Stress-induced Antibiotic Susceptibility Testing on a Chip

Summary

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Abstract

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

Introduction

The rise of bacterial resistance intensifies the need for fast phenotype-based antibiotic susceptibility tests in order to safeguard our drugs of last resort. Standard susceptibility tests are based on bacterial growth inhibition in the presence of antibiotics that take multiple (8-24) hours to complete. We have developed a novel antibiotic susceptibility test on a microfluidic platform that relies on the stress-activation of biosynthetic pathways to accelerate the action of antibiotics.

Antibiotic susceptibility tests at the microfluidic scale carry the advantage of effective sample usage, since they require small numbers of bacteria. Addi....

Protocol

1. Make the PDMS Layer (Figure 1)

1. Vigorously mix PDMS and curing agent in a 10:1 ratio. To remove bubbles, degas the viscous mixture in a vacuum chamber for 1 hr at room temperature.
2. On a scale, pour the PDMS slowly over the aluminum mold. Pour from the center and keep the mold leveled. Make sure to leave the pins uncovered. Stop pouring once target weight is achieved.
   Our mold requires 4 g of PDMS and 0.4 g of curing reagent.
3. Level the mold inside an oven, and cure at 37 °C overnight.
   Alternative curing times are 2 hr at 60 °C or 1 hr at 90 °C.
4. Dissect the cured PDMS layer along the edge of the mol....

Results

The data presented in Figure 4 show the response of a susceptible Staphylococcus aureus strain over time in an antibiotic-containing microfluidic channel. Phase contrast images acquired at 1 min and at the end of the 1 hr experiment are shown in Figures 4A and B. The analyzed 1 hr data are shown in Figure 4C with the bacteria highlighted in red (5,828 total). Corresponding fluorescence images are shown in Figures 4D and.......

Discussion

The presented protocol was validated and optimized in a set of experiments with methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains¹⁰. Therefore, this protocol without modification should be directly applicable to other strains of S. aureus and other antibiotics with mechanisms of action affecting bacterial cell wall biosynthesis. Bacteria types other than S. aureus may require variation in the stress parameters: soluble (enzymatic) and mechanical stres.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SYTOX Green	Invitrogen Corporation	S7020	Dead cell fluorescence stain
Bovine Serum Albumin (BSA)	Sigma Aldrich, Inc	A9418-5G	Used for lysostaphin storage
Sodium Acetate	Sigma Aldrich, Inc	S8750-500G	Used for lysostaphin storage
Lysostaphin	Cell Sciences	CRL309A	Arrives as 1 mg solid. For storage: Dissolve in 20 mM sodium acetate. Mix with BSA solution to final concentration of 1% BSA and 100 µg/ml  lysostaphin for storage
Oxacillin salt	Sigma Aldrich, Inc	28221-1G	Antibiotic
Mueller Hinton Broth	Fisher Scientific	DF0757-17-6
Sodium chloride	Sigma Aldrixh	S3014-500G	2% added to Mueller-Hilton broth prior to autoclaving
1 ml, Luer-lock syringe	BD (Beckton, Dickinson and Comp.)	14-823-2F
2 oz, Luer-lock syringe	BD (Beckton, Dickinson and Comp.)	309653 - 60 mL	Overfill to ~65 ml
Microscope
Inverted Fluoresccence Microscope Olympus IX-70	Cambridge Scientific	9349
60X, Fluorescence/Phase contrast objective	Olympus Corp.	LCPlan F1 60x/0.70 Ph2
Retiga 12-bit monochrome CCD camera	QImaging	RET-4000R-F-M-12-C
Microscope automation
Shutters phase contrast/fluorescence	PRIOR Scientific	H204/H202
X/Y Stage	PRIOR Scientific	H107AENN
Focus motor	PRIOR Scientific	H122
Joystick for XYZ control	PRIOR Scientific	CS152EF
Proscan Controller	PRIOR Scientific	H3-XY2
Image Acquisition Software	Fraunhofer CMI
Flow Cell Assembly and PDMS
Flow Cell	BU Scientific Instruments Facility/Fraunhofer CMI	3333-1044	Engineering drawings were produced by Fraunhofer CMI
Glass window	Fraunhofer CMI	3333-1054	Glass window was cut to the proper size at Fraunhofer CMI
BOROFLOAT Window 50 mm x 50 mm	Edmund Optics Inc.	NT48-543
Sealing plate	BU Scientific Instruments Facility	3333-1045
Epoxide glass slide	Arrayit Corporation	SuperEpoxy 2
PDMS master	Fraunhofer CMI	3333-1053	Master machined in aluminum or brass with UPM-0005 (ultrapresicion fly-cutting machine)
PDMS slide design	Fraunhofer CMI	3333-1053
Tubing
Nut, Super flangeless Tinytight, headless, 1/16 in, PEEK, green	IDEX Health Science	M-644-03	Flow cell inputs/outputs are tapped for this ferrule
Ferrule, Tinytight, 1/16 in, 6-40, .030 in TH, PEEK w/ SS lock ring, black	IDEX Health Science	M-657
Nut, Super flangeless Tinytight, headless, 1/16-1/32 in, 1/4-28, PEEK, natural	IDEX Health Science	P-255
Ferrule, Super Falngeless, 1/16 in, Tefzel (ETFE), yellow	IDEX Health Science	P-259	Fits Luer-lock adapter
Tubing, Teflon FEP, .030 in x 1/16 in x 20 ft, green	IDEX Health Science	1520G
Adapter, quick connect female Luer to female 1/4-28, PEEK, red	IDEX Health Science	P-658