Research Express@NCKU - Articles Digest
Research Express@NCKU Volume 11 Issue 9 - December 11, 2009 [ http://research.ncku.edu.tw/re/articles/e/20091211/1.html ]
Antimicrobial susceptibility testing method using surface plasmon resonance
Chi-Hung Lin
1, Shean-Jen Chen
2*, How-Foo Chen
31Institute of Microbiology & Immunology, National Yang Ming University, Taipei, Taiwan
2Department of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan
3Institute of Biophotonics, National Yang Ming University, Taipei, Taiwan sheanjen@mail.ncku.edu.tw
Y.-L. Chiang, C.-H. Lin, M.-Y. Yen, Y.-D. Su, S.-J. Chen, H.-F. Chen, “Innovative antimicrobial susceptibility testing method using surface plasmon resonance,” Biosensors & Bioelectronics, vol. 24, no. 7, pp. 1905-1910, March 2009.
S
urface plasmons was first predicted by Ritchie in 1957 and demonstrated by Otto using optical excitation in 1968. For the past two decades, SPs excited by light has been widely applied to the study of biomaterial processes, including biosensors,immunodiagnostics, and kinetic analysis of antibody–antigen interaction. The main application of surface plasmon resonance, SPR, on the biomedical science is to analyze the binding dynamics between antibody and antigen and to sense specific antigen through the chemical binding of corresponding antibody. Although dynamic analysis is required
for measuring affinity of cognate molecules, most of the applications of SPR so far can be considered as stationary measurement. It is only used on detection or recognition of cognate targets or development of antibodies. Until recently, using SPR biosensor on detecting whole cells was explored. These efforts include an observation of cell/
substrate contacts, a reaction of cells to toxicity, and detection of cholesterol concentration in cell membranes.
Infectious diseases are a leading cause of morbidity and mortality in hospitalized patients. This fact places 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 organism. In agriculture, rapid diagnosis of drug resistance of bacteria also has enormous impact on agriculture economy. Besides, laboratories are asked not only to perform these tasks with efficiency, but also in a cost effective manner in an era of increasing emphasis on reduction of laboratory expenses. Two common methodologies for antimicrobial susceptibility testing in a clinical laboratory are Kirby-Bauer disk diffusion and variations of broth microdilution. These methods usually take from 1 day to weeks to complete the experiments and issue the reports. Such a waiting period is not short for patients and clinical doctors who urgently need the information to adjust the therapeutic strategy.
To our best knowledge, this paper is the first effort to detect the dynamical change of bacteria to antibiotics by SPR biosensor. While current antimicrobial susceptible testing methods execute antimicrobial test based on the detection of cell duplication ability under the influence of antibiotics, the SPR biosensing method directly detect chemical or physical change of bacteria subject to antibiotics. This research then focused on exploring new application of SPR biosensing technique on antimicrobial susceptibility test. It is expected to take much shorter time than the conventional methods do. Since SPR is highly sensitive to the change of the refractive index of cells near the cell–metal interface, ampicillin as the antibiotic inhibiting the synthesis of cell walls was used for the examination of E. coli JM109.
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Research Express@NCKU - Articles Digest
Since SPs penetrate into cells approximately in the order of 100 nm, the biosensor is very sensitive to the change of the refractive index of cell walls and membranes attached on the Au thin film within that range – no matter what causes the change. The mechanism of ampicillin is to interrupt the activity of transpeptidase, which assists the formation of peptide bond combined with peptidoglycan to constitute cell walls, and then to interfere cell growth and proliferation. Therefore, for the measurement of direct influence of antibiotics, ampicillin was used as the antibiotic for the antimicrobial susceptibility test. E. coli JM109 strains were used as the tested objects.
To test the drug resistance of E. coli JM109 using the SPR system, sterilized DI water was injected into the cell chamber for 30 min 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 fluidic cell chamber for about 60 min to cover the Au thin layer. Another wash procedure was followed to remove the bacteria that were not bound to the Poly-llysine layer. When the bacteria binding reached a steady state, the ampicillin solution with concentration 3μg/ml was injected. The resonance angle of surface plasmon through the entire procedure was recorded as a function of time.
Fig. 1 Kinetic plot of SPR angle shift for the antimicrobial susceptibility test of E. coli to ampicillin for 30 min: (a) angle shift of the ampicillin-resistant strain is around −0.00154◦ after the treatment for 30min; (b) angle shift of the ampicillin-susceptible strain is around −0.01608◦ after the treatment for 30min.
The SPR angle of antibiotic-resistant strain of E. coli JM109 over the operation procedure described above is shown in Fig. 1(a) and that of antibiotic-susceptible strain is shown in Fig. 1(b). The shift of the SPR angle was referred to the value of the SPR angle before the E. coli strain was injected into the cell chamber. As is shown in Fig. 1(a), the SPR angle increased when the bacteria were injected into the cell chamber. After the amount of the bacteria attached to the Au thin film coating was saturated, DI water was injected to remove the unbound bacteria.
The SPR angle dropped dramatically during this procedure. After bacteria binding reached steady state, the 3 μg/
ml ampicillin was injected into the cell chamber. The value of SPR angle, changed by the refractive index of the bacterium surface, was recorded over time. The same procedure was applied on the susceptible strain and the result is shown in Fig. 1(b). The result shows that, after 30min treatment of ampicillin, the decrease of the SPR angle for the resistant and the susceptible strains were −0.00154◦ and −0.01608◦, respectively. The angle shift is about 10 times difference between the resistant strain and the susceptible strain. The decrease of the refractive index, which is depicted by the negative angle shift, is possible from the structure loose of bacteria cell walls or even breakdown due to the effect of ampicillin. Since the antibiotic-resistant strain is more resistant to ampicillin, the refractive index of its cell walls did not decrease as much as that of the susceptible strain.
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Fig. 2. SEM scanning pictures: (a) E. coli without the treatment of ampicillin; (b) ampicillin-resistant strains after 30-min treatment of ampicillin; (c) ampicillin-susceptible strains after 30-min treatment of ampicillin.
The damage degree of the ampicillin on the cell walls of the strain susceptible to ampicillin was examined by scanning electron microscope (SEM). The E. coli before the treatment of the ampicillin is shown in Fig. 2(a). The antibiotic resistant and susceptible E. coli strains after the antibiotic treatment for 30min are shown in Fig. 2(b) and (c), respectively. A comparison between the SEM pictures revealed no significant change in the appearance of the resistant strain and that of the susceptible strain subject to the treatment of ampicillin for 30 min. However, after 5 h of treatment of ampicillin, the susceptible strain shrank, which was verified by SEM.
We have reported an innovative antimicrobial susceptible testing method utilizing SPR. Susceptible and resistant strains of E. coli (Gram-negative) JM109 to ampicillin were examined. Since ampicillin directly acts on the formation of cell walls, the susceptible strain of E. coli revealed monotonic decrement over time on the SPR angle.
The experimental result has shown the feasibility of utilizing SPR biosensors for fast antimicrobial susceptibility test. Quantitative analysis of drug resistance has to be studied and established.
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