Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunit

Article abstract of DOI:10.1039/c1cc10691k

A polymeric composite containing a 2-aminoimidazole derivative was synthesized. It was found that this polymer was resistant to biofilm colonization by Acinetobacter baumannii, no leaching of the 2-aminoimidazole derivative was observed after 2 weeks of treatment with deionized water, and the resulting polymer was not hemolytic.

Full text of DOI:10.1039/c1cc10691k

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COMMUNICATION
Inhibition of Acinetobacter baumannii biofilm formation on a
methacrylate polymer containing a 2-aminoimidazole subunitw
a
a
Lingling Peng,z Joseph DeSousa,z Zhaoming Su,a Bruce M. Novak,a
Alexander A. Nevzorov,a Eva R. Garlandb and Christian Melander*a
Received 4th February 2011, Accepted 10th March 2011
DOI: 10.1039/c1cc10691k
A polymeric composite containing a 2-aminoimidazole derivative
was synthesized. It was found that this polymer was resistant to
biofilm colonization by Acinetobacter baumannii, no leaching of
the 2-aminoimidazole derivative was observed after 2 weeks of
treatment with deionized water, and the resulting polymer was
not hemolytic.
mechanism.7,8 In this vein, we became interested in asking
whether polymeric formulations that contain a 2-aminoimidazole-
based antibiofilm agent would deliver a surface capable of
resisting bacterial attachment. The 2-aminoimidazole (2-AI)
class of anti-biofilm agents has demonstrated potent activity
against both Gram-positive and Gram-negative bacteria, fungi,
and mixed species biofilms.9 Herein we report on our efforts to
create methacrylate polymers that contain active 2-AI molecules
and, using Acinetobacter baumannii as our test organism,
demonstrate that these polymers effectively resist biofilm
formation.
Biofilms, defined as surface attached communities of micro-
organisms,1 play a significant role driving infections of indwelling
medical devices2 (IMD). Once colonized by opportunistic
bacteria, the abiotic nature of the IMD provides an ideal
surface for biofilm formation. Typical treatment for
IMD-related infections involve antibiotic-regimens; however,
bacteria within a biofilm are approximately 1000-fold more
resistant to antibiotics and are especially recalcitrant to antibiotic
treatment when attached to abiotic surfaces.3 Therefore, eradi-
cation of these infections is virtually impossible and necessitates
removal of the device to stem the infection.
The two 2-AI molecular scaffolds that we elected to investigate
for incorporation are depicted in Fig. 1. 2-Aminoimidazole/
triazole (2-AIT) conjugate 110 was the first compound reported
to inhibit and disperse both Gram-positive and Gram-negative
bacterial biofilms through non-microbicidal mechanisms while
2-AI 211 has also been reported as a potent antibiofilm agent.
Given that we were aiming for covalent incorporation into the
methacrylate polymer, we needed to first identify methacrylate
conjugates of either 1 or 2 that retained anti-biofilm activity.
We elected to first explore conjugates based upon 2-AIT 1.
This decision was based upon our previous success using the
2-AIT framework to generate a broad array of anti-biofilm
compounds.9 The two sites of methacrylate conjugation we
pursued were coupling through the 2-amino position as well as
through the para position of the b-methyl styrenyl appendage
(Fig. 2). Starting with the base 2-aminoimidazole, we first
acetylated the 2-amino group using acetic anhydride in trifluoro-
acetic acid as a solvent to determine if acyl substitutions were
tolerated. Unfortunately, 2-AIT 3 only inhibited A. baumannii
biofilm formation by o15% at 100 mM indicating that acylation
abrogates anti-biofilm activity.
Given that infections of IMDs are a leading cause of
hospital-acquired infections (HAIs),4 there has been significant
effort to fabricate surfaces that are resistant to biofilm formation.
One of the most common practices is to impregnate the IMD
with an antibiotic or biocide (such as silver);5,6 however there
are significant issues associated with efficacy lifetime, toxicity
of silver to mammalian cells, and selectivity of impregnated
antibiotics. These issues are further complicated by the rising
occurrence of multidrug resistant bacteria in which many
bacteria are resistant to antibiotics that are approved for
device impregnation. Therefore, alternative methods to create
materials capable of resisting biofilm formation are urgently
needed.
An alternative method to impregnating devices with
antibiotics/biocides is to fabricate non-leaching surfaces
that resist biofilm formation through a non-microbicidal
Next, we synthesized a 2-AIT 4, an analogue of 1 that
contained an amino methylene substituent at the para position.
We envisioned that the primary amine could be employed
as a handle for conjugation to an appropriate methacrylate
derivative.12 Unfortunately, 4 and all derivatives where the
a North Carolina State University, Department of Chemistry, Raleigh,
North Carolina, 27695-8204, USA.
E-mail: Christian_Melander@ncsu.edu
b Agile Sciences, 1791 Varsity Drive, Suite 150, Raleigh,
North Carolina, 27606, USA
w Electronic supplementary information (ESI) available: Synthesis
and characterization data for 2-AI analogues, dose–response curves,
growth curve analysis, and XPS data are provided. See DOI: 10.1039/
c1cc10691k
z These authors contributed equally to this work.
Fig. 1 2-AI-based anti-biofilm agents.
c
4896 Chem. Commun., 2011, 47, 4896–4898
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dose response studies revealed that 5 inhibited A. baumannii
formation with an IC50 of 13 mM. Follow up analysis of
A. baumannii grown in the presence of 13 mM 5 indicated no
defect in the growth curve in comparison to bacteria grown in
the absence of 5, indicating that the 2-aminoimidazole is
modulating biofilm formation through a non-microbicidal
mechanism. We subsequently synthesized the 2-AI/methacrylate
conjugate 9 via click chemistry (ESIw) and assayed for its
ability to inhibit biofilm formation. Paralleling the results
with 5, conjugate 9 inhibited biofilm formation by 495% at
100 mM. Follow up dose response studies and growth curve
analysis revealed that 9 inhibited A. baumannii formation with
an IC50 of 17 mM and was nontoxic to the bacteria.
Fig. 2 Potential 2-AIT 1 analogues for methacrylate conjugation.
primary alkyl amine was functionalized also displayed poor
anti-biofilm properties against A. baumannii (B10% at 100 mM).
Faced with the difficulties associated with obtaining appro-
priated functionalized derivatives of 1 that retained their anti-
biofilm activity, we investigated whether derivatization of 2
would generate 2-AI analogues that retained activity. Based
upon our failure with analogues of 1, coupled with previous
observations in our group that tertiary amides are tolerated
within the reverse amide framework11 and that click chemistry
can rapidly functionalize 2-AI derivatives,9 we focused on
generating tertiary amide analogues of 2 that could rapidly
be appended to methacrylate via click chemistry. With this
goal, we identified reverse amide 5 as a potential conjugate for
evaluation.
With an active methacrylate conjugate in hand, we then
turned to fabricating methacrylate co-polymers that contained
9. The methacrylate co-polymer film was synthesized by the
photo-polymerization of a formulation containing 4 wt.% 9,
35% isobornyl methacrylate co-monomer, and 59% poly-
urethane methacrylate (PUMA) crosslinker using 2-hydroxy-
40-(2-hydroxyethoxy)-2-methylpropiophenone at a concentration
of 2.0 wt.% as the photoinitiator.15 The formulation was exposed
to a 450 W broadband UV lamp for six minutes between two
planes of glass. Energy curable PUMAs are common in the
construction of IMDs16 and provide these desired properties,
therefore this was an appropriate model to study the efficacy of 9
grafted to a polymer surface.
Compound 5 was synthesized as outlined in Scheme 1.
Tridecylamine was Boc-protected (Boc2O/CH2Cl2), N-alkylated
with 8-iodo-1-octyne that, after removal of the Boc group
(TFA/CH2Cl2), delivered secondary amine 6. Amine 6 was
then employed in an aminolysis reaction with glutaric anhydride
to generate carboxylic acid 7 that was then converted to
a-bromo ketone 8 by conversion to the corresponding
acid chloride (oxalyl chloride/cat. DMF/CH2Cl2) followed
by reaction with diazomethane and quenching with HBr.
Finally, the 2-aminoimidazole was installed by condensation
of 8 with Boc-guanidine13 that, following Boc-deprotection
and counterion exchange, delivered 5.
The synthesis and characterization of the PUMA is outlined
in the ESI.w To validate the presence of 9 in the polymer,
matrix infrared spectroscopy and 13C solid state NMR were
both conducted; however the results were inconclusive owing
to the difficulty in distinguishing between the blank and the
polymer with 4% 9 (designated P9). We then turned to X-ray
photoelectron spectrometry (XPS), which proved to be more
fruitful. Samples were soaked in DI water for 24 hours to
remove any non-covalently bonded material, rinsed, and then
dried under reduced pressure. We examined the samples for
changes in the carbon (1s), nitrogen (1s), and oxygen (1s)
content and type between the blank and P9. We found a
marked increase in the nitrogen content (1.7% to 7.5%) along
with the emergence of 0.7% chloride (2p) not found in the
blank, both attributable only to the presence of the 2-amino-
imidazole HCl salt within the polymeric matrix.
Compound 5 was initially screened, in solution, for the
ability to inhibit A. baumannii biofilm formation using the
crystal violet reporter assay.14 Initial inhibition studies indicated
that 5 inhibited biofilm formation by 495% at 100 mM, while
Once fabricated, polymeric strips (both blank strips and P9)
were challenged with either media only or media containing
A. baumannii for 24 hours. After 24 hours, media containing
bacteria had reached the stationary phase (Fig. 3A) and each
polymeric strip was removed and washed thoroughly with
water to remove any loosely adherent bacteria. Each strip was
then stained with crystal violet to visualize the remaining
bacterial mass that was attached (Fig. 3B). The resulting
crystal violet was then solubilized with ethanol and quantified
spectrophotometrically (A540). In comparison to the blank
strips, P9 showed ca. 85% reduction in attached bacteria.
We then monitored bacterial growth in the absence or
Scheme 1 Synthesis of 5. (a) Boc2O, DCM, 0 1C to rt, 1 day, 98%; (b)
NaH, 8-iodooct-1-yne, DMF : PhMe (2 : 1), rt, overnight, 94%; (c)
DCM : TFA (2 : 1), 0–rt, 3 h, quant.; (d) glutaric anhydride, DMAP,
Et3N, DCM, rt, 2 days, 96%; (e) (COCl)2, DMF(cat.), DCM,
ꢀ20 1C–rt, 2 h; (f) CH2N2, Et2O, DCM, 0 1C, 1 h; (g) HBr, 0 1C,
0.5 h, 46% over 3 steps; (h) Boc-guanidine, DMF, rt, 3.5 days, 62%;
(i) DCM : TFA (2 : 1), rt, 2 h, then MeOH, HCl (conc.), quant.
c
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Chem. Commun., 2011, 47, 4896–4898 4897

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activity (ca. 75% reduction assessed by the crystal violet
assay). We then performed leaching experiments with either
methanol or hexanes for 24 hours. After solvent treatment,
P9 still inhibited biofilm development by ca. 75%. We then
analyzed both the methanol extract and hexane extract by
LCMS and were only able to detect the initiator from the
polymerization reaction.
Fig. 3 B = control polymer, S = P9. Subscripts refer to different
samples. (A) Visualization of bacterial growth after 24 hours when
challenged with polymeric composites. LB = media only. OD600 of
B1/B2 = 3.07, S1/S2 = 3.06. (B) Results of crystal violet staining after
polymers were exposed to bacteria or media for 24 hours.
We also probed the importance of co-fabricating the polymer
with a 2-AI/methacrylate conjugate by substituting 5 for 9 in
the polymerization reaction. The resulting polymeric composite
inhibited biofilm formation on its surface by ca. 85%. However,
leaching the polymer in methanol for 24 hours abrogated all
anti-biofilm activity.
Finally, we tested the hemolytic potential of P9. Control
methacrylate polymer or P9 was incubated in the presence of
red blood cells. Both polymers showed no detectable hemolysis,
equivalent to cells treated with phosphate buffered saline.
In conclusion, we have demonstrated that a 2-AI/methracrylate
conjugate retains its anti-biofilm activity when incorporated
into a methacrylate co-polymer. These polymers show only
minor reduction in activity under conditions designed to
remove non-covalently bound monomers. Current efforts in
our laboratory now focus on optimizing these polymeric
materials to achieve greater biofilm inhibition capabilities
and evaluating their efficacy in vivo.
Financial support for this work is gratefully acknowledged
from the National Science Foundation (0930480 to EG
and CM).
Fig. 4 SEM images. (A) Blank polymer. (B) P9. (C) Blank polymer
exposed to bacteria for 24 hours. (D) P9 exposed to bacteria for
24 hours. Images are 10 000ꢁ magnification.
Notes and references
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Given that the crystal violet assay: (1) only reports on the
amount of biomass attached to the surface and (2) may result
in non-specific staining, we wanted to further analyze the
polymer surfaces for biofilm inhibition. To achieve this, blank
polymers and P9 were again exposed to A. baumannii for
24 hours. The resulting strips were then washed and imaged
using scanning electron microscopy (Fig. 4). Blank polymers
allowed biofilm development, while we only observed a few
individually attached bacteria lacking any community structure
on P9, clearly demonstrating that P9 efficiently inhibited
biofilm development on its surface.
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how robust the polymer was to various conditions that would
promote leaching of any non-covalently bound 9 and erode
activity. P9 continually washed with distilled water for up to
14 days (longest time tested) and then exposed to A. baumannii
for 24 hours demonstrated minor reduction in antibiofilm
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4898 Chem. Commun., 2011, 47, 4896–4898
This journal is The Royal Society of Chemistry 2011