The need for accurate and efficient antibiotic susceptibility testing (AST) has been emphasized with respect to the emerging antimicrobial resistance of pathogenic bacteria which has increased over the recent decades. In this study, we introduce a microfluidic system that enables rapid formation of the antibiotic concentration gradient with convenient bacterial growth measurement based on color scales. Furthermore, we expanded the developed system to analyze combinatory effects of antibiotics and measured the collective antibiotic susceptibility of bacteria compared to single microfluidic AST methods. By injecting a continuous flow precisely into the channel, the system enabled the concentration gradient to be established between two parallel channels of different antibiotic concentrations within 30 min, before bacteria enter the exponential growth phase. Moreover, the local bacterial growth levels under antibiotic gradient were quantitatively determined by calculating the position-specific grayscale values from the microscopic images and were compared with the conventional optical density measurement method. We tested five antibiotic types on our platform for the pathogenic Gram-negative bacteria strain Pseudomonas aeruginosa, and we were able to determine the minimum inhibitory concentration (MIC) at which 90% to 95% of bacterial growth was inhibited. Finally, we demonstrated the efficacy of our system by showing that most of the antibiotic MICs determined in our platform show good agreement with the MIC range suggested by the Clinical and Laboratory Standards Institutes.

1.
M.
Balouiri
,
M.
Sadiki
, and
S. K.
Ibnsouda
,
J. Pharm. Anal.
6
(
2
),
71
79
(
2016
).
2.
C. L.
Ventola
,
Pharm. Ther.
40
(
4
),
277
(
2015
).
3.
S. L.
Gellatly
and
R. E.
Hancock
,
Pathog. Dis.
67
(
3
),
159
173
(
2013
).
4.
H.
Giamarellou
,
Int. J. Antimicrob. Agents
36
(
Suppl 2
),
S50
54
(
2010
).
5.
G.
Cottarel
and
J.
Wierzbowski
,
Trends Biotechnol.
25
(
12
),
547
555
(
2007
).
6.
P. D.
Tamma
,
S. E.
Cosgrove
, and
L. L.
Maragakis
,
Clin. Microbiol. Rev.
25
(
3
),
450
470
(
2012
).
7.
R.
Chait
,
A.
Craney
, and
R.
Kishony
,
Nature
446
(
7136
),
668
671
(
2007
).
8.
J. M.
Blair
,
M. A.
Webber
,
A. J.
Baylay
,
D. O.
Ogbolu
, and
L. J.
Piddock
,
Nat. Rev. Microbiol.
13
(
1
),
42
51
(
2015
).
9.
P. J.
Yeh
,
M. J.
Hegreness
,
A. P.
Aiden
, and
R.
Kishony
,
Nat. Rev. Microbiol.
7
(
6
),
460
466
(
2009
).
10.
S.
Harbarth
,
J.
Garbino
,
J.
Pugin
,
J. A.
Romand
,
D.
Lew
, and
D.
Pittet
,
Am. J. Med.
115
(
7
),
529
535
(
2003
).
11.
J.
Barenfanger
,
C.
Drake
, and
G.
Kacich
,
J. Clin. Microbiol.
37
(
5
),
1415
1418
(
1999
).
12.
J. J.
Kerremans
,
P.
Verboom
,
T.
Stijnen
,
L.
Hakkaart-van Roijen
,
W.
Goessens
,
H. A.
Verbrugh
, and
M. C.
Vos
,
J. Antimicrob. Chemother.
61
(
2
),
428
435
(
2008
).
13.
E. P.
Hyle
,
A. D.
Lipworth
,
T. E.
Zaoutis
,
I.
Nachamkin
,
W. B.
Bilker
, and
E.
Lautenbach
,
Arch. Intern. Med.
165
(
12
),
1375
1380
(
2005
).
14.
L.
Ebbing
,
J. P.
Metlay
,
W. B.
Bilker
,
P. H.
Edelstein
, and
N. O.
Fishman
,
Clin. Infect. Dis.
41
(
7
),
923
929
(
2005
).
15.
J. A.
Ayukekbong
,
M.
Ntemgwa
, and
A. N.
Atabe
,
Antimicrob. Resist. Infect. Control
6
(
1
),
47
(
2017
).
16.
S. G.
Jenkins
,
Arch. Med. Res.
36
(
6
),
697
705
(
2005
).
17.
M. R.
Pulido
,
M.
Garcia-Quintanilla
,
R.
Martin-Pena
,
J. M.
Cisneros
, and
M. J.
McConnell
,
J. Antimicrob. Chemother.
68
(
12
),
2710
2717
(
2013
).
18.
J. H.
Jorgensen
and
J. D.
Turnidge
,
Manual of Clinical Microbiology
, 11th ed. (
American Society of Microbiology
,
2015
).
19.
S. G.
Jenkins
and
A. N.
Schuetz
,
Mayo Clin. Proc.
87
(
3
),
290
308
(
2012
).
20.
S.
Sutton
,
J. Validat. Technol.
17
(
1
),
46
49
(
2011
).
21.
J. H.
Jorgensen
and
M. J.
Ferraro
,
Clin. Infect. Dis.
49
(
11
),
1749
1755
(
2009
).
22.
23.
J.
Choi
,
J.
Yoo
,
M.
Lee
,
E.-G.
Kim
,
J. S.
Lee
,
S.
Lee
,
S.
Joo
,
S. H.
Song
,
E.-C.
Kim
,
J. C.
Lee
,
H. C.
Kim
,
Y.-G.
Jung
, and
S.
Kwon
,
Sci. Transl. Med.
6
(
267
),
267ra174
(
2014
).
24.
F. R.
Cockerill
,
Antimicrob. Agents Chemother.
43
(
2
),
199
212
(
1999
).
25.
J. A.
Karlowsky
and
S. S.
Richter
,
Manual of Clinical Microbiology
, 11th ed. (
American Society of Microbiology
,
2015
), pp.
1274
1285
.
26.
J.
Avesar
,
D.
Rosenfeld
,
M.
Truman-Rosentsvit
,
T.
Ben-Arye
,
Y.
Geffen
,
M.
Bercovici
, and
S.
Levenberg
,
Proc. Natl. Acad. Sci. U.S.A.
114
(
29
),
E5787
E5795
(
2017
).
27.
J.
Choi
,
H. Y.
Jeong
,
G. Y.
Lee
,
S.
Han
,
S.
Han
,
B.
Jin
,
T.
Lim
,
S.
Kim
,
D. Y.
Kim
,
H. C.
Kim
,
E. C.
Kim
,
S. H.
Song
,
T. S.
Kim
, and
S.
Kwon
,
Sci. Rep.
7
(
1
),
1148
(
2017
).
28.
J.
Avesar
,
Y.
Blinder
,
H.
Aktin
,
A.
Szklanny
,
D.
Rosenfeld
,
Y.
Savir
,
M.
Bercovici
, and
S.
Levenberg
,
Anal. Chem.
90
,
7480
7488
(
2018
).
29.
J.
Dai
,
M.
Hamon
, and
S.
Jambovane
,
Bioengineering
3
(
4
),
25
(
2016
).
30.
J.
Campbell
,
C.
McBeth
,
M.
Kalashnikov
,
A. K.
Boardman
,
A.
Sharon
, and
A. F.
Sauer-Budge
,
Biomed. Microdevices
18
(
6
),
103
(
2016
).
31.
B.
Li
,
Y.
Qiu
,
A.
Glidle
,
D.
McIlvenna
,
Q.
Luo
,
J.
Cooper
,
H. C.
Shi
, and
H.
Yin
,
Anal. Chem.
86
(
6
),
3131
3137
(
2014
).
32.
J.
Choi
,
Y. G.
Jung
,
J.
Kim
,
S.
Kim
,
Y.
Jung
,
H.
Na
, and
S.
Kwon
,
Lab Chip
13
(
2
),
280
287
(
2013
).
33.
C.
Murray
,
O.
Adeyiga
,
K.
Owsley
, and
D.
Di Carlo
,
Lab Chip
15
(
5
),
1226
1229
(
2015
).
34.
F. H.
Kai
,
W. D.
Jeremy
, and
P. H.
Michael
,
Phys. Med. Biol.
52
(
19
),
6001
(
2007
).
35.
J. S.
McGrath
,
C.
Honrado
,
D.
Spencer
,
B.
Horton
,
H. L.
Bridle
, and
H.
Morgan
,
Sci. Rep.
7
(
1
),
2601
(
2017
).
36.
R.
Ali
,
V.
Walter
,
S.
Yi-Hsuan
, and
N. S.
Swami
,
Electrophoresis
35
(
12–13
),
1795
1802
(
2014
).
37.
R. E.
Fernandez
,
A.
Rohani
,
V.
Farmehini
, and
N. S.
Swami
,
Anal. Chim. Acta
966
,
11
33
(
2017
).
38.
K. I.
Udekwu
,
N.
Parrish
,
P.
Ankomah
,
F.
Baquero
, and
B. R.
Levin
,
J. Antimicrob. Chemother.
63
(
4
),
745
757
(
2009
).
39.
N.
Høiby
,
T.
Bjarnsholt
,
M.
Givskov
,
S.
Molin
, and
O.
Ciofu
,
Int. J. Antimicrob. Agents
35
(
4
),
322
332
(
2010
).
40.
Z.
Hou
,
Y.
An
,
K.
Hjort
,
K.
Hjort
,
L.
Sandegren
, and
Z.
Wu
,
Lab Chip
14
(
17
),
3409
3418
(
2014
).
41.
K.
Kim
,
S.
Kim
, and
J. S.
Jeon
,
Sensors
18
(
2
),
447
(
2018
).
42.
K.
Churski
,
T. S.
Kaminski
,
S.
Jakiela
,
W.
Kamysz
,
W.
Baranska-Rybak
,
D. B.
Weibel
, and
P.
Garstecki
,
Lab Chip
12
(
9
),
1629
1637
(
2012
).
43.
J.
Cao
and
J. M.
Köhler
,
Eng. Life Sci.
15
(
3
),
306
317
(
2015
).
44.
L.
Derzsi
,
T. S.
Kaminski
, and
P.
Garstecki
,
Lab Chip
16
(
5
),
893
901
(
2016
).
45.
W. B.
Lee
,
C. Y.
Fu
,
W. H.
Chang
,
H. L.
You
,
C. H.
Wang
,
M. S.
Lee
, and
G. B.
Lee
,
Biosens. Bioelectron.
87
,
669
678
(
2017
).
46.
J.
Dai
,
S. J.
Suh
,
M.
Hamon
, and
J. W.
Hong
,
Biotechnol. J.
10
(
11
),
1783
1791
(
2015
).
47.
A. B.
Theberge
,
F.
Courtois
,
Y.
Schaerli
,
M.
Fischlechner
,
C.
Abell
,
F.
Hollfelder
, and
W. T.
Huck
,
Angew. Chem. Int. Ed.
49
(
34
),
5846
5868
(
2010
).
48.
O.
Fridman
,
A.
Goldberg
,
I.
Ronin
,
N.
Shoresh
, and
N. Q.
Balaban
,
Nature
513
(
7518
),
418
(
2014
).
49.
B.
Li
,
Y.
Qiu
,
H.
Shi
, and
H.
Yin
,
Analyst
141
(
10
),
3059
3067
(
2016
).
50.
Y.
Shin
,
S.
Han
,
J. S.
Jeon
,
K.
Yamamoto
,
I. K.
Zervantonakis
,
R.
Sudo
,
R. D.
Kamm
, and
S.
Chung
,
Nat. Protoc.
7
(
7
),
1247
1259
(
2012
).
51.
W. A.
Farahat
,
L. B.
Wood
,
I. K.
Zervantonakis
,
A.
Schor
,
S.
Ong
,
D.
Neal
,
R. D.
Kamm
, and
H. H.
Asada
,
PloS One
7
(
5
),
e37333
(
2012
).
52.
A. G.
Ogston
,
B.
Preston
, and
J.
Wells
,
Proc. R. Soc. Lond. A
333
(
1594
),
297
316
(
1973
).
53.
M.
Cokol
,
H. N.
Chua
,
M.
Tasan
,
B.
Mutlu
,
Z. B.
Weinstein
,
Y.
Suzuki
,
M. E.
Nergiz
,
M.
Costanzo
,
A.
Baryshnikova
, and
G.
Giaever
,
Mol. Syst. Biol.
7
(
1
),
544
(
2011
).
54.
M.
Zwietering
,
I.
Jongenburger
,
F.
Rombouts
, and
K.
Van’t Riet
,
Appl. Environ. Microbiol.
56
(
6
),
1875
1881
(
1990
).
55.
B. L.
Roth
,
M.
Poot
,
S. T.
Yue
, and
P. J.
Millard
,
Appl. Environ. Microb.
63
(
6
),
2421
2431
(
1997
).
56.
T.
Dong
and
X.
Zhao
,
Anal. Chem.
87
(
4
),
2410
2418
(
2015
).
57.
M. A.
Kohanski
,
D. J.
Dwyer
, and
J. J.
Collins
,
Nat. Rev. Microbiol.
8
(
6
),
423
435
(
2010
).
58.
P. C.
Appelbaum
and
P. A.
Hunter
,
Int. J. Antimicrob. Agents
16
(
1
),
5
15
(
2000
).
59.
D. T.
King
,
S.
Sobhanifar
, and
N. C. J.
Strynadka
, Handbook of Antimicrobial Resistance (Springer, New York, 2017), pp.
177
201
.
60.
T. S. J.
Elliott
and
D.
Greenwood
,
J. Med. Microbiol.
16
(
3
),
351
362
(
1983
).
61.
T. S. J.
Elliott
and
D.
Greenwood
,
J. Med. Microbiol.
17
(
2
),
159
169
(
1984
).
62.
I.
Wiegand
,
K.
Hilpert
, and
R. E. W.
Hancock
,
Nat. Protoc.
3
,
163
(
2008
).
63.
C. T.
Keith
,
A. A.
Borisy
, and
B. R.
Stockwell
,
Nat. Rev. Drug Discov.
4
(
1
),
71
78
(
2005
).

Supplementary Material

You do not currently have access to this content.