Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

Summary

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Abstract

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

Introduction

Since the introduction of the first antibiotic drug penicillin, microbial antibiotic resistance has developed into a global health problem¹. Although the acquisition of antibiotic resistance can be retrospectively studied in vivo, the conditions of these experiments are often not controlled throughout the entire evolution². Alternatively, adaptive laboratory evolution can reveal the molecular evolution of a microbial species under environmental stresses or selection pressure from an antibiotic drug³. Recently, many well-controlled evolutionary experiments of antibiotic drug resistance have elucidated the emergence of antibioti....

Protocol

1. Assembly and Pretesting of the Morbidostat Device

1. Assembly of the Morbidostat
   1. Punch 3 holes on the cap of the culture vial with an 18 G syringe needle. Cut three pieces of polyethylene tubing ~7 cm in length. Insert these three pieces of polyethylene tubing on the cap.
   2. Use tape to wrap the edge of the cap to serve as the cast for the polydimethylsiloxane (PDMS) mixture. Mix 5 g of A component and 0.5 g of B component of the PDMS in a 150 ml plastic container by stirring manually with a toothpick. Load the mixture into a 10 ml syringe.
      1. Pour the PDMS mixture on the cap with the syringe. Bake the entire cap to cure the ....

Results

The above-described morbidostat is schematized in Figure 1. The common morbidostat operations, including experimental evolution, antibiotic susceptibility test and cell morphology checking, were validated in an E. coli MG1655 culture exposed to trimethoprim (TMP), a commonly used antibiotic drug^5,6. TMP induces very distinctive stepwise increases in drug resistance, and the mutations are clustered around the dihydrofolate reductase (DHFR) gene. Therefo.......

Discussion

A low-footprint morbidostat device from low-cost components is demonstrated. The increases in drug resistance level registered by the device are consistent with those of previous reports⁵. Designed for evolutionary studies of drug resistance, the device is potentially applicable to many other experiments. First, a comprehensive database of drug-induced mutations can be established for a large set of clinically relevant antibiotics. For example, the evolutionary pathway of multiple drug resistance can be studie.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Environmental Shaker Incubator	BioSan	ES-20
Arduino Leonardo board	Arduino	Leonardo
680 Ohm Carbon Resistor	Digikey		Bias resistor for LED
100k Ohm Carbon resistor	Digikey		Bias resistor for phototransistor
940 nm light emitting diode	Bright LED Electronic	BIR-BM13E4G-2	Optical density measurement
940 nm phototransistor	Kodenshi	ST-2L2B	Optical density measurement
Darlington pair IC Toshiba	Mouser	ULN2803APG	this IC drives micropumps and magnetic stirring unit
5 V DC brushless fan	ADDA	AD0405LX-G70	spec: 5 V supply voltage and 80 mA; available www.jameco.com
Piezoelectric micropump	CurieJet	PS15I-FT-5L	Pressure > 3 kPa; Flow rate >5 ml/min
Tygon 3350 Tuning	Saint Gobain	ABW00001	ID: 1/32" OD: 3/32" L:50'
Magnetic Stir bar	COWIE		tapered shape dim: 10 mm x 4 mm
Glass scintillation 20 ml vial	DGS		Pyrex glass 28 mm (diameter) x 61 mm (height)
Culture vial holder			Custom made from Polyformaldehyde
Silicone	Dow Corning	Sylgald 184	used to seal the glass vial
Medium bottle	VWR	66022-065
Difco M9 minimal salt 5x	BD		Medium
Cadamino Acid	BD		Medium
glucose	Sigma
Agar Bateriological	Oxoid		for agar plate
Luria Bertani medium
Inverted microscope	Leica Microsystems	Leica DMI-LED	used for microfluidic measurement Use 40X objective NA = 0.55
Microscope Incubator	Live Cell Instrument	CU-109	used for microfluidic measurement
Solenoidal valves	Pneumadyne	S10MM-31-12-3	Normally open 1.3 Watt 12 Vdc
USB interface card	Hobby Engineering	USBIO24-R Digital I/O Module	for microfluidics measurement
Air compressor	Rocker Scientific	ROCKER 440	Pressure source for microfluidcs; Max. Pressure 80 psi
Male luer-lock fittings to 1/8" barb	ValuePlastics.com	MTLL230-1	used for microfluidic control
1/8" barb to 10-32 threaded port	ValuePlastics.com	B-1	used for microfluidic control
Female luer-lock fittings to 10-32 threaded port	ValuePlastics.com	KFTL-1	used for microfluidic control
NPN darlington transistor 500 mA, 40 V (2N6427)	DigiKey.com	2N6427GOS-ND	used for microfluidic control
10 kOhm, carbon film resistor, 0.25 W	DigiKey.com	P10KBACT-ND	used for microfluidic control
Tantalum capacitor, 10 μF, 25 V, 10%	DigiKey.com	478-1841-ND	used for microfluidic control
Andor CCD camera	Andor	Zyla 4.2 Plus SCMOS	used for microfluidic on chip imaging
ELISA plate reader
two component Silicone	Momentive	RTV 615	used for microfluidic chip fabrication
SU-8 photoresist	Micrchem	SU8 2015	used for microfluidic chip fabrication
AZ4620 photoresist	Clariant	AZ 4620	used for microfluidic chip fabrication
Plasma cleaner	Harrick Plasma	PDC 32G	used for microfluidic chip fabrication
20 Gauge Syringe Needle	BD		used for microfluidic chip fabrication
Labcycler	Sensoquest	Labcycler	PCR
DNA polymerase	Toyobo	KDO Plus	PCR amplification
Trimethoprim	Sigma
Plate reader	Biotek	Synergy H1 hybrid	antibiotic resistane measurement