Maltotriose-based probes for fluorescence and photoacoustic imaging of bacterial infections

Article abstract of DOI:10.1038/s41467-020-14985-8

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).

Full text of DOI:10.1038/s41467-020-14985-8

A R T I C L E
https://doi.org/10.1038/s41467-020-14985-8
OPEN
M a l t o t r i o s e- b a sed p r o b e s f o r flu o r e s c e n c e a n d
p h o t o a c o us ti c i m a g i n g o f b a c t e r i a l i n f e c ti o ns
✉
1 , 2
1 , 2
1 , 2
1 , 2
1 , 2 , 3
A i me n Z l i tn i
, G a y a t r i G ow r i s h a n k a r , I d a n S t e i n b e r g
, T o m H a y w o o d & S a n j iv S a m G a m b h i r
C u rr e n t l y, t h e r e a re n o n o n - i n v a s i v e t o o l s t o a c cu r a t e l y d i a g n os e w ou n d a n d s u r g i c a l s i te
i n f ec t i o ns b e f o r e t h ey b e c o m e s y s t e m i c o r c a u s e s i g n ific a n t a n a t om i c a l d a m a g e . F l u or es -
c e n c e a nd p h o t o ac o u s ti c i m a g i n g a r e c o s t- e f f e ct i v e i m a g i n g m o d a l i t i e s t h a t c a n b e u s e d t o
n o ni n v as i ve l y d ia g n o s e b a ct e r i a l i n f e c ti o n s w h e n p a i r e d w i t h a m o l e c u l a r l y t a r g e t e d i n fe c t i o n
i m a g i ng a ge n t . H e r e , w e d e v e l o p a flu o r e s c en t d e r i v a t i v e o f m a l to t r i o s e ( Cy 7 - 1 - m a l t o tr i o s e ),
w h i c h i s s h o wn t o b e t a ke n u p i n a v a r i e t y o f g r a m - p o s i t i v e a n d g r a m - n e g a t i ve b a c t e r i a l
s t r a i n s i n v it r o . I n v iv o flu o r e s c en ce a n d p h o t o a co u s ti c i m a g i n g s t u d i e s h i g h l i g h t t h e a b i l i t y o f
t h i s p ro b e t o d e t e c t i n f e ct i o n , a s s e s s i n f e c ti o n b u r d en , a n d v i s u a l i z e t h e e f f e c t i v e ne s s o f
a n t i b i ot ic t r e at me n t i n E. coli- i n d u ce d m y o si ti s a n d a c l i ni c a l l y r e l ev a n t S. aureus w ou n d
i n f ec t i o n m u ri n e m o d e l . I n a d d i t i o n , w e s h o w t h a t m a l t ot r i os e i s a n i d ea l s c a ff o l d f or i n fe c t i o n
i m a g i ng a ge n t s e n c o m p a s s i n g b e t t e r p h a r ma co ki n e t i c p ro p er t i es a n d i n v i vo s t a b i l i ty t h a n
o t h e r m al t o d e x tr i ns ( e . g . m a l t o h e x o s e) .
1 Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA. 2 Department of Radiology, Stanford University, Stanford, CA
94305, USA. 3 Department of Bioengineering, Department of Materials Science & Engineering, Stanford University, Stanford, CA 94305, USA.
✉
email: sgambhir@stanford.edu
N A T U R E C OM M U N I C A T I ON S |
(2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww . n a t u r e . c o m / n a t u r e c o m mu n i ca t i o n s
1

A R T I C L E
N A T U R E C O M MU N ICA T IO N S | h t t p s : / /d o i .o r g / 1 0 .1 0 3 8 / s4 1 4 6 7 - 0 2 0 - 149 8 5 - 8
acterial infections are of mounting medical and public relies on detecting ultrasound signals produced upon thermal
concern worldwide. One primary reason for this epidemic expansion of tissue when exciting the fluorescent probe at an
is the overuse of antimicrobials, which enhanced the appropriate wavelength with an external laser13. PAI has been
B
number of drug-resistant bacteria1,2. Furthermore, there is an shown to be a standalone, portable tool capable of imaging
increase in human life expectancy, which contributes to the high endogenous signals such as melanin, and exogenous chromophores
number of individuals at risk for infection and the proliferation of from contrast agents, with deeper imaging capabilities (up to 4 cm)
necessary medical procedures (i.e. surgery, arthroplasty, fracture than FLI and comparable resolution to MRI (~250 μm)13–16. In
fixations, biomedical implantations)3. Such procedures and addition, PAI has been used to monitor tissue healing by imaging
associated implants are susceptible to infection4–7
.
blood vessels as well as utilizing its ultrasound component to
Wound and surgical infections create a substantial burden on provide anatomical information15–18. Hence, PAI can be an opti-
patients’ quality of life and result in delayed healing and can even mal cost-effective and non-invasive tool to quickly detect bacterial
lead to death7. For example, surgical site infections (SSIs) are one infections and monitor the effectiveness of treatment at local sites
of the most common types of healthcare-associated infections and (i.e. surgery and injury sites).
occur in 2–5% of patients undergoing surgery in the United
A number of FLI and/or PAI probes targeted to bacteria have
States. This translates to around 400,000 SSIs for an average of 15 been developed by using antibiotics (vancomycin19,20 or teico-
million procedures performed annually in the United States. In planin21, specific to Gram-positive bacteria), Concanavalin
addition to increasing the duration of hospitalization, SSIs (targeting bacterial cell-surface mannose)22, antibodies (targeting
increase treatment cost as well as mortality risk by 2–11-fold8. the immunodominant staphylococcal antigen A, specific to S.
Unfortunately, many of these infections are only diagnosed after aureus)23, boronic-acid (targeting bacterial cell-surface glyco-
becoming systemic or having caused significant damage to key proteins, specific to Gram-positive bacteria)24, enzyme-activated
organs, making it harder and more costly to treat due to the high nanoparticles (targeting gelatinase-expressing Gram-positive
bacterial burden. It is therefore important to develop tools to bacteria)20 or through electrostatic and hydrophobic interactions
noninvasively detect bacterial infections at an early stage with (specific to Gram-positive bacteria)25. Preclinical evaluation of
high sensitivity and specificity. Such tools will aid clinicians in these probes showed promising results in FLI19,22–24 or PAI20,21
deciding the optimal route of treatment after surgeries and can be of some bacterial infections. Unfortunately, targeting the bac-
used to monitor the effectiveness of the treatment regimen to terial cell wall potentially limits the amount of signaling agent
ensure proper management of wound and surgical infections. In taken up leading to lower sensitivity. In addition, strain-specific
the clinic, bacterial infections are diagnosed through a combi- probes will have minimal clinical impact in imaging surgery and
nation of clinical, laboratory (detecting signs of inflammation, injury-related bacterial infections since they usually occur from
microbiology, and histopathology), and imaging assessments the presence of a variety of pathogenic bacteria. A few other
which are invasive, time-consuming, and/or costly6,7,9. Currently, examples rely on genetically encoding bacteria with reporters,
imaging modalities such as X-ray, ultrasound, magnetic reso- such as photo-switchable chromoproteins26,27 and violacein28,
nance imaging (MRI), and computed tomography (CT) provide have been reported. While these strategies allow non-invasive
valuable anatomical information but are only useful in diagnosing imaging of bacteria in vivo using PAI, the application of such
delayed and late-stage infections9. In order to improve sensitivity, platform would be limited to visualizing biochemistry, patho-
specificity, and earlier detection, a molecular imaging strategy, physiological processes, and gene expression profiles in living
which necessitates the development of imaging probes that can subjects as well as imaging tumor homing bacteria29 and cannot
specifically target sites of bacterial infections, will be required. A be used for diagnosing bacterial infections.
variety of radio-imaging agents or positron emission tomography
A more promising bacterial-imaging strategy relies on the
(PET) tracers for whole-body bacterial imaging have been utility of large sugar molecules to deliver the signaling moiety into
developed and several of these are currently under evaluation in bacteria. These complex sugars (e.g. maltose, maltotriose, and
clinical trials10. These approaches are however dependent on maltohexose) are major sources of glucose for bacteria and are
proximity to cyclotrons and generators (for isotope production or taken up in millimolar quantities30. A considerable advantage of
collection, respectively) and experienced radiochemists for tracer such probes is their specific uptake by bacteria through the
production, thus limiting their availability on demand. Their use maltodextrin transporter which is not present in mammalian
will therefore likely be restricted to major hospitals for the cells, thus allowing differentiation of bacterial infections from
management of in-patients. In addition, such approaches are not other diseases such as cancer and inflammation. Murthy and co-
recommended for use with infants, children, and pregnant workers30 developed a fluorescent and an 18F-labeled31 derivative
women, who represent a population at high risk for infections, of maltohexose at the anomeric carbon and showed its effec-
due to radiation exposure. To date, there are no rapid and reliable tiveness in fluorescence and PET imaging of bacterial infections
diagnostic techniques that can detect implant, wound, and sur- in rats, respectively32. Recently, Pang and co-workers33 developed
gical infections at an early stage in outpatient clinics. More spe- theranostic nanoparticles loaded with purpurin 18 and targeted to
cifically, an imaging tool that can aid doctors in emergency rooms bacteria by surface functionalization to maltohexose. These par-
(ERs) and field hospitals to quickly diagnose bacterial wound ticles showed great potential in treating bacteria using sonody-
infections and determine the extent of infection can potentially namic therapy and assessed the specificity of their particles to the
change clinical management.
infection site using FLI imaging and showed an example of PAI
Fluorescence imaging (FLI) relies on the detection of emission using their particles. In parallel, our lab has developed an 18F-6”-
signals from fluorescent probes upon excitation at their appro- labeled maltose and maltotriose derivatives and showed their
priate absorbance wavelengths11,12. FLI of bacterial infections effectiveness in imaging bacterial infections through PET ima-
gained attention due to its many advantages such as high resolu- ging34–36. While both imaging agents were specifically taken up
tion, real-time imaging capabilities, ease of use, and low cost11. in bacterial infections, the maltotriose derivative showed superior
Constricted by its limited depth and penetration (~1 cm), we clearance from organs and better pharmacokinetic properties.
believe that FLI can only be implemented in superficial infection Hence, we decided to utilize maltotriose as a scaffold for a pho-
imaging (during surgery, superficial implants, or endoscopy) as toacoustic and FLI agent of bacterial infections.
well as intra-operative applications11,12. On the other hand, pho-
toacoustic imaging (PAI) is an emerging imaging technique that derivative of maltotriose for photoacoustic and fluorescent
In this work, we report the development and evaluation of a
2
N A T U R E C O M M U NI C A TI O N S |
( 2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 41 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 02 0 - 1 4 9 8 5 - 8
A R T I C L E
OH
OAc
O
OAc
OH
O
O
OAc
O
OAc
O
O
HO
HO
OH
O
AcO
AcO
AcO
AcO
OAc
O
Ac2O, Pyridine
RT, 72 h
HO(CH2)3N3, BF3.OEt2, DCM
–72° to RT, 18 h
OAc
O
O
OH
O
O
OAc
OAc
O
OH
O
HO
O
OAc
OAc
AcO
AcO
OH
OH
O
R
HO
1a; n = 1, 95%
1b; n = 4, 88%
2a; n = 1, 73%
2b; n = 4, 80%
OAc
OAc
AcO
N3
AcO
n
n
n
R = OH or OAc
N
OH
OH
O
OH
O
N
DBCO-Cy7
RT, 8 h
NaOMe/MeOH
O
Maltotriose, a; n = 1
Maltohexose, b; n = 4
N
N
N
O
RT, 3 h
O
O
HO
OH
OH
OH
H
HO
HO
3a; n = 1, 65%
3b; n = 4, 60%
HO
N
N
O
n
O
Fig. 1 Synthesis of Cy7-1-maltotriose and Cy7-1-maltohexose. DBCO d i b en zo y l c y c l o o c t yn e , RT r o o m t e m p e r a t u re , Me C H , Et C H C H , DCM d ic hl or o
3
3
2
m
e
t
h
a
n
e
.
imaging of bacterial infections. The fluorescent dye is attached to
A direct assessment of the ability of Cy7-1-maltotriose (3a) and
the anomeric carbon (reducing end) of maltotriose, which is Cy7-1-maltohexose (3b) to be specifically taken up by a variety
shown to have a smaller effect on the internalization of the sugar of bacterial strains containing the maltodextrin transporter was
than functionalization at the 6″-position (non-reducing end). In also conducted. Figure 2b showcased the ability of this probe to be
addition, we conduct a face to face comparison study between taken up by E. coli, Staphylococcus aureus, Bacillus subtilis, and
maltotriose and maltohexose to determine the optimum scaffold Pseudomonas aeruginosa. Control studies, where azide-inactivated
for an imaging agent targeting the maltodextrin transporter.
E. coli or E. coli mutations lacking components of the maltodextrin
transporter were also evaluated and showed minimal uptake (Fig. 2b
maroon, P < 0.0001, n = 3 per study).
Results
Synthesis of the fluorescent probes. Detailed synthesis of the
fluorescent probes is provided in Supplementary Methods.
Briefly, azide-functionalized maltotriose intermediate at the
anomeric carbon (compound 2a) was synthesized to allow ease of
functionalization with a variety of signaling agents using copper-
free click chemistry. Compound 1a was synthesized utilizing an
adapted procedure30,37 and collected as a colorless precipitate in
94% yield. Compound 2a was then produced from 1a using an
adapted procedure37 resulting in a mixture of compound 2a as
well as partially deacetylated 2a (Fig. 1, Supplementary Figs. 1 and
2) which was also previously reported37. Since the glycosylation
was successful and the final product was going to be fully dea-
cetylated, the reaction was carried on the mixture. The mixture
was then functionalized with a commercially available fluorescent
dye coupled to dibenzoyl cyclooctyne (Cy7-DBCO), through
strain-promoted azide–alkyne [3+2] cycloaddition reaction.
Sodium methoxide was then added to the crude mixture to
deprotect the acetate groups before purification by reverse phase
high-performance liquid chromatography (HPLC) to afford
compound 3a in 65% yield (Supplementary Fig. 3). In addition,
the Cy7 derivative of maltohexose was prepared following the
same synthetic route producing compound 3b in 60% yield
(Fig. 1, Supplementary Fig. 4).
In vivo evaluation in E. coli-induced myositis murine model.
Cy7-1-maltotriose was evaluated in vivo in an E. coli-induced
myositis murine model. Fluorescence images of the mice over time
showed rapid accumulation of Cy7-1-maltotriose in the right thigh
of the mice that was infected with E. coli. Such accumulation was
not observed in the left thigh of the mice that was injected with
heat-inactivated E. coli (Fig. 3a). In addition, significantly higher
signal intensity in the right thigh compared to the left thigh starting
at the 1h imaging time point was observed (P < 0.0310, n = 3), and
the signal difference increased over time (Fig. 3b).
The same animal model was then used to monitor the infection
site with PAI. As a control, photoacoustic images of both infected
and control thigh muscle were collected before and after probe
injection. Qualitatively, a higher photoacoustic signal in the
infected thigh was observed when imaged after probe injection
compared to that of the control muscle (Fig. 3c, Supplementary
Fig. 11). In addition, quantitative analysis of the photoacoustic
signal showed ~3-fold higher PA signal in the post-probe
injection images of the infected thigh muscle compared to that
before injection or to control thigh muscle (P = 0.0004 and P =
0.0002, respectively) (Fig. 3d).
In the same animal model, a comparison study between the
maltotriose and maltohexose derivatives (compound 3a and 3b,
In vitro evaluation. A competition binding assay between the respectively) was conducted. A higher fluorescence signal in the
synthesized derivatives and 3H-maltose, which is taken up in infected thigh (right) was observed in the mice injected with
bacteria through the maltodextrin transporter, was conducted. A compound 3a (Fig. 4a, top). In addition, fluorescence signal in the
significant reduction in 3H-maltose uptake in E. coli was observed infected muscle was quantified and normalized to control muscle
when pre-incubated with azide- or Cy7-functionalized mal- (Fig. 4b) and showed ~1.5 times higher fluorescence signal in the
totriose or maltohexose (Fig. 2a, P = 0.0002, n = 3 per study). infected thigh of mice injected with Cy7-1-maltotriose compared
Furthermore, addition of the azide moiety and Cy7 functional to those injected with Cy7-1-maltohexose. In PAI, significantly
groups on the anomeric carbon of maltotriose or maltohexose did higher PA intensity in the infected thigh compared to control
not show any effect on its ability to block the uptake of 3H- thigh was observed using either probes (P < 0.0013) (Fig. 4c, d,
maltose.
Supplementary Fig. 12). Slightly higher PA intensity in the
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
2
0
)
1
1
:
1
2
5
0
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
|
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s
3

A R T I C L E
N A T U R E C O M MU N ICA T IO N S | h t t p s : / /d o i .o r g / 1 0 .1 0 3 8 / s4 1 4 6 7 - 0 2 0 - 149 8 5 - 8
a
b
Bacteria competition assay
Bacteria binding assay
500
4000
E coli
400
300
200
100
E coli + NaN3
None
MalG + LamB mutant
3000
Azide-1-Maltotriose
Azide-1-Maltohexose
Cy7-1-Maltotriose
Cy7-1-Maltohexose
MalK + LamB mutant
PTS + LamB mutant
Staphylococcus aureus
Bacillus subtilis
2000
1000
0
* P = 0.0002
Pseudomonus aerginosa
*P < 0.0001
5
4
3
2
1
0
E. coli + 3H-Maltose
Cy7-1-Maltotriose
Cy7-1-Maltohexose
c
Influx study
800
ns
ns
30 min
60 min
700
600
500
400
240 min
1080 min
* P < 0.0137
** P < 0.0001
*** P < 0.0001
Cy7-1-Maltotriose
Cy7-1-Maltohexose
Fig. 2 In vitro evaluation of Cy7-1-maltotriose and Cy7-1-maltohexose. a B a r p l o t r e p r e s e n t a ti o n s h o w i n g t h e e f fe c t i v e n e s s o f 1 - m a l t o t r i os e a nd 1 -
3
3
m a l t o h e xo se d e r i v a t i v e s i n c o m p e t in g w it h t h e u p t a k e o f H -m a l t o s e i n E. coli. S i g n ific a n t r e d u ct i o n i n H - m a l to s e u p t a k e i n E. coli w a s o b s e r v e d w i t h p r io r
3
i n c ub a ti o n w i th a n y o f t h e 1 -m a lt o t r i os e o r 1 -m a lt o h ex o s e d e r i va ti v es c o m p a r e d t o E. coli i n c u b a te d w i t h o n l y H - m a l t o s e ( P = 0 .0 0 0 2 , n = 3 ). D a ta a r e
p re s e n t ed a s c o u n t s p e r m in u t e ( C P M ) i n e a c h s a m p l e n o r m a l i z e d t o p r o t e i n c o n t e n t ( µg o f p r o t ei n ) . b B a r p l o t r e p r e s e n t a t i o n s h o w i n g q u a nt ifie d
flu o re s ce n c e s i g n a l i n E. coli, Staphylococcus aureus, Bacillus subtilis, a n d Pseudomonas aeruginosa a f t e r i n c u b a ti o n w i t h C y 7 - 1 - m a l t o tr i o s e o r C y 7 -1 -
m a l t o h e xo se . A s a c o n t ro l , s o d i u m a z i d e - in ac t i v a t e d E. coli, a n d E. c o l i m u t a n t s l a ck i n g c o m p o n e n t s o f t h e m a l t o d e x t r i n t r a n s p o r te r w e r e t e s t e d. S ig n i fic a n t
r e d u ct i o n i n C y 7 - 1 - m a lt o t r i o s e u p t a k e i n i n a c t iv a t e d E. coli a n d E. coli m u t a t i o n s w a s o b s e r v e d (P < 0 .0 0 0 1, n = 3 ) . S i g n ific a n t r e d u ct i o n i n C y 7 -1 -
m a l t o h e xo se u p t a ke i n E. coli m u t a t io n s w a s a l s o o b s e r ve d ( P < 0 .0 0 0 1 , n = 3 ) . N o s i gn ific a n t d i f f e r e n c e i n u p t a k e b e t w e e n C y 7 - 1 - m a l t o tr i o s e a nd C y7 - 1-
m a l t o h e xo se i n a l l b a c t e r i a s t r a in s w a s o b s e r ve d ( P > 0 . 05 , n = 3 ) . c B a r p l o t r e p r e s e nt a t i o n o f i n flu x o f C y 7 - 1 - m a lt o t r i o s e a n d C y 7 - 1 - m a lt o h e x o s e i n E. coli
o v e r t i m e . Q u a n t ifie d flu o r e s c e n c e s i g n a l f r o m i nflu x s t u d y s h o wc a s es s i gn ific a n t i n c r e a s e i n u p t a k e o f t h e p r o b e s w h e n i n c u b a t e d f o r 6 0 m i n c o m pa r e d t o
3 0 m i n i n c u b a t i o n f o r b o t h p r o b es ( P < 0 . 01 3 7 a n d P < 0 . 0 00 1 f o r C y 7 - 1 - m a l t o tr i o s e a n d C y 7 - 1 - m a lt o h e x o s e , r e s p e ct i v e l y, n = 3 ) . I n a d d i t i o n , s i g nific a n t ly
h i g he r flu o r es c e n ce u p t a k e o f C y 7- 1 -m a l t o tr i o s e w a s o b s e r ve d c o m p a r e d t o C y 7 - 1 - m a lt o h e x o s e w h e n i n c u b a t e d f o r 3 0 m i n w a s o b s e r v e d ( P < 0 . 0 0 0 1, n =
3 ) . B a r g r a p h s s h o w m e a n a n d S . E. M . S t a t i s t i c al a n a l y s i s w a s p e r f o r me d u s i n g o n e - a n d t w o - w a y A N O V A . T h e s o u r c e d a t a u n d e r l y i n g F i g . 2 a–c a r e
p ro v i d e d i n a S o u rc e D a t a fil e .
infected thigh of mice injected with Cy7-1-maltotriose (n = 6) incubated with S. aureus solution followed by Cy7-1-maltotriose
was observed compared to ones injected with Cy7-1-maltohexose (Fig. 5a, left). In addition, minimal fluorescence signal in the
(n = 3), but this difference was not significant (Fig. 4d, left, P = control catheters which were not incubated with S. aureus was
0.7571). However, the PA signal ratio between infected and observed highlighting the low levels of nonspecific binding of
control muscle was significantly higher for Cy7-1-maltotriose Cy7-1-maltotriose to the catheter (Fig. 5a, b; P < 0.0001).
compared to Cy7-1-maltohexose (Fig. 4d, right, P = 0.0353).
In addition, the catheters which were only incubated with S.
To further assess the specificity of Cy7-1-maltotriose to aureus solution, only showed the BLI signal and no fluorescence
bacteria containing the maltodextrin transporter, a similar study signal (Fig. 5a, right). The axial photoacoustic images showed
where a MalG + LamB mutant of E. coli was injected instead in noticeable photoacoustic signals on the surface of catheters
the left thigh. In vivo FLI at 3 and 20 h post-injection also shows a incubated with both the bacteria and Cy7-1-maltotriose (Fig. 5c,
rapid and significantly higher accumulation of Cy7-1-maltotriose left). While significantly lower photoacoustic signal was quanti-
in the E. coli-infected thigh (right thigh muscle) compared to the fied on sterile catheters incubated only with Cy7-1-maltotriose
left thigh infected with E. coli mutation (n = 5, Supplementary (Fig. 5d; P < 0.0248).
Fig. 13, P < 0.0001).
In vivo evaluation in S. aureus-infected wound murine model.
In vitro imaging of biomaterial infection. To examine the In order to mimic a clinically relevant wound infection model, a
ability of our probe to detect bacteria on biomaterials, sterilized bioluminescent strain of S. aureus (Xen 36) was inoculated into a
catheters were incubated in a solution of 106 CFU of S. aureus superficial wound made on the back of the mouse. After con-
before incubation in a solution of Cy7-1-maltotriose. Evident FLI firmation of the presence of bacteria through BLI imaging, FLI
and BLI signals were observed on the catheters which were and PAI were conducted (Fig. 6, before treatment). Mice were
4
N A T U R E C O M M U NI C A TI O N S |
( 2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 41 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
A
R
T
I
C
L
E
3 cm
c
a
Epi-Fluorescence
Control muscle
Infected muscle
10 mm
10.0
1 h
3 h
5 h
20 h
Max
8.0
× 108
6.0
4.0
Radiant efficiency
p/s/cm2/sr
Min
(
)
µW/cm2
b
d
ns
1×1010
400
300
200
100
0
E coli (108 CFU)
Infected muscle
Control muscle
8×109
6×109
4×109
2×109
0
Heat inactivated E coli (108 CFU)
ns
* P = 0.0310
** P = 0.0651
*** P = 0.0346
**** P = 0.0055
* P = 0.0004
** P = 0.0002
1 h
3 h
5 h
20 h
Before injection
After injection
Fig. 3 In vivo validation of Cy7-1-maltotriose in an E. coli-induced myositis murine model. a F l u o r e s cen c e i m a gi n g s h o w s a c c u m u l a t io n o f C y7 - 1-
m a l t o tr i o se i n E. coli- i n f e ct e d t h i gh m u s c l e a s e a r l y a s 1 h p o s t s y s t em i c i n j e ct i o n ( r i g h t t h i g h m u s c l e ) . N o e v i de n t a c c u m u la t i o n o f t h e a g e n t i n t h ig h m u s c l e
8
i n je c te d w i th 1 0 C F Us o f h e a t- in a c t i v a t e d E. coli ( l e f t t h i gh m u s c l e ). b B a r p l o t r e p r e s e n t a t i o n o f q u a n t i fie d flu o r e s ce n ce s i gn a l i n r i g h t a n d l e f t t h ig h m u s c l e
o v e r t i m e . A s e a r l y a s 1 h p o s t- in je c t i o n , a s i g n i fic a n t l y h i gh er flu o r e s ce n ce s i gn a l w a s f o u n d i n m u s c l e i n f e c t e d w i t h E. coli ( r i g h t t h i g h ) c o m p a r e d t o m u s c l e
i n f e c t e d w i th h e a t- i n a c t i v a t e d E. coli ( l e f t t h i gh ) ( P = 0 . 03 10 , n = 3 ) . c 3 D r e n d e r e d p h o t o a co u s t i c i m a ge o v er l a i d o n u l t r a s o u n d i m a ge o f a m o us e’s l e f t
( c o n t r o l ) a n d r i g h t ( i n f ec t e d ) t h i gh m u s c l e b e f o r e a n d 2 0 h p o s t- i n j e c t i o n o f C y 7 - 1 - m a lt o t r i o s e . Q u a l i t a t i v e l y , t h e i m a ge o f t h e i n f e c t e d t h i g h m u s c l e
( b o t t o m r i g h t) s h o w s h i g h e r p h o to a co u s t ic s i g n a l r e la t i ve t o i m a g e o f t h e s a m e m u s c l e a c q u i r e d b e f o r e p r o b e i n j e ct i o n ( t o p r i g h t ) a n d i m a ge o f t h e c o n t r o l
t hi g h m u s c l e ( b o t t o m l e f t ) . d B a r p l o t r e p r e s e n t a ti o n o f t h e q u a n t i fie d p h o t o a co u st i c s i gn a l i n t e n s i t y i n t h e p h o t o a co u st i c i m a ge s a c q u i r e d b e f o r e a nd 2 0 h
a f t e r i n j e c t i o n o f C y 7 - 1 - m a lt o t r i o s e . T h e q u a n t ifie d s i g n a l o f t h e i n f e ct e d t h i g h m u s c l e w a s s i gn ific a n t l y h i g h e r p o s t- i n j e c t i o n o f t h e p r o b e c o m p a r e d t o t h at
b e f o re i n j e c ti o n ( n = 4 a n d 5 r e s p e c t iv el y , P = 0 . 0 00 4 ). I n a d d i ti o n , t h e p o s t- i n j e c t i o n s i gn a l i n t h e i n f e c t e d t h i g h m u s c l e w a s s i gn ific a n t l y h i g h e r t h an t h at
o f t he c o n tr o l m u s c l e ( n = 4 , P = 0 . 0 00 2) . B a r g ra p h s s h o w m e a n a n d S . E .M . S t a t i s t i c al a n a l y s i s w a s p e r f o r m e d u s i n g t w o - w a y A N O V A . n s n o s t a t is t i c a l ly
s ig n ific a n t d i f f e re n c e ( P > 0 . 05 ) . T h e s o u r c e d a t a u n d e rl y in g F i g . 3b a n d d a r e p r o v i d e d i n a S o u r c e D a t a fil e .
then divided into two groups where one was administered sub- Discussion
cutaneously a therapeutic dose of vancomycin twice daily After evaluating 18F-labeled analogs of maltose and maltotriose,
(Treated group, n = 5), while the other group was not treated our group demonstrated that an 18F-6″-labeled maltotriose
with vancomycin (untreated group, n = 4). After 7 days of showed the most optimal pharmacokinetic and pharmacody-
antibiotic treatment, Cy7-1-maltotriose was administered and namic properties to specifically image bacterial infections36. The
imaging completed 20 h post-injection. No BLI nor FLI signal and radio-label, in this case, was at the non-reducing end of the
minimal PAI signal in the wound were observed in the treated molecule and the addition of 18F did not interfere with its
group post-treatment (Fig. 6a, b, Treated group—After). While internalization through the maltodextrin transporter36. While the
the untreated group showed evident BLI, FLI, and PAI signal in PET tracer would have extensive applications in a hospital setting,
the wound. In addition, a significant decrease in the fluorescence an optical imaging approach would have advantages in an out-
and photoacoustic signal (4.33 0.96 × 108 vs 1.48 0.15 × 108 patient setting or in the ER for the local evaluation of suspected
radiance efficiency and 0.99 0.09 vs 0.37 0.06 a.u. respectively, sites of infection. PAI has the added advantage of increased depth
P < 0.0001, n = 4) was observed in the images collected post- penetration, translation into ultrasound-based settings and the
treatment compared to before treatment (Fig. 6c, d).
ability to provide valuable real-time anatomical information. We
In a similar animal model, different amounts of the biolumines- therefore set out to develop a photoacoustic version of the pre-
cent Xen 36 strain Staphylococcus aureus (104, 106, or 108 CFU; viously reported maltotriose imaging agent. Unfortunately, the
n = 3, 3, and 5 respectively) were inoculated subcutaneously in a ability of the azide-functionalized maltotriose to block 3H-
small wound formed on the back of the mice. FLI images 18 h post- maltose uptake in E. coli dropped when the azide was functio-
injection showed accumulation of Cy7-1-maltotriose in the wound nalized at the 6″ position (Azide-6″-Maltotriose) compared to the
in all three mice groups where the location of the FLI signal directly 1 position (Azide-1-Maltotriose) (Supplementary Fig. 5). These
correlated to that of the BLI signal (Fig. 7a). In addition, a results confirm previous reports stating the adverse effects of
significant increase in the quantified fluorescence signal was functionalization of the non-reducing end of the maltodextrin on
observed with an increase in quantified BLI signal (i.e. increase in its binding affinity to maltose-binding protein (MBP) which can
CFU in the wound) (Fig. 7b).
severely compromise internalization through the maltodextrin
N A T U R E C OM M U N I C A T I ON S |
(2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww . n a t u r e . c o m / n a t u r e c o m mu n i ca t i o n s
5

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
a
c
2 h
4 h
18 h
Control muscle
Infected muscle
10 mm
Max
Epi-Fluorescence
3.0
× 109
2.0
1.0
Min
Radiant efficiency
p/s/cm2/sr
(
)
µW/cm2
ns
d
400
300
200
100
0
4
3
2
1
0
Cy7-1-Maltotriose
Cy7-1-Maltohexose
b
* P < 0.0001
** P = 0.0013
*** P = 0.0353
6
Cy7-1-Maltotriose
Cy7-1-Maltohexose
5
4
3
2
1
* P = 0.3216
** P = 0.0518
*** P = 0.0275
Infected muscle Control muscle
0
2 h
4 h
18 h
Fig. 4 In vivo comparison between Cy7-1-maltotriose and Cy7-1-maltohexose in an E. coli-induced myositis murine model. a I n v i v o flu o r e s ce n ce i m ag e s
a t 2 , 4 , a n d 1 8 h p o s t- i n j e c t i o n o f e i th er C y 7- 1 -m a lt o t r i o s e ( t o p) o r C y 7 - 1 - m a lt o h e x o s e ( b o t t o m ) s h o w i n g a c c u m u l a t io n o f b o t h p r o b e s i n t h e r i g ht t h i g h
m u s cl e (E. coli) . Q u a l i t a ti v e l y h i gh er flu o r e s c e n ce s i g n a l i n t h e i n f e c t e d m u s c l e ( r i g h t t h i g h ) w h e n i n j e ct i n g C y 7 - 1 - m a lt o t r i o s e ( t o p) c o m p a r e d t o C y7 - 1-
m a l t o h e xo se ( b o tt o m ) i s o b s e r ve d. b B a r p l o t r e p r e s e nt a t i o n o f r a t i o o f flu o r e s ce n ce i n t h e i n f e c t e d t h i g h v s c o n t r o l t h i g h . H i g h e r flu o r e s ce n ce s i g na l i n t h e
i n f e c t e d m u s c l e ( r i g h t t h i g h ) w h e n i n j ec t i n g C y 7- 1 -m a lt o t r i o s e ( n = 6 ) w a s o b s e r v e d c o m p a r e d t o i n j e ct i n g C y 7 - 1 - m a lt o h e x o s e ( n = 4 ) a n d t h is w a s
s ig n ific a n t a t 1 8 h p o s t- i n j e c t i o n ( P < 0 . 02 7 5 ). c 3 D r e n d e r e d p h o t o a c o u st i c i m a ge o v e r l a i d o n u l t r a s o u n d i m a ge o f a m o u s e 2 1 h p o s t- i n j e c t i o n o f C y7 - 1-
m a l t o tr i o se ( to p ) a n d C y 7 - 1 -m a lt o h e x o s e ( b o t t o m ) . I m a ge s s h o w i n f e c t e d ( r i g h t t h i g h ) a n d c o n t r o l ( l ef t t h i g h ) m u s c l e . E v i d e n t P A s i gn a l i s o b s e r v e d i n t h e
i n f e c t e d t h i g h m u sc l e w h e n i n j ec t i n g e i th er c o m p o u n d s w h il e m i n i m a l s i gn a l i s o b s e r v e d i n t h e c o n t r o l t h i g h m u s c l e . d B a r p l o t r e p r e s e nt a t i o n o f t h e
q u a nt ifie d p h o t o ac o u st i c s i g n a l i n t e n s i t y i n t h e p h o to a co u s t ic i ma ge s a c q u i r e d 2 1 h a f t e r i n j e ct i o n o f C y 7 - 1 - m a lt o t r i o s e a n d C y 7 - 1 - m a lt o h e x os e. T he
q u a nt ifie d s i g n a l s h o w s s i g n ific a n t l y h i gh er P A s i g n a l i n t h e i n f e c t e d m u s c l e c o m p a r e d t o c o n t r o l m u s c l e w h e n i n j e ct i n g e i t h e r c o m p o u n d s ( l e f t ) . N o
s ig n ific a n t d i f f e re n ce i n P A s i g n a l w a s o b s e r ve d b e t we e n t h e t w o c o m p o u n d s. S i g n i fic a n t l y h i g h e r i n f e c t e d o v e r t h e c o n t r o l P A s i gn a l r a t i o w a s o bs e r v e d
w h e n i n j e c t i n g C y 7 - 1 - m a lt o t r i o s e (n = 6 ) c o m p a r e d t o C y 7- 1 -m a lt o h e x o s e (n = 3 ) ( P < 0 .0 3 5 3 ) ( r i g h t ) . B a r g r a p h s s h o w m e a n a n d S . E .M . S t a t is t ic al
a n al ys i s w a s p e r f o rm e d u s in g t wo -w a y A N OV A . ns n o s t a t is t i c a ll y s i gn ific a n t d i f f e r e n c e (P > 0 .0 5 ) . T h e s o u r c e d a t a u n d e r l y i n g F i g . 4 b a n d d a r e p r ov id e d
i n a S o u rc e D a t a fil e .
transporter38. Ning and co-workers30 previously reported the (Fig. 1). Developing such an intermediate simplifies the functio-
development of a fluorescent derivative of maltohexose which was nalization of the maltotriose and maltohexose scaffold to a variety
functionalized at the reducing end and showed promising results of signaling agents using copper-free click chemistry or copper
in optical imaging of bacterial infections. However, Axer et al.38 catalyzed azide–alkyne reaction. In this work, Cy7 dye was chosen
reported that maltotriose could potentially provide a more opti- due to its great photo and chemical stability and linked to mal-
mal scaffold in bacterial infection imaging agents compared to totriose (n = 1) or maltohexose (n = 4) through azide-DBCO
other maltodextrins (e.g. maltohexose). This led us to believe that copper-free click chemistry (Fig. 1).
a fluorescent derivative of maltotriose can potentially be a
Following synthesis of the probes, it was essential to assess the
superior bacterial infection imaging agent when functionalized at effects of the azide and Cy7 motifs on the ability of maltotriose
the reducing end. Accordingly, we sought to functionalize the dye and maltohexose to internalize into bacteria. This was assessed
at the anomeric side (reducing end) of maltotriose and decided to by running a competition assay between the tested derivatives
also synthesize the maltohexose derivative to properly identify the and 3H-maltose as well as uptake studies (Fig. 2a and b,
optimal scaffold for our bacterial infection imaging agent.
respectively). Both probes were taken up in a wide variety of
We have established a modified synthetic route that produces Gram-positive and Gram-negative bacterial strains (Fig. 2b). The
an azide-1-functionalized maltotriose or maltohexose using an specificity of the probes for imaging live bacteria that contain the
adapted glycosylation procedure37 in two high yielding steps maltodextrin transporter was demonstrated in uptake studies
6
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
2
0
)
1
1
:
1
2
5
0
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
|
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
A
R
T
I
C
L
E
a
c
S. sureus +
Cy7-1-maltotriose
Cy7-1-maltotriose
S. sureus
Epi-Fluorescence Luminescence
S. sureus +
Cy7-1-maltotriose
1.0
0.8
0.6
0.4
2.0
1.5
1.0
0.5
Cy7-1-maltotriose
Max
US
PA
× 109
× 106
Radiant efficiency
p/s/cm2/sr
Radiance
(p/s/cm2/sr)
1 mm
Min
20 mm
(
)
µW/cm2
b
d
2.0×105
2.0×109
1.5×109
1.0×109
40
30
20
10
0
Fluorescence
BLI
S. sureus + Cy7-1-maltotriose
Cy7-1-maltotriose
1.5×105
1.0×105
* P < 0.0001
* P = 0.0248
5.0×104
0.0
5.0×108
0.0
S. sureus + Cy7-
1-maltotriose
Cy7-1-
maltotriose
S. sureus
Fig. 5 In vitro evaluation of Cy7-1-maltotriose in a S. aureus-infected biomaterial model. a F L I a n d B LI i m a ge s s h o w ca s i n g t h e p r es e n ce a n d l ac k o f
S. aureus o n c a t h e te r s . H i g h F L I a n d B L I s i g n a l s w er e o b s e r ve d i n c a t h e t e r s t h a t w e r e i n c u b a t e d w i t h S. aureus f o l l o w e d b y C y 7 - 1 - m a lt o t r i o s e ( le f t )
c o m p a re d t o s t er i l e c a t h e t e r s t h a t w er e o n l y i n c u b a t e d w it h C y 7 - 1 - m a lt o t r i o s e ( m i d d l e ) . S i m i l a r l y , n o F L I s i gn a l a n d o n l y B LI s i gn a l w a s o b s e r ve d o n
c a th e t er s t h a t w e re i n c u b a t e d w it h S. aureus o n l y ( r i gh t ) . b T o t a l F L I a n d B LI s i gn a l s i n c a t h e t e r s i n f e c t e d w i t h b i o l u m i n esc e n t S. aureus a n d i n c u ba t e d w i t h
C y 7 - 1 - m a lt o t r i o se . F l u o r es c e n c e q u a n t ific a t i o n w a s p l o t t e d t o t h e l e f t y- a x e s ( r e d ) w h i l e B LI s i gn a l p l o tt e d t o t h e r i g h t y- a x e s ( b l u e ) a n d s h o w e d
s ig n ific a n t l y h i g h e r F LI s i g n a l i n i n f e c t e d c a t h e t e r s c o m p a r e d t o s t e r i l e c a t h e t e r s p o s t i n c u b a ti o n w i t h a s o l u t i o n o f C y 7 - 1 - m a lt o t r i o s e ( P < 0 .0 0 0 1, n = 3 ).
c A x ia l U S a n d P A i m a gi n g s h o wc a s i n g t h e p r e s e n c e a n d l a c k o f S. aureus o n c a t h e t e r s u p o n i n c u b a ti o n w i t h C y 7 - 1 - m a lt o t r i o s e . A n e vi d e n t P A s i g na l w a s
o b se r v ed i n a x i a l i m ag es o f c a t h e t e r s i n c u b a t e d w it h S. aureus f o l l o w e d b y C y 7 - 1 - m a l t o tr i o s e ( b o t t o m l e f t ) , c o m p a r e d t o s t e r i l e c a t h e t e r s t h a t w e r e o nl y
i n c ub a te d w i th C y 7 - 1 - m a lt o t r i o s e ( b o t t o m r i gh t ) . Y e l l o w a r r o w s m a r k t h e c a t h e t e r ’s o u t l i n e . d B a r p l o t r e p r e s e nt a t i o n o f t h e q u a n t ifie d p h o t o a co u s t ic s i g na l
i n te n si t y i n t h e a x i a l p h o t o a co u s t i c i m a g e s o f i n f e c t e d a n d s t e r i l e c a t h e t e r s p o s t i n c u b a t i o n w i t h C y 7 - 1 - m a lt o t r i o s e . T h e i n f e c t e d c a t h e t e r s ( n = 5 ) h ad
s ig n ific a n t l y h i g h e r P A s i g n a l c o m p a r e d t o s t e r i le c a t h e t e r s (n = 3 ) p o s t i n c u b a t i o n w i t h C y 7 - 1 - m a lt o t r i o s e (P = 0 .0 2 4 8 ) . B a r g r a p h s s h o w m e a n a nd S . E . M .
S ta t i s t i c al a n a l ys i s w a s p e r f o r m e d u s in g t wo -w a y A N OV A . T h e s o u r c e d a t a u n d e r l y i n g F i g . 5b a n d d a r e p r o v i d e d i n a S o u r c e D a t a fil e .
with azide-inactivated E. coli and E. coli mutants that lack maltodextrins in a variety of bacterial strains38,42–48. We con-
components of the maltodextrin transporter (P < 0.0001, n = 3) ducted a comparison between the maltotriose and maltohexose
(Fig. 2b, maroon).
analogs of our imaging agent. Fluorescent maltohexose was pre-
Preclinical evaluation of Cy7-1-maltotriose in E. coli-induced viously used by Ning and co-workers30,32 as a fluorescent imaging
myositis murine model using FLI and PAI was conducted. FLI was agent for bacterial infection and showed specificity to bacterial
used as the primary tool to assess the specificity and uptake kinetics infections in rat models. In vitro competitive and uptake studies
of the probe in bacterial infections because it allows whole-mouse looked similar to that of Cy7-1-malotriose (Fig. 2a, b, respec-
imaging and provides information that can be compared to pre- tively). A comparison study was then conducted in the E. coli-
vious optical imaging agents for infection. The kinetics of accu- induced myositis murine model. These studies demonstrated the
mulation of the probe were examined and illustrate rapid clearance pharmacokinetic advantages of maltotriose over maltohexose
of Cy7-1-maltotriose through the kidneys. Notably, specific signal in vivo. As shown in Fig. 4, both Cy7-1-maltotriose and Cy7-1-
accumulation at the site of the infected muscle occurred very maltohexose showed specific uptake in the infected muscle (right
rapidly (as early as 1 h), again illustrating the potential utility in thigh) which is distinguishable from the control muscle (left
field hospitals where quick decisions need to be made (Fig. 3a, b). thigh) (Fig. 4a, b). Eighteen hours post-injection, Cy7-1-
In addition, photoacoustic images were also able to show a sig- maltotriose had 2.6-fold higher fluorescence compared to that
nificant difference between the infected and control muscle of Cy7-1-maltohexose (15.5 5.0 × 109 and 6.02 0.5 × 109
(Fig. 3c, d, Supplementary Fig. 11). We believe PAI will have radiance efficiency, respectively; P < 0.0275, n = 6 and 4, respec-
several advantages over FLI, as we have discussed in the intro- tively). This can be attributed to the faster clearance of the Cy7-1-
duction particularly the enhanced imaging penetration depth. maltohexose as compared to Cy7-1-maltotriose from circulation
Another potential advantage lies with the instrumentation rapidly due to higher hydrophilicity (CLogP = −5.8 vs −12.3 for mal-
being developed39,40, and the ease with which these handheld totriose and maltohexose, respectively). The in vivo studies match
scanners can be integrated into existing ultrasound systems41.
the observations made in our in vitro influx studies where a much
It was then important to identify the optimum maltodextrin faster uptake was observed for the maltotriose derivative (Fig. 2c,
scaffold to utilize in our bacterial infection imaging agent. A P < 0.0001).
copious amount of work was conducted to assess the difference
Stability studies on both compounds in murine, rat, and
in internalization kinetics, stability, and retention between human plasma as well as phosphate-buffered saline (PBS) over
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
2
0
)
1
1
:
1
2
5
0
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
|
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s
7

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
a
b
Before treatment
After treatment
Treated group
Untreated group
Before After
Before
After
Max
3 cm
Luminescence
8.0
6.0
10 mm
x 104
4.0
2.0
Radiance
(p/s/cm2/sr)
Min
3 cm
Epi-Fluorescence
5.0
c
d
ns
ns
4×109
4.0
1.5
1.0
0.5
0.0
× 108
3.0
Untreated
group (n = 4)
3×109
2×109
Treated
group (n = 5)
2.0
* P < 0.0001
** P = 0.0003
1×109
0
Radiant efficiency
p/s/cm2/sr
µW/cm2
*** P = 0.0134
(
)
Before
After
Before
After
treatment treatment
treatment treatment
6
Fig. 6 In vivo evaluation of Cy7-1-maltotriose in a S. aureus wound infection murine model. a B LI a n d F L I i m a ge s o f m i c e w i t h a w o u n d i n f e c t e d w i t h 1 0
C F U s o f b i o l u m i n e sc e n t S. aureus 1 9 h p o s t- in j e c t i o n o f C y 7- 1 -m a lt o t r i o s e w i t h o u t ( U n t r e a t ed G r o u p) a n d w i t h ( T r e a t ed G r o u p) t r e a tm e n t w i t h v a nc om y c i n
f o r 7 d a y s a n d b e f o r e a n d a f t e r t r e a t m e n t . F L I ( b o t t o m -B ef o r e) s h o w s a c c u m u la t i o n o f t h e p r o b e i n t h e S. aureus l o c a t e d b y B LI ( t o p —B e fo r e ). I n t h e
u n tr e a t e d g r o u p ( l e f t p a n e l ) b o t h B L I a n d F L I s h o wc a s es p r e s e n ce o f S. aureus i n f e c t i o n i n t h e w o u n d a f t e r 7 d a y s ( U n t r e a t ed G r o u p—A f t e r ) . W h i le i n t h e
t re a t e d g r o u p ( r i g h t p a n e l) , c o m p l et e d i s a p p e a r a n ce o f S. aureus i n f e c t i o n w a s o b s e r v e d i n t h e F L I i m a ge a n d c o nfir m e d b y B LI ( T r e a t ed G r o up —A f t e r ).
b 3 D r e n d e r e d P A i m a ge o v e r la i d o n U S i m a g e o f a m o u s e f ro m U n t r e a t e d ( T o p r o w ) a n d T r e a t e d ( B o t t o m r o w) g r o u p s b e f o r e a n d a f t e r t r e a tm e nt . I m ag e s
w e re a c q u i r e d 2 0 h p o s t- i n j e c t i o n o f C y 7- 1 -m a lt o t r i o s e . S i m il a r t o o b s e r v a t i on s i n F L I , t h e T r e a t e d g r o u p s h o w e d l o w e r P A s i gn a l p o s t- t r e a t m e n t ( bo t t o m
6
r
i
g
h
t
)
t
h
a
n
t
h
a
t
b
e
f
o
r
e
t
r
e
a
t
m
e
n
t
(
b
o
t
t
o
m
l
e
f
t
)
.
c
T
o
t
a
l
i
n
v
i
v
o
fl
u
o
r
e
s
c
e
n
c
e
s
i
g
n
a
l
i
n
w
o
u
n
d
i
n
f
e
c
t
e
d
w
i
t
h
1
0
C
F
U
s
o
f
b
i
o
l
u
m
i
n
e
s
c
e
n
t
S
.
a
u
r
e
u
s
1
9
h
a
f
t
e
r
t a il v e i n i n j e c t i o n o f C y 7 - 1 - m a l t o tr i o s e . S i gn ific a n t r e d u c t io n o f F L I s i gn a l a f t e r t r e a ti n g t h e m i c e w i t h v a n co m y ci n f o r 7 d a y s i n t h e t r e a te d g r o u p ( n = 5 , P <
0 . 0 0 0 1 ) w a s o b s er v ed , w h il e n o d i f f e r en c e w a s o b s e r ve d i n t h e u n t r e a t e d g r o u p b e f o r e a n d a f t e r 7 d a y s ( n = 4 ) . d B a r p l o t r e p r e s e nt a t i o n o f t h e q ua nt ifie d
a v e r a g e P A s i g n a l i n t en s i t y i n t h e P A i m a g e s a c q u i r e d 2 0 h a f t e r i n j e ct i o n o f C y 7 - 1 - m a lt o t r i o s e b e f o r e a n d a f t e r a n t i b i o t i c t r e a tm e n t. P A q u a n ti fic a t i on d at a
s ho w ed a s i m i l a r t r en d t o t h a t o b t a i n e d f ro m F L I i m a g e s . B a r g ra p h s s h o w m e a n a n d S . E .M . S t a t i s t i c a l a n a l y s i s w a s p e r f o r m e d u s i n g t w o - w a y A N O V A . n s
n o s ta ti s ti c a ll y s i g n ific a n t d i f f e r en ce (P > 0 . 05 ) . T h e s o u r c e d a t a u n d e r ly i n g F i g . 6c a n d d a r e p r o v i d ed i n a S o u r c e D a t a fil e .
time showed substantial differences in stability between the lower plasma stability provide proof that Cy7-1-maltotriose is the
maltotriose and maltohexose derivatives. Specifically, Cy7-1- superior bacterial-imaging probe. Further investigation into the
maltohexose is shown to rapidly break down into what we exact mechanism of uptake of the two compounds, their meta-
hypothesize to be smaller sugar forms in mere minutes in plasma bolism and their ability to be taken up by metabolically inactive
(<2% intact by 2 h in rat and murine and around 10% in humans, bacteria is necessary. In addition, there is some evidence in
Supplementary Fig. 6a, right, Supplementary Fig. 7b). While Enterococcus that maltotriose and maltohexose could be taken up
around 70% of Cy7-1-maltotriose was intact in murine and rat by different transporters at different rates46. More experiments
after 2 h and no degradation of maltotriose in human plasma was are underway such as using CRISPR-based knockouts of indivi-
observed (Supplementary Fig. 6a, left, Supplementary Fig. 7a). In dual subunits of the two different transport systems to address
addition, both maltotriose and maltohexose were stable in PBS this question.
for up to 24 h (Supplementary Figs. 6a and 7, right). These results
We believe one potential application of Cy7-1-maltotriose is to
directly correlate with previous reports by Axer et al.38. In the PA detect and monitor the treatment of bacterial infections in sus-
imaging study, slightly higher PA intensity in the infected thigh ceptible sites (i.e. wounds, surgical sites, and medical implants). It is
using Cy7-1-maltotriose (n = 6) was observed compared to Cy7- shown that device associated infections account for around 25.6% of
1-maltohexose (n = 3) (288.2 vs 264.6 a.u., respectively) yet this all healthcare-associated infections in the United States6. For
difference was not significant (P = 0.139). But when looking at example, it is projected that the total number of knee arthroplasties
the PA intensity ratio of infected over control muscle, a sig- (TKA) performed per year in the United States will be around three
nificantly higher ratio using the maltotriose derivative vs mal- million by 2030 (refs. 49,50). One of the leading reasons for TKA
tohexose was observed (2.5 vs 2, respectively, P = 0.0353). This failure is due to periprosthetic joint infection (PJI) which can occur
further resembles the data shown in the FLI study and is most in 1–2% of the cases. Unfortunately, current PJI diagnostic tools
likely due to the lower sensitivity of PA in detecting Cy7 which is necessitate sample collection from the prosthetic site and are divi-
more geared for FLI rather than photoacoustic detection (Sup- ded into culture-based tools (ex. peri-implant tissue culture, syno-
plementary Figs. 8–10).
vial culture, and histology)51 and culture-independent tools (ex. Ibis
Evidently, the slower bacterial uptake of the maltohexose PLEX-ID technology52, MALDI-TOF mass spectroscopy53, next-
derivative, its faster clearance due to its hydrophilicity, and its generation sequencing54). The latter tools have not yet been
8
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
( 2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 41 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
A
R
T
I
C
L
E
a
b
104 CFU
106 CFU
108 CFU
Luminescence
4.0
2.0×109
1.5×109
1.0×109
5.0×108
0.0
1.5×106
1.0×106
5.0×105
3.0
Fluorescence
BLI
× 106
2.0
* P < 0.0001
** P = 0.0139
6×104
4×104
2×104
0
*** P = 0.0665
1.0
Radiance
104 CFU
106 CFU
108 CFU
(p/s/cm2/sr)
Epi-Fluorescence
9.0
8.0
× 107
7.0
6.0
Radiant efficiency
p/s/cm2/sr
(
)
µW/cm2
Fig. 7 In vivo evaluation of Cy7-1-maltotriose in a S. aureus wound infection murine model. a F L I a n d B LI o f m i c e w i t h a w o u n d i n f e c t e d w i t h d if f e r e n t
a m o u n t s o f b i o l u m i n esc e nt S. aureus 2 2 h p o s t- in je c t i o n o f C y 7- 1 - m a lt o t r i o s e . F L I i m a ge s ( b o t t o m ) s h o w a c c u m u la t i o n o f t h e p r o b e i n t h e S. aureus i n t h e
4
6
8
s a m e a r ea a s i n d i c at e d b y B L I ( t o p) . b T o t a l i n v i vo F L I a n d B L I s i gn a l s i n w o u n d i n f e c t e d w i t h 1 0 , 1 0 , a n d 1 0 C F U s o f b i o l u m i n es ce n t S. aureus ( n = 3 , 3 ,
a n d 5 , r es p e ct i vel y) a n d 2 2 h a f t e r t a i l v e in i n j ec t i o n o f C y 7- 1 -m a lt o t r i o s e . F l u o r e s ce n ce q u a n t i fic a t i o n w a s p l o tt e d t o t h e l e f t y- a x e s ( r e d ) w h i l e B L I s i g na l
p l ot t e d t o t h e r i g h t y- a x es ( b lu e ) a n d s h o we d i n c r e a s e i n F L I s i g na l w i t h i n c r e a s e i n B LI s i gn a l ( r = 0 .9 28 ; r2 = 0 .8 6 1 2 ) . B a r g r a p h s s h o w m e a n a nd S . E . M .
S ta t i s t i c al a n a l ys i s w a s p e r f o r m e d u s in g t wo -w a y A N OV A . T h e s o u r c e d a t a u n d e r l y i n g F i g . 7b a r e p r o v i d ed i n a S o u r c e D a t a fil e .
adopted in the clinic due to high cost and lack of sufficient which allows serial imaging without having to administer the
validation55,56. Thus, a non-invasive tool for PJI diagnostic which probe repeatedly (Supplementary Fig. 14).
does not rely on sampling from the site will be very valuable and
In summary, a maltotriose-based infection imaging agent
allow differentiation from inflammation which is often confused functionalized with an optical dye at the anomeric carbon using
with infections in these situations as they present with similar copper-free click chemistry was developed. In vitro evaluation
symptoms. Since S. aureus is the most prevalent pathogen in showcases the efficacy and specificity of the probe to be taken up
medical device infections and accounts for ~32% of medical device by a variety of metabolically active Gram-positive and Gram-
infections6, we decided to assess S. aureus infections of biomaterials negative bacteria. In vivo assessments and in vitro stability studies
using PAI and FLI upon incubation with Cy7-1-maltotriose. As showed superior performance of the maltotriose-based probe
shown in Fig. 5, Cy7-1-maltotriose is able to differentiate infected compared to its maltohexose analog. Molecular fluorescence and
from uninfected catheters both using FLI and PAI. This provides PAI of bacterial infection was demonstrated and showed the
the possibility of using PAI to image implants particularly in the ability to specifically detect bacterial infections in myositis and
joints as well as fracture fixtures to screen for infections.
wound infection models and on biomaterial. Evaluations in S.
We then evaluated the ability of our imaging platform in aureus-infected wound model showcased the ability of the probe
assessing wound infections as well as determining the effective- to detect and differentiate between wounds infected with different
ness of antibiotic treatment in vivo. S. aureus is used in this study amounts of CFUs as well as determine the effectiveness of van-
since it is the most common cause of SSIs and results in a 5% comycin treatment. While the Cy7 dye used in this imaging agent
increase in mortality8. Antibiotic treatment reduced the bacterial possesses many great characteristics such as in vivo stability and
burden and the probe uptake reflected this decrease and showed optimum pharmacokinetics when bound to maltotriose, this dye
significant differences in signal between treated and untreated is not approved for clinical use and is more suitable for FLI than
groups in both FLI and PAI (Fig. 6). In addition, both FLI and PAI. This could explain the higher sensitivity in detecting this
PAI were able to distinguish between the treated and untreated probe using fluorescence imaging compared to PAI (Supple-
groups (Fig. 6c, d, P = 0.0003 and 0.0134, respectively) illustrating mentary Figs. 8–10). Hence, we are investigating attachment of a
the utility of this platform in assessing the effectiveness of variety of clinically approved dyes as well as dyes more geared for
treatment.
PAI to further enhance its performance. More specifically, the dye
Further FLI evaluation showed an increase in FLI signal (i.e. needs to have great plasma stability, high photostability, low
Cy7-1-maltotriose accumulation) with an increase in CFU of S. toxicity and ease of synthesis in a GMP facility. Furthermore,
aureus in the wound (i.e. increase in BLI signal). Wounds infected possessing more red-shifted as well as distinctive absorbance
with as low as 104 CFU were detectable by FLI of Cy7-1- characteristics from intrinsic signals (such as hemoglobin and
maltotriose and showed a strong correlation with the location of melanin) will further improve imaging depth and signal quanti-
the BLI signal (Fig. 7a, b). In addition, similar imaging done with fication, respectively. The substitution of the dye may affect the
the S. aureus wound model showed that once injected, the probe distribution of the imaging agent and a thorough in vivo eva-
remains at the infected wound for up to 144 h post-injection, luation of the new probes will be necessary. In addition, while
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
2
0
)
1
1
:
1
2
5
0
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
|
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s
9

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
whole-body PAI systems in humans do not exist, small-animal tubes were flushed with heparin sodium solution (1000 USP/mL, NDC 63739-931-
14) to avoid coagulation of blood inside the tube.
imaging systems have been reported and could provide better
means to evaluate the novel agents in a preclinical setting57,58
.
Bacterial cultures. E. coli was obtained from American Type Culture Collections
(ATCC 33456). E. coli mutants JW3992-1 (LamB and MalG deficient), JW3995
(LamB and Mal K deficient), and JW1613 (LamB and Mal X-PTS permease
deficient) were obtained from E. coli Genetic Resources at Yale (Yale University,
New Haven, USA). Bioluminescent strain of Pseudomonas aeruginosa (Xen 5),
Bacillus subtilis, and Staphylococcus aureus (Xen 36) were obtained from
Perkin Elmer.
We believe that we have identified the ideal scaffold for an
infection imaging agent and with our established synthetic
scheme, we can quickly synthesize and clinically translate these
new probes.
While PAI is growing to be a promising diagnostic tool, it
still possesses several limitations that are being addressed.
Namely, the limited field of view and imaging depth constricts
its utility in humans to localized imaging (no whole-body
imaging). Similar to ultrasound imaging, this tool is highly
dependent on the operator’s experience and means to automate
this technique will help overcome this issue and allow better
and more accurate reproducibility of the images (e.g. Robotic
handles and software tools to precisely delineate imaging plane
for accurate reproducibility)16. Alternatively, three-dimensional
(3D) volume scanning should also assist in reducing the
dependence of the operator’s experience. Furthermore, several
hurdles related to accurate signal quantification (such as dif-
ferentiating intrinsic signals from imaging agent, reduced
accuracy with increased depth and effects of disease progression
or tissue healing on PA signal intensity) need to be addressed to
conduct molecular PAI of disease16. Such limitations can be
somewhat mitigated by proper choice of the photoacoustic dye
(as mentioned above), choosing the best excitation wavelengths
that can assist in differentiating the signal (e.g. background
subtraction), the use of models for fluence correction16 and
other AI tools which can take into account any PA signal
intensity changes occurring during structural changes caused
by disease progression, inflammation, and healing.
Overnight culture conditions. E. coli overnight (O/N) culture was prepared by
inoculating a colony in Luria-Bertani (LB) broth (3 mL) in an incubator shaker at
37 ˚C. The mutant E. coli strains and Xen 36, the bioluminescent strain of S. aureus,
were grown in LB with 50 μg/mL of Kanamycin. After 16 h, 600 μL of the O/N
culture was added to 30 mL of LB in a 200 mL flask and placed in an incubator
shaker at 37 ˚C until the bacterial culture reached the log phase (OD600 = 0.5).
Metabolically inactive E. coli was prepared by either treating O/N culture (OD600
=
0.5) with sodium azide (10 mM) and incubating for 1 h in an incubator shaker at
37 °C or by heating the culture to 90 °C for 30 min. All cultures were harvested by
centrifugation and pellets washed three times with HBSS (1×) before suspending at
the concentration of interest.
Bacteria competition assay. Aliquots of 108 CFU of E. coli were first incubated
with test compounds (1 mM) for 1 h at 37 °C. After the first incubation, the bac-
teria culture was centrifuged at 9400 × g for 5 min, and the pellet washed with
HBSS (1×) three times. Pellets were then resuspended in a solution of 3H-maltose
(1 μCi in 200 μL HBSS (1×); American Radiolabeled Chemicals, Inc., St. Louis, MO,
USA) and incubated for 30 min at 37 °C. Aliquots were then centrifuged and
washed with HBSS (1×) three times before lysing in a bacterial lysis buffer
(BugBuster, EMD, Billerica MA USA). Activity in bacteria lysates was then counted
in a gamma-counter and protein concentration determined using a bicinchoninic
acid (BCA) assay (Pierce, Thermo Fisher Scientific). Test compounds included
maltose as a positive control, azide-1-maltotriose and Cy7-1-maltotriose. In
addition, aliquots incubated with only 3H-maltose were also included to assess
normal uptake. Results are shown as counts per minute (CPM) normalized to
protein content (μg of protein) per sample (n = 3 per study).
Nevertheless, ultrasound imaging has been widely used to diag-
nose late-stage infections by visualizing the anatomical changes
caused by the spread of infection9. Compact and easily transportable
PAI systems are being produced with state of the art technologies by
many research groups including our own16,41,59–61. This makes us
believe that the translation of PA for infection imaging can be
readily adapted with the pairing of the proper system and imaging
agent16,41,59,60. The development of a maltotriose-based PA agent
accompanied by rapid advances in PAI provides a readily available,
non-invasive, cost-effective, real-time imaging tool that can enable
early detection of bacterial infections during surgery and injury with
high resolution.
Bacteria uptake studies. Aliquots of 108 CFU of E. coli, E. coli mutants, azide-
inactivated E. coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus
aureus (Xen 36) were incubated with the same amount of compound 3a or 3b for
1 h at 37 °C. Aliquots were then centrifuged (9400 × g for 5 min) and washed with
HBSS (1×) three times before lysing in a bacteria lysis buffer (BugBuster for E. coli
and mutants, EMD; B-PER™ Complete Bacterial Protein Extraction Reagent for the
rest of strains, Thermo Scientific™). The fluorescence intensity in bacteria lysates
was measured in a SpectraMax GEMINI EM fluorescent plate reader (Molecular
Devices, San Jose, CA, USA).
In vitro influx studies. Aliquots of 108 CFU of E. coli were incubated with the
same amount of compound 3a or 3b (50 μM) at 37 °C. After each time point (30,
60, 240, and 1080 min), aliquots were then centrifuged (9400 × g for 5 min) and
washed with HBSS (1×) three times before lysing in a bacteria lysis buffer
(BugBuster for E. coli and mutants, EMD; B-PER™ Complete Bacterial Protein
Extraction Reagent for the rest of strains, Thermo Scientific™). The fluorescence
intensity in bacteria lysates was measured in a SpectraMax GEMINI EM fluor-
escent plate reader (Molecular Devices, San Jose, CA, USA).
Methods
Plasma stability studies. Compound 3a or 3b (100 μM) was incubated in either
PBS (1×), murine, human, or rat plasma at 37 ˚C for up to 24 h. At each time point
(0, 2, 4, 10, and 24 h), a 100 μL aliquot of the mixture was taken and added to an
ice-cold acetonitrile solution (200 μL) and vortexed for 10 s. Samples were then
centrifuged at max speed and the supernatant analyzed on the analytical HPLC
column (HPLC method). Compounds 3a and 3b had a retention time of 15.25 and
14.65 min, respectively. Monitoring was at 750 nm and the area of any observed
peak was assessed in each HPLC chromatogram. Bar plot representation of the
compound stability is shown as %intact (Eq. (1)), where
Animals and infection models. All animal models and in vivo experiments were
approved by the Stanford University Institutional Animal Care and Use
Committee.
E. coli-induced myositis murine model. Female nu/nu mice 6–7 weeks old were
anesthetized by isoflurane inhalation (2–3%). 108 CFU of E. coli in 50 μL of HBSS
was injected intramuscularly into the right thigh muscle of the mice. As a control,
108 CFU of heat-inactivated E. coli or E. coli MalG+LamB mutant in 50 μL of
HBSS (1×) was injected intramuscularly in the left thigh.
peak area of compound 3
P
% intact ¼
´ 100:
ð1Þ
Areas of all observed peaks
In vitro fluorescence and photoacoustic characterization. Mouse whole blood
was collected through cardiac puncture and stored in 1.5 mL heparin–lithium-
coated tubes (Eppendorf, cat no.: 022379208) on ice and used within 2 h of col-
lection. A serial dilution of Cy7-1-maltotriose was prepared in either PBS (fluor-
escence and PAI) or murine whole blood (PAI). In the IVIS spectrum instrument,
fluorescence images of 50, 25, 10, 1, 0.5, 0 μM solution of Cy7-1-maltotriose in PBS
were collected in a Corning® black clear bottom 96-well plate. While in PAI, the
samples were measured in a Vevo PHANTOM imaging chamber, where the
samples were injected inside the tubes and imaging conducted using the Vevo3100
LAZR imaging system. Similarly, photoacoustic measurements of 100, 50, 25, 10, 1,
and 0 μM solution of Cy7-1-maltotriose in whole blood were collected after the
Staphylococcus aureus wound infection murine model. Female CD1 or SKH1-
elite mice, 6–8 weeks old were anesthetized by isoflurane inhalation (2–3%). A
small wound on the upper back or lower back for the CD1 or SKH1 mice,
respectively, was formed using a sharp pair of scissors. Staphylococcus aureus in
20 μL saline was inoculated into a small pocket subcutaneously before sealing the
wound with Vetbond adhesive (1469SB; 3M, St. Paul, MN, USA).
Fluorescence imaging in E. coli-induced myositis model. Immediately after
E. coli-induced murine myositis (n = 3), Cy7-1-maltotriose (5 nmol in 2% DMSO/
saline, 100 μL) was injected via the tail vein. Fluorescence images were captured at
10
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
( 2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 41 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
A
R
T
I
C
L
E
1, 3, 5, and 20 h post-injection of Cy7-1-maltotriose. The fluorescence intensity in
the right thigh muscle (E. coli) and left thigh muscle (heat-inactivated E. coli) were
then quantified and analyzed.
prone position for PAI. PA and US images were then collected using a Vevo3100
LAZR imaging system (Vevo®LAZR, VisualSonics, Inc., Canada) with PA-mode
and B-mode, respectively. The PA system connected to an MX-250 transducer
that delivered the nanosecond laser pulses with a wavelength of 700 nm. A cross-
sectional 2D and 3D images of the right and left thigh muscle were acquired, and
3D rendered images presented as colored photoacoustic layer overlaid on top of
the ultrasound images were produced using VevoLAB software (VisualSonics,
Inc., Canada). The whole region of the wound was highlighted using VevoLAB
software and average PA signal intensity quantified and presented as a.u.
PAI in E. coli-induced myositis model. Upon inducing myositis by intramuscular
injection of 108 CFU of E. coli in the right thigh muscle of nu/nu mice, mice were
anesthetized and fixed in the prone position for PAI. PA and US images were then
collected using a Vevo2100 LAZR imaging system (Vevo®LAZR, VisualSonics, Inc.,
Canada) with PA-mode and Ultrasound B-mode, respectively. The PA system was
connected to an LZ-250 transducer that delivered the nanosecond laser pulses with
a wavelength of 700 nm. A cross-sectional two-dimensional (2D) and 3D images of
the right and left thigh muscle were acquired, and 3D rendered images presented as
colored photoacoustic layer overlaid on top of the ultrasound images were pro-
duced using VevoLAB software (VisualSonics, Inc., Canada). Using MATLAB, the
total PA signal intensity of the 3D volume was produced and quantified using Fiji
software and presented as a.u. PA and US images were collected before (n = 5) and
24 h after (n = 4) tail vein injection of Cy7-1-maltotriose (5 nmol in 2% DMSO/
saline, 100 μL injection).
Bacterial burden differentiation. 108 CFU (n = 5), 106 CFU (n = 3), and 104 CFU
(n = 3) of S. aureus were inoculated subcutaneously in the wound in CD1 mice.
Cy7-1-maltotriose (5 nmol in 2% DMSO/saline, 100 μL injection) was then injected
via the tail vein. To ensure no other infections occur, a daily dose of kanamycin
(800 mg/kg) was given to the mice intramuscularly. Fluorescence and biolumi-
nescence images were then captured at 5 and 20 h post-injection of the probe.
Image analysis was conducted using Living Image® software.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
Fluorescence imaging comparison between compounds 3a and 3b. Immedi-
ately after E. coli-induced murine myositis, Cy7-1-maltotriose (compound 3a, n =
6) or Cy7-1-maltohexose (compound 3b, n = 4) (5 nmol in 2% DMSO/saline,
100 μL injection) were injected via the tail vein. Fluorescence images were captured
at 2, 4, and 18 h post-injection of the probes. The fluorescence intensity in the right
thigh muscle (E. coli) and left thigh muscle (control) were quantified and data
analyzed. The data are presented as the ratio of fluorescence intensity in the right
thigh muscle (infected) vs left thigh muscle (control).
Data availability
Data supporting the findings of this work are available within the paper and its
Supplementary Information files. A reporting summary for this article is available as a
Supplementary Information file. The datasets generated and analyzed in the current
study are available from the corresponding author on request. The source data
underlying Figs. 2a–c, 3b, d, 4b, d, 5b, d, 6c, d, and 7b are provided as a Source Data file.
PAI comparison between compounds 3a and 3b. After FLI, PA, and US images
were collected post-injection of either 3a (n = 6) or 3b (n = 3) using a Vevo2100
LAZR imaging system (Vevo®LAZR, VisualSonics, Inc., Canada) with PA-mode
and Ultrasound B-mode, respectively. The PA system was connected to an LZ-
250 transducer that delivered the nanosecond laser pulses with a wavelength of
700 nm. A cross-sectional 2D and 3D images of the right and left thigh muscle
were acquired, and 3D rendered images presented as colored photoacoustic layer
overlaid on top of the ultrasound images were produced using VevoLAB soft-
ware (VisualSonics, Inc., Canada). Using MATLAB, the total PA signal intensity
of the 3D volume was produced and quantified using Fiji software and presented
as a.u.
Code availability
The codes that support the findings of this study are available from https://github.com/
idanstei/Maltotriose-based-probes-for-fluorescence-and-photoacoustic-imaging-of-
bacterial-infections.
Received: 12 November 2018; Accepted: 13 February 2020;
In vivo specificity study to maltodextrin transporter. Myositis in mouse was
induced by injecting 108 CFU of E. coli and 108 CFU of E. coli MalG+LamB
mutant in the right and left thigh, respectively (n = 5). Cy7-1-maltotriose (10 nmol
in 2% DMSO/Saline, 200 μL injection) was then injected via the tail vein. Fluor-
escence images were acquired 3 and 20 h post-injection. After imaging, mice were
sacrificed, both right and left thigh muscle collected, and ex vivo fluorescence
images acquired.
References
1. Fiore, D. C., Fettic, L. P., Wright, S. D. & Ferrara, B. R. Antibiotic
overprescribing: still a major concern. J. Fam. Pract. 66, 730–736 (2017).
2. McKenna, M. Antibiotic resistance: the last resort. Nature 499, 394–396
(2013).
3. Sporer, S. M., Weinstein, J. N. & Koval, K. J. The geographic incidence and
treatment variation of common fractures of elderly patients. J. Am. Acad.
Orthop. Surg. 14, 246–255 (2006).
4. Edwards, R. & Harding, K. G. Bacteria and wound healing. Curr. Opin. Infect.
Dis. 17, 91–96 (2004).
5. Bode, L. G. M. et al. Preventing surgical-site infections in nasal carriers of
Staphylococcus aureus. N. Engl. J. Med. 362, 9–17 (2010).
6. Arciola, C. R., Campoccia, D. & Montanaro, L. Implant infections: adhesion,
biofilm formation and immune evasion. Nat. Rev. Microbiol. 16, 397–409
(2018).
In vitro imaging of S. aureus-infected biomaterial. Sterile catheters were col-
lected from a BD Insyte™ Autoguard™ BC Shielded IV Catheter (ref: 382544, BD,
Franklin Lakes, NJ, USA) and placed in a 106 CFU of S. aureus per mL solution in
an incubator shaker at 37 °C for 2 h. Catheters were then placed in a Cy7-1-
maltotriose solution (50 nmol/mL) and incubated for 37 °C for 1 h before washing
by gently dipping in PBS (1×) solution. As a control, sterile catheters that were not
exposed to infection or infected catheters that were not incubated with the probe
were assessed. BLI and fluorescence images of the catheters were then collected and
analyzed. In addition, the catheters were placed inside a 4% agarose phantom and
axial ultrasound and photoacoustic images collected using a Vevo2100 LAZR
imaging system (Vevo®LAZR, VisualSonics, Inc., Toronto, Canada) and irradiation
at 700 nm. PA and US images were analyzed using VevoLAB software (Visual-
Sonics, Inc., Toronto, Canada).
7. Anderson, D. J. Surgical site infections. Infect. Dis. Clin. North Am. 25,
135–153 (2011).
8. Anderson, D. J. & Kaye, K. S. Staphylococcal surgical site infections. Infect.
Dis. Clin. North Am. 23, 53–72 (2009).
9. Trampuz, A. & Zimmerli, W. Diagnosis and treatment of infections associated
with fracture-fixation devices. Injury 37, S59–S66 (2006).
10. Jain, S. K. The promise of molecular imaging in the study and treatment of
infectious diseases. Mol. Imaging Biol. 19, 341–347 (2017).
11. Sevick-Muraca, E. M. Translation of near-infrared fluorescence imaging
technologies: emerging clinical applications. Annu. Rev. Med. 63, 217–231
(2012).
12. James, M. L. & Gambhir, S. S. A molecular imaging primer: modalities,
imaging agents, and applications. Physiol. Rev. 92, 897–965 (2012).
13. Wang, L. V. & Hu, S. Photoacoustic tomography: in vivo imaging from
organelles to organs. Science 335, 1458–1462 (2012).
14. Mallidi, S., Luke, G. P. & Emelianov, S. Photoacoustic imaging in cancer
detection, diagnosis, and treatment guidance. Trends Biotechnol. 29, 213–221
(2011).
Fluorescence and bioluminescence imaging of treatment study. 106 CFU (n =
9) of kanamycin-resistant S. aureus were inoculated subcutaneously in the wound
in SKH1-elite mice. Mice were then administered kanamycin (800 mg/kg) intra-
muscularly once daily to ensure no other bacterial infections occur. Two days post-
surgery, Cy7-1-maltotriose (10 nmol in 2% DMSO/saline, 200 μL injection) was
injected via the tail vein and bioluminescence and fluorescence imaging were
performed at 19, 45, 69, 93, and 144 h post-injection. Following imaging at 19 h,
the mice were divided into untreated (n = 4) and treated (n = 5) groups where the
first group only received kanamycin and the latter was administered vancomycin
(110 mg/kg) subcutaneously twice daily in addition to kanamycin. After 7 days of
antibiotic treatment, mice were injected again with Cy7-1-maltotriose (10 nmol in
2% DMSO/saline, 200 μL injection) and bioluminescence and fluorescence imaging
performed 24 h post-injection.
15. Beard, P. Biomedical photoacoustic imaging. Interface Focus 1, 602–631
(2011).
16. Steinberg, I. et al. Photoacoustic clinical imaging. Photoacoustics 14, 77–98
(2019).
PAI of treatment study. Twenty and 25 h post-injection of Cy7-1-maltotriose
(before and after treatment respectively), mice were anesthetized and fixed in the
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
2
0
)
1
1
:
1
2
5
0
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
|
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s
11

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
t
t
p
s
:
/
/
d
o
i
.
o
r
g
/
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
2
0
-
1
4
9
8
5
-
8
17. Zhang, E. Z., Laufer, J. G., Pedley, R. B. & Beard, P. C. In vivo high-resolution
3D photoacoustic imaging of superficial vascular anatomy. Phys. Med. Biol.
54, 1035–1046 (2009).
45. Oldham, M. L., Chen, S. & Chen, J. Structural basis for substrate specificity in
the Escherichia coli maltose transport system. Proc. Natl Acad. Sci. USA 110,
18132–18137 (2013).
18. Mari, J. M., Xia, W., West, S. J. & Desjardins, A. E. Interventional
multispectral photoacoustic imaging with a clinical ultrasound probe for
discriminating nerves and tendons: an ex vivo pilot study. J. Biomed. Opt. 20,
110503 (2015).
19. van Oosten, M. et al. Real-time in vivo imaging of invasive- and biomaterial-
associated bacterial infections using fluorescently labelled vancomycin. Nat.
Commun. 4, 2584 (2013).
46. Sauvageot, N. et al. Enterococcus faecalis uses a PTS permease and a host
colonization-related ABC transporter for maltodextrin uptake. J. Bacteriol.
199, e00878 (2017).
47. Licht, A. et al. Structural and functional characterization of a maltose/
maltodextrin ABC transporter comprising a single solute binding domain
(MalE) fused to the transmembrane subunit MalF. Res. Microbiol. 170, 1–12
(2019).
20. Li, L. L. et al. Pathological-condition-driven construction of supramolecular
nanoassemblies for bacterial infection detection. Adv. Mater. 28, 254–262
(2016).
48. Dumont, E. et al. Mechanistic aspects of maltotriose-conjugate translocation
to the gram-negative bacteria cytoplasm. Life Sci. Alliance 2, e201800242
(2019).
21. Wang, Y. et al. Preclinical evaluation of photoacoustic imaging as a novel
noninvasive approach to detect an orthopaedic implant infection. J. Am. Acad.
Orthop. Surg. 25, S7–S12 (2017).
22. Tang, E. N., Nair, A., Baker, D. W., Hu, W. & Zhou, J. In vivo imaging of
infection using a bacteria-targeting optical nanoprobe. J. Biomed.
Nanotechnol. 10, 856–863 (2014).
23. Romero Pastrana, F. et al. Noninvasive optical and nuclear imaging of
Staphylococcus-specific infection with a human monoclonal antibody-based
probe. Virulence 9, 262–272 (2018).
24. Kwon, H.-Y. et al. Development of a universal fluorescent probe for gram-
positive bacteria. Angew. Chem. Int. Ed. Engl. 58, 8426–8431 (2019).
25. Zhou, J., Qi, G.-B. & Wang, H. A purpurin-peptide derivative for selective
killing of gram-positive bacteria via insertion into cell membrane. J. Mater.
Chem. B 4, 4855–4861 (2016).
26. Yao, J. et al. Multiscale photoacoustic tomography using reversibly switchable
bacterial phytochrome as a near-infrared photochromic probe. Nat. Methods
13, 67–73 (2015).
27. Chee, R. K. W., Li, Y., Zhang, W., Campbell, R. E. & Zemp, R. J. In vivo
photoacoustic difference-spectra imaging of bacteria using photoswitchable
chromoproteins. J. Biomed. Opt. 23, 106006 (2018).
28. Jiang, Y. et al. Violacein as a genetically-controlled, enzymatically amplified
and photobleaching-resistant chromophore for optoacoustic bacterial
imaging. Sci. Rep. 5, 11048 (2015).
29. Peters, L. et al. Phototrophic purple bacteria as optoacoustic in vivo reporters
of macrophage activity. Nat. Commun. 10, 1191 (2019).
49. Kurtz, S. M., Ong, K. L., Lau, E. & Bozic, K. J. Impact of the economic
downturn on total joint replacement demand in the United States: updated
projections to 2021. J. Bone Jt. Surg. Am. 96, 624–630 (2014).
50. Pitta, M. et al. Failure after modern total knee arthroplasty: a prospective study
of 18,065 knees. J. Arthroplast. 33, 407–414 (2018).
51. Fernández-Sampedro, M. et al. Accuracy of different diagnostic tests for early,
delayed and late prosthetic joint infection. BMC Infect. Dis. 17, 592 (2017).
52. Arciola, C. R., Montanaro, L. & Costerton, J. W. New trends in diagnosis and
control strategies for implant infections. Int J. Artif. Organs 34, 727–736
(2011).
53. Harris, D. L. G. et al. Rapid identification of Staphylococci from prosthetic
joint infections using MALDI-TOF mass-spectrometry. Int J. Artif. Organs 33,
568–574 (2010).
54. Deurenberg, R. H. et al. Application of next generation sequencing in clinical
microbiology and infection prevention. J. Biotechnol. 243, 16–24 (2017).
55. Tagini, F. & Greub, G. Bacterial genome sequencing in clinical microbiology: a
pathogen-oriented review. Eur. J. Clin. Microbiol. Infect. Dis. 36, 2007–2020
(2017).
56. van Belkum, A., Welker, M., Pincus, D., Charrier, J. P. & Girard, V. Matrix-
assisted laser desorption ionization time-of-flight mass spectrometry in
clinical microbiology: what are the current issues? Ann. Lab. Med. 37, 475–483
(2017).
57. Li, L. et al. Single-impulse panoramic photoacoustic computed tomography of
small-animal whole-body dynamics at high spatiotemporal resolution. Nat.
Biomed. Engl. 1, 977–11 (2017).
30. Ning, X. et al. Maltodextrin-based imaging probes detect bacteria in vivo with
high sensitivity and specificity. Nat. Mater. 10, 602–607 (2011).
31. Ning, X. et al. PET imaging of bacterial infections with fluorine-18-labeled
maltohexaose. Angew. Chem. Int. Ed. Engl. 53, 14096–14101 (2014).
32. Takemiya, K. et al. Novel PET and near infrared imaging probes for the
specific detection of bacterial infections associated with cardiac devices. JACC
Cardiovasc. Imaging 12, 875–886 (2019).
58. Merčep, E., Herraiz, J. L., Deán-Ben, X. L. & Razansky, D.
Transmission–reflection optoacoustic ultrasound (TROPUS) computed
tomography of small animals. Light Sci. Appl. 8, 1–12 (2019).
59. Kim, C., Erpelding, T. N., Jankovic, L. & Wang, L. V. Performance
benchmarks of an array-based hand-held photoacoustic probe adapted from a
clinical ultrasound system for non-invasive sentinel lymph node imaging.
Philos. Trans. R. Soc. A 369, 4644–4650 (2011).
33. Pang, X. et al. Bacteria-responsive nanoliposomes as smart sonotheranostics
for multidrug resistant bacterial infections. ACS Nano. https://doi.org/
10.1021/acsnano.8b09336 (2019).
60. Sivasubramanian, K. & Pramanik, M. High frame rate photoacoustic imaging
at 7000 frames per second using clinical ultrasound system. Biomed. Opt.
Express 7, 312–323 (2016).
34. Gowrishankar, G. et al. Investigation of 6-[¹8F]-fluoromaltose as a novel PET
tracer for imaging bacterial infection. PLoS ONE 9, e107951 (2014).
35. Namavari, M., Gowrishankar, G., Hoehne, A., Jouannot, E. & Gambhir, S. S.
Synthesis of [18F]-labelled maltose derivatives as PET tracers for imaging
bacterial infection. Mol. Imaging Biol. 17, 168–176 (2015).
61. Steinberg, I., Shiloh, L., Gannot, I. & Eyal, A. First-in-human study of bone
pathologies using low-cost and compact dual-wavelength photoacoustic
system. IEEE J. Sel. Top. Quant. Electron. 25, https://doi.org/10.1109/
JSTQE.2018.2866702 (2018).
36. Gowrishankar, G. et al. Specific imaging of bacterial infection using 6ʹʹ -18F-
fluoromaltotriose: a second generation PET tracer targeting the maltodextrin
transporter in bacteria. J. Nucl. Med. 58, 1679–1684 (2017).
37. Khamsi, J., Ashmus, R. A., Schocker, N. S. & Michael, K. A high-yielding
synthesis of allyl glycosides from peracetylated glycosyl donors. Carbohydr.
Res. 357, 147–150 (2012).
38. Axer, A. et al. Harnessing the maltodextrin transport mechanism for targeted
bacterial imaging: structural requirements for improved in vivo stability in
tracer design. ChemMedChem 13, 241–250 (2018).
39. Deán-Ben, X. L. & Razansky, D. Portable spherical array probe for volumetric
real-time optoacoustic imaging at centimeter-scale depths. Opt. Express 21,
28062–28071 (2013).
40. Daoudi, K. et al. Handheld probe integrating laser diode and ultrasound
transducer array for ultrasound/photoacoustic dual modality imaging. Opt.
Express 22, 26365–26374 (2014).
Acknowledgements
Dr. Derek Holman for providing training on the mouse wound model. Dr. Mohammad
Namavari for providing azide-6″-maltotriose intermediate. Dr. Hui-Yen Chuang for
assisting with bacterial culture. Dr. Jonathan Hardy and Dr. Christopher H. Contag for
providing the Xen 36 strains. The Stanford University Small Animal Imaging Facility for
training and help with small-animal imaging studies. Dr. Naoki Nishio, Dr. Nynke S. van
den Berg, Dr. Katheryne Wilson, and Dr. Eben Rosenthal for assistance and allowing us
to use their Vevo3100 LAZR system.
Author contributions
A.Z. conducted all the synthesis and characterization of the probes, designed, carried out
and analyzed experiments, and wrote the manuscript. G.G. designed and carried out
experiments and contributed to the writing of the manuscript. I.S. wrote the code for PA
imaging analysis and assisted with PA image analysis, production, scientific discussion,
and manuscript editing. T.H. assisted with compound stability and metabolism studies
and manuscript editing. S.S.G. designed and supervised the project and contributed to
the writing of the manuscript.
41. Kim, J. et al. Programmable real-time clinical photoacoustic and ultrasound
imaging system. Sci. Rep. 6, 35137 (2016).
42. Dippel, R. & Boos, W. The maltodextrin system of Escherichia coli:
metabolism and transport. J. Bacteriol. 187, 8322–8331 (2005).
43. Dutzler, R., Wang, Y. F., Rizkallah, P., Rosenbusch, J. P. & Schirmer, T. Crystal
structures of various maltooligosaccharides bound to maltoporin reveal a
specific sugar translocation pathway. Structure 4, 127–134 (1996).
44. Quiocho, F. A., Spurlino, J. C. & Rodseth, L. E. Extensive features of tight
oligosaccharide binding revealed in high-resolution structures of the
maltodextrin transport/chemosensory receptor. Structure 5, 997–1015 (1997).
Competing interests
S.S.G. declares competing financial interests with Visualsonics Inc. The rest of the
authors declare no competing interests.
12
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
( 2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 41 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 02 0 - 1 4 9 8 5 - 8
A R T I C L E
Additional information
Open Access This article is licensed under
a
Creative Commons
Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
020-14985-8.
Correspondence and requests for materials should be addressed to S.S.G.
Peer review information Nature Communications thanks the anonymous reviewers for
their contribution to the peer review of this work. Peer reviewer reports are available.
Reprints and permission information is available at http://www.nature.com/reprints
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
© The Author(s) 2020
N A T U R E C OM M U N I C A T I ON S |
(2 0 2 0 ) 1 1 : 1 2 5 0 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 2 0 - 1 4 9 8 5 - 8 | w ww . n a t u r e . c o m / n a t u r e c o m mu n i ca t i o n s
13