Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11

Article abstract of DOI:10.1021/jacs.5b11357

Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.

Full text of DOI:10.1021/jacs.5b11357

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Article
Detection of pathogenic biofilms with bacterial
amyloid targeting fluorescent probe, CDy11
Jun-Young Kim, Srikanta Sahu, Yin-Hoe Yau, Xu Wang, Susana Geifman Shochat, Per
Halkjær Nielsen, Morten Simonsen Dueholm, Daniel E. Otzen, Jungyeol Lee, May Margarette
Salido Delos Santos , Joey Kuok Hoong Yam, Nam-Young Kang, Sung-Jin Park, Hawyoung
Kwon, Thomas William Seviour, Liang Yang, Michael Givskov, and Young-Tae Chang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11357 • Publication Date (Web): 18 Dec 2015
Downloaded from http://pubs.acs.org on December 21, 2015
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Department of Immunology and Microbiology, Faculty of Health and
Medical Sciences; Nanyang Technological University, Singapore Centre on
Environmental Life Science Engineering (SCELSE)
Chang, Young-Tae; National University of Singapore, Chemistry; Agency
for Science, Technology and Research, Singapore Bioimaging Consortium
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Detection of pathogenic biofilms with bacterial amyloid tarꢀ
geting fluorescent probe, CDy11
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a, b, c
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Jun-Young Kim
, Srikanta Sahu , Yin-Hoe Yau , Xu Wang , Susana Geifman Shochat , Per
b, e
e
f
a
Halkjær Nielsen , Morten Simonsen Dueholm , Daniel E. Otzen , Jungyeol Lee , May Mar-
b
b, g
c
c
garette Salido Delos Santos , Joey Kuok Hoong Yam , Nam-Young Kang , Sung-Jin Park ,
a, b
b
b, d
b, h
a,
Hawyoung Kwon , Thomas Seviour , Liang Yang , Michael Givskov , Young-Tae Chang
c,
*
a
Department of Chemistry & Med Chem Program, Life Sciences Institute, National University of Singapore, 3 Sci-
ence Drive 3, Singapore 117543, Singapore
b
Singapore Centre on Environmental Life Science Engineering (SCELSE), Nanyang Technological University,
6
37551, Singapore
c
Singapore Bioimaging Consortium, Agency for Science, Technology and Research, 11 Biopolis Way, # 02-02 Helios,
Singapore 138667, Singapore
d
School of Biological Sciences, Nanyang Technological University, SBS-04s-43, 60 Nanyang Avenue, Singapore
6
37551, Singapore
e
Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers
Vej 7H, 9220 Aalborg, Denmark
f
Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus Universi-
ty, 8000 Aarhus C, Denmark
g
Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637551, Singapore
h
Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences,
University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark
ABSTRACT: Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-
associated infections relies heavily on culturing methods which fail to detect non-culturable bacteria. Identification of
novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we
report a novel fluorescent probe, CDy11 (Compound of Designation Yellow 11), which targets amyloid in the Pseudomonas
aeruginosa biofilm matrix through a Diversity Oriented Fluorescent Library Approach (DOFLA). CDy11 was further
demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.
Introduction
Despite the discovery of antibiotics, bacterial infections
remain as one of the major threats to the human society.
This is partly caused by dissemination of resistance to-
wards the current assortment of antibiotics and an almost
30 year’s discovery void of new classes of antimicrobial
form biofilms. Microorganisms are able to form surface-
attached multicellular biofilm communities as a domi-
nant lifestyle in nature. These densely populated com-
munities are encased in large amount of self-generated
extracellular polymeric substances (EPS). The thick EPS
matrix offers protection to the biofilm cells against oth-
erwise harmful conditions, such as oxidative stress, ex-
2
1
drugs. Another important cause of antibiotic resistance
and persistent infections is the capability of bacteria to
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Images were acquired after 1 hour of incubation of dye
treme pH change and high dose of antibiotics. Biofilms
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are up to 1,000 times more resistant to antibiotics than
compounds with biofilms. The images were then analyzed
based on fluorescence intensity from two different sam-
ples of the PAO1 and PAO1∆fap biofilms. Based on the
clear distinction of the fluorescence images and reproduc-
ibility of the results, the best compound was selected and
named as CDy11 (Scheme 1). CDy11 shows strong selective
staining in PAO1 biofilms compared to those of PAO1∆fap
biofilms (Figure 1B) and of planktonic cells PAO1 (Sup-
porting Information Figure S1).
3
their free-living counterparts. What is more, biofilms are
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,5
able to survive and evade host immunity. According to
the National Institute of Health, more than 80 % of all
6
infections are associated with biofilms. Furthermore, bioꢀ
7
film bacteria are able to disperse and spread to new areas.
Bacteria in biofilm-associated infections often appear
8
non-culturable. Therefore, rapid and reliable identifica-
tion of biofilm-associated infections are important for
choosing the proper treatment strategy in the clinics,
such as antimicrobial administration or surgical removal
of the infected tissue.
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NO
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NO
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NO2
a
b,c,d
e
Biofilm EPS mainly consists of extracellular DNA, pro-
teins and polysaccharides. Several compounds have been
reported to specifically label EPS components. For exam-
ples, DNA staining dyes such as ethidium bromide, Syto9
or DAPI are frequently used to localize extracellular DNA
N
H
O
N
N
O
Cl
B
HN
F
F
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2
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O
in biofilms. Hippeastrum Hybrid (Amaryllis) Lectin,
NH2
NH
2
HN
HHA, that specifically binds to either 1,3- or 1,6-linked
mannosyl units in polysaccharides is used for biofilm
detection after conjugating with fluorophore due to its
binding specificity with Psl polysaccharide, a key compo-
g
f
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N
N
B
F
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N
F
nents of P. aeruginosa biofilms.
B
B
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F
F
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F
F
Recently, ligand targeted ultrasound contrast agents
3
BDN
CDy11
(
UCAs) were reported to detect biofilm in vitro under
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1
high-frequency scanning acoustic miscroscopy. Even
though it showed the possibility as a biofilm detecting
tool of application, there is no established method for in
vivo biofilm imaging so far.
MeO
MeO
Scheme 1. Synthesis of CDy11. (a) MeMgBr, THF, ꢀ78 ˚C to RT,
overnight; (b) NaBH , THF/H O (10:1), 0 ˚C to RT, 2h; (c) 2,4ꢀ
dimethyl pyrrole, InCl , CH Cl , RT, 3hr; (d) i. DDQ, CH Cl , RT,
5min. ii. BF .Et O, Et N, 0 ˚C to RT, 8h; (e) Pd/C, hydrazine monoꢀ
hydrate, EtOH, reflux, 2h; (f) 2ꢀFluoroꢀ4ꢀmethoxybenzaldehyde,
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We previously developed various imaging probes and
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13,14
sensors for stem cells , pancreatic alpha cells , beta
pyrrolidine, MeCN, 85 ˚C, 5 min.; (g) AcCl, NaHCO
to RT, 30 min.
3 2 2
, CH Cl , 0 ˚C
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cells, and neuron by screening our in house Diversity
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Oriented Fluorescent Library (DOFL). To develop clini-
cally relevant biofilm imaging probes, P. aeruginosa was
chosen, as the bacterium is a well known opportunistic
pathogen, associated with cystic fibrosis of hospitalized
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patients with immune-compromised conditions , and
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commonly found in catheters and in contact lens. In
this study, Functional amyloids in Pseudomonas (Fap)
was chosen as the molecular target (biomarker) of the
fluorescent probe, due to its importance for Pseudomonas
21-23
biofilm structure and function.
Results
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7
Discovery of CDy11. Thousands of DOFL compounds
were screened in two different P. aeruginosa strains, PAO1
wild type and PAO1∆fap, which lack the complete fap
24,25
operon, required for Fap biogenesis.
The primary
screening was performed in 96 well format with the two
strains of bacteria culture, and the relative fluorescence
®
intensity was compared under EVOS portable fluores-
Figure 1. Established screening format. CDy11 which was developed
as a bioimaging probe with established screening format. It was
targeted PAO1 biofilms specifically. (A) Screening format for finalizꢀ
ing primary hit candidates from 96 well plate based screening. Primaꢀ
ry hits were applied biofilms on cover glass for 1 hour and images are
taken under fluorescence microscope. (B) Phase contrast and fluoresꢀ
cent images acquired from CDy11 treated PAO1 biofilms and
cent microscope. Dyes with stronger signal to PAO1 wild
types were selected as primary hits. Then, secondary and
tertiary screenings were carried out by testing all of the
primary hit candidates with more advanced model of
cover-glass slide biofilms cultivated in 50 mL conical
tubes (Figure 1A).
PAO1ꢁfap biofilms. Scale bar, 10 ꢂm.
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CDy11
PseudomonasꢀGFP Merged
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Fap antibodyꢀCy5
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B
Name
CDy11 29 ± 2
ThT 85 ± 7
K
D
(ꢁM)
CDy11
Merged
Figure 2. Super resolution images of biofilms incubated with CDy11.
Images of CDy11 stained PAO1ꢀGFP biofilms acquired on a structure
illuminated microscope using a 100x objective oil lens. The merged
image shows that fluorescenct signal of CDy11 overlapped with GFP
signal of PAO1ꢀGFP biofilms. Image in white rectangle was magniꢀ
fied to show localization of CDy11. Scale bar, 10 ꢂm.
Concentration (M)
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Figure 3. Evaluation of target of CDy11. (A) Transmission electron
microscopic image of purified amyloid. Scale bar, 400 nm. (B) Surꢀ
face plasma resonance assay with CDy11 and purified amyloid after
immobilization on chips. Values were calculated by measurements for
their response unit by flowing ThT and CDy11. (C) Coꢀlocalization of
CDy11 and Fap antibody. Biofilms were incubated with primary Fap
antibody and secondary antibody linked with Cy5. Subsequently,
CDy11 was treated for 1 hour before acquiring images. The images
were captured using a fluorescence microscope equipped with x100
objective lens. Scale bars, 10 ꢂm. The images are shown in pseudoꢀ
colors.
To investigate the generality of CDy11 for other bacteri-
al amyloid of bioflim, we also tested two other species of
bacteria, E. coli and S. typhimurium in addition to P. ae-
ruginosa. Apparently, amyloid expressing wild type bacte-
ria showed much stronger staining by CDy11 in compari-
son to amyloid deficient mutant, demonstrating the gen-
eral applicability of CDy11 to broad range of bacterial
biofilm detection (Supporting Information S2).
1
hour, before treatment of 1 μM of CDy11. Each fluores-
cence signal was obtained either with a TRITC filter or a
Cy5 filter under a fluorescence microscope (Figure 3C).
For the specificity test of CDy11 to the amyloid, the colo-
calization of CDy11 and Fap antibody staining was ana-
lyzed by image comparison and high Pearson’s coefficient
of 0.91 was observed (Supporting Information S4). Based
on these data, we conclude that CDy11 specifically binds
to Fap and not to other cellular components.
The sensitivity of CDy11 has also been measured by in
vitro fluorescence response assay with serial dilution of
bacteria in biofilm, showing the apparent detection limit
8
of 8 x 10 CFU/mL (Supporting Information S3).
We further analyzed the staining pattern of CDy11 in
biofilms with structure illuminated microscope. Biofilms
were cultivated in 8 well chamber plates by incubating
PAO1-GFP strain with ABTGC media for 16 hours at 37°C
incubator. Biofilms were exposed to 1 μM of CDy11 for 1
hour before acquiring images. The GFP signal allowed for
observation of distribution of individual bacterial cells in
the biofilms and the CDy11 signal was recorded using the
TRITC (Tetramethylrhodamine) channel. Two 3D-SIM
images were acquired at two different fluorescent chan-
nels and merged. The signal from CDy11 was observed
outside of cells (Figure 2. merged). The PAO1 biofilms
showed much stronger staining intensity than the
PAO1∆fap biofilms, suggesting that the putative binding
target of CDy11 was Fap or Fap conjugated components of
the biofilm.
Application of CDy11 in an animal model of infection.
In general, P. aeruginosa is usually present in the lungs of
cystic fibrosis patients but also, in the inserted part of
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catheters removed by surgery and in contact lenses. To
investigate the feasibility of CDy11 application for detect-
ing in vivo biofilms, a corneal infection model was gener-
26
ated in mice. Black mice were anesthetized by injection
with ketamine/xylazine into peritoneal cavity and their
cornea scratched with a blade (Figure 4A). To generate an
eye infection, the GFP-tagged P. aeruginosa (PAO1-GFP)
7
8
suspended in PBS buffer (1-2 µL, 5 x 10 ~10 CFU) was
dropped in the eyes and mice were maintained for 2 days.
GFP signal was traced at different time points post infec-
tion to confirm the propagation of the P. aeruginosa in-
fection in the cornea. GFP signals could be detected with-
in the infected mouse cornea at 12 hours post infection
and appeared fully saturated at 36 hours post infection
(Supporting Information Figure S5).
Validation of the CDy11 binding target To prove that
Fap was the binding target of CDy11, P. aeruginosa Fap
was produced and purified from E.coli by expressing the
Fap operon from P. aeruginosa. Purified Fap fibril struc-
ture was verified under transmission electron microscopy
(
Figure 3A), before immobilization by an amine coupling
CDy11 was tested using this corneal infection model af-
ter 24 hour’s infection with PAO1-GFP. First, all images
from the control scratched eyes and P. aeruginosa infect-
ed eyes were acquired without CDy11 treatment. As a
result, only the GFP signal was detected in bacterial infec-
tion eye. Subsequently, 5 μM of CDy11 was dropped in
both eyes and incubated for 10 minutes where after the
eyes were briefly washed with PBS buffer prior to acquir-
ing images. While there was no obvious fluorescent signal
from CDy11 in the control eye (Figure 4B(-)CDy11), clear
fluorescence signals were detected in the infected eye3
reaction on a CM5 sensor chip for a binding assay using a
Biacore surface plasmon resonance biosensor. Thioflavin-
T, which is widely used for amyloid studies, was chosen as
a positive control. The binding constant (K ) for Thiofla-
D
vin T was 85±7 μM (Figure 3B). CDy11 had > 2 times high-
er binding affinity (K 29±2 μM) than Thioflavin T. Final-
D
ly, expression of the Fap subunit protein was checked
with Fap antibodies and CDy11 in PAO1 biofilms. Antise-
rum raised against the Fap amyloid protein from P. aeru-
ginosa was incubated with biofilms for 2 hours at 37˚C,
then Cy5-linked secondary antibody was incubated for
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A
Top view
B
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PAO1-GFP
CDy11
Merged
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CDy11
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Figure 4. Visualization of P. aeruginosa infection by CDy11 in corneal infection model. CDy11 was tested in corneal infection model from mouse
which was incubated for 24 hours post infection of P. aeruginosa (PAO1–GFP). (A) Overview of mouse head which has used for in vivo imaging.
B) In vivo imaging of bacterial infection eye with CDy11. Green fluorescence signals were detected P. aeruginosa (PAO1ꢀGFP) infected eye. No
signals were detected in P. aeruginosa infected eye without CDy11. P. aeruginosa infection eye was stained with CDy11. (C) CDy11 signals were
gone after 1 hour maintenance before imaging under stereomicroscope. (D) Quantification of remaining CDy11 signals in the bacterially infected
eye. Fluorescence intensity peaks after 10 minutes of incubation. (E) Quadrupleꢀfluorescence tissue image with DAPI/GFP/CDy11/Cy5ꢀFap antiꢀ
body. White arrows indicate the overlapped signals of Hoechst/PAO1ꢀGFP/Cy5ꢀFap/CDy11. Scale bars, 2 mm. Over view images were taken
under stereomicroscope with 4x magnification and eye images were taken under stereomicroscope with 34x magnification.
(
(
Figure 4B (+)CDy11). This observation revealed that the
All silicone tubes were collected after 2 hours incuba-
tion to observe CDy11 fluorescence of the inner surface of
the silicone tube by means of laser confocal scanning
microscopy. As a result, biofilm coated silicone tube using
propagated P. aeruginosa biofilms could be detected in
vivo by simple dropping of CDy11 without any surgical or
culture process. Also, retention time of CDy11 on eye was
investigated in a time dependent manner by acquiring
images under stereomicroscope. The fluorescence intensi-
ty of CDy11 peaked after 10 minutes incubation and most
of the signal was gone within 1 hour (Figure 4C, D). Sub-
sequently, eyes were extracted by sacrificing mice after
imaging and sectioning samples were prepared to check
P. aeruginosa infection in corneal regions with Fap anti-
body. No amyloid signal was observed from control eye
by incubating with Fap antibody (Supporting information
Figure S6A), but a clear signal was detected from the P.
aeruginosa infected eye by Fap antiserum (Supporting
information Figure 6B) and CDy11 signal. The GFP signal
from P. aeruginosa and Cy5 signal from Fap antibody
overlapped well (Figure 4E). Based on immunohisto-
chemical evidence with eye sectioning tissues, we con-
clude that CDy11 specifically targets biofilm forming re-
gions.
24
PAO1Δfap-gfp strain which does not produce Fap in the
EPS was not stained by CDy11 (Figure 5A). P. aeruginosa
biofilm (PAO1-GFP) coated silicone tubes were specifical-
ly stained with CDy11 (Figure 5B) while, biofilms under
same condition without CDy11 showed only the GFP sig-
nal (Supporting information Figure S7B). The silicone
tubes without P. aeruginosa pre-coating were also exam-
ined by injection of same amount of CDy11. Immune cells
had moved into the silicone tubes but those were not
stained by CDy11 (Supporting information Figure S7B).
In conclusion, CDy11 showed its feasibility as a bioimag-
ing probe for detecting biofilms of P. aeruginosa in two
different in vivo models of infection.
Discussion
In this paper, we have reported the development of
CDy11, a novel bioimaging probe for detecting biofilms
based on high throughput cell based screening using the
P. aeruginosa. SPR data and antibody staining demon-
strates that CDy11 binds to P. aeruginosa amyloid pro-
teins, one of the key components of biofilms. Bacterial
amyloids have important role in attachment of surface
Next, we applied a mouse implant model to test the ca-
pacity of CDy11 to detect P. aeruginosa biofilms in a surgi-
27
cal format. Silicone tubes were pre-colonized by bacteria
and subsequently inserted into the peritoneal cavity 1 day
before the experiment. BALB/c mice were assigned to
each of the two groups and 100 μM CDy11 (200 µL) was
injected into the mice via the peritoneal cavity 2 hours
before recovering the implants (Supporting information
Figures S7A). As a mock control, a similar volume of buff-
er was injected into the second set of mice. In addition,
CDy11 was injected into a set of mice which had been
installed with uncolonized silicone tubes in the peritoneal
cavity.
24,25
and initiation of biofilm formation.
Therefore, CDy11
provides an opportunity to investigate the early stage of
P. aeruginosa biofilm formation by interaction with Fap.
It is demonstrate that biofilm in mouse corneal infection
model is detected by CDy11 under the stereomicroscopy
(
Figure 4).
4
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A Phase contrast Pseudomonas CDy11
Merged
ability to visualize biofilm forming region in eyes and in
implants by CDy11 by invasive tracking of fluorescence
signals to biofilm-forming regions will be very useful
application.
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Conclusion
CDy11 was developed as a novel biofilm imaging probe
from DOFL and its working mechanism was elucidated by
testing putative target of amyloid, through surface plasma
resonance assay and antibody co-staining. Furthermore,
CDy11 was demonstrated as in vivo diagnostic tool for
detecting biofilm by testing it in two different animal
models, corneal infection model and implant model.
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Figure 5. Test of CDy11 in implant model. CLSM images of the P.
aeruginosa (PAO1ꢀGFP) colonizing silicone implants after removal
from BALB/c mice at day 1 after insertion. Green fluorescent areas
represent P. aeruginosa. (B) Insertion of silicone tube into mice
which was preꢀcoated with PAO1ꢁfapꢀGFP and PAO1ꢀGFP biofilms.
D) Images of the microꢀcolonies adhering to the silicone implants
with CDy11. Only PAO1ꢀGFP biofilms coated silicone tube was
stained by CDy11 Scale bars, 10 ꢂm.
(
Materials and Methods
General Procedure for the Synthesis of CDy11. To a
29
Additionally, in the clinical treatment, biofilm can be
formed on the surface of medical instruments such as
urinary catheter and implant device. One of main medical
materials is silicon which is highly utilized for medical
device because it has high chemical resistance, no allergic
solution of BODIPY Aniline (BDN) (x eq.) in dry ace-
tonitrile (ACN) was added with corresponding aldehyde
(4x eq.), followed by pyridine (6x eq.) and refluxed at 85
ºC for 5 minutes. The crude condensed BODIPY com-
pound was finally purified by silica gel chromatography in
7:3 hexane and ethyl acetate mixture. The purified com-
pound (0.02 mmol) from the above step was dissolved in
dichlorometnane (DCM) and added with 100 µL of satu-
rated solution of NaHCO3, followed by acetyl chloride (5
eq.) at 0 ºC. Then the reaction mixture was stirred at
room temperature for 30 minutes. The acetylated com-
pound was purified by silica gel chromatography in 7:3
hexane and ethyl acetate mixture.
3
0
reaction and very low immune respose . CDy11 was tested
with an implant model which mimicked biofilm forming
condition by insertion of biofilm coated silicone tube into
peritoneal cavity. The implants were inspected by laser
confocal scanning microscopy after removal from the
mice. The signal intensity of CDy11 was significantly high-
er from the silicone tube coated with biofilm than from
the non-coated silicone. Furthermore, CDy11 did not stain
cellular components adhering to the implant originated
from mouse (Supporting information Figure S7B). There-
fore, CDy11 can be applied for detecting biofilms on medi-
cal instruments safely with silicone coated artificial joint
or medical instruments, because CDy11 showed no back-
ground fluorescence when it was tested with in silicone
tube implant mouse model.
Characterization of CDy11. 1H NMR (CDCl3, 300 MHz):
δ 1.69 (s, 3H), 2.28 (s, 3H), 3.89 (s, 3H), 6.45 (m, 2H), 6.68
(
m, 1H), 6.8 (m, 2H), 7 (s, 1H), 7.38 (m, 3H), 7.74 (m, 5H).
1
1
1
1
3C NMR (DMSO-d6, 75 MHz): δ 15.5, 24.5, 56.4, 102.4,
12.1, 116.3, 116.4, 116.7, 118.8, 120.2, 126.4, 127.9, 130.0, 130.1,
30.7, 130.8, 133.6, 134.8, 138.3, 141.1, 142.0, 146.1, 158.0, 162.5,
62.6, 169.1, 174.6. HRMS: m/z (C27H22BF3N3O2) cal-
As mentioned in the introduction, there are several
methods to detect biofilm in vitro but, those are difficult
to transfer to in vivo conditions due to the lack of binding
culated: 488.1767, found: 488.1751 (M-H). Extinction coef-
ficient (ε): 12456 M-1cm-1 (Solvent: Ethanol, Wave-length
(λ): 558 nm).
9,28
specificity with biofilms. One exception is Fluorescence
Quantum Yield Measurements. Quantum yield were
calculated by measuring the integrated area of emission
spectra for CDy11 compound in comparison to the same
measurement for Rhodamine B (Φst = 0.5) as reference
compound in ethanol. Both CDy11 and Rhodamine B were
excited at 510 nm and emission spectra were collected
from 550 to 700 nm.
In Situ Hybridization (FISH), but this requires sample
fixing and therefore cannot be used for real-time moni-
toring.
Planktonic bacteria are easily neutralized by immune
systems in vivo, while the matrix encapsulated bacteria
are protected against immune systems and antibiotics.
Detection of biofilms enables their control and removal.
Therefore, there is existing demand for new methods to
detect biofilm in vivo with rapid and low cost process and
visualization of biofilms with new tools is very important
for clinical purpose.
Φx = Φst(Ix/Ist)(Ast/Ax)(ηx2/ηst2) (1)
Quantum yields were calculated using eq1, Where “Φst” is
the reported quantum yield of the standard, “I” is the
integrated emission spectrum, “A” is the absorbance at
the excitation wavelength, and “η” is the refractive index
of the solvents used. The subscript “x” denotes unknown
and “st” denotes standard. Emission was integrated be-
tween 550 and 700 nm, and quantum yield for CDy11 was
CDy11, developed as a P. aeruginosa biofilm imaging
probe, will be an option for detecting P. aeruginosa bio-
film in vivo. For non-invasive tracking of signals to detect
biofilm in deeper parts, CDy11 would have to be modified
due to the limitation of penetration depth. However, the
found to be 0.047.
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Cell Based Screening for Isolating Primary Hits. P.
aeruginosa strains used in cell based screening are PAO1
immobilised flow cell in a serial manner for association
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phase of 60 s followed by dissociation phase of 5 min.
Regeneration was not performed since all compounds
exhibit complete dissociation within the dissociation
duration. The chip surface was rinsed using HEPES buff-
er. All sensorgrams were double-referenced with respons-
es from unmodified channel and blank HEPES buffer
injections. Equilibrium responses from the compounds
were plotted against logarithmic of their corresponding
concentrations where the binding affinities were calculat-
ed from 50% saturation.
24
and PAO1Δfap. PAO1 and PAO1Δfap strains grown at
7˚C for 20 hours in ABTGC media were diluted 1:200 in
0 mL ABTGC media and aliquots were cultured in 96
3
1
well microplates (100 μL per well) for 20 hours at 37 ˚C
incubator. The next day, 1 μL (100 μM) of compound li-
brary dissolved in DMSO was incubated with cultured
cells for 1 hour before observation of images under
EVOS microscope (AMG, Mill Creek, WA).
fl
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Secondary and Tertiary Screening for Final Hits.
PAO1 and PAO1Δfap were cultured in 50 mL conical tube
with 2 pieces of cover glass by adding 5 mL ATBGC media
(1:200 dilution) at 37˚C incubator without shaking for
acquiring biofilm on cover glass. Cover glass was taken
and briefly washed with dH2O for removing planktonic
cells before put on slide glass. Subsequently, 5 μL of pri-
mary hits (1 μM) was incubated with biofilm for 1 hours
before taking better resolution of images under fluores-
cence microscope (Axio Observer Z1; Carl Zeiss).
Generation of Eye Infection Model. P. aeruginosa
(PAO1-GFP) was grown at 37˚C in ABTGC media for 16
hours. 500 μL of cultured PAO1-GFP was transferred to 1.5
mL ependorf tube and centrifuge at 14,000 g for 3
minutes. Supernatant was removed and pellet was wash-
ing with PBS buffer 3 times. Finally, pellet was resuspend-
ed in 250 μL of PBS buffer.
Mouse was anesthetized ketamine/xylazine by peritoneal
injection before making scratches in cornea with blade.
7
8
3
D-SIM Super Resolution Images. Pre-cultured P. ae-
PAO1-GFP (1 μL, 2X10 ~10 CFU) dissolved in PBS buffer
was treated in left eye and PBS buffer was treated in right
eye. P. aeruginosa inoculated mouse was incubated for 2
days before in vivo experiments. Animal handling is in
accordance with the Institutional Aminal Care and Use
Committee of Singapore Bio-imaging Consortium (Agen-
cy of Science, Technology and Research, Singapore).
24
ruginosa (PAO1-GFP) in ABTGC media was inoculated
in 8 well chamber plates with 1:200 dilution rates and
incubated for 20 hours at 37˚C incubator. Next day, 2 µL
of 100 µM CDy11 was treated in 200 µL cultured bacteria
and incubated in 37˚C incubator for 1 hour. All of super-
natants were removed before observation of images. Im-
ages were taken with 100x magnification oil lens (Zeiss
ELYRA PS.1, Jena, Germany). Images under FITC (Fluo-
rescein isothiocyanate) channel were taken before acquir-
ing images by CDy11 under TRITC channel. Two images
taken under different channels were processed to super
resolution images and finally merged.
Immunohistochemisty. Rabbit polyclonal antisera tar-
geting P. aeruginosa Fap was obtained from BioGenes
(
Berlin, Germany). Briefly, rabbits were immunized with
21
1
00 µg of purified FapC monomers at day 0 and again
after 7, 14, 28, and 70 days. The rabbits were sacrificed
after 77 days and antisera collected. The antisera showed
an ELISA titre of >1:200,000 against purified FapC com-
pared to a titre of 1:300 for the corresponding pre-
immune sera.
Surface Plasmon Resonance (SPR). Amyloids were
prepared in HEPES buffer (0.01 M HEPES, pH 7.4, 0.15 M
NaCl, 3 mM EDTA and 0.005% P20). Amyloid fibers were
fragmented with sonication at 80 Hz for 10 s and centri
fuged at 14,000 g for 30 seconds to remove any large fi-
bers. Con-centrate (40 µL) was mixed with 10 mM sodium
acetate (120 µL), pH 5.5 right before immobilisation.
The OCT freezing media embedded sections were cleared
by incubating with 1% gelatin PBS buffer for 30 minutes
and remaining solution was removed before incubating
with Fap anti-immune serum solution in 1% gelatin PBS
buffer for 2 hours at 37 ˚C. Samples were washed 3 times
with 400 µL washing buffer (1 % gelatin, 0.1 % triton x100
in PBS).before incubating with secondary antibody linked
with Cy5 fluorophore for 1 hour at 37 ˚C. Finally, samples
were examined with fluorescent microscope after 3 times
washing with washing buffer (2.5 % tween 20 in PBS).
SPR experiments were performed at 25°C using Biacore T-
200 biosensor with research grade CM5-S sensor chips
(
Biacore, GE Healthcare). Carboxymethylated CM5-S
chips were activated using 70 µL of 0.2 M 1-ethyl-3-[3-
dimethylaminopropyl] carbodiimide hydrochloride (EDC)
and 0.05 M N-hydroxysuccinimide (NHS) in a 1:1 ratio.
Amyloid in sodium acetate solution (100 µL) was injected
over the activated surface until an immobilization level of
Preparation of Bacterial Coating Implant. One bacte-
rial colony (PAO1-GFP) was picked up from the plate to
inoculate an overnight culture in LB (Luria Broth) media
in 37 ˚C incubator with 110 rpm/min for 20 hours. Silicone
tubes (Ole Dich) were cut with a thickness of 4 mm and
were sterilized in 0.5 % NaClO for overnight. Next day, 25
mL of overnight cultured media was transferred to 50 mL
conical tube and collected pallet by centrifuged at 3,000
7,700 response units (RU) was reached. Ethanolamine
hydrochloride (1.0 M), pH 8.5 was injected across the
surface for 5 min to block any residual activated-
unreacted carboxyl groups. Compounds were dissolved
into HEPES buffer containing final concentration of 5%
DMSO and serially diluted to 340 nM, 1.03, 3.09, 9.26,
2
7.78, 83.33 µM. Tht+ was prepared similarly as positive
control. Replicates of the diluted compounds were inject-
ed across unmodified flow cell and amyloid-
6
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rpm for 10 minutes. Subsequently, pellet was suspended
with 2 mL of LB media before observing optical density
under 600 nm wavelength.
nip, M.; Parsek, M.; Høiby, N.; Rasmussen, T.B.; Givskov,
M. Microbiology. 2009, 155(Pt 11), 3500.
6) Immunology of biofilms (PA-07-288). NIH, Depart-
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(
ment of Health and Human Services. 2007-01-09.
(
(
7) Research on microbial biofilms (PA-03-047). NIH, Na-
tional Heart, Lung, and Blood Institute. 2002-12-20.
8) Li, L.; Mendis, N.; Trigui, H.; Oliver, J. D.; Faucher, S. P.
Front Microbiol. 2014, 5, 258
Sterilized silicone was washed twice in 0.9% NaCl solu-
tion. Silicone tubes (8 pieces) in 50 mL flask was incubat-
ed with 10 mL of diluted bacteria (OD600 = 0.1) in 0.9%
NaCl solution in rotationary shaker at 37 ˚C with 110
rpm/min for 20 hours.
(9) Trachoo, N. Songklanakarin J. Sci. Technol. 2003, 25,
807.
(
(
10) Ma, L.; Lu, H.; Sprinkle, A.; Parsek, M. R.; Wozniak, D.
J. J. Bacteriol. 2007, 189, 8353.
11) Anastasiadis, P.; Mojica, K. D.; Allen, J. S.; Matter, ML. J.
Nanobiotechnology. 2014, 12, 24.
12) Im, C. N.; Kang, N. Y.; Ha, H. H.; Bi, X.; Lee, J. J.; Park,
S. J.; Lee, S. Y.; Vendrell, M.; Kim, Y. K.; Lee, J. S.; Li, J.;
Ahn, Y. H.; Feng, B.; Ng, H. H.; Yun, S. W.; Chang, Y. T.
Angew. Chem., Int. Ed. Engl., 2010, 49, 7497.
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Generation of Implant Model. After anesthesia of
mouse with Ketamine/Xylazine mix by intra peritoneal
injection, the mouse was placed ventral side up on plate.
Fur was clipped and the skin was swabbed with 70% eth-
anol. An incision of approximately 0.5-1 cm was made in
the left groin area. Then implant prepared the day before
was inserted into peritoneal cavity via the incision. The
incision will be closed with a suture and healed on warm
pad at 26-28˚C.
(
(13) Lee, J. S.; Kang, N. Y.; Kim, Y. K.; Samanta, A.; Feng, S.;
Kim, H. K.; Vendrell, M.; Park, J. H.; Chang, Y. T. J. Am.
Chem. Soc. 2009, 131, 10077.
(
14) Agrawalla, B. K.; Chandran, Y.; Phue, W. H.; Lee, S. C.;
Jeong, Y. M.; Wan, S. Y.; Kang, N. Y.*; Chang, Y. T.* J.
Am. Chem. Soc. 2015, 137, 5355.
ASSOCIATED CONTENT
Supporting Information.
Detailed synthetic procedure and building block structures
with characterization. This material is available free of
charge via the internet at http://pubs.acs.org.
(15) Kang, N. Y.; Lee, S. C.; Park, S. J.; Ha, H. H.; Yun, S. W.;
Kostromina, E.; Gustavsson, N.; Ali, Y.; Chandran, Y.;
Chun, H. S.; Bae, M. A.; Ahn, J. H.; Han, W.; Radda, G.
K.; Chang, Y. T. Angew. Chem., Int. Ed. Engl., 2013, 52,
8
557.
(
16) Er, J. C.; Leong, C.; Teoh, C. L.; Yuan, Q.; Merchant, P.;
Dunn, M.; Sulzer, D.; Sames, D.; Bhinge, A.; Kim, D.;
Kim, S. M.; Yoon, M. H.; Stanton, L. W.; Je, S. H.; Yun, S.
W.; Chang, Y. T. Angew. Chem., Int. Ed. Engl. 2015, 54,
2442.
AUTHOR INFORMATION
Corresponding Author
*
chmcyt@nus.edu.sg
(
17) Kang, N. Y.; Ha, H. H.; Yun, S. W.; Yu, Y. H.; Chang, Y.
T. Chem. Soc. Rev. 2011, 40, 3613.
Notes
(18) Ramsey, D. M.; Wozniak, D. J. Mol Microbiol. 2005, 56,
J.-Y.K., S.S., M.G., L.Y., and Y.-T.C. are the inventors of CDy11
for which a patent has been applied.
309.
(
19) Nickel, J. C.; Ruseska, I.; Wright, J. B.; Costerton, J. W.
Antimicrob. Agents. Chemother. 1985, 27, 619.
(20) Robertson, D. M.; Cavanagh, H. D. Clin. Ophthalmol.
2008, 2, 907.
(
ACKNOWLEDGMENT
The authors would like to acknowledge financial support
from the Singapore Centre on Environmental Life Sciences
Engineering (SCELSE), whose research is supported by the
National Research Foundation Singapore, Ministry of Educa-
tion, Nanyang Technological University and National Uni-
versity of Singapore, under its Research Centre of Excellence
Programme.
21) Seviour, T.; Hansen, S.H.; Yang, L.; Yau, Y.H.; Wang,
V.B.; Stenvang, M.R.; Christiansen, G.; Marsili, E.;
Givskov, M.; Chen, Y. J. Biol. Chem. 2015, 290, 6457.
22) Zeng, G.; Vad, B.S.; Dueholm, M.S.; Christiansen, G.;
Nilsson, M.; Tolker-Nielsen, T.; Nielsen, P.H.; Meyer,
R.L.; Otzen, D.E. Front Microbiol. 2015, 6, 1099.
(
(23) Herbst, F.-A.; Søndergaard, M.T.; Kjeldal, H.;
Stensballe, A.; Nielsen, P.H.; Dueholm, M.S. J. Proteo-
me Res.2014, 14, 72
ABBREVIATIONS
(
24) Dueholm, M.S.; Søndergaard, M.T.; Nilsson, M.; Christiꢀ
ansen, G.; Stensballe, A.; Overgaard, M.T.; Givskov, M.;
TolkerꢀNielsen, T.; Otzen, D.E.; Nielsen, P.H. Microbiolo-
gyOpen. 2013, 2, 365
25) Girgis, D. O.; Sloop, G. D.; Reed, J. M.; O'Callaghan, R.
J. Invest. Ophthalmol. Vis. Sci. 2003, 44, 1591.
CDy11, compound of designation yellow 11; Fap, functional
amyloid in Pseudomonas; DOFL, diversity oriented fluores-
cent library; EPS, extracellular polymeric substance; CFU,
colony-forming unit; FITC, Fluorescein isothiocyanate;
TRITC, Tetramethylrhodamine; CLSM, Confocal laser scan-
ning microscopy.
(
(26) Bjarnsholt, T.; Gennip, M. V.; Jakobsen, T. H.;
Christensen, L. D.; Jensen, P. Ø.; Givskov, M. Nature
Protocols. 2010, 5, 282
(
(
27) George, A. O. J. Vis. Exp. 2011, 47, pii: 2437.
28) Kang, N. Y.; Lee, S. C.; Park, S. J.; Ha, H. H.; Yun, S.
W.; Kostromina, E.; Gustavsson, N.; Ali, Y.;
REFERENCES
(
1) Stubbings, W.; Labischinski, H. F1000 Biol. Rep. 2009,
, 40.
2) Fröls, S. Biochem. Soc. Trans. 2013, 41,393.
1
Chandran,Y.; Chun, H. S.; Bae, M.; Ahn, J. H.; Han,W.;
Radda, G. K.; Chang, Y. T. Angew. Chem. Int. Edit. 2013,
(
(
(
3) Donlan, R. M. Emerg. Infect. Dis. 2002, 8, 881.
4) Alhede, M.; Bjarnsholt, T.; Givskov, M.; Alhede, M. Adv.
Appl. Microbiol. 2014, 86, 1.
5
2, 8557.
(
(
29) Brouwer, A. M. Pure Appl. Chem. 2011, 83, 2213.
30) Lawrence, E.L.; Turner, I.G. Medical Engineering &
Physics, 2005, 27, 443.
(5) Alhede, M.; Bjarnsholt,T.; Jensen, P.Ø.; Phipps, R.K.;
Moser, C.; Christophersen, L.; Christensen, L.D.; van Genꢀ
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CDy11
Bacteria
Bioflim
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Amyloid Fibrils
A novel biofilm imaging probe CDy11, which
targets to amyloid in biofilms was developed and
showed its feasibility as a diagnostic tool in imꢀ
plant and corneal infection mice models.
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