free, ultra-sensitive, and real-time detection capability. Thus this technique is considered as the candidate of the new platform. Surface plasmon polaritons (SPPs) was first theoretically predicted by Ritchie in 1957 (Ritchie,1957) based on the analysis of surface electromagnetic modes. The SPPs in general can be generated by electrons (Powell & Swan, 1959) or by light (Otto, 1968) under a proper excitation condition. For SPPs excited by light, in general, the dispersion characteristic of SPPs does not allow the energy of a propagation wave coupled into this surface mode: The spatial phase of a propagation wave is always smaller than that of the surface mode with the same optical frequency on a dielectric-metal interface. Thus an evanescence wave generated by a p-polarized light beam through a prism is suggested to obtain an extra spatial phase and then excite SPPs on the other surface of the metal layer. An alternative method to provide the additional spatial phase is through the aid of a grating, of which the sub-wavelength periodic structure can provide additional spatial phase. For the past two decades, SPPs excited by light has been widely applied to the study of biomaterial processes, which include biosensors, immunodiagnostics, and kinetic analysis of antibody-antigen interaction (Davies, 1996;Rich &Myszka,2005). The main application of SPR biosensors on biomedical science is to analyze the binding dynamics between specific antibody and antigen (Davies, 1996;Rich &Myszka,2005;Safsten et al., 2006;Misono & Kumar, 2005). Since the mode characteristics of SPPs depend on the refractive index of the material within the dielectric-metal interface of about one hundred nanometers, the refractive index of the material determines the resonance incident angle of light, the coupling efficiency, the coupling wavelength, and the optical phase of the reflected light. All the physical quantities can be measured by the reflected light, which is the uncoupled part of the incident light. Therefore, a SPR system does not require fluorescence labeling and provides real-time information with very high sensitivity (Chien & Chen, 2004). This also guarantees a very small amount of sample needed for the detection of the refractive index change through a SPR method.
Most of the biomedical applications of SPR focus on detection and identification of biomolecules. Extended applications have been applied to the detection and sorting of cells or bacteria based on the same principle (Takemoto et al., 1996). The capture of the desired biomolecules with or without cells or bacteria attached is achieved through antibodies or aptamers pre-coated on the metal thin film, where the SPR occurs. The enormous applications of SPR on biomedical science using antibody-antigen affinity can be found in Rebecca L. Rich and David G. Myszka’s Survay (Rich &Myszka,2005). For the methods using antibody-antigen binding, specific antibody is required and finding the specific antibody is usually not straight forward. This is the reason that characterization of antibody is still the main reports from utilization of SPPs. This is also an important reason that a method utilizing antibody-antigen interaction is difficult to use for antimicrobial susceptibility test. Different from the studies mentioned above, the method introduced in this chapter does not require pre-coating of specific antibodies. This method is then more versatile and can be used to detect reactions of drugs appearing on cell membranes or cell walls. While current antimicrobial susceptible testing methods take one day or more for a clinical laboratory to report the testing results (Poupard et al., 1994;Levinson&Jawetz, 1989), utilizing surface plasmon resonance significantly reduces the time duration to less than or about one hour of antibiotics treatment based on our experimental study. Antibiotics which modify or damage the cell walls of bacteria, thus, alternate the refractive index of bacterium surfaces.
Differentiation of susceptible strains of bacteria from resistant ones by using surface plasmon resonance (SPR) technique is discussed in this chapter. This technique detects the refractive index change of tested bacteria subject to antibiotics treatment in real time. Instead of detection the antimicrobial susceptibility through the cell doubling time, the SPR biosensor technology is used to detect the biochemical change of tested bacteria. A much shorter time to obtain the test result is achieved. Because of the feasibility of this antimicrobial test method using surface plasmon resonance biosensors, development of new biosensors is also very important.
2. Devices and methods
The detection principle can be realized on the detection of biochemical change of bacteria subject to antibioticsthrough the detection of their refractive index. This change on the refractive index of bacteria is achieved by an SPR biosensor. A chemical treatment of Poly-L-Lysine on the surface of the Au thin film in the SPR biosensor is used to trap bacteria. The Poly-L-Lysine layer does not provide specfic binding to select specific bacterium strain so that a pre-purification to select tested bacteria is required for the test. After the tested bacterium strain is trapped on the Poly-L-Lysine layer, antibiotic is appled to examine the antimicroial susceptibility.
2.1. Surface plasmon resonance biosensor
The experimental setup for the examination of drug resistance of the bacteria is shown in Fig. 1(a). The setup is the combination of the two parts: one is for the excitation of the surface plasmon and the other is the flow cell chamber. For the excitation of the surface plasmon, a Helium-Neon laser is used as the light source to provide the laser beam with wavelength 632.8 nm. Since surface plasmon can only be excited by p-polarized light, a polarized beam splitter is used to separate the p-polarized and s-polarized light. The s-polarized light is used as the normalization factor to eliminate the deterioration of measurement accuracy caused by the laser instability. After the polarized beam splitter, the p-polarized light is injected onto the Au thin film through a prism to generate surface plasmon. The required phase matching condition to excite the surface plasmon is provided by the proper incident angle and the prism, which provides an extraspatial phase along the gold film surface through its refractive index of the prism. Matching oil is applied between the prism and the glass substrate coated with the Au thin film to avoid occurrence of multiple reflection between the prism and the glass slide. The excitation efficiency of the surface plasmon by the p-polarized laser beam is measured through the silicon photodetector which receives the reflected p-polarized beam from the Au thin layer. When the surface plasmon resonance angle is reached, the energy of injected laser beam was transformed into the surface plasmonpolaritons. Thus, the laser beam reflected from the Au layer reaches minimum. The photocurrent generated from the photodetector is amplified and transformed into a voltage signal via 16-bit A/D converter（Adventech PCI-1716）. The intensity, normalized to the intensity of the s-polarized beam, of the reflected p-polarized beam as a function of the incident angle is obtained by the computer. Incident angle was controlled by a motorized rotation stage through a controller.The other arm that is for receiving reflection was controlled accordingly by another rotation stage to measure the power of the reflected beam. The resolution of the system on the change of refractive index of the dielectrics is refractive index unit (RIU), which corresponds to the value of the SPR angle shift as 0.00867 degree.
2.2. Cell chamber
A flow cell chamber was constructed on the SPR system described above to provide the bacteria for testing, DI water for washing, and the antibiotics for the examination of drug resistance. An O-ring is attached to the chamber to prevent the liquid leakage. A thermister of 10KΩ is used to monitor the temperature of the chamber and a TE cooler is used to control the temperature by receiving the temperature information from the thermister. The temperature of the cell chamber was controlled with the fluctuation less than 0.1 oC, which is achieved by a temperature controller usually used for controlling the temperature of laser diodes. As is depicted in Fig 2, the target bacteria are first injected into the chamber through the flow channel and attach on the gold film by the adhesion of the Poly-L-lysine. Antibiotics are then added to test if the cell walls or membranes are affected.
2.3. Bacterium adhesive coating
Poly-L-Lysine has been demonstrated as an effective tissue adhesive for use in various biochemistry procedures. Poly-L-Lysine solution is diluted with deionized water prior to the coating procedure.The flat glass deposited with Au thin film was immersed in poly-L-lysine solution (concentration = 200ug/ml) for from a couple of hours to 24hours to interact with Au thin film as the preparation of the biochips. Different time intervals provide different adhesion of Poly-L-Lysine to the bacteria and antibiotics. After incubation, cells can be immobilized on the Au-coated glass.
2.4. Bacterium preparation
2.5. Scanning Electron Microscope (SEM) imaging
The glass slide with Au thin film and bacteria was placed in critical point drying (CPD) machine (Samdri-PVT) and filled with Ethanol of 100%. After that liquid CO2 was used to replace the ethanol. The Au thin film with bacteria can then be detached from the glass slide for SEM imaging. Before taking the images, the sample was coated with Au for better conductivity. A scanning electron microscope JEOL JSM-5300 is used for the SEM images.
3. Antimicrobial susceptibility test
To test the drug resistance of bacteria using the SPR system, as depicted in Fig. 3, sterilized DI water was first injected into the flow cell chamber for 30 minutes to stabilize the system after the biochip coated with poly-L-lysine was assembled. Following the stabilization procedure, the incubated LA broth was injected into the cell chamberfor the bacteriato cover the Au metal film.Another washing procedure is applied to remove the bacteria that are not bound to the poly-L-lysine coating. After that an antibiotic solution was injected. The angle of surface plasmon resonance through the entire procedure was recorded as a function of time.
Antibiotics are classified into several categories depending on its mechanisms on the interruption of cell activities, namely cell wall synthesis, cell membrane synthesis, protein synthesis, folic acid biosynthesis, DNA gyrase, and RNA polymerase.
3.1. Gram negative bacterium–
3.1.1. Injection with LB
Since surface plasmon resonance is very sensitive to the refractive index change of the cells attached on the thin gold film, ampicillin as the antibiotics interrupting cell wall synthesis is chosen in this experiment. The mechanism of ampicillin is depicted in Fig. 4. As is shown in Fig. 4(a), the cell wall and membrane of
The SPR angle of antibiotic resistant strain of
This difference of the resonance angle shift can be more pronounced when the concentration of the ampicillin increases to 100ug/ml. As was shown in Fig. 6, the angle shift of the ampicillin-resistant strain of
The damage degree of theampicillin,with concentration of3 ug/ml, on the cell walls of the antibiotic susceptible strain is examined by SEM. The
In order to examine the reproducibility of the result, totally ten sets of resistant and susceptible strains of
3.1.2. Injection with DI water
In order to increase the accuracy of the antimicrobial susceptibility test. The coating time of Poly-L-Lysine was optimized from 24 hours to a few hours. Meanwhile, the LB injected with bacteria and for removing the unbound bacteria was replaced by DI water for reducing the interference of LB. After the adjustment, the amount of unbound or unstably bound bacteria was reduced significantly. As was shown in Fig. 9, the rinse procedure of DI water did not decrease the SPR angle from the saturation phase of bacterium adhesion as much as the situation in the injection with LB protocol. The ampicillin of 50ug/ml was applied from the time points indicated by the arrows. As shown in Fig. 9 (a), the resistant strain of
The result has demonstrated that the improved method has better accuracy in comparison with the method mentioned in the section 3.1.1. The same method using ampicillin of different concentrations, listed as 25 ug/ml, 50 ug/ml, and 100ug/ml, was also performed and the result was shown in Fig. 10. The resistant strain and susceptible strain of
3.2. Gram positive bacterium–
The protocol of DI water injection can also be used for gram positive bacteria. This tested object is
3.3. Differentantibiotics - tetracycline
An interesting question has arisen if the same method can be used to detection antimicrobial susceptibility by antibiotic with different mechanism. For this purpose and also served as a blind test, another bacterium,
It is import to mention that the serum is not supplied into the system, the growth rate of
We have reported two innovative antimicrobial susceptible testing methods utilizing surface plasmon resonance. One is injection with LB liquid. The other is injection with DI water. In the study, the drug resistance of the gram negative bacteria,
This work was supported by research grant NSC 97-2627-M-010-005- and NSC 99-2112-M-010-001-MY3 from National Science Council in Taiwan and by “A grant from Ministry of Education, Aim for the Top University Plan” from National Yang Ming University.