Simultaneous Identification and Antimicrobial Susceptibility Testing of

Subscriber access provided by UNIV OF WATERLOO

Article

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropahtogens on a Microfluidic Chip with Paper-supported Cell Culture Arrays Banglao Xu, Yan Du, Jinqiong Lin, Mingyue Qi, Bowen Shu, Xiaoxia Wen, Guangtie Liang, Bin Chen, and Dayu Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03052 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropahtogens on a Microfluidic Chip with Paper-supported Cell Culture Arrays Banglao Xu1,2§, Yan Du1§, Jinqiong Lin1, Mingyue Qi1, Bowen Shu1,2, Xiaoxia Wen1, Guangtie Liang1,2, Bin Chen1,2, Dayu Liu1,2*

1(Department of Laboratory Medicine, Guangzhou First People’s Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou 510180, China) 2(Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of Guangdong Province, Guangzhou 510180, China)

AUTHOR INFORMATION Corresponding Author *E-Mail: [email protected]. Tel: +86 20 81048084, Fax: +86 20 81048084. 1, Panfu Road, Yuexiu District, Guangzhou Author Contributions §These authors contributed equally and shared the first authorship. 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropahtogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 hours. The accuracy of the microchip method was in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggests this microfluidic approach will be a promising tool of being a simple and fast for uropathogen testing in resource-limited settings.

Keywords

Uropathogen; Bacterial identification; Antimicrobial Susceptibility

Testing; Hybrid microfluidic chip

2


Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Urinary tract infections (UTIs) are one of the most common infections encountered in the clinical practice1. Nearly 50% of the global population will experience a UTI2. UTIs are challenging because they cause significant discomfort and may associate with long term complications such as renal scarring, hypertension and chronic renal failure1. Therefore, early diagnosis, proper investigation and adequate therapy are of great importance in clinical disposition of patients with UTI. The UTI-causing organism includes a set of microbes3-5. The most common UTI-causing organism is Escherichia coli, with 80%−85% of the cases originating from these bacteria. Staphylococcus saprophyticus are responsible for 5%−10% of UTI cases, and in some rare cases, UTIs can also be caused by viral or fungal infections. Other groups of bacteria such as Klebsiella, Proteus, Pseudomonas, and Enterobacter can also cause UTIs. The treatment of UTIs is primarily dependent on antimicrobial administration, and the prescription of an antimicrobial agent relies on the pathogenic evidence, including the species of microbe and its susceptibility to a variety of antimicrobial agents. Shortening the time required to identify the particular pathogenic microbe and selecting an effective antibiotic regimen to treat UTIs could significantly reduce costs, prevent emergence of multidrug-resistant bacteria and lower mortality rates. The current standard for uropathogen testing is primarily based on microbial culture6, which involves multiple time-consuming steps: (1) isolation of pathogens from samples; (2) enriching the isolated bacteria to detectable levels; (3) identifying microbial pathogens using bacteria specie specific biochemical assays and incubation of cells with antibiotics in multiple-well plates; and (4) determination of bacterial growth using absorption spectroscopy or by visual assessment. Typically, the whole procedure takes 3-4 days thus cause delay of diagnosis. To reduce the time needed for uropathogen identification, several rapid detection methods have been developed, including polymerase chain reaction (PCR)7, immunoassays8-11, urinary flow cytometry12,

mass

spectrometry13,

DNA microarray14,

and

next-generation

sequencing15. However, each of these methods provides only a profile of the pathogenic

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

bacteria species, and no direct information about the susceptibility of the bacteria to a particular antimicrobial agent. The lack of a timely and definitive microbiological diagnosis has largely driven the overuse of broad-spectrum antibiotics, which in turn accelerates the selection of drug-resistant pathogens and decreases the lifespan of antibiotics. Therefore, there is significant interest in developing rapid diagnostics of UTI, comprising pathogen identification and antimicrobial susceptibility testing (AST). The rapid diagnostic assay should be adaptable to settings with limited medical resources16, providing factual, rather than empirical, management of infectious disease and allow more judicious use of antimicrobials. The emerging microfluidic techniques have provided versatile platforms for simple and rapid microbial pathogen testing. Generally, microfluidic technologies developed for microbial testing can be classified into two types: cell-culture assay and nucleic acids detection assay. Cell-culture assays give microbial identification and AST readings dependent on cell culture so that these assays need longer time. Nevertheless, these assays give AST readings closed to the in-vivo environment, as the test is based on factual results of bacterial growth inhibition. Microfluidic cell culture systems are superior to conventional multi-well plates in several aspects: (1) The scaling down of cell culture device results into enrichment of targets and enhanced reaction signals. For example, microdroplet-based systems have been utilized to confine bacterial cells in small volumes thus resulted into relative enrichment of the targets, as a consequence the analysis time was reduced due to the enhancement of reaction signals17-19; (2) The microstructures allow ease of cell manipulation and provide favorable conditions for cell growth. For example, Chen et al. 20 used high surface-to-volume microchannels to facilitate rapid cell growth, Choi and coworkers developed microfluidic agarose channel systems tailed to immobilize bacterial cells for tracking bacterium growth 21 or morphological changes22 at single-cell level. (3) The complexity of microfluidic network facilitates precise liquid handing and fluid control. As examples, microfluidic dilution/gradient generation chips were utilized to perform broth dilution for determining the minimum inhibitory concentration (MIC)

23-28

. In contrast to the cell

culture assay, nucleic acids detection assay targeted nucleic acids (16s RNA, gDNA 4


Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and aquired plasmid sequence) of microbial pathogens29-32, which is advantageous in terms of analysis time due to the free of cell multiplication. Taking advantages with respect to analysis time, reagents consumption, throughput, and functional integration, such microfluidic nucleic acids assays are able to achieve simple and rapid microbial testing even in the filed, and as an instances, fully integrated microfluidic devices were reported to give results in one-hour time frame32,33. Despite their definite value in microbial species identification, nucleic acids detection assays do not always provide relevant information on susceptibility, particularly that which is not genetically based34-36. Furthermore, it is worthy noting that the majority of microfluidic approaches30,37-41 developed for UTI diagnosis are designed either for AST or for microbial pathogen identification only. Therefore, a microfluidic system with the capability of performing microbial identification and AST simultaneously is desirable for clinical diagnosis of UTIs. As an attractive alternative, paper-based microfluidics is especially useful in environments with limited medical resources33,42-47. Recent studies demonstrated the superiority of the polymer/paper hybrid pattern microchips48-53. These hybrid microfluidic chips combine the microfluidic network with paper substrate, resulting in precise flow control and liquid delivery. The enclosed format microchip avoids evaporation and cross-contamination and thus well suited to cell culture assays. Polymer/paper hybrid microfluidic platforms are favorable as this format could draw benefits from multiple device substrates. As examples, polymer/paper hybrid microfluidic devices have emerged as a portable and cost effective platform for microbial pathogen identification or AST54,55.

As paper substrate in the hybrid

microchip allows for flexible reagent preloading, this format of microchip is especially suitable for performing the composite microbial testing. The current work aims to develop a simple-to-use, low-cost microfluidic assay for one-step identification and antimicrobial susceptibility testing of multiple uropathogens. A PDMS/paper hybrid microfluidic chip was designed with a paper-supported cell culture array. Paper substrates in the cell culture chambers allowed for a flexible combination of the chromogenic medium with the antimicrobial agents 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

required for the multiplexed colorimetric assay. Additionally, the integrated hydrophobic membrane valve allowed the urine sample to be equally distributed into individual chambers and consequently, crosstalk between neighboring chambers was eliminated. The multiplexed uropathogen identification and AST assay was obtained by combining the spatial resolution of the cell culture arrays and the color resolution benefited from the chromogenic reaction. Experimental results demonstrated that the developed microfluidic assay was able to complete the composite UTI diagnostics assay in 15 hours with more consistency than that of the conventional method. The developed microfluidic approach is simple and fast, and is a promising powerful tool for microbial testing when medical resources are limited.

2 Experimental Methods 2.1 Instruments and equipment Inverted fluorescence microscope (IX71, Olympus, Japan) and 4-channel syringe pump (TS-2A, Longer Pump Co. Ltd, Baoding, China) were used in the experiments. Vortex mixer (XW-80A) was from the Instrument Factory of Shanghai Medical University. Digital camera (D7100) was from Nikon (Japan). Vitek™ 2 compact system (Bio Mérieux, France).

2.2 Reagents and materials SU8 3035 photoresist (MicroChem, USA). Polydimethylsiloxane (PDMS) precursor and initiator (Dow Corning, USA). Polyvinylidene fluorid (PVDF) membrane（pore size 0.23 um，Millipore）；Medium-speed qualitative filter paper (Xinhua, Hangzhou, China); All the antimicrobial agents were from Sigma; Chromogenic medium for Staphylococcus aureus, Escherichia coli, and Dung enterococcus were from Hope Bio-technology (Qingdao, China). LIVE/DEAD BacLight Bacterial Viability Kits (Life Technologies). Blood plate, nutrient agar slant and McIntosh turbidimetric tube (0.5-5.0 McFarland) were purchased from Huankai Microbial Technology (Guangdong, China). All the reagents were of analytical grade, and the solutions were prepared in sterile

6


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


double distilled water.

2.3 Samples Control bacteria strains (Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Dung enterococcus ATCC29212) and clinical urine samples, were provided by Laboratory of Microbiology, Guangzhou First People’s Hospital. Urine samples from UTI patients and healthy controls were collected in tubes between September and December in 2015 at Guangzhou First Peoples’ Hospital. All samples were analyzed by the microchip and the conventional method in parallel, within 3 hours after reception. This study was approved by the Ethics Committee of Guangzhou First Peoples’ Hospital in accordance with the Helsinki Declaration.

2.4 Microfluidic System The microfluidic system contained an incubator and microfluidic chips (See SI, Fig. 1). The incubator had a heating block constructed by assembling an aluminum plate, a Peltier heating element and an aluminum base support. A PT-100 sensor was embedded in the aluminum plates, responsible for providing temperature readings for feedback thermal control. The temperature signal was received by a laptop, which determined the power input to the Peltier heater. The heating block had 3 chip holders, on which the microchips were mounted. The camera was installed above the heating plate, and transferred the photos to the laptop through a USB cable.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.1 Diagram showing the PDMS/paper hybrid microfluidic chip for uropathogen testing. The composite microchip contained 3 layers: (1) a top layer with inlet, outlet holes and air vents, (2) a middle layer with via holes and connected channels, and (3) a non-featured bottom layer. After bonding, the middle and the bottom layer formed the culture chambers where paper substrates were embedded. The orange areas in the left figure illustrate the location of paper substrates.

The microfluidic chip was fabricated using the standard soft lithography protocol56 . As shown in Fig.1, the microchip contained 3 layers: a top layer with a sample introduction channel (500 µm in width and 10 µm in height), and holes corresponding to inlet, outlet, and air vents (1 mm diameter), a middle layer with via holes (3 mm diameter) and a non-featured bottom layer. These layers were bonded together using a plasma assisted method. First, the middle and the bottom layer were bonded together and the formed wells were used to contain the paper substrates with preloaded antimicrobial agents and the chromogenic medium. Second, the top layer was bonded to the middle layer to produce the entire chip. Finally, air vents on the top layer were sealed with a PVDF membrane with punched double-layered adhesive tape. As shown in Fig.2, each of the culture chambers was linked to the sample introducing channel with a zigzag shaped upstream channel, and was connected to the air vent with a straight downstream channel.

8


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.5 Microchip Operation Before the experiment, the microchip, the syringe and the PTFE capillary were exposed to ultraviolet radiation for 1 hour. The microchip was then connected to the syringe through capillary tubing. Before sample injection, the outlet hole was blocked by a needle. Bacteria suspension (0.2 mL) was injected into the microchip at a flow rate of 50 µL·min-1. After all the culture chambers were filled, the needle was removed and excessive samples were expelled from the outlet. After sample loading, the inlet and outlet ports were sealed with tape to prevent contamination and evaporation. The microchip was then mounted on the heating plate, with a dual lighting source at 45° above. The microchip was maintained at 37°C for 15 hours. During this period, color changes in the culture chambers were monitored by the camera.

Fig. 2 Illustration of sample loading on the microchip. I. Sample introduced to the main channel preferably flows into the branched channels due to the difference in flow resistance; II. The microchambers are serially filled with sample fluid; III. All the microchambers are filled with sample fluid; and IV. Excessive fluid is expelled from the main channel, and the inlet and outlet ports are sealed with tape to prevent contamination.

2.6 Bacterial Viability Bacteria viability in the microchip culture environment was tested using Staphylococcus aureus (ATCC25923) as a model. Survival rates of bacteria cultured in flask and on chip were detected at successive time points of 0, 2, 4, 6, 8, and 10 hours. Bacteria were harvested from the microchip by uncovering the top layer and reclaiming the liquid in the chambers into a vial containing 0.8 mL of sterile saline solution. Then, the tubes were thoroughly mixed for 5 minutes. The bacteria were then stained with the LIVE/DEAD BacLight Bacterial Viability Kit, which used 2 nucleic

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acid stains: the green-fluorescent SYTO9 stain and the red-fluorescent propidium iodide (PI) stain. These stains differed in their ability to penetrate healthy bacterial cells. The SYTO 9 stain labeled both live and dead bacteria. In contrast, PI penetrated only bacteria with damaged membranes, reducing SYTO9 fluorescence. Thus, live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red. Live and dead bacteria were viewed and counted simultaneously by fluorescence microscopy.

2.7 Bacterial Identification and Quantitation The bacteria identification assay targeted 3 species of microbial pathogens. The presence of a pathogenic bacterium was indicated by a specific color change in a designated chamber. Photographs of the cell culture array were taken every 30 minutes, and the grey intensity (GI) of the colored spots was extracted using the Image J software (Version 1.46a, Rasband, 2011). Specifically, the captured images were first converted to 8-bit grayscale format. Then, the grey intensity was extracted from the circular area corresponding to the cell culture chamber as an averaged value. The averaged GI values were normalized by subtracting the background values. A semi-quantitative analysis was determined by generating a plot of averaged GI versus time. To facilitate the analysis, the threshold time (Tt) was defined as the time at which the GI value reached 3× standard deviations of the background signal, which was equivalent to a signal-to-noise ratio (SNR) of 3. The original bacteria density was estimated upon its Tt value.

2.8 Antimicrobial Susceptibility Testing Each of the microbial identification assays correlated with a six-antimicrobial agent test (SI, Table 1). The AST assay references “susceptible (S),” “intermediate (I),” and “resistant (R)” interpretive categories were assigned using the CLSI criteria57. Accordingly, 3 concentrations of each antimicrobial agent were added to the cell culture chambers, and the AST results were determined on basis of the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Results of the microchip AST assay were compared with those obtained from 10


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the conventional susceptibility test-KB method.

3 Results and Discussion 3.1 Liquid Distribution and Compartment The current work developed a microfluidic approach by designing a chamber-array format microchip particularly for microbial testing on the basis of multiplexed colorimetric assays. The multiplexed colorimetric assay required simultaneous implementation of multiple chromogenic reactions in individual chambers. During the sample loading step, reagents stored on the paper had the potential to enter neighboring

chambers

thus

cause

cross-contamination.

To

prevent

cross-contamination, the chip used a single direction flow. Each of the culture chambers were linked to a downstream air vent. The air vents were sealed by a hydrophobic membrane, which was air permeable but water repellant. When urine samples were loaded into the microchip, the sample fluid serially filled the microchambers with changes of flow resistance along the main and branched channels (See SI, Movie). Initially, flow resistance in the main channel was higher, so that the liquid flowed into the branch channel. At the same time, air inside the channel was purged through the air vents placed downstream the chamber (Fig. 2b). When the liquid contacted the hydrophobic membrane, flow resistance in the branch channel significantly increased so that the flow returned to the main channel and filled the next chamber. Because the hydrophobic membrane prevented the liquid from exiting the chip, the sample was retained in the chambers. By applying a syringe pump, samples were equally distributed in the chambers within a few tenths of second. After sample loading, excessive samples in the main channel were expelled, leaving the main channel empty. Therefore, the compartment of liquid eliminated cross-talk between neighboring chambers. The elimination of cross talk between neighboring chambers was confirmed using a bromophenol blue diffusion test (Fig. 3). Bromophenol blue (MW 670.02) has a molecular weight similar to the antibiotics. Paper substrates with preloaded 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

bromophenol blue were alternatively embedded in the chambers. A 0.1% NaOH solution was introduced into the main channel from the inlet. The diffusion of bromophenol blue to neighboring chambers was determined by color change. Upon contacting NaOH, the paper substrate with preloaded bromophenol blue appeared blue, while the blank paper remained white. Consecutive imaging showed that chambers without bromophenol blue were still white after 2 hours, indicating no contamination of reagents between neighboring chambers during the sample loading.

Fig. 3 Bromophenol blue diffusion test. Paper substrates with or without bromophenol blue were alternatively embedded in the chambers. Consecutive photos were taken after pouring 0.1% NaOH solution, showing no bromophenol blue diffusion between neighboring chambers.

3.2 Bacterial Cultures The microchip was placed on a heating plate and the cell culture chambers were maintained at 37°C. The cell culture chamber volume was 19 µL, which allowed enough space for bacterial growth. Bacteria were cultured on a paper substrate with preloaded culture medium. The paper substrate contained cellulose that was reported to be compatible with mammalian cell58,59 and bacteria55 cultures. For Staphylococcus aureus (ATCC25923), the counts of bacteria incubated in flask and on chip were comparably determined at successive time points of 0, 2, 4, 6, 8, and 10 hours. The live/dead test showed the survival rate of bacteria was ~100％, which indicated that the microchip provided an appropriate environment for bacteria culture. The results showed that the bacteria count increased exponentially with time (Fig. 4). 12


Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Although bacteria counts in the chip group were lower than those of the flask group, there were no significant differences between the two groups at all of the time points (t test, p>0.05). The relatively lower bacteria counts in the chip group were estimated to be caused by the detention of bacteria in the paper substrate.

Fig. 4 Comparison of growth of S.aureus cultured on microchips and in flasks (n=3). Bacterial counts were given as natural logarithmic in the y-coordinate.

3.3 Identification of Uropathogens The multiplexed colorimetric assays were conducted in a paper supported environment. The porous paper provided a simple 3D substrate for reagent storage so that the chromogenic media along with antimicrobial agents were allocated by design to the chambers. As such, the microchip had heterogeneous reaction conditions necessary for the multiplex microbial diagnostic assay. The chromogenic medium has been widely used for microbe screening and has superior sensitivity and specificity compared to the that of traditional cultures55. Colorimetric assays use the interaction between species-specific enzymes and chromogenic substrates60. These enzymes allow the chromogenic substrate to release the color gene so that the medium appears a specific color, thereby indicating the presence of certain type of bacteria. The white paper was a good medium for 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

colorimetric tests because it provided a strong contrast with a colored substrate. When a particular microbial pathogen was indicated by a color change, the result was easily interpreted using simple instrumentation. By combining the spatial resolution of the culture chamber array and the multiplexed chromogenic assay, multiple bacteria were simultaneously identified on the microchip. In our test using model samples containing Staphylococcus aureus, Escherichia coli, and Dung enterococcus, results of the colorimetric assay were consistent with manufacture’s instruction. Staphylococcus aureus, Escherichia coli, and Dung enterococcus appeared blue, red, and yellow, respectively. The multiplex colormetric assay allowed simultaneous identification of 3 uropathogens on a chip. With an bacteria input of 105 cfu·mL-1 or higher, bacteria was identified in 15 hours, which was significantly faster than the conventional method61.

3.4 Quantitative colorimetric assay As demonstrated62, urinary tract infections typically have bacterial counts in excess of 105 organisms per ml of urine, whereas contaminant bacteria should be less than 104 per ml. Therefore, a quantitative detection of uropathogens is essential to diagnose a UTI. Prior studies determined that the incubation time needed for a chromogenic test is depended on bacteria input55. In light of this determination, we observed the dynamic changes of color intensity in chambers with various bacteria. Staphylococcus aureus, Escherichia coli, Dung enterococcus suspensions of different densities (101-108 mL-1) were seeded on the microchip and cultured for 15 hours (Fig. 5a). The chip method detected the bacteria with a density as low as 101 cfu·mL-1. With bacterial inputs﹤102 mL-1, only a fraction of the chambers had color changes, indicating the random distribution of bacterium. Consecutive imaging showed the dependence of the Tt value on bacteria input: chambers with high bacteria had a faster color change while those with low bacteria had a slower color change. Therefore, a real-time colorimetric assay was developed to discriminate between pathogenic microbials and contaminants. As shown in Fig. 5b, Tt values correlated to bacteria inputs. The correlation coefficient (R2)

14


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was 0.9576 for Staphylococcus aureus, 0.9615 for Escherichia coli and 0.9464 for Enterococcus. The real-time colorimetric assay showed that Tt values of UTI cases (with bacteria input >105 mL-1) were 105 mL-1, and was determined as UTI (-) with bacteria counts

Simultaneous Identification and Antimicrobial Susceptibility Testing of

Recommend Documents