SINGLE CELL ANTIMICROBIAL SUSCEPTIBILITY TESTING USING ... · SINGLE CELL ANTIMICROBIAL...

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SINGLE CELL ANTIMICROBIAL SUSCEPTIBILITY TESTING USING CONFINED MICROCHANNELS AND ELECTROKINETIC LOADING Yi Lu and Pak Kin Wong * The University of Arizona, USA ABSTRACT This study reports a single cell antimicrobial susceptibility testing (AST) technique using confined microchannels and electrokinetic loading. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacteria (~2 µm), their growth can be monitored with high resolution at the single cell level. Furthermore, AC electrokinetics is successfully demonstrated in high-conductivity culture media to facilitate dynamic loading of bacterial pathogens into the confined microchannels with over 80% loading efficiency. The single cell AST technique allows antibiotic resistance profiling to be finished in less than one hour compared to days in standard culture based techniques. KEYWORDS: Single Cell, E. coli, Antimicrobial Susceptibility Testing, Microchannels, Electrokinetics INTRODUCTION Infectious disease caused by antibiotic resistant bacteria represents a major challenge in the healthcare system worldwide. To determine effective treatment for the patient and proper clinical management of the disease, AST is required to evaluate the antibiotic resistant of the bacterial pathogen. However, the standard culture based AST approach requires at least 48 hours from initial sample to result. The lack of rapid diagnostics has largely driven the overuse of antibiotics, which in turn accelerates the emergence of multidrug resistant pathogens or superbugs. One of the major challenges in current culture based AST is the long culture time required to grow the bacteria to proper concentration for genotyping or phenotyping testing. Furthermore, the bulk measurement of bacteria with diverse growth behaviors can mask the growth of the bacteria and increases the assay time required to obtain statistically significant data. To address this critical problem in clinical management of infectious diseases, we have previously shown that enhanced oxygenation using high surface-to-volume ratio PDMS microchannels facilitates bacterial growth at the point of care [1]. In this study, rapid AST is performed at the single cell level using confined microchannels to eliminate the uncertainty in bulk measurement and to reduce the total assay time [2]. EXPERIMENTAL To optimize the condition for culturing individual bacteria using confined microchannels, we designed and fabricateed microchannels with size from 1 µm to 10 µm. The height of the channels was 500 nm. PDMS, which is gas permeable and transparent, is chosen as the channel structural material. The microfluidic device was loaded onto a microscope stage hotplate to provide proper temperature (37°C) for continuous observation and cultivation of bacteria. For optical inspection, the microscope stage hotplate were directly mounted on a digital microscope equipped with phase contrast optics (Leica, DMI 4000B). The morphology and density of the bacteria were recorded by a CCD camera (Cooke DMI4000B SensiCAM QE). Uropathogenic E. coli, which accounts for more than 80% of uncomplicated urinary tract infection (UTI), was selected as the model pathogen in this study. Two E. coli strains (EC137 and EC132) isolated from urine samples of UTI patients were used. These strains were isolated as part of a research protocol approved by the Stanford University Institutional Review Board. In the antibiotic resistant profiling experiment, four conditions were tested: no antibiotic (Control), ampicillin (Amp), ciprofloxacin (Cipro), and trimethoprim/sulfamethoxazole (T/S). Figure 1: Schematics side view of a pair of parallel electrodes with the bacteria initially positioned by DEP force and trapped in the microchannel. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 1050 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA

Transcript of SINGLE CELL ANTIMICROBIAL SUSCEPTIBILITY TESTING USING ... · SINGLE CELL ANTIMICROBIAL...

SINGLE CELL ANTIMICROBIAL SUSCEPTIBILITY TESTING USING CONFINED MICROCHANNELS AND ELECTROKINETIC LOADING

Yi Lu and Pak Kin Wong* The University of Arizona, USA

ABSTRACT

This study reports a single cell antimicrobial susceptibility testing (AST) technique using confined microchannels and electrokinetic loading. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacteria (~2 µm), their growth can be monitored with high resolution at the single cell level. Furthermore, AC electrokinetics is successfully demonstrated in high-conductivity culture media to facilitate dynamic loading of bacterial pathogens into the confined microchannels with over 80% loading efficiency. The single cell AST technique allows antibiotic resistance profiling to be finished in less than one hour compared to days in standard culture based techniques. KEYWORDS: Single Cell, E. coli, Antimicrobial Susceptibility Testing, Microchannels, Electrokinetics

INTRODUCTION

Infectious disease caused by antibiotic resistant bacteria represents a major challenge in the healthcare system worldwide. To determine effective treatment for the patient and proper clinical management of the disease, AST is required to evaluate the antibiotic resistant of the bacterial pathogen. However, the standard culture based AST approach requires at least 48 hours from initial sample to result. The lack of rapid diagnostics has largely driven the overuse of antibiotics, which in turn accelerates the emergence of multidrug resistant pathogens or superbugs. One of the major challenges in current culture based AST is the long culture time required to grow the bacteria to proper concentration for genotyping or phenotyping testing. Furthermore, the bulk measurement of bacteria with diverse growth behaviors can mask the growth of the bacteria and increases the assay time required to obtain statistically significant data. To address this critical problem in clinical management of infectious diseases, we have previously shown that enhanced oxygenation using high surface-to-volume ratio PDMS microchannels facilitates bacterial growth at the point of care [1]. In this study, rapid AST is performed at the single cell level using confined microchannels to eliminate the uncertainty in bulk measurement and to reduce the total assay time [2]. EXPERIMENTAL

To optimize the condition for culturing individual bacteria using confined microchannels, we designed and fabricateed microchannels with size from 1 µm to 10 µm. The height of the channels was 500 nm. PDMS, which is gas permeable and transparent, is chosen as the channel structural material. The microfluidic device was loaded onto a microscope stage hotplate to provide proper temperature (37°C) for continuous observation and cultivation of bacteria. For optical inspection, the microscope stage hotplate were directly mounted on a digital microscope equipped with phase contrast optics (Leica, DMI 4000B). The morphology and density of the bacteria were recorded by a CCD camera (Cooke DMI4000B SensiCAM QE).

Uropathogenic E. coli, which accounts for more than 80% of uncomplicated urinary tract infection (UTI), was selected as the model pathogen in this study. Two E. coli strains (EC137 and EC132) isolated from urine samples of UTI patients were used. These strains were isolated as part of a research protocol approved by the Stanford University Institutional Review Board. In the antibiotic resistant profiling experiment, four conditions were tested: no antibiotic (Control), ampicillin (Amp), ciprofloxacin (Cipro), and trimethoprim/sulfamethoxazole (T/S).

Figure 1: Schematics side view of a pair of parallel electrodes with the

bacteria initially positioned by DEP force and trapped in the microchannel.

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 1050 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

E. coli samples were diluted with Mueller-Hinton medium and pipetted into the channels. The bacteria were inoculated at

37°C. For single cell AST, antibiotics were pre-warmed at 37 °C for 20 min. The antibiotics were then mixed with the bacteria sample before being injected into microchannels. To facilitate dynamic loading of bacteria into selected locations inside the channel, AC electrokinetic forces were applied to position the bacteria using a pair of microelectrodes aligned perpendicularly to the channel (Figure 1).

RESULTS AND DISCUSSION

Using confined microchannels with width from 1µm to 3µm, the bacteria can be physically trapped in the microchannel for continuous growth monitoring (Figure 1-3). We have applied the single cell AST technique to study the distribution of the growth rate of individual bacteria (Figure 4). The growths of bacteria under different antimicrobial concentrations were monitored for individual E. coli (Figure 5). The growth rate of the bacteria generally follows a Gaussian distribution, and the mean growth rate decreases with an increase in the antibiotic concentration. Remarkably, the distribution shifts smoothly with the drug concentration indicating analog response of the antimicrobial effect. Additionally, the variation in the growth rate generally increases with the growth rate. These observations allow us to determine the proper condition for single cell AST towards reducing the assay time for antimicrobial profiling. Using single-cell AST, we demonstrate that the growth of bacteria with antibiotics can be clearly distinguished in one hour (Figure 4) and results are consistent with clinical microbiology laboratory testing which requires days to obtain (Figure 6).

To facilitate dynamic loading of bacteria into selected locations inside the channel, electrokinetic force is applied using a microelectrode integrated perpendicularly in the channel. While AC electrokinetics is typically effective only in low conductivity buffers, we have optimized the electrokinetic conditions and found the electrokinetic force was able to trap the bacteria in high conductivity media for AST (~ 1 S/m) with over 80% efficiency. The high efficiency is likely due to the short distance between the electrode and the bacterium, which makes use of the strong electric field created. With electrokinetic loading, the growth of a large number of bacteria can be observed simultaneously (Figure 7). Moreover, we observed that the growth rate of bacteria is not affected by the electrokinetic loading procedure in our experimental condition (data not show).

CONCLUSION

This paper has demonstrated a microfluidic device for culture of single cells and rapid AST using confined microchannels and electrokinetic loading. This setting provides a simple and effective platform for rapid AST at the point of care. Our experimental results have shown that antibiotic resistance of bacteria can be determined in less than one hour. AC electrokinetics was also demonstrated successfully in high-conductivity bacterial culture media to facilitate dynamic loading of bacterial pathogens into confined microchannels with over 80% loading efficiency. These promising results form the foundation for using confined microchannels and AC electrokinetics for addressing the technical challenge in rapid AST at the point of care. With further development, we envision that single cell AST will be adopted in various situations for clinical management of infectious diseases.

Figure 2: Pathogenic E. coli loaded at different

locations in confined microchannels.

Figure 3:E. coli grows in a 1 µm channel at different

times.

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ACKNOWLEDGEMENTS

This work was supported by NIH Health Director's New Innovator Award (1DP2OD007161-01), NIAID (1U01AI082457-01, R43AI088756-01) and NICHD (R43HD065303-01). REFERENCES [1] C. H. Chen et al, "Antimicrobial Susceptibility Testing Using High Surface-to-Volume Ratio Microchannels,"

Analytical Chemistry, vol. 82, pp. 1012 -1019, Jan. 2010. [2] Q. Nathalie et al, " Bacterial Persistence as a Phenotypic Switch," Science, vol. 305, pp. 1622-1625, Sep. 2004. CONTACT *P.K. Wong, tel: +1-520-6262215; [email protected]

Figure 4. Growth of individual E. coli in confined microchannels. (*p <0.01, Student's t test; ciprofloxacin (CIP).)

Figure 5.Distribution of growth rates of E. coli 137 with different Ciprofloxacin concentrations. Inset: correlation between growth rate and variance of bacterial growth.

Figure 6. Rapid AST using confined microchannels. Two clinical isolates (EC137 and EC132) were tested in Mueller-Hinton media. Ampicillin (AMP); ciprofloxacin (CIP); trimethoprim/sulfamethoxazole (SXT). The superscripts “S” and “R” indicate sensitivity and resistance of the strain to the antibiotics.

Figure 7. E. coli trapped by AC electrokinetic force. Frequency 1MHz Square wave; Amplitude: 8 VPP; DC Bias: 100 mV

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