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Quanta Matrix Inc.

Rapid Antimicrobial Susceptibility Test Using Single Cell

Morphological Analysis (SCMA) in Plastic Microfludic Chips

Sunghoon Kwon
Professor, Department of Electrical Engineering, Seoul National University, Korea
CEO, Quanta Matrix Inc. , Korea

MAC 2.0

antibiotic
s
Bacteri
a

agaros
• The fastest DST
e * Image based DST
• Time lapse Single cell imaging- single cell imaging
• Observing cell response
Seoul National University
Biophotonics & Nano Engineering Lab.
 Antibiotics is one of the most important
invention saving lives

Alexander Fleming (1881-1955)

Nobel Prize, 1945

Discovery of penicillin in 1928
“It is not difficult to make microbes resistant to
penicillin and people may die as a result of bacteria
becoming resistant to antibiotics in the future.”
Seoul National University
-In Nobel Lecture, 1945-
Biophotonics & Nano Engineering Lab.
 Fleming’s prediction has come true :
Superbugs!

Drug-resistant bacteria (MRSA) have found refuge

in residences in New York outbreak. -Nature, 2014-
Seoul National University
Biophotonics & Nano Engineering Lab.
 Faster Antibiotic Susceptibility Test (AST) is needed

Rapid and accurate

Antibiotic treatment

Gentamicin
2μg/mL (MIC value)

P. aeruginosa

Choosing the proper

Identification of bacteria antibiotic (amount) out of
Among ~60 Clinical pathogens ~60 clinical antibiotics
Seoul National University
Biophotonics & Nano Engineering Lab.
 Resistance or Sensitive?
Blood Lab  Department of Infectious Diesease
* Takes 3-4 days
to get this report

<Vitek 2 System>
Seoul National University
인천 카톨릭
Biophotonics & Nano Engineering 성모병원 진단검사 의학과
Lab.
 Antibiotic Susceptibility Test (AST)
Using Turbidity

S R
Growth Inhibition Growth
At Over MIC Antibiotics At Under MIC

Bacteria

Minimum Inhibitory Concentration (MIC) :

the lowest concentration of an antimicrobial that will inhibit the visible growth
Seoul National University
Biophotonics & Nano Engineering Lab.
 AST for Determination of MIC
Inoculating Bacteria from Patients Gentamicin
ATM CAZ VAN
The most
Susceptible Different Antibiotics Control

Vancomycin

Growth
0.5 μg/mL
Control
MIC
0.5 μg/mL Points
Concentrations

1 μg/mL
1 μg/mL

2 μg/mL
2 μg/mL
4 μg/mL
Incubation for 16-20 hours
8 μg/mL
MIC 4 μg/mL

Non-Growth
16 μg/mL

8 μg/mL

16 μg/mL

Seoul National University

Biophotonics & Nano Engineering Lab.
 Conventional AST : SLOW!!!
Blood Culture (0.5~1 day) Pure Culture (1 day)
1st culture 2nd culture
105 CFU/mL

Blood culture Growth detecting

Blood-sampling Culture on
bottles machine
(8-10ml) Blood agar plate
(Pure culture)
- A few hours or 1 day -
≤ 10 CFU/mL

3rd culture AST (0.5 - 1 day) 108 cells/colony

concentration
antibiotics

Bacterial Colony
VITEK AST kit 108 CFU/
Overnight (16~24 hours)
Dilution tube
~108 CFU/mL Microdilution Test

Seoul National University 5ⅹ106 CFU/mL

Biophotonics & Nano Engineering Lab.
 Needs of ‘Same Day Result’
Rapid AST
• VITEK2 and Phoenix : the most AST’s time-to-result is 9~14h
• Antibiotics can be prescribed on following working day.

• QM RAST : Results come out within 4 hours

• Antibiotics can be prescribed on the same day.

Saving 20hrs

Seoul National University

Biophotonics & Nano Engineering Lab.
 Our RAST System : 3 hour AST
Bacteria
In the blood
sample

Bacteria in the Analyzer

agarose Antibiotics

Bacterial
Capture
and
Release

Single Cell
Imaging
Bacteria Agarose
Seoul National University v
Biophotonics & Nano Engineering Lab.
 The Idea : Single Cell Tracking AST

Seoul National University

Biophotonics & Nano Engineering Lab.
 Difficulty of Single Bacterium Imaging: It’s moving!

Seoul National University v

Biophotonics & Nano Engineering Lab.
 Integrating Microfluidic Chip with
96 Well Plate (MAC 2.0)

A) B) Antibiotic Microfluidic
Well Channel

Inlet
outlet

Imaging with
Temperature
96 well form Control
For covering whole
Range of candidate
Antibiotics

Seoul National University

Biophotonics & Nano Engineering Lab.
 Process of MAC 2.0 RAST

0 hr
- Cell and matrix (agarose) Loading

1 hr

- Liquid medium (chemical solution)

2 hr Loading

3 hr

Seoul National University

Biophotonics & Nano Engineering Lab.
 High Thoughput AST Chip

Seoul National University

Biophotonics & Nano Engineering Lab.
 Morphological Determination of Antibiotic
Susceptibility – Normal case
P. aeruginosa
A)
Amikacin
Susceptible

0h 1.5 h 3h

Amikacin
Resistance B)

0h 1.5 h 3h

Seoul National University

Biophotonics & Nano Engineering Lab.
 MIC Determination from Time Lapse Images
[0 h] [1 h] [2 h] [3 h]
P. aeruginosa
Concentration [μg/mL] 0 20 μm

0.5
Amikacin

Growth
1

MIC 4 Non-
Minimum
Inhibitory Growth
Concentration 8
(MIC)
17
 0 μg/ml v
MIC determination
through 0.5 μg/ml

imaging processing
1 μg/ml

2 μg/ml

4 μg/ml

8 μg/ml

CLSI MIC 0 μg/ml

1~4 1 μg/ml
ug/ml 0.5 μg/ml
2 μg/ml
MAC MIC
4 ug/ml
MIC
4 μg/ml
P. aeruginosa
8 μg/ml
ATCC 27853
with
Seoul National Amikacin
University v
Biophotonics & Nano Engineering Lab.
 Morphological Determination of Antibiotic
Susceptibility – Filament growth case

Aztreonam
(β-Lactam)
Susceptible

0h 1.5 h 3h

No dividing, No growth
Aztreonam
(β-Lactam)
Resistance

0h 1.5 h 3h

Seoul National University Current Opinion in Microbiology 2003, 6:594–599

Biophotonics & Nano Engineering Lab.
 RAST based on Single Cell Morphological Analysis
(SCMA)

Seoul National University

Biophotonics & Nano Engineering Lab.
 Validation of Morphological Analysis AST
using Standard Strains
E. coli S. aureus
Gram Negative Strains Gram Positive Strains
ATCC 25922 ATCC 29213
Antimicrobial MAC QC range Antimicrobial MAC QC range
Amikacin 0.5~1 0.5~4 Ampicillin 1 0.5~2
Amoxicillin/Clavulanic Acid 4/2 2/1~8/4 Amoxicillin/Clavulanic Acid 0.25/0.12~0.5/0.25 0.12/0.06~0.5/0.25
Ampicillin 2~4 2~8 Ciprofloxacin 0.5 0.12~0.5
Aztreonam 0.12 0.06~0.25 Clindamycin 0.12 0.06~0.25
Cefazolin 2 1~4 erythromycin 0.25 0.25~1
Cefepime 0.03~0.06 0.015~0.12 Gentamicin 0.25~0.5 0.12~1
Cefotaxime 0.06 0.03~0.12 Imipenem 0.03 0.015~0.06
Cefoxitin 2 2~8 Levofloxacin 0.12~0.25 0.06~0.5
Ceftazidime 0.5 0.06~0.5 Linezolid 1~4 1~4
Ciprofloxacin 0.004~0.008 0.004~0.015 Oxacillin 0.5 0.12~0.5
Gentamicin 0.25 0.25~1 Norfloxacin - -
Imipenem 0.12 0.06~0.25 Penicillin 0.5 0.25~2
Norfloxacin 0.03~0.06 0.03~0.12 Rifampin 0.004~0.008 (6 hr) 0.004~0.015
meropenem - - Streptomycin High Level - -
Piperacillin 2 1~4 Teicoplanin - -
Piperacillin/Tazobactam 1/4~2/4 1/4~4/4 Tetracycline 0.12~0.5 0.12~1
Tetracycline 1 0.5~2 Trimethoprim/sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5
Trimethoprim/Sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5 Vancomycin 1 0.5~2
Ticarcilin - - Time to Result 4 hr 16~20 hr
Ticarcillin/Clavulanic acid - -
Tobramycin - -
Time to Result 3 hr 16~20 hr

Seoul National University QC : Quality Control Range from

Biophotonics & Nano Engineering Lab. Clinical and Laboratory Standards Institute
 Clinical Samples Summary of Clinical Sample Test

mE ME VME CA
FDA
Requirement <1 < <1. >9
0% 3% 5% 0%
Methicillin-Resistant Staphylococcus aureus (MRSA) minor Error (mE)
Vancomycin-Resistant Enterococci (VRE) Major Error (ME)
Extended-Spectrum Beta-Lactamases (ESBL) Very Major Error (VME)
Categorical Agreement (CA)

J. Choi, S. Kwon et al., Science Translational Medicine, 2014 22

Seoul National University
Biophotonics & Nano Engineering Lab.
 Acknowledgements
• BiNEL & QuantaMatrix : Devices
• Prof. Eui Jong Kim & Prof. Sanghoon Song @ Seoul National
University Hospital, Korea : Clinical study
• Prof. Seungwuk Lee, Catholic Hospital, Korea : Clinical Study
• Prof. Hee Chan Kim & Jungchan Lee @ SNU : Image processing
• Dr. Sungwon Ryu @ Korean Tuberculosis Institutes: Clinical Study
QM & KTI

Sang-Woo Ha Jungil Choi

Yong-Gyun Jung Eun-Geun Kim

Sung-Weon Ryu

Hye-Jin Kim Sunghoon Kwon

Seoul National University

Seoul National University v Biophotonics & Nano Engineering Lab.
Biophotonics & Nano Engineering Lab.
 Thank you

Seoul National University

Biophotonics & Nano Engineering Lab.