Antimicrobial susceptibility test (AST) is often performed to determine the antibiotic sensitivity of bacterial pathogens in clinical samples such as urine, blood, sputum, or wound swabs1
. The current clinical practice requires sample transportation to a centralized microbiology laboratory and overnight culture of the infectious agents. AST results are not available for days after sample collection. These aspects have limited the accessibility at the point of care. Rapid determination of antimicrobial susceptibility is especially crucial towards judicious management of infectious diseases in emergency situations and high-risk areas such as hospitals, intensive care units, and clinics established in response to disasters2–4
. Without objective information of the drug resistance profile of the suspected pathogen, physicians have to select antibiotic therapy empirically based on the nature of the infection and antibiotic treatments are typically chosen based on the worst-case-scenario assumption. Injudicious use of broad spectrum antibiotics by clinicians, as a result of lack of objective diagnosis at the point of care and significant delay of standard procedures, has contributed to the emergence of resistant pathogens worldwide5, 6
. A point-of-care device for rapid AST in resource limited settings is, therefore, highly desirable. It will lead to evidence-based, rather than empiric, management of infectious diseases and will allow more judicious use of antibiotics, which in turn will reduce the emergence of multidrug-resistant pathogens7
While various genotypic markers have been identified for antibiotic resistance, measurement of the phenotypic response of bacteria to antibiotics is often superior to genotypic detection of antibiotic resistance genes due to the diverse resistance mechanisms and the continuous evolution of the pathogens1, 8
. In particular, growth-based phenotyping AST methods are the current gold standard in clinical microbiology laboratory. Conventional techniques for determining antibiotic resistance include broth dilution and disc diffusion9, 10
. For disc diffusion, the bacterial isolates are inoculated on the surface of an agar plate and a disc-shaped filter paper soaked with a standard amount of antibiotic is loaded onto the surface of the dish. With the diffusion of the antibiotic and the formation of an antibiotic concentration gradient into the adjacent medium, after 18~24 hours period of incubation, a zone of inhibition of bacterial growth appears depending on the effectiveness of the antibiotic. The size of the inhibition zone provides an indication of the potency of the antibiotic and is inversely proportional to the minimum inhibitory concentration1
. The disk diffusion and agar diffusion methods, while ‘low-tech’ and labor-intensive, are well established and still commonly used, particularly in resource-limited settings. To automate the labor intensive procedures and provide quantitative assessment of the antimicrobial sensitivity, various techniques that directly measure the concentration of the pathogens (e.g., optical density and micromechanical oscillators) or their activities (e.g., microcalorimetry, bioluminescence, and radioactive CO2
release) have been developed to facilitate the identification and evaluation of the antimicrobial resistance of bacteria in microtiter plate or other formats11–16
. All the current automated antimicrobial susceptibility techniques rely on first isolating the pathogens from the body fluid or tissue samples, which takes 18–24 hours, followed by phenotypic testing of the isolated bacteria for AST, which takes another 18–24 hours of incubation. Furthermore, these systems are expensive and have bulky footprints. Using a fluorescent viability indicator and an epifluorescence microscope, an emulsion-based microfluidic technique has been recently reported by observing the activity of individual bacteria confined in droplets17
. AST results can be obtained in 7.5 hours. Nevertheless, all these systems are difficult to be adapted to a point-of-care setting. In particular, the major hurdles for these techniques toward rapid, point-of-care testing are the time consuming bacterial growth step and the requirement of bulky supporting instrumentation.
The advent of microfluidics has the potential to revolutionize the clinical management of infectious diseases and the implementation of AST at the point of care18, 19
. An important requirement for rapid bacterial growth is sufficient oxygen in the microenvironment20
. In conventional bacteria culture, vigorous shaking with an orbital shaker is typically applied to facilitate oxygenation in the media to sustain the bacterial growth. Oxygenator systems are often included in perfusion circuits and bioreactors to supply adequate oxygen for tissue and cell culture21–23
. On the other hand, microfluidic devices have intrinsically a large surface-to-volume (S/V) ratio as a result of the small length scale. This provides a simple, yet effective, approach for oxygenation inside a microfluidic cell culture system24
. At a given concentration of bacteria, the amount of oxygen requires for sustaining the bacterial growth is proportional to the volume of the culture media (i.e., number of bacteria) while the oxygen flux is proportional to the surface area. This implies relatively abundant oxygen is available for bacterial culture at the microscale. illustrates the effect of the dimension of the microchannel for bacterial culture. The large S/V ratio of microfluidics for facilitating effective oxygenation has been utilized in various chip-based cell studies25–29
. Nevertheless, the relationship between the S/V ratio and bacterial growth has not been investigated systematically and the microfluidic approach has not been demonstrated for rapid AST.
Figure 1 Schematics illustrating the effect of the depth of a microchannel on the growth of E. coli. (a) For a microchannel with a large depth, the oxygen level is relatively low for supporting the growth of all E. coli as a result of the large S/V ratio. (b) (more ...)
In this study, we explore the use of gas permeable PDMS microchannels that has a large S/V ratio toward the implementation of rapid AST at the point of care. The growth of uropathogenic Escherichia coli (E. coli) in microfluidic channels was compared to other culture conditions, including an Erlenmeyer flask in an orbital shaker, a static Erlenmeyer flask, and a static Petri dish. The bacterial growth was investigated as a function of the S/V ratio of the apparatus by using laser-machined microchannels with different depths. Understanding the effect of the S/V ratio on bacterial growth helps to optimize the microfluidic design for rapid AST. Experimental results are presented to determine the dose dependence of ampicillin on an uropathogenic E. coli using microfluidic channels. Furthermore, the antimicrobial resistance profiles of four E. coli clinical isolates were also determined using the microfluidic channels with an optimized S/Vume ratio. These tests can be performed directly in urine mixed with bacteria culture media. The current study will potentially form the technological foundation of a microfluidic approach for performing rapid AST at the point of care without relying on a centralized clinical microbiology laboratory.